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PASPCR Annual Meeting 2019

2019

The Jackson Laboratory, Bar Harbor, Maine October 2-4, 2019

Melanin and melanocyte function in

development, photobiology and disease

Agenda and Abstracts

PASPCR Annual Meeting 2019

The Jackson Laboratory, Bar Harbor, Maine October 2-4, 2019

We recognize our sponsors who helped make

PASPCR 2019 Annual Meeting possible Funding for this conference was made possible (in part) by AR076230 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)*.

We thank L’Oreal and Colgate for financial support of the travel awards and

*The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention by trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

The Jackson Laboratory for

the hospitality and support

President Thomas Hornyak, M.D. Ph.D.

President-Elect

John D’Orazio, M.D. PhD.

Secretary/Treasurer Prashiela Manga, Ph.D.

PASPCR 2019 Annual Meeting Lead Organizer

Craig Ceol, Ph.D.*,†

Organizing Committee Members

Suzie Chen, Ph.D.* John D’Orazio, M.D. Ph.D.†

John Harris, M.D. Ph.D.* Thomas Hornyak, M.D. Ph.D.†

Deborah Lang, Ph.D.*,† Prashiela Manga, Ph.D.†

Manickam Sugumaran, Ph.D.*

*Local organizing committee, †Scientific organizing committee

We wish a special thank you to Erin McDevitt and

Charlie Wray, Ph.D. of The Jackson Laboratory for their contributions to organizing the PASPCR 2019 Annual Meeting.

PASPCR 2019 AGENDA

WEDNESDAY, OCTOBER 2 7:45am Shuttle pickup at Bar Harbor Regency Inn & Atlantic Oceanside Hotel 8:00am Shuttle pickup at Harborside Hotel & Quality Inn 8:15am – 8:45am Shuttle arrival at JAX Light breakfast, coffee/tea 8:45am – 9:00am Remarks from PASPCR President Tom Hornyak 9:00am – 10:35am PLENARY SESSION 1: MELANIN PHOTOBIOLOGY AND PHOTOCHEMISTRY

Invited Speaker: Yu-Ying He, University of Chicago

Zalfa Abdel-Malek, University of Cincinnati From bench to bedside: Unique MC1R selective small peptide analogs of a-MSH for skin cancer prevention and treatment of vitiligo and photosensitivity disorders

Dawn Watkins-Chow, National Human Genome Research Institute, NIH A comparative analysis of pheomelanin-specific mouse mutants

Manickam Sugumaran, University of Massachusetts, Boston Multifaceted melanin biosynthesis in insects is distinctly different from that of mammalian melanogenesis

Soumyadeep Sarkar, Washington State University Circadian clock protein BMAL1 regulates melanin levels through MITF

Jonathan Zippin, Weill Cornell Medical College Investigation of the interplay between melanosome pH and pigmentation in MC1R-deficient melanocytes. 10:35am – 11:00am Break 11:00am – 12:35am PLENARY SESSION 2: MELANOMA SIGNALING

Invited Speaker: Richard White, Memorial Sloan Kettering Cancer Center

Mayumi Fujita, University of Colorado Downregulation of ALDH2 increases MAPK activation and enhances resistance to MAPK inhibitors in human melanoma cells

Miriam Doepner, University of Pennsylvania Influence of melanocyte differentiation state on melanoma susceptibility

John D’Orazio, University of Kentucky Exploitation of the cAMP-enhanced DNA repair pathway as a potential melanoma-targeted strategy

Jason Lui, Boston University School of Medicine Transcriptional co-activator YAP1, but not TAZ, enhances melanoma metastatic potential through regulation of ARP2/3 complex subunit ARPC5

Ranjan Perera, Johns Hopkins All Children’s Hospital MicroRNA-211 promotes aggressive melanoma tumor growth in vivo and contributes to BRAFV600E inhibitor resistance via the DUSP6-ERK5 axis 12:35pm – 2:00pm Buffet Lunch (concurrent PASPCR Council meeting in ) 2:00pm – 3:10pm SPOTLIGHT SESSION

Rachel Belote, Huntsman Cancer Institute Anatomic specific epidermal melanocyte subpopulations are defined early in human skin development

Lee Huang, Boston University School of Medicine Inhibition of PAX3-ETS transcription factor interaction reduces MET expression in melanoma

Rolando Ruiz, University of California, Irvine Does “Oncogene Induced Senescence” explain nevus growth control?

Raj Shah, Rutgers University Concurrent targeting of glutaminolysis and metabotropic glutamate receptor 1 (GRM1) reduces glutamate bioavailability in GRM1+ melanoma

Michele Waters, New York University School of Medicine Melanoma chemoresistance to vemurafenib is mediated by adaptive integrated stress response

Takeshi Yamauchi, University of Colorado The role of ethanol and ALDH2 in activating melanocytes

Dalee Zhou, Weill Cornell Medical College Melanin synthesis is influenced by microenvironmental and metabolic changes in bicarbonate/CO2

3:10pm – 3:30pm Break 3:30pm – 4:30pm KEYNOTE ADDRESS: Ian Jackson, University of Edinburgh 4:30pm – 6:00pm Mentorship Mixer (JAX) 4:30pm Shuttle to hotels for those not attending Mentorship Mixer, dinner on your own 6:00pm Shuttle to hotels for Mentorship Mixer attendees, dinner on your own

THURSDAY, OCTOBER 3

7:45am Shuttle pickup at Bar Harbor Regency Inn & Atlantic Oceanside Hotel 8:00am Shuttle pickup at Harborside Hotel & Quality Inn 8:15am – 9:00am Shuttle arrival at JAX Light breakfast, coffee/tea 9:00am – 10:35am PLENARY SESSION 3: PIGMENT CELL DEVELOPMENT, DIFFERENTIATION & FUNCTION

Invited Speaker: Mayumi Ito, New York University School of Medicine

Pamela Cassidy, Oregon Health and Science University The selenoprotein thioredoxin reductase 1 controls melanin synthesis via redox modification(s) to Mitf

Ivana de la Serna, University of Toledo Bromodomain and extraterminal proteins in melanocyte differentiation

William Frantz, University of Massachusetts Medical School Cell-cell signaling in zebrafish melanocyte regeneration

Melissa Harris, University of Alabama, Birmingham Restoring melanocyte stem cells: A model of drug-mediated regeneration

Lionel Larue, Institut Curie The loss of dicer affects migration and homing of melanocytes and induces hair greying

10:35am – 11:00am Break 11:00am – 12:35am PLENARY SESSION 4: GENETICS AND GENOMICS OF MELANOCYTES AND MELANOMA

Invited Speaker: Mikhail Nikiforov, Wake Forest University

Stacie Loftus, National Human Genome Research Institute, NIH Defining the Spectrum of Mutations for Oculocutaneous Albinism

Revati Darp, University of Massachusetts Medical School Investigating the role of tetraploid intermediates in melanoma progression

Helen Michael, National Cancer Institute, NIH Event sequence of BRAF mutation and UV exposure is not a substantial factor in melanomagenesis in a mouse model of cutaneous melanoma

Eirikur Steingrimsson, University of Iceland MITF regulates the expression of extracellular matrix and focal adhesion genes

Catherine Van Raamsdonk, University of British Columbia Evolution of Endothelin independence in GNAQ driven melanoma 12:35pm – 2:00pm Buffet Lunch 1:00pm – 2:00pm PASPCR General Membership Meeting (lunch may be brought into the JAX Auditorium)

2:00pm – 4:00pm Poster Session

4:00pm Shuttle to hotels, time on your own 5:30pm Shuttle pickup at Harborside Hotel 5:45pm Shuttle pickup at Quality Inn 6:00pm Shuttle pickup at Atlantic Oceanside Hotel 6:00pm – 7:00pm Welcome Reception at the Stone House 7:00pm – 9:30pm Gala Dinner / Banquet at the Stone House

FRIDAY, OCTOBER 4 7:45am Shuttle pickup at Bar Harbor Regency Inn & Atlantic Oceanside Hotel 8:00am Shuttle pickup at Harborside Hotel & Quality Inn 8:15am – 9:00am Shuttle arrival at JAX Light breakfast, coffee/tea 9:00am – 10:50am PLENARY SESSION 5: VITILIGO AND PIGMENTARY DISORDERS

Invited Speaker: John Harris, University of Massachusetts Medical School

Stanca Birlea, University of Colorado Characterizing the Dysregulated Cellular and Molecular Pathways in Vitiligo Lesions that Do Not Respond to Phototherapy

Gisela Erf, University of Arkansas Melanocyte-specific Th1-like initiating- and recall-responses in growing feathers of Smyth chickens with autoimmune vitiligo

Jessica Shiu, University of California, Irvine Imaging and single cell transcriptomics reveal keratinocyte metabolic changes that may drive disease persistence in vitiligo skin

Caroline Le Poole, Northwestern University Adoptive transfer of antigen-specific Tregs controls depigmentation in vitiligo-prone mice

Michael Frisoli, University of Massachusetts Medical School BatF3-dependent dendritic cells are required for depigmentation in a mouse model of vitiligo 10:50am – 11:15am Break 11:15am – 1:05pm PLENARY SESSION 6: MELANOMA PREVENTION AND THERAPEUTICS

Invited Speaker: Christin Burd, Ph.D., Ohio State University

Arup Indra, Oregon State University

A Mitochondrial Complex I Inhibitor drives metabolic reprogramming and sensitizes vemurafenib resistant BRAFV600E mutation bearing metastatic melanoma cells

Sandeep Joshi, University of Maryland School of Medicine Hsp90 inhibition induces melanoma tumor antigens and enhances cellular immunotherapy against melanoma

Fabian Filipp, Helmholtz Zentrum München Targeting glutamate signaling in melanoma

Dinesh Jaishankar, Northwestern University HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Di Qu, Amway Research and Development Improving consistency of MED detection using objective method as the first step to improving the accuracy of SPF determination: A quantitative study

Jessica Flesher, University of California, Irvine Structure-based design of CDC42/RHOJ effector inhibitors for the treatment of cancer 1:05pm Closing remarks

PASPCR Annual Meeting October 2-4, 2019, Bar Harbor, Maine

INVITED SPEAKERS

Name Affiliation Date Time

Yu-Ying He University of Chicago Oct. 2 9 am

Richard White Memorial Sloan Kettring Cancer Center

Oct. 2 11 am

Ian Jackson (Keynote Speaker) University of Edinburgh Oct. 2 3.30 pm

Mayumi Ito New York University School of Medicine

Oct. 3 9 am

Mikhail Nikiforov Wake Forest University Oct. 3 11 am

John Harris University of Massachusetts Medical School

Oct. 4 9 am

Christin Burd Ohio State University Oct. 4 11.15 am

SESSIONS

Sessions Date Time Abstracts

Melanin Photobiology and Photochemistry

Oct. 2 9 am – 10.35 am 1 to 5

Melanoma signaling Oct. 2 11 am – 12.35 pm 6 to 10

Spotlight Oct. 2 2 pm – 3.10 pm 11 to 17

Pigment Cell Development, Differentiation and Function

Oct. 3 9 am – 10.35 am 18 to 22

Genetics and Genomics of Melanocytes and Melanoma

Oct. 3 11 am – 12.35 pm 23 to 27

Vitiligo and Pigmentary Disorders Oct. 4 9 am to 10.50 am 28 to 32

Melanoma Prevention and Therapeutics Oct. 4 11.15 am – 1.05 pm 33 to 38

POSTERS Oct. 3 2 pm – 4 pm 39 to 60


Abstract No: 1 From bench to bedside: Unique MC1R selective small peptide analogs of a-MSH for skin cancer prevention and treatment of vitiligo and photosensitivity disorders

Zalfa A. Abdel-Malek (1), Leonid Koikov (1), Renny Starner (1), Viki Swope (1), Pareth Upadhyay (1), Carrie Haskell-Luevano (2), and James J. Knittlel (3)

Presenting Author

Zalfa Abdel-Malek

[email protected]

Our studies on the a-MSH/MC1R axis in human melanocytes revealed that in addition to

stimulating eumelanin synthesis, activation of this axis enhances nucleotide and base

excision repair pathways, and inhibits UV-induced oxidative stress and apoptosis. The

above combined effects, which enable melanocytes to overcome genotoxic stress and resist

apoptosis, and sunless tanning, prompted us to develop small a-MSH analogs for

application to the skin as topical agents. We have developed tri- and tetrapeptide a-MSH

analogs with unprecedented selectivity for MC1R, and high melanogenic potency on

cultured human melanocytes and melanocyte-containing 3D-cultured skin. The

tetrapeptides are as potent as the first developed full length superpotent a-MSH analog

NDP-MSH (afamelanotide), and 10 fold more potency than a-MSH on tyrosinase activity.

The tripeptides are only slightly less (=10 fold) potent than a-MSH. The tri- and

tetrapeptide analogs are highly efficacious in enhancing repair of DNA photoproducts and

inhibiting UV-induced apoptosis, and have more prolonged melanogenic effects than a-

MSH on cultured human melanocytes. Additionally, similar to a-MSH, these analogs had

antioxidant effects, evidenced by increasing the protein levels of the enzymes

hemeoxygenase 1, peroxiredoxin 1 and NCUFB9, after challenging melanocytes with

H2O2. We expect these analogs to be clinically efficacious for prevention of skin cancer,

including melanoma, and for treatment of vitiligo and photosensitivity disorders.


Abstract No: 2 Circadian clock protein BMAL1 regulates melanin levels through MITF

Soumyadeep Sarkar (1), Kenneth I. Porter (1), Panshak Dakup (1) and Shobhan Gaddameedhi (1,2)

Presenting Author

Soumyadeep Sarkar

[email protected]

Solar ultraviolet radiation B (UVB) is a leading cause of various skin diseases including

photoaging and melanoma. As a protective response, the skin has inbuilt defense

mechanisms including DNA repair, cell cycle, apoptosis, and melanin biosynthesis, of

which, DNA repair and cell cycle have shown to be under circadian control. However, the

mechanism of circadian clock regulation of melanin biosynthesis is not well understood.

In this study, we report that the microphthalmia-associated transcription factor (MITF),

which is a rate-limiting protein in melanin biosynthesis, is expressed in a 24-hour rhythmic

fashion in the presence of core clock protein, BMAL1. Our results further demonstrate that

BMAL1 binds to the promoter region of MITF and transcriptionally regulates its

expression, which positively influences melanin synthesis. Finally, we report that increase

in melanin levels due to BMAL1 overexpression protects skin cells from UVB. In

conclusion, our studies provide novel insights into the mechanism of melanin synthesis and

its role in protection against UVB mediated genomic instability, photoaging, and

melanomagenesis.


Abstract No: 3 Multifaceted melanin biosynthesis in insects is distinctly different from that of mammalian melanogenesis

Manickam Sugumaran

Presenting Author

Manickam Sugumaran

[email protected]

Department of Biology, of Massachusetts Boston, Boston MA 02125

Insects, belonging to the phylum Arthropoda, constitute the largest percentage of animals.

Like mammals, they also use melanin as the exoskeletal pigment. In addition, they uniquely

employ melanin production for defense (innate immunity) and wound healing reactions.

Therefore, melanin is a very important and integral part of insects’ survival mechanism.

Insects use both tyrosinase, known as phenol oxidase or more correctly as o-diphenol

oxidase and laccase, p-diphenoloxidase for melanin biosynthesis. The first enzyme is

responsible for melanin production in the hemolymph, while the second enzyme causes

melanogenesis in the cuticle. Both these enzymes are quite different from mammalian

tyrosinases. Insect dopachrome converting enzyme is also significantly different from

mammalian dopachrome tautomerase as it decarboxylates dopachrome to 5,6-

dihydroxyindole, while simply causing the isomerization of dopachrome methyl ester to

5,6-dihydroxyindole-2-carboxylate methyl ester. Recent biochemical studies indicate that

unlike mammalian system which uses tyrosine and dopa for melanogenesis, insects use

dopamine, the decarboxylated product of dopa as the major precursor for cuticular melanin

formation. Such findings led the discovery of a new enzyme associated with insect

melanogenesis which converts dopaminechrome to 5,6-dihydroxyindole. Finally, insects

do not seem to produce 5,6-dihydroxyindole-2-carboxylic acid thus producing solely 5,6-

dihydroxyindole melanin and no 5,6-dihydroxyindole-2-carboxylic acid melanin.


Abstract No: 4

A comparative analysis of pheomelanin-specific mouse mutants.

Dawn E. Watkins-Chow, Colten Eberhard, Andrew Kemal Kirchmeier, Stacie K. Loftus, and William J. Pavan

Presenting Author

Dawn E. Watkins-Chow

[email protected]

Gray coat color has a multifaceted etiology, with one phenotype class represented by a set

of mouse mutants: dwarf grey (Ggt1dwg), grey-lethal (Ostm1gl), subtle gray (Slc7a11sut),

Clcn7, grizzled (gr) and grey intense (gri). These mutations disrupt the production of

pheomelanin and are phenotypically distinct from gray mutants which result from dilution

of the black eumelanin pigment in the hair, like ashen (Rab27aash), or mutants that display

an age-dependent loss of pigmentation (Mitfmi-vit). In humans, the ratio of eumelanin and

pheomelanin produced by melanocytes directly impacts skin cancer risk, thus

understanding the function and regulation of genes that specifically disrupt pheomelanin

production is of particular interest. We identified a deleterious mutation in grizzled mice

(Mfsd12gr) following a genome-wide association study linking the MFSD12 locus to

variation in skin pigmentation among African populations. To further extend our

knowledge of genes required for pheomelanin synthesis, we used exome sequencing to

identify a candidate mutation for grey intense (gri) and are currently confirming the

causative gene with a complementation test between a Crispr/Cas9-mediated knock-out

allele and the original gri allele. The pigmentation phenotype of gr and gri is being

characterized on a defined genetic background for comparison with previously published

pheomelanin mutants (Ggt1, Ostm1, Slc7a11, and Clcn7). Interestingly, several of these

mutations are associated with overlapping pleiotropic phenotypes, including altered

viability, body size, bone density, and tooth eruption. The varied genetic backgrounds of

the mutants and lack of comparative studies have made it difficult to distinguish cell-type

specific gene function from the influence of genetic background. Thus, we are establishing

each mutant on a defined, uniform genetic background to explore the reoccurring

association between the pleiotropic phenotypes and gray coat color.


Abstract No: 5 Investigation of the interplay between melanosome pH and pigmentation

in MC1R-deficient melanocytes.

Dalee Zhou (1), Qiuying Chen (1), Zalfa Abdel-Malek (2), John D’Orazio (3), Kazumasa Wakamatsu (4), Steven Gross (1), and Jonathan H. Zippin (1, 5)

Presenting Author

Jonathan H. Zippin

[email protected]

Melanosome pH plays an important role in the control of pigmentation. MC1R activity is

integral for pigmentation; however, the role of melanosome pH in the pigmentation of

MC1R deficient melanocytes remains unclear. We asked whether basal melanosome pH is

altered by MC1R activity and found that in mouse and human melanocytes, MC1R activity

does not affect melanosome pH. We recently demonstrated that inhibition of the soluble

adenylyl cyclase (sAC) pathway leads to the elevation in melanosome pH in melanocytes

with wild type MC1R. We now find that sAC has a similar effect on melanosome pH in

human and mouse MC1R deficient melanocytes. Elevated melanosome pH increases the

activity of the pH-sensitive enzyme tyrosinase. In melanocytes with wild type MC1R,

elevated melanosome pH leads to an increase in eumelanin and a decrease in pheomelanin

synthesis. We generated tyrosinase-CRE-ERT2; sAC floxed; Mc1r(e/e) mice to investigate

how melanosome pH impacts melanin synthesis. Mc1r(e/e) mice produce mostly

pheomelanin due to the low expression of eumelanin synthetic enzymes. sAC loss in

neonatal Mc1r(e/e) mice did not lead to darkening of the hair but did enhance the

yellow/red color of the hair. Chemical analysis confirmed that sAC loss leads to elevated

levels of pheomelanin in Mc1r(e/e) mouse hair with no significant change in eumelanin

levels. This suggests that similar to eumelanosomes, elevation of melanosome pH in

pheomelanosomes enhances tyrosinase activity, but in pheomelanosomes this leads to

increased pheomelanin synthesis due to low expression of eumelanin synthetic enzymes.

To explore the mechanism of melanosome pH induced changes to pigmentation in vitro,

we developed a mass spectrometry based technique to trace tyrosine metabolism using C13

labeled tyrosine. With this method, we are able to measure the conversion of tyrosine into

eumelanin and pheomelanin intermediates within hours allowing us to better understand

how melanosome pH affects pigmentation.


Abstract No: 6 Influence of Melanocyte Differentiation State on Melanoma Susceptibility

Miriam Doepner (1), Christopher A. Natale (1), Stuart Jarrett (2), John D'Orazio (2), Todd W. Ridky (1)

Presenting Author

Miriam Doepner

[email protected]

The risk of developing melanoma is substantially less for people with darkly pigmented

skin (1:1000 in the US) than those with lightly pigmented skin (1:38 in the US). Although

this discrepancy is traditionally attributed to the UV shielding effect of melanin pigment,

here we show that other mechanisms also serve to protect dark melanocytes (MCs) from

malignant transformation. At homeostatic baseline, darkly pigmented primary human

MCs are more fully differentiated than lightly pigmented MC, as defined by increased

melanin production, increased expression of classic MC differentiation antigens,

decreased proliferative capacity, and decreased stability of the stem cell marker and

melanoma oncodriver c-Myc. We show that this genetically-determined phenotype in

dark MCs is associated with increased cAMP and PKA signaling, which increases UV-

induced DNA damage repair and genomic stability. Pharmacologic activation of cAMP

and PKA signaling pathways in light MCs increases DNA repair capacity to levels

similar to the basal state of dark MCs. Further, we show that the more differentiated state

of dark MCs confers resistance to the tumorigenic effects of common melanoma

associated mutations. When incorporated into genetically engineered human skin tissues

xenografted to mice, light MCs expressing melanoma oncodrivers develop into

melanoma, whereas the dark MCs (with the same oncodrivers) do not. In summary, the

more differentiated cell state associated with dark MCs promotes genetic stability and

oncogenic resistance, which may explain the melanoma risk difference in darkly and

lightly pigmented skin.


Abstract No: 7

Exploitation of the cAMP-enhanced DNA repair pathway as a potential melanoma-targeted strategy

Stuart G. Jarrett (1), Nathaniel C. Holcomb (1), Katharine M. Carter(1) and John A. D'Orazio (1,2)

Presenting Author

John D'Orazio

[email protected]

cAMP signaling occurs downstream of interactions between the melanocortin 1 receptor

(MC1R) and melanocortins such as a-melanocyte stimulating hormone (a-MSH) in

melanocytes. This ligand-receptor interaction leads to activation of adenylyl cyclase,

generation of cAMP second messenger and downstream UV-adaptive responses such as

up-regulation of melanin production and enhancement of nucleotide excision repair (NER)

to more efficiently repair photodamage and reduce mutagenesis. We determined that the

key molecular event linking cAMP signaling to NER acceleration is phosphorylation of the

ataxia telangiectasia and rad3-related (ATR) protein by protein kinase A (PKA) on the

serine 435 (S435) residue that enhances ATR’s interactions with xeroderma pigmentosum

A (XPA) and augments the 5’ strand incision step of NER. Surprisingly, we found that

prolonged expression of the phosphomimetic form of ATR (S435D) proved toxic to

melanoma cells over time, even in the absence of exogenous damage such as UV radiation.

This suggested to us that PKA-mediated ATR phosphorylation alters ATR function in a

repair-directed way at the expense of critical ATR functions necessary for cellular viability.

Our studies using melanoma cells expressing either wild type (S435) or PKA-

phosphomimetic (S435D) ATR suggest that persistence of the PKA-phosphorylated form

of ATR is associated with DNA replication stress, altered cellular energetics and activation

of apoptosis. We hypothesize that phosphorylation of ATR by PKA alters its function

toward a DNA repair-enhancing phenotype that, unless inactivated in a timely manner,

leads to cell death by replication stress and disordered metabolism.


Abstract No: 8 Downregulation of ALDH2 increases MAPK activation and enhances

resistance to MAPK inhibitors in human melanoma cells.

Zili Zhai (1), Dinoop Ravindran Menon (1), Takeshi Yamauchi (1), Elizabeth J. Kovacs (2,3,4), and Mayumi Fujita (1,2,4)

Presenting Author

Mayumi Fujita

[email protected]

Aldehyde dehydrogenase 2 (ALDH2) is a detoxifying enzyme in ethanol metabolism.

ALDH2 deficiency has been causally linked to many human diseases including upper

aerodigestive track cancers, liver cancer, and colorectal cancer. Here, we investigated for

the first time the involvement of ALDH2 in melanoma biology. We found that ALDH2

expression was downregulated in melanoma tumors, and that ALDH2 was differentially

expressed in human metastatic melanoma cells. We then examined the role of ALDH2 in

regulating MAPK activation because MAPK signaling has a pivotal role in melanoma

biology and cellular response to ethanol and its byproducts. In ALDH2-high A375 and

HS294T melanoma cells, a short exposure of acetaldehyde, a metabolite of ethanol,

increased phosphorylation of MAPKs (ERK, p38, and JNK), whereas MAPK

phosphorylation was alleviated when cells were treated with ALDH2 activator alda-1. On

the other hand, ALDH2 knockout in A375 cells resulted in increased MAPK

phosphorylation and resistance to proliferation inhibition by MAPK/ERK pathway

inhibitors vemurafenib and trametinib. Moreover, Kaplan–Meier survival analysis showed

the association of decreased ALDH2 levels with poor prognosis in metastatic melanoma

patients. Gene set enrichment analysis (GSEA) of these cohorts revealed enriched tumor-

intrinsic pathways and decreased immune/inflammatory pathways. Our data demonstrate

that ALDH2 negatively controls MAPK activation but its deficiency facilitates MAPK

activation and endows tumor cells with greater capacity for therapy resistance.


Abstract No: 9 Transcriptional co-activator YAP1, but not TAZ, enhances melanoma metastatic potential through regulation of ARP2/3 complex subunit ARPC5

Jason W. Lui (1,2), Stephen P. Moore (1), Kelsey Ogomori (3), Deborah Lang (1)

Presenting Author

Jason W. Lui

[email protected]

Melanoma is a tumor with a high degree of metastasis. Transcriptional co-activators YAP1

(Yes associated protein 1) and TAZ (transcriptional coactivator with PDZ binding domain)

are implicated in cancer metastasis by acting as a drivers of pro metastatic genes. They are

often thought of as having overlapping functions and redundancy in their downstream

targets. ARP2/3 is a 7 subunit actin nucleating complex that is linked to cancer mobility

and metastasis through its regulation of actin dynamics. Previous studies show that two

different isoforms, ARPC5 and ARPC5L, can replace one another as a part of the ARP2/3

subunit. Additionally, it has been shown that actin dynamics can differ depending on which

isoform is in the complex. Here we show that YAP1 and TAZ are expressed in a panel of

melanoma cell lines, but only inhibition of YAP1 (and not TAZ) results in decreased cell

numbers, formation of focal adhesions, cell migration, and the ability to invade matrigel.

RNA-sequencing analysis revealed the ARP2/3 subunit ARPC5 but not the isoform

ARPC5L as a specific target of YAP1 upregulation. Direct inhibition of ARPC5

phenocopied YAP1 inhibition in terms of decreased cell migration and focal adhesion

formation but not growth in melanoma cells. Finally, only YAP1 and direct ARPC5

inhibition led to increased numbers of ARP2/3 complexes with ARPC5L as opposed to

ARPC5. Our findings suggest that YAP1, but not TAZ, is able to drive melanoma invasion

partly through its regulation of ARPC5, thus altering ARP2/3 downstream actin dynamics

towards a more invasive phenotype.


Abstract No: 10 MicroRNA-211 promotes aggressive melanoma tumor growth in vivo and contributes to BRAFV600E inhibitor resistance via the DUSP6-ERK5 axis

Bongyong Lee1,3, Anupama Sahoo3, Iqbal Mahmud8, Junko Sawada1,3, John Marchica1,3, Sanjay Sahoo3, Fabiana I. A. L. Layng4, Darren Finlay4, Joseph Mazar5, Piyush Joshi1, Masanobu Komatsu1,3, Kristiina Vuori4, Petrus R. de Jong4, Timothy Garrett8, Animesh Ray6,7, and Ranjan J. Perera 1,2,3*

Presenting Author

Ranjan J. Perera

[email protected]

The microRNA miR-211 is an important metabolic regulator of tumor cell behavior in the

skin cancer melanoma in human. To identify its tumor-promoter function, we ectopically

expressed miR-211 in non-pigmented melanoma cell lines A375 and LOX-IMV1

(BRAFV600E-mutants) in mouse xenografts and observed that, miR-211 forced-expressed

cells promote aggressive tumor growth accompanied by increased cellular proliferation and

angiogenesis. miR-211 directly inhibits DUSP6, a Dual Specificity Phosphatase 6, as

confirmed by RNA immunopurification with RNA-seq (RIP-seq) followed by target

cleavage assays. We provide supporting evidence that DUSP6 inhibition confers resistance

of melanoma cells to the BRAFV600E inhibitor vemurafenib and to the MEK inhibitor

cobimetinib, with associated increases in ERK5 phosphorylation. These findings are

consistent with a model in which miR-211 regulates melanoma tumor proliferation and

BRAFV600E inhibitor resistance by inducing ERK5 signaling. We reveal that miR-211

regulates cellular and xenograft lipid production that could play an important role in drug

resistance. Our results illustrate that miR-211-ERK5 axis represents an important and

sensitive regulatory arm in melanoma with BRAF kinase activating mutations, which occur

in approximately 60% of human melanomas. Finally, our current and previous studies

support that miR-211 is an important melanoma miRNA, and its behavior is context-

dependent and useful in theranostic applications.


Abstract No: 11 Anatomic specific epidermal melanocyte subpopulations are defined early in human skin development

Rachel L. Belote(1), Daniel Le(2), Ashley Maynard(2), Aaron D. Tward(3), Adriane Sinclair(4), Laurence Baskin(4), Spyros Darmanis(2), and Robert Judson-Torres(1)

Presenting Author

Rachel Belote

[email protected]

Epidermal melanocytes are often considered a homogenous group of cells. However,

human skin is phenotypically diverse with distinct morphological and functional

characteristics based on anatomic location. For example, fibroblasts have topographical

dependent gene expression patterns that are defined early in development and retained in

adulthood. Epidermal melanocytes also exist across all anatomical locations yet baseline

pigmentation differs from location to location. We hypothesize that, like fibroblasts,

distinct subtypes of human epidermal melanocytes occupy distinct anatomical locations.

In this study, we assessed the transcriptional diversity of freshly isolated human fetal,

neonatal, and adult melanocytes from multiple anatomical locations using single cell

RNAseq. Analysis of 6760 epidermal melanocytes from 22 patients identified distinct

transcriptional programs that change with development and age. Within age groups, we

have identified distinct subtypes of human melanocytes from lineages that diverge by day

67 of human development. At the single cell level, each subtype is enriched, but not

exclusive to, either hair-bearing, glabrous or foreskin. Our study is the first to profile

human epidermal melanocyte development and diversity at the single cell level and our

discoveries suggest that major subtypes of melanoma - including acral and cutaneous -

derive from distinct cells of origin.


Abstract No: 12 Inhibition of PAX3-ETS transcription factor interaction reduces MET expression in melanoma

Lee Huang, Yougang Zhai, Jennifer La, Adil Haresi, Stephen Moore, Jason Lui, Deborah Lang

Presenting Author

Lee Huang

[email protected]

Inhibition of PAX3-ETS transcription factor interaction reduces MET expression in

melanoma Lee Huang, Yougang Zhai, Jennifer La, Adil Haresi, Stephen Moore, Jason Lui,

Deborah Lang Department of Dermatology, Boston University School of Medicine,

Boston, MA, 02118 PAX3 and multiple members of the ETS family are transcription

factors that have been implicated in promoting oncogenesis in a variety of cancers. Our

experiments have demonstrated that YK-4-279, a small molecule inhibitor, interrupts the

interactions between these proteins in melanoma. Through treatment, we observed a

reduction in cell growth and migration in vitro and attenuated melanoma progression in a

clinically relevant mouse model (BrafCA/PtenloxP/Tyr::CreERT2). These findings

indicate potential for YK-4-279 as a melanoma therapeutic. However, the downstream

effects of the drug’s inhibition have yet to be elucidated. To investigate, we tested for

changes in the expression of MET, a receptor tyrosine kinase that is linked to driving

tumorgenesis. The interaction between PAX3 and ETS1 has been previously shown to

synergistically drive MET expression. The results of western analysis, luciferase assays,

and immunofluorescent staining show that MET expression decreases both in vitro and in

vivo after treatment with YK-4-279. Thus, inhibiting interactions between PAX3 and ETS

family members using YK-4-279 mitigates the effects of progression by affecting

downstream elements such as MET. We will test to see if YK-4-279 has similar effects on

other downstream receptor tyrosine kinases as well. Our experiments further support the

potential of YK-4-279 and the inhibition of PAX3-ETS interactions as a treatment option

for melanoma therapy.


Abstract No: 13 Does “Oncogene Induced Senescence” explain nevus growth control?

Rolando Ruiz(1,2), Chi-Fen Chen2, Emaad Razzak(2), Priya Vasudeva(3), Tatiana Krasieva(4), Jessica Shiu(3), Michael G. Caldwell(2), Huaming Yan(5), John Lowngrub(2,5), Anand Ganesan(2,3), Arthur Lander(1,2)

Presenting Author

Rolando Ruiz

[email protected]

Mutational activation of the BRAF proto-oncogene in melanocytes triggers a proliferative

burst followed by growth arrest, resulting in the formation of benign melanocytic nevi. As

the same mutation is the most common driver mutation in melanoma, it is important to

understand why nevi invariably stop growing, while melanomas do not. The idea that

oncogene expression directly triggers permanent withdrawal from the cell cycle—the

“oncogene-induced senescence (OIS) hypothesis”—is supported by classical in vitro

studies, but has been difficult to investigate in vivo. By analyzing nevus size distributions

in a mouse model in which the formation of large numbers of nevi can be induced by

conditionally activating Braf in melanocytes, we infer that nevus growth arrest cannot be

the result of any sort of cell-autonomous probabilistic process, but instead requires a

significant degree of synchronization among cells. Such a result is consistent with a

mechanism based on cell-cell communication. Indeed, we can easily fit nevus size

distribution data using mathematical models in which arresting cells secrete an arrest-

promoting factor. To assess whether such a factor might be among those commonly

associated with the “senescence associated secretory phenotype (SASP)”, we used single

cell RNA sequencing to characterize the transcriptome of mouse nevus melanocytes 50

days after induction, a time when they are fully growth arrested. Interestingly, we find that

such nevus melanocytes do not exhibit a strong SASP signature, especially when compared

with other melanocytes in the same skin. These results suggest that mechanisms of growth

arrest in nevi are independent of senescence, at least as traditionally defined, and more

related to the cooperative mechanisms that mediate size control of normal tissues. This

view is also consistent with the clinical observation that human nevi regrow to about their

original sizes after incomplete excision.


Abstract No: 14 Concurrent targeting of glutaminolysis and metabotropic glutamate receptor 1 (GRM1) reduces glutamate bioavailability in GRM1+ melanoma

Raj Shah (1, 2), Simar Singh (3), Keedrian Olmstead (3), Darling Rojas (1), Kevinn Eddy (1), Fabian Filipp (3, 4, 5) and Suzie Chen (1, 2, 6)

Presenting Author

Raj Shah

[email protected]

Aberrant glutamatergic signaling has been implicated in altered metabolic activity in

several types of cancer including melanoma. Previously, we illustrated the role of

metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in

vitro and spontaneous development of metastatic melanoma in vivo. Glutamate, the natural

ligand of GRM1, is one of the most abundant amino acids in humans and the predominant

excitatory neurotransmitter in the central nervous system. Using a set of isogenic cell lines,

we demonstrated correlations between GRM1 and glutaminase (GLS) expression.

Metabolomics reveals elevated levels of glutaminolytic mitochondrial tricarboxylic acid

(TCA) cycle intermediates especially glutamate in GRM1+ melanoma cells. Elevated

intracellular pool of glutamate to satisfy the demand to fuel the growing tumor cells is the

result of increased conversion of glutamine to glutamate via GLS. Using a rational drug-

targeting strategy, we critically evaluate metabolic bottlenecks with the goal to limit

glutamate availability in tumors. CB-839, a potent, selective, and orally bioavailable

inhibitor of GLS, plus riluzole, a functional inhibitor of glutamatergic signaling, inhibited

cell proliferation in vitro and significantly suppressed tumor growth in vivo in two

independent xenograft mouse models of melanoma, with no obvious symptoms of toxicity.

Western blot analysis on excised tumor lysates demonstrated reduced ERK and AKT

activities. Rigorous evaluation of the xenograft studies revealed gender biased responses

to treatment strategies. Using LCMS analysis, we determined that the circulating blood

plasma concentration of treatment modalities may provide explanations to differential

treatment responses. Currently we are performing stable isotope tracing to determine the

source of carbon fluxing into glutamate. These insights, combined with our data, support

the rationale to target glutamate bioavailability to combat GRM1+ human neoplasia.


Abstract No: 15 Melanoma Chemoresistance to Vemurafenib is Mediated by Adaptive Integrated Stress Response

Michele Waters , Martha Vega , Genevieve Torres , Seth Orlow , Prashiela Manga

Presenting Author

Michele Waters

[email protected]

Metastatic melanoma (MM) is highly refractory to treatment, making it the deadliest form

of skin cancer. Treatment with the BRAF inhibitor vemurafenib (VMF), while initially

effective, frequently fails long term due to acquired chemoresistance. We have previously

shown that melanocyte chemosensitivity is modulated by activation of the integrated stress

response (ISR); here, we investigate a role for this pathway in melanoma chemoresistance.

The ISR is triggered by various stressors and is characterized by increased eIF2a

phosphorylation, which blocks cap-dependent translation and activates cell survival

pathways via preferential expression of key transcription factors such as ATF4. In this

study, we developed VMF-resistant melanoma lines by step-wise conditioning of

SKMEL19 melanoma cells using 3-(SKMEL19V3) and 6µM (SKMEL19V6) VMF.

Prolonged treatment resulted in cell lines with significantly increased resistance to VMF-

induced cell death compared to parental lines, (p<0.0001 for SKMEL19V6 vs. control after

48hr, 18µM VMF). We investigated whether the ISR contributed to chemoresistance and

found that VMF sensitivity correlated with modulated eIF2a. VMF-resistant cell lines were

characterized by rapid phosphorylation/de-phosphorylation of eIF2a (between 1-3hrs),

while phosphorylation was only observed 24 hours after treatment in chemosensitive lines.

Pre-treatment with guanabenz (GB), a chemical inhibitor of eIF2a de-phosphorylation,

significantly increased chemosensitivity of resistant lines (p<0.0001 for SKMEL19V6 +/-

GB, dosed with 18µM VMF). Furthermore, GB treatment resulted in increased levels of

phospho-eIF2a (p=0.006, 6hrs), cleaved-PARP (a marker for cell death; p=0.043, 6hrs) and

phospho-p62 (a marker for modulated autophagy; p=0.0138, 3hrs) for SKMEL19V6.

Activation of the eIF2a/ATF4 pathway has previously been linked to stress-induced

autophagy. Thus ISR-mediated upregulation of autophagy soon after dosing may underlie

chemoresistance in these cells.


Abstract No: 16 The role of ethanol and ALDH2 in activating melanocytes

Takeshi Yamauchi (1), Akiko Matsumoto (2), Syosuke Ito (3), Kazumasa Wakamatsu (3), Tamio Suzuki (4), Zili Zhai (1), and Mayumi Fujita (1)

Presenting Author

Takeshi Yamauchi

[email protected]

Alcohol is metabolized to acetaldehyde, which is then oxidized to acetic acid by

mitochondrial aldehyde dehydrogenase 2 (ALDH2). In addition to its well-known role in

ethanol metabolism, ALDH2 dysfunction is associated with a variety of human diseases

including cardiovascular diseases, diabetes, neurodegenerative diseases and cancer.

However, the role of ALDH2 in the skin has not been explored. Recently, we reported

ethanol-induced skin pigmentation in mice with ALDH2 deficiency. In the current study,

we investigated the effects of ethanol and ALDH2 in melanocytes and other skin cells in

vitro and in vivo. When human primary melanocytes (African-American, Asian and

Caucasian) were cultured with ethanol (< 1% (v/v)), we observed increased melanin

production which was further enhanced in the addition of ALDH2 inhibitor in all types of

melanocytes. Next, we studied Aldh2 KO mice and control C57BL/6 (WT) mice fed with

a standard hard diet and bottled water with ethanol for 10 weeks. Consistent with our

previous report, we observed pigmentation in the ears, feet, tail and genital area in Aldh2

KO mice after ethanol administration. Fontana-Masson staining showed melanin pigment

deposit in the skin but not in other organs. HPLC analysis of pyrrole-2,3,5-tricarboxylic

acid (PTCA) and 4-amino-3-hydroxyphenylanine (4-AHP), degradation products of

eumelanin and pheomelanin, respectively, exhibited ethanol dose-dependent production of

PTCA but not 4-AHP in the skin of Aldh2 KO mice, demonstrating ethanol-induced

eumelanin production in these mice. Immunohistochemical analysis using melanocyte

markers revealed ethanol-induced proliferation of MART-1+ and TRP2+ melanocytes in

the epidermis and appendages of Aldh2 KO mice. These data demonstrate the role of

ethanol and ALDH2 in activating melanocytes, suggesting their potential involvements in

early transformation of melanocytes.


Abstract No: 17 Melanin synthesis is influenced by microenvironmental and metabolic

changes in bicarbonate/CO2.

Dalee Zhou (1), Anastasia Tsomides (2), Michael Reilly (3), Olivier Elemento (3), Laura Martin (3), Jonathan H. Zippin (1, 3)

Presenting Author

Dalee Zhou

[email protected]

Melanin possesses photoprotective as well as radioprotective and antioxidant properties.

While high melanin levels are thought to be protective against carcinogenesis, in melanoma

the presence of melanin is associated with more aggressive disease, treatment resistance,

and poorer prognosis overall. Pigmentation is dynamic in melanoma, it can be lost early on

and regained upon invasion and metastasis. Such changes in melanin synthesis in

melanoma are not always attributable to underlying genetic changes and instead appear to

be related to microenvironmental and metabolic factors. Here we show in a model organoid

system using human melanoma cells that microenvironmental and metabolic changes in

bicarbonate/CO2 dramatically influence melanin synthesis via melanosome pH, which is

known to be an important regulator of pigmentation. Growth in medium without

bicarbonate/CO2 (or treatment with the carbonic anhydrase inhibitor acetazolamide) led to

an increase in melanin production. We recently identified a novel cAMP signaling pathway

in melanocytes involving the bicarbonate/CO2 sensor, soluble adenylyl cyclase (sAC) that

is capable of dynamically regulating melanosome pH and pigment production in vitro and

in vivo. We demonstrate that incubation in bicarbonate/CO2-free medium leads to an

alkaline shift of melanosome pH in WT but not sAC-deficient melanocytes within hours.

Treatment with acetazolamide similarly alkalinizes melanosome pH in WT but not sAC-

deficient melanocytes. Finally, addition of membrane-permeable cAMP analogue

mitigates the melanosome pH change induced by bicarbonate/CO2 removal or

acetazolamide treatment in WT cells. In conclusion, it appears that microenvironmental

signals can alter the pigmentation of melanocytes by changing melanosome pH.


Abstract No: 18 The selenoprotein thioredoxin reductase 1 controls melanin synthesis via redox modification(s) to Mitf

Chelsey D. Kline (1), Madeleine Anderson (1), John W. Bassett(1), Matthew Honeggar (1), Shosuke Ito (3), Sancy A. Leachman (1,2), Arup Indra,(1,2, 4), Philip J. Moos (5) and Pamela B. Cassidy (1,2)

Presenting Author

Pamela Cassidy

[email protected]

The selenoprotein thioredoxin reductase 1 (TR1) in concert with glutathione reductase,

uses the reducing power of NADPH to control thiol-based redox signaling and oxidative

stress in cells. In a mouse model of UV-induced melanoma, we found that supplementation

with selenium, which is required for the catalytic activity of TR1, delays appearance of

melanomas when supplied at the initiation stage of tumorigenesis, but continued

application causes existing tumors to grow faster. In a second study we found TR1 is highly

expressed in human melanomas relative to melanocytic nevi (moles), and its expression

facilitates metastasis to the lung in a human melanoma xenograft model. Here we report

that melanocytes expressing an miRNA directed against TR1 (miTR1) grow more slowly

than control cell lines and contain significantly less melanin. In exploring this phenotype

we found that miTR1 cells have much lower tyrosinase activity as well as reduced protein

and mRNA levels for Tyrp1. qPCR and RNAseq analysis of a melanoma cell line in which

the TXNRD1 gene was disrupted using Crispr/Cas9 showed complete loss of expression

of the melanocyte-specific isoform of Mitf as well as many of the Mitf-controlled melanin

synthesis genes. Mitf-a, which differs from the –m isoform only in its first exon, is

unaffected by TXNRD1 loss in melanoma cells. We show evidence that these changes are

associated with increased sensitivity to oxidative modifications that appear to be specific

the Mitf,-m isoform. These findings indicate that redox signaling downstream of TR1

controls one of the major molecular effectors of cells in the melanocyte lineage, and that

these signals could have profound effects on both pigmentation and melanoma progression.


Abstract No: 19 Bromodomain and Extra-terminal proteins in melanocyte differentiation

Ivana de la Serna and Archit Trivedi

Presenting Author

Ivana de la Serna

[email protected]

Pharmacological Inhibition of the bromodomain and extra-terminal proteins (BET) family

of proteins (BRDT, BRD2, BRD3, and BRD4) is currently being explored as a novel

strategy for treating melanoma. However, nothing is yet known about the role of these

proteins in normal melanocytes. We found that BET inhibition suppresses the

differentiation of murine melanoblasts into pigmented melanocytes. Furthermore, BET

inhibition caused pigmented normal human epidermal melanocytes to de-pigment. Gene

expression profiling revealed that BET inhibition altered the expression of a large number

of pigmentation associated genes, many of which were MITF target genes. Depletion of

the BET family member, BRD4, inhibited expression of two MITF target genes that are

melanin synthesis enzymes, TYR and TYRP1. Furthermore, BRD4 occupied MITF

binding sites in the TYR and TYRP1 promoters, was associated with open chromatin

structure, and physically interacted with MITF. In conclusion, our data indicates that BRD4

has a critical function in the regulation of melanogenesis through interactions with MITF.


Abstract No: 20 Cell-Cell Signaling in Zebrafish Melanocyte Regeneration

William T. Frantz (1,2) and Craig J. Ceol (1,2)

Presenting Author

William T. Frantz

[email protected]

Stem cells are important in maintenance of adult tissues, and their dysfunction can

contribute to various diseases. Skin-resident melanocyte stem cells (MSCs) replenish dead,

damaged or exhausted melanocytes, and their loss contributes to vitiligo and other

hypomelanotic disorders. Additionally, MSCs are postulated to be a cell of origin for

melanoma. To understand the mechanisms that govern MSC activity, we have conducted

studies in zebrafish to identify genes and cells that regulate melanocyte regeneration.

Previously we discovered a novel mode of regeneration that is used to replenish

melanocytes in zebrafish; a subset of MSCs undergoes direct differentiation to generate

new melanocytes and another subset of MSCs divides symmetrically to maintain the pool

of MSCs. Initial experiments have found that zebrafish harboring deficiencies in kitlga/kita

signaling, the zebrafish orthologs of mammalian KIT ligand and receptor, are specifically

defective in the direct differentiation that occurs during melanocyte regeneration.

Subsequent transcriptional analyses in wild-type animals undergoing regeneration

demonstrated an upregulation of markers for macrophages (mpeg1) and neutrophils (mpx).

Furthermore, single cell RNA sequencing studies demonstrated that tissue resident

macrophages upregulate chemotactic cytokines in response to melanocyte destruction. In

follow up experiments, genetic ablation of macrophages produced a melanocyte

regeneration defect. Together these results suggest a role for kit signaling and immune cells

in melanocyte regeneration. To further explore these findings, we are currently

investigating the interplay between kitlga/kita signaling and macrophages during

melanocyte regeneration. An understanding of signals that regulate melanocyte stem cells

during regeneration has the potential to reveal therapeutic targets important in pigmentary

disorders as well as melanoma.


Abstract No: 21 Restoring melanocyte stem cells: A model of drug-mediated regeneration

Melissa Harris, Joseph Palmer, and Zoya Anderson

Presenting Author

Melissa Harris

[email protected]

In an attempt identify in situ methods by which endogenous stem cells can be used to

reverse aging phenotypes, we are investigating the novel drug RT1640 in hair

repigmentation in collaboration with Rivertown Therapeutics. In mice, sublethal exposure

to ionizing radiation results in a similar cellular phenotype to that observed in age-related

hair graying, namely, premature melanocyte stem cell differentiation. This similarity has

led others to suggest that genotoxic stress due to life-long exposure to ionizing radiation

may be a component of age-related hair graying, and thus an efficient and relevant model

to predict the potential for RT1640 in reversing age-related hair graying. We have found

that after the establishment of gray hair by exposure to ionizing radiation, topical treatment

with RT1640 results in increased numbers of melanocytes stem cells that are retained over

multiple hair cycles, and subsequently, permanent hair repigmentation. Our current work

is focused on identifying the molecular mechanism for RT1640’s function. We anticipate

that this compound will be relevant not only for the reversal of hair graying but also in the

treatment of hypopigmentation disorders. In addition, these studies will provide an example

of successful, non-invasive rejuvenation of endogenous stem cells in situ.


Abstract No: 22 The loss of dicer affects migration and homing of melanocytes and induces hair greying

Juliette Bertrand, Valerie Petit, Pierre Sohier, Franck Gesbert, & Lionel Larue

Presenting Author

Lionel Larue

[email protected]

Age related hair greying is due to exhaustion of the melanocyte stem cells (McSC) pool.

This phenomenon can be accelerated by genetic and/or environmental factors inducing

stress and the premature death and/or early differentiation of McSCs in the bulge. Since

Dicer is downregulated by stress, we inactivated this gene in the melanocyte lineage to

investigate its contribution to McSC survival. The absence of Dicer in McSC at birth led

to a progressive hair greying due to mis-localization and migration of melanocytes, and

exhaustion of the McSC pool. An un-supervised approach revealed that mRNAs

encoding integrins are enriched among the mRNAs modified by Dicer inactivation. More

specifically, we showed that altered ItgaV and Itgb5 expression impacted melanocyte

migration. Our data link Dicer, miRNAs (e.g., miR-92b), integrin expression (e.g.,

ItgaV) and Mc renewal. Altogether, we bring a novel cause of hair greying and its

associated mechanism.


Abstract No: 23 Investigating the role of tetraploid intermediates in melanoma progression

Revati Darp (1,2), Marc Vittoria (3), Neil Ganem (3), Craig Ceol (1,2)

Presenting Author

Revati Darp

[email protected]

Melanoma arises from altered cells in the melanocyte lineage, but the mechanisms by

which these cells progress to melanoma are unknown. To understand the early cellular

events that contribute to melanoma formation, we examined melanocytes in a melanoma-

prone zebrafish strain expressing BRAFV600E, the most common oncogenic form of the

BRAF kinase. We found that, unlike wild-type melanocytes, melanocytes in transgenic

BRAFV600E animals were binucleate and tetraploid. Furthermore, melanocytes in p53-

deficient transgenic BRAFV600E animals exhibited 8N and greater DNA content,

suggesting bypass of a p53-dependent arrest that halts cell cycle progression of tetraploid

melanocytes. These data implicate tetraploids generated by increased BRAF pathway

activity as contributors to melanoma initiation. To gain insight into the mechanism by

which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro system

by which such cells are generated following BRAFV600E expression. Using live-cell

imaging, immunofluorescence and flow cytometry approaches, we found that

BRAFV600E-generated tetraploids arose via cytokinesis failure during mitosis due to

reduced activity of the small GTPase RhoA. Additionally, BRAFV600E activity in the

prior G1/S phases of the cell cycle was essential in generating tetraploids. We are currently

determining the mechanism by which BRAFV600E activity in G1/S causes the cytokinesis

defect in mitosis and whether BRAFV600E-generated tetraploids serve as intermediates in

melanoma initiation and progression. These are important questions as recent studies have

shown that aneuploid progeny of tetraploid cells can be intermediates in tumor

development, and deep sequencing data suggest at least one third of melanomas and other

solid tumors have undergone a genome doubling event during their progression. Overall,

our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-

induced tetraploidy in the genesis of melanomas.


Abstract No: 24 Defining the Spectrum of Mutations for Oculocutaneous Albinism

S. K. Loftus (1), J. Choi (2), K. Brown (2), L. Lundh (3), NISC Comparative Sequencing Program (4), M. Brilliant (5), W. Oetting (6), D. R. Adams (3) and W. J. Pavan (1)

Presenting Author

S. K. Loftus

[email protected]

Oculocutaneous albinism (OCA) is a recessive disorder characterized by the reduction of

skin, hair and eye pigmentation. OCA individuals can present with a complex set of

developmentally-mediated visual impairments of diverse clinical severity and are at higher

risk for UV-induced skin cancers. OCA associated mutations are predominately found in

2 genes, TYR (OCA types 1A and 1B) or OCA2 (OCA type 2), with a small number of

cases attributed to mutations in TYRP1, SLC45A2, SLC24A5, C10orf11/LRMDA. Most

critically, 20-50% patients remain without identification of biallelic mutations after

traditional exon-based screening of known OCA genes. To better define the molecular

mechanisms underlying OCA, we are interrogating over 400 OCA proband individuals and

associated family members, using custom capture, short-read sequencing. This approach

queries coding, intronic, and cis-regulatory regions of 6 OCA genes and 31 genes identified

by GWAS pigmentation studies. Non-coding cis-regulatory regions include DNase1-HS

data (derived from primary and immortalized melanocytes, melanoma tumors, and retinal

pigment epithelium), H3K27Ac ChIP binding regions under conditions of MAPK

activation and inhibition in 501mel cells and regions of linkage disequilibrium for SNPs

from our primary melanocyte expression quantitative trait loci (eQTL) dataset. This dataset

identifies eQTLs for 4997 genes expressed in the melanocyte lineage. Here we find over

73,000 eQTLs for 128 genes associated with visible pigmentation-related phenotypes,

including 156 and 684 eQTLs for TYR and OCA2, respectively. Our family, trio-based

analysis and interrogation of coding, and non-coding sequence, will better define the

molecular spectrum of OCA mutations, facilitate assessment of non-coding DNA variation

function, and allow for the subsequent systematic evaluation of the molecular mechanisms

underlying both missing heritability and phenotypic variation observed among OCA

patients.


Abstract No: 25 Event sequence of BRAF mutation and UV exposure is not a substantial factor in melanomagenesis in a mouse model of cutaneous melanoma

Helen T. Michael (1), Patricia C. Bunda (1), Sung Chin (2), Huaitian Liu (1), Chi-Ping Day (1), Cari Smith (2), Maxwell Lee (1), Glenn Merlino (1)

Presenting Author

Helen T. Michael

[email protected]

Melanoma accounts for nearly 97,000 new diagnoses yearly and melanoma incidence has

been rising for several decades. Ultraviolet (UV) radiation exposure, particularly childhood

sunburn and intermittent sun exposure, are epidemiologically linked to melanoma

formation. Although mutations in BRAF and other MAPK pathway genes occur early in

melanocytic lesion development, these are not canonical UV mutations and there has been

little research into whether the order of sun exposure and driver mutation affect tumor

development or risk. Since these variables cannot be directly manipulated in humans, we

designed mouse models to look at the role of the relative timing of BRAF mutation and

UV exposure. Using mice with a tamoxifen-inducible BRAF mutation and heterozygous

CDKN2A loss on a C57BL/6 hepatocyte growth factor (HGF) transgenic background, we

exposed mice to one of four conditions, UV at 3 days and tamoxifen at 30 days, tamoxifen

at 3 days and UV at 30 days, tamoxifen at 3 days, or tamoxifen at 30 days. Control mice

with inducible heterozygous CDKN2A loss were exposed to UV at 3 days and tamoxifen

at 30 days. Although overall tumor incidence was similar for all groups, mice with both

BRAF mutations and UV exposure had significantly higher numbers of tumors per mouse

and faster tumor onset than mice with BRAF mutation or UV exposure alone. In fact, early

UV exposure followed by BRAF mutation had the fastest tumor onset. Additionally, UV

exposure resulted in histologic heterogeneity in the melanomas and increased frequency of

pigmented tumors relative to BRAF mutation alone. Pigmented melanomas had a higher

percentage of UV-associated signature 7 mutations and were transcriptomically distinct

from non-pigmented tumors, suggesting the possibility that they arose from a different cell

of origin. Surprisingly, our data suggest that, although more potent when combined, the

order sequence of BRAF mutation and UV exposure is not a major factor in the

development of melanomas.


Abstract No: 26 MITF regulates the expression of extracellular matrix and focal adhesion genes

Ramile Dilshat (1), Katrin Moller (1), Sara Sigurbjornsdottir (1), Valerie Fock (1), Lionel Larue (2) and Eirikur Steingrimsson (1)

Presenting Author

Eirikur Steingrimsson

[email protected]

The Microphthalmia-associated transcription factor (MITF) regulates the expression of

genes required for melanoma cell survival, proliferation, invasion, and differentiation. In

order to explain how a single transcription regulator can mediate all these effects, a model

has been proposed where the level of MITF activity determines the phenotypic state of

cells; high activity promotes proliferation whereas low activity leads to quiescent but

invasive cells. Here, we generated SKmel28 melanoma cells lacking MITF using CRISPR

technology and characterized the effects on the phenotype and gene expression. As

expected, the MITF knock out (KO) melanoma cells exhibit reduced proliferation rate

compared to the control cell line. Surprisingly, however, both the migration and invasion

potential of MITF-KO cells were significantly reduced compared to both control and

parental cells. The MITF-KO cells were flattened and increased in size compared to control

cell lines and had changes in cytoskeletal structure. RNA-sequencing followed by

differential gene expression analysis revealed that the MITF-KO cells had increased

expression of genes involved in the formation of the extracellular matrix (ECM) and focal

adhesion. The majority of these ECM and focal adhesion genes contained MITF ChIP-seq

peaks in their promoters or intronic regions suggesting that the regulation of these genes is

through direct MITF repression. Interestingly, among the MITF direct targets were the

major components of focal adhesion, paxillin (PAX) and focal adhesion kinase (FAK).

Consistent with this, immunofluorescent staining showed that the number of PAX positive

focal points was increased in MITF-KO and transient knockdown cell lines. Importantly,

a comparison of the transcriptomic profile of MITFlow and MITFhigh human melanoma

tumours from the TCGA revealed a gain of expression of ECM and focal adhesion genes

in the MITFlow tumour samples, suggesting that MITF is an important mediator of

melanoma-initiation.


Abstract No: 27

Evolution of Endothelin independence in GNAQ driven melanoma

Fagun Jain and Catherine D. Van Raamsdonk

Presenting Author

Catherine D. Van Raamsdonk

[email protected]

The melanocytic lesions that bear frequent oncogenic mutations in heterotrimeric G

protein alpha subunits include blue nevi in the dermis, meningeal melanocytomas of the

central nervous system, and uveal melanomas in the eye. Almost all uveal melanomas

exhibit substitution of the GNAQ or GNA11 Q209 or R183 residues, which contribute

to signal termination. Previous studies showed that Gaq/11 signaling is stimulated by

the G protein coupled receptor, Endothelin receptor B (EDNRB). Here, we determined

the effects of Ednrb gene knockout or pharmaceutical inhibition in a forced GNAQ

Q209L expression mouse model, which develops blue nevi, leptomeningeal

melanocytomas, and uveal melanomas. We found that the effects of GNAQ Q209L were

almost entirely blocked when the Ednrb gene was deleted prior to GNAQ Q209L

activation. When Ednrb was deleted coincident with GNAQ Q209L activation, the mice

remained healthy longer, with every oncogenic phenotype less severe. However, the

mice eventually developed large and ulcerated tumors on the trunk. Pharmaceutical

blockade of Ednrb activity using the small molecule inhibitor, A-192621, begun 6 weeks

after GNAQ Q209L expression was established, had no impact. These findings show

that GNAQ Q209L transformed melanocytes become Ednrb-independent, which is

consistent with the inverse relationship between prognosis and EDNRB expression in

uveal melanoma.


Abstract No: 28 Characterizing the Dysregulated Cellular and Molecular Pathways in Vitiligo Lesions That Do Not Respond to Phototherapy

Nathaniel B. Goldstein, Andrea Steel, Maranke I. Koster, Kavya Ganuthula, Velmurugan Balaiya, Dennis R. Roop, David A. Norris, Stanca A. Birlea

Presenting Author

Stanca A. Birlea

[email protected]

We have developed a human model of vitiligo repigmentation by combining the collection

of skin biopsies with fluorescent laser capture microdissection and whole transcriptome

RNA-sequencing of melanocytes (MCs) and keratinocytes from the hair follicle (HF) bulge

and epidermis. We identified RHO-GTPase (RHO), WNT/β-catenin, and p53 as essential

bulge pathways that control repigmentation in MCs. Next, we collected biopsies from a

different vitiligo phenotype: depigmented vitiligo lesions that do not respond to 6 months

of narrow band UVB (NBUVB) treatment, and we compared these to repigmenting lesions

from the same patients (N=3). Using transverse sections of formalin fixed paraffin

embedded skin, we performed staining for melanin, and fluorescent immunostaining using

MC specific markers (DCT, C-KIT, TYR), KI-67 (for proliferation), β-catenin (for

characterizing MCs precursors) and CD3 and CD8 (T cell markers). We identified that the

non-responding lesions present the following cellular deficiencies, as compared with

repigmented lesions (P<0.05): decreased number of MCs stem cells (DCT+/C-KIT-) in the

bulge, depletion of melanoblasts (DCT+/C-KIT+) in the INF and epidermis with fewer

proliferative sub-populations, decreased number of differentiated MCs (TYR+) and

reduced melanization in the epidermis, decreased β-catenin expression in the infundibulum

and epidermal MCs, and increased expression of CD3 and CD8 markers in the bulge and

INF. We believe that the above cellular dysregulation is the result of cytotoxic CD8+ T

cell attack to both epidermal and HF MCs, and by aberrant control of MC proliferation and

differentiation by WNT/β-catenin and MC migration by RHO. A better understanding of

the delayed repigmentation response during NBUVB can facilitate more robust

repigmentation phenotypes.


Abstract No: 29 Melanocyte-specific Th1-like initiating- and recall-responses in growing

feathers of Smyth chickens with autoimmune vitiligo.

Daniel M. Falcon (1), Marites A. Sales (2), and Gisela F. Erf (2)

Presenting Author

Gisela F. Erf

[email protected]

Vitiligo is a depigmentation disorder driven by the autoimmune targeting and elimination

of melanocytes in the skin. The Smyth line (SL) chicken is a well-established animal model

for autoimmune vitiligo. In addition to spontaneous onset of the disorder in 80-95% of SL

chickens, the easy access to the target tissue, namely the growing feather (GF) where

epidermal melanocytes and the autoimmune lesion are located, is another great advantage

of this model. To monitor immune activities associated with SLV initiation and

progression, GF collected from 14 SL chickens, twice per week, from hatch through 16

weeks of age, were used for leukocyte population analysis by flow cytometry and gene-

expression analysis by qRT-PCR. To determine establishment of a melanocyte-specific

autoimmune response in SL chickens with vitiligo, feather pulp-derived, syngeneic

melanocytes were injected (i.d.) into the pulp of multiple, completely depigmented GF. GF

were collected pre- and 0.25, 1, 2, 3, 5, 6, and 7 days post-injection for analysis as described

above. Independent of age, T- and B-cell infiltration was observed one- and two-weeks

prior to vitiligo onset, respectively. While both CD4+ and CD8+ T cells reached peak

levels at vitiligo onset, levels of CD4+ T helper cells declined gradually thereafter whereas

those of CD8+ cytotoxic T cells remained elevated until depigmentation (melanocyte loss)

was complete. Gene expression analysis suggested active recruitment (CCL19, CCR7) of

lymphocytes prior to onset and a sustained Th1-like gene signature (IFN-γ, FASLG,

GZMA) throughout disease progression. A response pattern similar to the effector phase

of the initiating-response was observed for the recall-response when feather-derived

melanocytes were injected into the dermis of the pulp of completely depigmented GF. The

SL-vitiligo model provides unique opportunities to dissect mechanisms underlying loss of

tolerance and establishment of the autoimmune response to epidermal melanocytes.


Abstract No: 30 BatF3-dependent dendritic cells are required for depigmentation in a

mouse model of vitiligo.

Michael L. Frisoli, Jillian M. Richmond, John E. Harris

Presenting Author

Michael L. Frisoli

[email protected]

BatF3 is a transcription factor that is required for the development of CD103+ dendritic

cells, and we found that BatF3-/- mice are protected from depigmentation in a model of

vitiligo. Migration of skin-resident CD103+ dendritic cells to skin-draining lymph nodes

is important for priming of T cells to skin-derived antigens, and this deficit in BatF3-/-

mice reduces development of Th1 skin immune responses in models of melanoma

challenge and cutaneous lupus erythematosus. Here we show that melanocyte-specific

CD8+ T cells have impaired IFN- production within BatF3-/- mice and express

significantly higher levels of the inhibitory receptor PD-1. Interestingly, melanocyte-

specific CD8+ T cells were found in equal numbers within the skin of BatF3-/- mice

relative to wild type mice, suggesting that T cell skin infiltration is unaltered within

BatF3-/- mice, and that the phenotype is instead due to altered T cell function.


Abstract No: 31 Adoptive transfer of antigen-specific Tregs controls depigmentation in vitiligo-prone mice

Zhussipbek Mukhatayev (1,2,3), Katherine Pontarolo-Maag (4), Jonathan M. Eby (4), Shikhar Mehrotra (5), Richard Daniel Peiffer, Richard Junghans (6), I. Caroline Le Poole (1,7).

Presenting Author

I. Caroline Le Poole

[email protected]

Regulatory T cells (Tregs) are crucial to peripheral self-tolerance in vitiligo. The

abundance of immunosuppressive Tregs in vitiligo lesions is greatly reduced compared to

healthy skin, suggesting that replenishing cutaneous Tregs will protect against

depigmentation. Previously, we showed that GD3 is highly expressed in vitiligo

perilesional skin. This prompted us to generate GD3-reactive CAR Tregs and in vitro,

transduced cells secreted significantly more IL-10 compared to untransduced Tregs and

suppress production of IFN-γ. Another target antigen of interest in vitiligo is tyrosinase.

Here we tested the hypothesis, that antigen specific Tregs will provide superior protection

from depigmentation in vivo. To generate GD3-specific CAR Tregs for adoptive transfer,

naïve CD4+ T cells originated from FoxP3 eGFP reporter mice has been polarized to

CD4+FoxP3+ Tregs in the presence of TGF-ß. To expand Tregs, we included anti-

CD3/CD28 T cell activator beads and a high concentration of IL-2, which amplified Treg

numbers >10-fold over 5 days. We also generated TYR-reactive Treg by crossing the h3T

mouse to the above reporter mice, and compared their activity to polyclonal Tregs. We

transferred polyclonal or tyrosinase-specific Tregs (h3T-Tregs) twice at a 2-week interval

into vitiligo-prone mice (h3TA2). Antigen-specific h3T-Tregs demonstrated a 29%

reduction in depigmentation compared to polyclonal Tregs and a 44% reduction compared

to untreated mice for 4 weeks (n=5 per group, p=0.0143). To compare the suppressive

activity of GD3-specific CAR Tregs versus untransduced Tregs, we adoptively transferred

Tregs (n=6) biweekly, 4 times total. After 10 weeks, transduced Tregs showed a 67%

decrease (p=0.0404) in depigmentation compared to polyclonal Tregs and a 75% decrease

compared to untreated animals (p=0.0002). Thus, these data demonstrate for the first time

that antigen specific Treg provide superior protection from depigmentation in mice.


Abstract No: 32 Imaging and single cell transcriptomics reveal keratinocyte metabolic changes that may drive disease persistence in vitiligo skin

Jessica Shiu (1), Griffin Lentsch (2), Jessica Flesher (1), Craig Mizzoni (3), Pezhman Mobasher (1), Bruce J Tromberg (2), Irene Georgakoudi (3), Mihaela Balu (2), Anand K Ganesan (1)

Presenting Author

Jessica Shiu

[email protected]

Vitiligo is an autoimmune skin disease characterized by the progressive destruction of

melanocytes, resulting in white patches that persist. Here we use a combination of in vivo

imaging and single cell genomics to identify factors that allow individual vitiligo lesions

to persist. Initial studies used in vivo multiphoton microscopy (MPM) and reflectance

confocal microscopy (RCM), to demonstrate that vitiligo lesions, but not adjacent skin,

were devoid of melanocytes. Treatment with phototherapy or skin grafting resulted in an

increased number of melanocytes, demonstrating the utility of these methods in visualizing

repigmentation. As vitiligo is thought to be initiated by oxidatively stressed melanocytes,

we used MPM to detect NADH intrinsic two-photon excited fluorescence in vitiligo and

perilesional normal skin before and after treatment with phototherapy or skin grafting.

Specifically, we used MPM images to determine the degree of mitochondrial clustering, a

sensitive measure of oxidative phosphorylation and glycolysis. Mitochondrial clustering in

the basal epidermis was altered in vitiligo skin compared to perilesional skin, and these

differences normalized after treatment. Next, we used single cell transcriptomics to identify

unique populations of keratinocytes present in vitiligo skin as compared to normal skin.

Two unique cell populations were found to be more abundant in stable vitiligo lesions as

compared to normal skin. The first cell cluster corresponds to immune cells, consistent

with the role of the immune system in disease pathogenesis. The second cluster

corresponds to a unique population of keratinocytes that exhibited increased expression of

keratins associated with cell stress (KRT6B) and increased expression of cytokines known

to induce vitiligo (CXCL9, CXCL10). Taken together, these observations suggest that a

metabolically altered population of keratinocytes drives vitiligo autoimmunity and may

control disease persistence.


Abstract No: 33 Structure-based Design of CDC42/RHOJ Effector Inhibitors for the Treatment of Cancer

Jessica L. Flesher (1), Sohail Jahid (2), Jose A. Ortega (3), Giuseppina La Sala (3), Nicolette Brindani (3), Jose M. Arencibia (3), Jacopo Manigrasso (3), Stephanie Hachey (4), Chi-Fen Chen (5), Chris Hughes (4), Marco De Vivo (3), Anand K. Ganesan (1,5)

Presenting Author

Jessica L. Flesher

[email protected]

Italian Institute of Technology, Genoa, IT

The CDC42 family of GTPases (RhoJ, RhoQ, Cdc42) control both the ability of tumor

cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors.

While recent studies have developed small molecules that target RAS GTPases, little

progress has been made in targeting the related CDC42 family of GTPases for cancer

treatment. Here, we use computer-aided drug design to identify a novel class of inhibitors

that act on an allosteric pocket in active CDC42 GTPases and block CDC42 effector

interactions. We show experimentally that these allosteric inhibitors prevent RHOJ and

CDC42 from binding to their downstream effector PAK and block PAK signaling in vitro,

while having no effect on the interactions between the closely related GTPase RAC1 and

PAK. Our lead compound ARN22089 has a druglike profile and can block both tumor

growth and tumor angiogenesis in a three-dimensional vascularized microtumor (VMT)

model, indicating that ARN22089 blocks RhoJ/Cdc42 signaling in both the tumor cell and

the tumor endothelium. Short term treatment of nascent melanoma tumors with ARN22089

halted the growth of BRAF mutant autochthonous mouse melanoma tumors and induced

tumor necrosis in melanoma patient-derived xenografts. In summary, we describe a

multidisciplinary structure-based drug discovery platform that can identify new RAS

family allosteric inhibitors and use this tool to develop RhoJ/CDC42 druglike inhibitors

that block tumor growth by simultaneously targeting cancer cells and the tumor

microenvironment.


Abstract No: 34 Targeting glutamate signaling in melanoma

Simar Singh, Raj Shah, Suzie Chen, Fabian V. Filipp

Presenting Author

Fabian V. Filipp

[email protected]

The glutamate metabotropic receptor 1 (GRM1) drives oncogenesis when aberrantly

activated in melanoma and several other cancers. Glutamate, the natural ligand of GRM1,

is one of the most abundant amino acids in the human body and the predominant excitatory

neurotransmitter in the vertebrate central nervous system. Stimulation of GRM1 by

glutamate leads to activation of mitogenic signaling pathways, which in turn increases the

production of glutamate, fueling autocrine feedback. Metabolomics reveals that patient-

derived xenografts with GRM1-positive melanoma cells exhibit elevated plasma glutamate

levels associated with spontaneous metastatic melanoma in vivo. Using a rational drug-

targeting strategy, we critically evaluate metabolic bottlenecks with the goal to cut off

tumor glutamate bioavailability. It has been thought that increased glutamine uptake and

glutaminolysis are solely responsible for increased glutamate production in tumors.

However, using stable isotope tracing and GCMS analysis, we determined that cells

expressing GRM1 fuel the proportion of glutamate derived from glucose and glutamine.

Furthermore, the glycolytic contribution into glutamate is increased compared to normal

cells. The increased dependence of glycolytic carbon for glutamate production is further

confirmed by the enhanced sensitivity of GRM1 expressing cells to inhibitors that block

the anaplerotic flow of carbon into the mitochondrial TCA pool. In vivo, xenografted tumor

burden and elevated circulating glutamate levels were significantly reduced by limiting

glutamate bioavailability through the combined treatment with riluzole and the glutaminase

inhibitor CB-839. Rigorous evaluation of sex differences in drug responses of PDX models

revealed higher tumor burden in same-sex tumor grafts compared to cross-sex models.


Abstract No: 35 A Mitochondrial Complex I Inhibitor drives metabolic reprogramming and sensitizes vemurafenib resistant BRAFV600E mutation bearing metastatic melanoma cells

Evan L. Carpenter1,2, Sharmeen Chagani1, Dylan Nelson1, Pamela B. Cassidy2,5, Madeleine Laws2, Gitali Ganguli-Indra1,5, Arup K. Indra1,2,3,4,5

Presenting Author

Arup K. Indra

[email protected]

There is an unmet need for alternative therapeutic strategies to treat vemurafenib-resistant

BRAFV600E metastatic melanomas due to a high incidence of drug resistance following

treatment. We have recently performed a high-throughput screening of two bioactive

compound libraries against a metastatic melanoma cell line and discovered two structurally

analogous compounds, deguelin and rotenone from a cell viability assay. Vemurafenib-

resistant melanoma cell lines, A2058R and A375R (containing the BRAFV600E

mutation), also showed reduced proliferation when treated with these two compounds.

Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen

consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly

activates AMPK signaling, which results in inhibition of mTORC1 signaling and

differential phosphorylation of mTORC1’s downstream effectors, 4E-BP1 and p70S6

kinase. Deguelin also significantly inhibited the MAPK pathway and cell proliferation

determined by reduced Ki67 expression without altering Akt activation in the same

timeframe in the vemurafenib-resistant melanoma cells. These data suggest that utility of

metabolic regulators such as deguelin can lead to energy starvation by altering the

intracellular metabolism and reducing survival of drug-resistant melanomas harboring

BRAFV600E mutations.


Abstract No: 36 HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Dinesh Jaishankar (1), Cormac Cosgrove (1), Jeffrey Mosenson (2) and I. Caroline Le Poole (1,3)

Presenting Author

Dinesh Jaishankar

[email protected]

Melanoma immunotherapies are often accompanied by autoimmune side effects, including

vitiligo. We generated an expression construct encoding inducible HSP70 with one amino

acid modification (HSP70iQ435A) to the DC activating moiety and in Sinclair swine, the

encoded protein showed a remarkable ability to treat vitiligo without interfering with anti-

tumor responses. To explain this, we postulate that HSP70iQ435A-encoding DNA elicits

a humoral response to the stress protein expressed selectively on the surface of melanoma

cells. We introduced HSP70iQ435A-encoding or empty vector DNA into CD8 knockout

mice by gene gun every 6 days, 5 times total, followed by B16 tumor challenge. Anti-

HSP70i titers and antibody isotypes were measured by ELISA in sera of C57BL/6 mice

exposed to HSP70iQ435A or empty vector DNA. Anti-HSP70-mediated ADCC was

measured in an NK cell degranulation assay. To identify the amino acid target sequence,

epitope mapping and substitution scanning was performed. Binding of serum antibodies to

extracellular HSP70i on melanoma cell lines was evaluated by FACS. We observed that

HSP70iQ435A-encoding DNA offered significant anti-tumor protection. Surface binding

to extracellular HSP70i on melanoma cell lines was detectable selectively with sera from

HSP70iQ435A-treated mice. Here, measurable anti-HSP70i titers of all isotype/subclasses

were observed and caused significant degranulation of NK cells. Epitope mapping and

substitution analysis revealed that sera from HSP70iQ435A-treated mice recognized the

C-terminal peptide SGLYQGAGGPGPGGF within HSP70i. Thus, besides reducing

autoimmunity, HSP70iQ435A-encoding DNA may elicit humoral responses that limit

tumor growth.


Abstract No: 37

Hsp90 inhibition induces melanoma tumor antigens and enhances

cellular immunotherapy against melanoma

Sandeep S. Joshi (1), Emmanual Unni (1), Stephen R. Goding (2),

Paul A. Antony (2), Thomas J. Hornyak (1,3,4)

Presenting Author

Sandeep S Joshi

[email protected]

(1)Department of Biochemistry and Molecular Biology, (2)Department of Pathology, (3)Department of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland, USA, and (4)Research and Development Service, VA Maryland Health Care System, Baltimore, Maryland, USA.

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with

important roles in tumor growth. Malignant melanomas have active RAF-MEK-ERK signaling

which can occur either through an activating mutation in BRAF (BRAFV600E) or activation

of wild-type BRAF (BRAFWT) and CRAF through mutations in NRAS or KIT. We and others

have shown that 17-AAG, a HSP90 inhibitor, inhibits cell growth in both types of melanomas

(mutant BRAF and NRAS) and induces degradation of BRAFV600E, which also is an HSP90

client protein. Recently, we showed that 17-AAG, in addition to inhibiting MAPK signaling

and cell growth, stimulated melanin production and enhanced TYRP1 and DCT expression

without stimulating tyrosinase production in 3 BRAFWT human melanoma cell lines as well

as in the mouse melanoma cell line, B16.F10. This finding demonstrates the importance of

accessory melanogenic enzymes in melanin biosynthesis and suggests a strategy to enhance

anti-tumor immunity. In vivo, the combination of 17-AAG and cellular immunotherapy

directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy

alone. Here, we have screened the effect of second generation HSP90 inhibitors, ganetespib,

onalespib and XL888, on the induction of melanogenic enzymes in melanoma cells. The second

generation HSP90 inhibitors, ganetespib, onalespib and XL888, similar to 17-AAG, inhibited

melanoma cell proliferation and enhanced TYRP1 and DCT expression in 3 human melanoma

cell lines, MelJuso, SK-Mel-30, SK-Mel-2, and in murine melanoma cells, B16.F10. In

addition, second generation HSP90 inhibitors showed significantly higher inhibition of

melanoma cell proliferation and induced higher TYRP1 and DCT expression in comparison to

17-AAG treatment in all melanoma cell lines. Thus, our study provides an alternative approach

of using an HSP90 inhibitor in targeting BRAFWT melanoma cells combined with

immunotherapy and is clinically significant for the treatment of the advanced melanoma

patients.


Abstract No: 38 Improving Consistency of MED Detection Using Objective Method as the First Step to Improving the Accuracy of SPF Determination: A Quantitative Study

Di Qu (1), Liu Jianwei (2), Liao Feng (2), Chen Yinbei (2), Gopa Majmudar (1), Lai Wei (3), Xie Xiaoyuan (3), Li Xiangzi (3), Zheng Yue (3), Ye Congxiu (3), Yi Jinling (3)

Presenting Author

Di Qu

[email protected]

Inconsistent determination of SPF has been a general concern to the industry, as seen in

reports of various testing labs in different part of world, which results in inadequate

protection of consumers. One key source of variation comes from the inconsistent detection

of minimal erythema dose (MED) upon which the entire SPF nomenclature is built. In this

study, we quantitatively analyzed the UV induced skin erythema against radiation dose and

developed two objectively measurable parameters to quantify MED: erythema intensity

(Delta a*) and %coverage of spot area. UV irradiation was performed on 31 Chinese

volunteers using Solar Simulator per the current industry guidelines. Two methods were

used to evaluate MED: clinical grading and digital image analysis. Computer algorithms

were developed to measure Delta a* and %coverage in six radiation sites of different UV

doses. A linear correlation with UV dose for Delta a* (mean r2=0.932), and a logarithmic

one for %coverage (mean r2=0.867), were obtained from the study population. Analyzing

the clinically detected MED spots with the above parameters we were able to characterize

the current subjective practice (mean Delta a*=2.62 and %coverage=50.63%) which were

subsequently set as clinically-relevant targets. Using the mathematical model developed in

this study, we objectively detected the MED spots from the 31 volunteers. Our results

showed a dramatic reduction in standard deviation from 1.24 to 0.37 for Delta a*, and from

12.85 to 7.85 for %coverage. We concluded that our objective method would significantly

improve the consistency of MED detection in future SPF tests.


Abstract No: 39

Slow-cycling melanoma cells demonstrate resistance to targeted

therapy

Dahihm Kim(1), Hyeongsun Moon(2), and Andrew White(1)

Presenting Author

Dahihm Kim

[email protected]

Identification of small molecules targeting driver mutations in melanoma has greatly

improved clinical responses. However, drug resistance is one of the most common

complications that occurs within a few months after targeted therapy, which is

responsible for tumor recurrence of an advanced cancer stage. Melanoma exhibits high

intra-tumoral heterogeneity and contains various cell subpopulations that demonstrate

different growth rates. This suggests that some cell subtypes could show different

responses to chemotherapy, enabling tumor recurrence. Here, we are using label

retaining methods to identify slow-cycling melanocytic tumor cells. We demonstrate

that slow-cycling cell subpopulations remain resistant to treatment by combined

BrafV600E/PI3K inhibition. Moreover, the percentage of slow-cycling labeling

retaining cells was decreased as tumor relapses during the cessation of chemotherapy.

This raises the possibility that drug-resistant slow-cycling cells may proliferate at the

onset of tumor recurrence. Thus, understanding the molecular mechanisms underlying

how slow-cycling cells acquire resistance under targeted therapy and obtain

proliferative properties in melanoma relapse could contribute to the development of

therapies that prevent chemotherapy resistance in recurrent tumors.


Abstract No: 40

Preclinical study using Trigriluzole and Anti-PD-1 in a spontaneous

melanoma-prone mouse model

Kevinn Eddy (1,2), Maryam Rasheed (1), Christina Marinaro (1),

Raj Shah (1) Suzie Chen (1,2,3,4)

Presenting Author

Kevinn Eddy

[email protected]

Much progress has been made in understanding melanoma pathogenesis within the last

few years through targeted therapies and immunotherapies. However, resistance to

small molecule inhibitors remains an obstacle in many patients. Immunotherapies such

as check point inhibitors against PD-1/PD-L1 lead to durable responses but only in a

subset of melanoma patients. Mouse models reflecting human cancers have provided

invaluable tools towards the translation of basic science discoveries to clinical therapies,

but many of these in vivo studies are short-term and do not accurately mimic

circumstances which patients undergo during treatments. Our lab has demonstrated that

ectopic expression of metabotropic glutamate receptor 1 (GRM1), in melanocytes leads

to cell transformation in vitro and spontaneous metastatic melanoma in vivo. In addition,

aberrant GRM1 expression is detected in > 90% of late-stage melanoma patients,

suggesting that aberrant GRM1 mediated signaling and expression may be involved in

some melanomas. Our ongoing longitudinal in vivo study is using our melanoma-prone

transgenic mice which are being treated with a functional inhibitor of GRM1-mediated

signaling plus anti-PD-1. Our preliminary results suggest that the addition of a

checkpoint inhibitor, anti-PD-1, does not provide any additional benefit in reducing

tumor progression. Molecular changes are being assessed for alterations in

cytokines/chemokines, in addition the immune cell population are being profiled within

these mice to better understand the long-term consequences of treatments. Our goal for

this longitudinal study is to mirror patient circumstances during treatment regimens, to

better design a rational treatment modality based on changes which transpire within

these animals.


Abstract No: 41

Targeting the colchicine binding site for advanced melanoma

Wei Li

Presenting Author Wei Li

[email protected]

Tubulin inhibitors (e.g., paclitaxel) are widely used chemotherapeutic drugs in treating

various types of cancers, including prostate cancer, breast cancer, lung cancer, ovarian

cancer, and pancreatic cancer. However, their clinical efficacy is often limited by the

ABC-transporter mediated drug efflux and non-specific tissue distribution, leading to

dose limiting toxicity. In addition, existing tubulin inhibitors cannot be taken orally and

must be administered by intravenous route in clinical facilities. We have discovered a

new generation of orally bioavailable tubulin inhibitors that: 1) target the colchicine

binding site in tubulin and have broad spectrum of potent anticancer activity; 2)

effectively circumvent major drug resistance mechanisms that hinder the clinical

efficacy with existing tubulin inhibitors; 3) are orally bioavailable and have excellent

drug-like properties; and 4) are efficacious against both drug sensitive and drug resistant

multiple types of tumors in vivo. We have solved the X-ray crystal structures for many

of these compounds to confirm their direct binding to tubulin. We have also developed

various nanoparticle formulations for these agents and showed that these targeted drug

delivery approaches can improve the anticancer efficacy for these tubulin inhibitors.

Currently the leading compound is under Phase I clinical trials. This work is mainly

supported by NIH grants R01CA148706 and the UTCoP Drug Discovery Center.


Abstract No: 42 The transcriptional co-regulators YAP1 and TAZ promote vascular mimicry in melanoma cells

Stephen P. G. Moore(1), Jason W. Lui(1), Rashi Purohit(1), Hiroshi Tokuo(2), Deborah Lang(1)

Presenting Author

Stephen Moore

[email protected]

Melanoma is the most severe of skin cancers, with a high mortality rate due to metastasis.

Vascular mimicry (VM) is the process whereby highly aggressive tumor cells form

vasculature like structures; a process mimicking angiogenesis that facilitates tumor growth

and metastasis. Indeed, VM formation is correlated with high levels of metastasis and poor

prognosis in cancer patients. It is currently unknown how VM is controlled molecularly.

The transcriptional co-regulators YAP1 (Yes-associated protein 1), or TAZ (transcriptional

co-activator with PDZbinding motif) play vital roles during angiogenesis. We recently

found inhibition of YAP1, and/or TAZ completely abrogates melanoma cells from forming

VM in vitro. Analysis of our RNA-seq data from YAP1 and/or TAZ inhibited melanoma

cells found 51 differently regulated genes, the majority of which are up-regulated, implying

that YAP1 and TAZ function as repressors of these genes. Of these, ~30% are known to

function in inhibiting vascular formation. From these results, we hypothesize that both

YAP1 and TAZ are required to suppress genes involved in impeding the process of

vasculogenesis thereby promoting VM formation. Currently, we are working to validate

the 15 identified genes through qPCR in multiple melanoma cell lines. The identification

of YAP1 and TAZ target genes involved in VM development is important as metastasis is

the main cause of mortality in melanoma; understanding the molecular pathways that

govern VM in melanoma could identify possible therapeutic targets.


Abstract No: 43 Targeting BMP signaling as therapy for advanced melanoma

Alec K. Gramann(1,2), Monir Ejemel(3), Arvind M. Venkatesan(1,2), Yang Wang(3), Craig J. Ceol(1,2)

Presenting Author

Alec K. Gramann

[email protected]

Melanoma is the leading cause of skin cancer death in the United States, with a 5-year

survival rate of 20% for patients with advanced disease. While improvements in therapy

have shown five-year survival rates as high as 60% in clinical trials, this still leaves many

patients that either do not respond to therapy or develop resistance and relapse, ultimately

succumbing to their disease. To continue improving patient outcomes in advanced

melanoma, novel targets must be identified and new therapies developed. Previously, we

identified a novel melanoma oncogene, growth differentiation factor 6 (GDF6), a bone

morphogenetic protein (BMP) ligand that promotes melanoma by suppressing

differentiation and apoptosis. We have shown that anti-apoptotic effects of GDF6 are

mediated by suppression of SOX9, a pro-apoptotic factor in melanoma, and the

dedifferentiation effects are in part mediated by suppression of MITF, the master regulator

of melanocyte differentiation. These results suggest GDF6-activated BMP signaling plays

a role in melanoma progression. We further determined that GDF6 is highly expressed in

approximately 80% of patient melanomas, and thus has potential as a therapeutic target to

broadly impact patient treatment options and outcomes. To target GDF6, we have

developed a panel of novel anti-GDF6 monoclonal antibodies. Here, we show these

antibodies are able to recognize GDF6 in melanoma cell lines and patient tumors

expressing GDF6. Furthermore, we determine a subset of these antibodies can block GDF6

activity in vitro, leading to decreased BMP signaling activity and, ultimately, death of

melanoma cells. Together, these results indicate anti-GDF6 antibodies exert anti-

melanoma effects in vitro and are candidates for treating advanced melanoma.


Abstract No: 44 Generating novel bone-tropic murine metastatic melanoma models

Patricia C. Bunda (1), Chi-Ping Day (1), Eva Perez Guijarro (1), Cari Smith (2), Sung Chin (2), Huaitian Liu (1), Maxwell Lee (1), Glenn Merlino (1), and Helen T. Michael (1)

Presenting Author

Patricia C. Bunda

[email protected]

Metastatic melanoma is responsible for the majority of deaths in melanoma patients. Five-

year relative survival for patients with distant metastatic melanoma is 22% versus 98% in

patients with localized tumors. About 40% of metastatic melanoma patients develop bone

metastasis, yet few tools are available to study mechanisms of bone metastasis. To address

this deficiency, we developed two novel bone metastasis mouse models using novel

C57Bl/6N murine metastatic melanoma cell lines developed in our laboratory. Through

serial intracardiac (IC) injections of cells harvested from bone lesions, we were able to

develop two bone-tropic cell lines which produce bone metastasis in 100% of recipients

and only metastasize to few other organs. In addition, melanoma cells derived from bone

have persistent in vitro morphologic differences compared to those derived from other

organs, suggesting alterations associated with the different tissue microenvironments.

Cytokine arrays revealed differential expression of multiple ECM-related and

inflammatory cytokines between bone and non-bone metastasis cell lines. Cytokines

involved in ECM structure and turnover such as MMP3 and Endostatin (Col18a1) were

upregulated and bone metabolism biomarkers Osteopontin and Osteoprotegerin were

differentially expressed. Results of RNA sequencing analysis from bone and non-bone

metastasis cell lines will be presented. Our novel models of melanoma bone metastasis are

promising tools for elucidating the mechanism of bone-tropism, identifying potential

treatment or prevention targets, and pre-clinical testing of pathway targeted and immune-

based drugs in melanoma.


Abstract No: 45 Tyrosinase-catalyzed oxidation of resveratrol produces a highly reactive ortho-quinone: implications for melanocyte toxicity

Kazumasa Wakamatsu (1), Makoto Ojika (2) and Shosuke Ito (1)

Presenting Author

Kazumasa Wakamatsu

[email protected]

(

trans-Resveratrol (3,5,4’-trihydroxy-trans-stilbene, RES), a naturally occurring

polyphenol, has recently attracted increased interest as a health-beneficial agent. RES is

also well known for its antioxidant, anti-platelet, anti-inflammatory, anti-aging,

cardioprotective and cancer chemopreventive properties. However, based on its p-

substituted phenol structure, RES was found to be a good substrate for tyrosinase and was

oxidized to produce a highly reactive o-quinone form. The recent incidence of

rhododendrol-induced leukoderma highlights the risk of using compounds having p-

substituted phenols because of their rapid conversion to toxic o-quinones. The results of

this study demonstrate that the oxidation of RES by tyrosinase produces 4-(3’,5’-

dihydroxy-trans-styrenyl)-1,2-benzoquinone (RES-quinone), which decays rapidly to an

oligomeric product (RES-oligomer). RES-quinone was identified after reduction to its

corresponding catechol, known as piceatannol. RES-quinone reacts with N-acetylcysteine,

a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS

analyses. The production of a triadduct is not common for o-quinones, suggesting a high

reactivity of RES-quinone. RES-quinone also binds to bovine serum albumin through its

cysteine residue. RES-oligomer can oxidize GSH to GSSG, indicating its pro-oxidant

activity. These results suggest that RES could be cytotoxic to melanocytes due to the

binding of RES-quinone to thiol proteins and show that the cosmetic use of RES should be

considered with caution.


Abstract No: 46

Determining the role of proinflammatory factors TNFa and Cox2 in UVB-induced melanocyte stem cell activation and migration

Luye An(1), Hyeongsun Moon(2), Leanne R. Donahue(1), Dah Ihm Kim(1) Andrew White(1)

Presenting Author

Luye An

[email protected]

Accurately controlled proliferation and differentiation of tissue stem cells are key to

maintaining tissue homeostasis. I will study how extrinsic environmental stimuli affect

tissue stem cell activities using melanocyte stem cells (McSCs) in the mouse dorsal skin

as a model. It is known that ultraviolet-B (UVB) irradiation can induce McSC activation

and migration to the inter-follicular epidermis (IFE). However, because McSCs are

located deeply within the hair follicle where UVB cannot penetrate, it is unclear how

the skin tissue responds to UVB in order to stimulate McSC activity. Blocking the

downstream pathway of TNFa, a proinflammatory factor, in mice shows a notable

change in McSC migration in response to UVB, suggesting a possible mechanism for

McSC activation. We have also characterized a mouse model which shows low McSC

migration following UVB and found that the expression level of cyclooxygenase-2

(Cox2), another proinflammatory factor, is reduced compared to wild type. To further

investigate this, I inhibited Cox2 catalytic activity and found that UVB-induced McSC

activation was restricted. Thus, I hypothesize that the proinflammatory factors TNFa

and Cox2 regulate McSC activation and interfollicular migration in response to UVB.

This study will help us to understand how tissues respond to environmental stresses and

regulate tissue stem cell activities to maintain tissue homeostasis. This may also

illuminate the potential mechanisms of UVB-induced melanoma and reveal vitiligo

treatments that are more efficient in human patients.


Abstract No: 47 Characterization of Dct-H2BGFP Bitransgenic Mouse Model with

C57BL/6J Background for Study of Melanocyte Lineage Cells.

Bishal Tandukar (1), Sandeep Joshi (1), Emmanual Unni (1), Li Pan (1), Thomas J. Hornyak (1,2,3)

Presenting Author Bishal Tandukar

[email protected]

Melanocyte stem cells (McSCs) are derived from neural crest during embryogenesis. They

are key components of the hair follicle (HF) stem cell system and are responsible for

regeneration of differentiated melanocytes during successive HF cycles. To facilitate

continued research on melanocyte development, McSCs, and melanocyte differentiation in

vivo, we generated Dct-H2BGFP inducible, bitransgenic mice that permit accurate

identification of McSCs and melanocytes in the murine HF through GFP expression. To

obtain Dct-H2BGFP mice, the Dct-tTA transgene was inserted via homologous

recombination into the neutral Hprt locus on the X chromosome and then intercrossed with

TRE-H2BGFP mice. The resultant mouse was backcrossed into the C57BL/6J background

(BL6-Dct-H2BGFP). We compared the expression pattern of BL6-Dct-H2BGFP to that of

Dct-H2BGFP mice on a mixed background reported previously. To characterize BL6-Dct-

H2BGFP mice, we confirmed not only the expression of H2BGFP in all melanocyte

lineage cells, but also doxycycline regulation of GFP expression. Gene expression analysis

of McSCs isolated by fluorescence-activated cell sorting (FACS) from telogen HFs of P56

murine skin showed higher expression of the melanocyte genes Kit, Dct, Tyr, and Tyrp1

compared to non-melanocytic cells of the skin. Furthermore, ex vivo culture of the McSCs

subsets showed propensity of bulge/CD34+ McSCs to differentiate with expression of non-

melanocytic, neural crest lineage markers including glia (Gfap and CNPase, 73±1% and

77±2%, respectively), neurons (Tuj1 26±5%), and smooth muscle (α-Sma, 31±9%; n=3).

In contrast, CD34-/secondary hair germ (SHG) McSCs differentiated into pigmented

melanocytes, with higher expression of melanogenic markers Tyr (71±1%), Tyrp1

(68±4%), and Mitf (75±7%; n=3). These results establish not only the utility of BL6-Dct-

H2BGFP bitransgenic mice for future in vivo studies of melanocytes but also the

reproducibility of our previous findings on McSCs and their subsets.


Abstract No: 48 Sound-induced color change in Koch’s giant day gecko from Madagascar

Randall L. Morrison (1) and Chiaka Nwachukwu (2)

Presenting Author

Randall L. Morrison

[email protected]

Koch’s giant day gecko is an abundant and widespread lizard in the dry deciduous forests

of northwestern Madagascar. At mid-day adults are a bright green color but can change to

a brown color. The mechanism of color change is likely similar to that of the anole where

melanosomes move over or under the xanthophores and iridophores to cause brown or

green colors respectively. Previous research has indicated that these lizards will change in

color lightness when hearing a variety of sounds, including the alarm call of the Paradise

flycatcher. We have conducted an analysis of color change in response to white noise, the

alarm call of the Paradise flycatcher and the call of the Madagascar buzzard (a gecko

predator). Color was quantified from standardized photographs and hue, saturation and

lightness values were compared before and after stimulation. The time required for color

to return from brown to green (latency) was calculated as well and varies depending on the

sound. Geckos change color from green to brown in response to a wide variety of sounds

and future research will quantify the color change response to different sounds, volumes

and sound frequency to determine the threshold levels required to elicit color-change

responses.


Abstract No: 49 Crasher is Critical for the Differentiation of Black Pigment Cells and the Production of Black Pigment

Samantha Neuffer (1), David Beltran (2), Lauren Clancey (2), Matt Lambert (2), and Cynthia Cooper (1)

Presenting Author

Samantha Neuffer

[email protected]

Pigment is essential to an organism’s quality of life. Zebrafish are an emerging model

organism used to study pigment development. By studying pigment mutations in zebrafish,

we can characterize the genes that are critical for normal development and how that

development may be altered in disease. Crasher is a mutant zebrafish line that is

characterized by a decrease in melanophores (melanocytes in zebrafish) and melanin.

Using in situ hybridization, we have shown that crasher does not affect genes important for

the specification of pigment cells such as microthalmia transcription factor 1a (mitfa) and

forkhead box d3 (foxd3). However, expression of genes important for the production of

melanin are reduced in crasher at 24 hours post fertilization (hpf). Tyrosinase (tyr) and

tyrosinase-related protein 1b (tyrp1b) are slightly reduced in crasher, while dopachrome

tautomerase (dct) is greatly reduced. All three of these genes are controlled by the same

transcription factor (mitfa), so the reason for the variation in reduction of tyr, tyrp1b, and

dct is unknown. Furthermore, tyrp1b is reduced significantly at 30 hpf when black pigment

cells differentiate indicating crasher plays a role in the differentiation of melanophores as

well. The mutation causing the crasher phenotype is mapped to one of seven possible genes

not previously known to cause a crasher-like phenotype. We will use CRISPR and RNA-

seq to identify the gene causing pigment and pigment cell loss in crasher. Characterization

of crasher will be a valuable tool to better understand how tyrosinase, dopachrome

tautomerase, and tyrosinase-related protein 1b are distinctly regulated and how these genes

interact in vivo to control pigment cell differentiation and pigment production.

Additionally, identification of the gene causing the crasher phenotype will help us better

understand a potentially novel protein necessary for full pigmentation and correct pigment

cell development.


Abstract No: 50 A CRISPR-based mutagenesis screen to identify novel mediators of

SOX10 function in melanogenesis.

Eric Domyan, Zach Dean, Jacob Kirkpatrick, Joshua Kirkpatrick, Tyrel Porter, and Mckenzie Roberts

Presenting Author

Eric Domyan

[email protected]

Domestic rock pigeons (Columba livia) homozygous for the “recessive red” mutation,

which causes downregulation of Sox10, display brilliant red colors instead of blue/black

feathers. Sox10 encodes a transcription factor important for melanocyte differentiation and

function, but the genes that mediate its promotion of black vs. red pigment are unknown.

Earlier transcriptomic comparisons of regenerating feathers from wild-type and recessive

red pigeons identified candidate SOX10 targets, the majority of which have not, to date,

been implicated in pigmentation. We have initiated a CRISPR-based mutagenesis screen

of these targets in the mouse melan-a cell line to identify novel mediators of SOX10

function in melanogenesis. Our preliminary results are encouraging, as mutations in both

genes tested thus far (Arsg, Tbx2) are correlated with a reduction in pigmentation. These

data highlight the value of using novel, emerging model organisms to gain insight into the

genetic basis of pigment variation.


Abstract No: 51

Iridophore morphogenesis during zebrafish stripe formation.

Emily Bain (1), Dvir Gur (2,3), Andrew Aman (1), Jennifer Lippincott-Schwartz (2), David Parichy (1)

Presenting Author

Emily Bain

[email protected]

Biological pattern formation, though pervasive, is not well understood especially during

later stages of development. Zebrafish stripes offer a tractable system to study cell

behaviors during post-embryonic pattern formation. In the adult, alternating dark stripes

and lighter interstripes made of black melanophores and yellow xanthophores,

respectively, comprise the stripe pattern. Iridescent iridophores are also found along the

flank in two different conformations: in a dense, epithelial-like mat in the interstripe region

and as loose, stellate cells associated with melanophores in the stripe. The prevailing model

of stripe pattern establishment and reiteration links dynamic behaviors in iridophores such

as shape change, proliferation, and migration. Dense iridophores in the first interstripe

change their shape to the mesenchymal-like loose configuration, migrate through the

developing stripe, and reaggregate to form the dense iridophores of the secondary

interstripe some specific distance away from their origin. To better understand this process,

we used time lapse analyses and individual cell tracking to further characterize iridophore

state and behavior. Interestingly, individual iridophores do not change their differentiation

state between loose and dense. Instead, iridophores assume a particular morphology at the

time of differentiation, according to presence or absence of melanophores, and this

morphology remains fixed thereafter. We further observed that interstripe and stripe

iridophores exhibit distinct organizations of guanine reflecting platelets conferring intrinsic

differences in color (yellow, blue), that stripe iridophores can modulate reflecting platelet

orientation physiologically (blue to yellow), and that iridophore types are associated with

distinct transcriptomic states. Our findings suggest a revised model of adult stripe pattern

formation and indicate that pattern phenotype depends not only on interactions among

pigment cells that affect their arrangements, but also factors that specify subcellular

organization and physiological responsiveness of specialized organelles.


Abstract No: 52 Investigating the neural crest gene regulatory network: identification of an enhancer nearby tfap2a

Angela Benton, Andrea Hallberg, Nathan Nowzari, Gregory Bonde, Colin Kenny, Robert Cornell

Presenting Author

Angela Benton

[email protected]

The gene regulatory network (GRN) governing specification of neural crest (NC) cells

from neural plate border (NPB) cells is poorly understood is believed to be activated in

melanoma initiating cells. Transcription factors in the TFAP2 family are essential

contributors to this GRN: in zebrafish tfap2a/tfap2c double mutants, markers of pre-

migratory NC, and all NC derivatives, are absent, while markers of the NPB are unaffected.

We hypothesize that tfap2a expression in the NC is mediated by one or more NC-specific

enhancers that respond to TFs expressed in the NPB. A published ATAC-Seq study of

isolated NC cells revealed several nucleosome-free regions in chromatin near tfap2a. We

found that one of them, i.e., zebrafish regulatory element 37 (ZRE37), drives reporter

expression in the NC in stable transgenic tg(ZRE37:GFP) embryos. To test the prediction

that ZRE37 is required for normal NC development we deleted it using guide RNAs

flanking it and Cas9; currently we are raising heterozygous carriers of this deletion. Our

hypothesis predicts that in tfap2a/c double mutants, driving tfap2a expression solely with

the ZRE37 enhancer will suffice to rescue NC development. In preliminary results

supporting this prediction, tfap2a/c double mutants transiently transgenic for

tg(ZRE37:tfap2a) exhibit some melanocytes. Finally, various signaling pathways are

employed at the NPB to NC transition. Treatment of tg(ZRE37:GFP) embryos with LiCl,

a potent activator of the Wnt pathway, expanded the GFP signal. We hypothesize that Wnt

signaling directly activates ZRE37 through the binding of Tcf and Lef. However, deletion

of high-scoring Tcf/Lef binding sites in ZRE37 did not diminish its NC enhancer activity

in transient transgenic reporter assays. We are currently deleting lower-scoring Tcf/Lef

sites and binding sites for TFs expressed in the NPB from ZRE37. These experiments

illuminate a GRN that controls NC specification GRN and possibly also melanoma

initiation.


Abstract No: 53 From latent space learning in dermatology to single cells and back

Fabian V. Filipp

Presenting Author

Fabian V. Filipp

[email protected]

Recent advances in systems biology including single cell transcriptomics enable precise

description of molecular, cellular, and immunological changes associated with pigmentary

disorders. However, uncovering the functional and clinical significance of these genomic

and cellular changes is a significant computational challenge. Originally our skin cells were

described as a uniform cell type by Ramony Cajal in 1900. Single cell technologies have

the ability to leverage the wealth of dermal constituents. During skin development, upon

wounding, at homeostasis, or in aging, various dermal subtypes showcase their specific

roles as functionally heterogeneous but distinct cells. Today, molecular insights from

genome and systems biology are influencing how disease is diagnosed and treated. Multi-

omics data is deep and complex, offering an opportunity for mechanistic data-driven

insights as well as automated disease classification in dermatology. Typical questions

asked to such data are how skin cells develop over time and after perturbation such as

malignant transformation, auto-immune attack, or wounding. Our approach integrates

hierarchical models of epigenetic and transcriptional control in skin differentiation and

disease with deep learning. The statistical tools to address these questions are techniques

from pseudotemporal ordering and lineage estimation, or latent space learning. Conversely,

links to transcriptional outcome and chromatin binding sites provide insight into genomic

regulation. By identifying links between complementary omics layers, it is possible to

mechanistically correlate oncogenic events with clinical phenotypes and functional

genomics impact.


Abstract No: 54 Leukocyte-Cancer Cell Fusion: Genesis of a Deadly Journey

Greggory S. LaBerge (1,2), Eric Duvall. (2), Kay Haedicke (3), Zachary Grasmik (4), Angela Galan (5) and John M. Pawelek (5)

Presenting Author

John M. Pawelek

[email protected]

The main cause of cancer mortality is not the primary tumor itself but metastatic

progression to distant organs and tissues. Yet the mechanisms of metastasis remain poorly

understood. Leukocyte–cancer cell fusion and hybrid formation as an initiator of metastasis

was proposed more than a century ago by the German pathologist Prof. Otto Aichel. This

prescient concept has since been confirmed in many animal models and, from our group,

in 2 patients with renal cell carcinoma and 3 patients with malignant melanoma. Numerous

reports from others have confirmed and extended these findings. Leukocyte–tumor cell

fusion provides a unifying explanation for metastasis. While primary tumors arise in a wide

variety of tissues representing not a single disease but many different diseases, metastatic

cancer may be only one disease arising from a common, non-mutational event: fusion of

primary tumor cells with leukocytes. From the findings to date, it would appear that such

hybrid formation is a major pathway for metastasis. Studies on the mechanisms driving this

process could uncover previously unexplored targets for therapeutic intervention.


Abstract No: 55 The chromatin underpinnings of the MITF/TFAP2A genetic interaction in melanocyte development

Colin Kenny (1), Andrea Hallberg (1), Hannah Seberg (1), Gregory Bonde (1), Ramile Dilshat (2), Eirikur Steingrimsson (2), and Robert Cornell (1)

Presenting Author

Colin Kenny

[email protected]

Activating-enhancer binding transcription factor (TFAP2) family members contribute to

the gene regulatory network that drives lineage specification of neural crest and the one

that promotes terminal differentiation of melanocytes. How one transcription factor family

promotes these distinct cellular events is unclear. Zebrafish embryos doubly homozygous

for loss-of-function alleles of tfap2a and tfap2c completely lack expression of definitive

neural crest markers. Using ATAC-seq we recently found that in such mutants in

comparison to wild types there is reduced chromatin accessibility at thousands of genomic

loci. These loci are enriched for Tfap2a binding motifs and a subset of such loci are near

genes whose expression is Tfap2a/c-dependent. At such loci Tfap2 may act as a pioneer

factor opening closed chromatin for access by other transcription factors. Previously we

showed that in human primary melanocytes, TFAP2A binds at about 70% of active

enhancers including many also bound by the melanocyte “master regulator” MITF. Given

our findings in zebrafish mentioned above, we predict that in the absence of TFAP2A,

MITF binding at loci normally co-bound by MITF/TFAP2A will be reduced. To test this,

we have deleted TFAP2A and its redundantly-acting paralog TFAP2C in human melanoma

cell lines and are assessing MITF binding using Cleavage Under Targets and Release Using

Nuclease (CUT&RUN). Conversely, we deleted MITF from a melanoma cell line and are

similarly profiling TFAP2A bound loci. These experiments will help to determine the

mechanism of TFAP2A in regulating chromatin accessibility during differentiation of

neural crest and its derivatives. This work was funded by the NIH (NIH/NIAMS

AR062547)


Abstract No: 56 The whitening effect of novel synthesized glucosamine fatty acid ester

Lingying Tong (1), Yong Han(1) and Shiyong Wu(1,2)

Presenting Author

Lingying Tong

[email protected]

Hyperpigmentation or skin pigment disorders affect the color of the skin by melanin

accumulation at specific spots. Glucosamine has been shown to have the potential for skin

whitening yet has obstacle of skin penetration. Coconut oil is a widely used active

component in skin care products, especially in moistures to keep skin hydrated. Here, we

synthesized a new compound by linking glucosamine to coconut oil. The novel synthesized

glucosamine fatty acid ester showed little toxicity in normal human melanocyte and

keratinocyte cells. On mouse B16F10 melanoma cells, which has a black color background,

the compound can significantly reduce the blackness in the cells by reducing the melanin

in the cells after about 7-day treatment. Based on our result and the fact that melanin

stimulation causes various skin problems including but not limited to letigo spots, moles,

birthmarks, overexposure to sunshine, etc., we hypothesize that the compound has the

potential to be applied to treat specific areas on the problematic skin surface.


Abstract No: 57 Dopaminechrome tautomerase – A new enzyme involved in melanin biosynthesis

Hanine Barek and Manickam Sugumaran

Presenting Author

Hanine Barek

[email protected]

It is well known that tyrosine and dopa are major precursors of melanin skin pigmentation

in animals. However, combined genetic, biochemical and chemical degradative studies

point out that dopamine and not dopa is the major cuticular melanin precursor in insects.

These catecholamine precursors are oxidized to their quinone and subsequently converted

to “chrome” by nonenzymatic reactions in all organisms. Two different enzymes acting on

dopachrome are characterized. The mammalian dopachrome tautomerase converts

dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and the insect dopachrome

decarboxylase/tautomerase converts dopachrome to 5,6-dihydroxyindole. Studies indicate

that dopaminechrome is a poor substrate for several insect dopachrome

decarboxylase/tautomerases. If dopamine is the specific melanin precursor in insect cuticle,

it calls for the presence of a specific enzyme that can isomerize dopaminechrome and not

dopachrome. In this report, we present our results on the characterization of such a novel

enzyme. Dopaminechrome tautomerase exhibits a narrow specificity and acts on

dopaminechrome and N-methyldopaminechrome and did not touch other chromes,

especially dopaminechrome. The partial purification and properties of this new enzyme

will be presented along with its importance in the melanogenesis occurring in insect cuticle.


Abstract No: 58 Investigating the role of neuropeptide Y in the vitiligo disease

Zoya Anderson (1), Joseph Palmer (1), Chris Keys (1), Lynn Dobrunz (2), Melissa Harris (1)

Presenting Author

Zoya Anderson

[email protected]

Vitiligo is an autoimmune disease that is characterized by destruction of the pigment-

producing cells called melanocytes, resulting in de-pigmentation of the skin and hair. This

disease is known to be associated with mutations in a number of genes and is thought to be

caused by a combination of intrinsic melanocyte stress, autoimmunity, and immune cell-

mediated melanocyte destruction. There is no cure for vitiligo, and the current treatments

that are available address different aspects of the disease in untargeted and often inefficient

manners. Because vitiligo is such a complex disease composed of multi-genic, multi-

cellular, and autoimmune factors, it is important to understand the molecules and signaling

pathways that are involved in the disease’s pathogenesis, progression, and maintenance in

order to design more efficient treatments for the disease. It has been shown that

neuropeptide Y (NPY), a neuropeptide with implications in stress-resiliency, has known

mutations that are associated with both increased NPY expression and increased

susceptibility for vitiligo. NPY has also been shown to be upregulated in the circulation of

vitiligo patients when compared to control patients and is also upregulated in vitiliginous

skin compared to unaffected skin of the same patient. In a mouse model with a targeted

mutation that results in the overexpression of endogenous NPY, our preliminary data has

shown that these mice have upregulated NPY expression in the skin, among other tissues,

and exhibit a premature and progressive loss of pigmentation in the hair, resulting in a

vitiligo-like hair-graying phenotype. The purpose of this project is to investigate how the

upregulation of NPY, as is seen in the circulation and skin of vitiligo patients, plays a role

in the vitiligo disease. Preliminary findings from RNA sequencing of skin from NPY-

overexpressing mice indicate that overexpression of NPY changes the expression of genes

involved in both keratinocyte and adipocyte biology, which implicates these two cell types

as potential mediators of hair graying in these animals. Future studies for this project aim

to identify whether and how these skin constituents are responsible for effecting NPY-

mediated signaling and to determine the pathogenic effects of upregulated NPY on the

regulation of follicular pigmentation.


Abstract No: 59 Type I Interferon signaling suppresses IFN-y mediated immune

responses in vitiligo and melanoma.

Rebecca L. Riding, Jillian M. Richmond, Keitaro Fukuda and John E. Harris

Presenting Author

Rebecca L. Riding

[email protected]

Type I interferons (IFNs), originally recognized for their anti-viral properties, can drive

protective or pathogenic adaptive immune responses in a context dependent manner.

Vitiligo is an autoimmune skin disease in which melanocytes are killed by autoreactive

CD8+ T cells specific for melanocyte/melanoma-shared antigens, such as premelanosome

protein (PMEL). Previous work has revealed IFNy as the central cytokine driving vitiligo

pathogenesis. Since type I IFN and IFNy can induce overlapping transcriptional programs,

we sought to determine the role of type I IFN in the development and progression of

vitiligo. We found that in contrast to IFNyR deficient host mice, which were protected from

disease, interferon alpha-receptor deficient (IFNAR-/-) host mice develop accelerated and

severe vitiligo compared to WT mice. IFNAR signaling was not required on PMELs, but

was required on radioresistant cells to suppress disease. IFNAR-/- mice had increased

CD8+ T cell activation characterized by reduced expression of PD-1, and increased effector

cytokine production TNFa and IFNy. Increased IFNy signaling in IFNAR-/- mice led to

significant skin chemokine ligand CXCL9 and CXCL10 expression, driving recruitment

of circulating PMEL to the epidermis. To determine whether this observation could be

useful in melanoma immunotherapy, we tested transfer of PMELs in B16F10 melanoma-

inoculated IFNAR-/- and WT mice and found that IFNAR-/- mice had reduced tumor

burden, elevated numbers of tumor-infiltrating PMELs and reduced PD-1 expression

compared to WT host mice. Collectively, these results reveal type I IFN mediated

suppression of IFNy anti-melanocyte immune responses in both autoimmunity and tumor

immunity.


Abstract No: 60 The target tissue in the Smyth line chicken model for spontaneous autoimmune vitiligo: relevance and opportunities for studies on disease etiology, progression, and treatment

Marites A. Sales and Gisela F. Erf

Presenting Author

Marites Sales

[email protected]

Autoimmune vitiligo is a pigmentary disorder characterized by immune system mediated

post-natal loss of epidermal melanocytes. The best-established animal model for

spontaneously developing autoimmune vitiligo is the Smyth line (SL) chicken. In addition

to spontaneous onset of this multifactorial disorder, the nature of the target tissue, namely

the growing feather (GF), is another valuable feature of this model. With few exceptions,

chickens do not have pigmented skin. Rather, plumage pigmentation occurs in the GF.

Specifically, the living portion (pulp) of a GF is an 8-10 mm x 2 mm (height x diam.)

column of skin, consisting mostly of inner dermis that is enveloped by epidermis. The

epidermis of the bottom third (newest growth) of the pulp is modified into barb ridges that

contain melanocytes and keratinocytes. The cell bodies of the melanocytes are facing the

dermis while their dendrites extend along columns of keratinocytes. Melanocytes from

neighboring barb ridges are in close association with each other, forming a ring of cell

bodies around the dermis. The GF dermis is a complex vascularized tissue capable of

recruitment of cells and proteins from the blood. Collection of GF is minimally invasive,

GF regenerate and can be collected before and throughout vitiligo development. The pulp

provides ample tissue for laboratory analyses [(immuno)-histology, flow cytometric cell

population analyses, gene-expression at RNA- and protein-level, measurements of enzyme

activity, melanogenesis and cell stress, cell culture, etc.] that have generated important

insights into the etiology and nature of the autoimmune response in vitiligo. Moreover, GF

can be injected (i.d.) with test-materials and in situ activities monitored in the same

individual. Hence, the skin-like, easily accessible target tissue in the autoimmune vitiligo-

prone SL chicken further underlines the suitability of this model for biomedical research

on mechanisms, treatment, and prevention of autoimmunity.

C1 - Internal use

Title: In Vivo Model for Visible Light Exposure

Hawasatu Dumbuya, Stephen Lynch, Kaili Ji, Qian Zheng, Janet Wangari-Talbot L’Oréal Research and Innovation, Clark, NJ Historically, sunscreens were developed to offer protection against the ultraviolet (UV) portion of

sunlight. It is now evident that visible light (VIS) irradiation, ranging from 400-700 nm, can cause

skin darkening and contribute to worsening of dyschromia, particularly in individuals with skin

phototype III and higher. Currently, sunscreens provide limited protection against visible light.

Various VIS irradiation protocols consisting of single or multiple exposures have been published.

Depending on the subjects’ skin phototypes and nature of exposure, the skin pigmentation

amplitude, persistence and make-up (i.e photo-oxidized melanin and/or de novo melanin

synthesis) will vary, which can influence products performance when evaluating for

photoprotection efficacy.

Due to the lack of standards in cosmetic industry, determining the appropriate VIS irradiation

protocol to induce skin darkening is crucial in order to properly develop and test new or existing

products for broadband protection ability and support claims. Here, we evaluate two different

VIS irradiation protocols: single and multiple doses, in order to compare the induced

pigmentation responses in subjects with skin phototype IV.

Our results highlight that both single or multiple doses of VIS can cause both immediate and persistent darkening in subjects with skin phototype IV. Whereas most of the photo-protection studies have focused on the effect of UV, we aimed to fill the current gap in sun protection by developing a new model for evaluating new actives with full visible light photoprotection properties. Future studies are warranted to test formulations containing pigments and/or metal oxides in this model in order to develop new prevention approaches against harmful effects of sunlight for multiple skin phototypes.

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