Founded in 1958October, 2010 Volume 7

www.grunebaumfoundation.org

Chairperson: Steven E. Wallach, Esq.Treasurer: Ms. Carol Grunebaum Kelly

Secretary: Ms. Shelby K. Schultz

Karen H. Antman, M.D.Provost of the Medical Campus and Dean,

Boston University School of MedicineAndrew C. Culbert, Esq., PartnerMasterman, Culbert and Tully, LLP

Michael J. Droller, M.D.Chairman Emeritus

Katherine and Clifford GoldsmithProfessor of Urology

Professor of Oncology, Department of Urology,The Mount Sinai Medical CenterDouglas V. Faller, Ph.D., M.D.

Grunebaum Professor inCancer Research

Director, Cancer CenterBoston University School of Medicine

Merrill I. Feldman, M.D.Professor of Radiology (ret.),

Boston University School of MedicineChairman, Department of Radiation Medicine,

University Hospital, Professor of Oral Oncology, Boston University

School of Graduate DentistryMichael A. Gimbrone, Jr., M.D.

Ramzi S. Cotran Professor of PathologyHarvard Medical School;Chairman, Department of

Pathology & Director,Center for Excellence in Vascular Biology,

Brigham & Women’s HospitalDavid E. Golan, M.D. Ph.D.Dean for Graduate Education

Harvard Medical SchoolFrank J. Hsu, M.D.

Senior Medical Director,Genzyme Oncology

Ms. Yvonne F. Grunebaum

Mrs. Geri j Wallach

Ms. Shawna Wallach

Mr. Christopher Kelly

BOARD OF TRUSTEES

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From the ChairDear Friends of the Karin Grunebaum Cancer Research Foundation;

Everyone loves a winner, and we are no exception. We are very pleased to tellyou that one of our three 2010 - 2011 Faculty Research Fellows, Michael H.A.Roehrl, M.D., Ph.D., has been awarded the Young Investigator Award by the Hu-man Proteome Organization (HUPO). This prestigious award was awarded to Dr.Roehrl at the organization’s international meeting in Sydney, Australia. There ismore detail about Dr. Roehrl’s pioneering work inside the Newsletter.

We salute Dr. Roehrl’s accomplishment as he joins the long list of KarinGrunebaum Fellows who have distinguished themselves in academic medicineand medical research. The “Focus on Research” section on the Foundation’swebsite (www.grunebaumfoundation.org) highlights their accomplishments. Wewould really appreciate hearing from otherpast or present Fellows who have alsoachieved unique recognition in their field.Please let me know so we can also highlightyour accomplishments.

As many of you know, our Foundation isunique in that the Board of Trustees is com-posed of pre-eminent figures in academicmedicine and cancer research, along withmembers of Karin Grunebaum’s immediatefamily. Thus, we are very proud to announcethat Karin’s two youngest grand-children(Shawna Wallach and Christopher Kelly) havejoined the Board of Trustees. We know thatthey will superbly complement the outstand-ing cadre of Board members.

One of the greatest benefits we receiveas Trustees (we are all unpaid volunteers) isto annually hear presentations from the cur-rent incoming and outgoing Fellows on theirresearch projects. The incoming Fellows tellus of their hypotheses and projects, while theoutgoing Fellows tell us of their accomplish-ments. This is such an enlightening experi-ence, that the Trustees decided to invite allformer Fellows to attend these presentations.The presentations are held annually on the

The incoming Fellowstell us of their hypothesesand projects, while theoutgoing Fellows tell us oftheir accomplishments.This is such an enlighten-ing experience, that theTrustees decided to inviteall former Fellows to at-tend these presentations.The presentations areheld annually on the sec-ond Friday in June at Bos-ton University School ofMedicine. If you wouldlike to attend, pleaseRSVP at least 2 weeksahead of time.

October, 2010 Volume 7Karin Grunebaum Cancer Research Foundation

From the Chair (continued from page one)

second Friday in June at Boston University School of Medi-cine. If you would like to attend, please RSVP at least 2weeks ahead of time. We would love to see you again, andwe think you would really enjoy the event.

The Board of Trustees is constantly looking at new av-enues to provide support for cancer research. In addition toannually sponsoring first-rate cancer researchers, we are cur-rently looking at ways to also help educate other budding sci-entists who want to focus their energy specifically on cancerresearch. We are hopeful that such a “seed” program can alsobe supported by the Foundation if sufficient additional fundscan be raised. Of course, adding to our mandate in these try-

ing economic times requires additional resources to help as-sure such expansion of our efforts. We sincerely appreciatethe donations received during the past year, and we ask eachof you to please contribute to our Foundation so that theseadditional dreams can be realized. Please make your tax de-ductible contributions by mail or on-line .We believe that whenmore people are focused on finding a cure for cancer, the soonerthat elusive dream can be realized.

Sincerely,Steven Wallach, Chairperson

(561) 750-7366E-mail: steven.wallach@grunebaumfoundation.org

Did you know that our very own trustee, Michael J. Droller, M.D.,Chairman Emeritus, was the first recipient of the KarinGrunebaum Cander Research Scholarship?

The following is the newspaper article announcing the award.

Dr. Droller is currently the Katherine and Clifford Goldsmith Professor of Urology, Professor of Oncology, De-partment of Urology, The Mount Sinai Medical Center.

From the Archives

October, 2010 Volume 7Karin Grunebaum Cancer Research Foundation

From the FellowsHarvard Medical School

Identification of Novel NF-kB Pathway Regulators in Lymphomagenesis

Figure 1. LMP1 stimulates NF- B through two cytoplasmic signaling domains. The membrane proximal domain triggers non-canonical NF- B activation, whereas the distal domain stimulates canonical NF- B.

Growth + Survival

NF- B

Non-CanonicalPathway

NF- B

LMP1

CanonicalPathway

Growth + Survival

NF- B

Non-CanonicalPathway

NF- B

LMP1

CanonicalPathway

Benjamin Gewurz, M.D., Ph.D. Instructor in Medicine,Brigham and Women’s Hospital, Department of Medicine

Nuclear Factor-kappa B (NF-kB) is a family of transcrip-tion factors that control lymphocyte proliferation, differentia-tion and survival. Immune receptors activate NF-kB througheither or both of two signal transduction cascades, termed‘canonical’ and ‘non-canonical’ pathways. Receptor cytoplas-mic domains recruit adaptor molecules that initiate down-stream signal transduction events. NF-kB pathways triggerproteasomal degradation of inhibitory proteins, NF-kB nucleartranslocation, and activating post-translational modificationsof NF-kB complexes. Activated NF-kB drives transcriptionprograms that promote cell growth and that block apoptosis.Multiple negative feedback loops tightly control NF-kB acti-vation and serve to terminate signaling in the absence ofpersistent stimuli. While NF-kB activity is integral to normalimmune system development and function, aberrant NF-kBhyperactivation underlies the growth of numerous lympho-

mas. Despite inten-sive study, potentiallydruggable enzymaticregulators of NF-kBand their importance inspecific malignanciesremain to be identified.

Gene profilingstudies have demon-strated particularlyhigh NF-kB activity lev-els in multiple aggres-sive lymphomas: dif-fuse large B-cell Lym-phoma, Hodgkin Dis-ease, primary medias-tinal B-cell lymphoma,and multiple myeloma.The genetic basis fortheir elevated NF-kBactivity remains largelyunknown, though ap-pears to arise from themutation of key canoni-cal and non-canonical

NF-kB pathway regulators. Notably, NF-kB pathway muta-tions may contribute to the development of malignanciesthroughout the body. A recent survey of 3131 human cancerspecimens spanning 26 histological types found NF-kB path-way genes to be among the most frequently targeted by so-matic copy number alterations. NF-kB activating mutations

span several classes of enzymes,including ubiquitin ligases,deubiquitinating enzymes, andkinases. Such mutations appearto collectively amplify activationpathways and diminish negativefeedback regulation. Aggressivelymphomas frequently becomedependent on constitutive NF-kBactivation, and undergoapoptosis upon NF-kB blockade.Unfortunately, there are no thera-peutic agents currently available for clinical use to specifi-cally block NF-kB activity. NF-kB pathways therefore con-tain promising targets for the development of rational che-motherapeutic agents to treat common lymphoid malignan-cies.

Epstein Barr Virus (EBV) is a ubiquitous herpesvirus thatis an important cause of Hodgkin disease and otherlymphoproliferative diseases, particularly in the setting ofimmune suppression. The primary EBV oncoprotein, LatentMembrane Protein 1 (LMP1), mimics NF-kB hyperactivationstates present in lymphoid malignancies. LMP1 is expressedin most EBV-associated lymphoproliferative malignancies,including in 40% of Hodgkin lymphoma samples. In a ligand-independent fashion, LMP1 constitutively activates both ca-nonical and non-canonical NF-kB pathways through two in-dependent cytoplasmic tail domains (Figure 1). EBV-trans-formed cells require persistent NF-kB activation for theirgrowth and survival, and undergo apoptosis upon NF-kBblockade. Small molecule inhibitors of LMP1/NF-kB couldtherefore potentially inhibit the malignant growth of EBV-in-fected human malignancies.

My central hypothesis is that EBV-transformed cells andlymphoid malignancies rely on shared cellular signal trans-duction pathway components to persistently activate NF-kB,the inhibition of which can be utilized to specifically treat hu-man malignancies. To elucidate novel NF-kB pathway com-ponents, I have recently completed a human genome-wideloss-of-function siRNA screen to comprehensively identify theLMP1/canonical NF-kB pathway. The screen successfullyidentified most pathway components, as well as many novelsuppressors and enhancers of the LMP1/canonical NF-kBpathway. Hits included numerous enzymes: kinases, phos-phatases, ubiquitin ligases, and deubiquitinating enzymes. Ihave found that 81% of screen hits likewise function in ca-

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October, 2010 Volume 7Karin Grunebaum Cancer Research Foundation

Andrew Liss M.D.Massachusetts General Hospital

Massachusetts General Hospital

Last year in the United States more that 42,000 peoplewere diagnosed with pancreatic ductal adenocarcinoma(PDAC) with at least 35,000 people dying from this disease.Currently, surgery provides the only hope for survival. How-ever, 70% of patients are not eligible for surgery since theyare diagnosed with locally advanced disease and distantmetasteses. While gemcitibine has been the standard of carefor PDAC for more than a decade, it has failed to dramaticallyimprove the survival of patients. Less than 5% of patients diag-nosed with PDAC survive five years after diagnosis.

Similar to other cancers, PDAC is composed of an as-sortment of phenotypically distinct tumor cells. Recent can-cer research has begun to focus on a subpopulation of tumorcells termed cancer stem cells. Cancer stem cells have theunique capacity for self-renewal, the ability to differentiateinto the cells that make up the bulk of the tumor, and effi-ciently initiate tumor formation in animal models. A numberof reports have demonstrated that cancer stem cells are re-sistant to chemotherapy. The resistance of these cells totraditional chemotherapeutics is consistent with a model inwhich chemotherapy fails to kill the cancer stem cell popula-tion, allowing for tumor recurrence. This model makes can-cer stem cells an attractive target for the development of noveltherapeutics.

Three populations of cancer stem cells have been re-ported in PDAC, CD44+/CD24+/ESA+, CD133+, and ALDH+cells. In addition to forming primary orthotopic tumors in mice,a subpopulation of these stem cells (CD133+/CXCR4+) hasmetastatic potential. PDAC-derived stem cell populations areresistant to treatment with gemcitibine. Importantly, recentstudies have demonstrated that combinatorial treatment oftumors with gemcitibine and inhibitors of stem cell signalingpathways (Shh and mTOR) effectively reduce the tumorgenicpotential of these cells in a mouse model.

My experiments will build upon this work to identify novelgenes and signaling pathways that are required for the tum-origenesis of PDAC-derived cancer stem cells. The ap-proaches described below will employ a genome-wide RNAiscreen specifically targeting the cancer stem cell populationof PDAC tumors and a molecular characterization approachto profile the stem cell population compared to its non-tum-origenic counterpart.

Specific Aim 1. Identification of genes required fortumorigenesis of cancer stem cells in PDAC.

A genome-wide shRNA screen will be employed to iden-tify genes whose expression is important for the ability ofPDAC-derived cancer stem cells to form tumors in vivo.shRNAs are small RNA molecules that can bind to cellularmRNAs, induce their degradation, and thereby down-regu-late gene expression. By utilizing tens of thousands ofshRNAs that target nearly every gene in the genome onecan evaluate the contribution of a single gene to the biologyof a cell in an unbiased manner. Similar screens have beenemployed in human systems to characterize genes impor-tant for cell survival, proliferation, and activated oncogene-dependent growth. These genome-wide screens have provento be powerful in identifying novel functions and genetic in-teractions for a variety of genes.

Purified cancer stem cell populations from PDAC tumorswill be infected with lentiviruses expressing shRNAs and in-jected into immunodeficient mice. Half of the injected micewill be maintained under normal growth conditions and theother half treated with gemcitibine. Once tumors have grown,they will be excised and the presence or absence of specificshRNAs will be determined by microarray analysis. ThoseshRNAs that are reproducibly depleted from the populationwill be considered anti-tumorigenic. Genes that are targetedby the shRNAs will be the focus of future analysis. We antici-pate that many of the depleted shRNAs will be in commonbetween mice treated with and without gemcitibine. How-ever, depleted shRNAs unique to the gemcitibine treatedanimals may identify novel mechanisms of chemo-resistancein PDAC.

Specific Aim 2. Molecular characterization of PDAC-derived cancer stem cells.

Transcriptome analysis will be simultaneously employedto identify genes that are differentially expressed betweenthe cancer stem cell and non-tumorigenic cell populations.RNA will be isolated from both the cancer stem cell and non-stem cell populations. Global gene expression as well asmicroRNA analysis will be conducted using GeneChip miRNAand Gene Arrays (Affymetrix). This non-functional approachwill allow the identification of genes and pathways that mightbe critical or aberrant in PDAC derived cancer stem cells.The results of these experiments may also identify genesthat, while not functionally important for cancer stem cells,may be amenable to targeted therapeutic approaches.

Molecular and Functional Characterization of Cancer Stem Cells in Pancreatic Cancer

October, 2010 Volume 7Karin Grunebaum Cancer Research Foundation

Boston University Medical School

Transformative Pathology and Systems Biology of Cancer

An overarching theme of my research is the experimen-tal investigation of human cancers, specifically colorectal ad-enocarcinomas. Colon cancer remains a leading cause ofcancer mortality in both men and women. Yet efficient meth-ods of detection and treatment remain difficult. We are par-ticularly interested in two areas: (1) Molecular characteriza-tion of global protein abnormalities in cancer tissues; and (2)isolation and therapy-predictive use of cancer stem cells fromprimary human tumor tissue retrieved intraoperatively.

1. Proteomic Biomarkers of Colorectal AdenocarcinomaWe have started to investigate the unbiased proteomic

composition of human tumor tissues using a combination of2-D gel electrophoresis, fluorescence labeling, and advancedbiophysical techniques (such as high-resolution tandem massspectrometry) in combination with computationalbioinformatics tools. By developing an innovative new sub-proteome enrichment technology, we were, for the first time,able to interrogate information-enriched tissue proteomes byovercoming the traditional bottlenecks of tissue proteomics,i.e., dynamic range, discoverable proteomic depth, and in-formative content. Using these newly developed methods,we went on to study clinical samples of colorectal cancer anddiscovered that PSB7 (proteasome subunit b7), PRDX1(peroxiredoxin-1), and SRP9 (signal recognition particle 9 kDaprotein) are important proteins in colorectal carcinomas, in-dicating a hypoxia-adaptive tumor response. We demon-strated that all three proteins are produced by the bona fidecancer cell compartment, not the surrounding induced stroma.Functional cell biological experiments are ongoing to define

the biological roles of these proteins. In particular, we haveexciting evidence that PSB7 equips the tumor with an im-mune escape mechanism to evade MHC class I peptide pre-sentation of tumor antigens, for the first time linking epithelialproteasome function to tumor immunology.

2. Cancer Stem Cells in Solid TumorsWe are particularly interested in isolating and studying

the proteomes of tumor-promoting stem cells within solid tu-mors. Within a solid tumor, there may co-exist a variety ofheterogeneous populations of neoplastic cells that, whilegenomically identical or highly related, differ widely in theirepigenetic preoteomic make-up. Furthermore, one or sev-eral of these subpopulations may possess self-renewing and/or differentiation-blocked properties rendering them tumor-propagating (“stem”-like) relative to other populations whichmay not possess propagating capability. The characteriza-tion of molecules that define cancer cells as tumor-propagat-ing cells will have significant impact on diagnosis, treatmentstrategy, and treatment response monitoring.

We have begun to isolate tumor-propagating cells fromsolid tumors by sorting for expressed surface protein mark-ers. One candidate is the the pentaspan glycoproteinprominin-1 (CD133). We are investigating how the functionalp r o t e o m i ccompositionsof CD133 low/negative andCD133 high/positive tumorcells differ andhow this maybe related totheir propagat-ing capacities.There is recentevidence thatsome liver me-tastases ofcolorectal carci-nomas displaylow levels or absence of CD133. By contrast, tumor-propa-gating cells in the primary colorectal neoplasm typically ex-press CD133. Thus, the comparison of proteomic networkalterations between these two types of lesions may providedirect clues about the functional context, interaction network,and downstream signaling partners of CD133. Through wholeproteome comparison, we also hope to identify new proteinmarkers that distinguish propagating from non-propagatingphenotypes.

Preliminary experimental data showing immunohistochemical localization of the new candidate biomarker PSB7 expression in a colon adenocarcinoma. Presence of the specific protein is indicated by the amount of brown staining. Nuclei were counterstained with hematoxylin for visualization purposes. (A, B) Normal colonic mucosa with low levels of PSB7 expression. (C) Markedly increased cytoplasmic and nuclear PSB7 expression in the patient’s adenocarcinoma. (D) Elevated PSB7 expression in carcinoma present in pericolonic lymphatic vessels; note the surrounding adipose tissue.

Potential approaches to tackle tumor cell heterogeneity. (i) Sorting an subpopulation analysis (top path). (ii) Engineering of homogeneous designer cancer stem cells (bottom path) using a currently hypothetical iPS-like reprogramming approach.

Michael H.A. Roehrl, M.D.

October, 2010 Volume 7Karin Grunebaum Cancer Research Foundation

www.grunebaumfoundation.org

Harvard Medical - Gewurz (continued from inside)

RECIPIENTS AT WORK

Dr. Roehrl – Human Proteome Organization2010 Meeting, Sydney, Australia

Dr. Roehrl was awarded a Young Investigator Awardfrom the International Forum of Proteomics at the 2010HUPO Meeting in Sydney, Australia. He presented on hislab’s work on tissue proteomics and protein biomarker dis-covery in colorectal and lung adenocarcinomas. The an-nual HUPO meeting is the world’s largest meeting of sci-entists engaged in human proteomics and biological massspectrometry.

nonical NF-kB activation by the prototypic canonical NF-kBpathway agonists Tumor Necrosis Factor-alpha andInterleukin-1-beta. Thus, LMP1 studies promise to uncovergeneral mechanisms that govern NF-kB activation. I am cur-rently characterizing screen hits with the most robust effectson LMP1/NF-kB, with a particular focus on potentiallydruggable enzymes. In parallel, I am performing compre-hensive siRNA analysis of the LMP1/non-canonical NF-kBpathway. To determine whether these LMP1/NF-kB hits alsofunction in the prototypic lymphocyte non-canonical NF-kBpathways, validated siRNAs will be retested for effects onNF-kB activation by two prototypic cytokine agonists.

Tumor genome characterization technologies are uncov-ering numerous genetic lesions in lymphoma cells. Differen-tiating biologically meaningful ‘driver’ mutations from the many

functionally inconsequential ‘bystander’ alterations remainsa key challenge. Integration of siRNA and tumor genetic al-teration data offers a powerful approach to identify noveloncogenes and tumor suppressor genes. I am currently de-termining whether the strongest LMP1/NF-kB pathway screenhits are targeted by copy number and/or coding region alter-ations in primary lymphoma samples and established cancercell lines. The convergence of multiple mutations on a givenNF-kB pathway component would provide compelling geneticevidence for its role in lymphoid malignancy.

Finally, in conjunction with the Broad Institute, I am per-forming high throughput chemical screens to discover novelsmall-molecule inhibitors of canonical and non-canonical NF-kB pathways. Potent NF-kB inhibitors will then be tested forselective effects on the growth and survival of NF-kB depen-dent lymphoma cells.

Your Support is Vitalto our Mission

The Karin Grunebaum Cancer ResearchFoundation and the support we are able to give tothe dedicated cancer researchers is solely depen-dent on donations from you.

To further demonstrate our dedication, we keepour overhead funding at 1% – meaning that 99% ofyour contribution will go directly to research.

Your tax-deductible contribution will directly helpfund the cancer research effort, since almost 99% ofour income is spent on supporting the researchers.Our Officers and Trustees are all unpaid volunteers,and the Foundation has no paid employees.

You can make your contribution via check by mail.Send to:

KGCRF85 Sherman Street, #8Cambridge, MA 02140

Online Support via PayPal atwww.grunebaumfoundation.org