George Adrian Calin, MD, PhD Associate Professor Experimental Therapeutics & Cancer Genetics Depts....

George Adrian Calin, MD, George Adrian Calin, MD, PhDPhD

Associate ProfessorAssociate ProfessorExperimental Therapeutics &Experimental Therapeutics &

Cancer Genetics Depts.Cancer Genetics Depts.

Co-Director, siRNA&ncRNA Center Co-Director, siRNA&ncRNA Center Univ. Texas, MD Anderson CCUniv. Texas, MD Anderson CC

Houston, USHouston, US

CLL: THE INTERPLAY BETWEEN NON-CODINGRNAS ANDCLL: THE INTERPLAY BETWEEN NON-CODINGRNAS ANDPROTEIN-CODING GENESPROTEIN-CODING GENES

Non-codingRNA Paradigms in Non-codingRNA Paradigms in Medical PracticeMedical Practice

Non-codingRNA Paradigms in Non-codingRNA Paradigms in Medical PracticeMedical Practice

Cytoplasm

ORF

mRNA cleavagemRNA cleavage

RISC RISC

RISCTranslation repressionTranslation repressionSome miRNAs

Pre-miRNAPre-miRNA(hairpin precursor)

DroshaDrosha

Genome - miRNA geneGenome - miRNA gene

Pri-miRNAPri-miRNA

Duplex

DicerDicer

miRNAmiRNA

NucleusExportin-5Exportin-5

Helicase

RISC

3’UTR

miRNAs make big splashmiRNAs make big splashmiRNAs make big splashmiRNAs make big splash

Lee RC, Feinbaum RL, Ambros V. Cell. 199 3 Dec 3;75( 5):843. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G. Natur e. 2000 Feb 24;403(6772 ):901.

MIRTRONS - nuclear pre-mRNA splicing

(Ruby et al, Nature 2007; Okamura et al, Cell 2007; Berezikov et al, Mol Cell 2007)

MiRNAs UPREGULATING translation

(Vasudevan et al, Science 2008)

MiRNAs targeting PROMOTERS of PCGS

(Place et al, PNAS, 2008)

MIRNAS acting in nucleus

(Hwang et al, Science 2007)

MiRNAs targeting ORF of PCGS

(Tay et al, Nature 2008)

A model for HE-miRNA-mediated control of human hematopoiesis

(Georgantas et al, PNAS 2007)

miR-155 inhibited generation of myeloid and erythroid colonies by normal primary human CD34+ cells

Normal PBSC CD34+ cells were transduced with FUGW (empty) or mir-155 lentivector. (A and B) Myeloid (A) and erythroid (B) colonies per 1,000 cells plated. (C) Representative colonies generated by control and mir-155-transduced cells. Results are representative of three separate experiments.

(Georgantas et al, PNAS 2007)

The non-codingRNA dictionary of the human genomeThe non-codingRNA dictionary of the human genomeThe non-codingRNA dictionary of the human genomeThe non-codingRNA dictionary of the human genome60-70% of the human genome is transcribed, while the total fraction of bases occupied 60-70% of the human genome is transcribed, while the total fraction of bases occupied

by known PCGS is only about 2% (Mattick & Makunin, Hum Mol Genet 2006)by known PCGS is only about 2% (Mattick & Makunin, Hum Mol Genet 2006) NAME FUNCTION REFERENCEs

tRNAs (transfer RNA)

Translation of genetic information.

tmRNA (transfer messanger RNA)

Trans translation. (Wower, Wower et al. 2008)

rRNA Ribosome component, catalysis of peptide bound formation.

snRNA (Small nuclear RNA)

Pre- mRNA splicing, telomerase maintenance; part of snRNPs (small nuclear ribonucleoproteins).

(Barrandon, Spiluttini et al. 2008)

snoRNA (Small nucleolar RNA)

RNA biogenesis; RNA modifications like 2’- O- methylation and pseudourydylation.

(Filipowicz and Pogacic 2002)

Telomerase RNA Telomeric DNA synthesis. (Theimer and Feigon 2006)

FC RNA Direct inhibition of RNA Polymerase I I and RNA transcription (Kettenberger, Eisenfuhr et al. 2006)

Centromeric repeat derived RNA

Kinetochore assembly and chromosome segregation. (Chen, Zhang et al. 2008)

RNase P Rybozymes that participate in tRNA processing. (Jarrous and Reiner 2007)

HOUSKEEPING RNAs

RNase MRP RNase mitochondrial RNA; biogenesis of ribosomes. (Martin and Li 2007)

Antisense- RNA Regulation of gene expression in CIS or in TRANS either at the DNA or mRNA level

(Amaral, Dinger et al. 2008)

LONG ncRNA Dosage Compensation and genomic imprintin; epigenetic control of development trajectories (HOTAI R); X- chromosome inactivation (XI ST and TSIX)

(Yang and Kuroda 2007) (Rinn, Kertesz et al. 2007) (Heard and Disteche 2006)

miRNA Mainly post- transcriptional regulation of gene expression. (Kent and Mendell 2006)

piRNAs (PIWI interacting RNAs)

Eterochromatin formation via RNAi pathways; Role in gametogenesis.

(Buhler and Moazed 2007) (Aravin, Gaidatzis et al. 2006; Brower- Toland, Findley et al. 2007) R

EGULATORY RNAs

Protein function modulators

6S RNA, SRA RNA (steroid receptor RNA activator). (Barrandon, Spiluttini et al. 2008)

Ultraconserved non-coding genes - the new chapter of the textbookUltraconserved non-coding genes - the new chapter of the textbookUltraconserved non-coding genes - the new chapter of the textbookUltraconserved non-coding genes - the new chapter of the textbook

1

3

4

2

(Bejerano et al, Science, 2004)

Ultraconserved genes - ncRNAs ubiquitously expressedUltraconserved genes - ncRNAs ubiquitously expressedUltraconserved genes - ncRNAs ubiquitously expressedUltraconserved genes - ncRNAs ubiquitously expressed

uc.352(N)

~20bp

~70bp

~70bp

~100bp

~20bp

uc.246(E)

U6

panc

reas

panc

reas

kidn

eyco

lon

brea

stbr

east

live

r

lung

kidn

ey

83

20 2340

226

3958

129

203

50 37

116

378

10691

181

0

50

100

150

200

250

300

350

400

1 2>9 10>17 18>19

Number of tissues

Number of UCGs expressed in tissues

Total Exonic Possibly exonic Non exonic

(Calin & Liu & Ferracin et al, Cancer Cell, 2007)

MICRORNAS AS ONCOGENES AND TUMOR MICRORNAS AS ONCOGENES AND TUMOR SUPPRESSORSSUPPRESSORS

MICRORNAS AS ONCOGENES AND TUMOR MICRORNAS AS ONCOGENES AND TUMOR SUPPRESSORSSUPPRESSORS

miRNAs as miRNAs as tumor sup pressorstumor sup pressors

(e.g.(e.g.miR-16/15a, let-7miR-16/15a, let-7) ) and/or oncogenes and/or oncogenes

(e.g. (e.g. miR-155, miR-21, miR-155, miR-21, miR17-92 cluster, miR17-92 cluster,

miR-372/373miR-372/373))

(Johnson et al, Cell 2005; He et al, Nature 2005; O’Donnell et al, Nature 2005; Cimmino et al, PNAS 2005; He et al, Nature 2007; Chang et al, Mol Cell 2007;

Voorhoeve et al, Cell, 2006; Raver-Shapira et al, Mol Cell 2007; Fabbri et al, PNAS 2007; Chang et al, Nat Genet 2008)

A non-codingRNA revolution in the cancer societyA non-codingRNA revolution in the cancer society

Non-coding RNAs (ncRNAs)

Protein coding genes (PCGs)

miR16-1 miR15-a

13q14.3 in CLL

DLEU 7LEU 1CLLD6 LEU 5

LEU 2KCNRG

KPNA3NY-REN-34 antigen

CLLD7CLLD8 ARLTS1

Centromere Telomere

100 200 400 500 700 1500 Kb0 300 600

D13S273 D13S1168 D13S1150 D13S319 D13S272

deletedregion in CLL, MMPr Ca, Pit Ad

(Calin et al, PNAS, 2002; Calin et al, N Engl J Med, 2005a; Calin et al, N Engl J Med 2005b)

miR15amiR15a and and miR16-1miR16-1 are deleted or are deleted or down-regulated in the majority of B-CLLsdown-regulated in the majority of B-CLLs

CLL13qLOH

1+/-

2ND

3+/-

4+/-

5+/?

6+/+

7+/-

8NI

9+/+

10+/+

11+/+

12+/-

13+/-

14NI

15+/-

16+/-

17+/-

18NI

miR16

miR15

CD

5 +

Precursor

Mature miR

Mature miR

EtBr

The LOH status for the presented samples is shown as: +/+ heterozygosity; +/- LOH; -/- homozygous deletion; The LOH status for the presented samples is shown as: +/+ heterozygosity; +/- LOH; -/- homozygous deletion; NI not informative; ND not done.NI not informative; ND not done.

(Calin et al, PNAS, 2002)

The Anatomy of CLL GenomeThe Anatomy of CLL GenomeThe Anatomy of CLL GenomeThe Anatomy of CLL Genome

(Calin & Sevignani et al, PNAS, 2004)

Cinzia

A Aberration# of

patients% of total

Male/

FemaleMean Age

RAI Stage

0 1 2 3 4

%

pts ZAP>20

13q deletion alone 115 68.9 68/47 57.43 60 39 10 2 4 24.35

11q deletion alone 5 3.0 4/1 58.88 2 2 1 0 0 40.00

17p deletion alone 5 3.0 3/2 47.10 4 1 0 0 0 40.00

11q plus 13q deletion 18 10.8 12/6 55.83 5 10 3 0 0 66.67

17p plus 13q deletion 15 9.0 10/5 54.07 6 1 4 3 1 53.33

11q plus 17p plus 13q deletion† 2 1.2 1/1 55.39 1 0 1 0 0 100.00

Normal Cytogenetics 7 4.2 6/1 52.83 2 3 1 1 0 42.86

TOTAL 167 100 104/63 54.50 80 56 20 6 5

B 6

0

1

2

3

4

5

miR-15a miR-16 TP53

Normal cytogenetics

13q-/-

Fold

Induct

ion (

R.U

.)

* *

* C

0

0.5

1

1.5

2

2.5

Normal cytogenetics

13q-/-

*

Tp5

3 p

rote

inExpre

ssio

n (

R.U

.)

Cytogenetic distribution and inverse correlation of miR-15a/16 and Tp53 in CLLsCytogenetic distribution and inverse correlation of miR-15a/16 and Tp53 in CLLs

(Fabbri and Botoni et al, submitted, 2009)

MCL1BCL2

ZAP-70

miR-15amiR-16-1

TP53

miR-34bmiR-34c

13q

17p

11q

MicroRNA/Tp53 pathogenetic and prognostic model for CLLMicroRNA/Tp53 pathogenetic and prognostic model for CLL

MCL1BCL2

ZAP-70

miR-15amiR-16-1

TP53

miR-34bmiR-34c

13qDEL

17pDEL

11qDEL

Normal B cellNormal B cell Malignant B cellMalignant B cell

(Fabbri and Botoni et al, submitted, 2009)

HMGA2Anchorage Independent Growth

PI3K

AKT

E2F

INSENSITIVITY to

ANTI-GROWTH SIGNALs

ONCOmiR+

PTEN

ECM

RAS

MYC

FAK

G1

S

SELF-SUFFICIENCY in

GROWTH SIGNALS

Cell Contact InhibitionTGFbGF DeprivationDifferentiation

TCL1

GF

P

RBP

P

CYC-CDKs

CKIs (p21, p27, p57)

-

E2F

miR-221/222miR-106b/93

miR-17~92miR-106b~25

miR-15miR-16miR-34amiR-124

let-7miR-34a

let-7

let-7

miR-21

let-7

miR-29miR-181

miR-143miR-145

miR-124

RTKs

RAF

MEK

ERK

SURVIVAL SURVIVAL RESPONSESRESPONSES

GF

IRS1

miR-140

(laminin

(Spizzo et al, Cell, 2009; Negrini et al, Current Oppin Cell Biol, 2009)

LIMITLESS LIMITLESS REPLICATIVE REPLICATIVE POTENTIALPOTENTIAL

Unscheduled Unscheduled ProliferationProliferation

Activated OncogeneActivated OncogeneDNA damaging agentsDNA damaging agentsTelomere ShorteningTelomere Shortening

EpigeneticTelomerehomeostasis

BCL6

BCL2MCL1

CaspaseCaspaseactivationactivation

Cyt C

p53

LATS2 BIMp21 TP53BP1 PDCD4

FasTnf-Trail

BAX/BAD

hTERT

p16

FADD

EVASION EVASION FROM FROM

APOPTOSIAPOPTOSISS

RBE2F

miR-372miR-373 miR-25miR-155miR-21

miR-106miR-93

miR-34

miR-34

miR-15amiR-16

let-7c miR-155miR-150miR-10amiR-144

APOPTOSISAPOPTOSISSENESCENCESENESCENCE

miR-148

miR-290

Rbl1

DNMT3a/3b

miR-24

miR-29 DeathReceptors

5’3’miR-127

(Spizzo et al, Cell, 2009; Negrini et al, Current Oppin Cell Biol, 2009)

microRNA

I am very small and non-coding!But I can do a big jobin your cells!Size doesn’t matter!

Really?! Who are you little thing? The son of a new genetics?

messengerRNAs

MIRNA PROFILING AS A NEW DIAGNOSTIC & PROGNOSTIC MIRNA PROFILING AS A NEW DIAGNOSTIC & PROGNOSTIC TOOL FOR CANCER PATIENTSTOOL FOR CANCER PATIENTS

MIRNA PROFILING AS A NEW DIAGNOSTIC & PROGNOSTIC MIRNA PROFILING AS A NEW DIAGNOSTIC & PROGNOSTIC TOOL FOR CANCER PATIENTSTOOL FOR CANCER PATIENTS

miRNAs expression miRNAs expression signatures associated signatures associated

with diagnosis and with diagnosis and prognostic factorsprognostic factors

(CLL, DLBCL, Lung,(CLL, DLBCL, Lung,Colon, Pancreas,Colon, Pancreas,

Brain ca.)Brain ca.)

(Michael et al, Molec Cancer Res 2003; Lu et al, Nature, 2005; Eis et al, PNAS, 2005 Lui et al, Cancer Res 2007, Bloomston et al, JAMA 2007; Mi et al, PNAS, 2007; Garzon et al, Blood 2008)

A unique miRNA signature is associated with CLL A unique miRNA signature is associated with CLL progression (time from diagnosis to therapy)progression (time from diagnosis to therapy)

Short

interval Long

interval microarray expression hsa-mir-181a High Low hsa-mir-155 High Low hsa-mir-146 High Low hsa-mir-024-2 High Low hsa-mir-023b High Low hsa-mir-023a High Low hsa-mir-222 High Low hsa-mir-221 High Low hsa-mir-029c Low High

Long interval Short interval

0.0

0.2

0.4

0.6

0.8

1.0

Months

Pat

ien

ts w

ith

ou

t tr

eatm

ent

(%)

(Calin et al, N Engl J Med, 2005b)

A unique miRNA signature is associated with CLL prognosisA unique miRNA signature is associated with CLL prognosis

Nr. Crt.

Compo nen t Map P value

Ag gre ss ive CLL **

Obs erv ation***

1 miR-15a 13q14.3 0,0 18 high cluster 15a/ 16-1 del CLL & Prosta te ca.

2 miR-195 17p13 0,0 17 high del HC C 3 miR-221 Xp11.3 0,0 10 high cluster 221/222 4 miR-23 b 9q22.1 0,0 09 high cluster 24-1/23b

FR A 9D; de l Urothelial ca. 5 miR-155 21q21 0,0 09 high a mp ch ild Burkitt’s lymphom a 6 miR-223 Xq12-

13.3 0,0 07 low normally expressi on restrict e d to

myelo id line age 7 miR-29a-2 7q32 0,0 04 low cluster 29a -2/29b-1

FR A7H; del Prostate ca. 8 miR-24-1 9q22.1 0,0 03 high cluster 24-1/23b

FR A 9D; de l Urothelial ca. 9 miR-29 b-2 1q32.2 -

32.3 0,0 007 low

10 miR-146 5q34 0,0 007 high 11 miR-16-1 13q14.3 0,0 004 high cluster 15a/ 16-1

del CLL, prostate ca. 12 miR-16-2 3q26.1 0,0 003 high ide ntical m iR-16-1 13 miR-29c 1q32.2 -

32.3 0,0 002 low

Note: * - All the me mbers of the s ignatu re a re mature mi croRN As; ** - Agg ressive CLL is rep res e nte d by group 1 includes pat ients with IgVh unm utat e d a nd Z a p-70 positive (high), bo th pre dictors of poor prognosis. *** - FRA = fra gile site; del = de letion; HCC = hepatoc e llular carci no ma; ca. = carci nom a.


17pDEL (64 pts MDACC & CRC)

FISH abnormal in 17p>20% cells (N=64)FISH abnormal in 11p, 12, 13q (N=43)

KARYO abnormal (N=64)

17pDEL (64 pts MDACC & CRC)

FISH abnormal in 17p>20% cells (N=64)FISH abnormal in 11p, 12, 13q (N=43)

KARYO abnormal (N=64)

NORM/NORM (40 pts MDACC & CRC)

normal FISH and normal KARYO (N=40)

NORM/NORM (40 pts MDACC & CRC)

normal FISH and normal KARYO (N=40)

104 pts

MicroRNAs signature associated with 17p deletion in CLL patientsMicroRNAs signature associated with 17p deletion in CLL patients

Associations Between miR-21 Expression in Tumors and Receipt of Adjuvant Chemotherapy With Prognosis

(Schetter et al. JAMA 2008)

Non-codingRNAfingerprints in ALL response to treatmentNon-codingRNAfingerprints in ALL response to treatment

UnsupervisedUnsupervised(all array probes)(all array probes)

SupervisedSupervised(array probes 0.01)(array probes 0.01)

SupervisedSupervised(most significant (most significant 20 array probes)20 array probes)

Cancer susceptibility and non-Cancer susceptibility and non-codingRNAscodingRNAs

But who cares about tumors

in mice?!

Germline mutations?SNPpppppssssss?MicroRNAs?non-coding RNAs?What about human cancer!

(Calin et al NEJM, 2005, Raveche et al, Blood 2007, Sevignani & Calin et al, PNAS, 2007, Wu et al, Carcinogenesis 2008, Landi et al, Carcinogenesis 2008, Hu et al, JCI 2008, Jazdzewsky et al PNAS 2008)

Cancer predisposition: from epidemiology to geneticsCancer predisposition: from epidemiology to geneticsCancer predisposition: from epidemiology to geneticsCancer predisposition: from epidemiology to genetics

SUSCEPTIBILITYSUSCEPTIBILITYGENES GENES

(ex, APC, (ex, APC, BRCA1, BRCA2)BRCA1, BRCA2)

““MODIFIER”MODIFIER”GENES GENES (MOM1)(MOM1)

SPORADIC (~60-75%)

FAMILIAL (~20-30%)

HEREDITARY (~5-10%)

A total of 1,444,920 new cancer cases and 559,650 deaths for cancer areprojected to occur in US in 2007 (CA Cancer J Clin 2007; 57:53)

Germline abnormalities in Germline abnormalities in miR15amiR15a//miR16-1 miR16-1 transcript are associated with transcript are associated with CLL and breast cancer aggregationCLL and breast cancer aggregation

mir-16-1 normal genome

precursor

mir-16-1 (CtoT)+7

+1 +7precursor

A

U6

A

P

miR

16 W

T

Em

pty

V

293

miR

16-1

M

UT

miR

16 W

T

Em

pty

V

293

miR

16-1

M

UT

GFP

miR-16-1 miR-15a

C

0

5

10

15

20

25

30

35

Mutant 15-16 Emptyvector

Nontransfected

Ct values

1ng RNA

5ng RNA

10ng RNA

miR-16-1 MEG01 cells

DB

NB Ratio 0.25 0.50 2.07 1.22

MAr Ratio 1.01 3.25 9.20 6.24

CLLCLL

CD5CD5

miR-16-1

NB Ratio 0.29 0.51 1.97 1.37

MAr Ratio 0.95 1.30 3.50 2.30

miR-15a

U6


About humans and mice: About humans and mice: same gene - same mutation - same diseasesame gene - same mutation - same disease

About humans and mice: About humans and mice: same gene - same mutation - same diseasesame gene - same mutation - same disease

* NZB strain naturally develop CLL-like disease during aging;

* Reduction of expression of miR-16 in hematopoietic tissues;

* Mutation in mir-16 gene, in the same location as the germline mutation described in humans, found only in NZB strain

Germline or Somatic mutations in Germline or Somatic mutations in Ultraconserved Genes in human cancersUltraconserved Genes in human cancers

Germline or Somatic mutations in Germline or Somatic mutations in Ultraconserved Genes in human cancersUltraconserved Genes in human cancers

ncRNA

Name Type

Chrom.

location

General

Population (175) Cancer pts. (72)

Neighbor coding genes Expressed in

normal tissues

ncRNA.21 N 1p33 NORM 190(TtoC) (CRC) SPATA6, FLJ 14442

FLJ 11588 Yes

ncRNA.206 N 7p15.3 NORM 324(GtoA) (CLL) SP8, \N, SP4 Yes

ncRNA.243 P 8q21.13 NORM 146(GtoA) (CLL) AF130052, ZFH4

PXMP3 No

ncRNA.276 P 9q33.3 335(GtoA) 90(AtoG) (CRC) LOC51145, PBX3

C9orf28 Yes

ncRNA.328 P 11p13 NORM 179(GtoT) (2xCRC) ELP4, PAX6

RCN1 No

ncRNA.483 P 3p24.3 NORM 166(AtoT) (CRC) AK125129, TBC1D5

SATB1 Yes

(Wojcik et al, Carcinogenesis, 2009)

UCG expression profiles classify human cancersUCG expression profiles classify human cancersUCG expression profiles classify human cancersUCG expression profiles classify human cancers

* 50 UCRs differentiatehuman cancers

(P<0.005)

* half of them Non-exonic

* 6/12 Exonic UCRs represent ANTISENSE transcripts with

host gene

CLL

CRC

HCC

133 cancer samples


miRNA::ncRNA interactionsmiRNA::ncRNA interactionsmiRNA::ncRNA interactionsmiRNA::ncRNA interactions

Days0 1 2

4e-8

8e-8

1e-7

2e-7

2e-7

0.000

0.004

0.008

0.012

0.016

Days

0 1 2

0

1e-6

2e-6

3e-6

4e-6

5e-6

0.000

0.004

0.008

0.012

0.016

Days

0 1 2

2.0e-7

4.0e-7

6.0e-7

8.0e-7

1.0e-6

1.2e-6

0.000

0.004

0.008

0.012

0.016

miR

-155

exp

ress

ion

miR

-155

exp

ress

ion

miR

-155

exp

ress

ion

Ucg

3. expressionU

cg2. expression

Ucg

.1exp

ression

ZAP-70 negative CLLs

ZAP-70 positive CLLs

UCG1

UCG2

miR-29b

miR-24-1

UCG3

miR-155


ncRNA::ncRNA interactions - miRNAs targeting UCGsncRNA::ncRNA interactions - miRNAs targeting UCGsncRNA::ncRNA interactions - miRNAs targeting UCGsncRNA::ncRNA interactions - miRNAs targeting UCGs

5’- ACTTCCCCCT:TCTATTA:TAGCATTAGCAACG ncRNA1 3’ -GGGGATAG:TGCTATACGTAATT miR-155

5’- TCAATGCACTATTGC:AAGAGCATTATTGCAT ncRNA2 3’ -GGGGATAG:TGCTATACGTAATT miR-155

5’- TGGAGATAC:AACAAGA:TAACATTAATGAGT ncRNA3 3’ -GGGGATAG:TGCTATACGTAATT miR-155

5’- CCGCCATGTACCTGC:CTACTTAGCCCAAGGG ncRNA2 3’- GACAAGGACGACTTGACTCGGT miR-24

5’- CTAATGAGACTGAGTTTACA::GTGCCATAGA ncRNA3 3’- TTGTGACTAAAGTTTACCACGAT miR-29b

UCG-miRNA direct interaction

0

1

2

3

4

5

6

7

8

155 + UCG3

scr + UCG3

29b + UCG3

scr + UCG3

24 + UCG2

scr+ UCG2

155 + UCG1

scr + UCG1


miR Amplification

miR Translocation

miR Mutation

miR Deletion/Hypermethylation/Abmormal processing

AAAA

AAAAAAAA

AAAA

AAAA

AAAAAAAA

AAAAPCG Amplification

PCG Translocation

AAAA

AAAA

PCG Mutation

PCG Deletion/Hypermethylation

AAAA

AAAA

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

Oncogenic proteins

NH2COOH

NH2COOH

Tumor-suppressor proteins

Low apoptosis Low apoptosis High proliferationHigh proliferation

Metastasis Metastasis High angiogenesisHigh angiogenesis

NH2COOH

Causing miRNA abnormal expression - Causing miRNA abnormal expression - the combinatorial “in cascade”modelthe combinatorial “in cascade”model

(Calin & Croce, Cancer Res, 2006; Calin & Croce, J Clinical Inv 2007)

(Calin & Croce, Nature Reviews Cancer, 2006)

MicroRNAs - the missing link in cancer predispositionMicroRNAs - the missing link in cancer predispositionMicroRNAs - the missing link in cancer predispositionMicroRNAs - the missing link in cancer predisposition

MICRORNAS AS NEW THERAPEUTIC TARGETS & NEW DRUGSMICRORNAS AS NEW THERAPEUTIC TARGETS & NEW DRUGS MICRORNAS AS NEW THERAPEUTIC TARGETS & NEW DRUGSMICRORNAS AS NEW THERAPEUTIC TARGETS & NEW DRUGS

ZEB1

VEGF

PTEN

RHOC

MAT

+

ZBTB10

EMT

miR-27amiR-210

miR-21

miR-16miR-15amiR-20amiR-20b

Laminin Collagen

miR-29cmiR-146

miR-373miR-520

TGFb1

MYCEFRINA

VEGF

CD44

miR-200miR-205

SP

miR-221miR-222

TPM1RECKTIMP3

MMPs

HOXD10

miR-10bTWIST+

SMAD4 miR-155+

RHOA

Tight junctions

Proliferation and motility at DISTANT

SITES

miR-101

EZH2ROCK

TRAF6IRAK

meme

CDH1

miR-18miR-19let-7f

miR-27bTSP1TSP1

CTGF

HYPOXIAHIF

Tumor cells Disseminatio

n

OFF

ON

C-KIT

SPRED1PIK3R2

miR-126

Let-7

HMGA2RAS

miR-221miR-222

p27CIP/KIP1

CRKunknown

miR-126

FGF

miR-335miR-206

Angiogenic

Switch

SOX4TNC

MicroRNAs - the micro-steering wheel of tumor metastasesMicroRNAs - the micro-steering wheel of tumor metastasesMicroRNAs - the micro-steering wheel of tumor metastasesMicroRNAs - the micro-steering wheel of tumor metastases

(Nicoloso et al, Nat Rev Cancer 2009)

Aberrant regulation of pVHL levels by microRNA promotes Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cellsthe HIF/VEGF axis in CLL B cells

Aberrant regulation of pVHL levels by microRNA promotes Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cellsthe HIF/VEGF axis in CLL B cells

(Ghosh et al, Blood 2009)

Vascular endothelial growth factor (VEGF) is highly expressed in B CLL cells

CLL B cells express constitutive levels of HIF-1alpha under normoxia and induces the VEGF expression

pVHL is responsible of HIF-1 alpha degradation and is downregulated in CLL cells versus normal B cells

miR-92-1 is overexpressed in CLL B cells and target pVHL and repress its expression

Principles of miRNA-based gene therapy and in vivo Principles of miRNA-based gene therapy and in vivo inhibition of miRNA in cancer cellsinhibition of miRNA in cancer cells

(Calin & Croce, J Clin Inv 2007)

WHY SHOULD MIRNAS BE MORE EFFICIENT IN WHY SHOULD MIRNAS BE MORE EFFICIENT IN SPECIFICALLY KILLING SPECIFICALLY KILLING

MALIGNANT CELLS?MALIGNANT CELLS?

WHY SHOULD MIRNAS BE MORE EFFICIENT IN WHY SHOULD MIRNAS BE MORE EFFICIENT IN SPECIFICALLY KILLING SPECIFICALLY KILLING

MALIGNANT CELLS?MALIGNANT CELLS?

(Calin, Cimmino & Fabbri et al, PNAS 2008)

Gene Symbol Map Gene name p value CLL

p value MEG-01

RAD51C 17q22-q23 RAD51 homolog C (S. cerevisiae) 0.0075 0.00334 MCL1 1q21 Myeloid cell leukemia sequence 1 (BCL2-related) 0.00863 0.011 HLC-8 17q25.1 lung cancer-related protein 8 0.0124 0.0164

PDCD6IP 3p23 programmed cell death 6 interacting protein 0.0214 0.00276 MSH2 2p22-p21 mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) 0.0242 0.00192 JUN 1p32-p31 v-jun sarcoma virus 17 oncogene homolog (avian) 0.0281 0.00059

Group Protein Gene description Z-Score Comments Anti-apoptotic Grp78 Heat shock 70kDa protein 5 (glucose-

related protein, 78 KDa) 2.43

Bcl2 B-cell CLL/lymphoma 2 2.43 Predicted and validated target Pdia2 Protein disulfide isomerase family A,

member 2 2.43

Oncogenesis Wt1 Wilms tumor 1 2.43 Predicted and validated target MageB3 Melanoma antigen family B, 3 2.43 Rab9B RAB9B member RAS oncogene family 2.16 Predicted target

miR-15 and miR-16 targeting multiple apoptosis- and miR-15 and miR-16 targeting multiple apoptosis- and cancer-related genescancer-related genes

TRANSCRIPTOMA

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miR-16 & miR-15 based Gene Therapy in CLLmiR-16 & miR-15 based Gene Therapy in CLLmiR-16 & miR-15 based Gene Therapy in CLLmiR-16 & miR-15 based Gene Therapy in CLL

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(Calin, Cimmino & Fabbri et al, PNAS, 2008)

Two strategies for the use of RNA inhibition Two strategies for the use of RNA inhibition in human cancersin human cancers

Two strategies for the use of RNA inhibition Two strategies for the use of RNA inhibition in human cancersin human cancers

““sandwich RNA inhibition”sandwich RNA inhibition” - to focus with multiple different agents on - to focus with multiple different agents on a major molecular alteration clearly linked to the pathogenesis of a a major molecular alteration clearly linked to the pathogenesis of a disease disease

* BCL2 overexpression in B cell malignancies - by using a combination * BCL2 overexpression in B cell malignancies - by using a combination of miR-15a, miR-16, ASO-BCL2.of miR-15a, miR-16, ASO-BCL2.

“multiplex RNA inhibition targeting” - targeting various molecular defects in the multistep pathways of specific cancers

* The apoptotic pathway in CLL by using a miR-15a, miR-16, miR-29s targeting BCL2 and MCL1

““sandwich RNA inhibition”sandwich RNA inhibition” - to focus with multiple different agents on - to focus with multiple different agents on a major molecular alteration clearly linked to the pathogenesis of a a major molecular alteration clearly linked to the pathogenesis of a disease disease

* BCL2 overexpression in B cell malignancies - by using a combination * BCL2 overexpression in B cell malignancies - by using a combination of miR-15a, miR-16, ASO-BCL2.of miR-15a, miR-16, ASO-BCL2.

“multiplex RNA inhibition targeting” - targeting various molecular defects in the multistep pathways of specific cancers

* The apoptotic pathway in CLL by using a miR-15a, miR-16, miR-29s targeting BCL2 and MCL1

Opening Pandora’s box: Revisiting the molecular oncology dogmaOpening Pandora’s box: Revisiting the molecular oncology dogmaOpening Pandora’s box: Revisiting the molecular oncology dogmaOpening Pandora’s box: Revisiting the molecular oncology dogma

Mad spiritMad spiritof cancerof cancer

proteinsproteins

miRNAsmiRNAssupressorssupressors

miRNAsmiRNAsactivatorsactivators

proteinsproteins

megaRNAsmegaRNAs

Ultraconserved genes

MicroRNAs and genomic dark matter alterations in human cancersMicroRNAs and genomic dark matter alterations in human cancersMicroRNAs and genomic dark matter alterations in human cancersMicroRNAs and genomic dark matter alterations in human cancers

microRNA

Calin’s laboratory

Milena S. Nicoloso, MDRiccardo Spizzo, MD

Masayoshi Shimizu, BSSimona Rossi, PhDSang Kil Lee, MD

Eun-Jung Jung, MDJana Ferdin (Slovenia,) PhD std

Maria Ines Almeida (Portugal), PhD std Juliana Edwards , PhD std

Past or present studentsElisa Barbarotto (Italy), Sumaiyah K. Rehman (India)

Angelica Cortez (Brasil), Frederica Dimitri (Italy)David Rushwood, Manrique Guerrero, Julie Fogarty (US),

Raul Terruel (Spain), Rosa Fabiola (Brasil)

FUNDINGSNIH-NCI & DOD & AACR MD Anderson Trust &

Reagents Research Scholar CLL Global Research Foundation

MDACCAnil Sood

Bogdan CzerniakChang-gong LiuDeepa Sampath

Garth PowisGordon Mills

Guillermo-Garcia ManeroHagop KantarjianHuaming Wang

James AbbruzzeseJeff Bartholomeusz

Jeffrey MyersLiana Adam

Lynne AbbruzzoMarina KonoplevaMichael AndreeffMichael Keating

Ray DuBois Robert BastScott KopetzShuxing Zang

Stanley HamiltonVarsha GandhiVinay Puduvali

Yiling LuWaldemar Priebe

Wei ZhangWilliam Plunkett

Xiao-Feng LeXiuping Liu

Univ of Ferrara Massimo NegriniManuela Ferracin

CNIO, MadridManel Esteller

Amaja Lujambio

Univ of GenoaSilvio de FloraAlberto Izzotti

Wistar Institute, PhillyRamana Davuluri

Hao Sun

TJU-KCCPhiladelphia

Cinzia SevignaniTerry Hyslop

Linda Siracusa

Fundeni Hospital, RomaniaVlad Herlea

Catalin Vasilescu

Carlo M. Croce Massimo Negrini

Dragos T. Stefanescu

Josephine Nefkens InstituteRotterdam

Riccardo FoddeIngrid De Vries

Anieta Sieuwerts

NIHCurtis HarrisStefan Ambs

OSU-PharmacologyTom Schmittgen

Eun Joo Lee

Johns Hopkins Schoolof MedicineCurt Civin

Rob Georgantas

How can we find targets?I am getting crazy to find them!

It is very difficult…we could do………………………I am not sure that is going to work and it will take for ever?!

Maybe we could askin the microarray facility to customize microchips for miRNA genes!

i hsa-miR-143ugagaugaagcacuguagcuca

Cinzia Sevignani 2005

Enjoy the ncRNA revolution!

George Adrian Calin, MD, PhD Associate Professor Experimental Therapeutics & Cancer Genetics Depts....

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Transcript of George Adrian Calin, MD, PhD Associate Professor Experimental Therapeutics & Cancer Genetics Depts....