George Adrian Calin, MD, PhD George Adrian Calin, MD, PhD Associate Professor Associate Professor Experimental Therapeutics & Experimental Therapeutics & Cancer Genetics Depts. Cancer Genetics Depts. Co-Director, siRNA&ncRNA Center Co-Director, siRNA&ncRNA Center Univ. Texas, MD Anderson CC Univ. Texas, MD Anderson CC Houston, US Houston, US CLL: THE INTERPLAY BETWEEN NON-CODINGRNAS AND CLL: THE INTERPLAY BETWEEN NON-CODINGRNAS AND PROTEIN-CODING GENES PROTEIN-CODING GENES Non-codingRNA Paradigms in Non-codingRNA Paradigms in Medical Practice Medical Practice
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George Adrian Calin, MD, PhD Associate Professor Experimental Therapeutics & Cancer Genetics Depts. Co-Director, siRNA&ncRNA Center Univ. Texas, MD Anderson.
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George Adrian Calin, MD, George Adrian Calin, MD, PhDPhD
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
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
(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
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
(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.
(Calin et al, N Engl J Med, 2005b)
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
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
(Calin et al, N Engl J Med, 2005b)
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
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
PROTEOMA
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
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
7 15 21
Days
Tu
mor
Volu
me (
mm
3)
Mock pRS-E pRS15/16A MEG01
MOCKMEG01pRS-E
MEG01pR15/16
B
C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Tu
mor
weig
ht
(gra
ms)
Mock pRS-E pRS15/16
Day 28
(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