Microsoft PowerPoint - microRNAs in Myeloma - Paris 2011

"microRNAs in Myeloma"
Carlo M. Croce, M.D.
The Ohio State University
Comprehensive Cancer Center
The John W. Wolfe Chair in Human Cancer Genetics
Director, Institute of Genetics
Director, Human Cancer Genetics Program

Occurrence of the most frequent and recurrent chromosomal
abnormalities in human CLL
18
55
%
%
13q14
11q23
12
7%
%
Trisomy 12
17p13

a
D13S273
D13S1168 D13S1150
D13S319
D13S272
Centromere
Telomere
100
200
300
400
500
600
700 kb
KPNA6
CLLD6
LEU 5 LEU 2 LEU 1
miR15/16
b
D13S1150
D13S272
GCT16C05
D13S25
100 kb
27
1150
ALT1
2
S31
18
LEU2
D
RFP2
D13S
ALU
ex1 ex2
ex3
ex7 ex6
ex5 ex4
ex3
ex2 ex1ex1 ex2
20 kb
27
18
2
Mir16
Mir15
ALU
D13S
ex5
ex4
ex3
ex2
ex1ex1
~ 31.4 kb
~ 29 kb

Target mRNA
overexpression
gene
trascript
miR
miR
promoter
miR
Proliferation
Apoptosis
Invasion
Target mRNA
downregulation
Angiogenesis
Specific effects
miR-142
c-myc mRNA
promoter

CLL - Cluster 2
CLL - Cluster 1
Le Ly CD5

Characteristics of patients analyzed with the miRNACHIP.
Characteristic
Value
Male sex no. of patients (%)
58 (61.7)
Age at diagnosis yrs.
median
57.3
range
38-78
Therapy begun
No
No. of patients
53
Time since diagnosis mo.
87
Yes
No. of patients
41
Time between diagnosis and therapy mo.
40
ZAP-70 level
20%
48
>20%
46
IgVH
Unmutated (98% homology)
57
Mutated (<98% homology)
37

miRNA signature associated with prognostic factors (ZAP70 and IgVH mutations) and disease progression in CLL patients*.
Nr.
Component
Map
P value
Group 4
Putative targets ***
Observation****
Crt.
expression**
1
miR-15a
13q14.3
0,018
high
NA
cluster 15a/16-1
del CLL & Prostate ca. (ref (10)
2
miR-195
17p13
0,017
high
NA
del HCC
3
miR-221
Xp11.3
0,010
high
HECTD2, CDKN1B, NOVA1,
cluster 221/222
ZFPM2, PHF2
4
miR-23b
9q22.1
0,009
high
FNBP1L, WTAP,
cluster 24-1/23b
PDE4B, SATB1, SEMA6D
FRA 9D; del Urothelial ca. (ref (13)
5
miR-155
21q21
0,009
high
ZNF537, PICALM, RREB1,
amp child Burkitt's lymphoma (ref (16)
BDNF, QKI
6
miR-223
Xq12-13.3
0,007
low
PTBP2,
SYNCRIP,
WTAP,
normally expression restricted to myeloid
FBXW7, QKI
lineage (ref (27)
7
miR-29a-2
7q32
0,004
low
NA
cluster 29a-2/29b-1
FRA7H; del Prostate ca. (ref (13)
8
miR-24-1
9q22.1
0,003
high
TOP1, FLJ45187, RSBN1L,
cluster 24-1/23b
RAP2C, PRPF4B
FRA 9D; del Urothelial ca. (ref (13)
9
miR-29b-2
(miR-
1q32.2-
0,0007
low
NA
102)
32.3
10
miR-146
5q34
0,0007
high
NOVA1, NFE2L1, C1orf16,
ABL2, ZFYVE1
11
miR-16-1
13q14.3
0,0004
high
BCL2, CNOT6L,
cluster 15a/16-1
USP15, PAFAH1B1, ESRRG
del CLL, prostate ca. (ref (10)
12
miR-16-2
3q26.1
0,0003
high
see miR-16-1
identical miR-16-1
13
miR-29c
1q32.2-
0,0002
low
NA
32.3
Note: * - All the members of the signature are mature miRNAs;
*** - top five predictions using TargetScan at http://genes.mit.edu/targetscan (32) were included. NA not available; for specific gene names see the
NCBI site at http://www.ncbi.nlm.nih.gov/entrez.
**** - FRA = fragile site; del = deletion; HCC = hepatocellular carcinoma; ca. = carcinoma.

Genetic variations in the genomic sequences of miRNAs in CLL patients *.
miRNA
Location **
CLL
Normals
miRNACHIP
Observation
expression
miR-16-1
Germline pri-miRNA
2/75
0/160
Reduced to
Normal allele deleted in CLL cells in both patients (FISH, LOH);
(CtoT)+7bp in 3'
15% and 40%
For one patient: Previous breast cancer; Mother died with CLL;
of normal,
sister died with breast ca;
respectively
miR-27b
Germline
pri-miRNA
1/75
0/160
Normal
Mother throat and lung cancer at 58. Father lung cancer at 57.
(GtoA)+50bp in 3'
miR-29b-2
pri-miRNA (GtoT)+212 in
1/75
0/160
Reduced to 75%
Sister breast cancer at 88 (still living). Brother "some type of
3'
blood cancer" at 70.
miR-29b-2
pri-miRNAs ins (+A)+107
3/75
0/160
Reduced to 80%
For two patients: Fam history of unspecified cancer
in 3'
miR-187
pri-miRNA (TtoC)+73 in 3'
1/75
0/160
NA
Unknown
miR-206
pre-miRNA
2/75
0/160
Reduced to 25%
Prostate cancer; mother esophogeal cancer. Brother prostate
49(GtoT)
cancer sister breast cancer
miR-206
Somatic pri-miRNA (AtoT)-
1/75
0/160
Reduced
to
25%
Aunt some type of leukemia (dead)
116 in 5'
(data only for one
pt)
miR-29c
pri-miRNA (GtoA)31 in 5'
2/75
1/160
NA
Paternal grandmother CLL; sister breast ca. (one pt).
miR-122a
pre-miRNA 53(CtoT)
1/75
2/160
Reduced to 33%
Paternal uncle colon cancer.
miR-187
pre-miRNA 34(GtoA)
1/75
1/160
NA
Grandfather polycythemia vera. Father a history of cancer but not
lymphoma.
Note: * - For each patient/normal control more than 12kb of genomic DNAs was sequenced and, in total, we screened by direct sequencing ~627kb of tumor DNA
and about 700kb of normal DNA. The position of the mutations are reported in respect with the precursor miRNA molecule. The list of 42 microRNAs
analyzed includes 15 members of the specific signature or members of the same clusters, miR-15a, miR-16-1, miR-23a, miR-23b, miR-24-1, miR-24-2, miR-
27a, miR-27b, miR-29b-2, miR-29c, miR-146, miR-155, miR-221, miR-222, miR-223 and 27 other microRNAs (randomly selected): let-7a2, let-7b, miR-17-
3p, miR-17-5p, miR-18, miR-19a, miR-19b-1, miR-20, miR-21, miR-30b, miR-30c-1, miR-30d, miR-30e, miR-32, miR-100, miR-105-1, miR-108, miR-122,
miR-125b-1, miR-142-5p, miR-142-3p, miR-193, miR-181a, miR-187, miR-206, miR-224, miR-346.
** - When normal correspondent DNA from bucal mucosa was available, the alteration was identified as germline when present or somatic when absent, respectively.
FISH = fluorescence in situ hybridization; LOH = loss of heterozygosity; NA = not available

Bcl2 protein expression is inversely correlated with miR-15a and miR-16-1 miRNAs expression in CLL patients. (A)The
unique site of complementarity miR::mRNA is conserved in human and mouse and is the same for all four human m
protein are inversely correlated with miR-15a and miR-16-1 expression. Five different CLL cases are presented, and the
normal cells were pools of CD5+ B lymphocytes. The T cell leukemia Jurkat was used as control for Bcl2 protein
expression. For normalization we used -actin. The numbers represent normalized expression on miRNACHIP. ND, not
determined. (C) The inverse correlation in the full set of 26 samples of CLL between miR-15a / miR-16-1 and Bcl2 protein
expressions. The normalized Bcl2 expression is on abscissa vs. miR-15a (Left) and miR-16-1 (Right) levels by miRNA
chip on ordinates. ACT, -actin.

BCL2
MCL1
_____
Patient #1
_____
Patient #2
_____
Patient #3
miR-15a
LV-E
LV-miR-15a
LV-miR-16
LV-miR-15a
LV-E
LV-E
LV-miR-15a
LV-miR-16
LV-miR-16
1
0.43
0.42
1
0.66
0.72
1
0.64
0.41
miR-16-1
Tp53
1
0.21
0.18
11
0.16
0.30
0.46
0.21
p21
TP53
1
0.71
0.26
11
0.42
0.78
0.41
0.20
Puma
1
0.51
0.14
11
0.41
0.23
0.15
0.16
miR-34b
Bcl2
miR-34c
Vinculin
ZAP-70
(Fabbri and Botoni et al, JAMA,)

a
b
c
d
e
f

Examples of proteins down-regulated by the miR-15a/16-1 cluster identified by proteomics in MEG-01 cells

Examples of the CLL signature of miR-15a/16-1 down-regulated genes by microarray

MiR15a/16-1 cluster inhibits the growth of MEG-01 tumor engraftments in nude mice. (A)
Growth curve of engrafted tumors in nude mice injected with MEG-01 cells pretransfected
with pRS-E or pRS15/16 or mock transfected. (B) Comparison of tumor engraftment sizes
of mock-, pRS-E-, and pRS15/16-transfected MEG-01 cells 28 days after injection in nude
mice. (C) Tumor weights SD in nude mice.

Table 7. miRNAs used to classify human cancers and normal tissues

Table 8