Download - PhD-Defense-May 13 2011
LDOC1, a Novel Biomarker of Prognosis in Chronic Lymphocytic Leukemia (CLL)
Hatice Duzkale
UT Graduate School of Biomedical Sciences at Houston UT MD Anderson Cancer Center Department of Hematopathology
Ph.D. Defense May 16, 2011
Chronic Lymphocytic Leukemia (CLL)
-most common leukemia
-disease of advanced age
-unknown etiology
-no curative treatment
-unpredictable prognosis
-1/3 indolent disease with normal survival -1/3 indolent initially, progresses -1/3 aggressive disease
Chronic Lymphocytic Leukemia Prognostic Subtypes
Aggressive CLL Indolent CLL
Good Prognosis Low Rai stage Low β2-microglobulin Low ZAP70 Del 13q Mutated IGHV
Poor Prognosis High Rai stage High β2-microglobulin High ZAP70 Del 11q, del 17p, tris 12 Unmutated IGHV
IGHV: Immunoglobulin Heavy chain Variable region
Immunoglobulin Somatic Mutation is a Physiologic Process
modified from Kuppers et al., Nature Reviews Cancer, 2005, 5:251-262
DNA modifications
IGHV Mutation Status Predicts Survival
Median survival: 8yr vs 25yr
8yr 15yr 25yr
Unmutated IGHV Mutated IGHV
Hamblin TJ, et al Blood. 1999 94(6):1848-54
Disease Subtype Classification by IGHV Somatic Mutation Status
Novel Biomarkers Novel Molecular Targets
Global Gene Expression Profiling
Genes Differentially Expressed Between CLL Subtypes
Klein U, et al. J. Exp. Med. 2001 194 (11):1625-1638
LDOC1 Distinguishes CLL Prognostic Subtypes, Based on IGHV Mutation Status
Abruzzo LV et al., J Mol Diagn. 2007, 9 (4):546-555
Screening of CLL literature for microarray studies for genes distinguishing
unmutated from mutated CLL
Evaluation of 88 genes in 49 untreated CLL patients
(MF-QRT-PCR)
36 genes significant
LDOC1 most significant unmutated CLL, mutated CLL
Can LDOC1 Predict Survival?
Evaluation of LDOC1 in 131 untreated CLL patients (MF-QRT-PCR)
Correlation with other prognostic parameters
Survival analysis
LDOC1 mRNA Expression is Bimodal in CLL (QRT-PCR)
high mRNA low mRNA
LDOC1 mRNA Expression Correlates with IGHV Somatic Mutation Status
LDOC1
mRN
A E
xpre
ssion
Poor prognosis Good prognosis
threshold
Duzkale H. et al., Blood 2011, 117(15):4076-84
LDOC1 mRNA Correlates with Some Poor Prognostic Parameters All Patients
(n = 131) LDOC1 positive (n = 46) LDOC1 negative
(n = 85) p value Age in years,
median (range) 59 (27 – 82) 56.4
(27 – 82) 56.1 (27 – 81) 0.68
Gender, n (%) Male 81 (62%) Female 50 (38%) Male 27 (59%)
Female 19 (41%) Male 54 (64%) Female 31 (36%) 0.71
Rai stage 0-2, n (%) 3-4, n (%)
102 (78%) 29 (22%) 35 (76%)
11 (24%) 67 (79%) 18 (21%) 0.83
WBC count ≤ 150x109/L, n (%) > 150x109/L, n (%)
118 (90%) 13 (10%) 38 (83%)
8 (17%) 80 (94%) 5 (6%) 0.06
β2m < 4, n (%) ≥ 4, n (%)
98 (75%) 32 (25%) 31 (67%)
15 (33%) 67 (80%) 17 (20%) 0.14
IGHV somatic mutation status,
n (%) Mutated 67 (52%)
Unmutated 63 (48%) Mutated 2 (4%) Unmutated 43 (96%) Mutated 65 (76%)
Unmutated 20 (24%) 2.20 x 10-16 ZAP70 protein status
n (%) Positive 51 (45%) Negative 62 (55%) Positive 30 (77%)
Negative 9 (23%) Positive 21 (28%) Negative 53 (72%) 1.06 x 10-6
Cytogenetic changes n (%)
(N=Total examined) None 27 (27%);
del(13q) 36 (36%); del(6q), del(11q), del(17p), +12, or
del(13q) with other abnormalities
37 (37%) (N=100)
None 14 (36%); del(13q) 5 (13%); del(6q), del(11q), del(17p), +12, or
del(13q) with other abnormalities
20 (51%) (N=39)
None 14 (23%); del(13q) 30 (49%); del(6q), del(11q), del(17p), +12, or
del(13q) with other abnormalities
17 (28%) (N=61)
0.0006
Duzkale H. et al., Blood 2011, 117(15):4076-84
LDOC1 mRNA Expression Independently Predicts Overall Survival
13.6 yr
not reached
Duzkale H. et al., Blood 2011, 117(15):4076-84
LDOC1 mRNA Expression Predicts Overall Survival Better than IGHV Somatic Mutation Status and ZAP70
ZAP70 IGHV
LDOC1
Multivariate Analysis
Duzkale H. et al., Blood 2011, 117(15):4076-84
-LDOC1 mRNA expression is associated with markers of poor prognosis in CLL
-unmutated IGHV status -high ZAP70 protein expression -cytogenetic abnormalities
-LDOC1 mRNA predicts overall survival
-LDOC1 mRNA predicts overall survival better than IGHV mutation status and ZAP70 protein
LDOC1 mRNA Expression as a Biomarker -Summary
Ellenberger et al., 1992
-putative Leucine zipper transcription factor
-ubiquitously expressed in normal tissues
-differentially expressed in cancer cell lines - pancreatic and gastric cancer - breast cancer
-anti-proliferative?
-pro-apoptotic?
-reduces NF-kB activity?
LDOC1 (Leucine Zipper Downregulated in Cancer)
500bp!
200bp
650bp wtLDOC1!
LDOC1S!
wtLDOC1!
Beta-Actin!
MCF-7
A! B!C!
A! B!
C!
CDS!5’UTR! 3’UTR! CDS!5’UTR! 3’UTR!
A+B A+C!
LDOC1S, a novel splice variant of LDOC1
Splice Variant transcript (LDOC1S)
Wild Type LDOC1 transcript (LDOC1)
CLL46 CLL49 CLL51 CLL53 CLL12 CLL37 CLL61 CLL67 CLL99 CLL42
Unmutated Mutated
MCF7 GA10 Jurkat NBC4 NBC6 CLL54 CLL58 CLL62 CLL60 Mutated
Differential expression of LDOC1 & LDOC1S (RT-PCR)
LDOC1
LDOC1S
NBC: Normal peripheral blood B cells
Βeta-
Actin
Βeta-
Actin
Unmutated
500bp!
200bp
650bp
500bp!
200bp
650bp
Duzkale H. et al., Blood 2011, 117(15):4076-84
LDOC1S, a novel splice variant of LDOC1
LDOC1 MVDELVLLLHALLMRHRALSIENSQLMEQLRLLVCERASLLRQVRPPSCPVPFPETFNGE 60
LDOC1S *******************************************-----------------
LDOC1 SSRLPEFIVQTASYMLVNENRFCNDAMKVAFLISLLTGEAEEWVVPYIEMDSPILGDYRA 120
LDOC1S ------------------------------------------------------------
LDOC1 FLDEMKQCFGWDDDEDDDDEEEEDDY 146
LDOC1S ------------------------------------------------------------ 180
LDOC1S ------------------------------------------------------------ 240
LDOC1S ------------------------------------------------------------ 300
LDOC1S ---------------------E
Leucine zipper
Duzkale H. et al., Blood 2011, 117(15):4076-84
LDOC1S is a Leucine zipper region alone
CDS!5’UTR! 3’UTR! CDS!5’UTR! 3’UTR!
Proline-rich region!
Acidic region!
Leucine zipper!
Leucine zipper!
Ellenberger et al., 1992
Splice Variant transcript (LDOC1S)
Wild Type LDOC1 transcript (LDOC1)
LDOC1S NCBI accession numbers mRNA: HQ343285 Protein: ADO32619
How much of the total LDOC1 mRNA is represented by LDOC1S?
CDS!5’UTR! 3’UTR! CDS!5’UTR! 3’UTR!
Common TaqMan Assay
Isoform-Specific TaqMan Assays
Isoform-Specific TaqMan Assays
Splice Variant transcript (LDOC1S)
Wild Type LDOC1 transcript (LDOC1)
Duzkale H. et al., Blood 2011, 117(15):4076-84
CDS!5’UTR! 3’UTR! CDS!5’UTR! 3’UTR!
Artificial Template LDOC1 Forward
Primer Reverse Primer
TaqMan Probe (Taq-LDOC1) Artificial
Template LDOC1S Forward
Primer Reverse Primer
TaqMan Probe (Taq-LDOC1S)
Isoform-Specific TaqMan Assays
Splice Variant transcript (LDOC1S)
Wild Type LDOC1 transcript (LDOC1)
Duzkale H. et al., Blood 2011, 117(15):4076-84
Common TaqMan Assay
Isoform-Specific TaqMan Assays
DCt TaqLD1sv (Ct target LD1svTemplate-Ct homolog LD1wtTemplate)]= 2^-(11.012-26.415) = 43,327.6 DCt TaqLD1wt (Ct target LD1wtTemplate-Ct homolog LD1svTemplate)]= 2^-(10.358-34.104) = 14,068,839.6
Taq-LDOC1S/LDOC1S Taq-LDOC1S/LDOC1
Taq-LDOC1/LDOC1 Taq-LDOC1/LDOC1S
Water/Taq-LDOC1
24 cycles
14.5 cycles
TaqMan Assays Highly Specific
for Their Target
Isoforms
Duzkale H. et al., Blood 2011, 117(15):4076-84
LDOC1 Isoforms are Differentially Expressed in Normal and Malignant B cells and Solid Tumor Cell Lines
[Error bars: SE of ΔΔ Ct values] Duzkale H. et al., Blood 2011, 117(15):4076-84
Rela
tive
Lev
els
of m
RNA
Relative
mRN
A E
xpre
ssion
LDOC1 Isoforms are Differentially Expressed in Normal and Malignant B cells and Solid Tumor Cell Lines
-LDOC1S mRNA constitutes a small portion of total LDOC1 mRNA
-Potential role for LDOC1 in B cell development, activation, differentiation and malignant transformation…
[Error bars: SE of ΔΔ Ct values] Duzkale H. et al., Blood 2011, 117(15):4076-84
Relative
mRN
A E
xpre
ssion
biomarker function
LDOC1
Functional studies
LDOC1 protein knock-down in
HeLa cells
Transcriptome
Biological Outcome
Proliferation? Cell cycle?
Network Construction
(Ingenuity Pathways)
Assessment in HeLa (QRT-PCR)
STRATEGY
Validation in CLL samples (MF-QRT-PCR)
Concordant Target Gene Assessment
Transient Transfection of HeLa Cells with siRNAs
Untransfected LDOC1
siRNA Pool Non-targeting siRNA Pool
Mock Transfection
siGlo
Cell number, viability & cell cycle Transfection Efficiency (IF, WB) RNA
3 biologic replicates/each
High Transfection Efficiency of HeLa Cells (24 hours)
(20X) (20X)
Transfection did not Affect Cell Number (93 hours)
Transfection did not Affect Cell Cycle (93 hours)
LDOC1
Beta-actin
Dramatic LDOC1 Protein Knock-down in HeLa Cells (93 hours)
Untransfected LDOC1 NonTargeting Mock siRNAs siRNAs Transfection
Global Gene Expression Profiling of HeLa Cells
Affymetrix Human Genome Chips U133 Plus 2.0 47,000 transcripts with 54,675 probe sets/array
LDOC1 siRNA pool NonTargeting siRNA pool
Biologic replicates Biologic replicates
Differentially Expressed Genes in HeLa Cells (n=122)
Gene Symbol Fold Change P value LDOC1 -5.24 0.000000144
SLC39A6 1.39 0.0000154 KRAS 1.35 0.0000199 WASL 1.28 0.0000209
STRADB 1.07 0.0000385 NOLC1 1.27 0.0000494
EIF4EBP1 -1.48 0.0000536 TADA2L 1.22 0.0000723
LOC552889 1.23 0.0000877 ORC5L 1.12 0.000088 GPM6B 1.17 0.0000883 CDV3 -1.27 0.0000927
SUSD1 1.43 0.0000968 WWTR1 1.20 0.000100038 RAB14 1.20 0.000109765
DAB1 /// OMA1 1.11 0.000139154 CLIC3 1.09 0.000139573 COTL1 -1.04 0.000141816 CNR1 -1.10 0.000156992
Networks by Ingenuity Pathways Analysis
1. Cardiac Arrythmia,Cardiovascular Disease, Gene Expression 2. Cellular Function and Maintenance, Cancer, GI Disease 3. Cell Cycle, Expression, Cellular Growth and Proliferation 4. Cellular assembly and Organization, Cardiac Necrosis/Cell death, Cell Death 5. Gene Expression, Cell Death, Tissue Development 6. DNA Replication, Recombination and Repair, Cardiovascular Disease, Cancer
Network 2: Cancer
HeLa cells (LDOC1 or )
30 CLL samples (LDOC1 or )
Concordant Target Gene Search- Intersection of Affymetrix Data
Intersection
Affymetrix Gene Chip Human Genome U133 Plus 2.0
47,000 transcripts
Affymetrix Gene Chip Human Genome U133A 2.0
18,400 transcripts (Abruzzo LV, et al., J Mol Diagn. 2005 7(3):337-45)
Definition of LDOC1 Positivity in Affymetrix Data, Aided by QRT-PCR Data
LDOC1
mRN
A E
xpre
ssion
(QRT
-PCR
)
LDOC1 mRNA Expression (Affymetrix arrays)
Concordant Target Genes in HeLa and CLL samples- Intersection of Affymetrix Data
(N=51; p<0.05)
CLL Fold Change = LDOC1 high CLL / LDOC1 low CLL
HeLa Fold Change = LDOC1 high (NT siRNAs) / LDOC1 low (LDOC1 siRNAs)
Concordant Target Genes in HeLa and CLL samples- Validation by QRT-PCR in HeLa
ITGAL (Integrin alpha-L) MAPKAPK2 (MAPK-activated protein kinase 2) CLCN4 (chloride channel 4) DNAJA1 (DnaJ homolog subfamily A member 1) UBE2N (E2 ubiquitin conjugating enzyme) NRIP1 (nuclear receptor interaction protein) LDOC1
(3 biologic replicates, triplicate/each)
Except for LDOC1, none of the genes was differentially expressed in HeLa cells
by QRT-PCR assay…
Abruzzo L.V. et al., submitted
Screening of CLL literature for candidate biomarkers of prognosis in CLL
Evaluation of 43 genes in 76 untreated CLL patients
(MF-QRT-PCR)
30 genes were validated as differentially expressed between LDOC1 CLL and LDOC1 CLL
Validation of Biomarkers of Prognosis in CLL with respect to LDOC1 Status
Assessment of 43 biomarkers of CLL prognosis in HeLa cells (LDOC1 or )
Concordant Target Gene Identification (MF-QRT-PCR)
Expression of 43 Biomarkers of CLL Prognosis in HeLa Cells (by MF-QRT-PCR)
Validation in HeLa cells
LDOC1 or [3 genes]
Validation in 76 CLL samples
LDOC1 or [30 genes]
Concordant Target Gene Identification (MF-QRT-PCR)
Growth Factor-Independent 1 (GFI1)
Growth Factor-Independent 1 (GFI1)
-zinc finger transcriptional repressor
-confers IL-2-independent proliferation to IL-2-dependent T cell lymphoma cell line
-enhances proliferation and inhibits apoptosis -targets: p21 & p15 (indirect), Bax (direct)
-induces accelerated lymphoma in combination with Pim-1 or L-myc in mice
-restricts hematopoetic stem cell proliferation; preserves self-renewal
-expressed highest in early B- and T-cells; low/absent in mature lymphocytes; increases by TCR stimulation
Zinc finger 268; by
Thomas Splettstoesser
How might LDOC1 and GFI1 contribute to CLL pathophysiology?
Bax
GFI1 Inhibits Apoptosis by Repressing Transcription of Pro-apoptotic Bax
GFI1
based on the data from: Grimes H.L. et al., 1996 PNAS 93: 14569-14573
Apoptosis X
CDKN1A (p21) CDKN2B (p15)
Miz1 Induces Transcription of CDKN1A and CDKN2B and Causes Cell Cycle Arrest
Miz1
adapted from: Liu Q. et al., 2010 Oncogene 29: 2843-2852 & Basu S. et al., 2009 PNAS 106(5): 1433-1438
Cell cycle arrest
GFI1
Miz1
c-Myc
GFI1 and c-Myc Promote Cell Proliferation by Repressing Transcription of CDKN1A and CDKN2B
through Miz1
CDKN1A (p21) CDKN2B (p15)
adapted from: Liu Q. et al., 2010 Oncogene 29: 2843-2852 & Basu S. et al., 2009 PNAS 106(5): 1433-1438
Cell cycle arrest X
LDOC1 may Directly Regulate GFI1 Transcription to Inhibit Apoptosis and Cell Cycle Arrest
Apoptosis Cell cycle arrest
GFI1
-In HeLa cells high LDOC1 expression is associated with high GFI1 mRNA, low Bax and CDKN1A mRNA expression -LDOC1 & GFI1 are highly expressed in aggressive CLL
LDOC1
Bax CDKN1A (p21)
X
GFI1
Miz1
c-Myc
LDOC1 might Indirectly Interact with GFI1 to Promote Proliferation
CDKN1A (p21)
LDOC1
-In HeLa cells high LDOC1 expression is associated with high GFI1 mRNA and low CDKN1A mRNA expression
Cell cycle arrest X
-LDOC1 & GFI1 are highly expressed in aggressive CLL
CONCLUSIONS
1. LDOC1 mRNA is a novel biomarker of survival in untreated CLL patients
2. LDOC1S is a new splice variant of LDOC1
3. LDOC1 knock-down in HeLa cells perturbs genes expressed in cancer-related networks
4. Interaction of LDOC1 and GFI1 may contribute to the aggressive behavior in a subset of CLL
FUTURE DIRECTIONS
1. Evaluate LDOC1 mRNA as a biomarker in a larger CLL patient cohort in a longitudinal study
2. Elucidate LDOC1-GFI1 interaction
3. Investigate dynamic interplay between GFI1 and LDOC1 in a system relevant to CLL pathogenesis -in vitro stimulation of CLL samples -BCR, CD40L/IL4,TLR…PI3K/Akt pathway
Acknowledgments
Abruzzo Lab Lynne V. Abruzzo, MD, PhD Lynn L. Barron, BS Katherine Lin, MD Roberto Nussenzveig, PhD Carmen D. Schweighofer, MD Wilkinson Lab
Wai Kin Chan, PhD Lulu Huang, PhD
Hematopathology Kaushali Patel, MS Marco Herling, MD Elias Drakos, PhD Lan Pham, PhD Evan Cohen, BS
Committee Members Shelly Barton, PhD Emil J Freireich, MD, DSci (Hon) Vicki Huff, PhD Kevin R. Coombes, PhD David McConkey, PhD Richard Ford, MD, PhD Malcolm Brenner, MD, PhD Gil Cote, PhD Craig Logsdon, PhD
UT GSBS George Stancel, PhD Jon R. Wiener, PhD
U.T. Center for Clinical and Translational Sciences (CCTS) NIH T32 Training Award
Microfluidics Card Design for 48 Genes (MF-QRT-PCR)
1 2 3 4 5 6 7 8
Microfluidics Card Assay Design for 48 Genes (MF-QRT-PCR)
1
2
3
4
5
6
7
8
LDOC1 siRNA pool
NonTargeting siRNA pool
LDOC1 siRNA pool
LDOC1 siRNA pool
NonTargeting siRNA pool
NonTargeting siRNA pool
NonTargeting siRNA pool
LDOC1 siRNA pool
(#1)
(#1)
(#2)
(#3)
(#2)
(#3)
(#1)
(#1)
Distinct Gene Expression Profile of B cell Subtypes
Klein U., et al. J. Exp. Med. 2001 194 (11):1625-1638
Potential LDOC1-Interacting Molecules in CLL Related Network
LDOC1 Directly Interacting Molecules (Ingenuity Knowledgebase)
Normal
Cancer
Nagasaki K. et al., 1999. Cancer Letters 140:227-234
LDOC1 mRNA expression in normal and cancer tissues
Duzkale H, et al., accepted to Blood
LDOC1 mRNA Expression Correlates with IGHV Somatic Mutation Status
mR
NA
Exp
ress
ion
Poor prognosis Good prognosis Poor prognosis Good prognosis
LDOC1 mRNA ZAP70 mRNA
threshold threshold