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


20 (51%) (N=39)

None 14 (23%); del(13q) 30 (49%); del(6q), del(11q), del(17p), +12, or


17 (28%) (N=61)

0.0006


LDOC1 mRNA Expression Independently Predicts Overall Survival

13.6 yr

not reached


LDOC1 mRNA Expression Predicts Overall Survival Better than IGHV Somatic Mutation Status and ZAP70

ZAP70 IGHV

LDOC1

Multivariate Analysis


-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


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


LDOC1S is a Leucine zipper region alone


Proline-rich region!

Acidic region!

Leucine zipper!

Leucine zipper!

Ellenberger et al., 1992



LDOC1S NCBI accession numbers mRNA: HQ343285 Protein: ADO32619

How much of the total LDOC1 mRNA is represented by LDOC1S?


Common TaqMan Assay

Isoform-Specific TaqMan Assays






Artificial Template LDOC1 Forward

Primer Reverse Primer

TaqMan Probe (Taq-LDOC1) Artificial

Template LDOC1S Forward

Primer Reverse Primer

TaqMan Probe (Taq-LDOC1S)





Common TaqMan Assay


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


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




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

PhD-Defense-May 13 2011

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