1. 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

2. 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

3. Chronic Lymphocytic Leukemia
Prognostic Subtypes
Aggressive CLLIndolent 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

4. Immunoglobulin Somatic Mutation is
a Physiologic Process
modified from Kuppers et al., Nature Reviews Cancer, 2005, 5:251-262
DNA modifications

5. 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

6. Disease Subtype Classification
by IGHV Somatic Mutation Status
Novel Biomarkers Novel Molecular Targets
Global Gene Expression Profiling

7. Genes Differentially Expressed Between CLL Subtypes
Klein U, et al. J. Exp. Med. 2001 194 (11):1625-1638

8. 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

9. Can LDOC1 Predict Survival?
Evaluation of LDOC1 in 131 untreated CLL patients
(MF-QRT-PCR)
Correlation with
other prognostic parameters
Survival analysis

10. LDOC1 mRNA Expression is Bimodal in CLL
(QRT-PCR)
high mRNA low mRNA

11. LDOC1 mRNA Expression Correlates
with IGHV Somatic Mutation Status
LDOC1mRNAExpression
Poor prognosisGood prognosis
threshold
Duzkale H. et al., Blood 2011, 117(15):4076-84

12. 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

13. LDOC1 mRNA Expression
Independently Predicts Overall Survival
13.6 yr
not reached
Duzkale H. et al., Blood 2011, 117(15):4076-84

14. LDOC1 mRNA Expression Predicts Overall Survival
Better than IGHV Somatic Mutation Status and ZAP70
ZAP70IGHV
LDOC1
Multivariate
Analysis
Duzkale H. et al., Blood 2011, 117(15):4076-84

15. -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

16. 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)

17. 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)

18. CLL46 CLL49 CLL51 CLL53 CLL12 CLL37 CLL61 CLL67 CLL99CLL42
Unmutated Mutated
MCF7 GA10 Jurkat NBC4 NBC6 CLL54 CLL58 CLL62CLL60
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

19. 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

20. 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

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

22. 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

23. 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

24. 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

25. 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
RelativeLevelsofmRNA
RelativemRNAExpression

26. 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
RelativemRNAExpression

27. biomarker function
LDOC1

28. 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

29. 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

30. High Transfection Efficiency of HeLa Cells
(24 hours)
(20X)(20X)

31. Transfection did not Affect Cell Number
(93 hours)

32. Transfection did not Affect Cell Cycle
(93 hours)

33. LDOC1
Beta-actin
Dramatic LDOC1 Protein Knock-down
in HeLa Cells
(93 hours)
Untransfected LDOC1 NonTargeting Mock
siRNAs siRNAs Transfection

34. 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

35. 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

36. 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

37. Network 2: Cancer

38. 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)

39. Definition of LDOC1 Positivity in
Affymetrix Data, Aided by QRT-PCR Data
LDOC1mRNAExpression(QRT-PCR)
LDOC1 mRNA Expression (Affymetrix arrays)

40. 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)

41. 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)

42. Except for LDOC1, none of the genes
was differentially expressed in HeLa cells
by QRT-PCR assay…

43. 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

44. Assessment of 43 biomarkers of CLL prognosis
in HeLa cells (LDOC1 or )
Concordant Target Gene Identification
(MF-QRT-PCR)

45. Expression of 43 Biomarkers of CLL Prognosis
in HeLa Cells (by MF-QRT-PCR)

46. 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)

47. 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

48. How might LDOC1 and GFI1 contribute
to CLL pathophysiology?

49. 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
ApoptosisX

50. 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

51. 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 arrestX

52. 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

53. 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 arrestX
-LDOC1 & GFI1 are highly expressed in aggressive CLL

54. 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

55. 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

56. 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

59. Microfluidics Card Design for 48 Genes
(MF-QRT-PCR)
1
2
3
4
5
6
7
8

60. 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)

62. Distinct Gene Expression Profile of B cell Subtypes
Klein U., et al. J. Exp. Med.
2001 194 (11):1625-1638

63. Potential LDOC1-Interacting Molecules
in CLL Related Network

64. LDOC1 Directly Interacting Molecules
(Ingenuity Knowledgebase)

65. Normal
Cancer
Nagasaki K. et al., 1999. Cancer Letters 140:227-234
LDOC1 mRNA expression in normal and cancer tissues

66. Duzkale H, et al., accepted to Blood
LDOC1 mRNA Expression Correlates with
IGHV Somatic Mutation Status
mRNAExpression
Poor prognosisGood prognosis Poor prognosisGood prognosis
LDOC1 mRNA ZAP70 mRNA
threshold
threshold