1. Research Focus: Inhibition of Syk in B-Cell Malignancies
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Research Focus: Inhibition of Syk in B-Cell Malignancies
This activity is supported by an independent educational grant from Gilead Sciences.
Image: Dr. Gopal Murti/Copyright©2014 Phototake All Rights Reserved

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3. Faculty
Jeff P. Sharman, MD Medical Director Hematology Research US Oncology Research Eugene, Oregon Jeff P. Sharman, MD, has disclosed that he has received consulting fees from Gilead Sciences and Pharmacyclics and funds for research support from Celgene, Genentech, Gilead Sciences, and Pharmacyclics.
This slide lists the faculty who were involved in the production of these slides.

4. B-Cell Function in the Immune System
Bone Marrow
Stem cell
Early pro-B cell
Late pro-B cell
Large pre-B cell
Small pre-B cell
Immature B cell
µ germline / germline
µ germline / germline
µDJ / germline
µVDJ / germline
µVDJ / germline
µVDJ / VJ
Secondary Lymphoid Organs and Circulation
Immature B cell (IgM+, IgD-)
Immature B cell (IgMhigh, IgDlow)
Mature naive B cell (IgMlow, IgDhigh)
Antigen-activated B lymphoblast
Antibody-secreting plasma cell
Memory cell
IgM
IgD
IgM
IgD
IgM
IgM
IgG
IgM
IgG
Leaves bone marrow and enters peripheral circulation
Alternative splicing to give both  and µ chains
Gains access to primary lymphoid follicle and matures
Enters circulation and binds specific antigen in lymphoid tissue draining infection
Alternative splicing to secrete lg Isotype switching Somatic hypermutation
Fighting the current infection
Preparing for future infection
1. © 2009 from The Immune System, Third Edition by Peter Parham. Reproduced by permission of Garland Science/Taylor & Francis LLC.

5. Tonic Signaling Time Ligand Dependent Activating threshold
Ligand Independent
Receptor Activity
Average (basal)
Individual (transient)

6. B-Cell Receptor Signaling and B Lymphocyte Survival
- Mx-cre 40 30 20 10
CD79
Splenic B Cells (x 106)
Syk
Survival of resting mature B lymphocytes depends on BCR signaling via the Ig alpha/beta heterodimer
3 x p(I)Vp(C)
Day 14
3. Adapted from Kraus M, et al. Cell. 2004;117:

7. B-Cell Malignancies: Cell Types and Associated Diseases
Lymphoid progenitor
Associated Diseases
Acute lymphoblastic leukemia
Blood and Marrow
Pre-B cell
Naive B cell
Mantle cell lymphoma,
marginal zone lymphoma, CLL
Activated B cell
DLBCL
Secondary Lymphoid Tissues
Follicular lymphoma, DLBCL,
Hodgkin’s lymphoma
Germinal-center B cell
CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma.
Memory B cell
Burkitt lymphoma, CLL
Waldenström’s macroglobulinemia
Lymphoplasmacytoid
Bone Marrow
Multiple myeloma, amyloidosis
Plasma cells

8. Diffuse Large B-Cell Lymphomas
Hierarchial clustering of DLBCL (blue, orange) and GC B cells (black) based on B-cell gene expression signature
Discovery of genes that are selectively expressed in GC B-like DLBCL and activated B-like DLBCL
Hierarchial clustering of genes selectively expressed in GC B-like DLBCL and activated B-like DLBCL
DLBCL, diffuse large B-cell lymphoma; GC, germinal center.
8. Alizadeh AA, et al. Nature. 2000;403:

9. Effect of Lymphoma on B-Cell Receptor SignalingAg Ag Ag c a b α α α β β β
CARD11
CARD11
CARD11
Tyrosine kinases
Tyrosine kinases
Tyrosine kinases
BCL10
BCL10
BCL10
MALT1
MALT1
MALT1
A20
A20
A20
ABC-DLBCL, activated B cell–like diffuse large B-cell lymphoma.
PI3K
MAPK/ERK
NF-κB
PI3K
MAPK/ERK
NF-κB
PI3K
MAPK/ERK
NF-κB
Normal B cell with
antigen stimulation
ABC-DLBCL with mutations in CARD11 or A20
ABC-DLBCL with mutations in CD79A and B
10. Reprinted by permission from Macmillan Publishers Ltd: Klein U, et al. Immunol Cell Biol ;88: © 2010.

10. Altered B-Cell Receptor Signaling Kinetics
α-µ/ + H202 Stimulated Cells, FL-P10, Gated CD20+
Stimulation Time (min): 4 16 30 60 90
100
101
102
103
104
P-Syk
100
101
102
103
104
 Light Chain
11. Irish JM, et al. Blood. 2006;108:

11. Summary Normal B cells require intact BCR to survive
Circumstantial evidence that this is true in B-cell malignancies
B-cell malignancies may depend on hyperactive BCR signaling
Could BCR signal inhibition be therapeutic?
BCR, B-cell receptor.

12. BCR Signaling: Syk’s Pivotal Role
IgG
CD79
BTK
Syk
PI3K
BLNK
BCR, B-cell receptor.
MEK
CARD11
AKT
ERK
NF-κB
MTOR

13. Syk Determines B-Cell Fate
Syk-deficient mice lack B cells
Constitutive Syk activity causes B-cell leukemia
13. Turner M, et al. J Exp Med. 1997;186: © 2006 Wossning et al. The Journal of Experimental Medicine. 2006;204: doi: /jem

14. Potential BCR Signal Inhibitors for B-Cell Malignancies
Syk
Fostamatinib, entospletinib (GS-9973), cerdulatinib (PRT062070), TAK-659
BTK
Ibrutinib, CC-292, ONO-4059, ACP-196
PI3K
Idelalisib, buparlisib, GDC-0941, BAY , IPI-145, BEZ-235, others
BCR, B-cell receptor.

15. Syk Inhibition With Fostamatinib in NHL and CLL/SLL: Responses and PFS
Lymphoma Subtype
100 80 60 40 20
-20
-40
-60
-80
-100
Best Responses
DLBCL FL CLL/SLL Other indications
% Change From Screening
Group 1, DLBCL
Group 2, FL
Group 3
Response
De novo (n = 17)
Transformed (n = 6)
(n = 21)
CLL/SLL (n = 11)
MCL (n = 9)
Other (n = 4)
CR, n 1
PR, n 3 2 6
SD, n 11 4
PD, n 8 7
Not evaluable, n
ORR (CR + PR), n (%)
4 (23.5)
1 (16.7)
2 (9.5)
6 (54.5)
1 (11.1)
CR + PR + SD, n (%)
6 (35.3)
3 (50.0)
13 (61.9)
8 (72.7)
5 (55.6)
1 (25.0)
PFS, mos (95% Cl) --
4.6 ( )
6.4 ( )
3.8 ( )
1.9 (1.8-NA)
CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; CR, complete response; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; NA, not available; NHL, non-Hodgkin’s lymphoma; ORR, overall response rate; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease.
15. Friedberg JW, et al. Blood. 2010;115:

16. Syk Inhibition With Fostamatinib
Before Treatment
After Treatment
Images courtesy of Jeff Sharman, MD.

17. Other Syk Inhibitor Options
Fostamatinib showed early promise as a Syk inhibitor in rheumatoid arthritis, but more recent studies showed disappointing results[18]
Other Syk inhibitors in the pipeline
Entospletinib (GS-9973)
Cerdulatinib (PRT062070)
TAK-659
18. Taylor PC, et al. Ann Rheum Dis. 2014;[Epub ahead of print].

18. Kinase Inhibition Analyses of Entospletinib (GS-9973) vs Fostamatinib
Entospletinib: Syk Specificity Profile
Fostamatinib: Syk Specificity Profile
Syk Kd = 7.6 nM
No other kinases with Kd < 100nM
Syk Kd = 15 nM
24 kinases with Kd < 15 nM
54 additional kinases with Kd < 100 nM
22. Sharman JP, et al. ASCO Abstract 7007. 18

19. Entospletinib (GS-9973), a Selective Syk Inhibitor in NHL: Phase II Study Design
Continue accrual to a max of 40 pts
Consider stopping accrual in that particular disease
LPL, SLL, MZL
Yes FL
Entospletinib
800 mg BID
10-40 pts in each arm
Results cross futility boundary
DLBCL
Continue accrual to a max of 40 pts
BID, twice daily; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; LPL, lymphoplasmacytic lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; NHL, non-Hodgkin’s lymphoma; SLL, small lymphocytic leukemia. No
MCL
CLL
23. Sharman JP, et al. ASCO Abstract 7007.

20. Entospletinib (GS-9973), a Selective Syk Inhibitor in NHL: Subject Characteristics
CLL
(n = 41)
NHL
(n = 104)
All
(N = 145)
Male, n (%)
28 (68.3)
60 (57.7)
88 (60.7)
Age, yrs (median range)
73 (51-89)
69 (41-91)
70 (41-91)
Previous therapies, median n (range)
2 (1-8)
3 (1-14)
Anti-CD20 antibody, n (%)
40 (97.6)
101 (97.1)
141 (97.2)
Any alkylating agent, n (%)
35 (85.4)
97 (93.3)
132 (91.0)
Bendamustine
26 (63.4)
44 (42.3)
70 (48.3)
Any purine analogue, n (%)
17 (16.3)
45 (31.0)
Fludarabine
15 (14.4)
43 (29.7)
Anthracyclines, n (%)
3 (7.3)
57 (54.8)
60 (41.4)
CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin’s lymphoma.
Susan M. O’Brien, MD:
Of note, these patients were less heavily pretreated than in previous B-cell inhibitor studies of fostamatinib, ibrutinib, or idelalisib, where patients typically had received a median of 3-4 prior regimens.
24. Sharman JP, et al. ASCO Abstract 7007.

21. Entospletinib (GS-9973), a Selective Syk Inhibitor: Responses in CLL
120
Very high risk (with 17p deletion and/or TP53 mutation)
High risk (with NOTCH1 and/or SF3B1 mutation and/or 11q deletion)
Low risk (none of the above deletion or mutation)
Undetermined
ORR rate: 49% (n = 41; 95% CI: 33% to 65%)
Hematologic response rate
Hb: 25% (n = 12)
Neutrophils: 86% (n = 7)
Platelets: 77% (n = 13)
100 80
Nodal response rate 62% (N = 39*; 95% Cl: 45% to 77%) 60 40 20
Best % Change in SPD
-20
CI, confidence interval; CLL, chronic lymphocytic leukemia; Hb, hemoglobin; ORR, overall response rate; SPD, sum of greatest perpendicular dimension.
Susan M. O’Brien, MD:
The waterfall plot displays the greatest change in lymphadenopathy. As with other B-cell receptor inhibitors, an improvement in adenopathy was seen in almost every patient. The overall response rate was 49%. Although not on the slide, lymphocytosis, which has occurred with all the B-cell receptor inhibitors, also occurs with this Syk inhibitor. Initially, the pattern of response is more nodal, then over time a true PR occurs as the lymphocyte count goes below baseline.
-40
-60
-80
-100
*2 subjects did not have postbaseline tumor assessment (1 died of pneumonia/1 withdrew consent prior to evaluation).
25. Sharman JP, et al. ASCO Abstract 7007.

22. Absolute Lymphocytes Count, Mean (± SE) (x 103/μL)
Entospletinib in Lymphoid Malignancies: Absolute Lymphocyte Count (CLL)
100 75 50 25
Absolute Lymphocytes Count, Mean (± SE) (x 103/μL)
CLL, chronic lymphocytic leukemia; SE, standard error. 2 1 3 4 6 8 12 16 20 24 28 32 36 40 44 48 52
Wks 37 38 34 32 26 24 20 21 13 6 4 2
Patients at Risk, n
26. Sharman JP, et al. ASCO Abstract 7007.

23. Entospletinib (GS-9973), a Selective Syk Inhibitor in CLL: PFS and DoR
100
100 75 75
PFS (%) 50
Continued Response (%) 50
Median PFS not yet reached 75% of subjects have PFS longer than 5.4 mos
Median DoR not yet reached 75% of subjects have DoR longer than 6.5 mos 25 25
PFS rate at 24 wks: 70% (95% CI: 51% to 83%)
CI, confidence interval; CLL, chronic lymphocytic leukemia; DoR, duration of response; PFS, progression-free survival. 2 4 6 8 10 12 2 4 6 8 10 12
Mos From Start of GS-9973
Mos From Response
Median PFS follow-up of 5.5 mos
27. Sharman JP, et al. ASCO Abstract 7007.

24. Entospletinib (GS-9973), a Selective Syk Inhibitor: Adverse Events
Fatigue
Nausea
Diarrhea
Constipation
Dec. appetite
Headache
Pyrexia
Dizziness
Anemia
Dehydration
Dyspnea
Rash
Hypotension
Pneumonia
Back pain
Chest pain
Atrial fibrillation
Feb. neutropenia
Hypoxia
Sepsis
Anemia
Neutropenia
Thrombocytopenia
Grade 1/2
Grade ≥ 3
Increased total bilirubin
Increased ALT
Increased AST 10 20 30 40 50
AE, adverse event; ALT, alanine transaminase; AST, aspartate aminotransferase; Dec, decreased; Feb, febrile; SAE, serious adverse events.
Incidence (%)
Reported AEs and lab abnormalities (N = 145)
Including treatment-emergent nonhematologic AE
Any grade (≥ 20%) or grade ≥ 3 (> 2%)
Any SAE (> 2%)
19 deaths
12 due to disease progression
7 due to AE
Grade 1/2
Grade ≥ 3
Serious 10 20 30 40 50
Incidence (%)
28. Sharman JP, et al. ASCO Abstract 7007.

25. Entospletinib (GS-9973), a Selective Syk Inhibitor in CLL: Conclusions
GS-9973 has an acceptable safety profile
14% of pts had reversible grade 3/4 transaminase elevations
Activity in CLL
ORR: 49% (95% CI: 33% to 65%)
PFS at 24 wks: 70% (95% CI: 51% to 83%)
Median PFS and DoR: not reached yet
75% of pts have PFS and DoR beyond 5.4 and 6.5 mos, respectively
Moving forward with new treatment cohorts in pts with CLL following prior treatment with BCR-targeted therapies in relapse
BCR, B-cell receptor; CLL, chronic lymphocytic leukemia; DoR, duration of response; ORR, overall response rates, PFS, progression-free survival.
30. Sharman JP, et al. ASCO Abstract 7007.

26. Open-Label Phase I Trial: Dual Syk/JAK Inhibitor Cerdulatinib (PRT062070)
3 x 3 dose-escalation study of cerdulatinib in patients with relapsed/refractory NHL or CLL/SLL
Dose started at 15 mg/day orally
Rationale
Syk is upstream of BTK and PI3k in BCR signaling pathway
JAK-mediated survival signals observed in leukemia and lymphoma
Combined knockdown of Syk and JAK synergistic in preclinical B-cell lymphoma cell-line studies
Cerdulatinib a selective inhibitor of Syk, JAK1, JAK3, Tyk2
BCR, B-cell receptor; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; NHL, non-Hodgkin’s lymphoma.
31. Flinn I, et al. ASCO Abstract 2619.

27. TAK-659: Novel Syk Inhibitor
A phase I study of the Syk inhibitor TAK-659 in patients with advanced solid tumors and lymphoma malignancies is currently recruiting participants[34]
TAK-659 showed antitumor activity correlated with Syk pathway inhibition in xenograft models of DLBCL subtypes[35]
DLBCL, diffuse large B-cell lymphoma.
34. ClinicalTrials.gov. NCT Huck J, et al. ASCO Abstract 8580.

28. Summary B-cell biology Syk
BCR determines B-cell fate: apoptosis (no BCR) or maturation (BCR present)
B-cell malignancies dependent on tonic signaling
Syk
Constitutive Syk activity causes B-cell leukemia
Syk inhibition may be effective in NHL and CLL/SLL
BCR, B-cell receptor; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; NHL, non-Hodgkin’s lymphoma.

29. Go Online for More CCO Coverage of B-Cell Malignancies!
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