scholarly journals Duohexabody-CD37, a Novel Bispecific Antibody with a Hexamerization-Enhancing Mutation Targeting CD37, Demonstrates Superior Complement-Dependent Cytotoxicity in Preclinical B-Cell Malignancy Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4170-4170
Author(s):  
Simone C. Oostindie ◽  
Hilma J. Van Der Horst ◽  
Marije B. Overdijk ◽  
Kristin Strumane ◽  
Sandra Verploegen ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Lymphocytic Leukemia ◽  
Single Point Mutation ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Healthy Human ◽  
Complement Dependent Cytotoxicity ◽  
Equity Ownership

Abstract CD37 is a tetraspanin plasma membrane protein abundantly expressed on B-cells and represents a promising therapeutic target for the treatment of B-cell malignancies. Although complement-dependent cytotoxicity (CDC) has proven to be a powerful Fc-mediated effector function for killing hematological cancer cells, CD37 antibody-based therapeutics currently in clinical development are poor inducers of CDC. Here we present DuoHexaBody-CD37, a novel humanized IgG1 bispecific antibody targeting two different CD37 epitopes, with an E430G hexamerization-enhancing mutation, for the potential treatment of B-cell malignancies. The natural process of antibody hexamer formation through intermolecular Fc-Fc interactions between IgG molecules after cell surface antigen binding can be improved by introducing a single point mutation such as E430G in the IgG Fc domain, thereby facilitating more efficient C1q binding and complement activation (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). The hexamerization-enhancing mutation E430G was introduced into two humanized CD37 monoclonal antibodies (mAbs) that bind non-overlapping CD37 epitopes. Different antibody formats and combinations, including the single antibodies, combinations of the mAbs and bispecific mAbs were tested for their capacity to induce CDC and antibody-dependent cellular cytotoxicity (ADCC). The bispecific hexamerization-enhanced antibody variant DuoHexaBody-CD37, showed superior CDC activity compared to the single hexamerization-enhanced mAbs and the combination thereof, both in vitro over a range of different B-cell lines, and ex vivo in tumor cell samples obtained from patients with chronic lymphocytic leukemia (CLL). In a CDC assay using tumor cells obtained from a relapsed/refractory CLL patient who received prior treatment with rituximab, ibrutinib and idelalisib, DuoHexaBody-CD37 induced almost complete lysis (84% lysis at a concentration 100 µg/mL), thereby outperforming the single HexaBody molecules (15% and 23% lysis) and the combination (57%) (Figure 1). In addition to its potent CDC activity, DuoHexaBody-CD37 was also capable of inducing potent ADCC of Daudi cells (EC50 = 12.3 ± 9.5 ng/mL), as assessed using peripheral blood mononuclear cells from 8 healthy human donors in a standard chromium release assay. In assays using whole blood from 6 healthy human donors, DuoHexaBody-CD37 showed efficient B-cell binding and potent and specific depletion of the B-cell population (98% ± 1.3% depletion at 10 µg/mL, EC50 = 0.85 ± 0.284 µg/mL). Furthermore, DuoHexaBody-CD37 induced significant inhibition of tumor growth in vivo in Daudi-luc Burkitt's lymphoma and JVM-3 CLL mouse xenograft models, at doses as low as 0.1 and 1 mg/kg (p<0.05), respectively. In summary, we present a novel therapeutic antibody that, for the first time, combines proprietary DuoBody® and HexaBody® platforms. DuoHexaBody-CD37 induced highly potent CDC and efficient ADCC in preclinical models, suggesting that DuoHexaBody-CD37 may serve as a potential therapeutic mAb for the treatment of human B-cell malignancies. Disclosures Oostindie: Genmab: Employment, Equity Ownership. Van Der Horst:Genmab: Research Funding. Overdijk:Genmab: Employment, Equity Ownership. Strumane:Genmab: Employment, Equity Ownership. Verploegen:Genmab: Employment, Equity Ownership. Lindorfer:Genmab: Research Funding. Cook:Genmab: Research Funding. Chamuleau:Gilead: Research Funding; BMS: Research Funding; celgene: Research Funding; Genmab: Research Funding. Mutis:Gilead: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding. Schuurman:Genmab: Employment, Other: Warrants. Sasser:Genmab: Employment, Equity Ownership. Taylor:Genmab: Research Funding. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Beurskens:Genmab: Employment, Equity Ownership. Breij:Genmab: Employment, Equity Ownership.

Inhibition Of PI3K-δ and -γ Isoforms By IPI-145 In Chronic Lymphocytic Leukemia Overcomes Signals From PI3K/AKT/S6 Pathway and Promotes Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4167-4167 ◽  
Author(s):  
Kumudha Balakrishnan ◽  
Marisa Peluso ◽  
Min Fu ◽  
Nathalie Y. Rosin ◽  
Jan A. Burger ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Therapeutic Potential ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Enhanced Production ◽  
Significant Difference ◽  
Equity Ownership

Abstract The functional relevance of the B cell Receptor (BCR) pathway and identification of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B cell malignancies. Inhibition of protein and lipid kinases (Bruton Tyrosine Kinase [BTK] and phosphoinositide 3-kinase [PI3K]) with ibrutinib and GS-1101 has been shown to be active in treatment of chronic lymphocytic leukemia (CLL). Importantly, differential expression and function of PI3K isoforms support isoform-selective inhibition of this kinase in CLL. Whilst PI3K-α and PI3K-β are ubiquitously expressed, PI3K-δ and PI3K-γ are primarily restricted to leukocytes. Since CLL cells generally express high levels of active PI3K-δ, great interest has been focused on inhibition of PI3K-δ. However, given the distinct and non-overlapping roles of PI3K-δ and PI3K-γ in immune cells, exploration of the therapeutic potential of combined inhibition of both PI3K-δ and PI3K-γ in CLL patients is warranted. IPI-145 is a potent, orally bioavailable, inhibitor of PI3K-δ and PI3K-γ isoforms with KD values of 0.023 nM and 0.24 nM, respectively. Treatment of primary CLL cells (n=51) with IPI-145 (1 µM) resulted in significant apoptosis (median 33%; range 12 – 40%). Patients with mutated (n=13) or unmutated IGHV gene status (n=13), previously untreated (n=21) or treated (n=8), displayed no significant difference in apoptosis from IPI-145. Samples with different prognostic markers such as 13q (del) or FISH negative samples were equally sensitive to IPI-145. Side by side studies of IPI-145 with ibrutinib and GS-1101, revealed that IPI-145 is comparatively potent (IC50 7.6 µM, compared to >10 µM) in promoting apoptosis. Crosslinking with anti-IgM enhanced the survival of primary CLL cells in association with activation of PI3K-δ,γ/AKTSer473/pBadSer136/S6Ser235/236 pathway, which was in turn mitigated upon treatment with IPI-145 (n=9). Consistent with cell death, cleavage of PARP and decrease in anti-apoptotic protein Mcl-1 (but not Bcl-2 or Bcl-xL) was observed. Measurement of the C-C chemokine, CCL3, a biomarker for BCR signaling inhibition in CLL, demonstrated 15 to 48 – fold increase upon anti-IgM stimulation, which was reversed when cells were treated with 1 µM IPI-145 (10 to 80-fold decrease; n=6). Alternatively, co-culturing CLL primary cells with bone marrow stromal cells to mimic the leukemic microenvironment induced the protein levels of all four Class I PI3K isoforms and downstream PI3K/AKT/S6 signaling axis, which was significantly attenuated by IPI-145. To mimic the proliferative state in lymph node pseudofollicles, CLL cells were stimulated to proliferate with CD40L/IL-2/IL-10 and the effect of IPI-145 was measured. Both pAKT and Ki-67 expression were markedly inhibited in primary CLL cells at concentrations of IPI-145 in the low nanomolar range (EC50<10nM; n=2), suggesting a potent anti-proliferative effect of IPI-145 on CLL cells in the nodal environment. Given the significant role of the chemo-attractant, SDF-1, in the directed migration of B-cells, chemotaxis assay demonstrated reduction in migration of CLL cells towards SDF-1 in presence of IPI-145 (% control reduction - median 23%; range 2-42%; n=8). Furthermore, IPI-145 treatment enhanced production of reactive oxygen species (n=6). Taken together, these results demonstrate the potential of combined inhibition of the PI3K-δ and -γ isoforms in CLL, and support clinical investigation of IPI-145 in B-cell malignancies, including CLL. Disclosures: Balakrishnan: Infinity Pharmaceuticals Inc: Research Funding. Peluso:Infinity Pharmaceuticals., Inc.: Employment, Equity Ownership. Faia:Infinity Pharmaceuticals., Inc.: Employment, Equity Ownership. Kutok:Infinity Pharmaceuticals., Inc.: Employment, Equity Ownership. McGovern:Infinity Pharmaceuticals., Inc.: Employment, Equity Ownership. Gandhi:Infinity Pharmaceuticals., Inc: Research Funding.

The Bruton's Tyrosine Kinase Inhibitor, PCI-32765, Is Well Tolerated and Demonstrates Promising Clinical Activity In Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL): An Update on Ongoing Phase 1 Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 57-57 ◽  
Author(s):  
Jan A. Burger ◽  
Susan O'Brien ◽  
Nathan Fowler ◽  
Ranjana Advani ◽  
Jeff Porte Sharman ◽  
...  
Keyword(s):  
B Cell ◽  
Phase 1 ◽  
Study Drug ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Bcr Signaling ◽  
Equity Ownership ◽  
Grade 3

Abstract Abstract 57 Introduction: Bruton's tyrosine kinase (Btk) is a downstream mediator of B-cell receptor (BCR) signaling and is not expressed in T-cells or NK-cells. As such, Btk represents an ideal therapeutic target for B-cell malignancies dependent upon BCR signaling. Chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) has been reported to have constitutively active BCR signaling. PCI-32765 is a potent, selective, irreversible and orally bioavailable small molecule inhibitor of Btk that has pre-clinical activity in B-cell malignancies (Proc Natl Acad Sci 2010;107(29):13075-80). PCI-32765 was therefore moved forward to a Phase 1 study in B-cell malignancies including patients (pts) with CLL/SLL. A subsequent CLL/SLL-specific Phase 1b study was initiated to further explore safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of PCI-32765. This report includes a composite summary of the CLL/SLL experience in both of these studies. Pts and Methods: Pts with CLL/SLL who had relapsed or refractory disease after >1 prior treatment regimens were eligible for treatment in each of the studies whereas the second Phase 1b study also included a cohort of elderly pts (aged ≥ 65 years) with CLL/SLL who required treatment and were “treatment-naive”. Responses were assessed by the investigator using the International Working Group CLL criteria (Hallek et al, Blood 2008 for pts with CLL) and the International Workshop to Standardize Response Criteria for Non-Hodgkin's Lymphomas (Cheson et al, J Clin Oncol 2007 for pts with SLL). Results: To date, 30 CLL/SLL patients (including 4 treatment-naive) have been enrolled across the 2 studies. Eighty-four percent of subjects are men with an overall median age of 68 (range 44–82) years. Of the subjects with prior therapy for CLL/SLL the median number of prior therapies is 3 (range 1–4). Treatment has been well-tolerated; Grade ≥ 3 toxicities have been infrequent (10/30 pts; 33%). Two study-drug related serious adverse events have been reported: 1 case of viral adenitis (Grade 3) and 1 case of viral infection (Grade 2). Two adverse events have led to discontinuation of study drug: a small bowel obstruction (Grade 3) and exacerbation of chronic obstructive disease (Grade 3); both events were reported as unrelated to study drug. No study-drug related deaths have reported. There has been no change in either NK cell or T cell counts. Target inhibition as measured by a probe of Btk drug occupancy showed inhibition of Btk at PCI-32765 exposure levels of ≥ 245 ng•h/mL. Of the 14 patients currently evaluable for response using the pre-defined criteria, the overall response rate is 64% (1 complete remission [CR], 8 partial remissions [PR], and 4 SD). Both studies are ongoing and open to enrollment. An update on response rate, response duration, safety, and PD information will be presented on enrolled patients based on a November 2010 database cut-off. Conclusion: PCI-32765 is a novel oral and selective “first-in-human” inhibitor of Btk that induces objective partial and complete responses in a substantial proportion of pts with CLL/SLL and has a favorable safety profile. These data support further studies of both monotherapy and also combination treatment with PCI-32765 in CLL/SLL. Disclosures: O'Brien: Pharmacyclics, Inc: Honoraria, PI grant. Fowler:Pharmacyclics: Consultancy, Research Funding. Advani:Pharmacyclics, Inc: Honoraria, PI grant. Sharman:Pharmacyclics, Inc: Honoraria, PI grant. Furman:Pharmacyclics, Inc: PI grant. Izumi:Pharmacyclics, Inc: Employment. Buggy:Pharmacyclics, Inc: Employment, Equity Ownership. Loury:Pharmacyclics: Employment, Equity Ownership. Hamdy:Pharmacyclics, Inc: Employment, Equity Ownership.

A Phase 2 Study of Idelalisib Monotherapy in Previously Untreated Patients ≥65 Years with Chronic Lymphocytic Leukemia (CLL) or Small Lymphocytic Lymphoma (SLL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1986-1986 ◽  
Author(s):  
Andrew D. Zelenetz ◽  
Nicole Lamanna ◽  
Thomas J. Kipps ◽  
Steven E. Coutre ◽  
Susan O'Brien ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Small Lymphocytic Lymphoma ◽  
Employment Equity ◽  
Treatment Naïve ◽  
Equity Ownership ◽  
Systemic Therapies

Abstract BACKGROUND: PI3K-delta (δ) is critical for activation, proliferation and survival of B cells and plays a role in homing and retention in lymphoid tissues. PI3Kδ signaling is hyperactive in many B-cell malignancies. Idelalisib (Zydelig, IDELA,), a potent and selective orally administered inhibitor of PI3Kδ, in combination with rituximab weekly x 8 has yielded an ORR of 97% in patients (pts) ≥65 years with previously untreated CLL or SLL (O’Brien, ASCO 2013). This report describes the preliminary experience in treating a similar cohort of pts with IDELA monotherapy. METHODS: Enrollment began in November, 2013. Treatment-naive pts ≥65 yrs with CLL or SLL, requiring treatment per IWCLL 2008 criteria, and with measurable lymphadenopathy, were treated with IDELA 150 mg bid continuously. Response assessment, at pre-determined time points, was investigator determined using either physical exam or CT scans per investigator discretion, using modified IWCLL guidelines (Hallek 2008, Cheson 2012). RESULTS: As of 21 July 2014, 37 pts were enrolled: 78% male; CLL/SLL in 92%/8%; median age 70 years; 73% Rai III or IV; WHO 0-1/2 in 97%/3%. 46% had ≥1 B-symptom. Hepatomegaly and splenomegaly were present in 14% and 57% respectively. Adverse prognostic factors: 14% del(17p) and/or TP53 mutated; 22% del(11q); 41% IGHV unmutated; β-2 microglobulin median 4.35 mg/L (range: 2.1-12.4). The median idelalisib exposure was 4.8 months (range 0.9-8.5). There has been one discontinuation at 3 mo for respiratory distress, assessed as related to a prior condition. The median absolute lymphocyte count was 59.7 K/µl (range: 0.8-294) at baseline peaking at 100 K/µl (range: 2-385) at week 4. 27 pts were evaluable for response, having reached the first evaluation time point of 8 weeks. Of these 27, the ORR was 81% with 9 (33%) PR and 13 (48%) PR with lymphocytosis. Splenomegaly has responded in 88% of 17 evaluable pts and hepatomegaly in 75% of 4 evaluable pts. The most frequent treatment emergent adverse events (TEAE) (% all Grade/% Grade ≥3) were rash (27/3), URI (16/0), constipation (14/0), cough (14/0), nausea (11/0), pyrexia (11/0), arthralgia (8/0), back pain (8/0), diarrhea (8/3), and pneumonia (8/5). Pneumonitis was observed in 2 pts (5%), Gr ≥3 in 1(3%). Gr ≥3 treatment emergent lab abnormalities included transaminase elevation (8%), anemia (5%), and neutropenia (20%); there was no Gr ≥3 thrombocytopenia. One pt had a TEAE leading to dose reduction. CONCLUSIONS: IDELA has substantial single agent activity in treatment-naïve pts with CLL or SLL. Early lymphocytosis is observed with monotherapy in this population, as opposed to an attenuation of lymphocytosis seen in the earlier cohort treated with IDELA plus weekly rituximab. IDELA was well tolerated and had a manageable safety profile in this preliminary analysis. Disclosures Zelenetz: Gilead Sciences: Research Funding. Off Label Use: Zydelig is a kinase inhibitor indicated for the treatment of patients with: 1) Relapsed chronic lymphocytic leukemia (CLL), in combination with rituximab, in patients for whom rituximab alone would be considered appropriate therapy due to other co-morbidities; 2) Relapsed follicular B-cell non-Hodgkin lymphoma (FL) in patients who have received at least two prior systemic therapies; and 3) Relapsed small lymphocytic lymphoma (SLL) in patients who have received at least two prior systemic therapies.. Lamanna:Gilead Sciences: Research Funding. Kipps:Gilead Sciences: Research Funding. Coutre:Gilead Sciences: Research Funding. O'Brien:Gilead Sciences: Research Funding. Aiello:Gilead Sciences: Employment, Equity Ownership. Cho:Gilead Sciences: Employment, Equity Ownership. Dubowy:Gilead Sciences: Employment, Equity Ownership. Flinn:Gilead Sciences: Research Funding.

scholarly journals Targeted Alpha Therapy with 212Pb-NNV003 for the Treatment of CD37 Positive B-Cell Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4422-4422
Author(s):  
Amal Saidi ◽  
Astri Maaland ◽  
Julien Torgue ◽  
Helen Heyerdahl ◽  
Jostein Dahle
Keyword(s):  
B Cell ◽  
Intravenous Dose ◽  
Lymphocytic Leukemia ◽  
Single Intravenous Dose ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Non Hodgkin Lymphoma ◽  
Equity Ownership ◽  
Daudi Cells ◽  
Alpha Therapy

Abstract Background Each year more than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US. The standard of care is immuno-chemotherapy with alkylating agents in combination with an anti-CD20 monoclonal antibody (mAb). While immuno-chemotherapy regimens are initially effective in inducing responses, most patients inevitably relapse and the same therapies show decreasing efficacy with repeated administration. In recent years, CD37 has been considered as a target for B-cell malignancies. CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Therapies targeting CD37 expressing cells may become a useful alternative to CD20 targeting agents. Alpha-emitting radionuclides have demonstrated good potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm). The absorbed energy cause irreparable DNA double-strand breaks and localized cytotoxicity while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-emitting radioisotope 212Pb. Materials and Methods Dose-dependent efficacy and tolerability of a single-dose 212Pb-NNV003 treatment was evaluated using escalating activity levels in human disseminated models of Burkitt's lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. A study of the tolerability of 212Pb-NNV003 was performed in R2G2 mice without tumor prior to the efficacy studies. Results 212Pb-NNV003 displays a favorable toxicity profile in tumor-free mice single intravenous dose injections up to 15 µCi showing 100 % survival 4 weeks post-injection. No acute hematological toxicity was observed and animals who received 5, 10 or 15 μCi doses of 212Pb-NNV003 presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered by 20 days after injection. A single intravenous dose of 10, 15 or 20 µCi of 212Pb-NNV003 lead to 70 %, 90 % and 100 % of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection (Figure 1). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively. A single intravenous dose of 2.5, 5 and 7.5 µCi 212Pb-NNV003 lead to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection (Figure 2). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively. Conclusion The results of preclinical studies suggest that TAT using 212Pb-NNV003 is a safe and effective method for the treatment of CD37 positive CLL and NHL. Disclosures Saidi: Orano Med: Employment. Maaland:Nordic Nanovector: Employment. Torgue:Orano Med: Employment, Membership on an entity's Board of Directors or advisory committees. Heyerdahl:Nordic Nanovector: Employment, Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties.

Correlation Between Serum Ofatumumab Concentrations, Baseline Patient Characteristics and Clinical Outcomes in Patients with Fludarabine-Refractory Chronic Lymphocytic Leukemia (CLL) Treated with Single-Agent Ofatumumab.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3433-3433 ◽  
Author(s):  
Anders Österborg ◽  
Birgitte Biilmann Ronn ◽  
Roxanne C Jewell ◽  
Thomas J Kipps ◽  
Jiri Mayer ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Pivotal Trial ◽  
Employment Equity ◽  
Baseline Factors ◽  
Equity Ownership ◽  
Univariate Analyses

Abstract Abstract 3433 Poster Board III-321 Introduction Monoclonal antibody (mAb) therapies represent an important clinical advance for patients (pts) with CLL, yet little is known about the pharmacokinetics (PK) and pharmacodynamics of mAb therapy in these pts. Ofatumumab is a human mAb targeting a membrane-proximal small-loop epitope on CD20 and mediates efficient complement-dependent cytotoxicity in vitro. Ofatumumab is being evaluated in a pivotal trial for pts with fludarabine-refractory CLL also refractory to alemtuzumab (FA-ref; n=59) or less suitable for alemtuzumab due to bulky (>5 cm) lymphadenopathy (BF-ref; n=79). Overall response rate (ORR; primary endpoint) was 58% in FA-ref and 47% in BF-ref pts at an interim analysis; median progression-free survival (PFS) was 5.7 and 5.9 months, respectively. We evaluated relationships between baseline factors and ofatumumab PK and between PK parameters and treatment outcomes from the pivotal trial. Patients and Methods Pts received 8 weekly infusions of ofatumumab followed by 4 monthly infusions (Dose 1, 300 mg; Doses 2-12, 2000 mg). Response (1996 NCI-WG criteria) was assessed by an Independent Review Committee over 24 weeks of therapy. Blood samples for PK analysis were collected at Dose 1, Dose 8 (last weekly dose), and Dose 12 (last monthly dose). A population PK model was employed that included data from a previous study (Coiffier et al, Blood 2008;111:1094). For Dose 1, Cmax was determined; for Doses 8 and 12, Cmax, Cmin, AUC, clearance (CL), volume of distribution (Vss) and t½ were determined. The relationships between baseline pt characteristics and disease factors and PK parameters were evaluated by multivariate regression analysis. Associations between PK and ORR or PFS were explored using univariate and multivariate logistic regression or Cox regression analyses. Results 90% of the 154 pts received 8 weekly infusions of ofatumumab and 55% received all 12 infusions. PK parameters were similar between FA-ref and BF-ref pts. In multivariate analysis, higher Cmax at Dose 1 was significantly associated with lower % of bone marrow infiltration (p<0.001), lower Rai stage (p=0.002), lower lymphocyte count (p=0.006), smaller BSA (p<0.001) and lower total bilirubin (p=0.013). The majority of responders and non-responders were still receiving treatment at Dose 8; thus, this dose represents an informative time point for analysis. Baseline factors that influenced PK parameters at Dose 8 are shown in the table. Based on univariate analyses, higher Cmax and Cmin at Dose 8 were associated with increased likelihood of response (Table); in addition, significantly higher Cmax, Cmin and AUC were observed in responders versus non-responders (p<0.05 for each; data not shown). Higher Cmax, AUC and Cmin and lower CL at both Doses 8 and 12 were significantly correlated with longer PFS (p<0.05 for each). Based on exploratory multivariate analyses, PK parameters were not independent predictors of ORR or PFS. Conclusions These data demonstrate that baseline factors reflecting disease burden significantly influenced ofatumumab PK. Additionally, higher serum concentrations of ofatumumab at Doses 8 and 12 were associated with positive clinical outcomes in univariate analyses. The pivotal study is ongoing, and further analyses of associations between disease-related factors, PK and treatment response will be performed at study completion. Such analyses will help us to better understand the response kinetics of biological therapy and to optimize the dose. Disclosures Österborg: Celgene: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding. Off Label Use: Ofatumumab is an investigational anti-CD20 monoclonal antibody, currently under development for the treatment of B-cell malignancies (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia and follicular lymphoma) as well as autoimmune diseases (rheumatoid arthritis and multiple sclerosis). Biilmann Ronn:Genmab: Employment. Jewell:GlaxoSmithKline: Employment, Equity Ownership. Kipps:Physicians' Educational Resource, Educational Concepts: Speakers Bureau; Genmab, Abbott Industries, Celgene, Biogen Idec, Cephalon, sanofi-aventis, Medimmune, Memgem, Genentech: Research Funding. Mayer:GlaxoSmithKline: Consultancy. Stilgenbauer:GlaxoSmithKline, Genmab: Consultancy, Honoraria, Research Funding. Hellmann:Novartis, BMS: Consultancy, Honoraria. Robak:GlaxoSmithKline, Roche: Advisory Board, Research Funding. Furman:GlaxoSmithKline: Consultancy, Speakers Bureau. Hillmen:GlaxoSmithKline: Consultancy, Honoraria for Advisory Boards. Trneny:GlaxoSmithKline: Honoraria. Padmanabhan:GlaxoSmithKline: Consultancy, Honoraria; Celgene, Genentech: Consultancy. Kozak:GlaxoSmithKline, Amgen: Consultancy. Chan:GlaxoSmithKline: Employment. Arning:GlaxoSmithKline: Employment, Equity Ownership. Losic:Genmab: Employment, Stock Ownership. Davis:GlaxoSmithKline: Employment, Stock ownership. Wilms:Genmab: Employment, Equity Ownership. Russell:Genmab: Employment, Equity Ownership. Wierda:Genmab, GlaxoSmithKline: Honoraria, Research Funding.

Target Engagement, Pathway Inhibition, and Efficacy Of The Bruton’s Tyrosine Kinase (Btk) Inhibitor CC-292

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4169-4169 ◽  
Author(s):  
Daniel W Pierce ◽  
Sabine Ponader ◽  
Kumudha Balakrishnan ◽  
Varsha Gandhi ◽  
William G. Wierda ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Phase 1 ◽  
Clinical Activity ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Post Dose ◽  
Equity Ownership

Abstract Introduction The B-cell receptor (BCR) and its downstream effectors have emerged as important therapeutic targets in B-cell malignancies. CC-292 is a novel, potent, covalent, and highly selective inhibitor of Btk (IC50apparent of 0.5 nM, kinact/KI ratio of 7.69 × 104 M-1s-1), that does not appreciably inhibit other kinases involved in BCR signaling (eg, IC50 Lyn kinase, 4401 nM) (Evans et al., J Pharmacol Exp Ther. 2013). Here, we report preclinical characterization and clinical data in CLL from a single-agent phase 1 dose-escalation trial of CC-292 in B-cell malignancies, with a focus on how target engagement and downstream events correlate with clinical activity. Results Pharmacodynamic effects of Btk inhibition by CC-292 can be monitored by occupancy of the Btk catalytic site, Btk autophosphorylation on Y223, and downstream phosphorylation of Plc-γ2 and Erk. We developed a sensitive (10 pg/mL lower limit of quantification) and quantitative assay to measure covalent binding of CC-292 to Btk (Evans et al., J Pharmacol Exp Ther. 2013), as well as Western and novel phos-flow assays to probe downstream signal transduction. These methods showed that CC-292 treatment blocks Btk autophosphorylation and downstream pathway activation in both tumor cells and human peripheral blood mononuclear cells (PBMCs). The extent of CC-292 binding to Btk correlated with its in vitro and in vivo effects. The occupancy assay demonstrated that CC-292 effectively targets Btk in tumor cell lines, PBMCs, spleen, and lymph nodes (LNs) in animal models, and in PBMC and lymph node samples from clinical trial subjects. In rats and non-human primates treated with CC-292, Btk occupancy in spleen and LNs was dose-dependent. Measured occupancy in rat spleen and axillary, mesenteric, and superficial cervical LNs was 94%, 92%, 90%, and 76% respectively, 4 hours (hrs) after a single 30-mg/kg dose. Interim data from the phase 1 CLL trial showed that PBMC Btk was completely occupied in the majority of subjects 4 hrs post-dose with both QD and BID dosing. Twenty-four hrs post-dose at 750 and 1000 mg QD, CC-292 exhibited 83% ± 17% Btk occupancy, whereas with BID dosing at 375 and 500 mg, occupancy was 94% ± 16% at the corresponding time point (12 hrs after the second dose). Thus, while both schedules achieved extensive and sustained Btk occupancy, residual free Btk levels were lower with the BID schedule, offering a rationale for an early trend towards more rapid nodal responses, lymphocytosis, and partial responses on the BID schedule observed to date in the phase 1 study. In the 10 clinical LN biopsies tested to date, no measurable levels of unoccupied Btk have been detected, although Btk protein was present as determined by Western blotting, showing that CC-292 was able to penetrate LNs and inhibit Btk in human subjects as it did in preclinical models. For monitoring downstream signal transduction, we developed reagents and assays including a phos-flow assay based on a novel rabbit monoclonal antibody to detect Btk pY223 levels in PBMC subsets. CC-292 effectively inhibited constitutive and induced phosphorylation of Btk and Plc-γ2 at low nanomolar concentrations. CC-292 also inhibited BCR activation and nurse-like cell–supported survival of CLL cells. Furthermore, CC-292 reduced CLL cell migration and actin polymerization in response to chemokines (CXCL12, CXCL13) and inhibited secretion of the chemokines CCL3 and CCL4 by CLL cells. These chemokines are essential for migration and retention of normal and neoplastic B cells in the marrow and secondary lymphatic tissues. Consistent with this preclinical data, CC-292 treatment resulted in rapid reductions in circulating CCL3 and CCL4 levels. In subjects treated at the 750 mg QD, 1000 mg QD, 375 mg BID, and 500 mg BID dose levels, plasma CCL3 was reduced from 99 ± 16 pg/ml before treatment to 28 ± 5 pg/ml (N = 48, mean ± SEM) at 24 hrs after the first dose, while CCL4 was reduced from 235 ± 59 pg/ml to 74 ± 16 pg/ml (N = 51). Conclusions These data demonstrate that CC-292 achieves significant and durable occupancy of Btk in vitro and in vivo, inhibits Btk-mediated downstream signaling events and chemokine production, and that these preclinical activities have translated into the clinic. Taken together, these results argue that Btk inhibition is necessary and sufficient for clinical activity in CLL. These emerging data support continued development of CC-292 for the treatment of B-cell malignancies. Disclosures: Pierce: Celgene: Employment, Equity Ownership. O'Brien:Genentech: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; CLL Global Research Foundation: Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Consultancy, Research Funding; Talon: Consultancy, Research Funding; Teva/Cephalon: Consultancy. Heise:Celgene: Employment, Equity Ownership. Nacht:Celgene: Employment, Equity Ownership. Aslanian:Celgene: Employment, Equity Ownership. Liu:Celgene: Employment, Equity Ownership. Hong:Celgene: Employment, Equity Ownership. Wu:Celgene: Employment, Equity Ownership. Zavodovskaya:Celgene: Employment, Equity Ownership. Marine:Celgene: Employment, Equity Ownership. Barnett:Celgene: Employment, Equity Ownership. Nava-Parada:Celgene: Employment, Equity Ownership. Mei:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Burger:Pharmacyclics: Research Funding; Gilead: Research Funding. Singh:Celgene: Employment, Equity Ownership.

scholarly journals Triple Degradation of BTK, IKZF1 and IKZF3 in B-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 263-263
Author(s):  
Sara Morrow ◽  
Dennis Dobrovolsky ◽  
Eric Wang ◽  
Radosław P. Nowak ◽  
Katherine Donovan ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Saturated Hydrocarbon ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Equity Ownership

Abstract Bruton's Tyrosine Kinase (BTK), a TEC-family non-receptor tyrosine kinase, plays a critical role in B-cell development and function. Targeting of BTK with covalent inhibitors like ibrutinib has become a standard approach for many B-cell malignancies, including chronic lymphocytic leukemia (CLL), Waldenstrom macroglobulinemia, mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). Yet, many patients demonstrate intrinsic or acquired resistance to covalent BTK inhibition. The prognosis of patients who relapse after ibrutinib treatment for MCL or CLL is dismal, highlighting the urgent need for new approaches that overcome resistance to current BTK inhibitors. We hypothesized that degradation of BTK could be a better alternative to inhibition alone, as it would both: 1) maintain efficacy in cells harboring the ibrutinib-resistant BTK C418S mutation and 2) target non-catalytic functions of BTK. To address this, we turned to a small molecule-mediated protein degradation platform that utilizes an E3 ligase-targeting moiety linked to the ligand of a target of interest, so that the target can be marked for ubiquitination and subsequent proteasomal degradation. We previously showed that BTK is one of the most robustly degraded kinase targets using a nonspecific kinase inhibitor linked to a imide-based core followed by agnostic proteomics. We synthesized highly potent and selective degraders of BTK using imides as a base. To do so, the parent BTK inhibitor CGI1746 was linked to thalidomide using either polyethylene glycol (DD-03-007) linkers or saturated hydrocarbon chain (DD-03-171) linkers. After verifying that these degraders induce dimerization of BTK and CRBN and penetrate cells, we explored the pharmacological effects in vitro. Both DD-03-007 and DD-03-171 reduced BTK levels at concentrations as low as 40 nM within 4h of treatment. Furthermore, cellular BTK levels remained low for 24h after washout, showing that these degraders are capable of sustaining depletion of BTK for an extended period of time (Figure). DD-03-007 and DD-03-171 are both ligase and proteasome-dependent, as shown by co-treatment with bortezomib or MLN-4924 as well as competition experiments with lenalidomide or CGI-1746. Moreover, the degraders exhibited strong synergy with the HCK inhibitor A419259, suggesting that it would be possible to recapitulate ibrutinib's previously reported polypharmacology with an HCK inhibitor. We overexpressed wild-type or C481S BTK in TMD-8 cells and ran an antiproliferation assay, which showed that DD-03-007 overcame ibrutinib resistance associated with BTK C481S mutation. Next, we explored the effects of the degraders in MCL specifically. Proteomic analysis showed that DD-03-171 is a triple-degrader, as it degrades BTK but retains degradation activity on IKZF1 and IKZF3. Finally, we performed In vivo efficacy studies in cell line and patient-derived xenograft (PDX) models of MCL. The latter was obtained from a patient who had progressed on ibrutinib. In both models, DD-03-171 caused a significant reduction in tumor burden at an early timepoint. DD-03-171 also markedly extended survival compared to treatment with ibrutinib or lenalidomide alone (Figure). In conclusion, we developed highly potent and selective BTK degraders with activity in vivo against human MCL that induce the degradation of multiple factors essential for MCL survival. Figure. Figure. Disclosures Treon: Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Johnson & Johnson: Consultancy; BMS: Research Funding. Weinstock:Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership.

scholarly journals Predicting Cerdulatinib Therapeutic Concentrations in Patients with Relapsed/Refractory B Cell Malignancies Using Preclinical Models and Clinical PK/PD and Tumor Response Correlates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3706-3706 ◽  
Author(s):  
Greg P. Coffey ◽  
Jiajia Feng ◽  
Glenn Michelson ◽  
Janet Leads ◽  
John T. Curnutte ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Whole Blood ◽  
Research Funding ◽  
Tumor Response ◽  
Plasma Concentrations ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Oral Dosing ◽  
Equity Ownership

Abstract Background: Preclinical data and early clinical studies demonstrate a reliance on B cell antigen receptor (BCR) signaling and/or cytokine-induced JAK/STAT signaling for survival of subsets of B cell malignancies. Increased serum concentrations of several cytokines are observed in CLL and NHL and predict a more aggressive disease progression (Yan, X. J., et al, 2011; Mahadevan, D., et al, 2009; el Far. M., et al, 2004; Lai, R., et al, 2002; Fayed, L., et al, 2001). The source of these cytokines may be derived from the tumor itself in an autocrine stimulation fashion, or from non-tumor leukocytes which have mounted an ineffective immune response within the tumor microenvironment. Experimentally, IL4 has been shown to promote the survival of CLL B cells in culture and protect them from death by treatment with fludarabine and chlorambucil (Steele, A.J., et al, 2010). The mechanism underlying this survival support appears to be cytokine-induced up-regulation of BCL2 family members (Castejon, R., et al, 1999; Jewell, A.P., et al, 1994; Panayiotidis, P., et al, 1993; Dancescu, M., et al, 1992). Combined SYK/JAK inhibition may therefore represent a powerful strategy to treat B cell malignancies. Cerdulatinib is a reversible ATP competitive inhibitor of SYK, JAK1, JAK3, and TYK2 that is currently in clinical development for the treatment of relapsed/refractory B cell lymphoma and leukemia. We review here cerdulatinib preclinical and clinical pharmacodynamic/pharmacokinetic relationships to tumor response in treated patients. Methods: Inhibition of SYK- and JAK-dependent and independent signaling was determined by intracellular phospho-flow following stimulation of human whole blood with various agonists against BCR and cytokine receptors. Anti-tumor activities of cerdulatinib and other selective inhibitors of BCR and JAK/STAT pathways were evaluated in fourteen DLBCL cell lines by CellTiter Glo, caspase 3 induction, and cell cycle arrest. In vivo anti-inflammatory activity of cerdulatinib was determined using the rat collagen induced arthritis model. The effective concentrations pre-clinically were compared to cerdulatinib-induced SYK and JAK inhibition, anti-inflammatory activity, and anti-tumor activity following oral dosing in relapsed/refractory CLL and NHL patients. Results: Cerdulatinib demonstrated broad anti-tumor activity in DLBCL cell lines, relative to other targeted agents. In a panel of 14 cell lines representing both ABC and GCB subtypes, 9 underwent apoptosis and an additional 2 underwent cell cycle arrest. Cooperative effects of SYK and JAK inhibition were observed in 4 of the cell lines, whereas 3 cell lines were sensitive to SYK but not JAK inhibition, and 1 cell line was sensitive to JAK but not SYK inhibition. Three of the 14 cell lines were resistant to cerdulatinib. Apoptosis was induced in the majority of DLBCL cell lines at 2 µM (achieved at clinical doses ≥40 mg). In healthy whole blood ex vivo spiking experiments, cerdulatinib selectively inhibited BCR/SYK and cytokine (IL2, IL4, IL6) JAK/STAT signaling with IC50's ranging from 0.2-0.9 µM (achieved at clinical doses of 15 mg-40 mg). Inflammation and joint destruction in the rat collagen-induced arthritis model was arrested at 0.3 µM average plasma concentration (achieved at clinical doses ≥30 mg). In cancer patients receiving cerdulatinib once daily, plasma concentrations above 2 µM have been safely achieved, while maintaining steady-state Cmin of ~1 µM. Following oral dosing in patients, SYK and JAK pathways were inhibited with similar potency as was observed in healthy volunteer ex vivo spiking experiments with >90% inhibition of SYK and JAK safely achieved for multiple cycles of therapy. Tumor response as measured by CT scan strongly correlated with percent inhibition of SYK and JAK signaling in patient-derived whole blood, and percent inhibition of serum markers of inflammation (e.g., β2M, CRP, IL10, VCAM1, sTNFR, CCL3). Conclusion: Once daily oral dosing of cerdulatinib at 40-65 mg achieves steady-state plasma concentrations in the 1-2µM range (trough to peak), which is sufficient to block SYK/JAK signaling, inflammatory mechanisms, and affect tumor viability both in DLBCL cell lines and in patients. We conclude that these exposure levels are sufficient to begin testing the clinical potential of cerdulatinib. We will begin enrolling phase Ib/IIa expansion cohorts this year. Disclosures Coffey: Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Feng:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Michelson:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Leads:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Curnutte:Portola Pharmaceuticals, Inc.: Employment; 3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy. Pandey:Portola Pharmaceuticals Inc: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment.

scholarly journals Genetic or CD40L-Mediated Loss of Iκbα Is Associated with Resistance to the Dual SYK/JAK Inhibitor Cerdulatinib in DLBCL Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2768-2768
Author(s):  
Greg P Coffey ◽  
Jiajia Feng ◽  
Sabah Kadri ◽  
Y. Lynn Wang ◽  
Pin Lu ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Stop Codon ◽  
Family Members ◽  
B Cell Malignancies ◽  
Employment Equity ◽  
Equity Ownership ◽  
External Stimuli

Abstract Abnormal upregulation of NFκB activity is observed in a variety of B cell malignancies, resulting in proliferative and survival signals that contribute to tumor progression. Under normal resting conditions, NFκB is negatively regulated principally via its physical association with IκB (inhibitor of NFκB) family members, thereby inhibiting nuclear transport or access to DNA. In B cells, NFκB is typically activated via various external stimuli (e.g., ligation of the B cell antigen receptor (BCR), toll-like receptors, cytokine receptors, CD40), leading to IκB kinase complex-dependent phosphorylation of IκB members and targeting them for ubiquitination and degradation. In some cases, the need for external stimuli is diminished or completely circumvented by mutations to critical regulators of NFκB, as has been described in the context of activating mutations to CD79A/B, MYD88, and CARD11, as well as inactivation of negative regulators such as A20 and IκB family members (reviewed by Staudt, 2010). Each of these mutations has been observed clinically in patients with B cell malignancies (Wilson et al, 2012; Norenberg et al, 2015; Mansouri et al, 2015), and can impact the anti-tumor activity of selective BCR pathway inhibition (Davis et al, 2010; Wilson et al, 2012) in part via induction of autocrine cytokine stimulation leading to JAK/STAT-dependent up-regulation of MCL1 (Lam et al, 2008). We previously reported that cerdulatinib, a small molecule kinase inhibitor that dually targets SYK and the JAK family members JAK1, JAK3, and TYK2, maintained anti-tumor activity in DLBCL cell lines bearing mutations to CARD11, MYD88, and A20 (Ma et al, 2015). The majority of DLBCL cell lines exhibit various degrees of reliance on SYK and JAK signaling for survival, however in a screen of 15 DLBCL cell lines we found 3 that were completely resistant to cerdulatinib and are described here. In one of the cerdulatinib-resistant cell lines, RCK8, next generation sequencing revealed bi-allelic inactivation of the IκBα gene. One allele carries a frameshift mutation in exon 1 resulting in the generation of a premature stop codon, and the second allele is a nonsense mutation in exon 3 at Gln154, also leading to a stop codon. In accord with a previous report (Kalaitzidis et al, 2002), the cell line lacks IκBα protein expression. We therefore proceeded to explore the possibility that loss of IκBα was responsible for resistance to cerdulatinib. Consistent with the loss of IκBα, the RCK8 cell line exhibited enhanced basal NFκB activity. Genetic re-introduction of wild type IκBα led to rapid suppression of NFκB, and ultimately cell cycle arrest and cell death, indicating that the cell line was dependent upon loss of this gene for survival. Associated with suppression of NFκB was decreased phosphorylation of cellular pAKT S473 and pERK Y202, but not of pSTAT3 Y705. We then attempted to knock down IκBα in cerdulatinib-sensitive cell lines using siRNA to determine if resistance to SYK/JAK inhibition could be generated. None of the DLBCL cell lines tested (n=4) could tolerate IκBα gene knock down, suggesting an additional mutation in RCK8 enables survival under conditions of homozygous loss of IκBα. Ligation of CD40 leads to a transient down-regulation of IκBα at the protein level (Oeckinghaus and Ghosh, 2009). We therefore examined the effect of CD40L on multiple DLBCL cell lines and found that IκBα was maximally suppressed within 30-60 minutes post CD40 stimulation, returning to pre-treatment levels by 2-4 hours. In contrast, the impact on NFκB activation was much longer, and 5 of 7 cerdulatinib-sensitive cell lines tested were made resistant by incubation with CD40L. Associated with this resistance was not only induction of NFκB, but also pERK Y204, pAKT S473, and pSTAT3 Y705. Interestingly, whereas the CD40L-induced NFκB activation was not inhibited by cerdulatinib, the other signaling events were, despite the generation of resistance. Loss of IkB family members has been described in the context of Hodgkin's lymphoma, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia (Cabannes et al, 1999; Norenberg et al, 2015; Mansouri et al, 2015). Here we demonstrate that loss of IκBα in multiple DLBCL cell lines generates resistance to cerdulatinib. We will be exploring the clinical relevance of these in vitro observations in cell lines as part of an ongoing phase II trial of cerdulatinib in patients with relapsed/refractory B cell malignancies. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Feng:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Wang:Portola Pharmaceuticals: Honoraria, Research Funding. Michelson:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Curnutte:3-V Biosciences: Equity Ownership; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding; Sea Lane Biotechnologies: Consultancy. Conley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Focal Adhesion Kinase Inhibitors Reverse the Stromal Adhesion Phenotype of Ikaros-Mutant B-ALL, Induce Apopotosis, and Synergize with ABL1 Tyrosine Kinase Inhibitors: A New Paradigm for Pathogenesis and Therapy of High-Risk B-ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 285-285 ◽  
Author(s):  
Ila Joshi ◽  
Nilamani Jena ◽  
Toshimi Yoshida ◽  
Leto Paraskevopoulou ◽  
Zhihong Zhang ◽  
...  
Keyword(s):  
High Risk ◽  
B Cell ◽  
Focal Adhesion ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Dominant Negative ◽  
Employment Equity ◽  
Equity Ownership ◽  
B Lymphoid ◽  
Inhibitor Treatment

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of precursor B-lymphocytes affecting both children and adults. Deletions and dominant-negative mutations in IKZF1, the gene encoding the Ikaros transcription factor, are found in ~85% of Ph+ B-ALL and in some cases of Ph– B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph– or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and imatinib have been added to chemotherapy regimens for Ph+ B-ALL, over half of these patients will still relapse, which correlates with residual disease burden in the bone marrow (BM) following induction therapy. Hence, new therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph– B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) where the recombined allele is similar to the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated recently that Ikaros DNA-binding function is required in the B-lymphoid lineage for transition from the large to small pre-B cell stage of differentiation, and that arrest at this stage of development can give rise to B-ALL (Joshi et al., Nat. Immunol. 2014;15:294). The survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK, downstream of integrins and growth factor receptors, which plays important roles in cancer stem cell biology, the tumor microenvironment and tumorigenesis. VS-4718 and VS-6063 (defactinib) are potent, orally bioavailable FAK inhibitors that inhibit tumor growth and metastasis in preclinical models, and are currently under evaluation in clinical trials in patients with various solid tumors. VS-6063 has demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I trials (ASCO, 2014). Here, we show that BCR-ABL1 cooperates with Ikzf1 mutation to accelerate B-leukemogenesis in mice. BCR-ABL1+ Ikaros-mutant B-ALLs exhibit stroma-mediated resistance to ABL1 TKIs, while the FAK inhibitors VS-4718 and VS-6063 are effective in blocking stromal adhesion and inducing apoptosis in both mouse and human Ikaros-mutant B-ALL samples. To test whether dysregulation of TK signaling cooperates with Ikzf1 mutation in the pathogenesis of high-risk B-ALL, we isolated BM B-lymphoid progenitor cells from wild-type (WT), IkE5fl/+ CD2-Cre, and IkE5fl/fl CD2-Cre donors, transduced them with BCR-ABL1 retrovirus and transplanted the cells into recipient mice. We observed a dramatic acceleration of precursor B-lymphoid leukemia induced by BCR-ABL1 in IkE5Δ/+ and particularly in IkE5Δ/Δ donor cells that correlated with a striking (~30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Relative to Ikzf1 WT BCR-ABL1+ leukemic cells, Ikzf1-mutant BCR-ABL1+ blasts showed significant resistance to imatinib and dasatinib that was dependent on the presence of OP9 stroma. The effect of FAK inhibition, using the FAK inhibitors VS-4718, VS-6062, and VS-6063 (Verastem), was first tested on murine B-ALL cells (genotypes Ikzf1 mutant, Ikzf1 mutant BCR-ABL1+, and Ikzf1 WT BCR-ABL1+) grown on OP9 stroma. FAK inhibitor treatment abolished stromal adhesion of Ikzf1-mutant B-ALL and induced apoptosis in non-adherent cells, but had little effect on Ikzf1 WT B-ALL cells. VS-4718 and VS-6063 were each synergistic with dasatinib in reducing the viability of Ikzf1-mutant BCR-ABL1+ B-ALL cells cultured on OP9 stroma. For primary human B-ALL samples grown on OP9 stroma, IKZF1-mutant cells were also more sensitive to FAK inhibitor treatment than WT IKZF1 WT B-ALL, with or without BCR-ABL1 expression. Collectively, these observations suggest a new model to explain the pathogenesis of high-risk B-ALL and its resistance to therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM LSCs. Inhibition of FAK signaling in Ph+ or Ph­–IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. Therefore, FAK inhibitors warrant further investigation for the treatment of high-risk IKZF1-mutant B-ALL patients. Disclosures Joshi: Verastem: Research Funding. Yoshida:Verastem, Inc.: Research Funding. Paraskevopoulou:Verastem, Inc.: Research Funding. Zhang:Verastem, Inc.: Research Funding. Krause:Glycomimetics. Inc.: Research Funding. Shapiro:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Georgopoulos:Verastem, Inc.: Research Funding.

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