CC-122 Exhibits Greater Preclinical Activity in Mantle Cell Lymphoma Than Lenalidomide through a Combination of Direct Cell-Autonomous and Increased Antibody Dependent Cell-Mediated Cytotoxicity

Abstract Introduction: CC-122 and lenalidomide (len) bind the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN) resulting in degradation of the transcription factor Aiolos, leading to direct anti-lymphoma activity and T and NK cell activation. CC-122 degrades Aiolos at a faster rate and to a greater extent compared to len. Len is currently indicated for the treatment of relapsed/refractory (R/R) mantle cell lymphoma (MCL) in the United States and European Union. CC-122 is in development for DLBCL, FL and CLL in combination with the anti-CD20 monoclonal antibodies, rituximab (Rtx) and obinutuzumab (GA101). We compared the ability of len and CC-122 to effect cell autonomous activity and enhance antibody dependent cell-mediated cytotoxicity (ADCC) in pre-clinical models of MCL. Methods: Proliferation was measured by 3H labeling. Apoptosis was measured by Annexin V/ToPro-3 flow cytometry. ADCC was measured by a 4 hour co-incubation of Rtx or GA101 labeled cells with stimulated PBMC treated with DMSO, len or CC-122 for 3 days followed by apoptosis assay. Inducible shRNA targeting luciferase or Aiolos were activated with 10 ng/ml doxycycline for 7 days followed by apoptosis assay. Results: Lenalidomide treatment (10μΜ) for 5 days resulted in a 31% and 49% decrease in proliferation of two of the six MCL cell lines investigated, whereas CC-122 treatment (1.25μΜ) decreased proliferation in four MCL cell lines by 37-81%. There was no increase in Annexin V and ToPro-3 staining in MCL cells treated with len (0.1-10μΜ) for 7 days. By contrast, CC-122 treatment (0.1-10μΜ) reduced viability in four of six cell lines examined by 32-95%. Examination of the biochemical activity of each drug in Mino and Rec-1 cells demonstrated CC-122 (0.1-10μΜ) induced rapid Aiolos degradation at 6 hours (33-88% and 38-85%, respectively) compared to 10μΜ len (27% and 25%, respectively). In Jeko-1 cells, two distinct doxycycline inducible shRNA targeting Aiolos led to 56-97% decreased Aiolos protein expression. Furthermore, shRNA targeting Aiolos led to a 2- to 3-fold increase in apoptosis relative to shLuciferase. In ADCC assays of Z138 and Granta-519 coated with Rtx (1μg/ml) or GA101 (1μg/ml), CC-122 (10-100nM) was more potent than len (0.1-1μΜ). CC-122 treatment of PBMC increased Rtx labeled Z138 and Granta-519 apoptosis (39-59% and 36-48%, respectively) compared to len (24-35% and 33-40%, respectively), versus vehicle controls (13% and 13%, respectively). Additionally, CC-122 treatment increased GA101 mediated ADCC of Z138 and Granta-519 (60-76% and 59-67%, respectively) compared to len (49-61% and 55-63%, respectively), versus vehicle controls (35% and 41%, respectively). Conclusions: CC-122 treatment of MCL cells resulted in considerable cell autonomous activity in in vitro proliferation and viability assays compared to len. Specific targeting of Aiolos, a substrate which is rapidly degraded by CC-122, through inducible shRNA results in increased levels of apoptosis compared to shLuciferase controls. Additionally, the combination of CC-122 with either Rtx or GA101 in in vitro co-culture ADCC assays resulted in greater apoptosis than len combined with either antibody. The combination of both enhanced cell-autonomous activity and α-CD20 mediated ADCC provide a rational combination strategy for CC-122 development in R/R MCL. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Chiu:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership.

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Lenalidomide Exhibits Activity in Mantle Cell Lymphoma through Increased NK Cell Mediated Cytotoxicity

Blood ◽

10.1182/blood.v126.23.821.821 ◽

2015 ◽

Vol 126 (23) ◽

pp. 821-821 ◽

Cited By ~ 1

Author(s):

Patrick Hagner ◽

Hsiling Chiu ◽

Maria Ortiz-Estevez ◽

Tsvetan Biyukov ◽

Carrie Brachman ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Employment Equity ◽

Mantle Cell ◽

Apoptosis Assay ◽

Equity Ownership

Abstract Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170). The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models. Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay. Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm. Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit. The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours. Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells. Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014). Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro-apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B. Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL. Disclosures Hagner: Celgene: Employment, Equity Ownership. Chiu:Celgene: Employment, Equity Ownership. Ortiz-Estevez:Celgene: Employment, Equity Ownership. Biyukov:Celgene: Employment, Equity Ownership. Brachman:Celgene: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Milpied:Celgene: Honoraria, Research Funding. Musto:Sandoz: Consultancy; Celgene: Honoraria; Roche: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Novartis: Honoraria; Janssen: Honoraria; Mundipharma: Honoraria. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Carmichael:Celgene: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Gandhi:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

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Forodesine (Immucillin H, BCX-1777) Shows Activity on Mantle Cell Lymphoma Primary Cells

Blood ◽

10.1182/blood.v112.11.4997.4997 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4997-4997

Author(s):

Andrea Rinaldi ◽

Emilia Ceresa ◽

Davide Rossi ◽

Gianluca Gaidano ◽

Shanta Bantia ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

B Cell Malignancies ◽

New Therapeutic Approaches ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) represents a subtype of B-cell lymphoma associated with a very unfavourable clinical outcome. Currently no therapy can be considered as standard, and new therapeutic approaches are needed. Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP), whose major role is to catalyze the cleavage of inosine, deoxyinosine guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. In the presence of deoxycytidine kinase, PNP inhibition leads to an increase in the concentration of dGuo triphosphate (dGTP), followed by inhibition of DNA synthesis and cell death by apoptosis. When combined with dGuo, forodesine has been shown to have in vitro cytotoxic activity on T-cell (T-ALL, T-PLL) and on B-cell malignancies (CLL, B-ALL), and Phase I/II trials are on going in CLL and CTCL patients. Here, we report the first data on in vitro activity of forodesine in MCL. Primary MCL cells, derived from six patients, were exposed to forodesine (0, 2, 20 μM) in combination with dGuo (0, 10, 20 μM), for 48 hrs. Cells were cultured in X-VIVO 10 medium (Cambrex) with 10% FBS. Cell viability was assessed by flow cytometry with the Annexin V - propidium iodide assay. Four patient samples (67%) showed an increase in the number of Annexin V positive cells ranging from 1.9 to 5.3 times compared to untreated cells. The effect was larger for 20 μM forodesine compared with 2 μM. There was no effect of dGuo alone and only a minimal effect of increasing dGuo concentration from 10 μM to 20 μM. Cell lines did not appear to be ideal models to evaluate the efficacy of forodesine in vitro. Three established MCL cell lines (Granta-519, Rec, JeKo1) were treated with escalating doses of forodesine, but the results were not reproducible, while the same cells showed expected IC50 values between 25–30 μM when exposed to bendamustine for 72 hrs. In conclusion, the in vitro data reported here with 4/6 MCL patients primary samples sensitive to forodesine and the results from various groups on other T- and B-cell malignancies suggest that clinical trials of forodesine in MCL may be warranted.

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Juxtaposing CD20 and CD74 with Novel Bispecific Antibodies Evokes Potent Cytotoxicity in Mantle Cell Lymphoma (MCL)

Blood ◽

10.1182/blood.v118.21.599.599 ◽

2011 ◽

Vol 118 (21) ◽

pp. 599-599

Author(s):

Pankaj Gupta ◽

David M. Goldenberg ◽

Edmund A Rossi ◽

Thomas M. Cardillo ◽

John C. Byrd ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

Chimeric Antibody ◽

Employment Equity ◽

Actin Reorganization ◽

Mantle Cell ◽

Equity Ownership ◽

Anti Cd20 ◽

Homotypic Adhesion

Abstract Abstract 599 Introduction: Initial response rates with first-line conventional or high-dose chemotherapy, are high in MCL, but most patients relapse. With 3,500 new cases detected every year in the US, there is an unmet medical need for new therapeutic interventions in MCL. Combination of two different targeting mAbs to achieve improved efficacy without increased toxicity was demonstrated first in NHL patients given both rituximab (anti-CD20 chimeric antibody) and epratuzumab (humanized anti-CD22 IgG). Recently, the potential advantage of targeting both CD20 and CD74 was reported in a preclinical study showing that combining rituximab and milatuzumab (humanized anti-CD74 IgG) together with a crosslinking antibody resulted in anti-tumor activity in MCL lines and patient samples in vitro (Alinari L, et al. Blood 2011; 117:4530-41). Here we describe the generation of two novel bispecific hexavalent antibodies [HexAbs; IgG-(Fab)4] constructed from veltuzumab (humanized anti-CD20 IgG) and milatuzumab (anti-CD74 IgG), and show both are capable of inducing potent cytotoxicity in mantle cell lymphoma (MCL) and other lymphoma/leukemia cell lines, as well as primary MCL and CLL patient samples, without requiring a crosslinking antibody. To identify these HexAbs, we assign each of them a code of X-(Y)-(Y), where × and Y are specific numbers given to differentiate the antibodies, and a designated number enclosed in a parenthesis representing the antibody as a stabilized Fab dimmer (e.g., 20-(74)-(74) designates the bispecific HexAb comprising a divalent anti-CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab). Methods: The two bispecific HexAbs, 74-(20)-(20) and 20-(74)-(74), as well as the two monospecific HexAbs, 74-(74)-(74) and 20-(20)-(20), were generated by the Dock-and-Lock method by reacting cognate CH3-AD2-IgG and CH1-DDD2-Fab modules under mild redox conditions and purified by Protein A. The in vitro activities were determined by cell viability and Annexin V binding assays. In addition, the role of signal transduction, homotypic adhesion, actin reorganization, and lysosomal volume changes were evaluated. Results: Each HexAb was purified to >95 % homogeneity and were stable in vitro and in serum. Both 20-(74)-(74) and 74-(20)-(20) potently inhibited the growth of MCL lines, JeKo-1, Granta-519 and Mino, at 10 nM. In contrast, neither parental antibody, alone or in combination, nor the two monospecific counterparts, 74-(74)-(74) and 20-(20)-(20), inhibited the growth of JeKo-1 under the same conditions, suggesting the requirement of apposing CD74 and CD20 for the observed cytotoxicity. Treatment of primary MCL and CLL patient cells with 74-(20)-(20) and 20-(74)-(74) induced 25–30% apoptosis, compared to 10–15% apoptosis with parental IgGs, alone or in combination. The two bispecific anti-CD20/CD74 HexAbs, but not the parental mAbs, induced strong homotypic adhesion, actin reorganization to cell-cell junctions, loss of mitochondrial membrane potential, generation of ROS, deactivation of the PI3K/Akt signaling pathway, as well as rapid and sustained activation of ERK and JNK MAPKs, and enlargement of lysosomes followed by release of cathepsin B in target cells. In SCID mice bearing JeKo-1 xenografts, both 20-(74)-(74) and 74-(20)-(20) were effective at the 370-μg dose level, resulting in 60% and 30% increases in median survival time, respectively, compared to saline controls (P = 0.001). Conclusion: Juxtaposing CD20 and CD74 in close proximity by HexAbs induces potent in vitro cytotoxicity in MCL lines and primary samples. These novel bispecific mAbs warrant further evaluation in B-cell lymphomas that are refractory or poorly responsive to anti-CD20 antibodies. Disclosures: Gupta: Immunomedics, Inc.: Employment. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Rossi:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment. Furman:Centocor Ortho Biotech Research & Development: Research Funding. Chang:Immunomedics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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PR-924, a Selective Inhibitor of the Immunoproteasome Subunit LMP-7 Blocks Multiple Myeloma Cell Growth Both in Vitro and In Vivo.

Blood ◽

10.1182/blood.v114.22.612.612 ◽

2009 ◽

Vol 114 (22) ◽

pp. 612-612

Author(s):

Ajita V. Singh ◽

Madhavi Bandi ◽

Monette Aujay ◽

Susan Demo ◽

Teru Hideshima ◽

...

Keyword(s):

Cell Lines ◽

Annexin V ◽

P Value ◽

Caspase 9 ◽

Employment Equity ◽

Catalytic Subunits ◽

Caspase 7 ◽

Equity Ownership

Abstract Abstract 612 Background: Therapeutic targeting of Ubiquitin-Proteasome pathway is exemplified by the recent FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of multiple myeloma (MM). As with other agents, dose-limiting toxicities and the development of drug resistance limit its long-term utility. The β5, β1 and β2 catalytic subunits within the 26S proteasome mediate chymotrypsin-like, caspase-like, and trypsin-like activities, respectively. Importantly, these catalytic subunits have corresponding immunoproteasome components LMP-7, LMP-2 and multicatalytic endopeptidase complex subunit-1 (MECL-1), which regulate immune cell function and cytokine production; however, the role of the immunoproteasome in MM cells is still unclear. Recent studies have therefore focused on the discovery and development of small molecule inhibitors of the immunoproteasomes, which will both delineate the function of immunproteasomes and allow for specific therapeutic targeting of the UPS in order to reduce off-target activities and associated toxicities. Here, we examined PR-924, an LMP-7-selective peptide-ketoepoxide proteasome inhibitor related to carfilzomib. PR-924, like carfilzomib, contains a ketopoxide pharmacophore that covalently modifies proteasomal N-terminal threonine active sites. We examined the effects of PR-924 in MM cell lines and primary patient cells in vitro. To determine the in vivo efficacy of PR-924, we utilized two xenograft models of human MM in SCID mice, a subcutaneous tumor plasmacytoma model and the SCID-hu model, which best reflects the human MM-BM microenvironment in vivo. Methods and Model: We utilized MM.1S, MM.1R, RPMI-8226, U266, DOX40, KMS12, LR-5, OPM1, OPM2 and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability and apoptosis assays were performed using Trypan blue, MTT and Annexin V staining. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, caspase-7, PARP, Bcl-2, BID, or GAPDH. For tumor xenograft studies, CB-17 SCID male mice (n = 10; 5 mice/each group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum-free RPMI-1640 medium. When tumors were measurable (∼150 mm3) 2-3 weeks after MM cell injection, mice were injected IV with either PR-924 (6 mg/kg BW) or vehicle twice weekly. Mice were sacrificed when their tumors reached >2 cm3. In the SCID-hu model, 2 × 106 INA-6 cells were injected directly into human bone chips implanted subcutaneously in SCID mice (n=10: 5 mice/EA group), and MM cell growth was assessed by serial measurements of circulating levels of soluble human interleukin-6 receptor (shulIL6R) in mouse serum. Statistical significance of differences observed in PR-924 vs. vehicle treated mice was determined using a Student t test. Results: PR-924 significantly inhibits growth of all the MM cell lines in a time- and dose-dependent manner (IC50 range: 3-5 μM; P <0.005 for all cell lineIt alsos. reduced the viability of primary patient cells (P < 0.05; n=5), without significant effects on normal peripheral mononuclear cells. The PR-924-triggered decrease in MM cell viability is due to apoptosis, as evidenced by Annexin V/PI staining. Moreover, PR-924-induced apoptosis in MM.1S and MM.1R MM cells is associated with activation of caspase-3, caspase-8, caspase-9, caspase-7, BID and PARP. In vivo PR-924 triggered significant tumor growth inhibition in tumor plasmacytoma xenografts (2.3 fold decrease in tumor volume in mice receiving PR-924 versus mice injected with vehicle alone; P value = 0.01). Similarly, a significant reduction in the shuIL6R levels (3.4 fold decrease; P value = 0.02) was observed in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated, as evidenced by the lack of weight loss even after three weeks of treatment. Importantly, treatment of tumor bearing mice with PR-924, but not vehicle alone, significantly prolonged survival (P < 0.005). Conclusion: Our preclinical findings establish immunoproteasome LMP-7 as a novel therapeutic target in MM. Disclosures: Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership.

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Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells

Blood ◽

10.1182/blood.v122.21.4411.4411 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4411-4411 ◽

Cited By ~ 4

Author(s):

Swaroop Vakkalanka ◽

Srikant Viswanadha ◽

Eugenio Gaudio ◽

Emanuele Zucca ◽

Francesco Bertoni ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Whole Blood ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

Primary Cells ◽

Employment Equity ◽

Human Whole Blood ◽

Equity Ownership

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas. Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining. Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples. Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies. Disclosures: Vakkalanka: Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.

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Overcoming CAR T Resistance with Non-Covalent BTK Inhibitor Loxo-305 in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-137645 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-10

Author(s):

Yang Liu ◽

Vivian Changying Jiang ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

Yijing Li ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

Annexin V ◽

Car T ◽

Mantle Cell ◽

Btk Inhibitor

Background: Mantle cell lymphoma (MCL) is a distinctive B-cell non-Hodgkin's lymphoma characterized by poor prognosis. Despite clinical success of the covalent Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, a subset of patients need to discontinue ibrutinib therapy due to treatment related adverse events, which are primarily caused by off-target effects. Furthermore, primary or acquired resistance to ibrutinib continues to emerge and often leads to dismal clinical outcomes. Therefore, exploration of more target-specific BTK inhibitors is crucial to minimize the adverse events and provide clinical benefit. CAR T therapy has achieved unprecedented response in patients with relapsed or refractory MCL. However, the development of resistant phenotypes is a new emerging medical challenge in MCL patients with unknown mechanisms. Here, we characterize the therapeutic efficacy of LOXO-305, a next generation non-covalent small molecule inhibitor with high selectivity for BTK. Preclinical efficacy of LOXO-305 alone or in combination with venetoclax (ABT199), a selective Bcl-2 inhibitor, was evaluated in MCL using in vitro and in vivo CAR T-resistant PDX models. Methods : In vitro cell viability was measured after 72 hour treatment with LOXO-305 alone and in combination with ABT-199 in MCL cell lines using Cell Titer Glo luminescent cell viability assay (Promega). To determine whether LOXO-305 induces cell death through cell apoptosis, we used annexin V/PI staining followed by flow cytometry analysis. To evaulate in vivo drug efficacy we used patient-derived xenograft (PDX) models established from primary patient samples. Results: LOXO-305 treatment, as a single agent, resulted in effective MCL cell growth inhibition in a panel of MCL cell lines including ibrutinib and/or ABT-199-resistant cell lines (IC50=6.6-24.4μM), except for JeKo BTK KD cells with BTK knockdown (KD) via CRISPR/Cas9 technology (IC50>30 μM). To improve the efficacy, we decided to investigate the potential of LOXO-305 in combination with ABT199, since the combo of ibrutinib and ABT199 is clinically beneficial in MCL patients. Indeed, LOXO-305 significantly improved the inhibitory effect of ABT-199 in the ABT-199 resistant Mino-R and JeKo BTK KD cells, suggesting that this combination could be further explored in overcoming ABT-199 resistance in MCL. The compelling synergistic effect was further confirmed by annexin V/PI apoptosis assay. Next, we assessed the in vivo efficacy of LOXO-305 in an ibrutinib-CAR T dual-resistant PDX model. LOXO-305 effectively reduced tumor size after 40 days of treatment as a single agent. Moreover, LOXO-305 treatment showed significant anti-tumor effects in an ibrutinib-ABT199-CAR T triple-resistant PDX model that recapitulates the most aggressive human MCL variants invivo. In this model, LOXO-305 treatment effectively decreased the tumor load in mice spleen and liver (p<0.05) as well as in bone marrow and peripheral blood, compared to vehicle-treated mice (p<0.001). Conclusions: By using various in vitro and in vivo multiple resistant MCL models we determined that LOXO-305 holds great promise for an effective single agent or combined treatment of the most eggressive forms of MCL, and that a continued investigation of the rationale for a combined therapy with ABT-199 is imperative to understand its role in overcoming ibrutinib-ABT199-CAR T triple resistance. Disclosures Wang: OMI: Honoraria, Other: Travel, accommodation, expenses; MoreHealth: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Targeted Oncology: Honoraria.

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BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽

10.1136/esmoopen-2018-000387 ◽

2018 ◽

Vol 3 (6) ◽

pp. e000387 ◽

Cited By ~ 10

Author(s):

Chiara Tarantelli ◽

Elena Bernasconi ◽

Eugenio Gaudio ◽

Luciano Cascione ◽

Valentina Restelli ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Antitumour Activity ◽

Single Agent ◽

Treatment Strategies ◽

Bet Inhibitor ◽

Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

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Clinical Pharmacodynamic Markers and Combinations with SY1425 (tamibarotene) in a Genomically-Defined Subset of Non-APL AML

Blood ◽

10.1182/blood.v128.22.2898.2898 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2898-2898

Author(s):

Michael R McKeown ◽

Christopher Fiore ◽

Emily Lee ◽

Matthew L Eaton ◽

Christian C. Fritz

Keyword(s):

Transcription Factor ◽

Cell Lines ◽

Binding Sites ◽

Clinical Studies ◽

High Cell ◽

Preclinical Models ◽

Employment Equity ◽

Equity Ownership ◽

Maximal Induction

Abstract SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

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Safety and Tolerability of Conatumumab in Combination with Bortezomib or Vorinostat in Patients with Relapsed or Refractory Lymphoma.

Blood ◽

10.1182/blood.v114.22.1708.1708 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1708-1708 ◽

Cited By ~ 3

Author(s):

Anas Younes ◽

Mark Kirschbaum ◽

Lubomir Sokol ◽

Lorrin Yee ◽

Jorge Romaguera ◽

...

Keyword(s):

Research Funding ◽

Cell Lymphoma ◽

Death Receptor ◽

Large Cell ◽

Employment Equity ◽

Expansion Phase ◽

Mantle Cell ◽

Equity Ownership ◽

Refractory Lymphoma ◽

Grade 3

Abstract Abstract 1708 Poster Board I-734 Conatumumab is an investigational, fully human, monoclonal antibody agonist of human death receptor 5 (DR5 [TRAIL receptor 2]) that activates caspases and triggers apoptosis in sensitive tumor cells. DR5 is expressed by a variety of lymphoma cell lines, and TRAIL receptor agonists have been shown to induce apoptosis in lymphoma cells and lymphoma xenografts. Bortezomib and vorinostat are active and approved agents in certain lymphoma subtypes. In addition, they enhance death receptor-mediated apoptosis in multiple tumor models. In this 2-part study, we evaluated conatumumab in combination with bortezomib or vorinostat to treat patients (pts) with relapsed or refractory lymphoma. The dose-escalation phase evaluated the safety and tolerability of escalating doses of conatumumab in combination with bortezomib or vorinostat; the dose-expansion phase was designed to estimate the efficacy of conatumumab plus bortezomib in pts with mantle cell lymphoma (MCL). Here we present data from the dose-escalation phase. Eligibility criteria included: relapsed or refractory low-grade lymphoma, mantle cell lymphoma (MCL), diffuse large cell lymphoma, or Hodgkin lymphoma; age ≥ 18 years; informed consent; ECOG performance status of 0 or 1; life expectancy of > 3 months; adequate organ function; no prior treatment with bortezomib or vorinostat; no evidence of CNS involvement by lymphoma; and no primary CNS lymphoma. Three to 6 pts were enrolled into 1 of 3 sequential dose cohorts (1.5, 5, or 15 mg/kg) of conatumumab administered intravenously every 3 weeks (on day 1 of every 21-day cycle) in combination with either bortezomib (1.3 mg/m2 IV twice weekly for 2 weeks followed by a 10-day rest period) or vorinostat (400 mg orally daily). Endpoints included safety, maximum tolerated dose (MTD) of conatumumab, pharmacokinetics (PK) of conatumumab, incidence of anti-conatumumab antibodies, and best tumor response (complete response [CR] and partial response [PR]). CRs were confirmed by FDG-PET and bone marrow biopsy per Cheson criteria (2007). Monocyte DR5 occupancy by conatumumab was determined as an exploratory endpoint. As of July 9, 2009, 27 pts were enrolled and 23 received ≥1 dose of conatumumab: 3, 3, and 6 pts at 1.5, 5, and 15 mg/kg conatumumab + bortezomib; 7, 3, and 1 pt at 1.5, 5, and 15 mg/kg conatumumab + vorinostat. 15 pts were men; median (range) age was 53 (23 to 81) years; ECOG PS 0 = 65%, 1 = 26%, unknown = 9%; disease stage I = 4%, II = 4%, III = 39%, IV = 48%, unknown = 4%. Nine pts are still receiving treatment. The most common treatment-emergent adverse events (AE) were: fatigue (13 pts), diarrhea (9 pts), constipation (8 pts), nausea (8 pts), thrombocytopenia (8 pts), headache (7 pts), anemia (5 pts), dizziness (5 pts), and peripheral neuropathy (5 pts). A total of 6 and 3 pts reported worst grade 3 and 4 AEs, respectively, with no apparent differences between the 2 drug combinations. There were 2 DLTs: grade 3 prolonged Qt at 1.5 mg/kg conatumumab + vorinostat and grade 4 pulmonary embolism at 15 mg/kg conatumumab + bortezomib. An MTD has not been reached. Anti-conatumumb antibodies have not been detected in any pt. After one dose of conatumumab at 1.5, 5, or 15 mg/kg after bortezomib or vorinostat, conatumumab exposures were slightly higher (< 2-fold) than those in the first-in-human monotherapy study, indicating minimal effect of bortezomib or vorinostat on PK of conatumumab. Two pts had a confirmed CR: 1 pt with diffuse large cell lymphoma (1.5 mg/kg vorinostat cohort) at day 97 and 1 pt with nodular sclerosis Hodgkin lymphoma (5 mg/kg vorinostat cohort) at day 169. Thirteen pts had stable disease as their best objective response, 10 of whom had tumor shrinkage (range [based on sum of nodal and extra-nodal at each visit], -1.74% to -68.24%]). Receptor occupancy data will be presented. The combination of conatumumab with either bortezomib or vorinostat did not result in an unacceptable rate of dose-limiting toxicities and showed preliminary evidence of anti-tumor activity in pts with relapsed or refractory lymphoma. The expansion phase in pts with MCL treated with conatumumab plus bortezomib is currently enrolling. Disclosures Younes: Seattle Genetics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Abbott Oncology: Consultancy; Genentech: Consultancy, Honoraria, Research Funding; Allos Therapeutics : Consultancy; Gloucester Pharm: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Tiba Oncology: Consultancy; Trubion Pharmaceuticals: Consultancy; Sanofi-Aventis: Honoraria, Research Funding; Methylgene: Honoraria, Research Funding; Pharmion: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Biogen Idec: Honoraria, Research Funding. Kirschbaum:Merck: Research Funding, Speakers Bureau. Romaguera:Wyeth: Research Funding; Millenium: Research Funding; Celgene: Research Funding. Goyal:Amgen Inc.: Employment, Equity Ownership. Hsu:Amgen Inc.: Employment, Equity Ownership. Hwang:Amgen Inc.: Employment, Equity Ownership. Gorski:Amgen Inc.: Employment, Equity Ownership. Wong:Amgen Inc.: Employment, Equity Ownership. Beaupre:Amgen Inc.: Employment, Equity Ownership.

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GST-n and Nitric Oxide: A Novel Approach to Mantle Cell Lymphoma Therapy.

Blood ◽

10.1182/blood.v114.22.3729.3729 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3729-3729

Author(s):

Heather Gilbert ◽

John Cumming ◽

Josef T. Prchal ◽

Michelle Kinsey ◽

Paul Shami

Keyword(s):

Nitric Oxide ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Xenograft Model ◽

Malignant Cells ◽

Mantle Cell ◽

Tumor Viability

Abstract Abstract 3729 Poster Board III-665 Mantle cell lymphoma (MCL) is a well defined B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13, which encodes cyclin D1 (CD1), next to the immunoglobulin heavy chain gene promoter on chromosome 14. The resulting constitutive overexpression of CD1 leads to a deregulated cell cycle and activation of cell survival mechanisms. In addition, the gene which encodes GST-n, an enzyme that has been implicated in the development of cancer resistance to chemotherapy, is also located on chromosome 11q13 and is often coamplified along with the BCL1 gene in MCL (1). These two unique biological features of MCL - the overproduction of cyclin D1 and GST-n – may be involved in the carcinogenesis, tumor growth and poor response of this disease to treatment, and they offer potential mechanisms for targeted anti-cancer therapy. Nitric oxide (NO) is a biologic effector molecule that contributes to a host's immune defense against microbial and tumor cell growth. Indeed, NO is potently cytotoxic to tumor cells in vitro (2–4). However, NO is also a potent vasodilator and induces hypotension, making the in vivo administration of NO very difficult. To use NO in vivo requires agents that selectively deliver NO to the targeted malignant cells. A new compound has recently been developed that releases NO upon interaction with glutathione in a reaction catalyzed by GST-n. JS-K seeks to exploit known GST-n upregulation in malignant cells by generating NO directly in cancer cells, and it has been shown to decrease the growth and increase apoptosis in vitro in AML cell lines, AML cells freshly isolated from patients, multiple myeloma cell lines, hepatoma cells and prostate cancer cell lines (3, 5–7). JS-K also decreases tumor burden in NOD/SCID mice xenografted with AML and multiple myeloma cells (5, 7). Importantly, JS-K has been used in cytotoxic doses in the mouse model without significant hypotension. To evaluate whether JS-K treatment has anti-tumor activity in MCL, the human MCL cell lines Jeko1, Mino, Granta and Hb-12 were grown with media only, with JS-K at varying concentrations and with DMSO as an appropriate vehicle control. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V and PI. Cell growth was evaluated using the Promega MTS cytotoxicity assay. Results show that JS-K (at concentrations up to 10 μM) inhibits the growth of MCL lines compared to untreated controls, with an average IC50 of 1 μM. At 48 hours of incubation, all cell lines showed a significantly greater rate of apoptosis than untreated controls. A human MCL xenograft model was then created by subcutaneously injecting two NOD/SCID IL2Rnnull mice with luciferase-transfected Hb12 cells. Seven days post-injection, one of the mice was treated with JS-K at a dose of 4 μmol/kg (expected to give peak blood levels of around 17 mM in a 20 g mouse). Injections of JS-K were given intravenously through the lateral tail vein 3 times a week. The control mouse was injected with an equivalent volume of micellar formulation (vehicle) without active drug. The Xenogen bioluminescence imaging clearly showed a difference in tumor viability, with a significantly decreased signal in the JS-K treated mouse. Our studies demonstrate that JS-K markedly decreases cell proliferation and increases apoptosis in a concentration- and time-dependent manner in mantle cells in vitro. In a xenograft model of mantle cell lymphoma, treatment with JS-K results in decreased tumor viability. Proposed future research includes further defining the molecular basis of these treatment effects; using this therapy in combination with other cancer treatments both in vitro and in vivo; and studying JS-K treatment in MCL patients. Disclosures: Shami: JSK Therapeutics: Founder, Chief Medical Officer, Stockholder.

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