Co-Targeting Intrinsic and Extrinsic Apoptosis to Maximize Cell Death Induction in Venetoclax-Resistant AML Cells

Abstract Bcl-2 family protein-regulated intrinsic and IAP-family protein-regulated extrinsic pathways are two major apoptotic cell death mechanisms. Components of both pathways are regulated by the tumor suppressor p53. Although combinations of Bcl-2 inhibitor venetoclax (VEN) and a hypomethylating agent induce high response rates in AML, most patients ultimately relapse. In addition, pre-clinical and clinical studies have shown that TP53-mutant AML cells are less sensitive to VEN (Carter BZ, ASH 2019 and 2020; DiNardo CD, Blood 2020). We investigated if simultaneous inhibition of Bcl-2 and IAPs and activation of p53, via MDM2 inhibition could maximize apoptosis induction in AML cells with acquired resistance to VEN-based therapy or in those carrying TP53 mutations. We treated MV4-11 cells with acquired resistance to VEN (VEN-R) with the Bcl-2 inhibitor APG2575, IAP inhibitor APG1387, or MDM2 inhibitor APG115, and with their combinations. As expected, VEN-R cells were more resistant to APG2575 compared to control cells. APG1387 alone had limited activity in both VEN-R and in control cells and the combination of APG2575 and APG1387 enhanced cell killing (P < 0.05 compared to each single agent). APG115 was active in both VEN-R and control cells and its activity was only increased by APG2575 in the control cells, but minimally affected by either APG2575 or APG1387 in VEN-R cells. However, maximal apoptosis induction was observed in both VEN-R and control cells when all three compounds were combined (P < 0.05 compared to any of the double combinations or single agent treatments). We next treated NSG mice harboring PDX cells derived from an AML patient who relapsed on the VEN/decitabine therapy with APG2575 (50 mg/kg, p.o., daily), APG1387 (10 mg/kg, i.v., once/wk), APG115 (50 mg/kg, p.o., daily at wk 1 and 5), or combinations. At the end of a 5-wk treatment, significant reductions of human CD45 + cells were observed in all treatment groups. At 4 wks post treatment, decreased circulating leukemia cells were found in the triple and APG115+APG2575 combination groups. APG115 (139 d, p=0.009), APG1387 (130 d, p=0.004), or APG2575 (132 d, p=0.004) significantly extended mouse survival compared to controls (116 d). Among two drug combinations, APG115 plus APG1387 did not further prolong survival, APG2575 plus APG1387 (144 d, p<0.01) was more effective, and APG2575 plus APG115 (180 d, p<0.01) most effectively extended survival compared to each drug alone. Triple combination treated mice lived longest (185 d), which was significantly longer than APG115 plus APG1387 and APG2575 plus APG1387 but did not reach statistical significance compared to APG2575 plus APG115. Data showed that triple and APG115+APG2575 combinations were most effective, followed by APG2575+APG1387, then APG115+APG1387, APG2575, or APG115, and finally APG1387. Finally, we treated Molm13 cells lacking TP53 or carrying TP53 mutations (R248W/R213*, R248Q, R175H, R282W, Y220C) with the three agents and their combinations. All mutant cells were insensitive to single drugs. Enhanced activity was observed when any of two agents were combined and combined inhibition of Bcl-2, IAPs, and MDM2 most effectively induced cell death in TP53 knockout and all TP53 mutant cells (P < 0.05 for the triple combination compared to any of the double combinations or single agent treatments, and double combinations compared to their respective single agent treatments). Western blot analysis showed that decreased cIAP1, cIAP2, XIAP, or p21 was observed in single agent or combination-treated cells. Only in the triple combination group, cIAP1, cIAP2, and XIAP as well as MDM2 were largely diminished and p21 was marked decreased. In conclusion, our study demonstrates that co-targeting intrinsic and extrinsic apoptosis maximizes cell death induction in AML cells with acquired resistance to VEN or with TP53 deletion/mutations by antagonizing Bcl-2, eliminating cIAPs and XIAP, as well as MDM2 and p21, a finding that needs to be validated clinically. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Andreeff: Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Medicxi: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Targeting Antibody Checkpoint Fcgriib Using Monoclonal Antibody BI-1206 to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140849 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-35

Author(s):

Alexa A Jordan ◽

Joseph McIntosh ◽

Yang Liu ◽

Angela Leeming ◽

William Lee ◽

...

Keyword(s):

Monoclonal Antibody ◽

Tumor Growth ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

Publicly Traded ◽

In Vivo Efficacy ◽

Mantle Cell ◽

Xenograft Models

Mantle cell lymphoma (MCL) is a rare but aggressive B-cell non-Hodgkin's lymphoma that represents 6% of all lymphomas in the United States. Recent therapies including anti-CD20 antibody rituximab, BTK inhibitors, and BCL-2 inhibitors alone or in combination have shown great anti-MCL efficacy. However, primary and acquired resistance to one or multiple therapies commonly occurs, resulting in poor clinical outcome. Therefore, resistance to such therapies is currently an unmet clinical challenge in MCL patients. Therapeutic strategies to overcome this resistance holds promise to significantly improve survival of refractory/relapsed MCL patients. Recent studies showed Fc gamma receptors (FcγRs) play important roles in enhancing the efficacy of antibody-based immunotherapy. In particular, FcgRIIB (CD32B), an inhibitory member of the FcγR family, is implicated in the immune cell desensitization and tumor cell resistance through the internalization of therapeutic antibodies such as rituximab. Based on our flow cytometry analysis, we demonstrated that FcgRIIB is highly expressed on the cell surface of MCL cell lines (n=10) and primary MCL patient samples (n=22). This indicates that FcgRIIB may play an important role in MCL malignancy and identifies FcgRIIB is a potential therapeutic target for the treatment of MCL. To address this, we tested the in vivo efficacy of BI-1206, a fully humanized monoclonal antibody targeting FcgRIIB, alone, or in combination with clinically approved or investigational drugs including rituximab, ibrutinib and venetoclax. In the first in vivo cohort, BI-1206, as a single agent, dramatically inhibited the tumor growth of ibrutinib-venetoclax dual-resistant PDX tumor models, suggesting that targeting FcgRIIB by BI-1206 alone has high anti-MCL activity in vivo. Next, we assessed whether BI-1206 can boost anti-MCL activity of antibody-based therapy such as rituximab in combination with ibrutinib or venetoclax using additional mice cohorts of cell line-derived xenograft and patient-derived xenograft models. BI-1206 significantly enhanced the in vivo efficacy of ibrutinib plus rituximab, and venetoclax plus rituximab, on tumor growth inhibition, including the JeKo-1 derived xenograft models, previously proven to be partially resistant to ibrutinib and venetoclax in vivo. This tumor-sensitizaton effect was further confirmed in the ibrutinib and venetoclax dual-resistant PDX models of MCL where BI-1206 was combined with venetoclax and rituximab. More detailed mechanistic studies are currently ongoing to reveal the mechanism of action of BI-1206-based combinations or as single therapy with the possibility that BI-1206 itself may have a cytotoxic anti-tumor direct activity in MCL. In conclusion, BI-1206 as single agent showed potent efficacy in overcoming ibrutnib-venetoclax dual resistance. Moveover, BI-1206 enhanced the in vivo efficacy of ibrutinib plus rituximab and venetoclax plus rituximab and overcomes resistance to these treatments resulting in enhanced anti-tumor effects. Disclosures Karlsson: BioInvent International AB: Current Employment. Mårtensson:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Kovacek:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Teige:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Frendéus:BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Wang:Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; Dava Oncology: Honoraria; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Acerta Pharma: Research Funding; VelosBio: Research Funding; MoreHealth: Consultancy; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; 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; Lu Daopei Medical Group: Honoraria.

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Dual SYK/JAK Inhibition Overcomes Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.5377.5377 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5377-5377

Author(s):

Yue Lynn Wang ◽

Pin Lu ◽

Greg P Coffey ◽

Anjali Pandey ◽

Ailin Guo

Keyword(s):

Cell Death ◽

Research Funding ◽

Single Agent ◽

Lymphocytic Leukemia ◽

Prognostic Indicators ◽

Employment Equity ◽

Btk Inhibitor ◽

Equity Ownership ◽

Ibrutinib Resistance ◽

Novel Therapeutic Strategies

Abstract Ibrutinib (a BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Further, cerdulatinib blocked proliferation of ibrutinib-sensitive and ibrutinib-resistant primary CLL cells and of BTKC481S-transfected cells. These anti-tumor effects are correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

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Preclinical Activity of Selective SYK Inhibitors, Entospletinib and Lanraplenib, Alone or Combined with Targeted Agents in Ex Vivo AML Models with Diverse Mutational Backgrounds

Blood ◽

10.1182/blood-2021-146180 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3356-3356

Author(s):

Melinda A. L. Day ◽

Philipp Sergeev ◽

Caroline A. Heckman ◽

Anna Schinzel ◽

Nikolaus D. Obholzer ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Viability ◽

Research Funding ◽

Ex Vivo ◽

Single Agent ◽

Clinical Activity ◽

Publicly Traded ◽

Aberrant Expression ◽

Syk Inhibitor ◽

Privately Held

Abstract Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that mediates integrin and Fc receptor signaling in myeloid cells. SYK has been implicated as an oncogenic driver in acute myeloid leukemia (AML) with aberrant expression of HOXA9 and MEIS1 and cooperates with FLT3 internal tandem duplication to drive leukemogenesis. The oral SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity in HOXA9/MEIS1 driven AML and is currently being investigated in a phase 3 trial of previously untreated patients with nucleophosmin1-mutated (NPM1 mut) AML. Lanraplenib (LANRA) is a next generation oral SYK inhibitor with potency and selectivity comparable to ENTO. In healthy volunteers and patients with autoimmune disease, LANRA has shown pharmacokinetic properties that compare favorably with ENTO. To support the clinical development of LANRA for the treatment of AML, ex vivo treatment of patient-derived AML cells was used to compare its activity to that of ENTO, both as a single-agent and in combination with other AML therapies. First, ENTO and LANRA single-agent activities were evaluated in peripheral blood-derived blasts from 15 AML patients, representing different mutational backgrounds including NPM1, FLT3, PTPN11, and NRAS mutations. AML cells were seeded into 96 well plates and treated with ENTO and LANRA for 6 days. Comparable effects on viability were observed across the 15 models with the 2 compounds, and in 11 of the models, the half maximal inhibitory concentration (IC 50) values were within 2-fold of each other. ENTO had a slightly lower IC 50 value than LANRA in the FLT3-mutated models possibly due to the direct FLT3 inhibitory activity of ENTO. Next, we tested the activity of ENTO and LANRA ex vivo in bone marrow-derived AML blasts from 29 AML patients representing diverse mutational backgrounds, including NPM1, IDH1, FLT3, and RAS mutations as well as MLL rearrangements. The models were treated for 9 days with either ENTO or LANRA, and viability was assessed using Annexin V and 7-aminoactinomycin D staining. Again, ENTO and LANRA showed comparable effects on cell viability with no significant differences between the compounds when compared across the different mutational backgrounds. Both studies suggest the potential for anti-leukemic activity in several different genetically defined subsets of AML. Matrix combination assays were performed by combining ENTO or LANRA with either cytarabine (NPM1 mut), gilteritinib (FLT3 mut), or trametinib (RAS mut) with cell viability and death assessed after a 3-day incubation period. Increased cell death in an additive manner was observed in all combinations tested, with results for ENTO and LANRA being similar, indicating the utility of both compounds in combinatorial treatment paradigms. These results support the clinical evaluation of LANRA in genetically defined subsets of AML. A phase 1b/2 study of LANRA in combination with the selective FLT3 inhibitor gilteritinib, in patients with relapsed or refractory FLT3 mut AML is planned for the end of this year. Disclosures Day: Cyteir Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding. Schinzel: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Obholzer: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lin: Kronos Bio, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Saffran: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company.

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Safety and Antitumor Activity Study Evaluating Loncastuximab Tesirine and Rituximab Versus Immunochemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2020-136614 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Yuliya Linhares ◽

Mitul D Gandhi ◽

Michael Chung ◽

Jennifer Adeleye ◽

David Ungar ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Advisory Board ◽

Publicly Traded ◽

Advisory Committees ◽

Large B Cell Lymphoma ◽

Large B Cell

Introduction: Patients with diffuse large B-cell lymphoma (DLBCL) who fail immunochemotherapy (IC) and are unsuitable for autologous stem cell transplantation (ASCT) and those who relapse shortly after ASCT have extremely poor prognosis and need additional treatment options. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate (ADC) composed of a humanized anti-CD19 antibody conjugated to a pyrrolobenzodiazepine dimer toxin. In a Phase 2 study (NCT03589469), Lonca demonstrated single-agent antitumor activity with manageable toxicity in patients with relapsed/refractory (R/R) DLBCL. Rituximab is a CD20-targeting monoclonal antibody used in front-line IC for DLBCL and in salvage regimens, such as rituximab/gemcitabine/oxaliplatin (R-GemOx). The addition of rituximab to a CD19-targeting pyrrolobenzodiazepine ADC appears to prolong tumor control in preclinical studies, providing the rationale for evaluating Lonca combined with rituximab (Lonca-R) as a treatment for R/R DLBCL. Study Design and Methods: This is a Phase 3, randomized, open-label, 2-part, 2-arm, multicenter study of Lonca-R versus standard IC in patients with R/R DLBCL (NCT04384484). Part 1 is a nonrandomized safety run-in with Lonca-R. The toxicity of Lonca-R will be compared with previous single-agent Lonca safety data after 20 patients have completed Cycle 1 in Part 1. Provided no significant increase in toxicity is observed, Part 2 will be initiated. Part 2 is a randomized study of Lonca-R versus R-GemOx (Figure 1). Key inclusion and exclusion criteria are reported in Table 1. The primary objective of Part 2 is to evaluate the efficacy of Lonca-R versus R-GemOx, using progression-free survival (PFS) as the primary endpoint. PFS will be defined as the time between randomization and first documentation of recurrence, disease progression or death (central review) and the primary analysis will compare PFS between treatment arms using stratified log-rank testing. Secondary objectives include evaluation of safety, pharmacokinetics, and immunogenicity of the combination, in addition to the impact of treatment on symptoms, patient-reported outcomes and patients' overall health. In Part 1 and in the Lonca-R arm of Part 2, patients will receive intravenous (iv) Lonca at 150 µg/kg on day 1 of each 21-day cycle for 2 cycles, then at 75 µg/kg on day 1 for up to 6 additional cycles. Rituximab 375 mg/m2 iv will be administered subsequent to Lonca infusion on day 1 of each cycle. Patients treated with Lonca-R will also be given dexamethasone 4 mg (oral, twice a day), where not contraindicated, on the day before, the day of, and the day after Lonca-R infusion. In the R-GemOx arm, patients will receive rituximab 375 mg/m2, gemcitabine 1000 mg/m2, and oxaliplatin 100 mg/m2 iv on day 1 of each 14-day cycle up to a total of 8 cycles. Patients will receive premedication and supportive care according to the respective prescribing information for rituximab, gemcitabine, and oxaliplatin. The trial is planned to open in Q3/Q4 2020, and target enrollment is 350 patients. Funding: This study is sponsored by ADC Therapeutics SA; https://clinicaltrials.gov/ct2/show/NCT04384484. Disclosures Linhares: Jazz Pharmaceuticals: Consultancy; ADC Therapeutics, Verastem Oncology, Bristol Myers-Squibb (Juno), AstraZeneca: Research Funding; Miami Cancer Institute, Baptist Health South Florida: Current Employment. Gandhi:TG Therapeutics (Advisory board), GlaxoSmithKline (Advisory board): Membership on an entity's Board of Directors or advisory committees. Adeleye:ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ungar:ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamadani:ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme, AstraZeneca: Speakers Bureau; Janssen R&D; Incyte Corporation; ADC Therapeutics; Celgene Corporation; Pharmacyclics, Omeros, AbGenomics, Verastem, TeneoBio: Consultancy; Takeda Pharmaceutical Company; Spectrum Pharmaceuticals; Astellas Pharma: Research Funding. OffLabel Disclosure: Rituximab is licensed for treatment of NHL but is being used in combination with an unlicensed drug (loncastuximab tesirine) in this study

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BCR-ABL+ Bp-ALL Exhibits Heightened Sensitivity to Simultaneous Induction of Energy and ER Stress Via Downregulation of Mcl-1 Expression

Blood ◽

10.1182/blood.v124.21.916.916 ◽

2014 ◽

Vol 124 (21) ◽

pp. 916-916

Author(s):

Guy J. Leclerc ◽

Joanna DeSalvo ◽

Gilles M. Leclerc ◽

Theodore J. Lampidis ◽

Julio C. Barredo

Keyword(s):

Cell Death ◽

Er Stress ◽

Cell Lines ◽

Aerobic Glycolysis ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Mtor Inhibition ◽

Energy Stress ◽

Simultaneous Induction ◽

And Control

Abstract Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with growth and survival. BCR-ABL+ acute lymphoblastic leukemia (BCR-ABL+ ALL) accounts for 25% of ALL and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG), induces simultaneous energy and ER-stress by inhibiting glycolysis (energy stress) via blocking HK and PGI enzymes, and interfering with N-linked glycosylation through its incorporation in place of mannose into the lipid-linked oligosaccharide (LLO) chain leading to premature termination of LLO synthesis (ER stress). We recently demonstrated that inhibition of both aerobic glycolysis and N-linked glycosylation by 2DG led to apoptosis in ALL (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2DG suggesting that in ALL cells BCR-ABL expression may be linked to heightened sensitivity to energy and/or ER stress. To test this premise, we constructed NALM6 (Bp-ALL) stable cell lines expressing the BCR-ABL p190 fusion and found that expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected controls. To investigate the contribution of each form of stress (energy and ER stress) in the increased sensitivity of BCR-ABL+ cells to 2DG, we examined the effects of agents known to preferentially induce energy stress (lowering ATP) such as 2-fluoro-2-deoxy-D-glucose and oxamate, and ER stress such as tunicamycin. We found no differences in cell death or ATP levels between BCR-ABL+ and control ALL cells treated with agents solely inducing energy or ER stress, indicating the increased susceptibility of BCR-ABL+ ALL cells to 2DG results from the simultaneous induction of both types of stress. Consistent with these data, Fluorophore-Assisted Carbohydrate Electrophoresis and immunoblotting demonstrated that 2DG induced equivalent inhibition of N-linked glycosylation and upregulation of the UPR signaling in BCR-ABL+ and control ALL cells. Similar findings were seen in primary ALL patient samples. Consequently, simultaneous energy and ER stress are required for the heightened vulnerability to these forms of stress conferred by BCR-ABL in ALL. Based on the known function of Mcl-1 promoting survival in BCR-ABL+ ALL, we investigated its role in the mechanism of cell death induced by energy and/or ER stress agents. We uncovered that BCR-ABL+ ALL cell lines exhibited the lowest levels of Mcl-1 expression compared to control BCR-ABL- ALL and CML cells, and this basal level of Mcl-1 expression directly correlated with their resistance to energy and/or ER stressing agents. Further, we found that Mcl-1 knock-down by shRNA sensitized ALL and CML cells to the dual induction of energy and ER stress by 2DG. We also found that Mcl-1 expression was further downregulated in BCR-ABL+ and - ALL cells treated with energy and/or ER stressors which correlated with the level of apoptosis. Using qRT-PCR or co-treatment with the pancaspase inhibitor qVD-OPH or the proteasome inhibitor bortezomib, we showed that Mcl-1 downregulation by energy and/or ER stressors is neither due to transcriptional, caspase dependent cleavage, or post-translational regulation mechanisms but likely due to translational control of the Mcl-1 protein via AMPK/mTOR and the UPR PERK/p-eIF2α. Finally, because TKIs lead to decrease ATP (energy stress) in BCR-ABL+ cells, we co-treated BCR-ABL+ ALL cells with TKIs plus ER stressors and found that the simultaneous induction of these forms of stress by these combinations significantly increased cell death compared to single drug. Further, we found that TKIs + ER stressors further downregulated Mcl-1 and GRP78 expression compared to single agent treatment. Taken together, our data demonstrate that simultaneous induction of energy and ER stress leads to significant induction of apoptosis in BCR-ABL+ ALL cells, and these heightened vulnerability to these forms of stress is likely mediated by downregulation of Mcl-1 expression via AMPK activation/mTOR inhibition and PERK/p-eIF2α. Our study demonstrates that strategies with agents that induce energy and/or ER stress plus BCR-ABL TKIs warrant further consideration as a potential approach for future clinical translation into trials for BCR-ABL+ ALL patients. Disclosures No relevant conflicts of interest to declare.

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Dreamm-5 Platform Trial: Belantamab Mafodotin (Belamaf) in Combination with Four Different Novel Agents in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Blood ◽

10.1182/blood-2020-139182 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Paul G. Richardson ◽

Ajay Nooka ◽

Hang Quach ◽

Suzanne Trudel ◽

David Routledge ◽

...

Keyword(s):

Board Of Directors ◽

Combination Treatment ◽

Research Funding ◽

Single Agent ◽

Gamma Secretase ◽

Publicly Traded ◽

Advisory Committees ◽

For Profit ◽

Secretase Inhibitor ◽

Gamma Secretase Inhibitor

Introduction: Single-agent belamaf (GSK2857916), a B-cell maturation antigen-targeting antibody-drug conjugate, induced deep and durable responses in patients with RRMM, with a manageable safety profile with 13 months of follow-up (DREAMM-2; NCT03525678; Lonial et al, ASCO 2020, Poster 436). A platform trial design allows efficient evaluation of belamaf in combination with other anti-myeloma agents, such as a humanized wild-type IgG1 anti-OX40 agonist, an IgG4-inducible T-cell co-stimulator (ICOS) agonist, a gamma-secretase inhibitor, and a humanized programmed cell death (PD)-1 antagonist. The unique, multimodal mechanisms of action (MoAs) of belamaf, in combination with MoAs of these agents, has the potential to achieve synergistic effects in RRMM to further enhance anti-myeloma activity without compromising safety. Methods: DREAMM-5 (NCT04126200) is a Phase I/II study that utilizes a master protocol with separate substudies comprised of sequential dose-exploration (DE) and cohort-expansion (CE) phases, to identify promising, effective belamaf combinations when compared with a shared single-agent belamaf control arm (CE phase only). The DE phase consists of multiple dosing cohorts with belamaf combinations in which patients are assigned to treatment slots by a predetermined algorithmic approach (N≤10 per cohort). A recommended Phase II dose (RP2D) for each combination treatment will be identified based on the safety and preliminary efficacy in the DE phase. At the end of the DE phase, an interim analysis of safety, pharmacokinetic, and efficacy data will also be performed for each substudy treatment combination to determine whether the combination should move forward at the RP2D to the CE phase. Patients in the CE phase (N≥35 per cohort) will be randomized to a substudy and within a substudy to either combination treatment or the belamaf monotherapy control arm; patients will also be stratified by number of prior therapies). Eligible patients will have RRMM and will have received ≥3 prior therapy lines, which includes a prior immunomodulatory agent, proteasome inhibitor, and anti-CD38 antibody; all patients will provide informed consent for participation. The primary objectives of the study are to identify the RP2D (DE phase) and the overall response rate (≥partial response, CE phase), along with safety and tolerability, for each combination treatment. Substudies 1 (combination with GSK3174998, OX40 agonist antibody), 2 (combination with GSK3359609, ICOS agonist antibody), and 3 (combination with nirogacestat [PF-03084014; SpringWorks Therapeutics], gamma-secretase inhibitor) are currently open to enrollment. Substudy 4 (combination with dostarlimab; PD-1 antagonist antibody) is under review. Additional substudies will be explored based on scientific rationale and/or preclinical combination study results. Funding: GSK (Study 208887); belamaf drug linker technology licensed from Seattle Genetics; belamaf monoclonal antibody produced using POTELLIGENT Technology licensed from BioWa; nirogacestat gamma-secretase inhibitor produced by and used in collaboration with SpringWorks Therapeutics. Figure: DREAMM-5 study design Figure 1 Disclosures Richardson: Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Nooka:GlaxoSmithKline: Consultancy, Honoraria, Other: Personal Fees: Travel/accomodations/expenses, Research Funding; Karyopharm Therapeutics, Adaptive technologies: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Spectrum Pharmaceuticals: Consultancy; Adaptive Technologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding. Quach:Amgen, sanofi, celgene, Karyopharm, GSK: Research Funding; Amgen, Celgene, karyopharm, GSK, Janssen Cilag, Sanofi.: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Consultancy; GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Honoraria. Trudel:Celgene, Janssen, Takeda, Sanofi, Karyopharm, Amgen Canada: Honoraria; Celgene, Amgen, GSK: Consultancy, Research Funding; GSK, Celgene, Janssen, Amgen, Genentech: Research Funding. Routledge:Celgene, Sandoz: Consultancy; Amgen, BMS, Celgene, Sandoz: Honoraria. Song:Otsuka: Honoraria; Janssen: Honoraria, Research Funding; Amgen, Celgene,Takeda: Consultancy, Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Paul:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Khan:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Brouch:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Ferron-Brady:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Yeakey:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Shelton:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Montes de Oca:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Smith:SpringWorks: Current Employment, Current equity holder in publicly-traded company. Im:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Ahlers:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Paul:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Holkova:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Gupta:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company; Novartis: Current equity holder in publicly-traded company. Kremer:GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Rodriguez-Otero:GlaxoSmithKline: Consultancy, Current Employment, Current equity holder in publicly-traded company, Honoraria; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Medscape: Membership on an entity's Board of Directors or advisory committees; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Sanofi: Consultancy, Honoraria.

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A Phase 1 Study to Evaluate the Safety, Pharmacokinetics (PK) and Pharmacodynamics (PD) of Lisaftoclax (APG-2575), a Novel BCL-2 Inhibitor (BCL-2i), in Patients (pts) with Certain Relapsed or Refractory (R/R) Hematologic Malignancies (HMs)

Blood ◽

10.1182/blood-2021-152235 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3730-3730

Author(s):

Mingyuan Sun ◽

Junyuan Qi ◽

Zi Chen ◽

Hongli Zhang ◽

Yongping Song ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

Phase 1 ◽

Single Agent ◽

Objective Response ◽

Tumor Lysis Syndrome ◽

B Cell Lymphoma ◽

Severe Neutropenia ◽

Publicly Traded

Abstract Background: Many B-cell malignancies evade apoptosis by overexpressing BCL-2 proteins. Studies of the BCL-2i venetoclax have demonstrated activity in certain HMs but show that venetoclax requires a slow dose ramp-up over several weeks to reduce the risk of tumor lysis syndrome (TLS), which may warrant frequent or intensive laboratory monitoring. Cases of severe neutropenia with venetoclax treatment have also been reported. Lisaftoclax is a novel, potent, selective BCL-2i that is active against HMs and offers a potential advantage of a daily clinical ramp-up (rather than weekly) to the target dose over a few days. Methods: This Chinese, multicenter, open-label, single-agent, phase 1 trial is evaluating the safety (including dose-limiting toxicity [DLT] and maximum tolerated dose [MTD]), efficacy, PK, and PD of lisaftoclax in adults with histologically confirmed diagnoses of R/R chronic lymphocytic leukemia (CLL) or non-Hodgkin's lymphoma. Eligibility criteria include an ECOG performance score of 0-1 (in dose escalation) or 0-2 (in dose expansion); life expectancy ≥ 3 months; and adequate bone marrow, renal, and liver function. Lisaftoclax was orally administered once daily in 4-week cycles across multiple dose cohorts. Results: As July 27, 2021, 31 pts had been enrolled and treated with lisaftoclax at doses ranging from 20 to 800 mg. Pts had a median (range) of 4 (1-14) prior lines of treatment and diagnoses of CLL/SLL (n = 9), mantle cell lymphoma (MCL; n = 6), marginal zone lymphoma (MZL; n = 3), follicular lymphoma (n = 8), diffuse large B-cell lymphoma (n = 2), lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (n = 1), angioimmunoblastic T-cell lymphoma (n = 1) or mycosis fungoides (MF; n = 1). DLT, MTD, and laboratory/clinical TLS have not been observed at doses up to 800 mg. The recommended phase 2 dose (RP2D) is 600 mg. Lisaftoclax was generally well tolerated. Treatment-related adverse events (TRAEs) were reported in 28 pts (87.5%), most of which were grade 1 to 2. Any grade TRAEs in > 10% of pts include thrombocytopenia (34.4%), neutropenia (21.9%), leukopenia (21.9%), anemia (28.1%), hyperuricemia (15.6%), hyperphosphatemia (12.5%), hypertriglyceridemia (12.5%), and diarrhea (15.6%). Grade 3-4 TRAEs were reported in 7 pts (21.9%), including thrombocytopenia (18.8%), neutropenia (12.5%), leukopenia (9.4%), and anemia (6.3%). Serious TRAEs occurred in 1 pt and included anemia and thrombocytopenia (in 3.1% each). With a median (range) treatment of 4 (1-14) cycles, 9 of 25 evaluable pts achieved an objective response (CR or PR), for an ORR of 39% with a median (range) time to response of 2 (2-4) cycles. The highest response rates were seen in pts with CLL/SLL (ORR 66.7% [6/9]). At doses ≥ 200 mg, an ORR of 100% (6/6 including 1 CR and 5 PR) was observed. Responses were also observed in MZL, with a PR in 2 of 3 pts (ORR, 66.7%), and MCL, with a PR in 1 of 4 pts (ORR, 25%). In 1 pt with MF, skin tumor shrinkage was observed after 1 lisaftoclax treatment cycle. Favorable absolute lymphocyte count (ALC) profiles included reductions at lisaftoclax doses as low as 100 mg/day. The preliminary PK profile showed that exposures increased with lisaftoclax doses from 20 to 800 mg, with an average half-life of 4 to 6 hours. On BH3 profiling, lisaftoclax rapidly triggered changes in BCL-2 complex in CLL/SLL pt samples, which were consistent with rapid clinical reductions in ALCs. Conclusions: Lisaftoclax was well tolerated up to 800 mg/day. No TLS was observed, even with the daily ramp-up schedule. There were no significantly new or unmanageable safety findings. Lisaftoclax showed single-agent antitumor activity in CLL/SLL, MZL, and MCL. The BCL-2i lisaftoclax offers a treatment alternative for pts with R/R HMs, with a daily ramp-up schedule that may be more pt friendly with a favorable preliminary safety profile. Internal study identifier APG-2575-CN-001; ClinicalTrials.gov identifier: NCT03913949. Figure 1 Figure 1. Disclosures Chen: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company. Zhang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Lu: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Ahmad: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company. Men: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Cen: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Wang: Astellas Pharma, Inc.: Research Funding; AbbVie: Consultancy. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding.

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Mechanisms of Acquired Resistance to Venetoclax in Preclinical AML Models

Blood ◽

10.1182/blood.v126.23.328.328 ◽

2015 ◽

Vol 126 (23) ◽

pp. 328-328 ◽

Cited By ~ 4

Author(s):

Qi Zhang ◽

Rongqing Pan ◽

Lina Han ◽

Ce Shi ◽

Stephen E. Kurtz ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Small Molecule ◽

Research Funding ◽

Synergistic Effects ◽

Acquired Resistance ◽

Parental Cell ◽

Resistant Cell ◽

Flt3 Inhibitors ◽

Resistant Cells

Abstract BH3-mimetic ABT-199 (venetoclax, VEN) is a selective small-molecule antagonist of the anti-apoptotic BCL-2 protein. It binds to BCL-2 specifically, causing the release of pro-apoptotic BAX and BH3-only proteins and induction of cell death. Our studies indicated that AML is a BCL-2 dependent disease that, in pre-clinical studies, responds robustly to VEN by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). As a single agent, VEN demonstrated clinical activity in relapsed/refractory AML, yet patients who initially responded ultimately developed resistance and progressed. In this study we investigated mechanisms of acquired resistance to VEN in preclinical AML models. First, we generated 5 VEN-resistant cell lines (OCI-AML2, Kasumi, KG-1, MV4;11 and Molm13; with VEN cell-killing IC50s of 0.021µM, 0.046µM, 0.073µM, 0.020µM and 0.050µM, respectively) by exposing the cells to gradually increasing VEN concentrations. The IC50s of resistant cells are 15.2µM, 5.7µM, 31.6µM, 11.4µM and 15.4µM (124-723-fold greater than their parental counterparts). Protein analysis of resistant cells using immunoblotting demonstrated increased expression of MCL-1, a known resistance factor to VEN, in 4 resistant cell lines (OCI-AML2, KG-1, Mv4;11 and Molm13); and BCL-XL increase in MV4;11 and Molm13 resistant cells. To characterize the functional role of MCL-1 and BCL-XL in resistance to VEN, we co-treated parental and resistant cells with novel MCL-1 and BCL-XL- selective inhibitors (A-1210477 and A-1155463). The combination of VEN with A-1210477 or A-1155463 showed synergistic growth inhibition in all 5 parental cell lines (combination indices (CI) for A-1210477 were 0.15-0.62; CI for A-1155463 were 0.33-0.51, except >3 for KG-1). Notably, 4 out of 5 resistant cell lines (OCI-AML2, Kasumi, MV4;11, Molm13) became more sensitive to MCL-1 selective inhibitor A-1155463 but not to BCL-XL inhibitor A-1210477. However, no further effects were seen in resistant cells when combined with VEN. We next compared sensitivity of three paired parental and resistant cell lines (OCI-AML2, MV4;11 and Molm13) to a library of 130 specific small-molecule inhibitors (Tyner, et.al.. Cancer Res. 2013). Cells were co-treated with VEN and each specific inhibitor, and drug target scores were calculated based on the IC50 of measured effectiveness of panel drugs against the cells. The screening revealed modulation of sensitivity to mTOR, MEK, and FLT3 pathways in resistant cells (Fig.1C). To confirm these findings, we next co-treated AML cells with VEN and specific inhibitors of the mTOR pathway (rapamycin and AZD2014) or MEK pathway (CI1040) in all 5 paired parental and resistant cell lines; or with FLT3 inhibitors (quizartinib and sorafenib) in parental and resistant MV4;11 and Molm13, which harbor FLT3-ITD. The combination of VEN and AZD2014 achieved synergistic effects in all 5 parental cell lines (CI AZD2014: 0.08-0.94), and VEN/rapamycin were synergistic in 3 parental cell lines (CI rapamycin: 0.00-0.55, except 1.76 for KG-1 and 1.59 for Molm13). Combination of VEN with CI1040 achieved synergy in OCI-AML2, Kasumi, MV4;11 and Molm13 parental cell lines (CI: 0.14-0.61). Finally, VEN/FLT3 inhibitors achieved synergistic effects in MV4;11 and Molm13 parental cell lines (CI quizartinib: 0.66-0.69; CI sorafenib: 0.64-0.71). The resistant cell lines exhibited sensitivity to these inhibitors as single agents, and no synergistic effects were seen when combined with VEN. We have further induced in vivo resistance in two primary AML xenografts by treating NSG mice engrafted with 2nd passage AML cells with 100 mg/kg Q.D. VEN for 4 weeks followed by harvest of leukemic cells that repopulated the mouse after treatment discontinuation. While the proteomics, gene expression (RNAseq) and drug screening assays are in progress, preliminary immunoblotting studies demonstrated decreased expression of BCL-XL and BCL-2 (Fig.1B). In summary, we identified multiple mechanisms of acquired resistance to VEN, which ultimately modulate the balance between pro- and anti-apoptotic BCL-2 family members. Our studies indicate that upfront combination of VEN with selective inhibitors of MCL-1, or with inhibitors of specific signaling pathways, can synergistically induce apoptosis in AML cells and conceivably prevent emergence of VEN resistance. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Tyner:Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Incyte: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

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Co-Targeting MCL-1 and BCL-2 Is Highly Synergistic in BH3 Mimetic- and Venetoclax/Hypomethylating Agent-Resistant and TP53 Mutated AML

Blood ◽

10.1182/blood-2020-141417 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Bing Z Carter ◽

Po Yee Mak ◽

Wenjing Tao ◽

Vivian Ruvolo ◽

Xuan Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Research Funding ◽

Single Agent ◽

Acquired Resistance ◽

Combination Group ◽

Bh3 Mimetics ◽

Bh3 Mimetic

Venetoclax (VEN), a highly selective BCL-2 inhibitor with limited single-agent activity in AML, has shown encouraging efficacy in combination with hypomethylating agents (HMA). Nevertheless, patients relapse and have limited treatment options. Like BCL-2, MCL-1 plays critical roles in the survival of AML cells and AML stem/progenitor cells. MCL-1 is also a known resistance factor to VEN. Preclinical studies have demonstrated that combined inhibition of BCL-2 and MCL-1 is highly effective in VEN-resistant AML cells. Diverse mechanisms contribute to the resistance to VEN, and likely also to BH3 mimetics targeting MCL-1 that are currently under clinical development in AML. The effectiveness of co-targeting BCL-2 and MCL-1 in the setting of various resistance mechanisms has not been fully explored. We investigated combined inhibition of BCL-2 and MCL-1 in AML cells resistant to apoptotic stimuli through various mechanisms and demonstrate that co-inhibition of BCL-2 with VEN and MCL-1 with AMG176 synergistically targets AML cells that exhibit intrinsic or acquired resistance to BH3 mimetics in vitro and in vivo. We generated AML cells with acquired resistance to VEN (VEN-R) or AMG176 (AMG-R) by exposing the cells to increased doses of the drug and we also generated the cells genetically overexpressing BCL-2, MCL-1, or BCL-2A1. We found that both VEN-R and AMG-R MV4-11 cells expressed increased levels of MCL-1, BCL-2, and BCL2A1, but decreased BAX. Although BCL-XL levels decreased in AMG-R MV4-11 cells, BAK, PUMA, and BID levels were also markedly lower in these resistant cells compared to the parental controls. VEN or AMG176 as single agents had diminished activity against AML cells with acquired resistance not only to VEN, but also to AMG176 and AML cells genetically overexpressing MCL-1, BCL-2, or BCL2-A1. In addition, we found that TP53 mutated primary AML cells expressed low levels of BAX and that Molm13 cells acquired a TP53 mutation (R248W) expressed lower levels of BAX and were more resistant to VEN, consistent with clinical observations, and they were also more resistant to AMG176. However, when VEN and AMG176 were combined, synergy was observed (combination index < 1). We next treated AML patient samples and found that combined inhibition of BCL-2 and MCL-1 was synergistic in primary AML cells and stem/progenitor cells obtained from patients with various cytogenetics/mutations, including TP53 mutations, and from patients resistant to/relapsed from VEN- or VEN/HMA-based therapy, even when AML cells were co-cultured with bone marrow-derived mesenchymal stromal cells that mimic the bone marrow microenvironment. To demonstrate potential clinical relevance, we developed a PDX model from a clinically-acquired VEN/HMA resistant AML patient and treated the PDX-bearing mice with VEN, AMG176, and the combination. Remarkably, the combination of VEN and AMG176 demonstrated strong antileukemia activities, markedly diminished not only AML blasts but also AML stem/progenitor cells, as determined by CyTOF analysis, and significantly extended survival (median 336 vs 126 d for controls, P < 0001), while VEN (129 d) alone and even AMG176 (131 d) alone had minimal efficacy. Several mice in the combination group survived over 400 d and died probably from old age with only minimal residual leukemia. In conclusion, we demonstrate the alteration of multiple BCL-2 family proteins contributes to BH3 mimetic resistance that can be overcome by combined inhibition of MCL-1 and BCL-2. The striking effectiveness of co-targeting BCL-2 and MCL-1 in AML resistance to a BH3 mimetic via various mechanisms or to VEN/HMA suggests broad clinical applications of this strategy, and warrants clinical evaluations. Disclosures Carter: Amgen: Research Funding; Ascentage: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding.

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Voruciclib Sensitizes High Risk Diffuse Large B Cell Lymphoma to BCL2 Inhibition Mediated Cell Death and Tumor Regression

Blood ◽

10.1182/blood.v128.22.4167.4167 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4167-4167

Author(s):

Joyoti Dey ◽

William Kerwin ◽

Joseph Casalini ◽

Angela Merrell ◽

Marc Grenley ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

Treatment Options ◽

Single Agent ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Diffuse Large B Cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma in adults. Although upfront chemotherapy leads to favorable survival outcomes, relapsed or refractory patients continue to have poor prognosis with limited treatment options. In DLBCL, evasion of apoptosis - a key hallmark of cancer is mediated by functionally redundant BCL family members: BCL2, BCLxL and MCL-1. The BCL2 specific inhibitor venetoclax is approved for treating high-risk CLL, but responses in DLBCL have been limited, potentially due to compensatory upregulation of MCL-1. Currently a well-tolerated drug for inhibition of MCL-1, is unavailable in the lymphoma clinic. Voruciclib, is a novel clinical stage oral CDK inhibitor with potent activity (<10 nM) against CDKs 9, 4, 6 and 1. Multiple mechanisms for downregulation of MCL-1 activity have been described for CDK inhibitors. Arguably best characterized is transcriptional inhibition of MCL-1, a short half-life transcript, via inhibition of transcriptional regulator CDK9. We evaluated MCL-1 expression in the FFPE lymphatic tissues from 33 patients with DLBCL, and found that it was expressed in 52% of cases, of both GC (germinal center) and ABC (activated B-cell)-like type. We therefore investigated whether voruciclib could synergize with venetoclax in pre-clinical models of DLBCL via inhibition of MCL-1. In cell-based assays, exposure of DLBCL cells to voruciclib as a single agent resulted in apoptosis which was preceded by context-dependent downregulation of MCL-1. To further explore the impact of voruciclib on MCL-1 activity and DLBCL viability in vivo, we utilized Presage's CIVO tumor microinjection technology. CIVO enables investigation of multiple drugs and drug combinations simultaneously in a living tumor facilitating in vivo assessment of anti-tumor drug synergy (Klinghoffer et al. Sci. Transl Med. 2015; Dey et al. PLOS One 2016). Voruciclib was introduced as a single agent or in combination with venetoclax to DLBCL xenografts. Microinjection, resulting in localized tumor exposure to voruciclib, led to MCL-1 downregulation in vivo across multiple models of DLBCL. In contrast, tumor exposure to venetoclax led to MCL-1 upregulation. Co-exposure to voruciclib and venetoclax demonstrated that the ability of voruciclib to downregulate MCL-1 is dominant to the upregulation by venetoclax. Consistent with the hypothesis that MCL-1 compensates for loss of BCL2 function in DLBCL, synergistic cell death was observed when voruciclib was combined with venetoclax. Synergy between voruciclib and venetoclax was observed in vivo in models representing both ABC (RIVA: CI value 0.5) and GC subtypes (NUDHL1 and Toledo: CI values 0.4). Similar activity was noted when venetoclax was combined with A1210477, an investigational MCL-1 inhibitor thereby suggesting MCL-1 downregulation to play a role in the observed synergy between venetoclax and voruciclib. Consistent with these results, preliminary studies on xenografted mice have shown that systemic administration of a sub-efficacious dose of venetoclax in combination with voruciclib led to impediment of tumor growth which was greater than the effect observed with each single agent. Additional systemic studies are ongoing with venetoclax in combination with voruciclib in a panel of DLBCL models to further strengthen this observation. Based on the above findings, a Phase 1b clinical trial has been designed to evaluate the combination of voruciclib and venetoclax in patients with the goal of expediting future treatment options for relapsed/refractory DLBCL. We expect to initiate this trial at multiple centers in early 2017. Disclosures Dey: Presage Biosciences: Employment. Kerwin:Presage Biosciences: Employment. Casalini:Presage Biosciences: Employment. Merrell:Presage Biosciences: Employment. Grenley:Presage Biosciences: Employment. Ditzler:Presage Biosciences: Employment. Dixon:Presage Biosciences: Employment. Burns:Presage Biosciences: Employment. Danilov:ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy; Takeda: Research Funding; Dava Oncology: Honoraria; Prime Oncology: Honoraria. Klinghoffer:Presage Biosciences: Employment.

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