CC-92480, a Novel Cereblon E3 Ligase Modulator, Is Synergistic with Dexamethasone, Bortezomib, and Daratumumab in Multiple Myeloma

Introduction: CC-92480 is a novel cereblon E3 ligase modulator (CELMoD) with enhanced autonomous cell-killing and immunomodulatory activity against multiple myeloma (MM) cells. CC-92480 is currently in phase 1 development in a late-line myeloma patient population (NCT03374085). Here, we sought to characterize the antitumor activity of CC-92480 in combination with dexamethasone (DEX), bortezomib (BORT), or daratumumab (DARA) in MM cell lines in vitro and xenograft mouse models in vivo. Methods: CC-92480 activity in combination with DEX was evaluated in MM cell lines. Apoptosis was measured by quantification of caspase-3 activation. The effect of BORT on CC-92480-induced Ikaros and Aiolos degradation was determined by concurrent treatment of MM cells with BORT and CC-92480. β5-site proteasome activity was also determined in the same experiment. The in vitro activity of CC-92480 in combination with BORT was characterized using washout experiments to more faithfully model the short in vivo exposure but more prolonged, gradually diminishing proteasome inhibitory activity of BORT. Apoptosis and cell viability of CC-92480 with BORT were analyzed by flow cytometry. The effect of CC-92480 on CD38 expression was also evaluated across a panel of MM cell lines. The effect of CC-92480 in combination with DARA was characterized with antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. CC-92480 in combination with DEX or BORT was tested in a lenalidomide-resistant (H929-1051) xenograft mouse model. Female SCID mice were inoculated with H929-1051 cells in the right hind leg. For the DEX combination, groups of tumor-bearing mice (n = 9-10) were dosed with vehicle, DEX, or CC-92480 once daily (QD), or CC-92480 in combination with DEX throughout the study, starting when the tumor volumes reached approximately 115 mm3. For combination with BORT, mice (n = 9-10/group) were dosed with vehicle, CC-92480, or BORT, or the CC-92480 and BORT combination starting when the tumor volumes reached approximately 500 mm3. CC-92480 was administered orally QD for 3 days and BORT as a single intravenous dose. Tumor volumes were measured twice a week for the duration of the studies. Results: CC-92480 synergized with DEX in reducing cell viability and potentiated DEX-induced apoptosis in a concentration-dependent manner in MM cell lines. Of note, the combination showed activity at concentrations of both DEX and CC-92480 that had minimal activity as single agents. In the xenograft model with H929-1051 cells, the combination of CC-92480 and DEX significantly inhibited tumor growth (−84%) when compared with either agent alone (−34% and −20% for CC-92480 and DEX, respectively) and was classified as a synergistic effect using the fractional product method. Although proteasome activity is required for CC-92480-induced degradation of Ikaros and Aiolos, CC-92480 nevertheless maintained its ability to efficiently degrade Ikaros and Aiolos in the presence of doses of BORT that cause clinically relevant levels of proteasome inhibition. The in vitro combination of CC-92480 with BORT resulted in greater cytotoxic activity on MM cells than either single agent alone. The in vivo efficacy of CC-92480 and BORT, administered concurrently, showed a strongly synergistic effect with a near complete or complete tumor regression in every animal, and 6 of 9 animals remained tumor-free through an observation period extending 157 days after the control group was terminated. Anti-CD38 therapies, including DARA and isatuxumab, target CD38-expressing MM cells for killing by immune cells through cytotoxic and phagocytic mechanisms. In a panel of MM cell lines, CC-92480 treatment caused increased cell surface expression of CD38 (2-3 times that of control). Pretreatment of MM cells with CC-92480 resulted in increased DARA-mediated ADCC and ADCP compared with DMSO-treated controls. Conclusions: The strong preclinical synergy in MM cell killing exhibited by CC-92480 in combination with DEX, BORT, and with an anti-CD38 antibody (DARA), highlights its potential to bring clinical benefit to patients with MM in combination with these agents and supports the rationale for testing these combinations in clinical studies. Disclosures Wong: Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment. Bauer:Celgene Corporation: Employment, Equity Ownership. Groza:Celgene Corporation: Employment, Equity Ownership. Gaffney:Celgene: Employment. Havens:Celgene: Equity Ownership; Pfizer: Employment, Equity Ownership. Choi:AnaptysBio Inc: Employment, Equity Ownership; Celgene Corporation: Equity Ownership, Other: Formerly Employed. Lopez-Girona:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment. Cathers:Celgene Corporation: Equity Ownership; Global Blood Therapeutics (GBT): Employment. Carmichael:Celgene plc: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.

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Preclinical Combination Of The Oral Investigational Agents ACY-1215, a Selective HDAC6 Inhibitor, and Ixazomib, a Proteasome Inhibitor, Demonstrates Combination Benefit In Multiple Myeloma Cell Lines and Xenograft Models

Blood ◽

10.1182/blood.v122.21.4437.4437 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4437-4437

Author(s):

Allison J Berger ◽

Bret Bannerman ◽

Steven N Quayle ◽

Jie Yu ◽

Khristofer Garcia ◽

...

Keyword(s):

Multiple Myeloma ◽

Pharmaceutical Company ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Hdac Inhibitors ◽

Proteasome Inhibitors ◽

Employment Equity ◽

Equity Ownership

Introduction The combination of HDAC inhibitors and proteasome inhibitors has demonstrated preclinical benefit in several settings, including multiple myeloma and lymphoma, and is being explored in clinical trials testing various HDAC inhibitors in combination with proteasome inhibitors. ACY-1215 is an investigational, orally available HDAC6-selective inhibitor that has demonstrated preclinical combination benefit with bortezomib in vitro and in vivo (Santos et al, Blood 2012; 119: 2579). These preclinical studies also support the hypothesis that the improved selectivity of ACY-1215 for HDAC6 over class I HDACs (HDAC1,2,3) may provide an improved tolerability profile compared to pan-HDAC inhibitors, while still providing the anti-myeloma effect of other HDACi/proteasome inhibitor combinations. ACY-1215 is currently in a Phase I/II trial in multiple myeloma with bortezomib (VELCADE) and dexamethasone to test this hypothesis (NCT01323751). Ixazomib citrate (MLN9708) is an investigational oral proteasome inhibitor in Phase III clinical trials in multiple myeloma (NCT01850524, NCT01564537). To examine the potential efficacy of the all-oral combination of ixazomib citrate and ACY-1215, we evaluated the combination of these agents in cell lines and xenograft models of multiple myeloma. Results In vitro viability experiments in 2 multiple myeloma cell lines (RPMI-8226 and MM.1S) using a dose matrix format demonstrated a combination benefit of ACY-1215 and ixazomib over a range of concentrations, very similar to the previously reported benefit of ACY-1215 plus bortezomib. Likewise, the combination benefit of the selective HDAC6 inhibitor ACY-1215 with ixazomib was similar to the combination effect observed with the pan-HDAC inhibitor SAHA (vorinostat). Together, these in vitro studies support the hypothesis that the combination of ACY-1215 and ixazomib provides similar levels of benefit as do combinations including other HDACi/proteasome inhibitors. Furthermore, experiments in MM.1S xenograft-bearing mice demonstrated an in vivo combination benefit of ACY-1215 and ixazomib. An all-oral regimen was well tolerated when ACY-1215 was dosed at 100 mg/kg PO twice daily for 5 days per week in combination with ixazomib dosed at 5 mg/kg PO twice weekly, and the combination regimen demonstrated additive antitumor activity (Figure 1). The in vivo combination benefit of ACY-1215 and ixazomib was further demonstrated in MM.1S xenograft-bearing mice using alternate routes of administration (IV dosing of ixazomib and IP dosing of ACY-1215). The combination of ACY-1215 dosed at 30 mg/kg IP once daily for 5 days per week with ixazomib dosed IV at 1.5 mg/kg twice-weekly was also well tolerated and had striking antitumor activity. This combination regimen in fact caused regression of the MM.1S xenograft tumors below the starting volumes, and this level of activity was maintained throughout the entire 17 day dosing period (Figure 2). In an accompanying pharmacodynamic (PD) study of the PO and IP doses of ACY-1215, we confirmed selective HDAC6 inhibition in MM.1S xenograft tumors as evidenced by elevated acetylation levels of the HDAC6 substrate tubulin, with little if any change in the levels of acetylated histone H3, a class I HDAC substrate. In vivo experiments in a second xenograft model, RPMI-8226, also demonstrated a combination benefit of ACY-1215 (30 mg/kg IP for 5 days per week) with ixazomib (0.75 mg/kg IV twice-weekly). Conclusion The combination benefit of ACY-1215 and ixazomib observed here in preclinical experiments utilizing in vitro and in vivo models of multiple myeloma provides rationale for clinical evaluation of this first all-oral combination of a proteasome inhibitor with an HDAC inhibitor. Disclosures: Berger: Takeda Pharmaceutical Company Ltd: Employment. Bannerman:Takeda Pharmaceutical Company Ltd: Employment. Quayle:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yu:Takeda Pharmaceutical Company Ltd: Employment. Garcia:Takeda Pharmaceutical Company Ltd: Employment. Ciavarri:Takeda Pharmaceutical Company Ltd: Employment. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽

10.1182/blood.v114.22.2744.2744 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2744-2744

Author(s):

Xiaochuan Chen ◽

Rhona Stein ◽

Chien-Hsing Chang ◽

David M. Goldenberg

Keyword(s):

T Cell ◽

B Cells ◽

Cell Lines ◽

Hairy Cell ◽

Employment Equity ◽

Cross Presentation ◽

Equity Ownership ◽

Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Phase 1 Study of CB-839, a First-in-Class, Glutaminase Inhibitor in Patients with Multiple Myeloma and Lymphoma

Blood ◽

10.1182/blood.v126.23.3059.3059 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3059-3059 ◽

Cited By ~ 6

Author(s):

Dan T. Vogl ◽

Anas Younes ◽

Keith Stewart ◽

Keith William Orford ◽

Mark Bennett ◽

...

Keyword(s):

Multiple Myeloma ◽

Research Funding ◽

Blood Platelets ◽

Safety Data ◽

Employment Equity ◽

Efficacy Data ◽

Trough Concentrations ◽

Equity Ownership

Abstract Background: Malignant cells alter metabolism in order to enable their highly anabolic state. In addition to a massive increase in glycolysis, malignant cells frequently become dependent on glutamine to feed the TCA cycle and provide key building blocks for cell growth and proliferation. CB-839 is a first-in-class potent and selective inhibitor of glutaminase (GLS), the first step in glutamine metabolism, that has broad in vitro and in vivo anti-tumor activity in solid and heme malignancies, including multiple myeloma. GLS inhibition with CB-839 induces apoptosis and/or growth arrest in multiple myeloma and lymphoma cell lines and is synergistic with pomalidomide and lenalidomide in vitro and as well as in multiple myeloma xenograft models in vivo. Methods: CX-839-002 is an ongoing Ph1 evaluation of escalating doses of CB-839 in patients with relapsed/refractory multiple myeloma (MM) or non-Hodgkins lymphoma (NHL) with the primary objective of assessing the safety profile and selecting a recommended Phase 2 dose (RP2D). Pharmacokinetics (PK) was monitored on Days 1 and 15. Initially, CB-839 was given three times daily (TID) without food, but based on PK and safety data generated across three Ph1 studies in patients with solid and heme malignancies, the drug is now being given twice daily (BID) with meals. Results: Safety data are available for a total of 14 patients (9 MM, 4 follicular lymphoma, 1 diffuse large B cell lymphoma) that have enrolled to date during the dose escalation (100-400 mg TID and 600 mg BID). The patients have received a median of 7 prior lines of systemic therapy. CB-839 has been well tolerated with only three subjects experiencing a Gr3/4 AEs considered possibly related to study drug and there have been no discontinuations due to AEs. A similar tolerability profile has been observed across three Ph1 studies for CB-839. With a total of 119 pts treated with CB-839 across the three studies, Gr3/4 drug-related AEs have occurred in 16 subjects (13%) and 4.3% of discontinuations were due to AEs. Reversible, asymptomatic elevations in transaminases have been the primary Gr3 AEs, occurring primarily on the TID schedule in 6/59 (10.2%) pts; only one occurred among 60 pts (1.7%) receiving the BID regimen. BID dosing with 600 mg was determined to be the RP2D and combination studies with pomalidomide and dexamethasone have been initiated. The half-life of CB-839 is ~4 hr, exposure increases with dose, and trough concentrations generally remain above the target threshold of 200 ng/mL for patients receiving the RP2D. Six of 8 MM pts that received ≥ 400 mg TID achieved steady state (D15) trough concentrations above the PK target threshold while 0 of 5 pts that received ≤ 250 mg TID achieved the PK threshold. Pharmacodynamic assessment of GLS activity in MM patients was consistent with a broader PK/PD assessment (across all 3 Ph1 studies), which established clear exposure-dependent inhibition of the target in peripheral blood platelets 4 hr after the first dose of CB-839, with >90% inhibition being maintained for most patients at the RP2D. Preliminary efficacy data include confirmed stable disease in 4 of 9 evaluable MM patients. Updated efficacy data and correlative studies on clinical samples will also be presented. The first pt treated with the combination of CB-839 and pomalidomide/dexamethasone (Pd) during dose escalation received 400 mg CB-839 BID, pomalidomide at 4 mg/day (D1-21) and dexamethasone at 40 mg on Days 1, 8, 15 and 22 of each 28-day cycle. This pt had a 71% decreased in urine M-protein and an 83% reduction in serum free light chain after the first 2 cycles of treatment. This pt had 11 prior lines of therapy but not pomalidomide and had two stem cell transplants and was progressing rapidly prior to study entry. The pt has tolerated the combination well and is continuing on study. Conclusions: CB-839 has been well tolerated at and above doses that produced robust inhibition of GLS in blood platelets and in tumors. Dosing BID with food has improved the PK profile and mitigated the frequency and severity of LFT elevations, which was the primary safety signal using TID dosing. Strong preclinical combination data, an excellent clinical safety profile, and initial data with CB-839 combined with Pd provide a strong rationale for continued development of CB-839 this combination in pts with relapsed/refractory multiple myeloma. Disclosures Vogl: Constellation Pharmaceuticals: Research Funding; Calithera Biosciences: Research Funding; Celgene Corporation: Consultancy; Acetylon Pharmaceuticals, Inc.: Research Funding; Millennium Pharmaceuticals: Research Funding; GSK: Research Funding. Younes:Celgene: Honoraria; Curis: Research Funding; Sanofi-Aventis: Honoraria; Seattle Genetics: Honoraria, Research Funding; Novartis: Research Funding; Janssen: Honoraria; Takeda Millenium: Honoraria; Bristol Meyer Squibb: Honoraria; Bayer: Honoraria; Incyte: Honoraria; Johnson and Johnson: Research Funding. Orford:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Berdeja:Curis: Research Funding; Acetylon: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Takeda: Research Funding; BMS: Research Funding; Array: Research Funding; MEI: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; Onyx: Research Funding.

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Coltuximab Ravtansine (SAR3419) Demonstrates Enhanced Activity in Combination with Bendamustine, Gemcitabine and Novel Targeted Agents Such As PI3K Inhibitors in Pre-Clinical Models of Relapsed and/or Refractory Non-Hodgkins Lymphoma (NHL)

Blood ◽

10.1182/blood.v126.23.5125.5125 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5125-5125

Author(s):

Callum M Sloss ◽

Katie O'Callaghan ◽

Jutta Deckert ◽

Jenny Tsui ◽

Leanne Lanieri ◽

...

Keyword(s):

Cell Lines ◽

Single Agent ◽

Targeted Agents ◽

Employment Equity ◽

Pi3k Inhibitors ◽

Xenograft Models ◽

Equity Ownership ◽

Chemotherapy Agents

Abstract Introduction: Relapsed/refractory B-cell NHL remains an area of significant medical need. CD19 is broadly expressed on B-cell malignancies making it an ideal target for antibody-drug conjugate (ADC) based therapy. Coltuximab ravtansine is a CD19-targeting ADC consisting of a CD19-targeting antibody conjugated to the maytansinoid anti-mitotic DM4. In preclinical studies, coltuximab ravtansine has shown potent, targeted activity against NHL cell lines and xenograft models. In early clinical trials, it has been well tolerated and has shown promising signs of efficacy as both a single agent and in combination with rituximab. In the STARLYTE Phase 2 trial coltuximab ravtansine monotherapy resulted in an ORR of 44% in R/R-DLBCL that included an ORR of 21% in hard-to-treat primary refractory patients (NCT01472887). Here we describe studies aimed at the identification of combination partners for coltuximab ravtansine to further optimize clinical benefit to R/R-NHL patients. We are employing a dual approach where we investigate combination of coltuximab ravtansine with multiple, novel targeted therapy partners whilst in parallel also investigating the combination of coltuximab ravtansine with chemotherapies commonly used in the late stage R/R-NHL setting. Methods: Coltuximab ravtansine and the DM4 payload were evaluated in a high throughput screen both as single agents and in combination with a selection of novel, emerging targeted agents across a panel of twenty NHL cell lines. The combinations were evaluated in a dose-response matrix and a statistical method was used to identify combination synergies significantly superseding baseline additivity values. The in vivo efficacy of coltuximab ravtansine was additionally assessed in combination with various clinically relevant chemotherapy agents in subcutaneous xenograft models of NHL. Results: Coltuximab ravtansine and DM4 both showed potent single agent activity against the entire panel of NHL cell lines with median GI50's of 770pM and 100pM, respectively. We observed a significant correlation in the cell line sensitivity of the two compounds suggesting that sensitivity to coltuximab ravtansine is driven, at least in part, by inherent sensitivity of cells to the cytotoxic effects of the DM4 payload. In vitro combination studies for coltuximab ravtansine were performed to identify targets or pathways that result in the most prominent combination effects across the cell line panel. Analysis of the in vitro combination dose-matrix revealed particularly strong synergy between coltuximab ravtansine and various inhibitors of the PI3K/AKT/mTOR axis. Studies to examine the synergism between coltuximab ravtansine and PI3K inhibitors in in vivo models of NHL are ongoing. In order to further determine the utility of coltuximab ravtansine as part of a potential combination regimen for the treatment of R/R-NHL, we assessed the combination of coltuximab ravtansine with the chemotherapy agents bendamustine and gemcitabine in vivo. As gemcitabine is typically used in combination we assessed the efficacy of a coltuximab ravtansine with rituximab and gemcitabine in vivo. In both cases the combination with coltuximab ravtansine was significantly more efficacious than the standard-of-care alone arms. Conclusions: Coltuximab ravtansine demonstrates synergistic activity in combination with multiple PI3K pathway inhibitors across a large panel of NHL cell lines. Additionally, we have shown that combination of coltuximab ravtansine with clinically relevant late stage treatments such as bendamustine and rituximab + gemcitabine is more efficacious than the chemotherapy regimens alone. These results support the continued development of coltuximab ravtansine in R/R-NHL in combination with chemotherapy regimens and suggest that a combination of coltuximab ravtansine with PI3K inhibitors may also be of interest in the clinical setting. Disclosures Sloss: ImmunoGen, Inc.: Employment, Equity Ownership. O'Callaghan:ImmunoGen, Inc.: Employment, Equity Ownership. Deckert:ImmunoGen, Inc.: Employment, Equity Ownership. Tsui:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

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Preclinical Development of an Anti-CD38 Antibody-Drug Conjugate for Treatment of Hematological Malignancies

Blood ◽

10.1182/blood-2019-132062 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5621-5621 ◽

Cited By ~ 1

Author(s):

Lingna Li ◽

Wenyong Tong ◽

Megan Lau ◽

Katherine Fells ◽

Tong Zhu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Cell Binding ◽

Antibody Drug Conjugate ◽

Employment Equity ◽

Cd38 Expression ◽

Equity Ownership ◽

Anti Tumor Activity ◽

Antibody Drug

CD38 is a validated target for the treatment of multiple myeloma (MM). Daratumumab (Darzalex®), an anti-CD38 monoclonal antibody (mAb), has shown great clinical efficacy and has been approved for multiple myeloma treatment. However, both primary refractoriness and development of resistance to daratumumab therapy have been reported. Based on the therapeutic benefits of this CD38 antibody, we developed a CD38-targeting antibody-drug conjugate (ADC), employing a fully human anti-CD38 antibody STI-6129, identified from Sorrento's G-MAB® antibody library, and proprietary linker-toxin technology. The toxin payload is duostatin 5.2 (Duo.5.2), a microtubule inhibitor, conjugated to STI-6129 via a non-polyethylene glycol linker resulting in our lead ADC CD38-077. Cell binding studies showed that it specifically binds to CD38-positive tumor cells but not CD38-negative cell lines. The cell binding was proportional to the CD38 expression level on the cell surface. The ADC was internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody, indicating that conjugation did not change the binding characteristics of STI-6129 to its antigen. In cytotoxicity studies, CD38-077 exhibited a CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines and was more potent in cells with high CD38 expression. The cytotoxic effect of CD38-077 was also examined against human PBMC cells, as it has been reported that certain types of the immune cells express CD38. The result indicated that normal PBMC cells were generally insensitive to the ADC up to 1 µM following 120 hr exposure. We investigated the anti-tumor activity of CD38-077 in xenograft animal models of Burkitt's lymphoma and two different multiple myeloma (MM) cell lines. The studies evaluated different dose levels and dosing regimens, including single dose and multiple doses at various intervals. The data showed that the ADC has a broad, potent and CD38-dependent in vivo efficacy in all three xenograft tumor models examined. In a pharmacokinetic study in naïve mice, CD38-077 was found to be stable, with T1/2 of about 7-11 days, comparable to that of the unconjugated STI-6129 antibody. In summary, CD38-077 exhibits strong anti-tumor activity in vitro and in vivo. The ADC showed specific activity towards CD38-expressing tumors but was less active against CD38-expressing normal PBMC cells, which express relatively low levels of CD38 level and where internalization was not detectable. These results warrant further development exploration of CD38-077. Disclosures Li: Concortis Biotherapeutics: Employment, Equity Ownership. Lau:Levena Biopharma: Employment, Equity Ownership. Fells:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhu:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties. Sun:Levena Biopharma: Employment, Equity Ownership. Kovacs:Levena Biopharma: Employment, Equity Ownership. Khasanov:Levena Biopharma: Employment, Equity Ownership. Yan:Levena Biopharma: Employment, Equity Ownership. Deng:Levena Biopharma: Employment, Equity Ownership. Takeshita:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Li:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties; Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Concortis Biotherapeutics: Employment, Equity Ownership, Patents & Royalties.

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Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽

10.1182/blood.v120.21.5028.5028 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5028-5028 ◽

Cited By ~ 1

Author(s):

Deepak Sampath ◽

Elizabeth Punnoose ◽

Erwin R. Boghaert ◽

Lisa Belmont ◽

Jun Chen ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Cell Lines ◽

Single Agent ◽

Employment Equity ◽

Bone Marrow Biopsies ◽

Xenograft Models ◽

Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

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The Novel Human Double Minute (HDM)-2 Inhibitor DS-5272 Is Active Both Alone, and In Combination With Other Novel Agents, Against Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.1927.1927 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1927-1927

Author(s):

Dongmin Gu ◽

Hua Wang ◽

Zhiqiang Wang ◽

Richard E. Davis ◽

Shengli Cai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Viability ◽

Cell Lines ◽

Annexin V ◽

Myeloma Cell ◽

Novel Agents ◽

Type I ◽

Employment Equity ◽

Equity Ownership ◽

Combination Regimens

Abstract Background The ubiquitin-proteasome pathway is now a validated target for myeloma therapy given the regulatory approvals of proteasome inhibitors such as bortezomib and carfilzomib. Another logical set of targets are the E3 ligases, whose role is to facilitate the protein poly-ubiquitination typically needed for recognition by the constitutive or immunoproteasome prior to proteolysis. One of the more attractive such targets is HDM-2, the E3 ligase best known for its role in turnover of the p53 tumor suppressor, in part because p53 deletion or mutation is less common in myeloma than in solid tumors, and because it has been possible to develop agents targeting the HDM-2 p53 binding pocket. Methods Activity of the specific HDM-2 inhibitor DS-5272 (Daiichi-Sankyo) was evaluated using a panel of p53 wild-type (wt) and mutant (mut) myeloma cell lines and also against primary patient samples. Studies were performed with DS-5272 both alone, and also in combination regimens with novel agents. Tetrazolium dye-based assays were employed to determine cell viability, Annexin V staining was used to examine apoptosis, and quantitative PCR and Western blotting were used to study selected transcripts and gene products, respectively. This study was supported in part by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results DS-5272 induced potent cytotoxicity in wt p53 MM1.S, H929, and MOLP-8 myeloma cell lines, with a median inhibitory concentration (IC50) in the single micromolar range, which was reproduced in studies of primary CD138+ plasma cells but not CD138- cells from myeloma patients. This cytotoxicity was both time- and concentration-dependent, and DS-5272 was more potent than the prototypical HDM-2 inhibitors, Nutlin-3a and MI-219. A dependence of DS-5272 activity on wt p53 was demonstrated by the much lower IC50 in the wt p53 than mut p53 cell lines, and the finding that suppression of p53 with a shRNA, as well as mutation of p53 with a sequence-specific zinc finger nuclease, significantly increased the IC50. Notably, DS-5272 remained active in the presence of conditioned media from stromal cells, or key myeloma cytokines such as IL-6. The reduced viability after exposure to DS-5272 was due at least in part to activation of type I cell death, as determined by increased staining for Annexin V, activation of caspases 9 and 3, and cleavage of PARP. Other downstream effects included induction of transcription of p21, Bax, HDM-2, NOXA, and PUMA. These proteins, as well as p27 and p53 were induced, while the abundance of Survivin, CHK1, Aurora A and B, PLK1, and KIF11 was reduced. Consistent with these latter effects, DS-5272 induced some accumulation of cells at the G2/M phase, and the appearance of cells with a disordered spindle. Suppression of KIF11 (kinesin spindle protein (KSP; Eg5)), which is responsible for centromere separation and bipolar spindle formation, seemed to occur through the binding of p21 to the cell cycle genes homology region (CHR) within the KIF11 promoter. Finally, combinations of DS-5272 with the specific KIF11 inhibitor Ispinesib produced enhanced G2/M arrest, as well as a synergistic reduction in cell viability with increased levels of apoptosis. Conclusion DS-5272 is a potent and novel agent with activity against multiple myeloma, and is active both alone and in rationally designed combination regimens with other drugs. These findings provide a rationale for the clinical translation of HDM-2 inhibitors as monotherapy, and possibly with other agents such as the KSP inhibitor ARRY-520, which is also active against myeloma, for patients with wt p53 relapsed and/or refractory myeloma. Disclosures: Cai: Daiichi-Sankyo Pharma Development: Employment, Equity Ownership. Seki:Daiichi Sankyo Co., Ltd.: Employment, Equity Ownership. Tse:Daiichi-Sankyo Pharma Development: Employment, Equity Ownership.

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Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.237.237 ◽

2013 ◽

Vol 122 (21) ◽

pp. 237-237 ◽

Cited By ~ 2

Author(s):

Michael P. Rettig ◽

Matthew Holt ◽

Julie Prior ◽

Sharon Shacham ◽

Michael Kauffman ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Nuclear Export ◽

Employment Equity ◽

Advisory Committees ◽

Ic50 Values ◽

Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

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Alvocidib Potentiates the Activity of Venetoclax in Preclinical Models of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.1652.1652 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1652-1652

Author(s):

Clifford J. Whatcott ◽

James M Bogenberger ◽

Wontak Kim ◽

Hillary Haws ◽

Nanna Hansen ◽

...

Keyword(s):

Clinical Trials ◽

Cell Viability ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Employment Equity ◽

Fold Reduction ◽

Remission Rates ◽

Equity Ownership

Abstract Introduction Venetoclax (ABT-199) is an approved BCL-2 inhibitor for the treatment of patients with chronic lymphocytic leukemia (CLL). Multiple clinical trials are underway to explore its efficacy in additional indications. While venetoclax demonstrated high remission rates in combination with azacitidine in early stage clinical trials, the question of durability of responses and primary and acquired resistance remain, especially given the modest activity and rapid development of resistance as a single agent. One reported mechanism of intrinsic resistance is high expression of other BCL-2 family proteins, including MCL-1. We and others have demonstrated that the CDK9 inhibitor, alvocidib, can mediate transcriptional repression of anti-apoptotic MCL-1. It has also been shown that alvocidib can increase pro-apoptotic BIM, a dual activator and sensitizer BH3-only protein that can directly induce apoptosis and simultaneously inactivate anti-apoptotic BCL-2 family proteins such as MCL-1 and BCL-2, thus having the same effect on mitochondria-associated apoptosis as MCL-1 down-regulation, with the potential to directly induce apoptosis. An alvocidib-containing cytotoxic chemotherapy regimen demonstrated favorable remission rates in high-risk AML patients over standard therapy in a randomized Phase 2 trial indicating its potential role and safety in AML. We hypothesized that alvocidib and venetoclax would synergize against AML cells by shifting the overall balance of pro- and anti-apoptotic BCL-2 proteins in favor of apoptosis and thus represent a novel active treatment regimen in AML. Aims This study seeks to examine the efficacy of a treatment regimen containing alvocidib and venetoclax in multiple preclinical studies, including in vivo models of AML. Methods Cell viability assays interrogating alvocidib and venetoclax activity in cell lines were performed using CellTiter-Glo according to manufacturer's protocol. mRNA/miRNA expression changes were assessed using standard RT-PCR technique. Protein expression changes were assessed using standard western immunoblotting technique. To assess the efficacy of an alvocidib and venetoclax combination on tumor growth in an in vivo model, the OCI-AML3 xenograft mouse model and ex vivo studies with AML patient samples were performed. Results Herein we demonstrate that alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion in 3 out of 4 AML cell lines analyzed, while in cells where alvocidib did not reduce MCL-1 protein levels (i.e. MOLM-13) a dose-dependent decrease in miR17-92, and concomitant increase in BIM protein was observed after 24 hours of alvocidib treatment. The alvocidib-venetoclax combination resulted in very strong synergistic reductions of cell viability (with combination indices [CI] of 0.4 to 0.7), both in venetoclax-sensitive and resistant cells. The venetoclax-sensitive lines, MV4-11 and MOLM-13, exhibited 5- to 10-fold reduction of venetoclax EC50 values in the low nM range when combined with only 80 nM alvocidib. Importantly, venetoclax-resistant lines, OCI-AML3 and THP-1, exhibited at least 20-fold reduction of venetoclax EC50 values from near 1 µM to 10-50 nM, when combined with 80 nM alvocidib.In the venetoclax-resistant OCI-AML3 xenograft model, single agent alvocidib and venetoclax achieved tumor growth inhibition (TGI) of 9.7 and 31.5%, respectively, while the combination achieved 87.9% TGI at the same dose levels of individual drugs. Conclusions Taken together, our data suggest that the combination of alvocidib with venetoclax is highly synergistic in vitro and in vivo, in both venetoclax-sensitive and -resistant AML across a heterogeneous genomic background. The particularly high level of synergy achieved in venetoclax-resistant cell lines highlights the central importance of both BCL-2 and MCL-1-mediated cell survival in AML. Importantly, the addition of alvocidib to venetoclax treatment reduced IC50s to clinically achievable concentrations. Therefore, we conclude that an alvocidib/venetoclax combination may be a novel approach for the treatment of AML and warrants further pre-clinical and clinical validation. Disclosures Whatcott: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Mesa:Celgene: Research Funding; Galena: Consultancy; Promedior: Research Funding; Ariad: Consultancy; Novartis: Consultancy; CTI: Research Funding; Incyte: Research Funding; Gilead: Research Funding. Peterson:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

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