scholarly journals Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1442-1442
Author(s):  
Xiangmeng Wang ◽  
Po Yee Mak ◽  
Wencai Ma ◽  
Xiaoping Su ◽  
Hong Mu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

scholarly journals The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4044-4044
Author(s):  
Blake S Moses ◽  
Jennifer Fox ◽  
Xiaochun Chen ◽  
Samantha McCullough ◽  
Sang Ngoc Tran ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
Leukemia Cell ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Equity Ownership

Abstract Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549). Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs. Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers. Disclosures Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.

scholarly journals In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3100-3100 ◽  
Author(s):  
Ken Flanagan ◽  
Muntasir M Majumder ◽  
Romika Kumari ◽  
Juho Miettinen ◽  
Ana Slipicevic ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Plasma Cell ◽  
Light Chain ◽  
Research Funding ◽  
Plasma Cells ◽  
Al Amyloidosis ◽  
Advisory Committees

Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function. Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population. To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells invitro and invivo. Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored. Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing. We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress. As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain invitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo. Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis. Figure 1 Disclosures Flanagan: Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3830-3830
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Jeffrey Ecsedy ◽  
Giada Bianchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Research Funding ◽  
Thymidine Incorporation ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Abstract 3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Pooled Safety Analysis From Phase (Ph) 1 and 2 Studies of Carfilzomib (CFZ) In Patients with Relapsed and/or Refractory Multiple Myeloma (MM)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1954-1954 ◽  
Author(s):  
Seema B. Singhal ◽  
David Samuel diCapua Siegel ◽  
Thomas Martin ◽  
Ravi Vij ◽  
Michael Wang ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Single Agent ◽  
Study Drug ◽  
Underlying Disease ◽  
Tolerability Profile ◽  
Treatment Schedule ◽  
Advisory Committees

Abstract Abstract 1954 Background: Carfilzomib (CFZ) is a novel, highly selective epoxyketone proteasome inhibitor that produces potent and sustained proteasome inhibition both in vitro and in vivo. CFZ appears to lack many of the off-target activities frequently associated with bortezomib (BTZ). This lack of off-target activity may account for observed differences in tolerability seen with CFZ including lack of significant neuropathy and minimal neutropenia and diarrhea. To date, single agent CFZ has been evaluated in Ph 1 and 2 studies in >600 patients, and the vast majority of patients treated had relapsed and/or refractory (R/R) MM. In these settings, CFZ has demonstrated durable single-agent activity and was well-tolerated in patients with advanced stage disease with co-morbidities including baseline neuropathy or renal insufficiency. Here we present the results of parallel safety analyses of patients from four Ph 1 and 2 studies with CFZ. Materials and Methods: The present safety analyses were based on data accumulated from patients enrolled in the following trials: PX-171-003 A0 (R/R MM), PX-171-003 A1 (R/R MM), PX-171-004 (relapsed MM), and PX-171-005 (R/R MM with varying degrees of renal function). In all studies, the treatment schedule was based on a 28-day cycle, dosing CFZ QDx2 each week for 3 weeks (Days 1, 2, 8, 9, 15, 16) with 12 days of rest. Doses of CFZ ranged from 15–20 mg/m2 in cycle 1 (005 [15 mg/m2], 003 A0 and A1, 004 [20 mg/m2]). In three studies CFZ was escalated to 27 mg/m2 after the first cycle, as tolerated (003- A1, 004-BTZ naïve subset and 005). In PX-171-005, low-dose dexamethasone was added in the majority of patients. Results: CFZ was well-tolerated by patients across the 4 studies analyzed. The most common treatment-emergent adverse events (AEs) included fatigue, anemia, nausea, dyspnea, and thrombocytopenia. Detailed descriptions of the incidence of treatment-related AEs (all Grades (G) in ≥25% of pts; ≥G3 in ≥5% of pts) across studies are presented in the table. Peripheral neuropathy (PN) occurred infrequently across all 4 studies (N= 517), with only 20 patients (3.9%) experiencing PN of any G and only 2 patients (0.4%) with G3 PN. Febrile neutropenia was likewise uncommon, occurring in only 3 patients (0.6%). Serious treatment emergent AEs (SAEs) occurring in ≥1% of patients and considered possibly/probably related to study drug across all 4 studies included: pneumonia (3.5%), congestive cardiac failure (2.5%), acute renal failure (1.7%), pyrexia (1.2%), and dyspnea (1%). Conclusions: Despite a substantial disease burden present in the patient populations described here, CFZ was well-tolerated by patients with MM across all studies examined. The excellent safety/tolerability profile of CFZ has permitted prolonged administration (in some cases over 24 mos of continuous therapy including extension study) with minimal dose modifications or discontinuations due to toxicity. The low levels of neuropathy seen with CFZ make this agent a potentially important treatment option for patients with pre-existing neuropathy from either underlying disease or prior neuropathic anti-myeloma therapy. Disclosures: Singhal: Celgene: Speakers Bureau; Takeda/Millenium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding. Siegel:Millenium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Martin:Celgene: Honoraria; Onyx: Consultancy. Vij:Onyx: Honoraria. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotec: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Kukreti:Celgene: Honoraria; Roche: Honoraria; Ortho Biotech: Honoraria. Zonder:Millenium: Consultancy, Honoraria, Research Funding; Cephalon: Research Funding; Celgene: Honoraria. Wong:Onyx Pharmaceuticals: Employment. McCulloch:Onyx Pharmaceuticals: Employment. Kauffman:Onyx Pharmaceuticals: Employment. Niesvizky:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millenium: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding. Stewart:Millennium: Consultancy; Celgene: Honoraria. Jagannath:Millenium, OrthoBiotec, Celgene, Merck, Onyx: Honoraria; Imedex, Medicom World Wide, Optum Health Education, PER Group: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 237-237 ◽  
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.

scholarly journals Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3950-3950
Author(s):  
Christopher A. Eide ◽  
Stephen E Kurtz ◽  
Andy Kaempf ◽  
Nicola Long ◽  
Jessica Leonard ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership

Background: In patients with acute lymphoblastic leukemia (ALL), patient outcomes vary considerably by patient age group, specific genetic subtypes, and treatment regimen. Large-scale sequencing efforts have uncovered a spectrum of mutations and gene fusions in ALL, suggesting that combinations of agents will be required to treat these diseases effectively. Previous preclinical studies have shown efficacy of the BCL2 inhibitor venetoclax alone or in combination in ALL cells (Chonghaile et al., Can Disc 2014; Leonard et al, STM 2018), and the multi-kinase inhibitor ibrutinib (approved for patients with chonic lymphoblastic leukemia (CLL)) has also shown potent activity in subsets of ALL (Kim et al., Blood 2017). However, the combination of ibrutinib and venetoclax has not been evaluated to date in patients with ALL. Methods: We used a functional ex vivo screening assay to evaluate the potential efficacy of the combination of ibrutinib and venetoclax (IBR+VEN) across a large cohort (n=808) of patient specimens representing a broad range of hematologic malignancies. Primary mononuclear cells isolated from leukemia patients were plated in the presence of graded concentrations of venetoclax, ibrutinib, or the combination of both FDA-approved drugs. IC50 and AUC values were derived from probit-based regression for each response curve. A panel of clinical labs, treatment information, and genetic features for tested ALL patient specimens was collated from chart review. Single and combination drug treatment sensitivity were compared within groups by Friedman test, across groups by Mann-Whitney test, and with continuous variables by Spearman rank correlation. Results: Consistent with clinical data and previous literature, IBR+VEN was highly effective in CLL specimens ex vivo (median IC50=0.015 µM). Intriguingly, among specimens from 100 unique ALL patients, we also observed that IBR+VEN demonstrated significantly enhanced efficacy by AUC and IC50 compared to either single agent (p<0.001; median IC50=0.018 µM). In contrast, evaluation of this combination on primary mononuclear cells from two healthy donors showed little to no sensitivity. Breakdown of combination sensitivity (as measured by AUC) by a variety of clinical and genetic features revealed no associations with gender or specimen type. Among continuous variables tested, age was modestly correlated with combination AUC (Spearman r = 0.26) and increased blasts in the bone marrow were associated with increased sensitivity to the combination (Spearman r = -0.41; p = 0.0068). More broadly, specimens from patients with B-cell precursor disease (B-ALL) were more sensitive to IBR+VEN than those with T-cell precursor leukemia (T-ALL) (p = 0.0063). Within the B-ALL patient samples, those harboring the BCR-ABL1 fusion were significantly less sensitive to IBR+VEN than other subtypes of B-ALL (p = 0.0031). Within the T-ALL subset, there was a trend toward reduced sensitivity in patients with evidence of mutations in NOTCH1, though statistical significance was not reached. Evaluation of the combination using an expanded 7x7 concentration matrix in human ALL cell lines revealed varying degrees of sensitivity. For example, IBR+VEN showed enhanced efficacy in RCH-ACV B-ALL cells and showed synergy for the majority of drug-pair concentrations by the highest single agent (HSA) method (ibrutinib, venetoclax, and combination IC50: 0.60, 3.4, and 0.28 uM, respectively). Conclusion: Our findings suggest that the IBR+VEN combination, currently approved for patients with CLL, also demonstrates impressive efficacy against primary leukemia cells from ALL patients, warranting further investigation as a treatment strategy in the clinic to continue to improve outcomes for patients. Disclosures Leonard: Amgen: Research Funding. Druker:Cepheid: Consultancy, Honoraria; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Gilead Sciences: Other: former member of Scientific Advisory Board; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees. Tyner:Petra: Research Funding; Agios: Research Funding; Array: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Array: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Syros: Research Funding; Takeda: Research Funding; Petra: Research Funding; Agios: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Gilead: Research Funding; Incyte: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Janssen: Research Funding; Genentech: Research Funding.

scholarly journals Acid Ceramidase (ASAH1) Mediates Intrinsic and Intercellular Transfer of Proteasome Inhibitor Resistance in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1206-1206
Author(s):  
Ryan T Bishop ◽  
Tao Li ◽  
Raghunandan R Alugubelli ◽  
Oliver Hampton ◽  
Ariosto Siqueira Silva ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Resistant Cell ◽  
Clinical Samples ◽  
In Vivo Model ◽  
Acid Ceramidase ◽  
Advisory Committees

Abstract INTRODUCTION: Despite proteasome inhibitors (PIs) improving multiple MM (MM) outcomes, patients often become resistant. Identifying mechanisms of resistance with translational potential are an urgent unmet clinical need. Preliminary studies from our group have identified that the therapeutically targetable acid ceramidase, ASAH1, is a key mediator of PI resistance and its presence in extracellular vesicles (EVs) derived from resistant MM cells, confers PI resistance on drug naïve MM cells. METHODS: Nanosight technology, transmission electron microscopy and immunoblot were used to define EVs. Viability and apoptosis assays were used to determine the effects of EVs and inhibitors on resistance acquisition/sensitization to PIs. LC-MS was used to interrogate EV cargo contents. Clinical relevance of ASAH1 was determined in multiple human data cohorts (M2GEN and MMRF CoMMpass). Genetic (shRNA) and pharmacological (ceranib-2) approaches were used to assess the role of ASAH1 mechanistically in vitro and in vivo using multiple isogenic naïve and PI resistant cell lines, patient derived CD138+ MM cells and NSG mouse models. RESULTS: Co-culture of sensitive MM cells with resistant MM-EVs alone significantly protected against PI cytotoxicity. Proteomic profiling revealed high levels of ASAH1 in EVs derived from PI resistant MM cells. Further, we observed ASAH1 is abundant in lysates of multiple PI resistant cell lines compared to their isogenic drug sensitive counterparts. In human datasets, high ASAH1 expression was noted in PI resistant MM patients compared to those newly diagnosed and correlated with significantly shorter survival times. Mechanistically, knockdown of ASAH1 led to reduced conversion of ceramide to sphingosine 1-phosphate (S1-P) and decreased expression/activity of the anti-apoptotic proteins MCL-1, BCL2 and BCL-xL and increases in pro-apoptotic BIM and NOXA. Notably, ASAH1 knockdown also significantly sensitized the cells to PI treatment and this effect was rescued by addition of exogenous S1-P. Pharmacological inhibition of ASAH1 with ceranib-2 also sensitized resistant cells to PI treatment and prevented EV mediated resistance transfer in vitro. This was recapitulated ex vivo with human clinical samples. Our orthotopic in vivo model using PI-resistant U266-PSR cells show that ceranib-2 is highly effective in limiting the growth of PI-resistant disease, protecting against MM induced bone disease, and increasing overall survival compared to both bortezomib and vehicle controls. CONCLUSION: We define the ceramidase ASAH1 as a novel, druggable target for the treatment of PI resistant MM. Disclosures Hampton: M2Gen: Current Employment. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Exploring the Possibility of Using Herpes Simplex Virus in Oncolytic Virotherapy of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4467-4467 ◽  
Author(s):  
Jayeeta Ghose ◽  
Luke Russell ◽  
Enrico Caserta ◽  
Ramasamy Santhanam ◽  
Alena Cristina Jaime-Ramirez ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Flow Analysis ◽  
Vero Cells ◽  
Oncolytic Virotherapy ◽  
Advisory Committees ◽  
Simplex Virus

Abstract Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Approximately, 80,000 people have died of the disease in the United States and 25,000 new cases are registered every year. Majority of patients develop resistance to current therapeutic treatments and die within 5-10 years of diagnosis. Thus, need of novel therapeutic intervention is extremely urgent. Although the field of oncolytic virotherapy (OV) based on using viruses with natural or engineered tumor selective replication to intentionally infect and kill tumor cells has been extensively explored for the treatment of solid tumors, only few data are available for the treatment of hematopoietic malignancies. Our laboratory was one of the first to show that OV using Reovirus can be an effective therapeutic strategy for the treatment of MM in vitro and in MM patients. In this work we aim at exploring the possibility of using genetically engineered HSV1 (Herpes Simplex Virus) for the treatment of MM. HSV1 is an enveloped, double stranded DNA virus. Engineered HSV1 (HSVQ) has both copies of viral gene important in viral replication in normal cells viz., ICP 34.5 gene deleted and has one copy of GFP inserted into viral ICP6 gene. Such engineered virus has been used for cancer cell selective killing in preclinical and clinical studies for the treatment of several types of solid tumors including melanoma and glioblastoma multiforme. In this study, we investigated the biological and preclinical impact of HSVQ on MM cell in vitro and in vivo. Method: Recombinant HSVQ was amplified in African green monkey kidney epithelial Vero cells, purified by sucrose density gradient centrifugation and titrated by plaque assay on Vero cells. Several MM cell lines (MM1.S, U266, RPMI8226, L363, NIH-H929) were infected with HSVQ at Multiplicity of Infection (MOI) 0.01 to 5. Fluorescence microscopy and flow cytometry analysis were used to assess MM cell infectivity with the virus. RT-PCR was performed to detect presence of viral genome in MM cell lines. Viral replication assays were also performed. Cell proliferation and apoptotic assays including MTT Assay, Tryphan Blue exclusion test, LIVE/DEAD cell viability staining and Annexin/7-AAD assays were done to determine viability of virus infected MM cells. Western Blot analysis was carried out to determine endoplasmic reticulum (ER) stress response mediated by ERK, Hsp90, Bip/GRP78, Hsp40 and apoptosis in HSVQ treated MM cells. Total bone marrow (BM) cells obtained from MM patients were infected with HSVQ and multi parametric flow analysis was performed to determine infectivity and specific killing of CD138+ MM cells by the virus. To study in vivo anti-tumorigenic properties of HSVQ, 12.5 x106 GFP/Luc + MM1.S or NIH-H929 cells were subcutaneously injected into the right flank of 20 NOD-SCID mice. Two weeks after injection, mice with comparable size tumors were randomly divided (5 animal for each treatment group) and treated twice with 1x107PFU (Plaque Forming Unit) HSVQ for 2 weeks or with saline. Tumor growth was measured to determine anti tumorigenic effect of HSVQ on MM tumors. Results and Conclusion: Fluorescence microscopy and flow cytometry revealed that MM cell lines can be effectively infected with and killed by HSVQ even at MOI as low as 0.1. Under such conditions, Western Blot analysis revealed increased BAX expression, decreased BCL2 expression and cleavage of Caspase 3 and PARP indicating apoptosis of virus infected cells. Interestingly, multi parametric flow analysis revealed that HSVQ specifically infects and kills CD138+ MM plasma cells in a total population of BM cellular fraction isolated from MM patients. Moreover in vivo preclinical data show that HSVQ dramatically reduces tumor volume (p<0.001) in both MM.1S and NIH-H929 xenograft mouse models. Thus, from the preliminary observations, it can be concluded that HSVQ can selectively infect and induce apoptosis in myeloma cells. Mechanisms of HSVQ replication in MM cells and induced MM cell killing are being currently investigated. Here for the first time we are providing clear evidences that HSVQ can infect and specifically kill MM cells supporting the idea of the use of HSV for the treatment of MM. Moreover, since the backbone of HSVQ can be further engineered, it can be used to specifically deliver anti-angiogenic and anti-inflammatory genes to MM cells for the treatment of MM. Disclosures Hofmeister: Arno Therapeutics, Inc.: Research Funding; Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Incyte, Corp: Membership on an entity's Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Signal Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1974-1974 ◽  
Author(s):  
Adam D. Cohen ◽  
J. Joseph Melenhorst ◽  
Alfred L. Garfall ◽  
Simon F Lacey ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

scholarly journals Synergistic Anti-Leukemic Activity with Combination of FLT3 Inhibitor Quizartinib and MDM2 Inhibitor Milademetan in FLT3-ITD Mutant/p53 Wild-Type Acute Myeloid Leukemia Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2720-2720 ◽  
Author(s):  
Michael Andreeff ◽  
Weiguo Zhang ◽  
Prasanna Kumar ◽  
Oleg Zernovak ◽  
Naval G. Daver ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Combination Treatment ◽  
Research Funding ◽  
Single Agent ◽  
Wild Type ◽  
Advisory Committees ◽  
Mouse Xenograft Model ◽  
Equity Ownership ◽  
Mdm2 Inhibitor

Abstract Background: MDM2, a negative regulator of the tumor suppressor p53, is overexpressed in several cancers including hematological malignancies. Disrupting the MDM2-p53 interaction represents an attractive approach to treat cancers expressing wild-type functional p53. Anticancer activity of small molecule MDM2 inhibitor milademetan (DS-3032b) has been demonstrated in preclinical studies and in a phase 1 trial in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome. Quizartinib is a highly selective and potent FLT3 inhibitor that has demonstrated single-agent activity and improvement in overall survival in a phase 3 clinical study in relapsed/refractory AML with FLT3-internal tandem duplication (FLT3-ITD) mutations. We present here the preclinical studies exploring the rationale and molecular basis for the combination of quizartinib and milademetan for the treatment of FLT3-ITD mutant/TP53 wild-type AML. Methods: We investigated the effect of quizartinib and milademetan combination on cell viability and apoptosis in established AML cell lines, including MV-4-11, MOLM-13 and MOLM-14, which harbor FLT3-ITD mutations and wild type TP53. Cells were treated with quizartinib and milademetan at specified concentrations; cell viability and caspase activation were determined by chemiluminescent assays, and annexin V positive fractions were determined by flow cytometry. We further investigated the effect of the combination of quizartinib and the murine specific MDM2 inhibitor DS-5272 in murine leukemia cell lines Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD+F691L and Ba/F3-FLT3-ITD+D835Y, which harbor FLT3-ITD, ITD plus F691L and ITD plus D835Y mutations, respectively. F691L or D835Y mutations are associated with resistance to FLT3-targeted AML therapy. In vivo efficacy of combination treatment was investigated in subcutaneous and intravenous xenograft models generated in male NOD/SCID mice inoculated with MOLM-13 and MV-4-11 human AML cells. Results: Combination treatment with milademetan (or DS-5272) and quizartinib demonstrated synergistic anti-leukemic activity compared to the respective single-agent treatments in FLT3 mutated and TP53 wild type cells. Combination indices (CIs) were 0.25 ± 0.06, 0.61 ± 0.03, 0.62 ± 0.06, 0.29 ± 0.004 and 0.50 ± 0.03, respectively, in MV-4-11, MOLM-13, MOLM-14, Ba/F3-FLT3-ITD+F691L and D835Y cell lines, all of which harbor FLT3-ITD or ITD plus TKD point mutations. The combination regimen triggered synergistic pro-apoptotic effect in a p53-dependent manner as shown by annexin-V staining and caspase 3/7 assays. Mechanistically, the combination treatment resulted in significant suppression of phospho-FLT3, phospho-ERK and phospho-AKT and anti-apoptotic Bcl2 family proteins (eg, Mcl-1), as well as up-regulation of p53, p21 and pro-apoptotic protein PUMA, compared to single agent treatments. Of note, the combination regimen also exerted a synergistic pro-apoptotic effect on venetoclax (BCL-2 inhibitor)-resistant MOLM-13 cells (CI: 0.39 ± 0.04) through profound suppression of Mcl-1. In an in vivo study using the MOLM-13 subcutaneous mouse xenograft model, quizartinib at 0.5 and 1 mg/kg and milademetan at 25 and 50 mg/kg demonstrated a significant tumor growth inhibition compared with vehicle treatment or respective single-agent treatments. In MV-4-11 intravenous mouse xenograft model, the combination of quizartinib plus milademetan showed a significantly prolonged survival, with no animal death in the combination group during the study period, compared to respective single agent treatments and untreated control (Figure). Conclusion: Synergistic anti-leukemic activity was observed for quizartinib plus milademetan combination treatment in preclinical AML models. A phase I clinical trial of quizartinib/milademetan combination therapy in patients with FLT3-ITD mutant AML is underway. Figure. Effects of quizartinib, milademetan and their combination on survival of mice intravenously inoculated with human MV-4-11 AML cells Disclosures Andreeff: Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Oncolyze: Equity Ownership; Astra Zeneca: Research Funding; Reata: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; SentiBio: Equity Ownership. Kumar:Daiichi Sankyo: Employment, Equity Ownership. Zernovak:Daiichi Sankyo: Employment, Equity Ownership. Daver:Pfizer: Research Funding; ImmunoGen: Consultancy; Otsuka: Consultancy; Karyopharm: Research Funding; Alexion: Consultancy; ARIAD: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Karyopharm: Consultancy; Novartis: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Kiromic: Research Funding; Sunesis: Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Sunesis: Consultancy. Isoyama:Daiichi SANKYO CO., LTD.: Employment. Iwanaga:Daiichi Sankyo Co., Ltd.: Employment. Togashi:Daiichi SANKYO CO., LTD.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment.

Sign in / Sign up

Export Citation Format

Share Document