Development of Astatine-211 (211At)-Based Anti-CD123 Radioimmunotherapy for Acute Leukemias and Other CD123+ Hematologic Malignancies

Abstract Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked "on-target, off tumor cell" toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells. Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting ("biodistribution") and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line. Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4>5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P<0.0001 for Ctrl vs. 10 µCi, P<0.004 for 10 µCi vs. 40 µCi), demonstrating potent in vivo anti-leukemia efficacy of a single dose of 211At-CD123 RIT. Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. Figure 1 Figure 1. Disclosures Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

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

Blood ◽

10.1182/blood-2018-99-113040 ◽

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.

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Selective Targeting of Histone Deacetylase 11 Disables Metabolism of Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-127235 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 474-474

Author(s):

Vasundhara Sharma ◽

Lanzhu Yue ◽

Nathan P. Horvat ◽

Agni Christodoulidou ◽

Afua Adutwumwa Akuffo ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Hdac Inhibitors ◽

Leukemia Cell ◽

Cell Types ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Leukemia Cell Lines

Introduction: Acetylated histone and non-histone proteins are pharmacologic targets for both solid and hematological cancers including myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies driven by aberrant JAK2/STAT signaling. MPNs are characterized by epigenetic alterations, including aberrant acetylation, which makes this disease particularly interesting for targeting with HDAC inhibitors. Four classes of histone deacetylases (Class I-IV HDACs) regulate gene transcription and modulate cellular processes that drive the initiation and progression of cancer. Pan-HDAC and class I-selective HDAC inhibitors have gained traction in clinical settings, yet we reasoned that specific targeting of the 18 distinct HDAC proteins may establish roles for select HDACs as therapeutic vulnerabilities in MPNs. Methods: To explore the roles of individual HDACs in MPN, we first conducted an inhibitor screen of compounds having distinct HDAC selectivity based on electrophoretic mobility shift assays with full-length human HDAC proteins expressed in baculovirus and unique peptide substrates. Ultra-specific HDAC6 compounds were initially targeted for analysis based on its previously defined role in HSP90-mediated JAK2 stabilization and translation. Survival of MPN cell line models, MPN patient samples, leukemia cell lines, and MPN disease progression in mice transplanted with Hdac6-/-, and Hdac11-/- hematopoietic stem cells (HSCs) transduced with the MPLW515L oncogene, as well as Tg-Hdac11-eGfp mice were used to show the role of HDAC6 and HDAC11 in oncogene-driven and homeostatic hematopoiesis. As further proof of specificity, HDAC6 and HDAC11 were genetically ablated in MPN model cell lines using either RNA interference or inducible shRNA. For HDAC11 substrate identification, a combination of RNA-seq, acetylated proteome (SILAC), global metabolomics (LC-MS), Seahorse metabolic assays (Agilent Technologies), enzymatic assays, and acetylation-specific immunoblotting and mutation profiling were performed (Fig. 1). Results: Despite the established interplay between HDAC6, HSP90 and JAK2, neither a highly selective HDAC6 inhibitor, HDAC6 silencing, nor the Hdac6 deficiency suppressed MPN pathogenesis, although there were clear effects on the acetylation of α-tubulin, a well characterized HDAC6-selective substrate. Intriguingly, both inhibition of HDAC11 activity with highly-specific HDAC11 inhibitors and silencing HDAC11 using an inducible validated shRNA, identified HDAC11 as a therapeutic vulnerability for multiple human MPN cell lines. The Tg-Hdac11-eGFP reporter mice showed that HDAC11 is expressed in several hematopoietic cell types, including myeloid cells, erythroblasts, and megakaryocytes. Thus, Hdac11-/- and Hdac11+/+MPLWT bone marrow were examined for steady-state hematopoiesis and transplantation chimerism. These studies demonstrated that HDAC11 does not contribute to homeostatic or transplantated bone marrow reconstitution. However, in the oncogenic MPL model, recipient mice transplanted withoncogenic MPLW515L-expressing Hdac11-deficient HSCs displayed markedly impaired cytokine-independent colony-formation, had less fibrosis, and displayed improved survival in primary and secondary MPN hematopoietic stem cell transplantation; thus HDAC11 contributes to MPN pathogenesis (Fig. 1). Studies in additional leukemia cell lines, including THP-1, HL-60, and mantle lymphoma cell lines, but not in Ramos or K562 cells, established that HDAC11 contributes to oncogene-driven events in other cell types. Mechanistically, RNA-seq, SILAC proteomics, and metabolic profiling revealed that HDAC11 controls aerobic glycolysis by deacetylating Lys343 of the glycolytic enzyme enolase-1 (ENO1), functionally inactivating ENO1. Finally, the effects of targeting HDAC11 on metabolism were augmented by blocking compensatory pathways of oxidative phosphorylation that are induced via JAK2V617Fand MPLW515L oncogenic signaling. Conclusions: Our comprehensive screens of HDAC inhibitors, coupled with our biological, in vivo and molecular studies, indicate that HDAC11 is an attractive and potent target for disabling MPN metabolism and pathogenesis. These finding support the rationale for further development of clinical HDAC11 inhibitors for the treatment of metabolically-active cancers such as MPNs. Disclosures Pinilla Ibarz: Teva: Consultancy; TG Therapeutics: Consultancy; Sanofi: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Reuther:Incyte Corporation: Research Funding. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Lilly: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria. Verma:BMS: Research Funding; Janssen: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Epling-Burnette:Incyte Corporation: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Forma Therapeutics: Research Funding.

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Development of Cirmtuzumab Antibody-Drug Conjugates (ADCs) Targeting Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

Blood ◽

10.1182/blood-2018-99-119447 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1862-1862 ◽

Cited By ~ 1

Author(s):

Yousaf A. Mian ◽

George F. Widhopf II ◽

Thanh-Trang Vo ◽

Katti Jessen ◽

Laura Z. Rassenti ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Half Life ◽

Research Funding ◽

Leukemia Cell ◽

Volume Distribution ◽

Advisory Committees ◽

High Affinity

Abstract ROR1 is an onco-embryonic surface antigen expressed on chronic lymphocytic leukemia (CLL) and a variety of other cancers, but not on most normal adult tissues. We generated a humanized IgG1 monoclonal antibody (mAb) cirmtuzumab (formerly UC-961) that binds with high affinity to a specific extracellular epitope of human ROR1 and that can block Wnt5a-induced ROR1 signaling (Yu, J et al, J Clin Invest126:585, 2016; Yu, J et al, Leukemia31:1333, 2017). Preclinical studies found that cirmtuzumab did not react with normal post-partem cells and had a pharmacokinetic (PK) volume distribution in primates consistent with a lack of off-target binding to normal tissues. We evaluated cirmtuzumab in a phase I clinical trial involving patients with relapsed-refractory CLL (Choi MY, et al, Cell Stem Cell22:951, 2018); the drug was well-tolerated at doses ≤20 mg/kg (highest dose tested) without dose-limiting toxicity. PK studies showed cirmtuzumab had a half-life of 32.4 days with no evidence for development of neutralizing antibodies or off-target sequestration of infused antibody. Furthermore, cirmtuzumab effected partial down-modulation of leukemia-cell ROR1 in patients treated with doses ≥2 mg/kg. In vitro confocal microscopy studies showed that this down-modulation was caused by internalization of cirmtuzumab-ROR1 complexes into lysosomal compartments and concomitant steady-state re-expression of nascent surface ROR1. Because of its high specificity, in vivo stability, long serum half-life, and potential capacity to concentrate conjugated drugs into lysosomal compartments, cirmtuzumab appeared ideally suited to serve as the targeting moiety in anti-ROR1 ADCs. We therefore examined cirmtuzumab-based ADCs in collaboration with VelosBio Inc., evaluating multiple linker/payload chemistries, both as single agents and in combinations. We selected for further testing cirmtuzumab-ADC-7, a cirmtuzumab-linker-monomethyl auristatin E (MMAE) ADC that preserves the high-affinity binding specificity of cirmtuzumab and allows for ROR1-targeted intracellular release of MMAE. We found cirmtuzumab-ADC-7 was selectively cytotoxic for ROR1+ CLL and mantle-cell lymphoma (MCL) cell lines at nM concentrations in vitro. Moreover, cirmtuzumab-ADC-7 caused dramatic and sustained in vivo clearance of adoptively-transferred ROR1+ leukemia cells generated from ROR1xTCL1 transgenic mice (Widhopf G, et al, PNAS111:793, 2014), ROR1+ MCL-xenografts, or ROR1+ cancer patient-derived xenografts (PDX). Further, treatment caused dose-dependent and statistically significant decreases in total cancer burden with complete regressions of tumor in multiple animals; no effect on tumor-clearance was observed in mice treated with a control MMAE-ADC of irrelevant specificity. Recently we identified that miR-15a/16-1, which commonly are deleted/downregulated in CLL, target both BCL2 and ROR1, thereby accounting in part for the direct relationship we observed between the levels of BCL2 and levels of surface ROR1 expressed by CLL of different patients (Rassenti, LZ, et al,PNAS114:10731, 2017). Because high level expression of BCL2/ROR1 may mitigate the cytotoxic activity of the BCL2-antagonist venetoclax, but potentially enhance the cytotoxicity of cirmtuzumab-ADC-7, we treated ROR1+ leukemia/lymphoma cell lines with venetoclax and/or cirmtuzumab-ADC-7. Chou-Talalay combination indices were <0.5 in all ROR1+ cell lines tested, indicating strong antitumor synergy with these two agents. Collectively these data support the rationale for clinical development of a cirmtuzumab-based ADC for treatment of patients with ROR1+ malignancies. Disclosures Vo: VelosBio: Employment. Jessen:VelosBio: Employment. Kipps:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Bromodomain and Extra Terminal Domain (BET) Inhibitors Sensitize Chronic Myelomonocytic Leukemia (CMML) to PIM Inhibition Via Downregulation of Mir-33a

Blood ◽

10.1182/blood-2019-128165 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4220-4220

Author(s):

Christopher Letson ◽

Alexis Vedder ◽

Ariel Quintana ◽

Phillip Liu ◽

Brett Reid ◽

...

Keyword(s):

Combination Therapy ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Leukemia Cells ◽

Advisory Committees ◽

Leukemia Cell Lines ◽

Bet Inhibitors ◽

Dose Dependent

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

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Loss of KDM6A Confers Drug Resistance in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114959 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3935-3935

Author(s):

Sophie M. Stief ◽

Anna-Li Hanneforth ◽

Raphael Mattes ◽

Sabrina Weser ◽

Binje Vick ◽

...

Keyword(s):

Drug Resistance ◽

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Cell ◽

K562 Cells ◽

Rna Seq ◽

Advisory Committees ◽

Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML. In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells. To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1. To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity. In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML. Disclosures Hiddemann: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.

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RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽

10.1182/blood.v124.21.2187.2187 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2187-2187

Author(s):

Xiaomei Yan ◽

Yoshihiro Hayashi ◽

Xinghui Zhao ◽

Aili Chen ◽

Yue Zhang ◽

...

Keyword(s):

Protein Expression ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Down Regulation ◽

Leukemia Cell Lines ◽

Flanking Regions ◽

The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

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

Blood ◽

10.1182/blood-2018-99-118367 ◽

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.

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In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽

10.1182/blood-2019-125952 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3100-3100 ◽

Cited By ~ 2

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.

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

Blood ◽

10.1182/blood.v114.22.3830.3830 ◽

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.

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MLN4924, a Novel Investigational NEDD8 Activating Enzyme Inhibitor, Exhibits Preclinical Activity In Multiple Myeloma and Waldenström's Macroglobulinemia through Mechanism Distinct From Existing Proteasome Inhibitors

Blood ◽

10.1182/blood.v116.21.2988.2988 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2988-2988

Author(s):

Douglas W. McMillin ◽

Zachary Hunter ◽

Jake Delmore ◽

Val Monrose ◽

Peter G Smith ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Normal Donor ◽

Advisory Committees ◽

Safety Studies ◽

Ubiquitin Proteasome ◽

Efficacy Studies

Abstract Abstract 2988 Background: Multiple myeloma (MM) and Waldenström Macroglobulinemia (WM) have both shown clinical responses to Bortezomib therapy which blocks the elimination of ubiquitin tagged regulatory proteins by the proteasome. The NEDD8 activating enzyme (NAE)-inhibitor MLN4924 is a novel agent which demonstrates selective inhibition of the proteins for degradation in the ubiquitin pathway and may offer benefits to MM and WM patients through the more targeted approach. Methods: A panel of human MM and WM cell lines were tested for their in vitro response to MLN4924 using MTT colorimetric survival assays. MM and WM cell lines tested exhibited dose and time dependent decrease of their viability upon exposure to MLN4924 (IC50=25-150 nM). In addition, miRNA and gene expression studies in response to MLN4924 were compared to treatment of the same cells with bortezomib. In vivo safety studies were performed in mice and animal efficacy studies are ongoing in both MM and WM engrafted mice. Results: A panel of MM and WM cells were treated with MLN4924 for 72hrs and compared to the colon carcinoma line HCT116 and normal cell lines HS-5 (stroma) and THLE-3 (hepatocytes). In addition, a longitudinal assessment of viability of MM1S (MM) and BCWM1 (WM) cells during a 72hr incubation with MLN4924 (500nM) showed commitment to death <48hrs. This result, coupled with the observation that normal donor peripheral blood mononuclear cells (PBMCs) and HS-5 stromal cells were less sensitive (IC50 >1000 nM) than the MM or WM cell lines tested, suggest that this compound exhibits a rapid, tumor-selective effect at clinically relevant conditions. We also evaluated primary MM (CD138+) and WM (CD19+) patient bone marrow cells and observed sub-μ M activity by MLN4924. In addition, we tested a series of combinations of MLN4924 with dexamethasone, doxorubicin and bortezomib in both MM1S and BCWM1 cells lines and observed additive activity or greater with MLN4924. Gene expression profiling revealed distinct signatures, in MM1S and BCWM1 lines, as well as distinct patterns of gene expression changes which were induced by MLN4924 vs. bortezomib. For instance, while bortezomib potently induces a compensatory upregulation of transcripts for ubiquitin/proteasome and heat shock protein genes which, in MM1S or BCWM1 cells, were not observed in response to MLN4924 treatment. Additional studies with the proteasome inhibitor MLN9708 revealed similar patterns of expression as bortezomib. These results indicate that MLN4924 does not induce pronounced proteotoxic stress in MM or WM cells, highlighting the distinct effect of MLN4924 on the ubiquitin/proteasome pathway compared to inhibitors which target the 20S proteasome subunit. Longitudinal miRNA profiling revealed a distinct pattern of miRNA expression in MLN4924-treated vs. bortezomib-treated MM and WM cells. Lastly, animal safety studies showed that MLN4924 was tolerated at doses up to 60mg/kg 2x daily for 1 week. Efficacy studies in MM and WM are ongoing. Conclusions: MLN4924 induces cell killing at sub-μ M concentrations for both MM and WM cells with higher sensitivity of tumor cells compared to normal tissues, exhibits selective gene expression and miRNA regulation and can be safely administered to mice. These studies provide the framework for the clinical investigation of MLN4924 in MM and WM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Smith:Millennium: Employment. Birner:Millennium: Employment. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

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