Preclinical Models Of Acute Myeloid Leukemia Research Articles

Dual Targeting of Mitochondrial Vulnerability Using Complex I Inhibitor Iacs-010759 with Bcl-2 Inhibitor Venetoclax and Azacitidine in Pre-Clinical Acute Myeloid Leukemia (AML) Models

Despite recent approval of hypomethylating agent/venetoclax (HMA+VEN) therapy for older patients (pt) with AML unfit for induction chemotherapy, their outcomes remain suboptimal. Such pts have a median overall survival of only 14 months and only approximately 35% of pts enjoying long-term survival (DiNardo, EHA 2020). Approximately 20-40% of newly diagnosed older pts with AML do not respond to the HMA+VEN regimens, with higher rates of refractory disease as well as early relapse in high-risk pts. Metabolic reprogramming and dependence on mitochondrial oxidative phosphorylation (OxPhos) is a core feature of AML leukemia stem cells (LSC). Recent reports have shown that upregulation of OxPhos confers intrinsic and acquired resistance to VEN in AML, multiple myeloma and lymphoid malignancies, which can be reversed by disrupting OxPhos; mutated TP53 has been shown to confer intrinsic resistance to venetoclax through increased OxPhos (Nechiporuk T, et al. Cancer Discovery 2019). We hypothesized that combined blockade of mitochondrial fitness by OxPhos inhibitors and of BCL-2 with VEN/azacitidine (AZA) will perform synergistically in pre-clinical AML models. To test this hypothesis, we utilized a novel complex I inhibitor IACS-010759 that effectively inhibits cell respiration and leukemia progression in the in vitro and in vivo AML pre-clinical models (Molina, Nat Med 2018). Priming of MOLM-13 cells with 20nM VEN for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while single agents reduced OCR by <15%. This translated into accelerated loss of the mitochondrial membrane potential measured by JC-1 flow cytometry, cleavage of caspase 3 and synergistic reduction (>70%) of viable cell numbers in several AML cell lines tested (OCI-AML2, MV-4-11, and MOLM-13) (Figure 1). By co-immunoprecipitation VEN disrupted interaction of BCL-2 with the mitochondrial protein VDAC, known to regulate ADP/ATP exchange during electron transport across mitochondria membrane; this resulted in dramatic reduction of the intracellular ATP and CTP levels measured by MS-based metabolomics. Further, VEN increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of VEN and IACS-010759, possibly because of RNA degradation. In primary AML samples (n=3) and AML PDX cells (n=4) cultured ex vivo, combined VEN and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. We next tested the efficacy of the "triple" combination of VEN, AZA and IACS-010759 in the in vivo AML PDX models. We injected AML PDX cells 3871344 (with no mutations identified by targeted sequencing) and 4404778 (harboring IDH1, NPM1, FLT3-ITD mutations) into NRG mice and upon engraftment, randomized mice into 4 groups to receive 2 cycles of treatment with 3 weeks interruption between cycles: vehicle, VEN (50mg/kg daily, 5 days on/2 days off, day 1-21) with AZA (2.5 mg/kg daily, day 1-7), IACS-010759 (1mg/kg ?daily, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. In the less aggressive model 3871344, all therapies reduced circulating leukemia burden, and the triple treatment achieved best efficacy, with average circulating tumor burden at 10 weeks after cycle 2 of 90.8%, 38.7%, 68.8% and 7.4% in vehicle, VEN+AZA, IACS-010759, and triple-therapy cohorts, respectively. In the aggressive model 4404778, the treatments were less effective, but the combination offered highest activity, with average circulating tumor burden of 71.0%, 52.3%, 88.7% and 39.9% in vehicle, IACS-010759, VEN+AZA and triple-therapy cohorts, respectively (Figure 1). Analysis of survival and additional PDX models are ongoing and will be reported. In summary, our study demonstrates that BCL-2 modulates mitochondrial respiration and mitochondrial ATP generation in addition to its established anti-apoptotic role. VEN disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitro and in vivo. Disclosures Konopleva: Ascentage: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; AstraZeneca: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding.

Phase 1/1b Trial of Talazoparib and Gemtuzumab Ozogamicin in Adult Patients with Relapsed or Refractory Acute Myeloid Leukemia

Background: Poly (ADP-ribose) polymerase (PARP) enzymes are involved in repair of single-strand DNA breaks through base excision repair pathways. Inhibitors of PARP are approved for the treatment of BRCA1/2-mutant malignancies where they result in compromise of dual DNA damage repair pathways, leading to induction of double-strand DNA breaks during replication and ultimately cell death. We have previously demonstrated (Portwood et al, ASH 2019 abstract) that PARP inhibitors can reduce in vitro acute myeloid leukemia (AML) growth and can synergistically enhance the anti-leukemic activity of antibody-drug conjugates containing DNA-damaging cytotoxic agents in preclinical human AML models. Talazoparib (Tala) is a novel selective PARP inhibitor which has been shown to exert more potent growth inhibitory effects across a panel of human AML cell lines than four other PARP inhibitors (veliparib, olaparib, rucaparib, niraparib). Gemtuzumab ozogamicin (GO) is an approved drug consisting of an antibody against human CD33 (gemtuzumab) linked to a cytotoxic component (calicheamicin). Hypothesis: We hypothesize that the combination of Tala + GO will be well tolerated and will result in improved clinical efficacy as compared with historical outcomes of GO monotherapy in patients with relapsed or refractory (R/R) AML. Objectives: The primary objectives of this study are (a) to determine the safety and tolerability of Tala + GO therapy in R/R AML; (b) to determine the overall response rate (ORR = complete remission (CR) + complete remission with incomplete hematologic recovery (CRi)) of combination therapy in patients with R/R AML. Additional anti-leukemic effects will be reported including complete remission rate, best response rate, duration of remission, leukemia-free survival, transfusion independence, and overall survival. Exploratory aims include measurable residual disease, evidence of PARP inhibition and DNA damage, feasibility of subsequent allogeneic stem cell transplant, and quality of life. Study Design: This open-label multi-center, Phase 1/1b trial is designed to determine whether the combination of Tala and GO represents a safe and effective approach for R/R AML. Eligible patients are adults aged ≥18 years with CD33-positive AML whose disease has failed to respond to and/or has recurred following at least one prior line of chemotherapy. There are two parts of the study: dose escalation and dose expansion. The dose escalation portion will establish the recommended Phase 2 dose (RP2D) of Tala in combination with GO. Cohorts of 3 subjects will be treated at one of three dose levels of Tala (0.5 mg, 0.75 mg, and 1 mg orally daily) in combination with fixed doses of GO (3 mg/m2/day on days 1,4, and 7, capped to one 4.5 mg vial) using a standard 3+3 subject cohort dose evaluation. Treatment will be given in 28-day cycles. The dose limiting toxicity (DLT) window will be Cycle 1 Day 1 to 28. Therapy may be given in the outpatient setting. Following determination of the RP2D, an expansion cohort of patients will be treated with the combination of Tala + GO for preliminary assessment of toxicities and anti-leukemic efficacy. Bone marrow assessment will be performed on Day 28 of Cycles 1 and 2. Safety and activity will be assessed using descriptive statistical analysis. If a patient achieves CR/CRi after cycles 1-2, the GO dose will be reduced to a maintenance dose of 2 mg/m2 on day 1 only of every subsequent cycle until disease progression or for a maximum of 6 total cycles of therapy. Patients not achieving a clinical response after 2 cycles will stop treatment. Treatment duration will be up to six months. This trial was activated in July 2020 and is registered at ClinicalTrials.gov (NCT04207190). Accrual is ongoing. Disclosures Griffiths: Novartis: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Boston Biomedical: Honoraria; Persimmune: Research Funding; AbbVie Inc: Honoraria; Genentech Inc: Research Funding; Astex Pharmceuticals: Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding. Emadi:Jazz Pharmaceuticals: Research Funding; NewLink Genetics: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor. Baer:AbbVie: Other: Institutional research funding; Astellas: Other: Institutional research funding; Forma: Other: Institutional research funding; Incyte: Other: Institutional research funding; Kite: Other: Institutional research funding; Oscotec: Other: Institutional research funding; Takeda: Other: Institutional research funding. Wang:Stemline: Speakers Bureau; Astellas: Consultancy; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Jazz Pharmaceuticals: Consultancy; Bristol Meyers Squibb (Celgene): Consultancy; Abbvie: Consultancy; Genentech: Consultancy; Pfizer: Speakers Bureau.

Acute Myeloid Leukemia Chemo-Resistance Is Mediated by E-selectin Receptor CD162 in Bone Marrow Niches.

The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-(endothelial)-selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance in vitro. Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.

CXCR4 Inhibition Enhances Efficacy of FLT3 Inhibitors in FLT3-Mutated AML Augmented by Suppressed TGF-b Signaling.

Given the proven importance of the CXCL12/CXCR4 axis in the stroma–acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-β signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-β in stromal cells or TGF-β-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-β signaling.

A phase Ib/II study of the CD123-targeting antibody-drug conjugate IMGN632 as monotherapy or in combination with venetoclax and/or azacitidine for patients with CD123-positive acute myeloid leukemia.

TPS7564 Background: Overexpression of CD123 is characteristic of a number of hematological malignancies, including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) with a novel anti-CD123 antibody coupled to a unique DNA-alkylating payload of the recently developed IGN (indolinobenzodiazepine pseudodimer) class of payloads. In preclinical models of AML, IMGN632 exhibited potent anti-leukemia activity, with a wide therapeutic index. Confirming preclinical expectations, encouraging single-agent activity and favorable tolerability have emerged for IMGN632 in the ongoing Phase I trial in patients with CD123-positive AML (ASH 2019, NCT03386513). Preclinical data from AML xenograft models have demonstrated synergy in IMGN632 combinations with azacitidine and venetoclax (EHA 2019), supporting the exploration of these combinations in AML patients. Methods: This Phase Ib/II study is designed to determine the safety, tolerability, and preliminary anti-leukemia activity of IMGN632 when administered in combination with azacitidine and/or venetoclax to patients with relapsed and frontline CD123-positive AML, and the single-agent activity of IMGN632 in patients with minimal residual disease (MRD)-positive AML after frontline treatment. Study Design: Adult patients with CD123-positive relapsed or refractory AML, who are deemed appropriate for experimental therapy, are eligible to enroll as part of the dose escalation phase. Key exclusion criteria for all regimens include active central nervous system disease, and history of sinusoidal obstruction syndrome/venous occlusive disease of the liver. Three combination regimens are being evaluated: Regimen A, IMGN632 plus azacitidine (632+AZA); Regimen B, IMGN632 plus venetoclax (632+VEN); and Regimen C, IMGN632 plus azacitidine and venetoclax (632+AZA+VEN). For each regimen, a Phase Ib dose escalation cohort will determine the recommended Phase II dose (RP2D) of IMGN632 for the specific combination. This will be followed by a Phase II dose expansion stage to further characterize the safety profile and assess antileukemia activity in frontline or relapsed AML patients, depending on combination regimen. In addition, IMGN632 monotherapy is being explored in expansion cohorts of MRD-positive patients to assess conversion rate from MRD+ to MRD-, in fit and unfit AML subpopulations. Clinical trial information: NCT04086264 .

Discovery of a novel and highly selective CDK9 kinase inhibitor (JSH-009) with potent antitumor efficacy in preclinical acute myeloid leukemia models.

Acute myeloid leukemia (AML) is reported to be vulnerable to transcription disruption due to transcriptional addiction. Cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation, has attracted extensive attention as a drug target. Although several inhibitors, such as alvocidib and dinaciclib, have shown potent therapeutic effects in clinical trials on AML, the lack of high selectivity for CDK9 and other CDKs has limited their optimal clinical efficacy. Therefore, developing highly selective CDK9 inhibitors is still imperative for the efficacy and safety profile in treating AML. Here, we report a novel highly selective CDK9 inhibitor, JSH-009, which exhibited high potency against CDK9 and displayed great selectivity over 468 kinases/mutants. It also demonstrates impressive in vitro and in vivo antileukemic efficacy in preclinical models of AML, which makes JSH-009 a useful pharmacological tool for elucidating CDK9-mediated transcription and a novel therapeutic candidate for AML.

RIG-I-based immunotherapy enhances survival in preclinical AML models and sensitizes AML cells to checkpoint blockade

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5′-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4+ and CD8+ T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.

HDAC Inhibitors in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by uncontrolled proliferation, differentiation arrest, and accumulation of immature myeloid progenitors. Although clinical advances in AML have been made, especially in young patients, long-term disease-free survival remains poor, making this disease an unmet therapeutic challenge. Epigenetic alterations and mutations in epigenetic regulators contribute to the pathogenesis of AML, supporting the rationale for the use of epigenetic drugs in patients with AML. While hypomethylating agents have already been approved in AML, the use of other epigenetic inhibitors, such as histone deacetylases (HDAC) inhibitors (HDACi), is under clinical development. HDACi such as Panobinostat, Vorinostat, and Tricostatin A have been shown to promote cell death, autophagy, apoptosis, or growth arrest in preclinical AML models, yet these inhibitors do not seem to be effective as monotherapies, but rather in combination with other drugs. In this review, we discuss the rationale for the use of different HDACi in patients with AML, the results of preclinical studies, and the results obtained in clinical trials. Although so far the results with HDACi in clinical trials in AML have been modest, there are some encouraging data from treatment with the HDACi Pracinostat in combination with DNA demethylating agents.

First-in-Human Study of Abbv-621, a TRAIL Receptor Agonist Fusion Protein, in Patients (Pts) with Relapsed/Refractory (RR) Acute Myeloid Leukemia (AML) and Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Apoptotic cell death can be triggered by activation of extrinsic and intrinsic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, binds to its death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) to selectively activate the extrinsic apoptotic pathway in cancer cells. ABBV-621 is a second-generation TRAIL receptor agonist with antitumor activity as monotherapy (621-mono) in preclinical models of AML and DLBCL. The intrinsic apoptotic pathway is regulated by the BCL2 protein family, commonly overexpressed in hematologic malignancies. Venetoclax (VEN), a highly selective small-molecule BCL2 inhibitor, has shown antitumor activity in combination therapy in AML and DLBCL. In preclinical and xenograft models of AML and DLBCL, the ABBV-621 and VEN combination (621-VEN) had antitumor activity superior to either agent alone. This first-in-human study evaluated ABBV-621 as single agent and in combination in pts with advanced solid tumors and hematologic malignancies (NCT03082209). Safety and tolerability of ABBV-621 in advanced solid tumors have previously been presented (Ratain et al. J Clin Oncol 2019;37[suppl]: abstr 3013). Here, we report preliminary data for 621-mono in pts with RR AML and for 621-VEN in pts with RR AML and DLBCL. Methods: Adult pts with RR AML or DLBCL (ECOG 0-2) were enrolled. Pts with AML received ABBV-621 at 1.25-, 3.75-, 7.5-mg/kg doses in the 621-mono and at 3.75-mg/kg dose in the 621-VEN arms. Pts in the DLBCL 621-VEN arm received ABBV-621 at 3.75- and 7.5-mg/kg doses. ABBV-621 was administered intravenously on D1, 8, and 15 of a 21-D cycle; in 621-VEN cohorts pts received 400 mg oral VEN daily, and could be escalated to 800 mg. The primary endpoint was safety. In addition, preliminary antitumor efficacy and ABBV-621 binding to decoy receptors on neutrophils from peripheral blood were assessed. Results: As of Jun 2019, 17 pts were enrolled. Pts in AML 621-mono arm (1.25 [1], 3.75 [1], 7.5 [2] mg/kg): 1 male; median (med) age, 75 yr (range 71-82); med prior treatments, 2.5 (range 1-4); med time on treatment, 15 D (range 1-70). Pts in AML 621-VEN arm (3.75 mg/kg [7]): 5 male; med age, 71 yr (60-79); med prior treatments, 2 (1-2); med time on treatment, 26 D (1-77). Pts in DLBCL 621-VEN arm (3.75 [3], 7.5 [3] mg/kg): 4 male; med age, 57 yr (40-75); stage 4 (3); med prior treatments, 2.5 (1 to ≥5); med time on treatment, 26 D (8-36). One pt in the AML 621-VEN 3.75-mg/kg dose cohort had increases in alanine aminotransferase, aspartate aminotransferase and bilirubin as dose-limiting toxicities. Sixteen pts experienced adverse events (AEs) irrespective of causality. A summary of AEs is shown in Table. One pt in the AML 621-mono 7.5-mg/kg dose cohort died due to AML progression, unrelated to ABBV-621. Antitumor activity was observed in 1 pt in the AML 621-VEN arm (with complex cytogenetics and TP53 mutation) who reached complete remission (CR). One pt in the DLBCL 621-VEN 3.75-mg/kg cohort had stable disease, 2 with AML had resistant disease (1 in 621-mono [7.5 mg/kg] and 1 in 621-VEN), and 7 had progressive disease (PD; 1 in AML 621-mono [1.25 mg/kg], 2 in AML 621-VEN, and 4 in DLBCL 621-VEN [2 each in 3.75- and 7.5-mg/kg] cohorts). Using flow cytometry, saturation of ABBV-621 binding to decoy receptors on neutrophils was observed at 2 h postdosing, followed by dose-dependent desaturation of receptors in pts with DLBCL at 48-168 h. In AML 621-VEN pts, ABBV-621 remained bound to decoy receptors for up to 168 h; in DLBCL 621-VEN pts, the duration of binding was higher at ABBV-621 7.5 mg/kg compared with 3.75 mg/kg. In AML, the baseline frequency of myeloblasts was higher in pts with PD than CR, while that of myelomonocytes was higher in the pt with CR. The frequency of myeloblasts and myelomonocytes expressing DR4 and DR5 at baseline was highest in the pt with CR (Figure). Conclusions: ABBV-621 was tolerated and showed antitumor activity in combination with VEN in pts with RR AML. Disclosures de Jonge: Faron Pharmaceuticals Ltd.: Consultancy. Carneiro:Actuate Therapeutics: Research Funding; Bayer: Research Funding; Pfizer: Research Funding; AstraZeneca: Research Funding; Medimmune: Research Funding; Astellas: Research Funding; AbbVie: Research Funding. Devriese:MSD: Consultancy. Penugonda:AbbVie: Employment, Other: Stock/stock options. Petrich:Abbvie: Employment, Equity Ownership. Nuthalapati:AbbVie: Employment, Other: Stock/stock options. Motwani:AbbVie: Employment, Other: Stock/stock options. Modi:AbbVie: Employment, Other: Stock/stock options. Chang:AbbVie: Employment, Other: Stock/stock options. Calvo:Celgene: Consultancy; Roche/Genentech: Consultancy, Other: travel/accommodations/expenses; Seattle Genetics: Consultancy; AstraZeneca: Consultancy, Research Funding; PsiOxus: Consultancy; Amcure: Consultancy; START: Other: stock/ownership interests, Research Funding; Janssen-Cilag: Consultancy; EUSA Pharma: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Oncoart Associated: Other: stock/ownership interests; Guidepoint Global: Consultancy; Nanobiotix: Consultancy; HM Hospitales Group: Honoraria; AbbVie: Consultancy; BeiGene: Research Funding; Servier: Consultancy; International Cancer Consultants: Other: stock/ownership interests; Foundation INTHEOS: Other: president and founder; Gerson Lehrman Group: Consultancy; Pfizer: Consultancy. Moreno:Puma Biotechnology: Consultancy; Sanofi/Regeneron: Other: travel/accommodations/expenses.

A Phase 1 Dose Escalation Study of Milademetan in Combination with 5-Azacitidine (AZA) in Patients with Acute Myeloid Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS)

A Phase 1 Dose Escalation Study of Milademetan in Combination with 5-Azacitidine (AZA) in Patients with Acute Myeloid Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS)

Pevonedistat, a Nedd-8 Activating Enzyme Inhibitor, Upregulates NOXA to Increase Effectiveness of Azacitidine and Venetoclax in Preclinical Models of Acute Myelogenous Leukemia

Introduction: Venetoclax (VEN), a selective BCL-2 inhibitor, has yielded exceptional response rates in patients with acute myeloid leukemia (AML). VEN binds BCL-2 to directly inhibit sequestration of pro-apoptotic proteins such as the activator BIM. Free BIM can bind to BAX, enabling its oligomerization with BAK, mitochondrial outer membrane permeabilization (MOMP) and subsequent apoptosis (PMID 24074954, 9687260, 19641500, 20164920). VEN has limited efficacy in relapsed-refractory AML as a single agent, but when used in combination with DNA methyltransferase inhibitors (DNMTi; 5'azacitidine-AZA-or decitabine) or low-dose cytosine arabinoside (LDAC), 50-70% of untreated patients in recent clinical trials achieved complete remission (PMID 27520294, 30361262, 30892988). Despite this progress in AML therapy, the majority of AML patients treated with VEN ultimately relapse, and a large subset of patients never respond (PMID 30361262, 30892988). One postulated route of resistance to VEN is cellular upregulation of myeloid cell leukemia-1 protein (MCL-1), which, similarly to BCL-2, functions as an anti-apoptotic protein. VEN in combination with selective MCL-1 inhibitors has demonstrated added benefit over either agent alone in AML cells in vitro and in xenograft models but efficacy of this combination in the clinic has yet to be reported (PMID 30185627). Both MCL-1-dependent and MCL-1-independent mechanisms of VEN resistance are emerging (PMID 31048321, 30148320, 31262744) and new approaches aimed at addressing these are moving toward the clinic. Pevonedistat (PEV) was developed as a targeted inhibitor of NEDD-8 activating enzyme (NAE), which disrupts protein turnover mediated by Cullin-RING ligases (PMID 19360080) and has been shown to have activity in combination with AZA (PMID 29348128). PEV and AZA both upregulate NOXA, a BCL-2 family member that is known to suppress MCL-1 (PMID 26045051, Jin, Cojocari, Purkal et al.,unpublished data), so we postulated that PEV/AZA in combination synergizes with VEN in a triple combination with efficacy superior to VEN/AZA alone. Methods/Results: AML cell lines were treated with PEV, AZA and VEN alone, and compared with various VEN/PEV/AZA combinations revealing improved combinatorial activity with the triplet in the majority of cell lines and associated NOXA induction. Similar results were seen in primary AML patient samples ex vivo (Figure 1). These combinations were compared in vivo in an OCI-AML2 cell line xenograft model, also illustrating significantly improved response with the PEV/AZA/VEN combination (Figure 2). CRISPR/Cas 9 deletion of PMAIP1 in AML cell lines demonstrated that NOXA deletion abrogates this synergy. Discussion: Together, these results demonstrate that, in several pre-clinical models of AML, the PEV/AZA/VEN triple combination provides stronger anti-tumorigenic activity than either agent alone or the VEN/PEV and VEN/AZA combinations. Importantly, the three-drug combination may be effective in AML cells which do not respond to VEN/AZA alone. Further studies to delineate this mechanism of action, and a clinical trial (NCT03862157) testing the combination in newly diagnosed AML are underway. Figure 1 Disclosures Cojocari: AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Purkal:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Leverson:AbbVie Inc: Employment, Other: Stock or options. Boghaert:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Phillips:AbbVie Inc: Employment, Other: DC, JP, ERB, JDL, and DCP are employees of AbbVie. JP, ERB, JDL & DCP are stockholders of AbbVie Inc. The design study conduct, and financial support for this research were provided by AbbVie. AbbVie Inc. participated in the interpretation of data, review. Savona:AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees.

Targeting DHODH with AG-636 Induces Apoptosis and Differentiation and Inhibits Mitochondrial Function in AML, Translating into Anti-Tumor Efficacy in Vitro and in Vivo

Targeting DHODH with AG-636 Induces Apoptosis and Differentiation and Inhibits Mitochondrial Function in AML, Translating into Anti-Tumor Efficacy in Vitro and in Vivo

The Combination of CUDC-907 and Gilteritinib Shows Promising Antileukemic Activity in Vitro and In Vivo in Preclinical Models of FLT3-ITD AML

Introduction FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are found in approximately one quarter of acute myeloid leukemia (AML) cases. Its presence results in constitutive activation of the FLT3 receptor tyrosine kinase and its downstream growth/pro-survival pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT, and confers a poor prognosis. Gilteritinib is a selective inhibitor of FLT3 recently approved by the Food and Drug Administration for treatment of patients with relapsed/refractory AML and a FLT3 mutation. However, gilteritinib exposure induces upregulation of FLT3 - a mechanism of resistance. Previously, we showed that CUDC-907, a dual PI3K/histone deacetylase inhibitor, downregulates FLT3 expression (Li X, et al. Haematologica. 2019; epub ahead of print). We therefore hypothesized that combining CUDC-907 with gilteritinib would abrogate FLT3 upregulation and expression, resulting in synergistic antileukemic activities against FLT3-mutated AML. Methods FLT3-ITD AML cell lines and primary patient samples were treated with gilteritinib or CUDC-907, alone or in combination at clinically achievable concentrations, and subjected to annexin V/propidium iodide staining and flow cytometry analysis to quantify apoptosis. Protein levels of FLT3, Bcl-2 family proteins, and key components of the MAPK/ERK, PI3K/AKT, and JAK/STAT pathways were examined using western blotting. The impact of the observed alterations upon apoptosis were confirmed via overexpression, knockdown, and targeted inhibitor experiments. Real-time RT-PCR was used to determine FLT3 transcript levels. The FLT3-ITD AML cell line MV4-11 was used to generate a xenograft mouse model to assess in vivo efficacy of the two agents. Results CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines in vitro and patient samples ex vivo, with combined therapy. CUDC-907 abolished gilteritinib-induced expression of FLT3 in both cell lines and primary patient samples. Gilteritinib treatment reduced p-AKT, p-S6, and p-STAT5 and increased p-ERK, while CUDC-907 reduced p-AKT and p-ERK, and upregulated p-STAT5. The combination of gilteritinib and CUDC-907 decreased not only p-AKT and p-S6, but also p-ERK and p-STAT5. Targeted inhibition of ERK and JAK2/STAT5 signaling by SCH772984 and AZD1480, respectively, confirmed their roles in resistance to gilteritinib and CUDC-907 monotherapies, respectively. Combined gilteritinib and CUDC-907 treatment reduced expression of the anti-apoptotic BCL-2 family member Mcl-1 and increased expression of the pro-apoptotic protein Bim. MCL-1 overexpression and BIM knockdown partially rescued FLT3-ITD AML cells upon drug treatment, confirming their role in the antileukemic activity of combined gilteritinib and CUDC-907. To determine in vivo efficacy of the two agents, NSGS mice were injected with MV4-11 cells. Three days later, the mice were randomized into vehicle control (n=5), 40 mg/kg gilteritinib (oral gavage; n=5), 100 mg/kg CUDC-907 (oral gavage; n=5) or combination (40 mg/kg gilteritinib + 100 mg/kg CUDC-907; n=6) groups. CUDC-907 was given daily for 5 days on, 2 days off, for a total of 4 cycles. Gilteritinib was administered daily for 28 days. Both agents were well tolerated; maximal weight loss was 5.5%, 0.9%, and 6.7% in the CUDC-907, gilteritinib, and combination groups, respectively. Median survival of mice in the vehicle control group was 43 days. Median survival in the CUDC-907 monotherapy and gilteritinib monotherapy arm was 40.5 days and 104 days, respectively. One mouse in the combination therapy arm died on day 138, while the remaining 5 mice in the combination therapy arm continue to survive, as of time of writing (day 168), and are asymptomatic (Figure 1). Conclusion We confirmed that the combination of CUDC-907 plus gilteritinib synergistically induces apoptosis in both FLT3-ITD AML cell lines and primary patient samples, and that gilteritinib-induced FLT3 expression is abolished by CUDC-907. Cooperative inhibition of the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, as well as upregulation of Bim/downregulation of Mcl-1 all appear to contribute to this observed antileukemic synergy. Our cell line-derived xenograft mouse model provides strong evidence of in vivo efficacy and robust grounds for clinical translation of this therapeutic combination. Disclosures No relevant conflicts of interest to declare.

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Acute Myeloid Leukemia Models

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for ~25% of all AMLs, carries a poor prognosis, and is prone to relapse despite targeted therapy. FLT3 mutations are associated with aberrant activation of the Wnt signaling pathway, which itself is implicated in AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. These studies examined the antitumor activity of SM09419 as a single agent and in combination with targeted and standard therapies in preclinical models of FLT3-ITD AML. In MV-4-11 and MOLM-13 AML cells carrying the FLT3-ITD mutation, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt pathway-related genes (CCND1, MYC, TCF7, DVL2). The effect on cell proliferation was tested in 8 AML cell lines with varying mutation profiles as well as 26 different leukapheresis-derived primary human AML cells. Proliferation was strongly impaired by SM09419 across all tested cell lines (average EC50=0.2 + 0.048 µM]); MV-4-11 and MOLM-13 cells had EC50 of 0.049 and 0.144 µM, respectively. SM09419 also potently inhibited proliferation in all primary AML cells (average EC50=0.048 + 0.0097 µM) regardless of FLT3 mutation status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). SM09419 also induced apoptosis in MV-4-11 and MOLM-13 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with either midostaurin (FLT3 inhibitor) or venetoclax (BCL2 inhibitor) and/or azacitidine were assessed in FLT3-ITD xenograft models (n=5-6/group). In MOLM-13 xenografts, SM09419 (12.5 and 25 mg/kg) induced strong tumor growth inhibition (TGI) vs. vehicle at Day 14 (TGI 52% [p<0.05] and 74% [p<0.001], respectively). Midostaurin (50 mg/kg) induced significant TGI vs. vehicle (50%, p<0.05), which was increased when administered in combination with 12.5 mg/kg SM09419 (81%, p<0.001). In MV-4-11 xenografts, single-agent SM09419 (6.25, 12.5, and 25 mg/kg) induced significant TGI vs. vehicle (56% [p<0.05], 94%, and 95% [p<0.001], respectively) at Day 26 with tumor regression in all mice dosed at 12.5 mg/kg and 25 mg/kg. In a subsequent experiment, midostaurin (50 mg/kg) alone and combined with 6.25 mg/kg SM09419 for 23 days induced tumor regression in MV-4-11 xenografts (100% TGI vs. vehicle, p<0.0001). After treatment discontinuation, tumor regression was maintained in all mice (6/6) treated with the combination for 26 days, whereas tumor regrowth was immediately observed in midostaurin-treated mice. In another MV-4-11 xenograft study, the combination of 6.25mg/kg SM09419 with azacitidine (0.8 mg/kg QD) and/or venetoclax (25 mg/kg QD) induced significant TGI (95-98% vs. vehicle, p<0.001) with tumor regression at Day 26. Azacitidine + venetoclax induced 79% TGI (p<0.001), but no tumor regression was observed. The triple combination induced tumor regression in all mice and complete regressions in 4/6 mice (67%); it had a greater effect on slowing tumor regrowth after treatment discontinuation vs. a single agent or doublet. SM09419 alone or in combination was well tolerated in these xenograft models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation and Wnt signaling pathway activity and induced apoptosis in FLT3-ITD cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of FLT3 status. The strong in vivo antitumor effects observed as combination treatment suggest that SM09419 combined with standard therapies may provide a clinical benefit by slowing or preventing relapse in AML with a marker of poor prognosis such as FLT3-ITD. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

Enhanced expression of the sphingosine-1-phosphate-receptor-3 causes acute myelogenous leukemia in mice.

Acute myeloid leukemia (AML) carries a 10-100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P3) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P3 signaling to leukemogenesis that warrants the exploration of S1P3 antagonists in preclinical AML models.

Abstract 3833: Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel targets for the therapy of various malignant diseases. Also LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we have characterized a novel LSD1 inhibitor and assessed its potential as a novel therapy for AML and SCLC patients. Materials and Methods: Novel LSD1 inhibitor, HM97211 and its derivatives were designed and synthesized as the active biologic inhibitory compound against the AML and SCLC cells. Biochemical enzyme assay was performed to identify enzyme selectivity of HM97211 derivatives. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM97211 derivatives in AML and SCLC cell lines. In vivo anti-tumor activity was evaluated in AML and SCLC cell xenograft mice models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. Results: HM97211 derivatives showed higher selectivity toward LSD1 compared to other FAD-utilizing enzyme as well as other epigenetic enzymes. HM97211 derivatives alter expression of various genes and induce differentiation and apoptosis, resulting in growth suppression in a panel of AML and SCLC cells. Moreover, HM97211 derivatives significantly induced programmed cell death in AML and SCLC cells. Oral administration of HM97211 derivatives inhibited tumor growth of human AML and SCLC xenograft models as a single agent at doses exhibiting significant. Efficacy of HM97211 derivatives was further evaluated in combination treatment with standard chemotherapies in human AML and SCLC xenograft model. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211 derivatives, can be a strong therapeutic agent for AML and SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, WonJeoun Kim, JiSook Kim, JiYoung Song, JungSoo Nam, Moonsub Lee, Jooyun Byun, Hyeongki Kim, TaeHun Song, Seokhyun Hong, KyuHang Lee, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3833.

Abstract CT102: Phase Ib safety, preliminary anti-leukemic activity and biomarker analysis of the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with low-dose cytarabine or decitabine in patients with relapsed or refractory acute myeloid leukemia

Abstract Background: Despite recent advances in the understanding of the biology and development of new agents for patients with acute myeloid leukemia (AML), prognosis remains poor with most patients (pts) succumbing to their disease. PLK1 is a serine/threonine kinase, a master regulator of cell-cycle progression, and is overexpressed in numerous cancer types including AML. Onvansertib is a third generation, orally active and highly selective PLK1 inhibitor with a ~24-hour half-life that demonstrates activity in preclinical AML models both as a single agent and in combination with low-dose cytarabine (LDAC). A Phase I study showed that Onvansertib was well tolerated in pts with solid tumors. Methods: The goal of this Phase Ib (NCT03303339) study is to test the safety of Onvansertib in combination with either LDAC or Decitabine in relapsed or refractory AML. Efficacy, pharmacokinetics and pharmacodynamics of the combination are secondary endpoints. Pts are treated with Onvasertib for 5 days in combination with either LDAC (20 mg/m2 SC qd x 10d) or Decitabine (20 mg/m2 IV qd x 5d) over a flexible 21 to 28-day cycle, with the next cycle initiated based on recovery of cell counts. Each arm follows a standard dose escalation design with 50% increments in successive cohorts of 3 pts. Dose limiting toxicity (DLT) is evaluated during the 1stcycle. As of January 2, 2019, 21 pts have enrolled. The starting dose of Onvansertib was 12mg/m2 and was escalated to 18, 27 and 40mg/m2 in subsequent cohorts. In both arms, no treatment-related serious adverse events and deaths or DLTs occurred at the first 3 doses and treatment at 40mg/m2 is ongoing. Additional objectives include pharmacokinetics, preliminary anti-leukemic activity and correlative biomarker and pharmacodynamics analyses. Assessment of PLK1 inhibition is being determined in pts by changes in phosphorylation status of the translationally controlled tumor protein (TCTP), which is a PLK1 substrate, and is being evaluated not only in terms of response to treatment, but also as a potentially identifiable marker ex vivo for pts that may be more responsive to therapy. Genomic and gene expression analysis are assessed to identify biomarkers that may also be associated with response to treatment. Citation Format: Amer M. Zeidan, Pamela Becker, Alexander I. Spira, Prapti A. Patel, Gary J. Schiller, Michaela L. Tsai, Tara L. Lin, Maya Ridinger, Mark Erlander, Sandra L. Silberman, Jorge E. Cortes. Phase Ib safety, preliminary anti-leukemic activity and biomarker analysis of the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with low-dose cytarabine or decitabine in patients with relapsed or refractory acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT102.

Antileukemic activity and mechanism of action of the novel PI3K and histone deacetylase dual inhibitor CUDC-907 in acute myeloid leukemia.

Induction therapy for patients with acute myeloid leukemia (AML) has remained largely unchanged for over 40 years, while overall survival rates remain unacceptably low, highlighting the need for new therapies. The PI3K/Akt pathway is constitutively active in the majority of patients with AML. Given that histone deacetylase inhibitors have been shown to synergize with PI3K inhibitors in preclinical AML models, we investigated the novel dual-acting PI3K and histone deacetylase inhibitor CUDC-907 in AML cells both in vitro and in vivo. We demonstrated that CUDC-907 induces apoptosis in AML cell lines and primary AML samples and shows in vivo efficacy in an AML cell line-derived xenograft mouse model. CUDC-907-induced apoptosis was partially dependent on Mcl-1, Bim, and c-Myc. CUDC-907 induced DNA damage in AML cells while sparing normal hematopoietic cells. Downregulation of CHK1, Wee1, and RRM1, and induction of DNA damage also contributed to CUDC-907-induced apoptosis of AML cells. In addition, CUDC-907 treatment decreased leukemia progenitor cells in primary AML samples ex vivo, while also sparing normal hematopoietic progenitor cells. These findings support the clinical development of CUDC-907 for the treatment of AML.

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

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 [Display omitted] DisclosuresAndreeff: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.

Synergistic Anti-Leukemic Activity of PARP Inhibition Combined with IMGN632, an Anti-CD123 Antibody-Drug Conjugate in Acute Myeloid Leukemia Models

BackgroundCD123 (IL-3 receptor alpha-chain) is a therapeutic target for hematological malignancies based on high expression levels in acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), and other cancers. The anti-CD123 antibody-drug conjugate (ADC), IMGN632, comprises a humanized monoclonal antibody covalently linked to a DNA - alkylating cytotoxic payload which is currently in phase 1 evaluation for relapsed/refractory CD123-positive hematological malignancies (NCT03386513). Novel approaches to enhance the efficacy of ADCs are of significant therapeutic interest. Our laboratory has previously demonstrated that the Poly ADP Ribose (PARP) inhibitor, olaparib, synergistically enhances the activity of the CD33-targeted ADC, IMGN779, in preclinical AML models (Portwood S et al, ASH 2016). Based on the hypothesis that PARP inhibition will synergize with DNA damaging mechanism of IMGN632, we investigated the ability of olaparib and other PARP inhibitors (PARPi) in clinical development (talazoparib, niraparib, rucaparib, and veliparib) to enhance the therapeutic efficacy of IMGN632 across diverse human AML cell lines and primary relapsed/refractory AML samples.Materials and MethodsCD123 expression on human AML cell lines (HEL, HL60, MV411, Molm13, EOL-1, THP-1, and Kasumi-1) was quantified by flow cytometry using QuantriBrite beads. AML cells were continuously cultured for 72-96 hours with varying doses of IMGN632 (range 100pM - 100nM) and specific PARP inhibitors (range 100pM -15μM) alone and in combination. Cell viability was measured using a WST-8 colorimetric assay. Primary clinically annotated CD123+ AML cells from patients with relapsed/refractory disease were obtained under IRB-approved protocols from the Roswell Park Hematologic Procurement Shared Resource and cultured short-term in the presence of multiple cytokines plus IMGN632 +/- PARP inhibitors. Apoptosis (Annexin V/PI), cell cycle, and DNA damage (H2AX) were evaluated by flow cytometry. Additive vs. synergistic effects were determined by combination indices using Compusyn software. PARP trapping was evaluated by Western blot analysis in nuclear lysates obtained from IMGN632 +/- PARP inhibitors treated AML cells.ResultsHigh expression levels of CD123 (range 937 - 2231 CD123 molecules/cell) were detected on multiple human AML cell lines (HEL-luc, MV411, Molm13, EOL-1, and THP-1) relative to unstained negative controls. Western blot analysis of nuclear lysates from AML cells demonstrated that all PARP inhibitors had varying degrees of PARP trapping on DNA. Continuous single agent 5-day treatment with all tested PARP inhibitors resulted in dose dependent in vitro inhibition of AML cell line growth with IC50 values ranging from 360 nM (talazoparib, most potent) to 78uM (veliparib, least potent). Combination therapy using PARP inhibitors (doses ranging from 300nM - 15uM) and IMGN632 (10nM) consistently resulted in enhanced anti-leukemic effects over monotherapy (Figure 1 for example). Synergistic anti-proliferative effects were obtained across all tested AML cell lines (n=5) with combination indexes ranging from 0.3-0.7 by Compusyn analysis. Combination therapy correlated with enhanced DNA damage, tumor cell apoptosis, and cell cycle arrest of AML cells. Moreover, IMGN632 and PARPi (olaparib or talazoparib) resulted in single agent activity against clinically annotated primary relapsed/refractory AML patient samples with evidence of synergistic effects when combined in vitro.ConclusionsAddition of PARP inhibitors to IMGN632, a novel anti-CD123 antibody-drug conjugate, further enhances DNA damage effects and consistently results in synergistic in vitro anti-leukemic effects across multiple CD123+ AML cell lines and primary AML patient samples. Further studies investigating this novel combinatorial approach in specific molecular subtypes of AML with variable baseline sensitivities to PARPi are currently ongoing. Our results strongly support future investigation of PARPi as a novel class of agents with the potential to significantly enhance the efficacy of DNA-alkylating ADCs and/or cytotoxic chemotherapy for hematological malignancies. [Display omitted] DisclosuresSloss:ImmunoGen: Employment. Watkins:ImmunoGen Inc.: Employment. Kovtun:ImmunoGen Inc.: Employment. Adams:ImmunoGen Inc.: Employment. Wang:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.