ALBERT

Description: Introduction. Acute erythroid leukemia (AEL) is a high-risk leukemia subtype of poorly understood genetic basis. In integrated comparative genomic analysis of 159 AEL and 1509 non AEL myeloid tumors, we identified 5 age-related AEL subtypes with distinct genomic features and outcome: adult AEL with bi-allelic alterations in TP53 (31%), frequently co-occurring with alterations in DNMT3A,BCOR, EZH2, RB1, or NFIX; NPM1-mutated (12%); KMT2A-mutated/rearranged (11%), mostly co-mutated with STAG2; pediatric, NUP98-rearranged (5%) and adult, DDX41-mutated (3%). Thirty-eight percent of cases lacked an identifiable exclusive recurrent founding alteration but were enriched in mutations in ASXL1, TET2 and splicing factors. Methods. To explore the roles and cooperativity of the identified alterations in leukemogenesis we used CRISPR-Cas9 genome editing to induce combinations of loss-of-function mutations in 9 recurrently mutated genes in AEL (Tp53, Tet2, Dnmt3a, Asxl1, Ezh2, Stag2, Bcor, Ppm1d, Rb1 and Nfix). Based on patterns of mutation association, we generated 6 pools of lentiviral vectors (Table 1) with different combinations of single guide RNA (sgRNA) to induce multiplex genome editing in Cas9-eGFP-mouse lineage-negative hematopoietic stem cells (HSCs). Transduced cells were transplanted into lethally irradiated congenic mice. Tumors were characterized by morphology, immunophenotyping, and genomic analysis (sequencing of sites of editing, and exome, methylation and transcriptome sequencing). Ex vivo drug screening was performed to test sensitivity to 192 therapeutic agents, including conventional chemotherapeutic agents, and compounds targeting epigenetic regulators, protein kinases and cell cycle checkpoints. Results. In contrast to the uniform representation of guide RNAs observed in HSCs pre-transplant, tumors exhibited enrichment of specific sgRNAs with tumors of specific phenotype. We frequently observed bi-allelic editing of Tp53, Bcor, Tet2, Dnmt3a, Rb1 and Nfix in agreement with the presence of bi-allelic loss in patients. Concomitant editing and inactivation of Tp53/Bcor/Dnmt3a, or Tp53/Bcor/Rb1/Nfix promoted the development of acute erythroid leukemia (GATA1+/RUNX1+/GlyA+/-Ter119+/- and B220/CD19/PAX5/CD3/Mac1/Gr1-). Concomitant editing of Tp53/Bcor/Tet2 promoted the development of B-lineage ALL, and editing of Dnmt3a and Tet2 without Tp53 promoted T-cell ALL. Leukemic clones from primary tumors were serially transplantable across multiple different genetic backgrounds, with the same dominant clone present in all transplanted mice. Notably, mice that did not develop leukemia showed enrichment of different combinations of sgRNAs for Tet2, Dnmt3a and/or Asxl1, genes commonly mutated in clonal hematopoiesis of indeterminate potential, confirming that these mutations are alone not sufficient to induce leukemogenesis. Additional somatic mutations were acquired during clonal expansion of leukemic cells such as alterations of Notch1 and Ikzf1 in T-ALL; Setd2 at the serial passaging of T-ALL; Ptpn11, Kit (D816V), Kras (Q61L) and Lmo7 in the AEL models; and Sf3b3 in the B-ALL model (Fig 1A). Tumors with mutated Tp53 acquired aneuploidy whereas Tet2-mutated cells were genomically stable (Fig. 1B). Unsupervised hierarchical clustering of gene expression profiling identified 3 subgroups (Fig. 1C) associated with distinct genotypes and methylation profiles (Fig. 1D). Tet2-mutated tumors showed increase of hypermethylated sites and co-mutated Bcor/Dnmt3a leukemic cells showed loss of methylation. Eleven tumors representative of key AEL genotypes from the established models were selected to explore drug sensitivity. Response to individual drugs was associated with genotype with co-mutated Tet2/Dnmt3a T-ALL cells sensitive to bromodomain and histone methyltransferase inhibitors; co-mutated Bcor/Tp53/Dnmt3a or Bcor/Rb1 AEL cells to CDK9 inhibitor (LY2857785); Tp53 mutations alone were exclusively sensitive only to PARP inhibition (Talazoparib). In vivo mouse studies are ongoing to confirm ex vivo results. Conclusions: We successfully generated genetically defined models of AEL, demonstrated the role of Tp53 and Bcor mutations in driving the erythroid phenotype, and showed that sensitivity to different classes of compounds is genotype-dependent. These results provide the rational for testing new targeted agents in AEL. Disclosures Mullighan: Abbvie: Research Funding; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Description: Abstract 2047 Poster Board II-24 Introduction: The mitotic kinesin spindle protein (KSP) is required for the assembly of a normal bipolar spindle and cell cycle progression through mitosis. ARRY-520 is a potent, selective inhibitor of KSP that arrests cells in mitosis forming an abnormal monopolar spindle with subsequent onset of apoptosis. ARRY 520 has shown potent activity in preclinical models of hematological malignancies which support clinical investigation of this novel targeted antimitotic therapy in patients with leukemias. ARRY-520 was evaluated in a Phase 1 trial, administered as an intravenous (IV) infusion on Day 1 or on Days 1, 3 and 5 in patients with advanced/refractory leukemias. Objectives: The primary objectives of this study were to establish the safety and the maximum tolerated dose (MTD) of ARRY-520 when given as a 1-hour infusion in either a single dose or on a Days 1, 3 and 5 divided-dose schedule per cycle. Secondary objectives were to characterize the pharmacokinetics (PK) of ARRY-520 when given on these schedules, to assess evidence of preliminary clinical activity, and to explore potential biomarkers of KSP inhibition. Methods: ARRY-520 was administered as a 1-hour IV infusion as a single dose per cycle or on a divided dose schedule, in a “3 + 3” Phase 1 design. PK analyses for ARRY-520 were performed on plasma samples collected during Cycle 1 and Cycle 2. Results: A total of 33 patients with acute myelogenous leukemia (AML) and with a median age of 66 years (range 21-88 yrs) were enrolled: 15 in the single-dose schedule (dose levels 2.5, 3.75, 4.5 and 5.6 mg/m2) and 18 in the divided dose schedule (dose levels 0.8, 1.2, 1.5 and 1.8 mg/m2/day). All but one patient had disease refractory to and/or relapsed from prior therapy with a median of 4 prior regimens (range 0-7). The MTD was 4.5 mg/m2 for the single-dose schedule with the dose-limiting toxicity (DLT) of Grade 3 mucositis and was 1.5 mg/m2/day (cumulative dose per cycle of 4.5 mg/m2) for the divided dose schedule, with DLTs of Grade 3 mucositis, hand-foot syndrome and hyperbilirubinemia. ARRY-520 was well tolerated at the MTD and doses below. At the MTD, Grades 3 or 4 reversible drug-related leukopenia were observed in 4/8 evaluable patients (Grade 0 or 1 WBC at baseline) with a median nadir occurring on Day 7. In both schedules ARRY-520 showed promising signs of clinical activity as measured by significant decreases in leukemic blasts in both the peripheral blood and bone marrow. Four of 33 patients (12%) showed at least 50% reduction in bone marrow blasts and 12/33 patients (36%) showed 〉 1 log reduction in blasts in the peripheral blood. Preliminary plasma PK analyses revealed increasing ARRY-520 concentrations with increasing dose, a mean terminal t1/2 of ∼90 hours, with clearance values ranging from 1.6 to 8.0 L/hr. Conclusions: ARRY-520 showed promising signs of clinical activity and has been well tolerated in both schedules investigated in patients with AML. The most prominent DLTs were mucositis and hand-foot syndrome. The MTD was determined to be the same total dose per cycle for the single-dose and the divided-dose schedule. Data including the safety, PK, pharmacodynamics and preliminary activity of ARRY 520 from this study will be presented. Disclosures: Bethelmie-Bryan: Array BioPharma: Employment. Rush:Array BioPharma: Employment. Freeman:Array BioPharma: Employment. Simmons:Array BioPharma: Employment. Ptaszynski:Array BioPharma: Employment.

Description: Abstract 1959 Background: Kinesin Spindle Protein (KSP) is required for cell cycle progression through mitosis. Inhibition of KSP induces mitotic arrest and cell death. ARRY-520 is a potent, selective KSP inhibitor. Cancers such as multiple myeloma (MM) which depend on the short-lived survival protein MCL-1 are highly sensitive to treatment with ARRY-520. ARRY-520 shows potent activity in preclinical MM models, providing a strong rationale for its clinical investigation in this disease. Methods: This Phase 1 study was designed to evaluate the safety and pharmacokinetics (PK) of ARRY-520 administered intravenously (IV) on Day 1 and Day 2 q 2 weeks without/with granulocyte-colony stimulating factor (G-CSF). Patients (pts) with relapsed/refractory (RR) MM with 2 prior lines of therapy (including both bortezomib and an immunomodulatory agent, unless ineligible for or refusing to receive this therapy) were eligible. Cohorts of at least 3 pts were enrolled in a classical 3 + 3 dose escalation design. Pts were treated for 2 cycles (4 weeks) to evaluate safety prior to dose escalation. Results: Twenty five pts have been treated to date, with a median age of 60 years (range 44–79) and a median of 5 prior regimens (range 2–16). All pts received prior bortezomib or carfilzomib, 21 pts received prior lenalidomide, 17 pts prior thalidomide, and 18 pts had a prior stem cell transplant. Pts received ARRY-520 without G-CSF at 1 mg/m2/day (n = 3), and at 1.25 mg/m2/day (n = 7, 6 evaluable). A dose-limiting toxicity (DLT) of Grade 4 neutropenia was observed at 1.25 mg/m2/day, and this was considered the maximum tolerated dose (MTD) without G-CSF. As neutropenia was the DLT, dose escalation with prophylactic G-CSF support was initiated, at doses of 1.5 mg/m2/day (n = 7, 6 evaluable), 2.0 mg/m2/day (n = 6) and 2.25 mg/m2/day (n = 2) with G-CSF. Both the 2.0 mg/m2/day and 2.25 mg/m2/day dose levels were determined to be non-tolerated, with DLTs of febrile neutropenia (FN) (2 pts at 2.0 mg/m2/day and both pts at 2.25 mg/m2/day) and Grade 3 mucositis (both pts at 2.25 mg/m2/day). One out of 6 evaluable pts at 1.5 mg/m2/day also developed a DLT of FN. In an attempt to optimize the Phase 2 dose, an intermediate dose level of 1.75 mg/m2/day with G-CSF is currently being evaluated. The most commonly reported treatment-related adverse events (AEs) include those observed with other KSP inhibitors, such as hematological AEs (thrombocytopenia, neutropenia, anemia, leukopenia), fatigue, mucositis and other gastro-intestinal AEs. Pts displayed linear PK, a low clearance and a moderate volume of distribution, with moderate-to-high inter-individual variability in PK parameters. The median terminal elimination half life is 65 hours. The preliminary efficacy signal as a single agent is encouraging with 2 partial responses (PR) observed to date per IMWG and EBMT criteria in a heavily pretreated population (23 evaluable pts). A bortezomib-refractory pt with 8 prior lines of therapy, including a tandem transplant, treated at 1 mg/m2/day of ARRY-520 obtained a PR after Cycle 6, with urine protein and kappa light chain levels continuing to decline over time. He remains on-study after 15 months of ARRY-520 treatment. A pt with 2 prior lines of therapy, including prior carfilzomib, has obtained a PR after Cycle 8 at 2 mg/m2/day of ARRY-520, and she is currently ongoing after 4.5 months on therapy. Fifteen pts had a best response of stable disease (SD), including 1 pt with a thus far unconfirmed minimal response, and 6 had progressive disease. A total of 10 pts (43%) achieved a PR or SD lasting 〉 12 weeks. Several additional pts have shown other evidence of clinical activity, with decrease in paraproteins, increase in hemoglobin levels and regression of plasmacytomas. The median number of cycles is 4 (range 1–28+). Treatment activity has not correlated with any baseline characteristics or disease parameters to date. Conclusions: : The selective KSP inhibitor ARRY-520 has been well tolerated, and shows promising signs of single agent clinical activity in heavily pretreated pts with RR MM. Prophylactic G-CSF has enabled higher doses to be tolerated. No cardiovascular or liver enzyme toxicity has been reported. Enrollment is ongoing at 1.75 mg/m2/day with G-CSF support, and a planned Phase 2 part of the study will be initiated as soon as the MTD is determined. Complete Phase 1 data will be disclosed at the time of the meeting. Disclosures: Shah: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Research Funding. Off Label Use: Revlimid (lenalidomide) in combination with dexamethasone is indicated for the treatment of multiple myeloma patients who have received at least one prior therapy. Zonder:Millennium: Consultancy, Myeloma and Amyloidosis Patient Day Symposium – Corporate support from multiple sponsors for a one-day educational event, Research Funding; Celgene:; Novartis:; Proteolix: . Weber:novartis-unpaid consultant: Consultancy; Merck- unpaid consultant: Consultancy; celgene- none for at least 2 years: Honoraria; millenium-none for 2 years: Honoraria; celgene, Millenium, Merck: Research Funding. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Kaufman:Celgene: Consultancy, Honoraria, Research Funding; Millenium: Consultancy, Honoraria; Merck: Research Funding; Genzyme: Consultancy. Walker:Array Biopharma: Employment, Equity Ownership. Freeman:Array Biopharma: Employment, Equity Ownership. Rush:Array Biopharma: Employment, Equity Ownership. Ptaszynski:Array Biopharma: Consultancy. Lonial:Millennium, Celgene, Bristol-Myers Squibb, Novartis, Onyx: Advisory Board, Consultancy; Millennium, Celgene, Novartis, Onyx, Bristol-Myers Squibb: Research Funding.

Description: Introduction: Hypodiploid acute lymphoblastic leukemia (ALL) is a rare, high-risk subtype of B-ALL associated with poor outcome. Genomic analysis of over 130 Hypodiploid ALL patients defined the distinct genomic landscape of this disease, defining two principal subtypes including Ras-activating alterations and IKZF3 loss in near haploid tumors (24-31 chromosomes) and mutation of TP53 coupled with homozygous IKZF2 loss in low hypodiploid tumors (32-39 chromosomes). New therapies are needed to improve outcome of this disease, necessitating preclinical studies in hypodiploid tumors in vitro, ex vivo and in vivo. Hypothesis: The observation of frequent Ras pathway activating mutations in hypodiploid ALL tumors, as well as increased phosphorylation of downstream Ras signaling targets, suggests that these tumors may be susceptible to PI3K/MEK inhibition. Ras signaling target activation was observed in hypodiploid tumors irrespective of mutational status. Here we used cell lines and representative xenograft models to explore the therapeutic efficacy of inhibiting PI3K/MAPK signaling in hypodiploid ALL. Methods: The near haploid cell lines NALM-16 and MHH-CALL-2 and eight patient-derived xenograft (PDX) models (four near haploid and four low hypodiploid) were selected for preclinical studies. Xenografts were representative of the most common genomic lesions observed in hypodiploid ALL patients, including deletion of NF1, PAG1 and IKZF3 in near haploid and mutation of TP53 and IKZF2 deletion in low hypodiploid (Holmfeldt et al. Nat Genet 2013; Mullighan et al. Blood 2015). Next-gen sequencing of xenograft and diagnosis tumor DNA confirmed the presence of key genetic alterations in the xenografts. A lentiviral vector was used for stable expression of luciferase in xenograft tumors for in vivo disease burden monitoring. Cell lines were treated in vitro with PI3K, mTOR, MEK, CDK4/6 and BET inhibitors either as single agents, inhibitor combinations or combined with chemotherapeutic agents. Xenografts were treated ex vivo with single agents and in vivo with PI3K and MEK inhibitors with or without chemotherapy. Results: NALM-16 and MHH-CALL-2 cell lines were most sensitive to PI3K, mTOR and BET inhibitors with IC50 values in the sub-micromolar range (Fig. 1).Treatment of xenografts ex vivo showed near haploid and low hypodiploid tumors to be four-fold more sensitive to pan-PI3K inhibitors and PI3K/mTOR dual inhibitors compared to PI3K α and d isoform-specific inhibitors. Near haploid and low hypodiploid xenografts were sensitive to MEK and BET inhibition ex vivo, but MEK inhibitors showed weak activity in the near haploid cell lines. Treatment with CDK4/6 inhibitors either as single agents or in combination with MEK inhibitors revealed minimal efficacy. Changes in cell signaling upon treatment was assayed by phosphoflow (Fig. 2). Akt and Erk1/2 phosphorylation was variable between tumors, whereas 4E-BP1 and S6 phosphorylation was consistently elevated, suggesting strong signaling through mTOR pathways. Pan-PI3K and PI3K/mTOR dual inhibitors showed potent activity against hypodiploid ALL tumors in vitro and ex vivo, however PI3K inhibition alone was insufficient to eliminate tumors in vivo. Substantial weight loss in mice treated with PI3K/mTOR dual inhibitors limited prolonged study in vivo. MAPK inhibition showed activity in both near haploid and low hypodiploid tumors ex vivo and displayed a modest reduction in tumor growth as a single agent in vivo. Combining the MEK inhibitor GDC-0973 with dexamethasone treatment increased efficacy, but induced significantly more weight loss than either monotherapy. Conclusion: Treatment of hypodiploid cell lines and xenografts in vitro and ex vivo, respectively, with PI3K and MEK inhibitors showed promising results, however the anti-tumor effect in vivo appeared mostly cytostatic and inhibitors were not sufficient as single agents to kill the tumor cells. MEK inhibitors showed stronger activity than PI3K inhibitors on xenografts in vivo and ex vivo, but this was not significantly increased when combined with dexamethasone treatment. The notable resistance of hypodiploid cells to dexamethasone may underlie this limited efficacy in addition to the increase in GDC-0973 clearance observed upon combination. Our findings also highlight the need to consider the PK properties of agents in the preclinical setting, particularly in combinations. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.