Ambit Biosciences Research Articles

Effect of Avelumab to Immune Response in AML: A Phase I Study of Avelumab in Combination with Decitabine As First Line Treatment of Unfit Patients

Introduction: Treatment of AML remains challenging. Decitabine is a hypomethylating agent (HMA) commonly used for treating AML. However, its efficacy is limited. Recent success targeting immune checkpoints offers great promise and several agents blocking the PD-1 pathway have been FDA approved for cancer therapy. It has been demonstrated that HMA enhances PD-1 pathway in MDS and AML patients, providing a strong rationale for combining HMA and PD-1 inhibition in AML treatment. Avelumab is a PD-L1 antibody that blocks the interaction between PD-L1 and PD-1. It has been FDA approved for treating Merkel cell and urothelial carcinoma and in combination with axitinib for renal cell carcinoma. This phase I study aimed to investigate the safety of avelumab combining with decitabine in AML. In addition, extensive correlative studies were performed to define the effect of avelumab on the immune response. Methods: This is a single arm, open label phase I study to evaluate safety and tolerability of avelumab in combination with decitabine in patients with untreated AML, who are unfit for intensive chemotherapy. Decitabine was given at 20mg/m2 IV day 1-5, every 28 days. Avelumab was given at 10mg/kg IV day 1, every 14 days. Each treatment cycle was 28 days. Treatment length was up to one year or until progression. In the initial stage, classic "3+3" design was applied to determine safety. Patients were evaluated for DLTs in cycle 1. In the expansion stage, a cohort of 9 additional patients using the same regimen was planned. For correlative studies, blood and bone marrow samples were collected prior to and post initiation of avelumab. Flow cytometry-based assays for immune phenotype and functions were performed. Results: Seven patients have been treated on protocol. Median age was 71 (62-78). Most patients (6/7) carried adverse cytogenetics per ELN 2017 risk stratification. Median number of treatment cycles was 2 (range 1-12). No DLT was detected. The most frequent TEAEs (any grade or cause) were anorexia (86%), fatigue (71%), edema (71%), pleural effusion (71%), febrile neutropenia (57%) and respiratory failure (57%). Two treatment-related TRAEs were observed: fever (14%) and pneumonitis (14%). TRAEs led to treatment discontinuation in 1 patient (14%). The trial was closed before accrual met for the best interest of patients due to the recent FDA approval of venetoclax, a novel treatment for the same patient population. Among the 7 patients accrued, 1 patient achieved CR, 3 patients stable disease, 1 had progression of disease; response could not be assessed in 2 patients due to death before response assessment. To investigate the effect of avelumab on immune response, we conducted complex immune assays using blood samples collected from each patient prior to vs. 1 month post treatment. We observed no alteration in the frequency of each immune component (NK, NKT, B cells, DCs, Monocytes, CD4 T cells, CD8 T cells, and Treg) upon avelumab and decitabine treatment. However, there was an increase in effector memory differentiation while decreased terminal differentiated subsets in CD8 T cells. We next performed comprehensive phenotypic and functional analysis of CD8 T cells. Data of the first accrued 6 patients are shown here (Figure 1); the data from patient 7 is underway and will be integrated into the presentation at the meeting. We observed a strong trend of up-regulation of activation markers and co-stimulatory receptors (CD69, CD226, CD28, ICOS and 4-1BB) on CD8 T cells post treatment of avelumab and decitabine. In contrast, the expression of inhibitory molecules, including TIGIT, TIM-3, CD160, LAG-3, 2B4 and BTLA, were trending down. Consistently, CD8 T cell function was enhanced in majority of patients, manifested by higher expression of granzyme B, perforin and Ki67, as well as more cytokine release (IFN-γ and TNF-α) upon in vitro TCR engagement. Importantly, avelumab is likely the major contributor for the positive regulatory effect as studies on samples from patients who received decitabine alone did not show the same trend. Conclusion: In this phase I study, we demonstrated that avelumab in combination with decitabine as first line treatment of unfit patients with AML is safe and tolerable. CD8 T cell response trends up upon avelumab treatment. Our study strongly supports the addition of avelumab to AML treatment with a potential to improve anti-leukemia immunity. Disclosures Zheng: Pfizer: Research Funding. Claxton:Daiichi Sankyo Co. and Ambit Biosciences Corp, Astellas Pharma, Novartis Pharmaceuticals, Incyte Corporation, Cyclacel Pharmaceuticals, Inc, Celegene Corporation, Medimmune, Inc, Merck Sharp & Dohme Corp., Gilead Sciences, Inc.: Research Funding. Naik:Celgene: Other: Advisory board.

Combined Treatment with Lenalidomide and Epoetin Alfa Leads to Durable Responses in Patients with Epo-Refractory, Lower Risk Non-Deletion 5q [Del(5q)] MDS: Final Results of the E2905 Intergroup Phase III Study - an ECOG-ACRIN Cancer Research Group Study, Grant CA180820, and the National Cancer Institute of the National Institutes of Health

Combined Treatment with Lenalidomide and Epoetin Alfa Leads to Durable Responses in Patients with Epo-Refractory, Lower Risk Non-Deletion 5q [Del(5q)] MDS: Final Results of the E2905 Intergroup Phase III Study - an ECOG-ACRIN Cancer Research Group Study, Grant CA180820, and the National Cancer Institute of the National Institutes of Health

Non-Myeloablative Allogeneic Stem Cell Transplant in Acute Myeloid Leukemia: Graft-Versus-Host Disease Potentiates Graft-Versus-Leukemia Effect and Improves Overall Survival

Introduction: Non-myeloablative conditioning regimens enable elderly and younger patients with significant comorbidities to undergo allogenic stem cell transplant (allo SCT). These regimens allow for the benefit of graft-versus-leukemia (GVL) effect with reduced toxicity. A retrospective analysis of 94 patients with acute myeloid leukemia (AML) who received a non-myeloablative transplant with fludarabine (25 mg/m2 days -7 through -3)/cyclophosphamide (1000 mg/m2 days -7 and -6) (Flu/Cy) conditioning was conducted to evaluate patient outcomes. Methods: Ninety-four patients with AML underwent non-myeloablative allo SCT with Flu/Cy conditioning at Penn State Cancer Institute between 2007 and 2015 were retrospectively analyzed. The patients were deemed ineligible for myeloablative or reduced intensity conditioning transplants due to advanced age or significant comorbidities. Most unrelated donor patients (n=69) received tacrolimus/sirolimus/methotrexate as graft-versus-host disease (GVHD) prophylaxis. Overall survival (OS) and disease-free survival (DFS) were assessed with Kaplan-Meier survival curves and log rank tests, and non-relapse mortality (NRM) and relapse rates (RR) were assessed with cumulative incidence of competing events and Gray test. Cox proportional hazard regression with stepwise selection was used for multivariate analysis. Results: Patients with AML underwent Flu/Cy conditioning for allo SCT from either a matched related donor (MRD) (n =16), matched unrelated donor (MUD) (n=54), or mismatched unrelated donor (MMUD) (n=24). Evaluation of AML status immediately prior to allo SCT identified 79 patients in complete remission, 10 patients with non-remission, and 5 patients with primary induction failure. There were 11 patients with hematopoietic cell transplantation specific comorbidity index (HCT-CI): 0, 14 patients with HCT-CI: 1-2, and 69 patients with HCT-CI: ≥3. Post-transplant analysis identified: 48 patients with grade 0, 31 patients with grade I, and 15 patients with grade II-IV acute graft-versus-host disease (aGVHD). Furthermore, 52 patients were without chronic GVHD (cGVHD), 15 experienced limited cGVHD, and 13 experienced extensive cGVHD. One-year OS and DFS were 41.3% (95% CI: 31.3-51%) and 27.7% (95% CI: 18.7-37.4%), respectively. One-year NRM and RR were 12.9% (95% CI: 0.68-21%) and 59.5% (95% CI: 48.2-69.1%). There were no statistically significant differences in NRM between patients with varying comorbidity indices. One-year OS for patients in complete remission versus non-remission prior to transplant was 45.3% versus 20.0% (p<0.01), and was significantly greater in patients with grade I versus grade II-IV aGVHD (45.2% versus 17.8%, p<0.05). Patients with cGVHD showed a lower RR compared to patients without cGVHD (1-year RR 20.4% vs 48.5%, p<0.05). Patients who achieve a chimerism ≥90% within 100 days of transplant achieved a better OS and lower RR compared to patients with a chimerism <90% (1-year OS 51.3% versus 21.4%, p<0.01; 1-year RR 44.0% vs 89.3%, p<0.01, respectively). Multivariate analysis identified grade I aGVHD (versus grade II-IV, p<0.01), cGVHD (versus no cGVHD, p<0.05), and chimerism ≥90% (versus <90%, p<0.05) as independent factors for improved OS. Conclusions: The primary endpoint for this retrospective analysis was OS following non-myeloablative allo SCT for AML utilizing Flu/Cy conditioning. Although RR was high (59.5%), this conditioning regimen resulted in rapid stem cell recovery and low NRM. There were no significant differences in NRM between patients with lower (0, or 1-2) and higher (≥3) HCT-CI and patients receiving MRD, MUD or MMUD. This confirms that Flu/Cy conditioning is safe, especially for patients with multiple comorbidities and receiving MMUD. This analysis also identified a significant improvement in OS for patients with grade I as compared to grade II-IV aGVHD. Also, patients with cGVHD showed a significantly reduced RR. Collectively, the improved survival for patients with grade I aGVHD and cGVHD may be associated with a more robust GVL effect. Additionally, patients who achieve a higher chimerism showed a reduced RR. This study highlights the tolerability of Flu/Cy and the importance of balancing GVHD and GVL effects; future studies are needed to evaluate the addition of maintenance therapies to overcome higher RR occurring with this regimen. Disclosures Naik: Celgene: Other: Advisory board. Zheng:Pfizer: Research Funding. Claxton:Daiichi Sankyo Co. and Ambit Biosciences Corp, Astellas Pharma, Novartis Pharmaceuticals, Incyte Corporation, Cyclacel Pharmaceuticals, Inc, Celegene Corporation, Medimmune, Inc, Merck Sharp & Dohme Corp., Gilead Sciences, Inc.: Research Funding.

Maintenance Decitabine (DAC) Improves Disease-Free (DFS) and Overall Survival (OS) after Intensive Therapy for Acute Myeloid Leukemia (AML) in Older Adults, Particularly in FLT3-ITD-Negative Patients: ECOG-ACRIN (E-A) E2906 Randomized Study

Maintenance Decitabine (DAC) Improves Disease-Free (DFS) and Overall Survival (OS) after Intensive Therapy for Acute Myeloid Leukemia (AML) in Older Adults, Particularly in FLT3-ITD-Negative Patients: ECOG-ACRIN (E-A) E2906 Randomized Study

Recurrent Pathogenic Cyclin D2 Mutations in Myeloid Malignancies

Introduction: Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) are rare hematologic neoplasms characterized by leukocytosis, a hypercellular bone marrow with granulocytic predominance, absence of the Philadelphia chromosome (t(9;22); BCR-ABL1), and absence of PDGFRA/B or FGFR1 gene rearrangements. While oncogenic mutations in colony stimulating factor 3 receptor (CSF3R) are commonly reported in CNL and, to a lesser extent, in aCML, a significant percentage of aCML and CNL cases lack well-defined pathogenetic lesions. In this study, we utilized whole exome sequencing and a functional genomics approach to identify recurrent cyclin D2 (CCND2)mutations in aCML/CNL.Methods: To achieve a more complete understanding of the pathogenic variants underlying these leukemias, genomic DNA was isolated from peripheral blood or bone marrow aspirates from 116 patients with aCML/CNL. Whole exome sequencing was then performed, allowing for the identification of novel and potentially deleterious recurrent mutations. Variants in CCND2 were prioritized for validation based on proximity to functional targets in protein interaction network databases and subsequently confirmed via Sanger sequencing. CCND2 expression was assessed by immunoblotting and immunofluorescence in NIH-3T3 cells. Transforming potential of the CCND2 mutations was assessed by examining IL-3-independent growth of Ba/F3 cells and murine bone marrow colony formation assays. Sensitivity of these mutations to CDK4/6 inhibition was examined via a colorimetric viability assay (MTS) at 72 hours following drug treatment.Results: Out of 116 suspected cases of aCML/CNL, we identified 4 cases (3.4%) that harbored recurrent mutations in CCND2, the gene encoding the cell cycle regulator cyclin D2. The mutations occurred in two nucleotides in a conserved region of the C-terminal PEST degradation domain and encoded either a P281S or a P281L (3 P281S, 1 P281L) variant. These variant proteins exhibited resistance to both degradation and to export from the nucleus, effectively resulting in accumulation of cyclin D2 protein. In turn, this accumulation was associated with increased murine bone marrow colony formation and sensitivity to CDK4/6 inhibition. Subsequent whole exome sequencing of 239 AML samples revealed the presence of the same variants (CCND2 P281S and P281L) in 3 samples (1.3%), suggesting that these variants may occur across multiple subsets of myeloid malignancy.Conclusions: Our study provides evidence of recurrent deleterious CCND2 mutations in aCML/CNL and AML. These mutations may contribute to oncogenesis in myeloid malignancy and are therapeutically targetable through CDK4/6 inhibition. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Vadastuximab Talirine Plus Hypomethylating Agents: A Well-Tolerated Regimen with High Remission Rate in Frontline Older Patients with Acute Myeloid Leukemia (AML)

Vadastuximab Talirine Plus Hypomethylating Agents: A Well-Tolerated Regimen with High Remission Rate in Frontline Older Patients with Acute Myeloid Leukemia (AML)

Mass Cytometry As a Modality to Identify Candidates for Immune Checkpoint Inhibitor Therapy within Acute Myeloid Leukemia

Introduction: Immune checkpoint inhibitors (ICIs) have shown significant efficacy in solid malignancies and recently in Hodgkin's disease. However, there are presently no biomarkers that predict which cancer types and/or individual patients will benefit from ICIs. Despite intensification of chemotherapeutic regimens, survival rates in acute myeloid leukemia (AML) have plateaued. We are therefore, performing studies to characterize the immune phenotype and genetic profile of AML patients who may respond to ICI therapy. Specifically, we are identifying cell subsets present in the marrow of patients with AML, evaluating their differentiation and activation status, and quantifying the density of immune modifiers. We are also performing functional assays to define the status of T cells in the AML microenvironment by evaluating the responsiveness of these cells to stimulation. Additionally, these studies assess the impact of several ICIs on the proliferative potential and cytokine production capacity of T cells resident in the bone marrow of AML patients.Methods: Immunophenotyping was performed on the diagnostic bone marrow (Dx BM), diagnostic peripheral blood (Dx PB), and remission bone marrow (CR BM) for a single patient with AML. These studies used a time-of-flight mass cytometer (CyTOF) by staining samples with a panel containing 35 antibodies to surface markers and cytokines (Table 1). T cells were defined and then differentiated based on the presence of CD3 and CD4 or CD8. T cells were further classified into naive, central memory (CM), effector memory (EM), and effector cells. Functional assays measured proliferative capacity and cytokine production profile of bone marrow T cells in response to CD3 stimulation for the Dx BM and Dx PB. Changes to the baseline T cell functional profile in the presence of antibodies against PD1, CTLA4, TIM3, and TIGIT were measured in the Dx BM.Results: T cell percentages were 3.14%, 5.46%, and 17.1% for the Dx BM, Dx, PB, and CR BM, respectively. CD4:CD8 ratios were 3.5, 2.9, and 4.5, respectively. EM cells made up the largest percentage of CD8+ T cells in the Dx BM (40.99%), Dx PB (57.06%), and CR BM (53.55%). Naïve (52.49%), EM (37.63%) and CM (44.3%) cells made up the largest percentages of CD4+ T cells in the Dx BM, Dx PB, and CR BM, respectively (Figure 1). Checkpoint expression was absent (median intensity < 1) on all T cells except for some CD8+ subsets (Figure 2). Within the Dx BM, the effector (1.93) and EM (4.34) cells expressed TIGIT and the EM cells expressed PD1 (1.92). Within the Dx PB, the effector (1.81) and EM (4.0) cells expressed TIGIT. Within the CR BM, the effector cells (2.3) expressed TIGIT. In response to CD3 stimulation, 57.5% of the Dx PB T cells divided while there was no proliferation observed in the Dx BM. In response to CD3 stimulation and PD1 blockade, 28.2% of the Dx BM T cells divided and there was a 4-fold increase in IFNγ production. There was no division in the Dx BM T cells in response to TIGIT blockade but there was a 10-fold and 121-fold increase in IL-2 and IFNγ production, respectively. There was no significant proliferation or cytokine production in response to TIM3 or CTLA4 blockade.Conclusion: We observed differential T cell responses to stimulation in the presence of ICIs in this patient. The Dx PB T cells were capable of proliferating in the presence of CD3 stimulation alone whereas the Dx BM T cells were only able to divide in the presence of PD1 blocking antibody. PD1 blockade elicited proliferation and low levels of IFNγ production whereas TIGIT blockade elicited negligible proliferation but high levels of IFNγ production. There was low but differential checkpoint expression on T cells, with PD1 and TIGIT having the highest expression, specifically on CD8+ T cell subsets. The predominant CD8+ T cell subset, along with differential checkpoint expression, persisted into the CR BM but there was a shift in the predominant CD4+ T cell subset. These data suggest the presence of immune suppression in the bone marrow microenvironment that may be amenable to checkpoint inhibition. [Display omitted] [Display omitted] [Display omitted] DisclosuresHuang:Janssen Research & Development, LLC: Employment, Equity Ownership. Sasser:Janssen Research & Development, LLC: Employment. Adams:Janssen Research & Development, LLC: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Lind:Janssen: Research Funding; Fluidigm: Honoraria.

FGF2 from Bone Marrow Stroma Protects Acute Myeloid Leukemia Cells from the FLT3 Inhibitor Quizartinib and Facilitates Acquisition of Resistance Mutations

Background: Acute myeloid leukemia (AML) patients with internal tandem duplication of FLT3 (FLT3-ITD AML) routinely develop resistance to FLT3 inhibitor monotherapy. Resistance to inhibitors such as quizartinib can develop through mutation of the FLT3 kinase domain, but not all resistant patients have mutations indicating that additional mechanisms of resistance are important. Although quizartinib induces a rapid reduction of circulating leukemia blasts, residual blasts in the marrow persist, suggesting that the microenvironment protects blasts from quizartinib and may serve as a reservoir for development of resistance.Results: Using a screen of molecules from the microenvironment, we found that FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) were the two most protective proteins of FLT3-ITD MOLM14 cells when treated with 10 nM quizartinib in vitro. FL protection has been previously described by Mark Levis' group, so we focused on the mechanism of FGF2 protection. FGF2 bound FGFR1, activating the MAPK pathway to mediate resistance. FGF2-mediated resistance could be overcome by concomitant inhibition of the FGFR receptor using the inhibitor PD173074 or by siRNA knock-down of FGFR1. FGFR1 is highly expressed in primary AML cells, and exogenous FGF2 protected primary FLT3-ITD AML cells from quizartinib ex vivo in a dose-dependent fashion (p=0.0007 at 100 ng/ml).MOLM-14 cells were cultured continuously with quizartinib in media alone, or media supplemented with 10 ng/ml FGF2 or FL to mimic the effects of the microenvironment. MOLM14 cells supplemented with FL or FGF2 resumed growth after 6-8 weeks, significantly faster than cells cultured in media alone, of which only 2/4 developed resistance after 12 weeks. To test the dependence of resistance cultures on exogenous ligand, we removed ligand from the FGF2- and FL-dependent cultures after 4 months and continued quizartinib treatment. After a temporary pause in growth, the cells regained exponential growth within 1 month. We assayed FLT3 resistance mutations by Sanger sequencing during this time, and again at 8 months with a targeted next-generation sequencing panel of commonly mutated genes in AML. Deep sequencing revealed multiple FLT3 mutations along with recurrent mutations of KRAS and NRAS, indicating the importance of the FLT3 and MAPK signaling pathway in resistance (Figure 1). Multiple FLT3 mutations were identified strongly suggesting that FLT3 mutations were not pre-existing but developed during culture with quizartinib (Figure 1). In contrast, the frequency of the KRAS G13D mutation suggested this mutation was present at a low frequency in MOLM14 cells.FGF2 expression was evaluated by immunohistochemistry in serial bone marrow core biopsies from 10 FLT3-ITD AML patients on the phase II quizartinib trial. Stromal FGF2 expression increased significantly with quizartinib therapy (p<0.01) and remained elevated until patients acquired FLT3 resistance mutations or other intrinsic mechanisms of resistance. Once stromal FGF2 became redundant for survival, expression decreased again. FGF2 protected cells in a paracrine fashion since FGF2 immunofluorescence did not overlap with CD45 (hematopoietic marker) in core biopsies of quizartinib-treated patients.To test the effect of combined FLT3 and FGFR inhibition in a more complex in vitro model of the microenvironment, MOLM14 cells were co-cultured in transwells over HS-5 (FGF2 high expression) or HS-27 stromal cell lines (FGF2 low). HS-5 stromal cells were highly protective of MOLM14 cells treated with quizartinib alone, but this protection was attenuated by combined FGFR and FLT3 inhibition (p<0.01). In contrast, protection of MOLM14 cells in HS-27 co-culture was minimal, and unaffected by combined FLT3 and FGFR inhibition.Conclusions: Our data supports a two-step model of resistance to quizartinib with initial resistance mediated by microenvironmental proteins such as FGF2 and/or FL, followed by kinase domain mutation of FLT3 and/or activating mutations of the RAS pathway. Early resistance mediated by FGF2-expressing stroma can be overcome by concomitant inhibition of FLT3 and FGFR suggesting that pre-emptively targeting extrinsic resistance pathways, in combination with newer FLT3 inhibitors that have activity against kinase domain mutations, will improve the durability of response to FLT3 inhibitors. [Display omitted] DisclosuresAgarwal:CTI BioPharma Corp: Research Funding. Kovacsovics:Seattle Genetics: Research Funding. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

A Genomic Signature Predicting Venetoclax Treatment Response in AML Identified By Protein Network Mapping and Validated By Ex Vivo Drug Sensitivity Testing: A Beat AML Project Study

Abstract Background: Data from a small clinical trial of venetoclax in acute myeloid leukemia (AML) recently supported FDA breakthrough therapy designation for use in combination with hypomethylating agents in treatment-naïve patients who are ineligible for high-dose induction chemotherapy. Approval of this BCL-2 inhibitor raises the question of mechanism of action in AML and patient selection for treatment. Unfortunately, no biomarkers or methods exist to predict venetoclax response in AML, making treatment selection challenging. Aim: To define a novel genomic signature rule to predict AML response to venetoclax therapy and to validate the rule with ex vivo drug sensitivity testing. Methods: The Beat AML project (supported by the Leukemia &amp; Lymphoma Society) collects clinical data and bone marrow specimens from AML patients. Bone marrow samples are analyzed by conventional cytogenetics, whole-exome sequencing, RNA-seq, and an ex vivo drug sensitivity assay. For 19 of these randomly chosen patients, every available genomic abnormality was inputted into a computational biology program (Cellworks Group) that uses PubMed and other online resources to generate patient-specific protein network maps of activated and inactivated protein pathways. Digital drug simulations with venetoclax were conducted by quantitatively measuring drug effect on a composite AML disease inhibition score (i.e., cell proliferation, viability, and apoptosis). Computational predictions of drug response were compared to venetoclax IC50 values from the Beat AML ex vivo testing. Results: Ten of the 19 AML patients were predicted by computer simulation to respond to venetoclax, and 9 of those 10 patients had the lowest IC50 values to venetoclax. Nine of the 19 patients were predicted to not respond to venetoclax, and 8 of those 9 patients had the highest IC50 values to venetoclax. Ex vivo venetoclax responses were correctly matched to their computer simulation prediction in 17 of 19 cases, and incorrectly matched in 2 cases. The positive predictive value of the computational method was 90%, negative predictive value was 89%, sensitivity was 90%, specificity was 89%, and accuracy was 89%. Amplification of the genes RB1CC1 and/or RB1was predicted by computational modeling to increase MCL1, which made venetoclax less responsive in the digital drug simulation. This genomic rule was validated with 3 AML patients: one who received venetoclax as treatment and showed refractory disease, and 2 patients who achieved complete remission after venetoclax treatment. Conclusion: We identified a new genomic signature, confirmed by functional testing, that predicts AML response to venetoclax treatment. This unique computer-based approach is intended to inform the design of phase 2 and 3 clinical trials of venetoclax in AML patients for a forthcoming precision enrollment clinical trial. Disclosures Abbasi: Cellworks: Employment. Vali:Cellworks Group: Employment. Radhakrishnan:Cellworks: Employment. Kumar Singh:Cellworks: Employment. Usmani:Cellworks: Employment. Parashar:Cellworks: Employment. Vidva:Cellworks: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents &amp; Royalties, Research Funding; Array: Patents &amp; Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents &amp; Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents &amp; Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents &amp; Royalties; Roche: Consultancy.

Pacritinib Targets IRAK1 and Shows Synergy with HDAC and BET Inhibitors in Acute Myeloid Leukemia

Background: The bone marrow microenvironment modulates response of acute myeloid leukemia (AML) to therapy. Thus necessitating the identification of microenvironment-driven signaling mechanisms that contribute to AML pathogenesis. We recently showed that AML growth depends on IL-1 signaling in a majority of cases that include a spectrum of genetic aberrations. IL-1 signals through the serine/threonine kinase IL-1 receptor-associated kinase (IRAK1), a central mediator of immune and inflammatory responses that was recently suggested as a potential therapeutic target in leukemia. Pacritinib is currently under development as a small-molecule JAK2 inhibitor for myelofibrosis; recently a kinome screen of 429 kinases showed that pacritinib also inhibits IRAK1 activity (IC50 = 13.6 nM). Here we provide further evidence to support targeting of IRAK1 by pacritinib as a therapeutic strategy in AML, and show synergistic activity with histone deacetylase (HDAC) or bromodomain (BET) inhibitors to overcome drug resistance across genetic subtypes.Methods: We used inducible shRNA to reduce IRAK1 levels in primary AML cells. To validate the selectivity of pacritinib for IRAK1, we performed targeted mutagenesis on key interacting residues. We compared the efficacy of pacritinib, an established dual FLT3/JAK2 inhibitor, to that of the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 12 cell lines and 46 primary AML samples with various genetic lesions. We tested the synergy of pacritinib in combination with various drugs, including HDAC, BET, and BTK inhibitors, and BH3 mimetic. Equimolar concentrations of both agents were tested at a dose gradient of 1.0-5000 nM. Synergy and combination index were calculated using Calcusyn software. The effects of drug treatments were evaluated on cell viability, survival, differentiation, and downstream signaling.Results: Genetic knockdown of IRAK1 reduced the viability of AML cell lines and primary AML cells by 70% and reduced p38 and ERK1/2 kinase activity. Molecular docking simulation showed that pacritinib forms favorable interactions with D298 and S295 residues in the IRAK1 kinase domain. IRAK1D298K and IRAK1S295D conferred significant pacritinib resistance compared to native IRAK1, confirming the selective binding and on-target inhibitory effect of pacritinib. Primary AML samples and cell lines showed sensitivity to pacritinib (median IC50 = 90 nm, range = 2.0-1000 nM, p<0.004), with a higher response rate (44/46 or 96%, or IC50 < 1000 nM) than that observed with ruxolitinib (15%) or quizartinib (80%). With regard to IC90 < 1000 nM, a more stringent measure of efficacy, a higher percentage of primary AML samples (33%) showed sensitivity to pacritinib than that for quizartinib (13%, p<0.0001). Mechanistic studies showed that pacritinib inhibits the activity of downstream effectors in the IL-1 pathway, including IRAK1 and p38, in addition to inhibiting the secretion of various inflammatory cytokines. Consistent with previous studies, pacritinib inhibited FLT3 and JAK2/JAK3 activity in FLT3- or JAK-dependent AML cells, respectively. Additionally, our data suggest that pacritinib reduced the viability of AML primary samples and cell lines harboring a variety of genetic abnormalities, including TET2, MLL, ASXL1, NPM1, CEBPA, and RAS mutations, by inhibiting IRAK1 activity. To identify potential combination therapies, we combined pacritinib with agents from several classes and found synergy with the HDAC inhibitor panobinostat and the BET inhibitor JQ1 with a combination index <1. Specifically, treatment with 50 nM pacritinib or 50 nM JQ1 alone reduced the viability of primary AML cells to 50%, while the combined treatment reduced viability to ~25%. Similarly, 50 nM pacritinib or 20 nM panobinostat alone reduced the viability of AML cells to 50%, while the combined treatment reduced viability to ~10%. Pacritinib blocked cell growth, while HDAC or BET inhibitors promoted differentiation of AML cells and reduced numbers of early progenitors. The combinations blocked not only IRAK1 and ERK activity, but also c-Myc.Conclusions: IRAK1 is a potential therapeutic target in AML across genetic subtypes. Pacritinib, in addition to suppressing JAK2 and FLT3 signaling, specifically blocks IRAK1 signaling in AML and synergizes with HDAC or BET inhibitors to block growth and promote differentiation, establishing its potential clinical utility. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Singer:CTI BioPharma Corp.: Employment, Equity Ownership, Other: Leadership . Agarwal:CTI BioPharma Corp: Research Funding.

A Phase 1b Study of Vadastuximab Talirine As Maintenance and in Combination with Standard Consolidation for Patients with Acute Myeloid Leukemia (AML)

A Phase 1b Study of Vadastuximab Talirine As Maintenance and in Combination with Standard Consolidation for Patients with Acute Myeloid Leukemia (AML)

Vadastuximab Talirine Monotherapy in Older Patients with Treatment Naive CD33-Positive Acute Myeloid Leukemia (AML)

BackgroundCD33is expressed in approximately 90% of AML cases, representing a promising target despite age, prior therapies, or mutational heterogeneity. Vadastuximab talirine (33A) is a CD33-directed antibody conjugated to 2 molecules of a pyrrolobenzodiazepine (PBD) dimer. Upon binding, 33A is internalized and transported to the lysosomes where PBD dimer is released via proteolytic cleavage of the linker, crosslinking DNA, and leading to cell death.MethodsThe dose-escalation portion of this phase 1 study (NCT01902329) was designed to evaluate the safety, tolerability, pharmacokinetics, and antileukemic activity of 33A as monotherapy. After dose escalation, 40 mcg/kg was identified as the recommended monotherapy dose. Eligible patients (ECOG status 0-1) in this expansion cohort must have had CD33-positive AML and considered ineligible for or declined conventional induction/consolidation. 33A monotherapy was administered outpatient IV every 3 weeks for up to 2 cycles, followed by optional low-dose maintenance treatment for patients who achieved a CR/CRi. Investigator assessment of response was per IWG criteria; CRi required either platelet count of ≥100,000/µL or neutrophils of ≥1,000/µL (Cheson 2003).ResultsTwenty-seven treatment naive patients (48% male) with a median age of 74 years (range, 67-89) were treated with 40 mcg/kg of 33A. Most patients had intermediate (70%) or adverse (26%) cytogenetic risk by MRC and 48% of patients had underlying myelodysplasia. Twenty-four patients were considered unfit for intensive therapy and 3 patients declined intensive therapy. At baseline, patients had a median of 47% BM blasts. Three patients remain on treatment. The most common Grade 3 or higher adverse events (AE) were thrombocytopenia (44%), febrile neutropenia (41%), anemia (33%), fatigue, and pneumonia (19% each). Other common treatment-emergent AEs regardless of relationship to 33A were decreased appetite, diarrhea, fatigue, peripheral edema, thrombocytopenia (44% each), febrile neutropenia (41%), dizziness (37%), anemia, chills, cough, dyspnea, and epistaxis (33% each). The 30- and 60-day mortality rates were 0% and 15%. Of the 26 efficacy evaluable treatment naive patients, 6 patients (23%) achieved a best clinical response of CR, 8 (31%) achieved CRi, and 5 patients (19%) achieved a morphologic leukemia-free state. Most remissions were achieved after 1 cycle and were observed in many patients with adverse risk including underlying myelodysplasia (6/12, 50%), FLT3/ITD+ (3/4, 75%), and in patients ≥75 years of age (8/12, 67%). Of the responding patients with available minimal residual disease (MRD) data, 6 of 13 (46%) achieved MRD negativity by flow cytometry. In patients who achieved at least a CRi, the median time to full count recovery from first dose was 6.1 weeks for neutrophils (≥1,000/µL) and 5.1 weeks for platelets (≥100,000/µL). Median OS continues to evolve with 10 patients (37%) alive at the time of this data cut. Pharmacokinetic data for treatment naive patients is consistent with previously reported results; 33A exhibits rapid elimination consistent with target-mediated drug disposition.ConclusionsIn an expansion cohort of 33A monotherapy at 40 mcg/kg, AEs observed were generally manageable and commonly associated with on-target myelosuppression. 33A has demonstrated favorable antileukemic activity as a single agent with 54% achieving a CR+CRi in this high risk treatment naive older AML population, more than doubling the response rate expected with standard non-intensive therapies such as hypomethylating agents or low-dose cytarabine. The rapid clearance of marrow blasts, high rate of MRD-negative remissions, and low early mortality rate are encouraging. These data support exploration of 33A in combination with standard induction, consolidation, and pre- and post-transplant regimens. DisclosuresStein:Celgene: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Argios: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics: Research Funding. Fathi:Agios Pharmaceuticals: Other: Advisory Board participation; Bexalata: Other: Advisory Board participation; Seattle Genetics: Consultancy, Other: Advisory Board participation, Research Funding; Celgene: Consultancy, Research Funding; Merck: Other: Advisory Board participation. Kovacsovics:Seattle Genetics: Research Funding. Levy:Seattle Genetics: Research Funding; Jansen: Speakers Bureau; Amgen: Speakers Bureau; Millennium: Speakers Bureau. Erba:Daiichi Sankyo: Consultancy; Jannsen: Consultancy, Research Funding; Celator: Research Funding; Incyte: Consultancy, DSMB, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Juno: Research Funding; Astellas: Research Funding; Ariad: Consultancy; Millennium Pharmaceuticals, Inc.: Research Funding; Pfizer: Consultancy; Agios: Research Funding; Novartis: Consultancy, Speakers Bureau; Gylcomimetics: Other: DSMB; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Sunesis: Consultancy. Jillella:Seattle Genetics: Research Funding. Ravandi:Seattle Genetics: Consultancy, Honoraria, Research Funding; BMS: Research Funding. Stein:Novartis: Consultancy; Seattle Genetics: Research Funding; Agios Pharmaceuticals: Other: Advisory Board, Research Funding; Celgene: Other: Advisory Board, Research Funding. Faderl:Astellas: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding; Celator Pharmaceuticals: Research Funding; BMS: Research Funding; Ambit Bioscience: Research Funding; Karyopharm: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; JW Pharma: Consultancy; Amgen: Speakers Bureau. DeAngelo:Baxter: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; Celgene: Consultancy. Ho:Seattle Genetics: Employment, Equity Ownership. O'Meara:Seattle Genetics: Employment, Equity Ownership. Voellinger:Seattle Genetics: Employment, Equity Ownership. Advani:Seattle Genetics: Consultancy, Research Funding.

Ponatinib in Chronic-Phase Chronic Myeloid Leukemia Patients: Final Report from a Phase 1 Trial

Ponatinib in Chronic-Phase Chronic Myeloid Leukemia Patients: Final Report from a Phase 1 Trial

MS4A3: A New Player in Leukemic Stem Cell Survival in Chronic Myeloid Leukemia

Background: We have previously demonstrated that the transcriptional profile of diagnostic CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients exhibiting primary cytogenetic resistance to imatinib overlaps with that of patients with myeloid blast phase CML (BP-CML) (McWeeney et al. Blood 2010). These data suggest that primary resistance to tyrosine kinase inhibitors (TKIs) and advanced disease are biologically related. The hematopoietic cell cycle regulator, MS4A3, was identified as a principal component of the gene expression classifier predicting response to imatinib. Low MS4A3 correlated not only with primary TKI resistance, but also with shorter overall survival in CP-CML (n=35). Consistently, microarray (n=19 CP-CML; n=16 BP-CML), qRT-PCR (n=22 CP-CML; n=17 BP-CML), and immunoblot (n=3 CP-CML; n=3 BP-CML) analyses demonstrated that MS4A3 mRNA and protein levels are reduced in CD34+ progenitor cells from BP-CML versus CP-CML patients, with no difference between CP-CML and normal CD34+progenitors (n=3) (Eiring et al. ASH 2015 #14). These data suggest that MS4A3 may play a role in both primary TKI resistance and blastic transformation of CML.Results: To assess the functional role of MS4A3 in CML and TKI response, we used ectopic MS4A3 expression and shRNA-mediated MS4A3 knockdown in CD34+ cells from BP-CML and CP-CML patients, respectively. Ectopic expression of MS4A3 in BP-CML CD34+ progenitors (n=5) markedly reduced colony formation in the presence and absence of imatinib, consistent with a tumor suppressor role for MS4A3 in CML. While re-expression of MS4A3 alone did not increase apoptosis compared to empty vector-expressing controls, imatinib-induced apoptosis in BP-CML CD34+ cells was increased by 62%, with no effect on normal CD34+ cord blood cells (n=2). Conversely, shRNA-mediated MS4A3 knockdown (shMS4A3) in CP-CML CD34+ cells (n=7) reduced the effects of imatinib in colony formation and apoptosis assays, with no effect on normal CD34+ progenitors (n=4). In contrast to a previous report (Donato JL, et al. J Clin Invest 2002), we detected no change in cell cycle status of CML or normal CD34+ cells upon MS4A3 ectopic expression or knockdown (n=3). Altogether, these data suggest that MS4A3 positively regulates patient survival and imatinib response in CML progenitor cells.To evaluate MS4A3 in the leukemic stem cell compartment, we performed qRT-PCR on primary CP-CML cells (n=5) and observed that MS4A3 mRNA levels are 22-fold higher in committed CD34+38+ progenitors compared to more primitive CD34+38- stem cells, suggesting a role for MS4A3 in differentiation. Consistently, qRT-PCR, immunoblot, and flow cytometry demonstrated that MS4A3 mRNA and protein were upregulated in CP-CML CD34+ cells upon G-CSF treatment (n=3). Flow cytometry also revealed that shMS4A3 in CP-CML CD34+ cells resulted in a reduction of CD11b+ cells by ~45% in the presence of G-CSF (n=3). To assess the function of MS4A3 in CML stem cells, we performed long-term culture-initiating cell (LTC-IC) assays and xenografts into NSG mice upon MS4A3 knockdown in CP-CML (n=3). shMS4A3 increased Ph+ LTC-IC colony formation in the absence, and even more so in the presence, of imatinib, with no effects on Ph- LTC-ICs. Consistent with these data, shMS4A3 enhanced engraftment of CD34+CD45+GFP+ cells into the bone marrow of NSG recipient mice. Preliminary data in primary TKI-resistant and BP-CML CD34+ cells suggests regulation of this gene by promoter hypermethylation.Conclusions: Altogether, these data suggest that MS4A3 plays a key role in imatinib response of 1) patients with primary TKI resistance, 2) patients with BP-CML, and 3) the CML stem cell compartment. Since the effects of MS4A3 in CML do not involve changes to the cell cycle, experiments are underway to identify the mechanism by which MS4A3 improves imatinib response and survival in CML. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Deininger:Incyte: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Gilead: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CTI BioPharma Corp.: Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

A Phase II Pilot Study of Kinase Inhibition in Relapsed/Refractory Acute Leukemias: Using an in Vitro Kinase Inhibitor Panel to Select Individualized, Targeted Therapies

A Phase II Pilot Study of Kinase Inhibition in Relapsed/Refractory Acute Leukemias: Using an in Vitro Kinase Inhibitor Panel to Select Individualized, Targeted Therapies

A Phase 1b Study of Vadastuximab Talirine in Combination with 7+3 Induction Therapy for Patients with Newly Diagnosed Acute Myeloid Leukemia (AML)

A Phase 1b Study of Vadastuximab Talirine in Combination with 7+3 Induction Therapy for Patients with Newly Diagnosed Acute Myeloid Leukemia (AML)

TP53 Mutations Determine Outcome of Patients (pts) with High-Risk Myelodysplastic Syndrome (HRMDS) and Acute Myelogenous Leukemia (AML) Regardless of Karyotype and ELN Risk Stratification

Background: Cytogenetic and molecular abnormalities are the backbone of risk stratification for pts with AML/MDS. Yet, the current stratification systems still fail to identify some pts at high risk for treatment failure. TP53 mutations have been linked to poor outcome and treatment resistance in many malignancies. We conducted a retrospective case-control study of pts with HRMDS and AML with TP53 mutations (TP53 m) to define disease characteristics, response, and outcome after transplant (HSCT).Methods: Twenty-one consecutive pts with HRMDS or AML were treated between January 2014 and March 2016 who wereTP53 m. The pts were compared to a control cohort of 46 pts with wild type TP53 (TP53 wt). Mutation analysis was performed using next generation sequencing. All AML pts were treated with a standard 3+7 induction protocol. Pts were excluded for incomplete cytogenetic and molecular data.Results: A total of 21 patients were identified with TP53 m (14 HRMDS, 11 AML). The median age of the 21 pts was 64 years (range 32-84). Pts with TP53 m were older (median age 64 vs 55 years, p=.0256) and exhibited a higher rate of adverse cytogenetics (71% vs 30.4%, p=.043) when compared to TP53 wt. Secondary AML was also more common with TP 53 m (57% vs 31%, p=.05). Pts with TP53 m were less likely to achieve CR (33% vs 74%, p=.009). Three pts with TP53 m proceeded to transplant (2 with HRMDS and 1 with AML).With a median follow up of 3.4 months (range 0.3-73) the estimated PFS and OS at 18 months was superior in TP 53 wt compared to TP 53 m (17% vs 69%, p=.037 and 17% vs 50%, p=.0095 respectively). HSCST was associated with a 70% reduction in mortality, but this was not statistically significant (p=.067).Conclusion: TP 53 mutations in pts with HRMDS and AML are associated with advanced age, high risk cytogenetic abnormalities, and secondary AML. Our findings demonstrate that TP53 m in HR MDS and AML is associated with poor clinical outcome including lower CR rates, and lower PFS and OS. Different treatment strategies need to be devised for these pts. DisclosuresMcCloskey:Incyte: Consultancy; Ariad: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Novartis: Speakers Bureau. Koprivnikar:Alexion: Consultancy, Speakers Bureau. Depadova:Amgen: Speakers Bureau. Stanislaus:Novartis: Consultancy, Speakers Bureau. Goldberg:COTA Inc: Employment; Bristol Myers Squibb, Novartis: Speakers Bureau; Neostem: Equity Ownership; Novartis: Consultancy; Pfizer: Honoraria. Faderl:Celgene: Consultancy, Research Funding; JW Pharma: Consultancy; BMS: Research Funding; Ambit Bioscience: Research Funding; Karyopharm: Consultancy, Research Funding; Celator Pharmaceuticals: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding; Amgen: Speakers Bureau.

Pharmacological IRS1/2 Inhibition Induces Apoptosis in BCR-ABL1T315I mutant Cells

Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 oncoprotein, which results in deregulated kinase activity-driven leukemic phenotype. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, but a significant percentage of patients develop drug resistance and/or progress to blast crisis, a disease stage that is refractory to therapy. Furthermore, recent studies have provided evidence that the CML leukemia stem cell is resistant to imatinib treatment. In this sense, the identification of other crucial proteins that cooperate with BCR-ABL1 signaling pathways may represent secondary therapeutic targets optimize treatment strategies. Increased IRS1 mRNA expression has been previously identified as positively correlated with worse overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 and is capable of activating the PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We have previously shown that NT157 treatment reduces colony formation of primary CML cells but not in normal cells, decreases cell viability and proliferation of K562 cells, and increases apoptosis of K562 cells in a time- and dose-dependent manner (ASH 2015 - Abstract #83876). To further characterize the mechanism of the sensitivity of CML cells to IRS1/2 inhibition, here we performed PCR-based gene expression analyses in CML cells in response to NT157 treatment and investigated the effects of IRS1/2 inhibition on cell proliferation and apoptosis in Ba/F3 cells expressing wild-type or T315I mutant BCR-ABL1.Materials and Methods: K562 cells treated with vehicle or with the IRS1/2 pharmacological inhibitor NT157 (1.6 µM) for 48 hours were submitted to PCR array analysis using the PI3K-AKT Signaling Pathway and CancerPathwayFinder RT2 Profiler PCR Array kit (#PAHS-033A and #PAHS-058A, respectively; SA Biosciences, Frederick, MD, USA). Each cDNA sample was processed in a 96-well plate containing 156 signaling pathway-related genes and 5 endogenous control genes. Ba/F3 parental, BCR-ABL1WT and BCR-ABL1T315I cells were subjected to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6 and 3.2 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI), and protein expression/activation (Western blot). Statistical analyses were performed by Student’s t-test or Mann-Whitney test, as appropriate.Results: Sixteen genes were found to be differentially expressed (fold change ≥ 2); 5 upregulated genes (FOXO3A, CDKN1A, FOS, JUN, VEGF) and 1 downregulated gene (E2F1) were chosen for validation in a larger number of experiments. Notably, NT157 treatment also resulted in significant dose- and time-dependent decrease in viability in BCR-ABL1-expressing cells regardless of BCR-ABL1 mutation status, compared to parental Ba/F3 cells. NT157 treatment (1.6 µM) for 48 hours reduced cell viability of Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells to 64% and 76%, respectively (p<0.05). Identical treatment with NT157 also significantly induced apoptosis as noted by the percentage of cells positive for annexinV/PI staining compared to vehicle in Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells (84 vs. 13% and 92 vs. 22%, respectively; p<0.05). Similar results were observed after 24 and 72 hours of NT157 treatment for both cell lines. More importantly, NT157 treatment reduced phosphorylation of STAT3 at both serine (S)727 and tyrosine (Y)705 in Ba/F3 BCR-ABL1T315I in the absence of IL3.Conclusion: Pharmacological inhibition of IRS1/2 decreases cell viability, increases apoptosis, and reduces STAT3 activation in cells expressing wild-type or T315I-mutant BCR-ABL1 in a time- and dose-dependent manner. These findings suggest that targeting of IRS1/2 alone or in combination with ABL1 TKIs may offer further improved treatment strategies and outcomes for maximizing control of disease resistance and persistence in patients with CML. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Enhanced VISTA Expression in a Subset of Patients with Acute Myeloid Leukemia

Introduction: Identification of tumor associated antigens (TAA) in acute myeloid leukemia (AML) will facilitate the use of targeted immunotherapies such as chimeric antigen receptor T cell therapy and bispecific antibodies. The characteristics of an ideal TAA include tumor specificity, high expression level, large percentage of positive tumor cells, and evidence for expression on putative cancer stem cells. V-domain Ig Suppressor of T cell Activation (VISTA) is a cell surface protein previously shown to be present on hematologic cells and inhibitory towards T cells. VISTA's role and presence within AML has yet to be defined. We performed detailed studies to characterize VISTA expression on tumor cells and mononuclear cell subsets in patients with AML.Methods: Immunophenotyping was performed on mononuclear cells from primary AML patient specimens using mass cytometry (CyTOF) by staining samples with a panel containing 34 antibodies to surface markers and cytokines (Table 1). This antibody panel was used to identify blast cells (variable immunophenotype), leukemic stem cells (LSCs) (CD34+, CD38-), T cells (CD3+), monocytes (CD33+, CD14+, CD11b+, HLA-DR+), myeloid derived suppressor cells (MDSCs) (CD33+, CD11b+, HLA-DR-), and M2 macrophages (CD11b+, CD163+). Raw median VISTA expression and percentage of cells expressing VISTA were calculated for all of these cell types. Samples were split into 2 categories based on blast VISTA expression. A sample was considered VISTA high if >10% of the blasts expressed VISTA, and VISTA low if <10% of the blasts expressed VISTA. Similar data was generated from a pool of healthy donors for comparison.Results: Fourteen bone marrow samples from patients with newly diagnosed AML and 10 from healthy donors were obtained. The average percentage of blasts expressing VISTA was 68% (range 28% - 81%) in the VISTA high group and 4.2% (range 0.3% - 6.8%) in the VISTA low group (p<0.05). VISTA expression was higher in the VISTA high group than in the VISTA low group (17.7 vs -0.1, p<0.05). The VISTA high group had a lower blast percentage in the bone marrow compared to the VISTA low group (56% vs 91%, p<0.05). The VISTA high and low groups had similar percentages of LSCs (2.5% vs 0.6%, p=0.11), but the LSCs in the VISTA high group maintained higher VISTA expression than did those in the VISTA low group (6.81 vs -0.11, p<0.05). The LSCs within the VISTA high group had similar VISTA expression to the blast population (6.81 vs 17.7, p=0.15). Within the non-blast population, VISTA expression was higher on the myeloid cell subsets than on the T cells for both the AML samples (11.5 vs 0.17, p<0.05) and the healthy donor samples (8.07 vs 0.24, p<0.05). VISTA expression was similar between the VISTA high and low groups on T cells (0.36 vs 0.02, p=0.08), monocytes (15.7 vs 19, p=0.7), MDSCs (6.3 vs 4, p=0.26), and M2 macrophages (7.4 vs 16.6, p =0.25). VISTA expression was similar between AML and healthy donor samples for T cells (0.17 vs 0.24, p=0.6), monocytes (15.7 vs 14, p=0.41), MDSCs (6.3 vs 3.6, p=0.22), and M2 macrophages (7.4 vs 6.6, p=0.19). Monocytes had the highest expression of VISTA within the healthy donor samples (p<0.05) and this difference was maintained in the AML samples.Conclusion: A subset of patients with AML has a large percentage of blasts, including LSCs, with high VISTA expression on their surface. VISTA is expressed on normal myeloid cells in the bone marrow of patients with AML, but at low levels and to a similar degree as in the marrow of healthy donors. [Display omitted] [Display omitted] DisclosuresHuang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Janssen Research & Development, LLC: Employment. Adams:Janssen Research & Development, LLC: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Lind:Janssen: Research Funding; Fluidigm: Honoraria.

Phase I Trial of Targeted Alpha-Particle Immunotherapy with Actinium-225 (225Ac)-Lintuzumab (Anti-CD33) and Low-Dose Cytarabine (LDAC) in Older Patients with Untreated Acute Myeloid Leukemia (AML)

Background: The use of short-ranged (50-80 µm), high-energy (~100 keV/µm) α particle-emitting isotopes for radioimmunotherapy may result in more specific tumor cell kill and less damage to normal tissues than β-emitters. 225Ac-lintuzumab consists of a radiometal that emits four α-particles linked to an anti-CD33 antibody. A phase I trial showed that 225Ac-lintuzumab is safe at doses ≤ 3 µCi/kg and has anti-tumor activity against relapsed/refractory AML across all dose levels studied (Jurcic et al. ASH, 2011). We are conducting a multicenter, phase I dose-escalation trial to determine the maximum tolerated dose (MTD), toxicity, and biological activity of fractionated-dose 225Ac-lintuzumab in combination with LDAC.Patients and Methods: Patients ≥ 60 years with untreated AML not suitable for standard induction chemotherapy (e.g., antecedent hematologic disorder, unfavorable cytogenetic or molecular abnormalities, and significant comorbidities) were eligible. Patients received LDAC 20 mg twice daily for 10 days every 4-6 weeks for up to 12 cycles. During Cycle 1, two fractions of 225Ac-lintuzumab were given one week apart, beginning 4-7 days following completion of LDAC. To prevent radiation-induced nephrotoxicity, patients were given furosemide while receiving 225Ac-lintuzumab and spironolactone for one year afterward. 225Ac doses were escalated using a 3+3 design. Four dose levels were studied with a total accrual of up to 24 patients. In planned analyses, dose escalation proceeded if < 33% of patients in a cohort experienced dose-limiting toxicity (DLT).Results: Fourteen patients (median age, 77 years; range, 68-87 years) completed therapy. An additional patient received only one of two planned fractions of 225Ac-lintuzumab due to technical issues and is excluded from analysis. Nine (64%) had prior myelodysplastic syndrome, for which seven received prior therapy with hypomethylating agents (n=6) or allogeneic hematopoietic cell transplantation (n=1). One patient (7%) had chronic myeloid leukemia in molecular remission prior to development of AML. Nine patients (64%) had intermediate-risk and five (36%) had unfavorable cytogenetics. Median CD33 expression was 81% (range, 45-100%). 225Ac-lintuzumab was given at 0.5 (n=3), 1 (n=6), 1.5 (n=3), or 2 (n=2) μCi/kg/fraction. Up to 4 cycles of LDAC were administered. DLT was seen in one patient at 1 µCi/kg/fraction who had grade 4 thrombocytopenia with marrow aplasia for more than 6 weeks following therapy. Hematologic toxicities included grade 4 neutropenia (n=4) and thrombocytopenia (n=6). Grade 3/4 non-hematologic toxicities included febrile neutropenia (n=7), pneumonia (n=4), bacteremia (n=1), cellulitis (n=1), transient creatinine increase (n=1), hypokalemia (n=1), rectal hemorrhage (n=1), and generalized weakness (n=2). Eight of 11 patients (73%) evaluated after Cycle 1 had bone marrow blast reductions (mean reduction, 72%; range, 34-100%). Seven (64%) had blast reductions of at least 50%. Objective responses (1 CR, 1 CRp, 2 CRi) were seen in four of the 14 patients (29%) after one cycle of therapy (Table 1). Responses were seen only at doses ≥ 1 µCi/kg/fraction (4 of 11 patients, 36%). Median progression-free survival (PFS) was 2.7 months (range, 1.7-16.9 months). Median overall survival (OS) was 5.5 months (range, 2.2-24 months).Conclusions: Fractionated-dose 225Ac-linutuzmab can be safely combined with LDAC and produce remission in older patients with untreated AML. Dose escalation continues to define the MTD. Additional patients will be treated at the MTD in the phase II portion of this trial to determine response rate, PFS, and OS.Table 1Objective ResponsesResponseDose Level (μCi/kg/fraction)Total (n=14)0.5 (n=3)1 (n=6)1.5 (n=3)2 (n=2)CR001 (33%)01 (7%)CRp0001 (50%)1 (7%)CRi01 (17%)1 (33%)02 (14%)Overall Response01 (17%)2 (67%)1 (50%)4 (29%)Abbreviations: CR, complete remission; CRp, CR with incomplete platelet recovery; CRi, CR with incomplete count recovery. DisclosuresJurcic:Ambit Biosciences: Research Funding; Astellas Pharma US, Invc.: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Sunesis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Bayer Pharmaceuticals: Consultancy; Merck and Co.: Consultancy; Celgene Corp.: Research Funding; Actrinium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Ac-225-lintuzumab is an investigational agent being developed for the treatment of acute myeloid leukemia.. Pagel:Actinium Pharmacetuicals, Inc.: Equity Ownership. Park:Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy. Levy:Takeda: Consultancy. Perl:Actinium Pharmaceuticals, Inc.: Research Funding. Earle:Actinium Pharmaceuticals, Inc.: Employment. Cicic:Actinium Pharmaceuticals, Inc.: Employment, Equity Ownership. Scheinberg:Actinium Pharmaceuticals, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.