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Adding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day −8 for 6-7 doses) in combination with fludarabine 30 mg/m2 per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day −5 to day −2 (FluBu2). Transplant related–toxicity was evaluated from the first venetoclax dose on day −8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.gov as #NCT03613532.

Adding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day −8 for 6-7 doses) in combination with fludarabine 30 mg/m2per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day −5 to day −2 (FluBu2). Transplant related–toxicity was evaluated from the first venetoclax dose on day −8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.govas #NCT03613532.

Inhibition of the anti-apoptotic machinery of cancer cells is a promising therapeutic approach that has driven the development of an important class of compounds termed “BH3 mimetics”. These novel small molecules mimic BH3-only proteins by antagonizing the pro-survival function of anti-apoptotic proteins, thereby inducing apoptosis in cancer cells. To qualify as an authentic BH3 mimetic, a compound must function directly on the mitochondria of a cell of known anti-apoptotic dependence, must directly and selectively inhibit the anti-apoptotic protein with high-affinity binding, and must induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis in a BAX/BAK-dependent manner. While many BH3 mimetics have entered clinical trials, the lack of a reliable validation assay to directly test the mitochondrial activity of new BH3 mimetic candidates has resulted in many misleading reports of agents touted as BH3 mimetics despite their off-target mechanisms of action. BH3 profiling probes the activity of a compound at the mitochondrial level by measuring cytochrome c release as a surrogate marker for MOMP. We propose a comprehensive biochemical toolkit consisting of BH3 profiling in parallel with high-throughput Annexin V/Hoechst viability testing to validate BH3 mimetic candidates. We tested our toolkit on eighteen different putative BH3 mimetics using a set of standardized cell lines of known anti-apoptotic dependence. Included in this set of cell lines is an apoptosis refractory BAX/BAK DKO control line to detect compounds that function independently of the BCL-2 family. Taken together, this rapid, efficient means of testing will prove advantageous as the demand for BH3 mimetics increases, particularly in the quest to identify and develop more potent MCL-1 inhibitors for use in the clinic. We strongly urge researchers utilizing BH3 mimetics in their work to use the potent and selective compounds identified with this validation toolkit instead of those lacking such potency and selectivity.

•Venetoclax plus low-intensity chemotherapy can be safely administered in ALL.•Venetoclax plus low-intensity chemotherapy is a promising therapy in older patients with newly diagnosed, high-risk ALL.

•Maintenance treatment was safe with low rates of infections and chronic GVHD, and minimal impact on T cell-immune reconstitution.•With 2-year follow-up, ven/aza maintenance showed a manageable safety profile and encouraging responses in high risk MDS/AML.

Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1–dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2–mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.

Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1–dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2–mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.

Despite of remarkable changes in therapeutic landscape of AML in past 5 years, relapsed AML patients still succumb to disease. In majority of patients, the common cause of death ultimately is the emergence multi-drug resistance at relapse. Using patient-derived xenograft (PDX) as our models, we investigated the basis of multi-drug resistance and methods for identifying drug sensitivity in relapsed AML. We hypothesized that there is differential activation of drug-induced mitochondrial apoptotic signaling between treatment-naïve and relapsed myeloblasts. To study mechanisms of drug resistance in AML, we developed PDX models (n=18 models) of in vivoacquired resistance to single agents (BCL-2/MCL-1 inhibitor, FLT3 inhibitor, SMAC mimetics and BRD-4 inhibitor) of distinct mechanisms, which caused board chemoresistance across all models. We tested whether multidrug resistance is associated with reduction in apoptotic signaling, using BH3 profiling, a method for measurement of mitochondrial sensitivity to apoptosis via exposure to synthetic BH3 domain peptides, on matched pre- and post-resistant PDXs. Using BH3 profiling, we found that a reduction in mitochondrial apoptotic priming consistently accompanied the acquisition of multidrug resistance irrespective of the mechanism of drug action and genetic status of PDX models. Next we created pharmacologic sensitivity landscape for 22 AML PDX models by exposing splenic myeloblasts isolated after confirmed engraftment (>70% circulating hCD45+ blasts) to drug a drug panel (n=40) for 14 hours. We first demonstrate that ex vivo drug-induced mitochondrial sensitivity singling measured viadynamic BH3 profiling (DBP) segregates PDXs according to prior treatment. By using unsupervised hierarchical clustering we found that AML PDXs developed from treatment naïve patients clustered together and showed greater apoptotic priming responses to a larger number of targeted agents than PDXs from R/R patients, which also formed discrete clusters. We replicated drug-response patterns from PDX models in primary and relapsed AML patients by analyzing ex vivo sensitivity responses to >500 drugs across relapse and diagnosis patients (Malani et al, 2021 and Tyner et al, 2018). We show that human AML patients at relapse also broadly lose sensitivity to agents in a similar pan-resistant manner as PDX models.

Alpha helices form a critical part of the binding interface for many protein–protein interactions, and chemically stabilized synthetic helical peptides can be effective inhibitors of such helix-mediated complexes. In particular, hydrocarbon stapling of peptides to generate constrained helices can improve binding affinity and other peptide properties, but determining the best stapled peptide variant often requires laborious trial and error. Here, we describe the rapid discovery and optimization of a stapled-helix peptide that binds to Mcl-1, an antiapoptotic protein that is overexpressed in many chemoresistant cancers. To accelerate discovery, we developed a peptide library synthesis and screening scheme capable of identifying subtle affinity differences among Mcl-1-binding stapled peptides. We used our method to sample combinations of non-natural amino-acid substitutions that we introduced into Mcl-1 inhibitors in the context of a fixed helix-stabilizing hydrocarbon staple that increased peptide helical content and reduced proteolysis. Peptides discovered in our screen contained surprising substitutions at sites that are conserved in natural binding partners. Library-identified peptide M3d is the most potent molecule yet tested for selectively triggering mitochondrial permeabilization in Mcl-1 dependent cell lines. Our library approach for optimizing helical peptide inhibitors can be readily applied to the study of other biomedically important targets.

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

Mcl-1 is overexpressed in many cancers and can confer resistance to cell-death signaling in refractory disease. Molecules that specifically inhibit Mcl-1 hold potential for diagnosing and disrupting Mcl-1-dependent cell survival. We selected three peptides from a yeast-surface display library that showed moderate specificity and affinity for binding to Mcl-1 over Bfl-1, Bcl-xL, Bcl-2, and Bcl-w. Specificity for Mcl-1 was improved by introducing threonine at peptide position 2e. The most specific peptide, MS1, bound Mcl-1 with 40-fold or greater specificity over four other human Bcl-2 paralogs. In BH3 profiling assays, MS1 caused depolarization in several human Mcl-1-dependent cell lines with EC50values of ∼3 μM, contrasted with EC50values of >100 μM for Bcl-2-, Bcl-xL-, or Bfl-1-dependent cell lines. MS1 is at least 30-fold more potent in this assay than the previously used Mcl-1 targeting reagent NoxaA BH3. These peptides can be used to detect Mcl-1 dependency in cells and provide leads for developing Mcl-1 targeting therapeutics.

Human embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.

Garcia: Eli Lily: Research Funding; Pfizer: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stone:Astellas: Consultancy; Argenix: Other; Syros: Consultancy; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Celgene: Consultancy, Other; Daiichi-Sankyo: Consultancy; Syndax: Consultancy, Research Funding; Macrogenics: Consultancy; Stemline: Consultancy; Pfizer: Consultancy; Trovagene: Consultancy; Aztra-Zeneca: Consultancy; Jazz: Consultancy; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy, Research Funding; Biolinerx: Consultancy; Takeda: Other: DSMB; Syntrix: Other: DSMB; Agios: Consultancy, Research Funding; Gemoab: Consultancy; Abbvie: Consultancy, Research Funding. Koreth:EMD Serono: Consultancy; Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; Equillium: Consultancy; Biolojic Design Inc: Consultancy; Therakos: Membership on an entity's Board of Directors or advisory committees; BMS: Other: Research Support; Amgen: Consultancy; Moderna Therapeutics: Consultancy; Miltenyi: Other: Research Support; Clinigen: Other. Nikiforow:Nkarta Therapeutics: Honoraria; Novartis: Honoraria; Kite/Gilead: Honoraria. Steensma:Takeda: Consultancy; BMS/Celgene: Consultancy; Onconova: Consultancy; Arena: Current equity holder in publicly-traded company; CRISPR: Current equity holder in publicly-traded company; Aprea Therapeutics: Research Funding; Arrowhead Pharmaceuticals: Current equity holder in publicly-traded company; Astex Pharmaceuticals, Onconova Therapeutics, Pfizer, Stemline Therapeutics, Summer Road: Consultancy; H3 Biosciences: Research Funding. Letai:AbbVie: Consultancy; AstraZeneca: Consultancy; Chugai: Other; Dialectic: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Zentalis: Membership on an entity's Board of Directors or advisory committees. Lindsley:Jazz Pharmaceuticals: Consultancy, Research Funding; MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Bluebird Bio: Consultancy. Soiffer:Celgene: Other; Juno: Other; Alexion: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Gilead: Consultancy; Be The Match/National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees. DeAngelo:Forty-Seven: Consultancy; Shire: Consultancy; Blueprint Medicines Corporation: Consultancy, Research Funding; Takeda: Consultancy; Abbvie: Research Funding; Glycomimetics: Research Funding; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy; Jazz: Consultancy; Agios: Consultancy; Amgen: Consultancy; Autolos: Consultancy.venetoclax with conditioning chemotherapy in the context of a clinical trial

Objective: To alert clinicians to the hepato‐toxic potential of Augmentin (amoxycillin and clavulanic acid), a widely prescribed antibiotic, in susceptible patients, and to point out that the hepatitic illness may be delayed but serious and protracted. Design and setting: Case reports of patients with Augmentin‐induced jaundice referred to the gastroenterology departments in three major teaching hospitals, and a review of cases reported to the Australian Adverse Drug Reactions Advisory Committee (ADRAC). Patients: Eight patients with nine episodes of Augmentin‐induced jaundice personally treated by the authors from March 1988 to February 1990 are described. A further 19 patients reported to ADRAC from May 1987 to November 1989 are discussed. All patient histories were carefully reviewed to ensure that there was a temporal relationship between the course of Augmentin and the onset of the hepatitic illness and that other causes of jaundice were reasonably excluded. Results: Jaundice developed in some of these patients several weeks after drug treatment was completed. The illness may be protracted over many weeks. As yet, there has been no case of progressive disease leading to liver failure. Conclusions: The data suggest that a hypersensitivity reaction to clavulanic acid is the likely cause of the jaundice. Therefore, Augmentin, although an important antibiotic, should be reserved for severe infections for which amoxycillin is unsuitable.

1. Cirrhosis is often complicated by disturbances in the systemic circulation. We have previously demonstrated decreased vascular responses to vasoconstrictors in forearm resistance arteries in subjects with alcoholic cirrhosis. In the current study we investigate the role of the potent endogenous vasodilator nitric oxide in the peripheral circulation of these patients. 2. Ten patients with alcoholic cirrhosis (Pugh grade A) and 10 age-matched control subjects were studied. The effect of blockade of nitric oxide synthesis was studied both in vivo in forearm resistance arteries using forearm venous occlusion plethysmography and in vitro in veins isolated from the forearm. The role of endothelium-derived nitric oxide was studied in vivo using the endothelium-dependent vasodilator acetylcholine. 3. Mean arterial pressure and forearm basal flow in vivo were similar in the two groups. The constrictor response (percentage decrease in forearm blood flow) to noradrenaline (100 ng/min) was 26% smaller in patients with cirrhosis (31.65 ± 2.64%) than in control subjects (42.75 ± 3.87%, P = 0.037). Constrictor responses to the nitric oxide synthase inhibitor NG-monomethyl-l-arginine were not different in the two groups. Dilator responses to acetylcholine were significantly attenuated in cirrhotic patients compared with control subjects. 4. To investigate the role of smooth muscle-derived nitric oxide in vitro, all veins were stripped of their endothelium. Responses to noradrenaline were significantly diminished in veins isolated from patients with cirrhosis compared with control subjects. Incubation with the nitric oxide synthase inhibitor Nω-nitro-l-arginine had no effect on responses to noradrenaline in veins from control subjects but significantly enhanced the maximal response to noradrenaline by 23.95% (range 3.77–100%, P = 0.043) in veins from patients with cirrhosis. 5. Responses to noradrenaline were attenuated in vivo in forearm resistance arteries in patients with alcoholic cirrhosis. This impairment was also apparent in forearm isolated veins, stripped of the endothelium. Our data exclude a major role for endothelium-derived nitric oxide but highlight a possible role for smooth muscle-derived nitric oxide.

Background:Studies of the in vivo vascular reactivity of the peripheral circulation to pressor agents in cirrhosis have produced conflicting results, possibly because of changes in mean arterial pressure that make it difficult to clearly separate peripheral and central effects. The aim of the present study was to assess the reactivity of the forearm circulation to pressor agents in vivo without activating central control systems. Methods:Forearm blood flow was measured by venous occlusion strain gauge plethysmography in the basal state and during the infusion of subpressor doses of norepinephrine and angiotensin II into the brachial artery in 10 male patients with well-compensated alcoholic cirrhosis and 10 male age-matched controls. Plasma renin activity and aldosterone and angiotensin II concentrations were assayed. Forearm and systemic sympathetic nervous system activity was estimated using a norepinephrine spillover technique. Results:Basal forearm blood flow, renin angiotensin aldosterone system activity, and forearm sympathetic nervous system activity were similar in both the control and cirrhotic groups. The cirrhotic patients showed an impaired response to both norepinephrine and angiotensin II. Conclusions:There is impaired reactivity of the peripheral vasculature to pressor agents in cirrhosis, indicating that the control of vascular tone is disturbed even in well-compensated cirrhosis.

Wang: Novartis Institutes for Biomedical Research: Current Employment. Qiu:Novartis Institutes for Biomedical Research: Current Employment. Halilovic:Novartis Institutes for Biomedical Research: Current Employment, Current equity holder in publicly-traded company.

Pharmacodynamic (PD) biomarkers provide information about the pharmacologic effects of a drug on its target, i.e. to assess whether a given agent is engaging its target in the expected manner. There is currently no convenient PD marker for BH3 mimetics. BH3 profiling (BP) is a functional assay developed to measure cells susceptibility to apoptosis and cell's dependence on certain Bcl-2 anti-apoptotic proteins for survival. Dynamic BH3 profiling (DBP) measures the changes of BP signals after drug perturbations. In this study, we studied whether DBP with lymphoid cells can serve as a PD biomarker for the activity of BH3 mimetics.

Effective treatments might be identified by rapidly profiling drug sensitivities in patient biopsies from solid tumors.

Garcia: Abbvie: Research Funding; Genentech: Research Funding. Ryan:Vivid Biosciences: Consultancy. Letai:Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Lindsley:Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding; Jazz Pharmaceuticals: Research Funding. Ho:Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Omeros Corporation: Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Consultancy; Amgen: Consultancy; Equillium: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Galinsky:ABIM: Other: Member on specialty oncology board; AbbVie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Merus Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Steensma:Summer Road: Consultancy; Arrowhead: Equity Ownership; H3 Biosciences: Other: Research funding to institution, not investigator.; Onconova: Consultancy; Astex: Consultancy; Pfizer: Consultancy; Stemline: Consultancy; Aprea: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Soiffer:Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Kiadis: Other: supervisory board; Mana therapeutic: Consultancy; Cugene: Consultancy; Jazz: Consultancy. Cutler:BiolineRx: Other: DSMB; Cellect: Other: DSMB; Jazz: Consultancy; BMS: Consultancy; Genentech: Consultancy; ElsaLys: Consultancy; Kalytera: Other: DSMB; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy; Incyte: Consultancy; Kadmon: Consultancy; Omeros: Consultancy. DeAngelo:Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Novartis: Consultancy, Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Blueprint: Consultancy, Research Funding.Venetoclax is a BH3 mimetic and selective BCL-2 inhibitor that was added to standard conditioning chemotherapy (fludarabine and busulfan) on a clinical trial.

BCL-2 antagonist venetoclax combined with hypomethylating agents or low-dose cytarabine is a new standard of care for treatment-naive elderly or unfit AML patients. Despite the striking overall response of 70%, only 30% achieved MRD negative status with a short remission duration of 11.3 months. This highlights the need for new agents that can overcome venetoclax resistance. We exploited mitochondrial functional assay called BH3 profiling to identify predictors of clinical responses and to discover combination partners of venetoclax. BH3 profiling measures apoptotic priming of a cell (proximity to the apoptotic threshold) by measuring mitochondrial response to a standardized panel of pro-apoptotic BH3 oligopeptides. By using pretreatment myeloblasts of patients (N=16) from clinical trial of venetoclax and azacitidine, we found that response to HRK + MS1 peptides (infers BCL-XL and MCL-1 dependency, respectively) inversely correlates with the achievement of remission. The serial sampling on treatment revealed the emergence of differential patterns of anti-apoptotic dependency that correlated with patient response. Of note, mitochondrial sensitivity to the MS-1 peptide, an indication of MCL-1 dependence, increased from 0% at diagnosis to 40% at relapse, suggesting an opportunity for MCL-1 antagonism in the relapsed setting.

Ryan: Vivid Biosciences: Consultancy. Erick:Novartis: Employment. Weinstock:Celgene: Research Funding. Garcia:Abbvie: Research Funding; Genentech: Research Funding. Letai:Novartis: Research Funding; Flash Therapeutics: Equity Ownership; Abbvie: Consultancy, Research Funding; Vivid Bioscience: Equity Ownership; Astrazeneca: Research Funding.

Background:Patients (pts) with myeloid malignancies characterized by high risk mutations or cytogenetics who undergo allogeneic hematopoietic cell transplantation (HCT) have poor outcome primarily due to risk of relapse. Strategies to limit relapse include post-transplant maintenance or novel conditioning regimens. The selective BCL-2 inhibitor venetoclax (VEN), a BH3 mimetic, promotes mitochondrial apoptosis in myeloblasts in the presence of cytotoxic stress and is associated with high response rates when combined with hypomethylating agents or low dose cytarabine in untreated elderly AML. We found that VEN could induce apoptotic priming in myeloblasts regardless of poor risk mutations such as TP53by BH3 profiling, which measures the mitochondrial response to perturbation by a panel of BH3 domain peptides. We thus hypothesized that VEN would promote the anti-leukemic effect of conditioning chemotherapy and therefore reduce the risk of relapse without undue toxicity.

Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

Johnson: Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; AbbVie Inc.: Consultancy, Honoraria, Research Funding; Lundbeck: Consultancy, Honoraria, Other: travel, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, Research Funding. Steidl:Juno Therapeutics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding. Scott:Janssen: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria; Roche: Research Funding. Letai:Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership; AbbVie: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AstraZeneca: Consultancy, Other: Lab research report.

Background:The anti-apoptotic protein BCL2 is expressed in most non-Hodgkin lymphomas (NHL) including chronic lymphocytic leukemias (CLL), mantle cell lymphomas (MCL), follicular lymphomas (FL), diffuse large B cell lymphomas (DLBCL) and marginal zone lymphomas (MZL). BCL2 expression is associated with an inferior survival when co-expressed with MYC, an oncogene that induces cellular proliferation, especially in high-grade lymphomas with translocations in MYCand BCL2(HGBL-DH). Venetoclax, a selective BCL2 inhibitor, is effective in CLL, but has modest clinical activity in other NHLs. CLL has been shown to be “primed” and BCL2-dependent, predicting for a favorable response to venetoclax using BH3 profiling. This technique measures mitochondrial outer membrane depolarization (MOMP) after exposure to synthetic pro-apoptotic BH3 peptides that have different affinities to anti-apoptotic proteins BCL2, BCLXL, MCL1 and BCLW. Cells with functional BAX/BAK undergo MOMP after exposure to pro-apoptotic proteins BIM/BID. “Primed” cells undergo MOMP after exposure to BIM, BID and PUMA, where the latter inhibits all anti-apoptotic proteins. Thus, inhibiting the anti-apoptotic proteins would initiate apoptosis. We hypothesized that BH3 profiling of NHLs could help us understand the mechanisms of venetoclax-resistance in BCL2+ NHL. This has not yet been performed due to the rarity of samples archived as a viable cell suspension.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1.

ABT-263 blocks BCL-XLto induce apoptosis in human dermal fibroblasts from patients with scleroderma and in myofibroblasts in mice, reversing dermal fibrosis.

Background:The anti-apoptotic protein BCL2 is expressed in chronic lymphocytic leukemias (CLL), 85% of follicular lymphomas (FL) and 60% of diffuse large B cell lymphomas (DLBCL). It is associated with an inferior survival when it is co-expressed with MYC, an oncogene inducing cellular proliferation, especially when MYC and BCL2 expression is caused by translocations, as in “double hit lymphomas” (DHL). Venetoclax, a selective BCL2 inhibitor, is effective in CLL, but has modest activity in other BCL2-expressing non-Hodgkin lymphomas (NHL). CLL cells are considered “primed” to die and BCL2-dependent, i.e. they are closer to the apoptotic threshold because they express high levels of pro-apoptotic BIM, which is sequestered by BCL2. Venetoclax binds to BCL2 allowing BIM to initiate mitochondrial outer membrane depolarization (MOMP) and apoptosis. We hypothesize that other NHLs may not be “primed” (e.g. low BIM), express other anti-apoptotic proteins not targeted by venetoclax (eg. MCL1, BCLXL, BFL1, BCLW) or have dysfunctional effector proteins BAX/BAK.

ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models.

Tyner: Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Hofmann: Novartis Institutes for BioMedical Research: Employment. Baffert:Novartis: Employment. Vangrevelinghe:Novartis Institutes for BioMedical Research: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Weinstock:Novartis: Consultancy, Research Funding.

Off Label Use: The presentation discuss the sensitivity of myeloma cells to the Bcl-2 specific BH3 mimetic ABT-199. Le Gouill:Roche: Consultancy; Janssen: Consultancy. Letai:Abbvie: Consultancy.

Davids: Genentech: Consultancy; Infinity Pharmaceuticals: Consultancy. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Letai:Abbvie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.

No relevant conflicts of interest to declare.

Despite significant improvement in the treatment of Multiple Myeloma (MM), patients still experience relapse and the disease remains incurable. Cellular fate decision in response to drug therapy is mainly determined by the interactions among BCL-2 family members. Thus anti-apoptotic BCL-2 family proteins represent an attractive target for therapy. BH3 profiling is a functional assay that identifies the tumor cell’s addiction to anti-apoptotic members, such as BCL-2, BCL-XL or MCL-1. In the present study, we determined the BCL-2, BCL-XL and MCL-1 dependency of both MM cell lines and primary myeloma samples, and then tested the ability of BH3 profiling to predict the in vitrosensitivity to BH3 mimetics.

BH3 profiling is a functional assay that assesses the degree to which cells are primed to die via mitochondrial apoptosis. We previously used this technique to show that pre-treatment CLL cells from patients who responded to frontline chemoimmunotherapy had higher levels of apoptotic priming than cells from refractory patients (Davids et al., Blood, 2012). BH3 profiling also detects dependence on individual anti-apoptotic Bcl-2 family proteins. Recently, ABT-199, a selective, potent, small molecule antagonist of the anti-apoptotic protein Bcl-2, has been found to be highly active in patients with relapsed or refractory CLL, even those with high-risk markers such as del(17p) (Seymour et al., EHA, 2014). Unlike its Bcl-XL/Bcl-2-targeting predecessor navitoclax (ABT-263), ABT-199 spares platelets, which rely on Bcl-XL for survival. We hypothesized that baseline levels of apoptotic priming would be associated with the depth of initial clinical response to ABT-199 in CLL patients.

Approximately 10% of B-ALLs harbor CRLF2rearrangements, which may portend a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity in vitroor in vivo(Weigert et al. J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al. Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation, which may overcome this resistance. When assayed in MHH-CALL4 cells harboring a CRLF2/IGH rearrangement and JAK2 I682F mutation, the type II JAK2 inhibitors NVP-BBT594 and NVP-CHZ868 were 10-35-fold more potent than the type I JAK2 inhibitors NVP-BSK805 and NVP-BVB808. Similarly, in Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the IC50 for CHZ868 was 5-20-fold lower than the IC50s for BSK805 and BVB808. Unlike type I inhibitors, which induce paradoxical hyperphosphorylation of JAK2, CHZ868 completely blocks JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not alter sensitivity to CHZ868. CHZ868 abrogates STAT5 phosphorylation in Ba/F3 cells expressing CRLF2 with JAK2 R683G/Y931C while BVB808 does not. CHZ868 is the first type II JAK2 inhibitor amenable to in vivouse.We assessed its efficacy in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/-or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Splenocytes from patient-derived xenografts (PDXs) treated with CHZ868 in vivofor 3 days are more primed for apoptosis as demonstrated by a 2-6-fold EC50 reduction for PUMA permeabilizing activity compared to vehicle. Transcriptional profiling of splenocytes from CHZ868-treated PDXs revealed downregulation of critical survival pathways including E2F1, STAT3, and AKT-mediated signaling. Of note, 2 of the most downregulated genes are STAT targets, PIM1and Myc. Mice treated for 5-6 days with CHZ868 had significant reductions in spleen size and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p<0.001). BH3 profiling of splenocytes from PDXs treated until moribund showed a 2-4-fold increase in the EC50 for BIM compared to vehicle, consistent with decreased priming for apoptosis in the relapsed setting. To study mechanisms of resistance to type II JAK2 inhibitors, we screened a randomly mutagenized JAK2 R683G library expressed in Ba/F3-CRLF2 cells for clones resistant to BBT594. All >30 clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P displayed cross-resistance to CHZ868, while sensitivity to type I inhibitors was not affected. Structural modeling of the JAK2 JH1 domain suggested that L884P alters the binding pocket for type II inhibitors. JAK2 L884P is homologous to an EGFR L747P activating mutation, which destabilizes the P-loop and C-helix portion of the kinase domain (He et al. Clin Cancer Res 2012). The fact that L884P was reported in two B-ALL patients lacking additional JAK2 mutations (Torra et al. Blood (ASH Annual Meeting Abstracts) 2010) raised the possibility it was also an activating mutation. We confirmed L884P is an activating allele, as Ba/F3 cells expressing CRLF2, IL7R, and JAK2 L884P proliferated in the absence of TSLP ligand. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents currently used in B-ALL treatment. Dexamethasone was the most highly synergistic with CHZ868 in MHH-CALL4 cells. To assess the combination in vivo,we treated mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL with vehicle, CHZ868, dexamethasone, or CHZ868 + dexamethasone for 14 days post engraftment. CHZ868 treatment prolonged survival compared to vehicle (p<0.0001) or dexamethasone (p<0.01), and the combination prolonged survival beyond CHZ868 monotherapy (p<0.0001). In summary, the type II JAK2 inhibitor CHZ868 potently kills JAK2-dependent B-ALL and overcomes genetic resistance to type I inhibitors. CHZ868 prolongs survival in murine transgenic and human xenograft models and synergizes with dexamethasone in vivo. Thus, combination strategies using dexamethasone with type II JAK2 inhibitors merit testing in patients with relapsed or refractory JAK2-dependent B-ALL.

CD4+CD25+Foxp3+ regulatory T cells (Treg) play a critical role in establishment of immune tolerance and prevention of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The recovery and maintenance of Treg after HSCT is dependent on homeostatic factors including the generation of naïve Treg from hematopoietic precursor cells, the proliferation and expansion of mature Treg and the survival of Treg in vivo. We previously reported that Treg in healthy donors are more susceptible to mitochondrial apoptotic priming than conventional T cells (Tcon). Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T cell subsets and particularly in patients with chronic GVHD. Interleukin-2 (IL-2) is a critical homeostatic cytokine that is required for Treg development, expansion, activity and survival. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo, clinical improvement of chronic GVHD (Koreth et al. NEJM 2011), increased Treg Bcl2 expression and increased Treg resistance to apoptosis in vitro (Matsuoka et al. SciTM, 2013). These observations led us to undertake a more detailed analysis of apoptotic pathways in functionally and phenotypically distinct Treg subsets including naïve Treg, recent thymic emigrants (RTE) Treg and memory Treg.

Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.

CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the maintenance of self-tolerance and immune homeostasis. Interleukin-2 (IL-2) is critical for Treg development, expansion, activity and survival. Lack of either IL-2 or IL-2Rα (CD25) results in Treg deficiency and Treg impairment is associated with loss of tolerance, autoimmunity, and chronic GVHD. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo and clinical improvement of chronic GVHD (Koreth et al. NEJM 2011). We also reported that low-dose IL-2 enhanced Treg resistance to FAS-mediated apoptosis through the extrinsic pathway (Matsuoka et al. Sci Transl Med 2013). However susceptibility of Treg to apoptosis through the intrinsic pathway has not been examined. To examine the mechanisms whereby IL-2 affects susceptibility of Treg to apoptosis through the intrinsic pathway, we used a functional flow cytometry-based assay (BH3 profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). Mitochondria that are more sensitive to the BH3 peptides are more 'primed' for apoptosis, and more sensitive to pro-apoptotic signaling. This assay allowed us to compare “priming” which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization in Treg and Tcon. We also examined cytoplasmic expression of Bcl-2, BclxL, Mcl1, Bim, XIAP, Ki67 and Staurosporine (STS) induced apoptosis as additional measures of susceptibility to apoptosis and proliferation in each subset. In resting blood obtained from healthy donors (n=25), Treg were more “primed” than Tcon when exposed to several BH3 peptides (Bim, Bad, PUMA and BMF). This BH3 peptide pattern suggests that the anti-apoptotic protein, Bcl2 plays a primary role in the priming of these cells. Analysis of pro- and anti-apoptotic proteins revealed that Treg expressed higher levels Bim and BclxL and lower levels of Bcl2 and XIAP than Tcon, but there was no significant difference in Mcl1. Treg expressed higher levels of Ki67 and were more susceptible to both STS- and FAS-induced apoptosis than Tcon. Thus, Treg in healthy individuals have higher proliferative activity and are more susceptible to apoptosis than Tcon through both mitochondrial and death receptor pathways.

Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.

Most patients with acute myeloid leukemia (AML) become resistant to chemotherapy at some point in their course and succumb to their disease. It is necessary to prevent chemo-resistance or enhance chemosensitivity in a selective fashion to lead to a higher cure rate and a lower toxic burden. The B-cell leukemia /lymphoma 2 (BCL-2) protein acts to prevent commitment to programmed cell death initiated in the mitochondrion. Inhibiting the function of this protein is an attractive approach to cancer therapy, but it remains a challenge to identify those tumors that will best be treated with such a strategy. Here, we test the sensitivity of AML cell lines and primary patient samples to the selective BCL-2 inhibitor, ABT-199, and test for correlation between ABT-199 sensitivity with BCL-2 dependence measured by BH3 profiling. We hypothesized that cells that are more dependent on BCL-2 will be more sensitive to inhibition by ABT-199.

Abstract 3280

No relevant conflicts of interest to declare.