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Background CGM097 inhibits the p53-HDM2 interaction leading to downstream p53 activation. Preclinical in vivo studies support clinical exploration while providing preliminary evidence for dosing regimens. This first-in-human phase I study aimed at assessing the safety, MTD, PK/PD and preliminary antitumor activity of CGM097 in advanced solid tumour patients (NCT01760525). Methods Fifty-one patients received oral treatment with CGM097 10–400 mg 3qw (n = 31) or 300–700 mg 3qw 2 weeks on/1 week off (n = 20). Choice of dose regimen was guided by PD biomarkers, and quantitative models describing the effect of CGM097 on circulating platelet and PD kinetics. Results No dose-limiting toxicities were reported in any regimens. The most common treatment-related grade 3/4 AEs were haematologic events. PK/PD models well described the time course of platelet and serum GDF-15 changes, providing a tool to predict response to CGM097 for dose-limiting thrombocytopenia and GDF-15 biomarker. The disease control rate was 39%, including one partial response and 19 patients in stable disease. Twenty patients had a cumulative treatment duration of >16 weeks, with eight patients on treatment for >32 weeks. The MTD was not determined. Conclusions Despite delayed-onset thrombocytopenia frequently observed, the tolerability of CGM097 appears manageable. This study provided insights on dosing optimisation for next-generation HDM2 inhibitors. Translational relevance Haematologic toxicity with delayed thrombocytopenia is a well-known on-target effect of HDM2 inhibitors. Here we have developed a PK/PD guided approach to optimise the dose and schedule of CGM097, a novel HDM2 inhibitor, using exposure, platelets and GDF-15, a known p53 downstream target to predict patients at higher risk to develop thrombocytopenia. While CGM097 had shown limited activity, with disease control rate of 39% and only one patient in partial response, the preliminary data from the first-in-human escalation study together with the PK/PD modeling provide important insights on how to optimize dosing of next generation HDM2 inhibitors to mitigate hematologic toxicity.

Introduction: Uveal melanoma (UM) is a rare and malignant intraocular tumour with a dismal prognosis. Despite a good control of the primary tumour by radiation or surgery, up to 50% of patients subsequently develop metastasis for which no efficient treatment is yet available.Methodology: To identify therapeutic opportunities, we performed an in vitro screen of 30 combinations of different inhibitors of pathways that are dysregulated in UM. Effects of drug combinations on viability, cell cycle and apoptosis were assessed in eight UM cell lines. The best synergistic combinations were further evaluated in six UM patient-derived xenografts (PDXs).Results: We demonstrated that the Bcl-2/X-L/W inhibitor (ABT263) sensitised the UM cell lines to other inhibitors, mainly to mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase (MEK) and murine double minute 2 (MDM2) inhibitors. mTOR (RAD001) and MEK1/2 (trametinib) inhibitors were efficient as single agents, but their combinations with ABT263 displayed no synergism in UM PDXs. In contrast, the combination of ABT263 with MDM2 inhibitor (HDM201) showed a trend for a synergistic effect.Conclusion: We showed that inhibition of Bcl-2/X-L/W sensitised the UM cell lines to other treatments encouraging investigation of the underlying mechanisms. Furthermore, our findings highlighted Bcl-2/X-L/W and MDM2 co-inhibition as a promising strategy in UM. (C) 2019 The Author(s). Published by Elsevier Ltd.

BackgroundResistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer.MethodsWe used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer.ResultsWe demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model.ConclusionsWe conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.

BackgroundResistance to endocrine therapy is a major clinical challenge in the management of estrogen receptor (ER)-positive breast cancer. In this setting p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment resistant ER-positive breast cancer.MethodsWe used the MDM2 inhibitor NVP-CGM097 to treatin vitroandin vivomodels alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate antitumor effects in p53 wildtype and p53 mutant ER positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and -resistant ER positive breast cancer.ResultsWe demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtypein vitroandin vivobreast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant resistant patient derived xenograft model.ConclusionsWe conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programs.

Activation of p53 by inhibitors of the p53–MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53–MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo. Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2–p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure–response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53–MDM2 inhibitors. Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257–67. ©2018 AACR.

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.

Background: Treatment options for patients (pts) with relapsed/refractory (r/r) AML and HR-MDS who cannot tolerate standard therapy are limited, and innovative combination approaches are needed. Dysregulation of the tumor protein 53 [p53, (TP53 gene)], Murine Double Minute-2 (MDM2) interaction leads to uncontrolled cell proliferation and tumor growth (Chene 2003). Siremadlin is an orally administered small molecule inhibitor of the p53-MDM2 interaction, which has a well-characterized safety profile and shows antitumor activity in pts with AML (Stein 2021). Combination of siremadlin with BCL-2 inhibitors (eg, venetoclax [VEN]) results in synergistic activity in p53 wild-type AML cell lines and leads to complete and durable antitumor responses in a variety of p53wt AML patient-derived xenograft models (Wang 2019). Thus, combined MDM2 and BCL-2 inhibition may result in improved outcomes for pts with hematologic malignancies. Here we report the preliminary results of a phase Ib, open-label study of siremadlin in combination with VEN for the treatment of pts with AML and HR- MDS (NCT03940352). Methods: Pts were adults with r/r AML after ≥1 but ≤3 prior therapies who were not suited for standard therapy, or pts with AML who were unfit for standard chemotherapy. HR-MDS pts (per Revised International Prognostic Scoring System) who have previously failed hypomethylating agent therapy were also eligible. AML pts with TP53-mutant tumors (determined as the presence of any mutations in exons 5,6,7, and 8 at minimum) were excluded. Pts with prior treatment with an MDM2 or MDM4 inhibitor in combination with a BCL-2 inhibitor were excluded. Siremadlin (20 or 30 mg) was administered daily from day 1-5 during each 28-day cycle in combination with VEN at a starting dose of 50 mg daily, followed by a gradual ramp-up period over 4 days to a target daily dose of 400 mg (dose level [DL] 1: siremadlin 20 mg + VEN and DL2: siremadlin 30 mg + VEN]. Pts continued study treatment until unacceptable toxicity, disease progression, or investigator/patient decision. The primary objective is to evaluate safety/tolerability; secondary objectives are to test preliminary efficacy (per Cheson 2003) and pharmacokinetics (PK). Results: As of data cutoff March 1, 2021, 18 total pts underwent treatment. The median age was 72.5 y (range, 29-84). Most pts had an Eastern Cooperative Oncology Group performance status of 0 (n=13, 72%). Of the 17 (94%) pts with AML, 3 pts had first-line and 14 pts had r/r AML. One (6%) pt had HR-MDS (DL1); efficacy data for this pt are not presented. Eleven pts were treated at DL1 and 7 at DL2. Treatment is ongoing for 2 pts (both DL2). The mean treatment duration was 3.8 mo (range, 0.9-10.3; DL1) and 1.8 mo (range, 0-3.8; DL2). Two pts (DL1) had ≥9 mo treatment duration. The most common grade ≥3 adverse events (AEs) suspected to be treatment-related were neutropenia (n=5, 28%) and thrombocytopenia (n=4, 22%); 11 pts (61.1%) experienced grade ≥3 serious AEs regardless of study-treatment; only febrile neutropenia was experienced by >20% of pts (n=8, 44%). One case of tumor lysis syndrome was reported. Notably, gastrointestinal grade ≥3 serious AEs were uncommon (diarrhea [n=2, 11%] and nausea [n=1, 6%]). Dose modification or interruption due to an AE occurred in 10 (56%) pts, mostly due to febrile neutropenia (n=4) and neutropenia (n=3), and discontinuation due to an AE occurred in 4 (22%) pts, mostly due to febrile neutropenia (n=2). Among the 11 pts in the dose-determining set, 2 experienced dose limiting toxicities (1 with anemia, bone marrow failure, and thrombocytopenia and 1 with febrile neutropenia). Two on-treatment deaths occurred; however, none were treatment-related. Among 10 pts with AML in DL1, 1 had complete remission (CR), 3 had CR with incomplete blood count recovery (CRi) and had 1 partial remission. Among 7 pts with AML in DL2, 3 pts had CRi (Table). One pt in DL2 currently has an ongoing CRi. PK exposure and parameters for siremadlin and VEN for both cohorts were comparable to historical single-agent data. No drug-drug interaction between siremadlin and VEN was observed. Conclusions: Siremadlin + VEN is well-tolerated in pts with AML and shows promising antileukemic activity in r/r AML pts. These results validate the MDM2-p53 interaction as a therapeutic target and provide support for further development of siremadlin + VEN in pts with AML or HR-MDS. Dose escalation is ongoing to determine the recommended dose for expansion. Figure 1 Figure 1. Disclosures Wei: Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Breccia: Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria; Abbvie: Honoraria; Bristol Myers Squibb/Celgene: Honoraria. Cedena Romero: Janssen: Honoraria; BMS: Honoraria. Ciceri: IRCCS Ospedale San Raffaele: Current Employment. Erba: AbbVie Inc; Agios Pharmaceuticals Inc; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Incyte Corporation; Jazz Pharmaceuticals Inc; Novartis: Speakers Bureau; AbbVie Inc: Other: Independent review committee; AbbVie Inc; Agios Pharmaceuticals Inc; Astellas; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Daiichi Sankyo Inc; Genentech, a member of the Roche Group; GlycoMimetics Inc; Incyte Corporation; Jazz Pharmaceuticals Inc; Kura Oncology; Nov: Other: Advisory Committee; AbbVie Inc; Agios Pharmaceuticals Inc; ALX Oncology; Amgen Inc; Daiichi Sankyo Inc; FORMA Therapeutics; Forty Seven Inc; Gilead Sciences Inc; GlycoMimetics Inc; ImmunoGen Inc; Jazz Pharmaceuticals Inc; MacroGenics Inc; Novartis; PTC Therapeutics: Research Funding. Gaur: Novartis: Current Employment. Sechaud: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Halilovic: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kuenzle: Novartis: Current Employment. Fabre: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. OffLabel Disclosure: Siremadlin is a small molecule inhibitor of the p53-MDM2 interaction under investigation for the treatment of patients with myeloid malignancies.

Intrinsic resistance of unknown mechanism impedes the clinical utility of inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) in malignancies other than breast cancer. Here we used melanoma patient-derived xenografts (PDXs) to study the mechanisms for CDK4/6i resistance in pre-clinical settings. We observed that melanoma PDXs resistant to CDK4/6i frequently displayed activation of phosphatidyl inositol 3 kinase (PI3K)-AKT pathway, and inhibition of this pathway improved CDK4/6i response in a p21-dependent manner. We showed that a target of p21, CDK2, was necessary for proliferation in CDK4/6i-treated cells. Upon treatment with CDK4/6i, melanoma cells up-regulated cyclin D1, which sequestered p21 and another cyclin-dependent kinase inhibitor, p27, leaving a shortage of p21 and p27 available to bind and inhibit CDK2. Therefore, we tested whether induction of p21 in resistant melanoma cells would render them responsive to CDK4/6i. Because p21 is transcriptionally driven by p53, we coadministrated CDK4/6i with a murine double minute (MDM2) antagonist to stabilize p53, allowing p21 accumulation. This resulted in improved anti-tumor activity in PDXs and in murine melanoma. Furthermore, co-administration of CDK4/6 and MDM2 antagonists with standard of care therapy caused tumor regression. Notably, the molecular features associated with response to CDK4/6 and MDM2 inhibitors in PDXs were recapitulated by an ex vivo organotypic slice culture assay, which could potentially be adopted in clinic for patient stratification. Our findings provide a rationale for co-targeting CDK4/6 and MDM2 in melanoma.

This work was supported by the Spanish ISCIII-FIS and FEDER Funds (PI 15/00067 and PI 15/02156) and the Regional Health Council of Castilla y León (GRS 1604/A/17). EMA was supported by a grant from the Regional Education Council of Castilla y León co-financed by the European Social Fund.

// Guillaume Carita 1,* , Estelle Frisch-Dit-Leitz 2,* , Ahmed Dahmani 1 , Chloe Raymondie 1 , Nathalie Cassoux 3 , Sophie Piperno-Neumann 4 , Fariba Nemati 1 , Cecile Laurent 5 , Leanne De Koning 6 , Ensar Halilovic 7 , Sebastien Jeay 7 , Andrew Wylie 7 , Caroline Emery 7 , Sergio Roman-Roman 2 , Marie Schoumacher 2,* and Didier Decaudin 1,4,* 1 Laboratory of preclinical investigation, Department of Translational Research, PSL University, Institut Curie, Paris, France 2 Department of Translational Research, Institut Curie, PSL University, Paris, France 3 Department of Ophthalmological Oncology, Institut Curie, Paris, France 4 Department of Medical Oncology, Institut Curie, Paris, France 5 Residual Tumor & Response to Treatment Lab, Department of Translational Research, Institut Curie, PSL University, Paris, Paris, France 6 RPPA Platform, Department of Translational Research, Institut Curie, PSL University, Paris, France 7 Novartis Institutes for Biomedical Research, Cambridge, MA USA * These authors have contributed equally to this work Correspondence to: Didier Decaudin, email: // Keywords : xenograft models, cellular response to anticancer drugs, uveal melanoma, AEB071 combinations, synergy Received : April 04, 2016 Accepted : May 10, 2016 Published : May 22, 2016 Abstract Uveal melanoma (UM) is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified. Novel therapeutic approaches are therefore urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071. The most promising approaches were further investigated in vitro using our unique panel of UM cell lines. When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs. Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death. While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease. Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients.

Background: Resistance to endocrine therapy is a major clinical challenge in the management of estrogen receptor (ER)-positive breast cancer. In this setting p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment resistant ER-positive breast cancer. Methods: We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate antitumor effects in p53 wildtype and p53 mutant ER positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and -resistant ER positive breast cancer. Results: We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant resistant patient derived xenograft model. Conclusions: We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programs. Citation Format: Neil Portman, Heloisa Milioli, Sarah Alexandrou, Rhiannon Coulson, Aliza Yong, Kristine Fernandez, KeeMing Chia, Ensar Halilovic, Davendra Segara, Andrew Parker, Sue Haupt, Ygal Haupt, Wayne Tilley, Alex Swarbrick, Liz Caldon, Elgene Lim. Mdm2 inhibition synergises with endocrine therapy or cdk4/6 inhibition for the treatment of estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-17.

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. First, we exposed 6 AML cell lines with a BCL-2 (venetoclax) and MCL-1 antagonist (S63845). As previously reported, combination treatment resulted in synergistic cell line killing. Venetoclax treatment (within an hour) led to a dynamic increase in mitochondrial sensitivity to the MCL-1 selective MS-1 peptide, while S63845 caused increased mitochondrial susceptibility to the BCL-2 and BCL-XL selective BAD peptide. There was a correlation between % of priming caused by MS-1 and BAD peptide with Loewe synergy score (Spearman r=0.79, p We next investigated combination MCL-1 and BCL-2 antagonism in vivo in venetoclax resistant AML PDXs. We transplanted NSG mice with human AML cells and started them on venetoclax (100mg/kg, PO) until leukemia relapsed (N=6 models). We subjected resistant myeloblasts to BH3 profiling and identified dependency on BCL-2 and MCL-1 to guide therapeutic choices. As a single agent, S63845 showed limited activity in venetoclax resistance models (p Next, we applied the dynamic BH3 profiling technique that measures drug-induced changes in mitochondrial priming to discover additional agents that may overcome venetoclax resistance. We compared mitochondrial priming signals of 40 targeted agents in isolated parental and resistant cells. Although venetoclax resistant cells gained cross resistance to most of the agents, inhibitors of the FLT-3 pathway, including quizartinib, crenolanib, and gilteritinib retained similar priming responses as in parental cells. To validate this, we re-transplanted venetoclax resistant cells and found that quizartinib treatment maintained anti-leukemia efficacy compared to venetoclax or vehicle-treated mice (p In conclusion, our study illustrates the power of mitochondrial measurements as a predictive biomarker for BH3 mimetic based therapy. We provide an evidence for the persistent activity of FLT-3 inhibitors in venetoclax resistance settings, a discovery made possible by dynamic BH3 profiling. Disclosures 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.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy, characterized by uncontrolled proliferation and impaired differentiation of myeloid cells. With the exception of certain subtypes, the average long-term survival rate remains low, thus underlining the need to further improve the outcome of AML patients. Since AML is one of the least mutated cancer types, the majority of AML patients may not carry targetable genetic alterations. However, the anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2 and Mcl-1, are often overexpressed in AML, allowing deregulated survival; hence pro-apoptosis priming with small molecule inhibitors of Bcl-2 and Mcl-1 may provide a broader therapeutic benefit across the disease. In addition, a majority of AML patients carry wild-type p53, providing therapeutic opportunity for Hdm2 inhibitors to stabilize p53 and lead to expression of pro-apoptotic molecules (e.g., PUMA & BAX). Therefore, targeting the combined apoptosis mechanisms by inhibiting different anti-apoptotic Bcl-2 family of proteins and activating p53 concomitantly may synergistically enhance apoptotic cell death of AML tumor cells. We tested the combination of Bcl-2 inhibitors (BCL201/S55746 or venetoclax) with either MIK665/S64315, a novel and selective inhibitor of Mcl-1 or HDM201, a selective small molecule inhibitor of p53:Hdm2 interaction, in a series of in vitro and in vivo studies in AML. In vitro, strong combination synergy was observed with a remarkable induction of cell death for both combinations. In vivo, the combination of Bcl-2 inhibitors with MIK665/S64315 or HDM201 lead to complete and durable antitumor responses in a variety of p53wt AML patient-derived xenograft models of heterogeneous genetic profiles. Notably, lowering the dose of HDM201 by 4 fold from its most efficacious dose, resulted in a high degree of tumor regressions while mitigating the toxicity effects on platelets. Taken together, these data demonstrate that a combination of Bcl-2 inhibitor (BCL201/S55746 or venetoclax) with MIK665/S64315 or HDM201 provide therapeutic benefit over the monotherapy, and support a rationale for testing these apoptosis enhancing combination approaches in AML patients. Citation Format: Youzhen Wang, Shumei Qui, Sneha Sanghavi, Iain Mulford, Gaëlle Lysiak, Maïa Chanrion, Prakash Mistry, Ulrike Pfaar, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Claire Fabre, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Ensar Halilovic. Targeting AML through apoptosis activation using Bcl-2/Mcl-1 or Bcl-2/Hdm2 inhibitor combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 257.

Mcl-1 is highly expressed in a variety of human cancers (including those of hematopoietic and lymphoid origin) and is exploited by cancer cells to evade cell death and to develop resistance to diverse chemotherapeutic agents. We disclose, for the first time, the structure of S64315 (also named MIK665) a highly potent and selective inhibitor of Mcl-1 with improved potency over its predecessor S63845 (Kotschy et al, Nature, 2016). S64315/MIK665 is currently in phase 1 in AML (Acute Myeloid Leukemia) and MDS (Myelodysplastic Syndrome) (EudraCT 2016-003768-38, NCT 02979366) and in MM (Multiple Myeloma) and lymphoma (NCT02992483). A fragment-based, structure-guided drug discovery effort led to the identification of S64315/MIK665 that binds to human Mcl-1 with a sub-nanomolar affinity (Ki 0.048 nM) and selectively over other anti-apoptotic Bcl-2 family members. It has similar affinity for human, rat, dog and monkey Mcl-1 but about a ten-fold lower affinity for mouse Mcl-1. S64315/MIK665 causes dose-dependent activation of the intrinsic apoptosis pathway in a Bax/Bak-dependent manner, as measured by increased caspase activity and cleaved PARP. S64315/MIK665 shows strong cell killing activity in a diverse panel of human hematological tumor cell lines, including AML, lymphoma and MM. The activity profile of S64315/MIK665 is distinct from that of venetoclax, a selective Bcl2 inhibitor. In vivo, S64315 as single agent demonstrated potent and dose-dependent apoptotic and antitumor response after intravenous administration in several human hematological tumor models grafted in immuno-compromised mice and rats. Complete regression of established tumors, at well tolerated doses, was achieved using different intravenous dosing regimens in rats as well as in mice. Finally, dual BH3-mimetic targeting approach combining S64315/MIK665 with BCL2 inhibitors showed strong and durable antitumor responses in several hematological tumor models both in vitro and in vivo. Citation Format: Ana Leticia Maragno, Prakash Mistry, András Kotschy, Zoltán Szlavik, James Murray, James Davidson, Gaëtane Le Toumelin-Braizat, Maïa Chanrion, Alain Bruno, Audrey Claperon, Heiko Maacke, Erick Morris, Youzhen Wang, Alix Derreal, Márton Csekei, Attila Paczal, Zoltán Szabo, Szabolcs Sipos, Agnes Proszenyak, Balázs Balint, Allan Surgenor, Pawel Dokurno, Natalia Matassova, Ijen Chen, Gaëlle Lysiak-Auvity, Anne-Marie Girard, Fabienne Grave, Frédéric Colland, Ensar Halilovic, Olivier Geneste. S64315 (MIK665) is a potent and selective Mcl1 inhibitor with strong antitumor activity across a diverse range of hematologic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4482.

One of the hallmarks of cancer is evasion of apoptosis. The B-cell lymphoma-2 (Bcl-2) family of proteins represents a crucial point of control of apoptosis. The Bcl-2 family comprises both pro- and anti-apoptotic members, the latter of which (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-2A1) are often overexpressed in cancer cells, supporting their aberrant survival. Thus, these anti-apoptotic proteins have become an attractive target for cancer therapy. BH3 mimetics have been shown to bind to the BH3 binding groove of anti-apoptotic Bcl-2 family members and inhibit their function, resulting in apoptotic cell death, and one such BH3 mimetic, ABT-199 (venetoclax), has recently been approved for treatment of relapsed or refractory Chronic Lymphocytic Leukemia. We have developed two novel and potent BH3 mimetics: MIK665/S64315, a highly selective inhibitor of Mcl-1 and BCL201/S55746, a selective Bcl-2 inhibitor. Both compounds, individually induce apoptosis in hematological cancer cell lines, primary patient samples and demonstrate anti-tumor efficacy in xenograft models. MIK665/S64315 is currently in phase 1 clinical development in AML and MDS (NCT 02979366) and in MM and lymphoma (NCT02992483). Here, we describe the activity of the combination of MIK665/S64315 with BCL201/S55746 or venetoclax, both in vitro and in vivo, across a range of hematological indications (AML, MM and DLBCL). In vitro, a strong synergy was observed with these combinations, resulting in a remarkable induction of cell death in majority of cell lines tested. In vivo, MIK665/S64315 and BCL201/S55746 combinations lead to complete and durable antitumor responses in many different xenograft models in mice and rats. Taken together, these data demonstrate that a combination of MIK665/S64315 and BCL201/S55746 provide strong therapeutic benefit over either monotherapy, and support a rationale for testing Mcl-1 and Bcl-2 inhibitor combinations in patients with hematological malignancies. Citation Format: Ensar Halilovic, Maïa Chanrion, Prakash Mistry, Markus Wartmann, Shumei Qiu, Sneha Sanghavi, Yan Chen, Gaëlle Lysiak, Ana Leticia Maragno, Ulrike Pfaar, Felix Huth, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Youzhen Wang. MIK665/S64315, a novel Mcl-1 inhibitor, in combination with Bcl-2 inhibitors exhibits strong synergistic antitumor activity in a range of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4477.

Over 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publically available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment called the Public Repository of Xenografts (PRoXe; www.proxe.org). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional and proteomic biomarkers in both treatment-naïve and relapsed/refractory disease.

Background: Venetoclax is a BCL-2 inhibitor particularly effective in patients with multiple myeloma (MM) harboring the t(11;14). However, resistance to venetoclax has been linked to MCL-1 overexpression. On the other hand, it is wellknown that MM cells depend on MCL-1 rather than BCL-2 for survival, and this dependence has recently been reported to be enhanced by the tumor-associated microenvironment. Therefore, the combination of venetoclax with the potent MCL-1 inhibitor S63845 arises as a promising and novel approach for the treatment of MM. Aims: To evaluate the efficacy and mechanism of action of S63845 alone and in combination with venetoclax in absence and presence of the bone marrow tumor microenvironment in preclinical in vitro, ex vivo and in vivo models of MM. Methods: S63845 was provided by an agreement with Servier and Novartis. In vitro activity of S63845 and venetoclax alone and in combination was evaluated by bioluminescence on a MM cell line expressing luciferase (MM.1S-luc) in absence and presence of mesenchymal stromal cells isolated from bone marrow aspirates of MM patients (pMSCs). MM.1S cells cultured in absence or presence of pMSCs were analyzed for MCL-1 and BCL-2 protein levels by Western blot. Interactions between these anti-apoptotic proteins with the pro-apoptotic protein BIM were assessed by immunoprecipitation assays. The efficacy of S63845 and venetoclax alone and in combination was also evaluated ex vivo in MM cells and normal lymphocytes from MM patients. Finally, a disseminated MM model in BRG mice was used for in vivo studies. Results: S63845 and venetoclax showed a strong antimyeloma dose-dependent effect on MM.1S-luc cells co-cultured with pMSCs. However, whereas the presence of tumor-associated MSCs increased the IC50 value of venetoclax in MM.1S-luc cells from 6.2 to 9.8 mM, it reduced that of S63845 from 94.1 to 81 nM, suggesting a mild sensitization to this drug in the context of the microenvironment. Neither S63845 nor venetoclax affected pMSC viability even at high concentrations by MTT assay. The co-culture with the BM stromal microenvironment increased MCL-1 expression on untreated MM.1S cells in two out of four experiments performed with MSCs from different MM patients, whereas it surprisingly induced a decrease on BCL-2 levels in all of them. Treatment with S63845 completely blocked MCL-1 binding to BIM, both in the absence or presence of pMSCs but did not induce the compensatory increase of BCL-2/BIM complexes observed in MM.1S cells in monoculture. Venetoclax also completely blocked the binding of BCL-2 to BIM in MM.1S alone or in co-culture, and induced a similar compensatory increase of MCL-1/BIM complexes in both situations. Importantly, the double combination S63845 + venetoclax was significantly superior to both drugs in monotherapy in killing MM.1S-luc cells co-cultured in the presence of the stromal microenvironment. BIM immunoprecipitation assays showed that the double combination was able to counteract the compensatory upregulation of MCL-1 bound to BIM observed on MM.1S cells treated with venetoclax and to entirely disrupt BCL-2/BIM complexes, both in the absence and presence of pMSCs. Furthermore, S63845 + venetoclax increased the percentage of apoptotic MM plasma cells from three MM patients with respect to single treatments with moderate toxicity detected on normal lymphocytes, suggesting the existence of a therapeutic window for the double combination. Finally, the combination of S63845 + venetoclax clearly delayed tumor growth as compared with the agents in monotherapy in a disseminated model of MM with statistically significant differences from day 19 of treatment. This in vivo effect translated into a significatively improved survival for mice treated with the double combination (median 60 days) vs control mice (median 32 days; log-rank test P=0.045). Conclusion: Our preclinical data demonstrate the potent activity of the combination of venetoclax with S63845 in MM even in presence of the stromal associated-tumor microenvironment, and provides the rationale for the clinical development of this combination in relapsed or refractory MM patients. This project was supported by Novartis Pharmaceuticals and by the Spanish , ISCIII-FIS PI15/00067 and PI15/02156, GRS 1604/A/17 and CRMRTC de Castilla y León. Predoctoral grant to EMA by Consejería de Educación de Castilla y León. Disclosures Schoumacher: Servier: Employment. Banquet:Servier: Employment. Kraus-Berthier:servier: Employment. Kloos:Servier: Employment; Novartis: Other: Partnership. Halilovic:Novartis: Employment, Equity Ownership. Maacke:Novartis: Employment. Mateos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ocio:AbbVie: Consultancy; Novartis: Consultancy, Honoraria; BMS: Consultancy; Seattle Genetics: Consultancy; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pharmamar: Consultancy; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding.

Background: Well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS) is characterized by a consistent coamplification of human double minute 2 homolog (HDM2) and cyclin-dependent kinase 4 (CDK4) which inactivates the tumor suppressor pathways p53 and Rb, respectively. HDM201 is a selective inhibitor of the p53-HDM2 interaction and ribociclib is a CDK4/6 inhibitor. Preclinical studies suggested a synergy in in vitro and in vivo models of WDLPS/DDLPS and both agents have demonstrated single-agent clinical activity in solid tumors. We aim to determine the optimal dose and regimen of HDM201 + ribociclib and to assess the preliminary antitumor activity of this combination in patients (pts) with liposarcoma. Methods: In this multicenter, open-label, Phase Ib ongoing study, pts with locally advanced or metastatic liposarcoma that had unequivocally progressed on, or despite prior systemic therapy were treated orally with HDM201 + ribociclib. Three treatment regimens were explored: Regimen (Reg) 1 (HDM201 + ribociclib daily for the first 2 wks [QD 1st 2 wks] in a 4-wk cycle), Reg 4 (HDM201 every 3 wks + ribociclib QD 1st 2 wks in a 3-wk cycle), and Reg 5 (HDM201 every 4 wks + ribociclib QD 1st 2 wks in a 4-wk cycle). Results: As of Nov 21, 2017, 74 pts received HDM201 + ribociclib (Reg 1 n=26; Reg 4 n=29; Reg 5 n=19); 12 pts (6 each in Reg 4 and 5) were still receiving treatment. Ten pts (Reg 1 n=3; Reg 4 n=6; Reg 5 n=1) discontinued treatment due to adverse events (AEs) and 2 pts (1 each in Reg 4 and 5) died. The most common AE of any grade, regardless of cause, reported across regimens (Reg 1; Reg 4; Reg 5) was nausea (81%; 76%; 63%) which was mainly Grade 1/2 and not dose limiting. Common Grade 3/4 AEs regardless of cause included neutropenia (39%; 52%; 42%), thrombocytopenia (35%; 45%; 42%), anemia (27%; 17%; 21%), leukopenia (27%; 28%; 37%), and lymphopenia (15%; 14%; 21%). Dose-limiting toxicities were reported in 16 pts (2; 9; 5) and all except 1 (prolonged QT) were hematologic (including neutropenia [n=5], thrombocytopenia [n=4], febrile neutropenia, and anemia [n=2 each]). Partial responses were observed in 3 (4%) pts (2 in Reg 4; 1 in Reg 5). Stable disease was achieved by 36 (49%) pts (11 in Reg 1; 16 in Reg 4; 9 in Reg 5). The median progression-free survival (PFS) was 2.7 mo (95% confidence interval [CI]: 1.9-8.2 mo) in Reg 1, 4.8 mo (95% CI: 3.9 mo-not reached) in Reg 4, and 2.1 mo (95% CI: 1.4 mo-not reached) in Reg 5. Conclusions: HDM201 + ribociclib demonstrated a manageable safety profile and preliminary efficacy in pts with locally advanced or metastatic WDLPS/DDLPS, with hematologic toxicities being dose limiting. The median PFS in Reg 4 compares favorably with single-agent CDK4 inhibitors and, alongside a tolerable safety profile, suggests further exploration of this regimen may be warranted in Phase II studies of HDM201 + ribociclib in this patient population. Citation Format: Albiruni Abdul Razak, Sebastian Bauer, Jean-Yves Blay, Richard Quek, Cristina Suárez, Chia-Chi Lin, Marie L. Hütter-Krönke, Ricardo Cubedo, Stephane Ferretti, Christophe Meille, Ensar Halilovic, Giorgia Clementi, Maria Santos-Rosa, Nelson Guerreiro, Astrid Jullion, Claire Fabre, Antoine Italiano. Results of a dose- and regimen-finding Phase Ib study of HDM201 in combination with ribociclib in patients with locally advanced or metastatic liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT009.

p53 is a transcription factor that plays a central role in guarding genomic stability of the cell through cell cycle arrest or induction of apoptosis. It has also been reported that p53 participates in the regulation of tumor immunity and in homeostatic regulation of immune responses. However, the immunomodulatory effect of p53 in tumor and the tumor microenvironment is not well understood. From gene expression and immunohistochemical analysis of pre- and post-treatment biopsies from patients treated with the MDM2 inhibitor NVP-CGM097, we observed upregulation of immune checkpoint transcripts (PD1 and PDL1), and an increase in the number of CD8+ tumor infiltrating lymphocytes. To investigate the immunomodulatory effect of p53 in more detail, we studied murine syngeneic models treated with NVP-HDM201, a potent and selective second generation MDM2 inhibitor. We performed multi-color FACS analysis on tumors and tumor draining lymph nodes. With NVP-HDM201 treatment, we observed increase in numbers of CD103+ DC cells, capable of antigen cross-presentation. Furthermore, NVP-HDM201 increased the percentage of Tbet+EOMES-T cells in tumors as well as tumor draining lymph nodes and also resulted in increased ratios of CD8+ Tcells/Treg in tumor. In addition, levels of both PD-L1 on CD45- cells and PD-1 on CD4+ T cells were increased. Importantly, PD-1 and PD-L1 blockade enhanced HDM201 activity in p53 WT syngeneic mouse models but not in p53 mutated models. The rate of complete tumor regressions (CR) was significantly increased with combination treatment as compared to either treatment alone. The animals that achieved complete regressions also developed long lasting anti-tumor memory against the specific tumor cells as evidenced by re-challenge experiments. Taken together, these results demonstrate that MDM2 inhibition triggered adaptive immunity which was further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in cancer patients with wildtype p53. Citation Format: Hui Qin Wang, Jinsheng Liang, Iain Mulford, Fiona Sharp, Swann Gaulis, Yan Chen, Gina Trabucco, David Quinn, Joseph D. Growney, Matthew J. Meyer, Juliet Williams, Peter Hammerman, Francesco Hofmann, Glenn Dranoff, Jeffrey Engelman, Jennifer Mataraza, Ensar Halilovic. PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5560.

Background: NVP-HDM201 is a selective inhibitor of the p53-HDM2 interaction, and has demonstrated potent single-agent activity in various in vitro and in vivo tumor models dependent on wild-type (wt) TP53. This study aims to determine the optimal dose and schedule of NVP-HDM201 for treating patients (pts) with TP53 wt tumors for further clinical study. Here we report results from pts with solid tumors. Methods: In this multicenter, open-label, Phase I, ongoing study, pts with advanced TP53 wt tumors progressing on standard therapy or for whom no standard therapy exists were treated with single-agent oral NVP-HDM201. Four treatment regimens of NVP-HDM201 are explored: two high-dose intermittent regimens, Regimen (Reg) 1A (single dose [SD] on Day 1 in a 3-week [wk] cycle) and Reg 1B (SD on Days 1 and 8 in a 4-wk cycle); and two low-dose extended regimens, Reg 2A (SD every day for first 2 wks in a 4-wk cycle) and Reg 2C (SD every day for first wk in a 4-wk cycle). Results: As of the data cut-off (Sep 19, 2016), 85 pts received NVP-HDM201 (Reg 1A n=26; Reg 1B n=20; Reg 2A n=20; Reg 2C n=19); 13% were still receiving treatment. Common Grade 3/4 adverse events (AEs) suspected to be treatment related (Reg 1A; Reg 1B; Reg 2A; Reg 2C) included neutropenia (23%; 25%; 15%; 5%), thrombocytopenia (23%; 10%; 20%; 11%), and anemia (12%; 0%; 20%; 16%); the first two were dose limiting in 4 pts (2; 1; 0; 1). Gastrointestinal toxicity was predominantly low grade, and not dose limiting; the most common treatment-related AE reported was nausea (62%; 60%; 40%; 42%). Median duration of exposure across all regimens was 8.5 weeks (range: 2-86 weeks). Partial responses were observed in 2 (2%) pts (1 in Reg 1A and 1 in Reg 1B). Stable disease was achieved by 29 (34%) pts (8 in Reg 1A, and 7 each in Reg 1B, Reg 2A and Reg 2C). Furthermore, the average plasma concentration per cycle reached with Reg 1A/Reg 1B was closer to the predicted preclinical target efficacious levels required for tumor regression compared with Reg 2A/Reg 2C, and is associated with the observed clinical activity. NVP-HDM201 showed approximate dose-proportional pharmacokinetics, and exposure correlated with blood concentrations of the GDF-15 biomarker on day 1. Conclusions: NVP-HDM201 demonstrated a manageable safety profile and clinical activity in a heavily pretreated population. Dose-limiting toxicities consisted primarily of neutropenia and thrombocytopenia. Reg 1B was chosen for the expansion phase as it achieved the most favorable therapeutic index: the lowest incidence of Grade 3/4 thrombocytopenia while achieving therapeutically relevant exposures. The recommended dose for expansion was declared as 120 mg NVP-HDM201 and the expansion phase is enrolling. To enhance the safety and efficacy of NVP-HDM201, a separate cohort combining NVP-HDM201 with eltrombopag to mitigate thrombocytopenia is being investigated and will be reported. Citation Format: David M. Hyman, Manik Chatterjee, Filip de Vos, Chia-Chi Lin, Cristina Suárez, David Tai, Philippe Cassier, Noboru Yamamoto, Vincent A. de Weger, Sébastien Jeay, Christophe Meille, Ensar Halilovic, Luisa Mariconti, Matthieu Klopfenstein, Nelson Guerreiro, Rajkumar Radhakrishnan, Emil T. Kuriakose, Sebastian Bauer. Optimizing the therapeutic index of HDM2 inhibition: Results from a dose- and regimen-finding Phase I study of NVP-HDM201 in pts with TP53 wt advanced tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT150. doi:10.1158/1538-7445.AM2017-CT150

Background: NVP-HDM201 is a selective inhibitor of the p53-HDM2 interaction and has demonstrated potent single-agent activity in various in vitro and in vivo tumor models, dependent on wild-type (wt) TP53. This study aims to determine the optimal dose and schedule of NVP-HDM201 for treating patients (pts) with TP53 wt tumors for further clinical study. Here we focus on pts with advanced, TP53 wt acute leukemias. Methods: In this multicenter, open-label, dose-finding, Phase I study, pts with advanced, TP53 wt tumors who had relapsed or refractory acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) were treated with single-agent oral NVP-HDM201. Four treatment regimens were explored: two high-dose intermittent regimens (reg), Reg 1A and 1B (1A: Day 1 of a 3-week [wk] cycle; 1B: Days 1 and 8 of a 4-wk cycle) and two low-dose extended regimens, Reg 2A and 2C (2A: once daily for the first 2 wks of a 4-wk cycle; 2C: once daily for the first wk of a 4-wk cycle). Results: As of Dec 07, 2016, a total of 37 pts, comprising 35 pts with AML and 2 pts with ALL, had been enrolled in the study (Reg 1A n=16; Reg 1B n=6; Reg 2A n=7; Reg 2C n=8); treatment is ongoing in 3 pts (2 in Reg 1B and 1 in Reg 2C). The most common Grade 3/4 adverse events (AEs) suspected to be treatment-related (occurring in ≥25% of pts; Reg 1A; Reg 1B; Reg 2A; Reg 2C) were thrombocytopenia (50%; 50%; 29%; 50%), tumor lysis syndrome (TLS; 44%; 0; 14%; 13%), neutropenia (38%; 17%; 0; 25%), anemia (25%; 33%; 29%; 38%), febrile neutropenia (25%; 33%; 29%; 38%), and decreased white blood cell count (0; 0; 14%; 25%). Six dose-limiting toxicities (DLTs) were observed in 4 pts at 400 mg in Reg 1A: G4 hypophosphatemia (n=2), G3 infection (n=1), G3 chronic graft versus host disease (n=1), G3 stomatitis (n=1), and G4 subarachnoid hemorrhage (n=1). One DLT each occurred in Reg 1B (G4 acute kidney injury at 150 mg) and Reg 2C (G4 TLS at 45 mg). Importantly, there were no dose-limiting gastrointestinal (GI) toxicities. NVP-HDM201 also showed approximate dose-proportional pharmacokinetics (PK) and pharmacodynamics. Investigator-assessed overall response rate (CR + CRi + PR) for all pts with AML who had ≥1 post-baseline assessment (n=34) was 20.6% (95% confidence interval: 8.7-37.9%). There were 3 CRs (2 in Reg 1A; 1 in Reg 2C) and 4 CRis (1 in Reg 1B; 3 in Reg 2C). CRs/CRis were observed in pts receiving a cumulative dose of 250 mg within the first wk of treatment. Conclusions: Across all regimens, the AEs reported were overall expected and manageable, with no dose-limiting GI toxicities. The recommended dose for expansion (RDE) was declared as 45 mg in Reg 2C, based on the manageable safety profile, therapeutically relevant exposures determined by PK modeling, and meaningful antitumor activity seen at this dose level of NVP-HDM201. RDE determination for Reg 1A and Reg 1B is ongoing. Preliminary anti-leukemic activity is promising in these pts and warrants further study of this agent in AML. Citation Format: Eytan Stein, Joerg Chromik, Daniel J. DeAngelo, Manik Chatterjee, Richard Noppeney, Filip de Vos, Hironobu Minami, Sébastien Jeay, Christophe Meille, Ensar Halilovic, Luisa Mariconti, Matthieu Klopfenstein, Nelson Guerreiro, Rajkumar Radhakrishnan, Emil T. Kuriakose, Cecilia Carpio. Phase I dose- and regimen-finding study of NVP-HDM201 in pts with advanced TP53 wt acute leukemias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT152. doi:10.1158/1538-7445.AM2017-CT152

Tumors are complex biological systems that often retain proliferative capacity even when challenged with drug treatment. Given this resiliency, drug combinations may provide greater therapeutic benefit, however, which molecules to combine and how many to include in combinations for effective responses is not clear yet. Using image-based proliferation and apoptosis assays in colorectal cancer cell lines we systematically investigated combinations that ranged in number from two to six drugs and targeted critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds, but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In many cases, where cell lines were resistant to two- and three-way drug combinations, increased expression of anti-apoptotic proteins was observed and induction of cytotoxic responses required up to fourth-order combinations. Our results demonstrate that high-order drug combinations might be needed to kill cancers and show how systematic drug combination screening together with a molecular understanding of drug responses can guide their identification. Citation Format: Thomas Horn, Stephane Ferretti, Nicolas Ebel, Angela Tam, Samuel Ho, Fred Harbinski, Ali Farsidjani, Matt Zubrowski, William R. Sellers, Robert Schlegel, Dale Porter, Erick Morris, Jens Wuerthner, Sebastien Jeay, Joel Greshock, Ensar Halilovic, Levi A. Garraway, Giordano Caponigro, Joseph Lehar. High order drug combinations are required to effectively kill colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1311.

Uveal melanoma (UM), although rare in incidence, is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified and therefore novel therapeutic approaches are urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway. This has led to the use of PKC inhibitors as a rational targeting strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models, several PKC inhibitor-based combination studies were performed using the PKC inhibitor AEB071 (Sotrastaurin). When combined with AEB071, the targeted agents CGM097 (p53-MDM2 inhibitor), RAD001 (Everolimus, mTORC1 inhibitor) and MEK162 (Binimetinib, a MEK inhibitor) demonstrated greater activity in the UM patient-derived xenograft models than their activity as single agents. Importantly, tumor regressions were observed in several UM models with AEB071 + RAD001 and AEB071 + CGM097 co-treatments. Follow-up in vitro studies in UM cell lines using AEB071 combined with either CGM097 or RAD001 provided a more detailed mechanistic understanding of their combination activity and confirmed their ability to induce cell death. Together, these preclinical studies reveal that combining PKC and p53-MDM2 inhibitors or PKC and mTORC1 inhibitors may provide significant clinical benefit for patients with UM. Citation Format: Guillaume Carita, Estelle Frisch Dit Leitz, Ahmed Dahmani, Chloe Raymondie, Nathalie Cassoux, Sophie Piperno-Neumann, Fariba Némati, Ensar Halilovic, Sebastien Jeay, Andrew Wylie, Caroline Emery, Sergio Roman-Roman, Marie Schoumacher, Didier Decaudin. Dual inhibition of PKC and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3027.

Stabilization of p53 protein by preventing its interaction with the negative regulator Mdm2 leads to selective induction of the p53 pathway, thus offering a promising cancer therapeutic strategy in p53 wild-type tumors. In the present study, we show the identification of NVP-HDM201, a novel, highly optimized, and selective inhibitor of the p53-Mdm2 interaction. NVP-HDM201 activates p53 in human cells and induces robust p53-dependent cell cycle arrest and apoptosis, selectively in p53 wild-type tumor cells. Its activity and selectivity has been tested and confirmed across a large panel of cancer cell lines from the Cancer Cell Line Encyclopedia. In vivo, NVP-HDM201 shows a dose-proportional pharmacokinetic (PK) profile and a clear PK/PD relationship, resulting in tumor growth inhibition and regression in SJSA-1 tumor-bearing rats at well-tolerated oral (p.o.) doses. The validation and understanding of its mechanism of action, the overall favorable drug-like properties and the characterization of its on-target toxicological profile in preclinical species strongly supported the initiation of Phase I clinical trials with NVP-HDM201 in pre-selected patients with p53 wild-type tumors. Citation Format: Sébastien Jeay, Patrick Chène, Stéphane Ferretti, Pascal Furet, Bjoern Gruenenfelder, Vito Guagnano, Nelson Guerreiro, Ensar Halilovic, Francesco Hofmann, Joerg Kallen, Michelle Léonard, Robert Mah, Keiichi Masuya, Rita Ramos, Caroline Rynn, Stephan Ruetz, Thérèse Stachyra-Valat, Stefan Stutz, Andrea Vaupel, Jens Wuerthner, Philipp Holzer. NVP-HDM201: cellular and in vivo profile of a novel highly potent and selective PPI inhibitor of p53-Mdm2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1225.

The majority of drugs tested in clinical trials for hematologic malignancies ultimately fail to confer benefit. In many cases, this results from the inability to accurately identify patients most likely to benefit and mechanisms that mediate resistance. At the same time, clinical trials commonly fail to capture essential specimens at early and late timepoints that could be used to identify predictors of response, pharmacodynamic markers of on-target activity and targetable resistance pathways. To overcome these challenges, we have developed a repository of >200 patient-derived xenografts (PDXs) of leukemias and lymphomas now available through the DFCI Public Repository of Xenografts (PRoXe; http://www.proxe.org). Because PDX models can be developed and propagated from a large number of patients, randomized in vivo studies using adequate numbers of PDXs offer the first opportunity to capture the diversity of disease biology in pre-clinical drug testing. We tested the novel MDM2 inhibitor CGM097, which is currently undergoing clinical testing in solid tumors, in 24 B-cell acute lymphoblastic leukemia (B-ALL) PDXs (including hypodiploid, near haploid, MLL- rearranged, CRLF2-rearranged, and BCR-ABL models) in a randomized, phase II-like trial. Only a small subset of B-ALLs harbor de novo TP53 mutations, suggesting that MDM2 antagonists may have broad activity in this disease. Each PDX was injected into 4 NOD.SCID.IL2Rɣ-/- (NSG) mice. Upon engraftment (>2% hCD45+/hCD19+ cells in the peripheral blood), mice were randomized to vehicle or CGM097 treatment arms. One animal from each treatment arm was sacrificed 26 hours after beginning treatment to examine pharmacodynamic endpoints. The remaining two mice continued on daily therapy until moribund. CGM097 markedly improved overall survival (median 73 vs 28 days for vehicle; p=0.0008). All 19 models with survival benefit from CGM were TP53 wild-type. Among 6 models (all TP53 wild-type) derived from patients with relapsed disease, the median survival improvement compared to vehicle was 53 days (p=0.0059), consistent with robust single-agent activity in relapsed disease. Specimens at the 26 hour timepoint and upon progression to moribund were captured from the majority of mice in the trial, allowing for comprehensive characterization of the trial population. Dynamic BH3 profiling (Montero et al. Cell 2015), in which CGM097 or vehicle is added to leukemia cells harvested from mice and the effect of CGM097 on "priming" for apoptosis was performed on 10 models and demonstrated 100% accuracy in predicting response to CGM097. To characterize the effects of MDM2 inhibition on p53-dependent gene expression, we measured expression of 120 p53-related genes using a custom Nanostring gene expression panel. Differential expression analysis identified 11 genes that were significantly upregulated (p≤0.05) by CGM097 treatment at the 26 hour timepoint, including the canonical p53 targets BBC3, CDKN1A and MDM2. All mice treated with CGM097 ultimately became moribund from progressive leukemia. Targeted deep sequencing identified acquired TP53 mutations in only 2 leukemias after progression on CGM097. This indicates that p53 mutation is not the primary genetic driver of resistance to MDM2 inhibition in B-ALL PDXs. Despite this, CGM097-dependent transcriptional changes were largely abolished in the majority of leukemias collected from mice upon progression on CGM097. In summary, we established a paradigm for "Phase II-like" trials in panels of human leukemia PDX models. With this approach, we defined CGM097 as a highly active agent across the diverse spectrum of TP53-wildtype B-ALL, and established 19 independent models of acquired resistance that are the ideal reagents for defining mechanisms and then testing combinations in vivo that overcome those mechanisms. The same paradigm could be applied as a new standard for pre-clinical testing of drugs to minimize the empiric nature of current drug development strategies. Disclosures Barzaghi-Rinaudo: Novartis Institutes for Biomedical Research: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding. Jeay:Novartis: Employment, Equity Ownership. Wuerthner:Novartis: Employment, Equity Ownership. Halilovic:Novartis: Employment, Equity Ownership.

Neuroblastoma is the most common cancer in infancy, accounting for 15% of all childhood cancer-related death. MYCN amplification is the major genetic aberration in high-risk neuroblastoma and is associated with poor outcome. Genome-wide association studies have identified activation mutations and high-level amplification of ALK in approximately 10% of neuroblastoma patients. In addition, ALK mutations can coexist with MYCN amplification, which defines a subset of ultra-high-risk neuroblastoma patients. In contrast to the high frequency of p53 mutations observed in many human cancers of adults, mutations of p53 are less common in childhood cancers and have been reported in less than 2% of neuroblastomas. Wild-type (WT) p53 is required for the activation of p53 signaling by Mdm2 inhibitors. This suggests that neuroblastoma could be amenable to intervention with Mdm2 inhibitors. In this study, we demonstrated that the ALK inhibitor, NVP-LDK378, in combination with a novel Mdm2 inhibitor, NVP-CGM097, promoted apoptosis in ALK mutant and p53 WT neuroblastoma cell lines. NVP-LDK378 inhibited ALK phosphorylation and NVP-CGM097 caused induction of p53 and its downstream target genes in these cell lines. Meanwhile, Mdm2 inhibition in MYCN-amplified neuroblastoma cell lines significantly decreased the levels of Mycn protein. In addition, NVP-LDK378 and NVP-CGM097 combination resulted in complete tumor regression and markedly prolonged survival in neuroblastoma xenograft models. Overall, NVP-LDK378 and NVP-CGM097 combination may provide an effective treatment for ALK mutant and p53 WT neuroblastoma patients. Citation Format: Hui Qin Wang, Linda Battalagine, Jinsheng Liang, Ensar Halilovic, Robert Schlegel, Alan Huang, Z. Alexander Cao, John Monahan, Fang Li. The Mdm2 inhibitor NVP-CGM097 enhances the anti-tumor activity of NVP-LDK378 in ALK mutant neuroblastoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2929. doi:10.1158/1538-7445.AM2014-2929

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Patient selection biomarkers are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study we have identified a novel patient selection strategy for NVP-CGM097, a p53-Mdm2 inhibitor currently in a Phase I clinical trial ([NCT01760525][1]). We have analyzed the sensitivity of over 500 cell lines from the “Cancer Cell Line Encyclopedia” to the p53-Mdm2 inhibitor in cell viability assays, and intersected response data with information on gene expression and genomic alterations. This analysis has led to the identification of a gene signature consisting of 13 genes, as a predictor for sensitivity to NVP-CGM097. Interestingly, these 13 genes are known p53 downstream target genes, supporting the hypothesis that the identified gene signature is reflective of the p53 pathway functionality in sensitive cell lines. We show the performance of the signature as ROC and Precision-Recall curves in cell lines as well as in a number of human primary tumor xenografts, both unselected as well as pre-selected for p53 wild-type status. Work is now ongoing to validate this gene signature using baseline tumor biopsy samples and RECIST-based efficacy readouts in the current Phase I clinical trial. Citation Format: Sebastien Jeay, Swann Gaulis, Stephane Ferretti, Genevieve Albrecht, Louise Barys, Daniel Guthy, Ensar Halilovic, Moriko Ito, Masato Murakami, Astrid Pornon, Stephan Ruetz, Kavitha Venkatesan, Jianjun Yu, Michael Jensen, Marion Wiesmann, Jens Wuerthner, Diana Graus-Porta. A gene signature composed of 13 p53 target genes predicts for response to NVP-CGM097, a novel p53-Mdm2 inhibitor, in cell lines and in human primary tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2909. doi:10.1158/1538-7445.AM2014-2909 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01760525&atom=%2Fcanres%2F74%2F19_Supplement%2F2909.atom

RAF inhibitors have remarkable activity in melanomas harboring BRAFV600E mutations. We previously found that active Ras induces RAF dimers in tumors with wild-type RAF, and, that RAF inhibitors transactivate these dimers. Because BRAFV600E melanomas have enhanced ERK signaling, they are hypothesized to have feedback-mediated suppression of Ras in order to maintain monomeric and inhibitor-sensitive BRAFV600E. To test this hypothesis we evaluated the response of these tumors to RAF inhibitors over time. We found that in the treatment-naive state, BRAFV600E melanomas had suppressed RTK signaling and maintained a low level of Ras-GTP. This occurred through a multifactorial negative feedback process driven by active ERK. In this baseline state, BRAFV600E was sensitive to RAF inhibitors. After treatment with RAF inhibitors, however, inhibition of ERK signaling lead to loss of feedback, a process that restored signaling from RTKs and resulted in Ras activation. In turn, this diminished the effect of RAF inhibitors and lead to a partial, but sustained, reactivation of ERK signaling. In this post-treatment state, ERK signaling was insensitive to RAF inhibitors but sensitive to MEK inhibitors. Targeting the regulatory components of this new steady state, either through co-administration of a MEK inhibitor or a RTK inhibitor along with a RAF inhibitor resulted in improved antitumor activity. These results highlight the need to understand the adaptive response to ERK pathway inhibition in order to design better combinatorial therapies and achieve maximal antitumor effects. Citation Format: Piro Lito, Christine Pratilas, Eric Joseph, Madhavi Tadi, Ensar Halilovic, Matthew Zubrowski, Alan Huang, Elisa de Stanchina, Sarat Chandarlapaty, Poulikos Poulikakos, Neal Rosen. Feedback dependent suppression of mitogenic signaling and its effect on RAF inhibition in BRAFV600E melanomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4630. doi:10.1158/1538-7445.AM2013-4630