Search

Supplementary Figure Legends

Supplementary Table III: AML patient samples tested with HDM2 inhibitor, CGM097: IC50 values for CGM097.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.

Supplementary Table I. AML patient information.

Supplementary Figures 1-7 Supplementary Figure 1. Amplification and sequencing primers used for genotyping of p53 in AML patient samples. Supplementary Figure 2. Apoptosis data corresponding to representative bar graphs shown in Figure 1C. Supplementary Figure 3. Cell cycle analysis data corresponding to representative bar graphs shown in Figure 1D. Supplementary Figure 4. Inhibition of growth of mutant FLT3-positive AML cells by CGM097. Supplementary Figure 5. Potentiation of effects of PKC412 by CGM097 against mutant FLT3-positive AML cells. Supplementary Figure 6. Potentiation of effects of AC220 by CGM097 against mutant FLT3-positive AML cells cultured in the absence and the presence of cytoprotective SCM. Supplementary Figure 7 (A-F). Apoptosis data corresponding to representative bar graphs shown in Figure 5C. Supplementary Figure 7 (G-L). Apoptosis data corresponding to representative bar graphs shown in Figure 5C. Supplementary Figure 7 (M-R). Apoptosis data corresponding to representative bar graphs shown in Figure 5C.

Supplementary Table II: Cell lines tested with HDM2 inhibitor, CGM097: IC50 values for CGM097.

Supplementary Figure from Co-Targeting of MDM2 and CDK4/6 with Siremadlin and Ribociclib for the Treatment of Patients with Well-Differentiated or Dedifferentiated Liposarcoma: Results from a Proof-of-Concept, Phase Ib Study

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Purpose:This phase I, dose-escalation study investigated the recommended dose for expansion (RDE) of siremadlin, a p53–MDM2 inhibitor, in patients with wild-type TP53 advanced solid or hematologic cancers.Patients and Methods:Initial dosing regimens were: 1A (day 1; 21-day cycle; dose 12.5–350 mg) and 2A (days 1–14; 28-day cycle; dose 1–20 mg). Alternative regimens included 1B (days 1 and 8; 28-day cycle) and 2C (days 1–7; 28-day cycle). The primary endpoint was incidence of dose-limiting toxicities (DLT) during cycle 1.Results:Overall, 115 patients with solid tumors and 93 with hematologic malignancies received treatment. DLTs occurred in 8/92 patients with solid tumors and 10/53 patients with hematologic malignancies. In solid tumors, an RDE of 120 mg was defined in 1B. In hematologic tumors, RDEs were defined in 1A: 250 mg, 1B: 120 mg, and 2C: 45 mg. More patients with hematologic malignancies compared with solid tumors experienced grade 3/4 treatment-related adverse events (71% vs. 45%), most commonly resulting from myelosuppression. These were more frequent and severe in patients with hematologic malignancies; 22 patients exhibited tumor lysis syndrome. Overall response rates at the RDEs were 10.3% [95% confidence interval (CI), 2.2–27.4] in solid tumors and 4.2% (95% CI, 0.1–21.1), 20% (95% CI, 4.3–48.1), and 22.2% (95% CI, 8.6–42.3) in acute myeloid leukemia (AML) in 1B, 1A, and 2C, respectively.Conclusions:A common safety profile was identified and preliminary activity was noted, particularly in AML. Comprehensive investigation of dosing regimens yielded recommended doses/regimens for future combination studies.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target 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 some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950–63. ©2016 AACR.

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.

Compounds used in this study.

Median of the shRNAs and RSA values. File containing median counts and log fold changes of each gene from Supplementary Data File S1. According significance values of each gene as activator or sensitizer compared to DMSO control (see Methods) are included.

Supplementary information containing six supplementary figures (Figure S1-S6), one supplementary table (Table S1) and supplementary materials and methods. Supplementary Figure S1. Flow cytometric gating strategies for analysis of intra-tumoral T cells in parental and p53KO Colon26 tumors. Supplementary Figure S2. Flow cytometric gating strategies for analysis of intra-tumoral myeloid cells in parental and p53KO Colon26 tumors. Supplementary Figure S3. Anti-tumor activity of HDM201 in Colon26 syngeneic tumor model requires intact immune system. Supplementary Figure S4. p53 knockout in Colon 26 cells results in loss of response to HDM201 in vitro and in vivo. Supplementary Figure S5. HDM201 in combination with PD-1/PD-L1 blockade enhances anti-tumor responses in p53 wild-type tumors, but not in KO tumors. Supplementary Figure S6. Flow cytometric analysis of CD80, GITR, PD-L1, and CD86 expression after HDM201 treatment in vitro. Supplementary Table S1. Antibodies for flow cytometry analysis.

Purpose:Well-differentiated (WDLPS) and dedifferentiated (DDLPS) liposarcoma are characterized by co-amplification of the murine double minute-2 (MDM2) and cyclin-dependent kinase-4 (CDK4) oncogenes. Siremadlin, a p53–MDM2 inhibitor, was combined with ribociclib, a CDK4/6 inhibitor, in patients with locally advanced/metastatic WDLPS or DDLPS who had radiologically progressed on, or despite, prior systemic therapy.Patients and Methods:In this proof-of-concept, phase Ib, dose-escalation study, patients received siremadlin and ribociclib across different regimens until unacceptable toxicity, disease progression, and/or treatment discontinuation: Regimen A [4-week cycle: siremadlin once daily (QD) and ribociclib QD (2 weeks on, 2 weeks off)], Regimen B [3-week cycle: siremadlin once every 3 weeks; ribociclib QD (2 weeks on, 1 week off)], and Regimen C [4-week cycle: siremadlin once every 4 weeks; ribociclib QD (2 weeks on, 2 weeks off)]. The primary objective was to determine the maximum tolerated dose (MTD) and/or recommended dose for expansion (RDE) of siremadlin plus ribociclib in one or more regimens.Results:As of October 16, 2019 (last patient last visit), 74 patients had enrolled. Median duration of exposure was 13 (range, 1–174) weeks. Dose-limiting toxicities occurred in 10 patients, most of which were Grade 3/4 hematologic events. The RDE was siremadlin 120 mg every 3 weeks plus ribociclib 200 mg QD (Regimen B). Three patients achieved a partial response, and 38 achieved stable disease. One patient (Regimen C) died as a result of treatment-related hematotoxicity.Conclusions:Siremadlin plus ribociclib demonstrated manageable toxicity and early signs of antitumor activity in patients with advanced WDLPS or DDLPS.

From images to synergies (S1); Reproducibility of data from two screens (S2); Single agent responses and selectivity (S3); Screen-wide comparison of Caspase 3/7 activation and growth inhibition (S4); Heatmaps of growth inhibition and Capase 3/7 activation, and examples of broadly synergistic combinations targeting RAS/MAPK and/or PI3K/AKT pathways (S5); Combinations targeting RAS/MAPK and PI3K/AKT pathways at different nodes show similar efficacies (S6); Combinations involving RTKs (S7); Heatmaps of growth inhibition and Capase 3/7 activation, and examples of combinations targeting RAS/MAPK or PI3K/AKT pathways and other cellular processes (S8); Synergies of triple combination increase with synergies of underlying drug pairs (S9); Heatmap of synergies, growth inhibition, and Caspase 3/7 activation for triple combinations after hierarchical clustering (S10); Triple combinations targeting RAS/MAPK and PI3K/AKT pathways (S11); Triple combinations targeting RAS/MAPK and/or PI3K/AKT pathways and other cellular processes (S12); Combination targeting MDM2 and MEK in p53 wild-type models (S13); Sequential treatment of p53 wild-type lines with triple combination targeting MDM2, MEK, and BCL-2/-XL (S14); High order combinations to kill 'robust' cell lines (S15).

Summarized results for high-order combinations in DLD-1, RKO, HT-29, and LS-180 cell lines.

Supplementary Data from Co-Targeting of MDM2 and CDK4/6 with Siremadlin and Ribociclib for the Treatment of Patients with Well-Differentiated or Dedifferentiated Liposarcoma: Results from a Proof-of-Concept, Phase Ib Study

Selective single agents and combinations.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors.Significance:This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

Summarized results of pair-wise and triple combination screens.

Supplementary material and methods include the description for the bioanalytical method for HDM201 detection in plasma and tumor, the human and mouse gene expression analysis in vitro and in vivo, the live-cell quantification of cleaved-caspase activation, the western blot analysis, the immunohistochemistry, the splinkerette PCR for the amplification of transposon integration sites and the tumor sequencing, mapping of insertion sequences to the mouse genome and identification of common integration site, additional information on the shRNA screen and the tumor models and supplementary references. Supplementary figures include: • Fig S1: the SJSA-1 inhibition growth curves when treated with HDM201 at different doses and for different times and the data for MOLM-3. • Fig S2: the cumulative percentage of cleaved-caspase-3/7 positive cells over the time, the GI50 of HDM201, CGM097 or nutlin-3a on SJSA-1 cells and the cellular apoptosis, as judged by AUC of cleaved-caspase-3/7 positive cells, induced by these compounds. • Fig S3: the PK profile in plasma and tumor of HDM201 in SJSA-1 tumors-bearing rat after p.o. and i.v. treatment, the Bcl-xl mRNA levels in tumors after HDM201 treatment, representative images of SJSA-1 tumors stained with p53 and cleaved-caspase 3 antibodies after HDM201 treatment and the individual data for the efficacy experiment in SJSA-1 tumor-bearing rats. • Fig S4: the PD of HDM201 in PB tumor bearing nude mice after single dose administration. Supplementary tables include: • Table S1: Biochemical profile of HDM201. • Table S2: List of cell lines tested for their sensitivity to HDM201 (n=291) • Table S3: Contingency table indicating association between sensitivity to HDM201 and TP53 wild-type status. • Table S4: List of cell lines tested for their sensitivity to both MDM2 knock-down by shRNA and HDM201 (n=261) • Table S5: Contingency table indicating association between sensitivity to HDM201 and sensitivity to MDM2 shRNA. • Table S6: List of significant rescuer and sensitizer genes following both HDM201 treatment types • Table S7: Pharmacokinetic parameters for HDM201 after p.o. and i.v. dosing in rat. • Table S8: Summary of primary PK parameters for HDM201 daily regimen after single dose (Day 1) in patients. • Table S9: Summary of primary PK parameters for HDM201 daily regimen on Day 14 in patients. • Table S10: Summary of primary PK parameters for HDM201 q3w regimen after single dose in patients.

Legend for Supplementary Tables S1-S6 and Supplementary Figures S1-S15.

Data for characterization of triple combination targeting MDM2, MEK, and BCL-2/-XL.

Cell lines used in this study.

List of individual shRNA scores. File containing raw data of individual shRNAs quantified by barcode sequencing of DMSO- and HDM201- treated samples. Log fold changes of individual shRNAs compared to plasmid library and of HDM201-treated samples compared to DMSO-control are also listed.

Supplementary Methods, Figures 1-5 from PIK3CA Mutation Uncouples Tumor Growth and Cyclin D1 Regulation from MEK/ERK and Mutant KRAS Signaling

Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small-molecule MEK inhibitor, decreases MEK/ERK pathway signaling and destabilizes cyclin D1, resulting in significant anticancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor–resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores cyclin D1 expression and G1-S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and phosphoinositide 3-kinase mutations are unlikely to respond to the inhibition of the MEK pathway alone but will require effective inhibition of both MEK and phosphoinositide 3-kinase/AKT pathway signaling. Cancer Res; 70(17); 6804–14. ©2010 AACR.

Supplementary Figures 1-3, Tables 1-9, Methods from Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Supplementary Materials from Genetic Predictors of MEK Dependence in Non–Small Cell Lung Cancer

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)–targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3. Cancer Res; 70(14); 5901–11. ©2010 AACR.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors. Significance: This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

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.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target 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 some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950–63. ©2016 AACR.

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.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.

Myeloid cell leukemia 1 (Mcl-1) is one of the key anti-apoptotic Bcl-2 family proteins that binds and neutralizes pro-apoptotic BIM, BAX and BAK at the mitochondrial outer membrane, preventing cytochrome c release and caspase activation. Selective upregulation of Mcl-1 functionally contributes to resistance of acute myeloid leukemias (AML) with FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) to chemotherapy (Kasper S. et al. 2012, Blood Cancer J. 2:e60, doi:10.1038/bcj.2012.5). Here we show that a novel Mcl-1 inhibitor S63845 (Kotschy A. et al. 2016, Nature 538, 477-482), has synergistic proapoptotic activity in combination with FLT3-ITD kinase inhibitor midostaurin in pre-clinical models of AML. Our studies demonstrate that S63845 has potent single agent activity in AML cell lines and primary AML samples harboring FLT3-ITD with IC50 values in low nanomolar range. Co-targeting of Mcl-1 and FLT3-ITD with S63845 and midostaurin, respectively, significantly increased apoptosis in FLT3-ITD cells with caspase-3 activation and PARP cleavage occurring rapidly within 6 hours of treatment. Consistent with markedly reduced cell growth and viability, analysis of drug combinations efficacy using Bliss independence model revealed strong synergistic interactions between S63845 and midostaurin in FLT3-ITD cell lines and primary AML samples. Midostaurin caused de-phosphorylation of FLT3-ITD and its downstream targets such as STAT5, AKT and MAPK. This was accompanied by significant downregulation of MAPK-mediated phosphorylation of Mcl-1 at Thr163 required for Mcl-1 stability. Consequently, midostaurin reduced Mcl-1 protein levels, with no major changes in antiapoptotic Bcl-2 or Bcl-XL. Importantly, midostaurin increased expression of pro-apoptotic Bim, which could in turn bind and negate residual Mcl-1 pro-survival activity. Elevated Bim was sustained upon S63845 co-treatment, suggesting that Bim plays functional role in midostaurin/S63845-mediated lethality. Dynamic BH3 profiling showed that midostaurin primed FLT3-ITD cells to Mcl-1 and Bcl-2 inhibitors and facilitated general apoptosis priming in response to Bim peptide. Importantly, given that Mcl-1 is a major contributing factor to resistance of AML to Bcl-2 selective BH3-mimetic venetoclax, S63845/midostaurin treatment induced cell death in venetoclax-resistant FLT3-ITD mutants. In summary, S63845/midostaurin is highly synergistic in FLT3-ITD mutated AML cells including those resistant to venetoclax. In vivo experiments of tolerability and efficacy are ongoing and will be reported. Citation Format: Anna Skwarska, Qi Zhang, Shelley M. Herbrich, Natalia Baran, Ensar Halilovic, Peter Ruvolo, Vivian Ruvolo, Erick Morris, Andrew Wei, Donia Moujalled, Michael Andreff, Marina Konopleva. S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations [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 342.

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.

Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting anti-apoptotic regulators such as Bcl-2 and Bcl-xL has shown efficacy in pre-clinical models but has not resulted in successful clinical trials (Rudin et al., Clin Cancer Res. 2012). Although SCLC cell-lines do not reflect the clinical impact of these inhibitors, patient-derived xenograft (PDX) models may more accurately recapitulate Bcl-2 family expression profiles and BH3 mimetic efficacy. One promising hypothesis is that the fellow anti-apoptotic protein MCL-1 rescues viability in the presence of Bcl-2/Bcl-xL antagonists. Here we evaluate the efficacy of the MCL-1 inhibitor S63845 in combination with a novel specific inhibitor of Bcl-2, BCL201/S55746, in SCLC patient-derived xenografts. Methods: BH3 mimetic compounds were tested for synergy in vitro in SCLC cell lines. A set of ten cell lines was chosen based on relative expression of BCL2, MCL1, and BCL2L1 (Bcl-xL) mRNA. Single agent and and pair-wise combinations of Bcl2 family inhibitors were compared in three-day growth inhibition assays. Loewe synergy scores were plotted versus Bcl2 family mRNA expression to identify the determinants of drug sensitivity. Based on the cell line synergy assays, a combination of BCL201/S55746 and S63845 was selected to test in PDX models of SCLC. Bcl-2 family expression was profiled across a panel of 37 SCLC PDX models generated at MGH by quantitative western blot, and standardized to the most sensitive SCLC cell line, NCI-H211. Ten models were selected based on absolute expression of Bcl-2, Bcl-xL and MCL-1. Mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling precise measurement of tumor regression and time to tumor regrowth. Findings: Bcl-2 family dependency in SCLC cell lines was profiled with selective inhibitors as single agents or combinations. Maximum synergy was found between BCL201/S55746 and S63845 in cell lines with the highest Bcl-2:Bcl-xL expression ratio. Bcl-2 family expression was profiled across a panel of 37 PDX models of SCLC, and a representative set of 10 models was selected for in vivo testing. Consistent with cell line results, the two most sensitive models to BCL201/S55746+S63845 demonstrated the highest Bcl-2:Bcl-xL ratios, with moderate to high expression of MCL-1. In these models BCL201/S55746+S63845 resulted in a 44-70% tumor regression that was stable throughout 4 weeks of treatment. Efficacy was not dependent on MCL-1 expression, and was not strongly correlated with PDX sensitivity to platinum-etoposide. Conclusions: Combined inhibition of Bcl-2 with BCL201 and MCL-1 with S63845 is effective in SCLC tumors with relatively low Bcl-xL expression. This combination overcomes MCL-1 mediated resistance to Bcl-2 inhibitors, and represents a promising strategy to target anti-apoptotic dependency in SCLC. Citation Format: Benjamin J. Drapkin, Sneha Sanghavi, David T. Myers, Jun Zhong, Sarah Phat, Youzhen Wang, Ensar Halilovic, Javad Golji, Anna Farago, Erick Morris, Nicholas J. Dyson. Combined inhibition of Bcl-2 and MCL-1 in small cell lung cancer (SCLC) is most effective in tumors with low Bcl-xL expression [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 381.

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.

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.

Tumors are complex and robust biological systems that harbor the potential to proliferate against various drug treatments. Drug combinations provide a promising therapeutic strategy, but it is not clear which and how many drugs are required to overcome cancers. Using image-based proliferation and apoptosis assays in colorectal cancer cells we systematically investigated complex treatments composed of two to six drugs targeting critical oncogenic pathways. Drug pairs targeting growth signaling resulted in synergies across a broad spectrum of genetic backgrounds, but often yielded cytostatic responses and failed to induce apoptosis. Enhanced, and sometimes genotype-specific cytotoxicity was seen after targeting additional mechanisms including apoptosis or cell cycle. Cells that resisted all tested drug pairs and drug triples were protected by a mechanism that prevented apoptosis. Targeted inhibition of this mechanism using combinations of up to four compounds induced cytotoxic responses in cells in vitro and in vivo. Our results demonstrate that complex combinations of targeted drugs might be required to induce killing in cancers and show how the cells' genetic alterations and a molecular understanding of drug responses can guide their identification. The identification of resistance mechanisms that are pre-existing in subpopulations of tumor cells also opens the exciting avenue of sequenced treatments, with each drug or drug combination targeting and eradicating a specific cell population. Citation Format: Thomas Horn, Stéphane Ferretti, Nicolas Ebel, Angela Tam, Samuel Ho, Fred Harbinski, Ali Farsidjani, Matthew Zubrowski, Ensar Halilovic, Erick Morris, William R. Sellers, Robert Schlegel, Jens Wuerthner, Levi A. Garraway, Sébastien Jeay, Joel Greshock, Giordano Caponigro, Joseph Lehár. Complex drug combinations can induce apoptotic killing in robust colorectal cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B04.

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