Abstract P6-21-07: The stapled peptide ALRN-6924, a dual inhibitor of MDMX and MDM2, and the CDK4/6 inhibitors palbociclib or abemaciclib synergistically enhance each other's <i>in vitro</i> and <i>in vivo</i> anticancer activity
Authors: A Annis, LA Carvajal, J-G Ren, D Sutton, S Santiago, N Narasimhan, V Guerlavais, M Aivado
Journal: Cancer Research (2019)

Abstract Background ALRN-6924 is a cell-penetrating α-helical stapled peptide that disrupts the interaction of the p53 tumor suppressor protein and its inhibitors, MDMX and MDM2. Reactivation of p53 with ALRN-6924 in TP53-wild-type tumors triggers cell cycle arrest and apoptosis resulting in antitumor efficacy. CDK4/6 inhibitors induce apoptosis, senescence, and cell growth arrest via the interrelated Rb pathway, and co-amplification of MDM2 and CDK4 (both on chromosome 12q13) is a known oncogenic driver, suggesting that combinations of ALRN-6924 and CDK4/6i's may be synergistic. This study evaluates the antitumor efficacy and pharmacodynamics (PD) of ALRN-6924 combined with palbociclib or abemaciclib. Methods ALRN-6924 was tested in combination with palbociclib or abemaciclib in MCF-7 breast cancer cell lines and MDM2- and CDK4-co-amplified SJSA-1 sarcoma cell lines using WST-1 cell viability assays. Synergy was quantified by the Chou-Talalay combination index method. Single agents and combinations were evaluated in cell culture using assays for apoptosis (Caspase 3/7 cleavage), proliferation (BrdU), senescence (ß-Galactosidase), colony growth (Giemsa), and Western blot analysis of p53, p21, Rb, phospho-Rb, FOXM1, and phospho-FOXM1; and E2F1 mRNA. In vivo combinations were tested in athymic nude mouse MCF-7 and SJSA-1 xenograft models, with cell cycle assays (EdU) measured in tumor samples by flow cytometry. Results ALRN-6924 combinations with palbociclib or abemaciclib display synergistic in vitro anti-proliferative activity in MCF-7 and SJSA-1 cells. ALRN-6924 induces senescence in vitro as a monotherapy and in combination with CDK4/6i's. Western blot assays show that ALRN-6924/palbociclib combinations trigger sustained on-mechanism biomarker activation, vs. transient activation with single agents. Phospho-Rb and phospho-FOXM1 down-regulation, p53 and p21 up-regulation, and repression of E2F1 mRNA are sustained after wash-out in combination, but not in single agent-treated cells. MCF-7 and SJSA-1 tumor growth inhibition was improved in mice treated with ALRN-6924 combinations with either palbociclib or abemaciclib vs. single agent. EdU assays show that ALRN-6924/palbociclib combinations inhibit SJSA-1 tumor cell proliferation in vivo. Body weights and mortality data show the combination of ALRN-6924 with palbociclib 75 mg/kg/day was well tolerated; the combination with abemaciclib 100 mg/kg/day was tolerated with interruption and dose-reduction. No pharmacokinetic (PK) drug-drug interactions were noted in nude mice due to different modes of metabolism for ALRN-6924 (proteolysis) and palbociclib (CYP3A). Conclusions This study demonstrates that ALRN-6924 and CDK4/6i combinations show synergistic activity. PD biomarkers indicate on-mechanism in vitro activity that is sustained after wash-out. In vivo efficacy, biomarker, PK, and tolerability results, plus clinical evidence that the most frequent and concerning safety issues for CDK4/6i's (neutropenia, leukopenia, infections) do not overlap with ALRN-6924's reported safety profile (Meric-Bernstam et al., ASCO 2017) support the development of combination regimens for breast cancer and other malignancies. Citation Format: Annis A, Carvajal LA, Ren J-G, Sutton D, Santiago S, Narasimhan N, Guerlavais V, Aivado M. The stapled peptide ALRN-6924, a dual inhibitor of MDMX and MDM2, and the CDK4/6 inhibitors palbociclib or abemaciclib synergistically enhance each other's in vitro and in vivo anticancer activity [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-07.