Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor A (TNFA) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFA signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFA induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFA-mediated apoptosis.

Background: Combined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/ HER2− breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance. Methods: In this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance. Results: We show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2− breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2− breast cancer models. Conclusions: These data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2− breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

Loss-of-function studies offer a potential for validation of known and unrecognized tumor-associated targets. RNA interference-mediated gene knockdown can be exploited to study the reprogrammed circuitry of genes, discover gene interactions restricted to cancer cells and identify mechanisms of chemoresistance in cancer cells. In addition, the simultaneous use of cancer drugs and RNA interference also provides a paradigm to develop strategies to inactivate essential genes promoting neoplastic growth.

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6. Citation Format: Neil A. O'Brien, Martina SJ McDermott, Dylan F. Conklin, Alex Gaither, Tong Luo, Raul Ayala, Suruchi Salgar, Emmanuelle DiTomaso, Naveen Babbar, Faye Su, Sara A. Hurvitz, Ronald Linnartz, Kristine Rose, Samit Hirawat, Dennis J. Slamon. Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer 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 3825.