No abstract

Purpose: Altered PI3K/mTOR signaling is implicated in the pathogenesis of a number of breast cancers, including those resistant to hormonal and HER2-targeted therapies.Experimental Design: The activity of four classes of PI3K/mTOR inhibitory molecules, including a pan-PI3K inhibitor (NVP-BKM120), a p110a isoform-specific PI3K inhibitor (NVP-BYL719), an mTORC1-specific inhibitor (NVP-RAD001), and a dual PI3K/mTORC1/2 inhibitor (NVP-BEZ235), was evaluated both in vitro and in vivo against a panel of 48 human breast cell lines.Results: Each agent showed significant antiproliferative activity in vitro, particularly in luminal estrogen receptor-positive and/or HER2 þ cell lines harboring PI3K mutations. In addition, monotherapy with each of the four inhibitors led to significant inhibition of in vivo growth in HER2 þ breast cancer models. The PI3K/ mTOR pathway inhibitors were also effective in overcoming both de novo and acquired trastuzumab resistance in vitro and in vivo. Furthermore, combined targeting of HER2 and PI3K/mTOR leads to increased apoptosis in vitro and induction of tumor regression in trastuzumab-resistant xenograft models. Finally, as previously shown, targeting mTORC1 alone with RAD001 leads to consistent feedback activation of AKT both in vitro and in vivo, whereas the dual mTOR1-2/PI3K inhibitor BEZ235 eliminates this feedback loop. However, despite these important signaling differences, both molecules are equally effective in inhibiting tumor cell proliferation both in vitro and in vivo.

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers.Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib.Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2-and ER+/HER2+ subtypes. However, a subset of triple negative breast cancer (TNBC) cell lines with intact Rb-signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2-, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved anti-tumor activity. CDK4/6 inhibition with abemaciclib combined with anti-mitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic-biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on CyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as mono-therapy or as a combination partner in selected ER+/HER2-, HER2+/ER+ and TNBCs.

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.

Approximately 60-70% of invasive breast cancers express estrogen receptor (ER) and/or progesterone receptor and are termed hormone receptor positive (ER+). Endocrine therapy remains the therapeutic backbone for the treatment of ER+ cancers and although anti-estrogen therapies are initially frequently effective, 50% of ER+ patients develop resistance to hormonal manipulation within their lifetime, ultimately leading to therapeutic failure. Two emerging mechanisms of endocrine resistance include activation of growth signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway and more recently, the decoupling of cell cycle control from ER-signaling, via deregulation of the cyclinD-cyclin dependent kinase (CDK-4/6:INK4:Rb) pathway. In this study we hypothesized that combining an anti-estrogen (letrozole or fulvestrant) with a CDK-4/6 inhibitor (LEE011) and PI3K inhibitors (buparlisib [BKM120; pan-PI3K inhibitor] or BYL719 [α-specific PI3K inhibitor] would elicit an improved tumor response over agents inhibiting either pathway alone. Four ER+ BC mouse models including, an ER+ patient primary human letrozole sensitive model (HBX34, PTEN/PIK3CA wild-type) and three ER+ cell lines; ZR75-1 (PTEN null), MCF7 (PIK3CA mutant) and KPL1 (PTEN/PIK3CA wild-type) were used for this study. Treatment was carried out daily at doses relevant to clinical exposures for a period of 4 weeks with fulvestrant combinations and 8 weeks with letrozole combinations followed by observation for tumor progression. Treatment doses were as follows: LEE011 (75 mg/kg), BKM120 (30mg/kg), BYL719 (35mg/kg), Letrozole (2.5mg), Fulvestrant (5mg/wk) either as single agents or in combinations (Hormone therapy (HT)+LEE011, HT+PI3Ki, HT+LEE011+PI3Ki). Significant tumor growth inhibition (TGI) was observed for single agent treatment with the CDK-4/6 inhibitor in each of the four ER+ xenograft models. The addition of letrozole (HBX34) or fulvestrant (MCF7, ZR751 and KPL1) with LEE011 increased the TGI observed with single agent. The triplet combination with each PI3K inhibitor increased the (TGI) even further, inducing tumor regressions in each of the four models. Response was independent of PI3K/PTEN mutation status. Complete tumor regressions were observed for a subset of mice within each of the triple combination arms. Tumor regressions were maintained for up to four weeks post-interruption of treatment. No significant toxicities were observed with any of the triplet combinations. These preclinical data highlight the potential efficacy and safety of targeting the ER, CDK-4/6 and PI3K signaling pathways in ER+ breast cancer. LEE011 in combination with an anti-estrogen is sufficient to inhibit tumor growth in vivo, while the addition of a PI3K inhibitor results in robust tumor regressions. The combination of LEE011 with an anti-estrogen and a PI3K inhibitor is a rational therapeutic approach that should be investigated in the clinic. Citation Format: Neil A. O'Brien, Emmanuelle Di Tomaso, Raul Ayala, Luo Tong, Shawnt Issakhanian, Ronald Linnartz, Richard S. Finn, Samit Hirawat, Dennis J. Slamon. In vivo efficacy of combined targeting of CDK4/6, ER and PI3K signaling in ER+ breast cancer. [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 4756. doi:10.1158/1538-7445.AM2014-4756