Abstract P5-04-02: The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation
Authors: Torres-Adorno AM , Lee JJ , Kogawa T , Bartholomeusz C , Pitner MK , Ordentlich P , Lim B , Tripathy D , Ueno NT 
Published: May 2016

Purpose: Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the two most aggressive types of breast cancer whose molecular mechanisms remain unclear, representing a challenge for the development of effective targeted therapies. Single agent targeted therapies are of limited effectiveness in these types of breast cancer. Therefore, we aim to identify new combination therapeutic candidates for these aggressive diseases by comprehensive genomic screening.

Experimental Design: We screened kinome siRNA libraries with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor [pimasertib], and genome-wide functional mRNA expression with the histone deacetylase (HDAC) type I inhibitor [entinostat] in TNBC and IBC cell lines. We evaluated the relationship between targets of interest and breast cancer patient survival using the IBC consortium database composed of breast cancer patient samples with clinical follow up. After identifying the targets, we assessed the combinational synergistic effect and its mechanism via cytotoxicity assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, western blotting, and mammary fat pad xenograft mouse models.

Results: We identified that knock-down of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, enhanced the anti-proliferative effect of pimasertib. We observed that entinostat induced the expression of Noxa, a pro-apoptotic BH3-only member of the Bcl-2 family that is known to bind and degrade Mcl-1. Interestingly, in a breast cancer patient cohort (N = 389), high Mcl-1/low Noxa co-expression was associated with poorer survival outcomes than low Mcl-1/high Noxa co-expression (P = 0.0038). We found that combination with pimasertib and entinostat enhanced the inhibition of tumor cell proliferation (P &lt; 0.001) compared with entinostat or pimasertib alone. We also observed significant in vivo tumor growth inhibition in both IBC (SUM190, P &lt; 0.0001) and TNBC (SUM149, P &lt; 0.05) xenograft models. Specifically, TNBC and IBC cell lines that overexpressed Noxa after treatment with entinostat were observed to be selectively sensitive to combination treatment with pimasertib. The synergistic antitumor activity of the entinostat- pimasertib combination was due to increased expression of Noxa, which induced the degradation of Mcl-1, resulting in the induction of mitochondrial cell death.

Conclusion: Our data provide evidence that entinostat has enhanced antitumor effect in combination with pimasertib, resulting in the induction of apoptosis by Noxa-mediated Mcl-1 degradation. These findings provide a novel preclinical rationale for developing a clinical trial based on combinatorial HDAC and MEK inhibition therapy for TNBC and IBC with high Mcl-1 expression.

Citation Format: Torres-Adorno AM, Lee JJ, Kogawa T, Bartholomeusz C, Pitner MK, Ordentlich P, Lim B, Tripathy D, Ueno NT. The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-02.