Abstract 1058: Synergistic activity of p97 inhibitors with histone deacetylase 6 inhibitors in mantle cell lymphoma

Abstract

Abstract p97, or valosin-containing protein (VCP), is an ATPase whose function is essential to restore protein homeostasis in cells. Working in concert with the ubiquitin proteasome system, p97 promotes the retrotranslocation of misfolded proteins from the endoplasmic reticulum and/or their degradation. Consequently, p97 inhibition has emerged as a novel therapeutic target in cancer cells, especially those that depend on high protein turnover and/or ER function. p97 is also present in a complex with many protein complexes including the HSP90, histone deacetylase (HDAC) 6 and heat shock factor 1 (HSF1)-also called the repressive complex. p97 participates in the disaggregation of the repressive complex to regulate HSP90 and HSF1 function under stressed conditions. Accumulation of misfolded polyubiquitylated proteins in the cytosol or on damaged organelles promotes the binding of p97 to polyubiquitylated proteins in an HDAC6-dependent manner and promotes their turnover by autophagic degradation. Given that perturbation of protein homeostasis and ER function induces apoptosis as well as autophagy in B cell malignancies such as multiple myeloma and mantle cell lymphoma (MCL), we hypothesized that inhibition of p97 function in combination with HDAC6 inhibitors would induce proteotoxic stress and/or apoptotic cell death in MCL cells. In this study, we report that the p97 inhibitors DBeQ, ML240 and NMS-873 induce a dose-dependent loss of cell viability in cultured and primary MCL cells. Treatment of MCL cells with ML240 induces the accumulation of polyubiquitylated proteins and induces markers of ER stress such as glucose regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation factor 2 (eIF2) α as well as the autophagic markers LC3-B and p62 (protein whose accumulation is suggestive of reduced autophagic clearance of misfolded proteins). Co-treatment with ML240 and the HDAC6 inhibitor ACY-1215 induces more accumulation of polyubiquitylated proteins and markers of enhanced ER stress as well as autophagy, than either agent alone. Mechanistically we determined that co-treatment with ML240 and ACY-1215 inhibits the binding of p97 to polyubiquitylated proteins and HDAC6, resulting in the reduced clearance of cytotoxic protein aggregates in the cells. Co-treatment with ML240 and ACY-1215 also induces the accumulation of cytosolic polyubiquitylated proteins and their co-localization with LC-3B puncta as demonstrated by immunofluorescent microscopy. These observations are suggestive of enhanced initiation of autophagy but not its completion. Consequently, treatment of MCL cells with ML240 and ACY-1215 resulted in enhanced proteotoxic stress and synergistic apoptotic cell death in MCL cells. Collectively our studies create a strong rationale to test efficacy of the combination of p97 inhibitors in combination with HDAC6 inhibitors in MCL. Citation Format: Rekha M. Rao, Pratikkumar H. Vekaria, Trisha Home, Anusha Vallurupalli, Abdulraheem Yacoub, Frank Schoenen, Joseph McGuirk. Synergistic activity of p97 inhibitors with histone deacetylase 6 inhibitors in mantle cell lymphoma [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 1058. doi:10.1158/1538-7445.AM2017-1058