Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells.

Jane L. Roberts,Andrew Poklepovic,Laurence Booth,Paul Dent

doi:10.3389/fonc.2021.723966

Abstract

We have extended our analyses of HDAC inhibitor biology in sarcoma. The multi-kinase inhibitor axitinib interacted with multiple HDAC inhibitors to kill sarcoma cells. Axitinib and HDAC inhibitors interacted in a greater than additive fashion to inactivate AKT, mTORC1 and mTORC2, and to increase Raptor S722/S792 phosphorylation. Individually, all drugs increased phosphorylation of ATM S1981, AMPKα T172, ULK1 S317 and ATG13 S318 and reduced ULK1 S757 phosphorylation; this correlated with enhanced autophagic flux. Increased phosphorylation of ULK1 S317 and of Raptor S722/S792 required ATM-AMPK signaling. ULK1 S757 is a recognized site for mTORC1 and knock down of either ATM or AMPKα reduced the drug-induced dephosphorylation of this site. Combined exposure of cells to axitinib and an HDAC inhibitor significantly reduced the expression of HDAC1, HDAC2, HDAC3, HDAC4, HDAC6 and HDAC7. No response was observed for HDACs 10 and 11. Knock down of ULK1, Beclin1 or ATG5 prevented the decline in HDAC expression, as did expression of a constitutively active mTOR protein. Axitinib combined with HDAC inhibitors enhanced expression of Class I MHCA and reduced expression of PD-L1 which was recapitulated via knock down studies, particularly of HDACs 1 and 3. In vivo, axitinib and the HDAC inhibitor entinostat interacted to significantly reduce tumor growth. Collectively our findings support the exploration of axitinib and HDAC inhibitors being developed as a novel sarcoma therapy.

Highlights

Soft tissue sarcomas (STS) are a rare mesenchymal-derived and diverse group of tumors, with over 50 subtypes
The present studies were performed to define the molecular mechanisms by which low clinically achievable concentrations of axitinib interact with histone deacetylase (HDAC) inhibitors to kill sarcoma cells
The drug combinations activated an ATM-AMP-dependent protein kinase (AMPK) pathway which was responsible for phosphorylation of Raptor, inactivation of mTORC1 and mTORC2, activation of ULK1 and elevated ATG13 S318 phosphorylation

Summary

INTRODUCTION

Soft tissue sarcomas (STS) are a rare mesenchymal-derived and diverse group of tumors, with over 50 subtypes. We recently demonstrated that the multi-kinase inhibitor pazopanib interacted with HDAC inhibitors in vitro and in vivo to kill sarcoma cells and suppress tumor growth [1, 2]. In multiple prior studies combining multi-kinase inhibitors such as sorafenib and pazopanib with HDAC inhibitors, we noted that three important alterations in cell signaling occurred which were all vital for causing tumor cell death [7,8,9,10]. Knock down of autophagy regulatory genes such as Beclin, ATG5 and ATG16L1 or lysosomal proteases such as cathepsin B significantly reduced drug combination lethality. Our findings provide compelling evidence that axitinib combines with HDAC inhibitors to kill sarcoma cells

MATERIALS AND METHODS

Methods

RESULTS

DISCUSSION

ETHICS STATEMENT