Abstract 3892: EZH2 mediated kinome reprogramming drives AR phosphorylation and activation in receptor tyrosine kinase inhibitors resistant renal carcinoma models
Authors: Rupert Christopher , Metcalf Stephanie , Rajendran Saranya , Colligan Sean , Hollenhorst Peter , Pili Roberto 
Published: April 2023

Systemic treatment of recurrent/metastatic renal cell carcinoma (RCC) largely uses receptor tyrosine kinase inhibitors (RTKI), as single agents or in combination with immune checkpoint inhibitors. Unfortunately, patients commonly develop resistance to RTKI’s, leaving few options for further treatment. Therefore, understanding the mechanisms responsible for drug resistance is critical to improve the clinical outcome in this disease. Our previous studies of acquired resistance to the RTKI sunitinib identified two major regulators, EZH2 and androgen receptor (AR). Inhibition of EZH2 or AR in combination with sunitinib re-sensitizes cells to sunitinib both in vitro and in vivo. To further explore the link between EZH2 and AR in drug resistance, 786-0 RCC cells with acquired resistance to sunitinib (786-0RS) was compared to drug naive 786-0 cells with exogenous AR (786-0AR). Chromatin immunoprecipitation sequencing (ChIP-seq) showed that AR and EZH2 bind the same genomic locations, but only in the resistant strain. Interestingly, treatment of 786-0AR cells with sunitinib caused AR to translocate to the nucleus observed by the presence of AR at EZH2 bound sites. Additionally, mass spec of AR purified from 786-0RS cells and 786-0AR cells found high phosphorylation of S81 and S213, two markers associated with ligand independent activation, in 786-0RS cells, but not in 786-0AR cells. Previously, our lab has reported that 786-0RS cells display a reprogramming of the tyrosine kinome which is dependent on EZH2 likely via a non-canonical mechanism. Consistent with this, EZH2 with a phosphomimetic mutation in Serine 21 (S21D) could promote sunitinib resistance when over-expressed with AR, whereas wild-type and phosphonull (S21A) EZH2 could not. We have also expanded the serine/threonine phosphoproteomic analysis and found extensive rewiring of these signaling networks in 786-0RS cells with an enrichment of EGFR mediated MAPK and AKT signaling when compared to 786-0 cells. Finally, our lab has generated 786-0 strains resistant to other RTKI including pazopanib and lenvatinib, and found that AR is expressed in these resistant models, solidifying AR activation as a conserved feature of RTKI resistance. Overall, our results suggest that AR activation is driven by phosphorylation induced by EZH2 dependent kinome reprogramming. Untangling the cross talk between AR and EZH2 in RTKI resistant RCC will provide the rationale for novel therapeutic strategies to improve the clinical outcome in patients with this disease.

Citation Format: Christopher Rupert, Stephanie Metcalf, Saranya Rajendran, Sean Colligan, Peter Hollenhorst, Roberto Pili. EZH2 mediated kinome reprogramming drives AR phosphorylation and activation in receptor tyrosine kinase inhibitors resistant renal carcinoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3892.