Tazemetostat is a small molecule inhibitor of the histone methyltransferase EZH2 and is currently in phase 2 clinical trials including relapsed refractory Non-Hodgkin Lymphomas (RR-NHL) Diffuse Large B Cell Lymphoma and Follicular Lymphoma. In the phase 1 clinical trial RR-NHL patients demonstrated positive clinical activity with a favorable safety profile. Acquired mutations in the D1 domain (I109K, Y111D, Y111L) and the SET domain (Y661D) of EZH2 have recently been reported as a mechanism of resistance to non-tazemetostat small molecule EZH2 inhibition. Given the clinical activity observed in the phase 1 tazemetostat clinical trial and these reports of pre-clinical EZH2 inhibitor induced resistance we embarked on investigations of the potential of tazemetostat to induce resistance in NHL cell lines. An EZH2 Y641F mutant DLBCL cell line WSU-DLCL2 was exposed to 1 μM tazemetostat, a dose which is 100 fold greater than the naïve line's 11 day growth IC50. After 8 weeks, growth of the tazemetostat treated cells matched that of the control cells. Subsequent increases in dose of tazemetostat up to 10 μM did not yield any changes in growth rate of the treated cells. EZH2 wild-type PMBCL cell line U2940 was exposed to a step wise increase in tazemetostat concentration for 7 weeks and finally maintained at the naïve cell line 11 day proliferation IC50 of 10 μM, with minimal effects on cell growth. Tazemetostat resistant cell lines were screened for acquired EZH2 mutations using a full coding next generation sequencing assay, with a mean depth of 17,126 across all positions. Sequencing results showed the resistant U2940 had gained mutations in EZH2 similar to those previously identified, a heterozygous Y661N mutation and a low frequency mutation of Y111H, consistent with a subclonal mutation. These acquired mutations have been reported to interfere with the binding of EZH2 inhibitors, which supports the minimal reduction of H3K27me3 as measured by ELISA in the resistant U2940 after treatment with tazemetostat. In contrast, after induction of tazemetostat resistance WSU-DLCL2 retained equipotent sensitivity to reduction of H3K27me3 by ELISA. Correspondingly, sequencing of EZH2 in the resistant WSU-DLCL2 line did not identify any additional mutations. These findings suggest continued target engagement with tazemetostat in the resistant WSU-DLCL2, and that a novel bypass mechanism may be engaged to confer resistance. In an attempt to identify the mechanism of resistance in the WSU-DLCL2 line, whole exome sequencing and RNA sequencing has been performed. Detailed mutational, gene expression and pathway analysis will be performed on these data to investigate mechanisms of treatment emergent resistance to tazemetostat. Understanding these mechanisms may guide hypotheses for rational combinations and provide direction for future preclinical and potentially clinical studies.

Citation Format: Carly T. Campbell, Jeff S. Jasper, Scott R. Daigle, Scott A. Ribich, Heike Keilhack, Jesse S. Smith, Peter T. Ho, Stephen J. Blakemore. Evidence of EZH2 dependent and independent mechanisms of tazemetostat treatment emergent resistance in models of diffuse large B cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 312.