# Pharmacologic and Oncohistone Inhibition of SETD2 Converge on Genomic Instability

## TL;DR

This study shows that both drug inhibition and a specific histone mutation disrupt genomic stability by inactivating the tumor suppressor SETD2, which could explain cancer progression and treatment risks.

## Contribution

The study identifies genomic instability as a shared outcome of pharmacologic and oncohistone-mediated SETD2 inhibition.

## Key findings

- Pharmacologic inhibition of SETD2 increases chromatin bridges and micronuclei in human cells.
- H3.3K36M oncohistone expression similarly reduces SETD2 activity and causes mitotic defects.
- Genomic instability is a canonical feature of SETD2 inactivation through multiple mechanisms.

## Abstract

SETD2 is a tumor suppressor that trimethylates histone H3 at lysine 36 (H3K36me3). When inactivated, it drives the development of several cancers, most notably clear cell renal cell carcinoma (ccRCC). Our study looked beyond genetic loss of SETD2 activity, as occurs during oncogenesis, to other, clinically relevant means for inactivation of this methyltransferase: pharmacologic inhibition and a histone mutation that binds and sequesters SETD2. We found that both pharmacologic inhibition and sequestration made cells more genomically unstable. Identification of loss of genomic stability as a canonical feature of SETD2 inactivation is important as it reveals a potential mechanism for progression of cancers expressing oncohistone mutations, and potential liability associated with targeting this methyltransferase.

Background/Objectives: SETD2 is a dual-function methyltransferase important for methylation of histone H3 at lysine 36 and α-tubulin in spindle microtubules. Genetic inactivation of SETD2 during oncogenesis drives loss of H3K36me3, genomic instability, and cancer progression. This study asked if disruption of genomic stability was a canonical feature of SETD2 inactivation via different pathways. Methods: We evaluated the impact of EPZ-719, a pharmacologic SETD2 inhibitor, and an H3.3K36M mutant histone (“oncohistone”) that binds and sequesters SETD2, on methylation activity and genomic stability in human cell lines. SETD2 activity was measured using in vitro methylation assays, H3K36me3 loss confirmed by Western analysis, and mitotic defects, specifically micronuclei and chromatin bridges, quantified with cytogenetic analysis. Results: EPZ-719 caused a dose- and time-dependent reduction in SETD2 activity on both histone and tubulin substrates, accompanied by significant increases in chromatin bridges and micronuclei in retinal pigmented epithelial (RPE-1) and 786-O ccRCC cells. Similarly, oncohistone expression markedly decreased SETD2 function, as determined by H3K36me3 levels, and induced comparable mitotic defects in 786-O cells, and aneuploidy in two chondrocyte cell lines expressing the H3.3K36M oncohistone. Combining EPZ-719 with H3.3K36M expression did not exacerbate mitotic defects beyond either oncohistone or pharmacologic inhibition alone, consistent with inhibition of SETD2 as their shared underlying mechanism of action. Conclusions: Pharmacologic inhibition and oncohistone-mediated sequestration of SETD2 converge on the induction of mitotic defects, underscoring SETD2’s essential role in maintaining genomic stability. Identification of loss of genomic stability as a canonical feature of SETD2 inactivation points to a potential therapeutic liability associated with targeting SETD2 in cancers where it is overexpressed and reveals a mechanism that could contribute to the progression of cancers expressing oncohistone mutations.