Durga Nand Tripathi

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.

Supplementary Table S3 lists the baseline demographics, clinical and prior treatment characteristics of the prospective RMC cohort used in the study.

This pdf file contains Supplementary Figures S1-S5 and their legends.

Supplementary Table S1 provides detailed information on the patient samples used for RNA-seq and ChIP-Seq.

The supplementary methods file contains additional details on the chromatin extraction and immuno-precipitation sequencing approaches.

Supplementary Table S4 is the “Representativeness of Study Participants”

Table S5 lists the full results of the pathways enrichment over-representation analyses shown in main Figures 2i and 5e, as well as supplementary Figure 4c.

Supplementary Table S2 provides detailed information on differentially expressed genes and peaks from the tissue and cell line comparisons performed in the study.

Purpose: Renal medullary carcinoma (RMC) is a highly aggressive malignancy defined by the loss of the SMARCB1 tumor suppressor. It mainly affects young individuals of African descent with sickle cell trait, and it is resistant to conventional therapies used for other renal cell carcinomas. This study aimed to identify potential biomarkers for early detection and disease monitoring of RMC. Experimental Design: Integrated profiling of primary untreated RMC tumor tissues and paired adjacent kidney controls was performed using RNA sequencing and histone chromatin immunoprecipitation sequencing. The expression of serum cancer antigen 125 (CA-125), was prospectively evaluated in 47 patients with RMC. Functional studies were conducted in RMC cell lines to assess the effects of SMARCB1 reexpression. Results: MUC16, encoding for CA-125, was identified as one of the top upregulated genes in RMC tissues, with concomitant enrichment of active histone marks H3K4me3 and H3K27ac at its promoter. Elevated serum CA-125 levels were found in 31 of 47 (66%) patients with RMC and correlated significantly with metastatic tumor burden (P = 0.03). Functional studies in RMC cell lines demonstrated that SMARCB1 reexpression significantly reduced MUC16 expression. Conclusions: The correlation between serum CA-125 levels and metastatic burden suggests that CA-125 is a clinically relevant biomarker for RMC. These findings support further exploration of CA-125 for disease monitoring and targeted therapeutics in RMC.