Evaluation of Mechanisms Underlying Therapeutic Susceptibility and Immunotherapeutic Potential in SETD2 Mutant Kidney Cancers
Open Access
- Author:
- Uzzo, Robert
- Area of Honors:
- Science
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- David James Degraff, Thesis Supervisor
Ronald Albert Markle, Thesis Honors Advisor - Keywords:
- CRISPR
CAS9
SETD2
Synthetic Lethality
H3K36me3 - Abstract:
- OBJECTIVE: SETD2 mutations occur in approximately 10% of all renal epithelial malignant neoplasms. As a histone H3 lysine methyltransferase, SETD2 is implicated in the proper splicing of mRNA. Its loss induces intron retention. We hypothesized that SETD2 deficient renal cell carcinomas (RCC) retain introns, which are aberrantly translated, resulting in protein misfolding. We hypothesize that this would trigger the unfolded protein response (UPR). This mechanism may offer two potential therapeutic approaches, a synthetically lethal strategy as well as mechanisms based on increased immunogenicity secondary to upregulated neopeptide expression. METHODS: We analyzed KIRC tumors from TCGA with gene set enrichment analysis, comparing transcriptomes of SETD2 mutant versus wild type (WT) tumors. Next, we established SETD2 knockout cell lines in RENCA, a naturally occurring murine RCC line, using CRISPR edits. Sanger and next-generation sequencing were performed to confirm SETD2 deficient isogenic cell lines. Western blotting was then used to measure three sensors of the UPR pathway (PERK, IREα, ATF6) to investigate pathway activation. This was further investigated using immunofluorescence microscopy. Finally, we analyzed a panel of UPR inhibitors to determine if SETD2 loss results in increased therapeutic susceptibility. RESULTS: In SETD2 deficient TCGA tumors, the UPR gene set was significantly enriched (q=0.01). Sanger sequencing and immunoblot confirmed the successful knockout of SETD2 at the gene and protein levels. In addition, H3-K36 trimethylation was lost, confirming loss of SETD2 function. SETD2 deficiency-induced activation of UPR through upregulation of protein kinase PERK and the enzyme IREα. Additionally, transcription factor ATF6 was noted to be cleaved and localized to the nucleus using immunofluorescence microscopy in SETD2 deficient cells indicating that UPR pathway was activated. These results demonstrate a potential therapeutic sensitization that occurs with SETD2 mutations in RCC. CONCLUSION: We show that SETD2 loss in RCC induces activation of the UPR and offers a potential therapeutic mechanism that may be druggable using synthetic lethality. Furthermore, candidate therapeutic susceptibilities could lead to a biomarker-based clinical trial using repurposed FDA-approved therapies.