SETD2 Deficiency Enhances The Synergistic Effect Of Dnmt Inhibitor And Parp Inhibitor In Renal Cell Carcinoma

Purpose: SETD2(SET domain-containing 2)deficiency,a driving force of depleted levels of H3K36me3 due to impaired methyltransferase activity,plays an important role in diverse forms of cancer,most notably in aggressive and metastatic clear cell renal cell carcinomas(cc RCC).Recent studies have shown that H3K36me3 catalyzed by SETD2 is related to active transcriptional genes and invasiveness of renal cancer,and is involved in important cellular physiological processes such as DNA methylation,chromatin stability and DNA damage repair.The relative 5-year survival rate for localized cc RCC is 91%,but once cc RCC metastasizes or invades,the survival rate drops to 11%.This is why it is so urgently needed to develop new therapies to target SETD2-deficient cancers,as these alterations may be an attractive target for diagnostic and therapeutic purposes.DNA hypomethylation agents(HMAs)have shown clinical anti-tumor efficacy and are approved by FDA to treat myelodysplastic syndrome(MDS).Poly(ADP-ribose)polymerase inhibitor(PARPi)is a new drug used in the treatment of solid tumors such as ovarian cancer and breast cancer.Meanwhile,studies have shown that the combination of HMA and PARPi can significantly inhibit cell growth by enhancing DNA damage and immune responses.The purpose of this study is to verify whether SETD2 gene defects have a synergistic anti-tumor effect on RCC upon the combination therapy of HMA and PARPi,and to explore the mechanism and provide novel ideas for cancer treatment.Methods: Human kidney cancer cell lines with or without SETD2 deficiency were treated with HMA,PARPi,and both(HMA + PARPi)in combination in both in vitro and in vivo settings.Changes in cell proliferation,cell cycle arrest and apoptosis related to H3K36me3 expression levels induced by drugs were determined by western blot,cell proliferation assay,colony formation assay and flow cytometry.Changes in DNA damage and repair functions were verified by western blot and comet assay.RNA-seq and other bioinformatics analysis were performed to detect the changes of various pathways in the transcription level of RCC cells with different SETD2 expression levels.Last but not least,the inhibition of the combination therapy on RCC xenografts with different SETD2 expression levels was determined in vivo.Results: Considering that SETD2 is involved in DNA methylation and DNA repair,we found the combination treatment of an HMA(Decitabine/DAC)and a PARPi(Talazoparib/BMN-673)caused increased cytotoxicity in vitro in SETD2-deficient RCC cell lines than in SETD2-proficient cell lines.Furthermore,our results demonstrate synergetic anti-tumor effects in SETD2-deficient RCC cell lines aftertreatment with DAC and BMN-673 that driven by apoptotic induction,increased DNA damage,insufficient DNA damage repair,upregulation of immune responses by STING and transposable elements(TEs)and increased genomic instability.Finally,it was further validated that combination efficacy of DAC and BMN-673 has a stronger effect on SETD2-deficient than SETD2-proficient RCCs using in vivo mouse models.Conclusions: The SETD2 deficiency leads to a vulnerable epigenetic status that generates effective anti-tumor effects on RCCs in vitro and in vivo via the combination treatment of DAC and BMN-673,making it a precision medicine approach based on SETD2-compromised cancers.This project further provides evidence for clinical trials using precision medicine-based approaches in patients with aggressive RCCs with defective SETD2 genes or impaired H3K36me3 levels.