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• W4380086931 abstract "Abstract Background: Structural variants are common in cancer that drive tumor formation and typically occur early in carcinogenesis. Despite being a common and well-known feature of cancer, this cancer hallmark has not yet been broadly explored therapeutically in the clinic. We present a novel precision-cancer approach, KLIPP, which consists of guide RNAs that target cancer-specific structural variant junctions (SVJs) to nucleate two parts of dCas9-conjugated endonuclease, Fok1, leading to its activation and induction of toxic DSBs. Furthermore, this approach reduced tumor growth in an orthotopic bladder cancer model in mice. Methods and Materials: HCT116 colon cancer cells and UMUC3 bladder cancer cells were engineered to express combinations of SVJ-targeting sgRNAs and express Fok1-dCas9 under doxycycline (dox) regulation. The HCT116 and UMUC3 cells were treated with dox for 24 hours and assessed for DSBs at SVJs by γH2AX foci assay and γH2AX ChIP-PCR. The cells were assessed for cell death using flow cytometry measuring sub-G1 DNA content, the clonogenic survival assay (12 days), and PARP1 and caspase cleavage (after 48 h) using Western blot. Tumor growth of orthotopic xenografts of UMUC3 cells expressing Fok1-dCas9 alone or with 2 pairs of SVJ-targeting sgRNAs were assessed weekly after addition of dox to the drinking water. Bioluminescence assessment of tumor growth was monitored weekly. Results: Expression of SVJ-targeting sgRNA and the induction of Fok1-dCas9 by dox for 24 h in HCT116 colon cancer and UMUC3 bladder cancer cells, resulted in one or two γH2AX foci per cell. Using ChIP-PCR, the identity of the site of the DSB was validated to occur at the targeted SVJ. Furthermore, dox treatment for 12 days caused &gt;50% reduced clonogenic survival that was augmented by the DNA-PK inhibitor NU7441. Further, we observed &gt;50% induction of apoptosis when assessed by subG1 DNA content cells and PARP1 cleavage. When UMUC3 cancer cells were grown orthotopically in mice, activation of Fok1-dCas9 and together with two pairs of SVJ-targeting sgRNAs led to significantly reduced tumor growth with 7/11 mice showing no signs of tumor. Conclusions: We have developed a precision CRISPR approach based on the homodimerization of Fok1 endonuclease at sites of cancer specific SVJs to specifically induce toxic DSBs in cancer cells. Here we show that this approach led to efficient targeting of cancer cells both in cell culture and in vivo. We are currently testing the efficacy of the KLIPP approach in preclinical models of bladder and ovarian cancers using lipid nanoparticle delivery techniques. On successfully implementation of this approach, it will be transformative for cancer therapy. Further, KLIPP will be generated in a personalized way for each patient in a rapid and cost-effective way. Citation Format: Radhika Suhas Hulbatte, Huibin Yang, Natalie Gratsch, Alan Kellher, Phillip L. Palmbos, Mats Ljungman. A precision CRISPR approach to target structural variant junctions in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1068." @default.
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• W4380086931 date "2023-04-04" @default.
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• W4380086931 title "Abstract 1068: A precision CRISPR approach to target structural variant junctions in cancer" @default.
• W4380086931 doi "https://doi.org/10.1158/1538-7445.am2023-1068" @default.
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