In vivo CRISPR-screening of novel cancer cell-intrinsic targets that sensitize to local ionizing radiation, and possible combination with systemic checkpoint blockade.

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Project Summary While many promising candidate radiosensitizers have been pursued, the development of a clinically approved radiosensitizer remains a “holy grail” of clinical radiobiology. Our proposal represents an exciting union of traditional translational basic science with state-of-the-art technology: the use of a CRISPR screening library to identify new molecular targets that would radiosensitize cancer cells in vivo, both intrinsically and through the involvement of the immune system (Aim 1), and testing in both in vivo mouse tumor models and using a unique clinical dataset (Aim 2). This collaboration between the Weichselbaum lab, which has pioneered radiation therapy breakthroughs for over 40 years, and the Manguso lab, featuring the next generation of investigators who are world leaders in the nascent field of in vivo CRISPR screenings, makes us uniquely qualified to use cutting-edge technology to solve a long-standing problem that would immediately improve clinical radiotherapy. Specifically, we plan to use in vivo CRISPR-screening for novel cancer cell-intrinsic genetic targets that increase or decrease the efficacy of radiation combined or not with checkpoint blockade, directly or through the participation of the immune system. By determining which sgRNAs are depleted in treated vs. untreated mice, which would indicate that a sensitizing loss-of-function was introduced, we can identify putative targets for RT alone or in combinational strategies. sgRNAs will be ranked by degree of depletion. Candidate targets will be selected based on (i) having highest cumulative scores, (ii) novelty, and (iii) being depleted in both MC38 and LLC tumor models. We will use SBRT-like and fractionated IR schemes +/- checkpoint blockade (anti- PD1/anti-CTLA-4) to test in vivo three targets that increase the therapeutic effect of radiation directly, i.e., in immunodeficient mice, and three targets that increase the therapeutic effect of IR combined with checkpoint blockade in immunocompetent mice. Finally, we will examine target amplification in exome sequencing data from patients treated in our institution with RT or radio-immunotherapy. Additionally, although not the main focus for this proposal, an inspection of sgRNAs enriched instead of depleted in irradiated mice will point at genes whose targeting could play an unknown role in cancer radio-resistance. The findings we present supporting our proposal highlight the key role of the local interaction between cancer cells and immune cells in the irradiated tumor microenvironment to determine treatment outcome after radiotherapy alone, or combined with immunotherapy. Unbiased approaches are required to discover novel targets and better prioritize combination strategies to improve treatment. Successful completion of our studies will address the long sought unmet need of a radiosensitizer to improve outcomes for cancer patients. This would be the first in vivo CRISPR-based screening of genetic targets that radiosensitize tumors. An in vivo screening in mice will for the first time enable the discovery of potential indirect radio-sensitizing targets that would require the involvement of the immune system, in addition to direct (intrinsic) radio-sensitizer targets.