Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.

A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation.

BACKGROUND: CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. METHODS: We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. RESULTS: Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. CONCLUSIONS: This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.

CD47 expression is critical for CAR T-cell survival in vivo.

BACKGROUND: CD47 is an attractive immunotherapeutic target because it is highly expressed on multiple solid tumors. However, CD47 is also expressed on T cells. Limited studies have evaluated CD47-chimeric antigen receptor (CAR) T cells, and the role of CD47 in CAR T-cell function remains largely unknown. METHODS: Here, we describe the development of CD47-CAR T cells derived from a high affinity signal regulatory protein α variant CV1, which binds CD47. CV1-CAR T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo. The role of CD47 in CAR T-cell function was examined by knocking out CD47 in T cells followed by downstream functional analyses. RESULTS: While CV1-CAR T cells are specific and exhibit potent activity in vitro they lacked antitumor activity in xenograft models. Mechanistic studies revealed CV1-CAR T cells downregulate CD47 to overcome fratricide, but CD47 loss resulted in their failure to expand and persist in vivo. This effect was not limited to CV1-CAR T cells, since CD47 knockout CAR T cells targeting another solid tumor antigen exhibited the same in vivo fate. Further, CD47 knockout T cells were sensitive to macrophage-mediated phagocytosis. CONCLUSIONS: These findings highlight that CD47 expression is critical for CAR T-cell survival in vivo and is a 'sine qua non' for successful adoptive T-cell therapy.

Disruption of NSD1 in Head and Neck Cancer Promotes Favorable Chemotherapeutic Responses Linked to Hypomethylation.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) represents a distinct classification of cancer with worse expected outcomes. Of the 11 genes recurrently mutated in HNSCC, we identify a singular and substantial survival advantage for mutations in the gene encoding Nuclear Set Domain Containing Protein 1 (NSD1), a histone methyltransferase altered in approximately 10% of patients. This effect, a 55% decrease in risk of death in NSD1-mutated versus non-mutated patients, can be validated in an independent cohort. NSD1 alterations are strongly associated with widespread genome hypomethylation in the same tumors, to a degree not observed for any other mutated gene. To address whether NSD1 plays a causal role in these associations, we use CRISPR-Cas9 to disrupt NSD1 in HNSCC cell lines and find that this leads to substantial CpG hypomethylation and sensitivity to cisplatin, a standard chemotherapy in head and neck cancer, with a 40% to 50% decrease in the IC50 value. Such results are reinforced by a survey of 1,001 cancer cell lines, in which loss-of-function NSD1 mutations have an average 23% decrease in cisplatin IC50 value compared with cell lines with wild-type NSD1Significance: This study identifies a favorable subtype of HPV-negative HNSCC linked to NSD1 mutation, hypomethylation, and cisplatin sensitivity. Mol Cancer Ther; 17(7); 1585-94. ©2018 AACR.