Background Immunotherapy with checkpoint blockade is effective in only a subset of cancer patients and additional treatment strategies are needed. Here we used a pooled in vivo CRISPR-based genetic screening approach to discover cancer cell intrinsic regulators of immune resistance and sensitization. A two-step approach was taken to uncover tumor suppressor gene loss driving immune evasion as step one, and immune sensitizers specifically reversing the immune evasion driven by certain tumor suppressor loss as step two. Through this approach, we are determined to identify genetic context specific immune sensitizers, also known as synthetic lethal immune targets.

Methods We first performed a pooled CRISPR-Cas9-based in vivo genetic screen targeted to a pre-defined set of tumor suppressor genes to mimic loss-of-function mutations in syngeneic tumor models. CRISPR edited tumor cells were implanted into immune-deficient or immune-competent C57BL/6 mice, a subset of which were treated with anti-PD1 to simulate increased immune pressure in vivo. Tumor samples at the endpoint were subjected to next generation sequencing and statistical analysis to identify tumor suppressor genes driving immune evasion. Furthermore, a second pooled CRISPR-Cas9-based in vivo genetic screen targeted to a set of potentially druggable genes was performed in MC38/STK11-/- model vs MC38 wild type model in the presence of increasing immune pressure in vivo. Next generation sequencing and statistical analysis were conducted to uncover STK11 loss specific immune sensitizers.

Results The screen confirmed previously identified immunotherapy targets such as CD47 as well as known drivers of immune resistance in the interferon signaling and antigen presentation pathways. Importantly, STK11 loss was identified as a driver of immune evasion in MC38 and 3LL models. STK11 knockout in MC38 or 3LL tumor cells significantly accelerated tumor growth in immune-competent mice and in these mice treated with anti-PD1. Tumor infiltrating lymphocyte profiling suggests that STK11 knockout induces an immune suppressive tumor microenvironment. Our second step target identification screen discovered HDAC1 as an immune sensitizer specific for STK11 loss context.

Conclusions CRISPR-based in vivo genetic screen is an effective approach to identify tumor cell-intrinsic drivers of immune resistance and sensitization. HDAC1 was identified as an immune sensitizer specifically reversing immune resistance driven by STK11 loss. STK11 mutations are found in ~15% of non-squamous non-small cell lung cancer and have been reported as a predictor of primary resistance to PD-1 blockade. Inhibitors selectively targeting HDAC1 & 2 in combination with anti-PD1 represent a promising therapeutic opportunity for non-small cell lung cancer patients with STK11 mutations.