Abstract How the genetic landscape of a tumor governs the tumor’s response to immunotherapy remains largely elusive. Here, we established a direct in vivo CRISPR/Cas9 gene editing methodology to assess the immune-modulatory capabilities of 573 putative cancer genes associated with altered cytotoxic activity in human cancers. Using Kras G12D - and Braf V600E -driven mouse lung cancer models, we identify Serpinb9 and Adam2 as our top immune suppressive and immune enhancing genes, respectively. Mechanistically, we show that Serpinb9 ablation in Kras G12D - and Braf V600E -mutant lung tumor cells greatly enhances the efficacy of cytotoxic T-cells in vitro and in vivo . ADAM2 is a cancer testis antigen broadly expressed in human cancers such as lung adenocarcinoma (13.9%), renal (74.7%), prostate (72.4%), uterine (28.6%) and invasive breast (9.5%) cancer. In our mouse models, we show that Adam2 expression is induced in Kras G12D - but not Braf V600E -driven murine lung tumors and that its expression is further enhanced by immunotherapy. We show that loss of Adam2 significantly decreases Kras G12D -lung tumor burden but blocks the efficacy of cytotoxic T-cells. Consistently, Adam2 overexpression dramatically increases tumor growth and enhances immunotherapy efficacy. Mechanistically, we find that Adam2 ’s oncogenic function depends on modulating the tumor immune microenvironment by restraining productive type I and type II interferon responses as well as cytokine signaling, reducing the presentation of tumor-associated antigen, and modulating surface expression of several immunoregulatory receptors within Kras -driven lung tumors. Adam2 expression also leads to reduced levels of immune checkpoint inhibitors such as Pd-l1, Lag3, Tigit and Tim3. This reduced exhaustion within the tumor microenvironment may explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy against Adam2 overexpressing lung tumors. Together, our study highlights the power of integrating cancer genomic with in vivo CRISPR/Cas9 screens to uncover how cancer-associated genetic alterations control responses to immunotherapies.

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer