Introduction
Despite radical improvements in survival of some patients treated with immune checkpoint inhibitors (ICIs), many NSCLC patients do not respond to treatment. In particular, never smoker (NS) patients with NSCLC derive inferior benefit from ICIs compared to those with a smoking history (ES). Immune evasion is a hallmark of cancer. Tumour cell-specific MHC-II (tsMHC-II) expression may augment neoantigen visibility and is a predictive biomarker of response to ICI in multiple cancers. We used CRISPR-Cas9 knockout screens to identify novel regulators of tsMHC-II.
Methods
H1975 (NS) and A549 (ES) are lung adenocarcinoma cell lines carrying the EGFR L858R/T790M and KRAS G12S mutations respectively. Cas9-expressing H1975 and A549 cell lines were transduced (6 replicates each) with a genome-wide sgRNA library and a boutique nuclear factor sgRNA library. Cells with increased surface expression of MHC-II compared to the parental cell line were sorted and sequenced to identify enriched sgRNAs. For each sgRNA, Z-scores were calculated according to Log2Fold change between sorted populations and controls based on counts per million. STARS v1.3 analysis was performed with hits in-silico cross validated using publicly available datasets.
Results
Screening of H1975 cells identified 5 genes (STARS score >2.5 on pooled analysis) as potential negative regulators of tsMHC-II expression. The top 2 candidates- identified in all replicates- are implicated in protein ubiquitination and transcriptional regulation. They were found to be expressed in NSCLC samples from the TRACERx patient cohort. The capacity of these
candidates to regulate tsMHC-II expression will be validated in multiple cell lines using flow cytometry and in vitro CD4 + T cell stimulation assays. Their mechanisms of action will be
interrogated using chromatin immunoprecipitation and RNAseq experiments. We will also evaluate the correlation between the expression of hits with tsMHC-II expression
in treatment-naïve and ICI-treated NSCLC samples. These analyses will be combined with profiling of the immune infiltrate in tumours using the MIBIscope™ to evaluate the
interplay between tsMHC-II cell surface expression and the immune microenvironment in response to therapy.
Conclusion
Using pooled CRISPR-Cas9 knockout screening approaches, we identified multiple tsMHC-II regulators. Targeting these pathways may unleash anti-tumour immunity in a population with an unmet need for effective therapies.