Oleksandr Kyrysyuk

Despite promise in preclinical models, most immuno-oncology drug candidates fail in clinical trials. These failures reflect limitations in our ability to directly model the response of human tumor and immune cells to immunotherapies. To address this gap and test the effect of innate immune agonists, we developed PERCEPT, an approach that uses ex vivo perturbational single-cell RNA sequencing to compare the response of immunomodulatory treatments with unstimulated controls directly in patient samples. Using PERCEPT, we tested cytokines and innate immune agonists in melanoma and Merkel cell carcinoma (MCC) and identified the dsRNA mimetic, RIG-I agonist, Stem Loop RNA (SLR) 14 as a powerful inducer of anti-viral states and enhancer of T cell activation. We compared transcriptional responder and non-responder patient samples and identified midkine (MDK), a multifunctional cytokine, as a potent repressor of IFN signaling in both tumor and immune cells. MDK expression dampened MHC-I presentation in human tumor cells and reduced activation of antigen-presenting cells, disrupting tumor immunity at multiple levels. In contrast to prior studies, we identified MDK as specifically enriched in neuroendocrine cancers such as MCC and small cell lung cancer compared with melanoma, suggesting the importance of lineage- and context-specific targeting. Our results demonstrate the utility of high-dimensional controlled perturbation of patient samples to identify mechanisms of innate immune response and resistance and demonstrate an actionable path towards clinical development of MDK-inhibiting therapies including FDA-approved ALK inhibitors in neuroendocrine cancers.

Supplementary Fig. S7 shows effect of PD-1 inhibition on treatment of tumors containing a B2m-/- population.

Supplementary Fig. S8 shows CD8 T cell-dependent control of tumors containing a B2m-/- population with inactivated Rnf31 and Atg5 genes.

Supplementary Fig. S3 generation of tumor cell lines with multiple gene edits.

Supplementary Fig. S5 shows association of Atg5-KO and Rnf31/Atg5-dKO signatures with patient survival.

Supplementary Fig. S10 shows pharmacological sensitization to T cell cytokines in vitro and in vivo.

Supplementary Fig. S6 shows functional links between autophagy and cytokine signaling pathways.

Supplementary Fig. S11 shows pharmacological sensitization of human melanoma cell line to T cell cytokines.

Supplementary Fig. S4 shows differentially expressed genes revealed by RNA-seq analysis.

Supplementary Table S1: # Raw count of the genome-wide CRISPR screen for identifying T cell-mediated tumor killing in B2m-deficient cells. # MAGeCK results (Gene summary) for the genome-wide CRISPR screen from the B2m-deficient cells. # Gene log2FC in CRISPR screens from B2m-WT cells and B2m-KO cells. ## Common regulators of T cell mediated tumor killing in both B2m-WT and B2m-KO cells