Unlabelled

Significance

Immune checkpoint blockade (ICB) targeting programmed cell death-1 (PD-1) has led to prolonged clinical responses among several patients with melanoma and other cancer types. ICB response relies on effective immune recognition and elimination of tumor cells. This depends on the successful presentation of tumor-specific antigens on surface major histocompatibility complex (MHC) class I molecules by tumor cells to the T-cell receptor of cytotoxic CD8+ T cells. Currently, both primary and acquired mechanisms of resistance remain significant barriers to expanding the therapeutic benefits of anti-PD-1 treatment. Several studies have stressed the critical role of antigen processing machinery (APM) defects in mediating resistance to ICB therapies. More specifically, aberrations of the β-2-microglobulin (B2M) gene, which encodes a critical component of the MHC class I molecule, were found to be enriched in patients with melanoma who progressed during therapy. Nevertheless, there are cases of patients with B2M-deficient tumors that respond to ICB, but the exact mechanism mediating this response is currently unknown. Presently, the exact effect of MHC class I APM alterations on anti-PD-1 therapy response requires further characterization and the cells mediating the anti-tumorigenic effects of PD-1 blockade in B2M-defective tumors have not been fully elucidated. In this work, we aim to holistically characterize alterations in components involved in class I antigen processing, presentation, and regulation in melanoma tumors, as well as to elucidate the main cell mediators of class I-deficient tumor removal in patient-derived biopsies and mouse models. Using a clinical dataset of patients with melanoma treated with ICB, whole-exome sequencing and bulk RNA-sequencing data from baseline tumor samples were analyzed to identify somatic class I APM alterations and evaluate correlative clinical outcomes, infiltrating immune cells, and gene expression patterns. Additionally, human melanoma cell lines and B2M-null mouse tumor models were utilized to validate these findings and further explore the impact of B2M and MHC class I defects on tumor intrinsic and extrinsic factors. Collectively, our findings may help offer alternative therapeutic targets and avenues for the treatment of tumors with class I-mediated ICB resistance.

Melanoma dedifferentiation has been reported as a state of cellular resistance to targeted therapy and immunotherapy as cancer cells revert to a more primitive cellular phenotype. In a patient with metastatic melanoma who received adoptive T-cell transfer therapy using T cells with receptors against the melanoma antigen recognized by T cells 1 (MART-1/ Melan-A), we observed dedifferentiation as a resistance mechanism after initial response. However, biopsies obtained from responding patients during anti-programmed cell death receptor 1 (PD-1) therapy had decreased expression of melanocytic markers and increased neural crest markers. When modeling the effects in vitro, we documented that melanoma cell lines that were originally melanocytic differentiated underwent a process of neural crest dedifferentiation when continuously exposed to interferon gamma (IFNγ), through a global chromatin landscape change leading to enrichment in specific hyperaccessible chromatin regions. The IFNγ-induced dedifferentiation signature corresponded with improved outcomes in patients with melanoma, challenging the notion that neural crest dedifferentiation is an adverse phenotype.

Immune checkpoint blockade therapies constitute one of the major advances in cancer treatment. Despite the long-lasting responses observed in a wide range of tumor types, the majority of patients do not respond or relapse shortly after. In order to increase response rates, we need to better predict patients that would benefit from the treatment as well as identify resistance mechanisms to find novel treatment strategies. However, the interplay between the immune system, the cancer cells and the tumor microenvironment is complex and dynamic, rendering the understanding of resistant mechanisms particularly challenging.

Lack of immune cell infiltration constitutes one of the main mechanisms of primary resistance to checkpoint blockade. The absence of T cells on the tumor margin leaves the therapeutic targeting of immune checkpoints ineffective. Interestingly, cancer cell-intrinsic mechanisms could actively participate in the process of immune evasion and importantly, could be pharmacologically targeted to reverse immune exclusion. The finding of actionable molecules that increases T cell infiltration in the tumor could be used in combination with PD-1 blockade, in order to reverse adaptive immune resistance.

Here, we characterized the tumors of melanoma patients treated with PD-1 blockade. Our analyses further validated lack of immune cell infiltration as one mechanism of resistance. Importantly, we identified PAK4 as a novel and actionable target that is enriched in biopsies with poor immune infiltration and lack of response to PD-1 blockade. We show how genetically and pharmacologically that inhibition of PAK4 increases immune cell infiltration and improves checkpoint blockade therapy in several in vivo mouse models. We further characterized the impact of inhibiting PAK4 expression. The transcriptomic profiles of PAK4 KO tumors revealed the profound effect that has on the tumor microenvironment, particularly in the genes related to the extracellular matrix and blood vessel formation. This work serves as an example of how direct changes in cancer cells impact the tumor microenvironment and influence the anti-tumor immune response. Importantly, this work also provides the scientific rationale for a novel treatment strategy to combine PAK4 inhibitor with checkpoint blockade.