Jerby-Arnon and Izar et al. used single-cell RNAseq of human melanoma to identify a cancer cell-intrinsic genetic program informed by cell-cell interactions and associated with T cell exclusion and immune checkpoint inhibitor (ICI) resistance. The program was validated at the protein level in situ through immunofluorescence of patient tumor sections and provided improved predictive capacity for ICI treatment response in several patient cohorts and animal models. CDK4 was implicated as a master regulator, and CDK4/6 inhibition reduced program expression and enhanced antitumor efficacy in combination with ICI in mouse melanoma.

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

Author Info: (1) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (2) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (3) Broad Institute of

Author Info: (1) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (2) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (3) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (4) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (5) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA. (6) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (7) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA. (8) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA. (9) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Laboratory for Systems Pharmacology, Harvard Medical School, Boston, MA, USA. (10) Laboratory for Systems Pharmacology, Harvard Medical School, Boston, MA, USA. (11) Laboratory for Systems Pharmacology, Harvard Medical School, Boston, MA, USA. (12) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (13) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (14) Molecular & Cellular Oncogenesis Program and Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA. (15) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (16) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (17) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (18) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (19) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (20) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (21) Massachusetts General Hospital Cancer Center, Boston, MA, USA. (22) Massachusetts General Hospital Cancer Center, Boston, MA, USA. (23) Massachusetts General Hospital Cancer Center, Boston, MA, USA. (24) Massachusetts General Hospital Cancer Center, Boston, MA, USA. (25) Massachusetts General Hospital Cancer Center, Boston, MA, USA. (26) Massachusetts General Hospital Cancer Center, Boston, MA, USA. (27) Molecular & Cellular Oncogenesis Program and Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA. (28) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Massachusetts General Hospital Cancer Center, Boston, MA, USA. (29) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (30) Broad Institute of MIT and Harvard, Cambridge, MA, USA; Celsius Therapeutics, Cambridge, MA, USA. (31) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (32) Department of Dermatology, University Hospital Essen, West German Cancer Center, University Duisburg-Essen and the German Cancer Consortium, Essen, Germany; Department of Dermatology, Venereology and Allergology, University Hospital Wurzburg, Wurzburg, Germany. (33) Center for Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA. (34) Center for Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA. (35) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (36) Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (37) Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (38) Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (39) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (40) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (41) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (42) Laboratory for Systems Pharmacology, Harvard Medical School, Boston, MA, USA. (43) Center for Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA. (44) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. (45) Department of Dermatology, University Hospital Essen, West German Cancer Center, University Duisburg-Essen and the German Cancer Consortium, Essen, Germany. (46) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA. (47) Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA. (48) Broad Institute of MIT and Harvard, Cambridge, MA, USA. (49) Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA; Ludwig Center for Cancer Research at Harvard, Boston, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. (50) Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Brigham and Women's Hospital, Department of Surgical Oncology, Boston, MA, USA. (51) Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Center for Cancer Precision Medicine of Dana-Farber Cancer Institute, Boston, MA, USA; Laboratory for Systems Pharmacology, Harvard Medical School, Boston, MA, USA; Center for Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA; Ludwig Center for Cancer Research at Harvard, Boston, MA, USA. Electronic address: benjamin_izar@dfci.harvard.edu. (52) Broad Institute of MIT and Harvard, Cambridge, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA; Ludwig Center for Cancer Research at MIT, Boston, MA, USA; Massachusetts Institute of Technology, Department of Biology, Cambridge, MA, USA.

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