Our findings provide a rationale for combining ASFA inhibition and antiPD immunotherapy in patients with LUAD.On day, mice were treated with antiPD or isotype control.To identify changes in sgRNA abundances across treatment groups, tumors were harvested on day, genomic DNA was isolated, and amplified sgRNA samples were prepared for nextgeneration sequencing. This is consistent with their dichotomous roles in regulating tumor immunity. We also performed the treatment study in another KP model. Twelve tumors from mice were included in each group of the screen.Similarly, there was minimal correlation between ASFA and CDD, CD, or CDA expression in LUAD samples in TCGA. Furthermore, analysis of mousederived organotypic tumor spheroids. These data support that ASFA may directly inhibit the transcription of GMCSF, independent of nucleosome assembly.For all flow cytometry experiments, the whole tumorbearing lungs from an intravenous injection model were harvested and processed after week of treatment. Genomic DNA from H cells was used as input control.reasch Leupeptin%20Hemisulfate Moreover, GMCSF blockade also Targetmol’s Leupeptin%20Hemisulfate decreased the expression of M macrophage markers CD, CD, and IAIE. These data further support that GMCSF upregulationmediated M macrophage polarization plays pivotal roles in the antitumor immunotherapy.A, UMAP plot showing clusters of tumor cells and intratumoral immune cell populations.C, UMAP plot showing secondary clusters of macrophagesmonocytes.E, UMAP plots show the expression of M macrophage marker genes and M macrophage marker genes in the macrophagemonocyte subpopulations.Asfa deficiency andor antiPD treatment markedly expanded the cluster population.Asfa deficiency andor antiPD immunotherapy decreased T and T populations. Asfa deficiency plus antiPD immunotherapy increased the T population, which implies the memory and effector T cells started to expand from an early time point. Asfa deficiency plus antiPD immunotherapy increased the T population, supporting the enhanced M macrophage polarization.Particularly, using small focused libraries is a practical strategy for in vivo CRISPR screens.Manguso and colleagues first used an in vivo CRISPR screen using an sgRNA sublibrary containing, selected genes to identify novel immunotherapy targets in melanoma. Our current study is the first in vivo CRISPR screen to identify novel immunotherapy targets in lung cancer utilizing an epigenetic sgRNA library containing epigenetic genes.Internal quality controls indicated that our in vivo CRISPR screen functioned successfully, and our study provided further proof of principle for the utilization of in vivo CRISPR screens for detecting potential targets for cancer immunotherapy.The modest response rates highlight an urgent need for new therapeutic approaches to augment the antitumor immune response.Our results thus provide a rationale for combination therapy consisting of ASFA inhibition and antiPD immunotherapy for patients with LUAD.Unfortunately, we could not access any human lung cancer patient data with immunecheckpoint blockade treatment to analyze whether ASFA loss is a biomarker for immunecheckpoint blockade efficacy.ASFA plays a role in regulating gene transcription, but its function in cancer has been only scarcely studied.Individual reports showed that ASFA inhibition elicits DNA damage in cancer cells. The function of GMCSF in antitumor immunity is controversial.Moreover, GMCSF was reported to promote M macrophage polarization, and M macrophages promote antitumor immunity.It is hard to segregate tumorderived GMCSF versus overall GMCSF systemically in the current study.