In Vivo Epigenetic CRISPR Screen Identifies Asf1a as an Immunotherapeutic Target in Kras-Mutant Lung Adenocarcinoma

Abstract

Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras ^G12D/Trp53 ^-/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.

Figure 1.. In vivo epigenome-wide CRISPR screen identifies Asf1a as a negative regulator of response to anti-PD-1 therapy.

A, Strategy of in vivo epigenome-wide CRISPR screen. 12 tumors from 6 mice were included in each group of the screen. B, Volcano plot illustrating the comparison of IC-IgG and IC-PD1 genes whose knockout (KO) can enhance (blue) or inhibit (red) sensitivity to anti-PD-1 treatment. Some top candidates are highlighted, along with positive control genes whose KO is expected to enhance or inhibit anti-PD-1 treatment. C, Illustration of the top 10 candidates from (B). D, Scatter plot showing the performance of 8 Asf1a sgRNAs in the comparisons indicated “ID-IgG VS IC-IgG”, “ID-IgG VS ID-PD1” and “IC-IgG VS IC-PD1”. E, Detailed information on the performance of 8 Asf1a sgRNAs in the comparison “IC-IgG VS IC-PD1”. ID, immunodeficient B6 Rag1−/− mice; IC, immunocompetent B6 mice; IgG, IgG treatment; PD1, anti-PD-1 treatment.

Figure 2.. Asf1a deficiency synergizes with anti-PD-1 treatment to inhibit tumor progression.

A, Representative MRI scans (1 of 24 scanned images of each mouse) showing mouse lung tumors before and after treatment. The red arrow indicates the single tumor nodule on the left lobe. “H” indicates the heart. B, Waterfall plot showing percentages changes in tumor volume in response to treatment. Each column represents one mouse. C, Dot plot illustrating the tumor volume across the different treatment groups. (Ctrl, n=10; Ctrl + PD-1 ab, n=4; Asf1a KO, n=5; Asf1a KO + PD-1 ab, n=6). 4 mice in Ctrl group, 1 mouse in Ctrl + PD-1 ab group, and 5 mice in Asf1a KO group died prior to week 3 MRI imaging, and hence are excluded here. D, Survival curve for each group in the treatment study. (Ctrl, n=14; Ctrl + PD-1 ab, n=5; Asf1a KO, n=10; Asf1a KO + PD-1 ab, n=6). All data are mean ± SEM. *p < 0.05, **p < 0.01, *** p <0.001

Figure 3.. Asf1a deficiency and anti-PD-1 treatment promotes T cell activation, inflammatory response and M1-like macrophage polarization.

A, Representative flow cytometry analysis of CD62L⁺ (naive T cell marker) and CD69⁺ (T cell activation marker) populations of CD4⁺ T cells across all treatment groups. B, Bar graphs comparing the expression of CD44⁺ (T cell activation marker), CD62L⁺ and CD69⁺ populations of CD4⁺ T cells across all treatment groups. C, Representative flow cytometry analysis of CD62L⁺ and CD69⁺ populations of CD8⁺ T cells. D, Bar graph comparing the expression of CD44⁺, CD62L⁺ and CD69⁺ populations of CD8⁺ T cells. E–H, Flow cytometry analysis on changes in the expression of inflammatory monocytes (CD11b⁺/Gr1⁻/SelecF⁻/Ly6c⁺) (E), macrophages (CD11b⁺/Gr1⁻/F4/80⁺) (F), M1-like macrophages (CD11b⁺/Gr1⁻/F4/80⁺/MHC-II⁺/CD206⁻) (G), and M2-like macrophages (CD11b⁺/Gr1⁻/F4/80⁺/MHC-II⁻/CD206⁺) (H) in CD45⁺ cells. For all flow cytometry experiments, the whole tumor-bearing lungs from an IV injection model were harvested and processed after 1 week of treatment. (Ctrl, n=5; PD-1 ab, n=5; Asf1a KO, n=5; Asf1a KO + PD-1 ab, n=5). All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p <0.001

Figure 4.. Asf1a deficiency activates TNFA signaling and upregulating GM-CSF.

A, Gene set enrichment analysis (GSEA) showing the top 8 enriched pathways in KP cells with Asf1a KO. B, Enrichment of genes associated with TNFA_SIGNALING_VIA_NFKB in KP cells with Asf1a KO. C, Enrichment of genes associated with INFLAMMATORY_RESPONSE in KP cells with Asf1a KO. D, Enrichment of genes associated with TNFA_SIGNALING_VIA_NFKB in human lung ADC tumors with low ASF1A expression. E, Enrichment of genes associated with INFLAMMATORY_RESPONSE in human lung ADC tumors with low ASF1A expression. Top 25% and bottom 25% of ASF1A expression levels were determined using RNA-seq data. F, Heatmap of the genes that comprise the TNFA_SIGNALING_VIA_NFKB gene set in KP cells with or without Asf1a KO. Red star marks the Csf2 gene. G, Relative Csf2 transcripts in KP cells with or without Asf1a KO from RNA-seq data. H, Expression of Csf2 in KP cells with or without Asf1a KO as determined by real-time qPCR. I, Luminex analyses of chemokines/cytokines secreted in cell culture medium harvested 30 hours after the cells were seeded. J, ChIP-seq data of HeLa cells showing that ASF1A occupies the CSF2 promoter. K, ChIP-seq data of H2009 cells showing that ASF1A occupies the CSF2 promoter. Genomic DNA from H2009 cells was used as input control. All data are mean ± SEM. **p < 0.01, ****p <0.0001

Figure 5.. Asf1a deficiency promotes M1-like macrophage polarization and T cell activation through upregulation of GM-CSF.

A, Flow cytometry analysis of IA/IE and CD206 expression in macrophages co-cultured with KP-Ctrl cells or KP-Asf1a KO cells for 7 days. B, Bar graph showing the percentages of IA/IE⁺CD206⁻ (M1-like) macrophages and IA/IE⁻CD206⁺ (M2-like) macrophages from the co-culture experiment shown in (A). (Ctrl, n=4; Asf1a KO, n=6). C, Flow cytometry analysis of IA/IE and CD206 expression in macrophages co-cultured with KP cells in the presence of IgG or anti-GM-CSF for 7 days. D, Bar graph showing the percentages of IA/IE⁺CD206⁻ (M1-like) macrophages and IA/IE⁻CD206⁺ (M2-like) macrophages from the co-culture experiment shown in (C). (IgG, n=3; Anti-GM-CSF, n=3). E-F, Flow cytometry analysis of changes in expression of CD62L (E), CD69 (F) in OT-I T cells co-cultured with macrophages which were sorted from the co-culture system shown in (A). (Ctrl, n=3; Asf1a KO, n=7). G-I, Flow analysis of the expression of CD44⁺(G), CD62L⁺(H) and CD69⁺(I) populations in CD4⁺ T cells. J-L, Flow analysis of the expression of CD44⁺ (J), CD62L⁺ (K) and CD69⁺ (L) populations in CD8⁺ T cells. For flow cytometry analyses in (G-L), whole tumor-bearing lungs from the trans-thoracic injection model were harvested and processed for flow cytometry analysis after 3 weeks of treatment. (Ctrl + PD-1 ab, n=4; Asf1a KO + PD-1 ab, n=4; Asf1a KO + F4/80 ab + PD-1 ab, n=5; Asf1a KO + GM-CSF ab + PD-1, n=4). All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p <0.001, **** p <0.0001 (MFI, mean fluorescence intensity).

Figure 6.. Single-cell analyses of intratumoral immune cell populations confirm the alterations of macrophage and T cell populations.

A, Umap plot showing clusters of tumor cells (center) and intratumoral immune cell populations. B, Changes in the different immune compartments in response to indicated treatments. C, Umap plot showing secondary clusters of macrophages/monocytes. D, Changes in different macrophage/monocyte subpopulations in response to indicated treatments. E, Umap plots show the expression of M2 macrophage marker genes (Arg1, Thbs1, Fn1 and Mrc1) and M1 macrophage marker genes (H2.Aa, H2.Ab1, H2.DMb1, H2.Eb1, Aif1, Tmem176a, Tmem176b, Cd86, Ass1 and Cxcl9) in the macrophage/monocyte subpopulations. F, Umap plot showing secondary clusters of T cell population. G, Changes in different T cell subpopulations in response to indicated treatments. H, Umap plots showing the expression of T cell marker genes (Cd4, Cd8, Sell, Cd44, Gzma, Gzmb, Gzmk, Prf1, Eomes, FasI, Mki67, Icos, Tbx21 and Ifng) in T cell subpopulations.

Figure 7.. Working model for Asf1a deficiency combined with anti-PD-1 combination therapy.

Tumor cell-intrinsic Asf1a deficiency promotes an inflammatory response and GM-CSF secretion, which promotes M1-like macrophage polarization and T cell activation. Anti-PD-1 therapy also promotes T cell activation. Thus, Asf1a KO synergizes with anti-PD-1 treatment to promote anti-tumor immunity.