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Primary clustering of tumor cells and immune cell populations

MAGeCK analysis of CRISPR screen

Loss of Asf1a shows no significant effect on DNA damage and tumor cell proliferation

Asf1a knockdown combined with anti-PD-1 therapy shows significant inhibition of tumor growth

Asf1a KO and anti-PD-1 combination therapy primes and activates T cells that can recognize specific tumor-associated antigen

Secondary clustering of T cells

Gating strategy for immune profile analyses in this study

Macrophages pre-cultured with Asf1a KO tumor cells show potential effect to activate T cells

In vivo F4/80 or GM-CSF blockade attenuates anti-tumor immune response in Asf1a KO and anti-PD-1 combination therapy

Library and cells used for in vivo CRISPR screen

Luminex analysis of chemokine/cytokine secretions

Reads count of CRISPR screen

Asf1a KO synergizes with anti-PD-1 to inhibit tumor progression in multiple models

Alterations of myeloid populations in an orthotopic KP lung tumor model with short-term treatment

Quality controls performed to validate the in vivo screening platform

Secondary clustering of macrophages/monocytes

Alterations of T cell populations in an orthotopic KP lung tumor model with short-term treatment

CRISPR screen to identify targets regulating the natural host antitumor response

Enriched gene sets in ASF1A-low tumors and Asf1a KO cells

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 KrasG12D/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

ATAC-seq in KP cells and the up-regulation of CSF2 by ASF1A knockdown in H2009 cells

GSEA analysis of mobocertinib and T-DM1 treated tumors versus mobocertinib-only treated tumors Genes involved in "INTERFERON ALPHA RESPONSE", "INFLAMMATORY RESPONSE", "INTERFERON GAMMA RESPONSE", "IL2 STAT5 SIGNALING", "TNFA SIGNALING VIA NFKB" and "IL6 JAK STAT3 SIGNALING" pathways of HALLMARK gene sets.

No targeted treatments are currently approved for HER2 exon 20 insertion–mutant lung adenocarcinoma patients. Mobocertinib (TAK-788) is a potent irreversible tyrosine kinase inhibitor (TKI) designed to target human epidermal growth factor receptor 2 (HER2/ERBB2) exon 20 insertion mutations. However, the function of mobocertinib on HER2 exon 20 insertion–mutant lung cancer is still unclear. Here we conducted systematic characterization of preclinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion–mutant cell lines, the IC50 of mobocertinib was higher than poziotinib and comparable with or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC50/wild-type (WT) EGFR IC50 ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20YVMA allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20G776>VC lung tumors exhibited a sustained complete response to mobocertinib, whereas HER2 exon 20YVMA tumors showed only partial and transient response. Combined treatment with a second antibody–drug conjugate (ADC) against HER2, ado-trastuzumab emtansine (T-DM1), synergized with mobocertinib in HER2 exon 20YVMA tumors. In addition to the tumor cell autonomous effect, sustained tumor growth control derived from M1 macrophage infiltration and CD4+ T-cell activation. These findings support the ongoing clinical development of mobocertinib (NCT02716116) and provide a rationale for future clinical evaluation of T-DM1 combinational therapy in HER2 exon 20YVMA insertion–mutant lung adenocarcinoma patients.Significance:This study elucidates the potent inhibitory activity of mobocertinib against HER2 exon 20 insertion–mutant lung cancer and the synergic effect of combined mobocertinib and T-DM1, providing a strong rationale for clinical investigation.

GSEA analysis of mobocertinib acquired resistant tumors versus response tumors Genes involved in "G2M CHECKPOINT", "MITOTIC SPINDLE" and "MTORC1 SIGNALING" pathways of HALLMARK gene sets.

Figure S1

Figure S2

Figure S3

Figure S4

Candidate lists for CRISPR screens

qRT-PCR primer sequences

Purpose:Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed.Experimental Design:We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines.Results:We show that multiplex gene editing of mouse lung organoids using the CRISPR–Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell–mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti–PD-1 therapy.Conclusions:We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage–inducing therapies in the treatment of LSCC.

CRISPR/Cas9 guides, primers and sequencing results

Antibodies used for Immunohistochemistry, Immunofluroesence and immunblotting

Despite advancements in treatment options, the overall cure and survival rates for non–small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC.Significance:Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.

Pathway enrichment of differentially expressed genes (DEG) between mouse LSCC and LADC.

Reads count for CRISPR screens

Cell lines models

Enriched gene sets in KP cells with or without Npm1 knockdown

12 supplementary figures

Figures S1-9

No targeted treatments are currently approved for HER2 exon 20 insertion–mutant lung adenocarcinoma patients. Mobocertinib (TAK-788) is a potent irreversible tyrosine kinase inhibitor (TKI) designed to target human epidermal growth factor receptor 2 (HER2/ERBB2) exon 20 insertion mutations. However, the function of mobocertinib on HER2 exon 20 insertion–mutant lung cancer is still unclear. Here we conducted systematic characterization of preclinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion–mutant cell lines, the IC50 of mobocertinib was higher than poziotinib and comparable with or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC50/wild-type (WT) EGFR IC50 ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20YVMA allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20G776>VC lung tumors exhibited a sustained complete response to mobocertinib, whereas HER2 exon 20YVMA tumors showed only partial and transient response. Combined treatment with a second antibody–drug conjugate (ADC) against HER2, ado-trastuzumab emtansine (T-DM1), synergized with mobocertinib in HER2 exon 20YVMA tumors. In addition to the tumor cell autonomous effect, sustained tumor growth control derived from M1 macrophage infiltration and CD4+ T-cell activation. These findings support the ongoing clinical development of mobocertinib (NCT02716116) and provide a rationale for future clinical evaluation of T-DM1 combinational therapy in HER2 exon 20YVMA insertion–mutant lung adenocarcinoma patients. Significance: This study elucidates the potent inhibitory activity of mobocertinib against HER2 exon 20 insertion–mutant lung cancer and the synergic effect of combined mobocertinib and T-DM1, providing a strong rationale for clinical investigation.

Despite advancements in treatment options, the overall cure and survival rates for non–small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. Significance: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.

Inactivating mutations in LKB1/STK11 are present in roughly 20% of nonsmall cell lung cancers (NSCLC) and portend poor response to anti-PD-1 immunotherapy. Unexpectedly, we found that LKB1 deficiency correlated with elevated tumor mutational burden (TMB) in NSCLCs from nonsmokers and genetically engineered mouse models, despite the frequent association between high-TMB and anti-PD-1 treatment efficacy. However, LKB1 deficiency also suppressed antigen processing and presentation, which are associated with compromised immunoproteasome activity and increased autophagic flux. Immunoproteasome activity and antigen presentation were restored by inhibiting autophagy through targeting the ATG1/ULK1 pathway. Accordingly, ULK1 inhibition synergized with PD-1 antibody blockade, provoking effector T-cell expansion and tumor regression in Lkb1-mutant tumor models. This study reveals an interplay between the immunoproteasome and autophagic catabolism in antigen processing and immune recognition, and proposes the therapeutic potential of dual ULK1 and PD-1 inhibition in LKB1-mutant NSCLC as a strategy to enhance antigen presentation and to promote antitumor immunity. Wong and colleagues show that LKB1-deficient lung tumors are sensitive to autophagy inhibition, which can restore impaired antigen presentation and antitumor immune responses, and propose dual targeting of ULK1 and PD-1 for these tumors.

No targeted treatments are currently approved for HER2 exon 20 insertion-mutant lung adenocarcinoma patients. Mobocertinib (TAK-788) is a potent irreversible tyrosine kinase inhibitor (TKI) designed to target human epidermal growth factor receptor 2 (HER2/ERBB2) exon 20 insertion mutations. However, the function of mobocertinib on HER2 exon 20 insertion-mutant lung cancer is still unclear. Here we conducted systematic characterization of pre-clinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion mutant cell lines, the IC(50) of mobocertinib was higher than poziotinib and comparable or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC(50) / WT EGFR IC(50) ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20(YVMA) allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20(G776>VC) lung tumors exhibited a sustained complete response to mobocertinib, while HER2 exon 20(YVMA) tumors showed only partial and transient response. Combined treatment with a second antibody-drug conjugate (ADC) against HER2, ado-trastuzumab emtansine (T-DM1) synergized with mobocertinib in HER2 exon 20(YVMA) tumors. In addition to the tumor cell autonomous effect, sustained tumor growth control derived from M1 macrophage infiltration and CD4(+) T cell activation. These findings support the ongoing clinical development of mobocertinib (NCT02716116) and provide a rationale for future clinical evaluation of T-DM1 combinational therapy in HER2 exon 20(YVMA) insertion-mutant lung adenocarcinoma patients.

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC.

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 KrasG12D/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

Purpose: Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed. Experimental Design: We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines. Results: We show that multiplex gene editing of mouse lung organoids using the CRISPR–Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell–mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti–PD-1 therapy. Conclusions: We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage–inducing therapies in the treatment of LSCC.

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.

KRAS/STK11 (LKB1) mutant lung cancers are a subtype of cancer that respond poorly in the clinic to immunotherapies with shorter progression-free survival and overall survival comparing with other KRAS mutant lung cancer patients. Interestingly, this group of patients have high tumor mutational burden (TMB) comparable with other KRAS mutant lung cancers, which usually is an indicator for better response to anti-PD1 treatment. By using genetically engineered mouse model (GEMMs), we found that LKB1 loss of function can cause high level of nonsynonymous mutations, as a consequence of both replication dependent and independent DNA repair machinery dysfunctions. This LKB1 mutant dependent increased mutational load might further synergize with smoking mutagen exposures. When LKB1 function is defective, the neoantigens are not properly presented to T cells, which is required for proper T cell activation and effector T cells expansion. This will lead to insensitivity to anti-PD1 antibodies in high TMB tumors and tolerance to neoantigens produced by cancer cells. We further demonstrated that using a small molecule drug that impacts on the LKB1 downstream effector pathway, we can reverse this process through activating innate immunity in tumors, which will restore the antigen presentation functions. Combinational treatment of this small molecule with anti-PD1 can reverse the tumor progression in vivo, through releasing the suppressive tumor infiltrating lymphocytes (TILs) in LKB1 mutant lung cancers. Our study not only helps to further our understanding of the mechanism that contributes to immune evasion in high TMB tumors, but also provides a potential solution for LKB1 mutant cancers to overcome resistance to anti-PD1 treatment in the clinic. Citation Format: Jiehui Deng, Aatish Thennavan, Yuanwang Pan, Igor Dolgalev, Ting Chen, Heather Silver, Matthew Harris, Val Pyon, Fei Li, Chelsea Lee, Aristotelis Tsirigos, Eli Rothenberg, Charles M. Perou, Kwok-Kin Wong. Overcome LKB1 mutated cancer resistance to anti-PD1 treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3223.