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Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers. Significance: Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH–mediated transformation in AML and glioma.

Identification of host determinants of coronavirus infection informs mechanisms of viral pathogenesis and can provide new drug targets. Here we demonstrate that mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) chromatin remodeling complexes, specifically canonical BRG1/BRM-associated factor (cBAF) complexes, promote severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and represent host-directed therapeutic targets. The catalytic activity of SMARCA4 is required for mSWI/SNF-driven chromatin accessibility at the ACE2 locus, ACE2 expression and virus susceptibility. The transcription factors HNF1A/B interact with and recruit mSWI/SNF complexes to ACE2 enhancers, which contain high HNF1A motif density. Notably, small-molecule mSWI/SNF ATPase inhibitors or degraders abrogate angiotensin-converting enzyme 2 (ACE2) expression and confer resistance to SARS-CoV-2 variants and a remdesivir-resistant virus in three cell lines and three primary human cell types, including airway epithelial cells, by up to 5 logs. These data highlight the role of mSWI/SNF complex activities in conferring SARS-CoV-2 susceptibility and identify a potential class of broad-acting antivirals to combat emerging coronaviruses and drug-resistant variants.

Tumor heterogeneity is a major barrier to cancer therapy, including immunotherapy. Activated T cells can efficiently kill tumor cells following recognition of MHC class I (MHC-I)–bound peptides, but this selection pressure favors outgrowth of MHC-I–deficient tumor cells. We performed a genome-scale screen to discover alternative pathways for T cell–mediated killing of MHC-I–deficient tumor cells. Autophagy and TNF signaling emerged as top pathways, and inactivation of Rnf31 (TNF signaling) and Atg5 (autophagy) sensitized MHC-I–deficient tumor cells to apoptosis by T cell–derived cytokines. Mechanistic studies demonstrated that inhibition of autophagy amplified proapoptotic effects of cytokines in tumor cells. Antigens from apoptotic MHC-I–deficient tumor cells were efficiently cross-presented by dendritic cells, resulting in heightened tumor infiltration by IFNγ-and TNFα-producing T cells. Tumors with a substantial population of MHC-I–deficient cancer cells could be controlled by T cells when both pathways were targeted using genetic or pharmacologic approaches. Significance: Tumor heterogeneity is a major barrier to immunotherapy. We show that MHC-I–deficient tumor cells are forced into apoptosis by T cell–derived cytokines when TNF signaling and autophagy pathways are targeted. This approach enables T cell–mediated elimination of tumors with a substantial population of resistant, MHC-I–deficient tumor cells. This article is highlighted in the In This Issue feature, p. 1027

Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non–B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.Significance:The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.

CRISPR-based technologies represent a major breakthrough in biomedical science as they offer a powerful platform for unbiased screening and functional genomics in various fields, including immunology. Pooled and arrayed CRISPR screens have uncovered previously unknown intracellular drivers in innate and adaptive immune cells for immune regulation as well as intercellular regulators mediating cell-cell interactions. Recent single-cell CRISPR screening platforms expand the readouts to the transcriptome and enable the inference of gene regulatory networks for better mechanistic insights. CRISPR screens also allow for mapping of genetic interactions to identify genes that synergize or alleviate complex immune phenotypes. Here, we review the progress in and emerging adaptation of CRISPR technologies to advance our fundamental immunological knowledge and identify novel disease targets for immunotherapy of infection, inflammation and cancer.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. Significance: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.

Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

Tumor heterogeneity is a major barrier to cancer therapy, including immunotherapy. Activated T cells can efficiently kill tumor cells following recognition of MHC class I (MHC-I)–bound peptides, but this selection pressure favors outgrowth of MHC-I–deficient tumor cells. We performed a genome-scale screen to discover alternative pathways for T cell–mediated killing of MHC-I–deficient tumor cells. Autophagy and TNF signaling emerged as top pathways, and inactivation of Rnf31 (TNF signaling) and Atg5 (autophagy) sensitized MHC-I–deficient tumor cells to apoptosis by T cell–derived cytokines. Mechanistic studies demonstrated that inhibition of autophagy amplified proapoptotic effects of cytokines in tumor cells. Antigens from apoptotic MHC-I–deficient tumor cells were efficiently cross-presented by dendritic cells, resulting in heightened tumor infiltration by IFNγ-and TNFα-producing T cells. Tumors with a substantial population of MHC-I–deficient cancer cells could be controlled by T cells when both pathways were targeted using genetic or pharmacologic approaches.Significance:Tumor heterogeneity is a major barrier to immunotherapy. We show that MHC-I–deficient tumor cells are forced into apoptosis by T cell–derived cytokines when TNF signaling and autophagy pathways are targeted. This approach enables T cell–mediated elimination of tumors with a substantial population of resistant, MHC-I–deficient tumor cells.This article is highlighted in the In This Issue feature, p. 1027

Supplementary Fig. S10 shows pharmacological sensitization to T cell cytokines in vitro and in vivo.

Supplementary Table S3: ## Raw count matrix of the RNA-seq data. ## Atg5-KO vs Ctrl-KO (Veh).

Characterization of isogenic TF-1 cell lines expressing TET2-targeting shRNAs.

Supplementary Table S9 shows the gene set enrichment analysis gene set that corresponds to Figure S6A.

List of supplementary figures and tables.

Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers.Significance:Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH–mediated transformation in AML and glioma.

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

Supplementary Figure from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Supplementary Table from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.Significance:Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.See related commentary by Beytagh and Weiss, p. 2730.See related article by Mo et al., p. 2906.This article is highlighted in the In This Issue feature, p. 2711

CRISPR screen in tumor-infiltrating T cells

Pool composition of in vivo screen.

A number of cancer drugs activate innate immune pathways in tumor cells but unfortunately also compromise antitumor immune function. We discovered that inhibition of CARM1, an epigenetic enzyme and cotranscriptional activator, elicited beneficial antitumor activity in both cytotoxic T cells and tumor cells. In T cells, Carm1 inactivation substantially enhanced their antitumor function and preserved memory-like populations required for sustained antitumor immunity. In tumor cells, Carm1 inactivation induced a potent type 1 interferon response that sensitized resistant tumors to cytotoxic T cells. Substantially increased numbers of dendritic cells, CD8 T cells, and natural killer cells were present in Carm1-deficient tumors, and infiltrating CD8 T cells expressed low levels of exhaustion markers. Targeting of CARM1 with a small molecule elicited potent antitumor immunity and sensitized resistant tumors to checkpoint blockade. Targeting of this cotranscriptional regulator thus offers an opportunity to enhance immune function while simultaneously sensitizing resistant tumor cells to immune attack.Significance:Resistance to cancer immunotherapy remains a major challenge. Targeting of CARM1 enables immunotherapy of resistant tumors by enhancing T-cell functionality and preserving memory-like T-cell populations within tumors. CARM1 inhibition also sensitizes resistant tumor cells to immune attack by inducing a tumor cell–intrinsic type 1 interferon response.This article is highlighted in the In This Issue feature, p. 1861

Supplementary Figure S1. Distribution of barcode read representation in pre-expansion and pre-injection samples. Supplementary Figure S2. Tumor composition of in vivo pooled screen, excluding the pools shown in Figure 2. Supplementary Figure S3. Gene expression differentiates functional alleles. Supplementary Figure S4. Validation of rare oncogenic alleles, excluding the ones shown in Figure 4. Supplementary Figure S5. Gene expression signatures of NFE2L2 wild type and gain-offunction mutants are correlated. Supplementary Figure S6. Comparison to in silico methods.

Contains 16 supplementary figures and references

Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR-Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2-encoding a phosphate importer-is correlated with sensitivity to loss of the phosphate exporter XPR1, both in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2, XPR1 copy number amplifications and XPR1 messenger RNA overexpression. Mechanistically, in SLC34A2-high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic "vacuolar" structures preceding cell death. These data point to the XPR1-KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.

Catalog of qRT-PCR primers, CRISPR guide sequences, and antibodies used in this study

Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non–B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.Significance:The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.

Supplemental figures 1-6

Supplementary methods and legends for supplementary figures

Table S2: A Whole-genome shRNA screen for inhibitors of macropinocytosis

Macropinocytosis has emerged as an important pathway of protein acquisition in cancer cells, particularly in tumors with activated Ras such as pancreatic and colon cancer. Macropinocytosis is also the route of entry of Bacillus Calmette-Guerin (BCG) and other microbial therapies of cancer. Despite this important role in tumor biology and therapy, the full mechanisms by which cancer cells can activate macropinocytosis remain incompletely defined. Using BCG uptake to assay macropinocytosis, we executed a genome-wide shRNA screen for macropinocytosis activators and identified Wnt pathway activation as a strong driver of macropinocytosis. Wnt-driven macropinocytosis was downstream of the β-catenin–dependent canonical Wnt pathway, was PAK1 dependent, and supported albumin-dependent growth in Ras-WT cells. In cells with activated Ras-dependent macropinocytosis, pharmacologic or genetic inhibition of Wnt signaling suppressed macropinocytosis. In a mouse model of Wnt-driven colonic hyperplasia via APC silencing, Wnt-activated macropinocytosis stimulated uptake of luminal microbiota, a process reversed by topical pharmacologic inhibition of macropinocytosis. Our findings indicate that Wnt pathway activation drives macropinocytosis in cancer, and its inhibition could provide a therapeutic vulnerability in Wnt-driven intestinal polyposis and cancers with Wnt activation.Significance: The Wnt pathway drives macropinocytosis in cancer cells, thereby contributing to cancer growth in nutrient-deficient conditions and, in the context of colon cancer, to the early phases of oncogenesis. Cancer Res; 78(16); 4658–70. ©2018 AACR.

Figure S1: Activation of the canonical Wnt pathway stimulates macropinocytic BCG uptake; Figure S2: Macropinocytosis Assay Validation; Figure S3: rWnt3A increased macropinocytosis in a Ras-wild type cell line; Figure S4: Expression of E(beta)P increases macropinocytosis in a Ras-wild type cell line; Figure S5: Activation of the Wnt pathway stimulates macropinocytosis in Ras-wild type cell lines; Figure S6: Wnt Pathway Inhibitors inhibit macropinocytic uptake of BCG; Figure S7: Treatment with ICG001 decreases macropinocytosis in a Ras-mutant cell line; Figure S8: WNT pathway activation is an essential cofactor in Ras driven macropinocytosis; Figure S9: Expression of pQCXIP-AXIN1 decreases macropinocytosis in a Ras-wild type cell line; Figure S10: Wnt pathway activation stimulates macropinocytosis and bacterial uptake in the mouse colon

Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery:https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available athttps://phenaid.ardigen.com/jumpcpexplorer/.