Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here, we present the first genomewide CRISPR interference and CRISPR activation screens in human neurons. We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons following perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.

Cancer cells commonly develop resistance to immunotherapy by loss of antigen expression. Combinatorial treatments that increase levels of the target antigen on the surface of cancer cells have the potential to restore efficacy to immunotherapy. Here, we use our CRISPR interference– and CRISPR activation–based functional genomics platform to systematically identify pathways controlling cell surface expression of the multiple myeloma immunotherapy antigen B-cell maturation antigen (BCMA). We discovered that pharmacologic inhibition of HDAC7 and the Sec61 complex increased cell surface BCMA, including in primary patient cells. Pharmacologic Sec61 inhibition enhanced the antimyeloma efficacy of a BCMA-targeted antibody-drug conjugate. A CRISPR interference chimeric antigen receptor T cells (CAR-T cells) coculture screen enabled us to identify both antigen-dependent and antigen-independent mechanisms controlling response of myeloma cells to BCMA-targeted CAR-T cells. Thus, our study shows the potential of CRISPR screens to uncover mechanisms controlling response of cancer cells to immunotherapy and to suggest potential combination therapies.

Cancer cells commonly develop resistance to immunotherapy by loss of antigen expression. Combinatorial treatments that increase levels of the target antigen on the surface of cancer cells have the potential to restore efficacy to immunotherapy. Here, we use our CRISPR interference and CRISPR activation-based functional genomics platform to systematically identify pathways controlling cell-surface expression of the multiple myeloma immunotherapy antigen - B cell maturation antigen, BCMA. We discovered that pharmacological inhibition of HDAC7 and the Sec61 complex increased cell-surface BCMA, including in primary patient cells. Importantly, pharmacological Sec61 inhibition enhanced the anti-myeloma efficacy of a BCMA-targeted antibody-drug conjugate. A CRISPR interference CAR-T coculture screen enabled us to identify both antigen-dependent and -independent mechanisms controlling response of myeloma cells to BCMA-targeted CAR-T cells. Thus, our study demonstrates the potential of CRISPR screens to uncover mechanisms controlling response of cancer cells to immunotherapy and to suggest potential combination therapies.Key PointsUsing CRISPR screens, we systematically identify mechanisms increasing expression of the immunotherapy target BCMA and ADC efficacy.We also identify antigen-independent mechanisms regulating response of cancer cells to BCMA-CAR-T cells.

SUMMARYSingle-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here, we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons.We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons following perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.

Background and rationale. Leukemia-initiating cells (LIC) were extensively studied in AML (Bonnet 1997) and CML (Graham 2002), while LIC populations in B-ALL remained elusive. In the absence of a functional definition, targeted approaches for LIC-eradication are not feasible in B-ALL. Some studies suggested immunophenotypes for B-ALL LIC-populations (Cox 2004, Castor 2005, Wang 2007). However, other groups demonstrated that tumor-initiation in B-ALL is not limited to rare populations or developmental hierarchy (Kelly 2007; Le Viseur 2008; Rehe 2013; Aoki 2015). Concept: We hypothesize that self-renewal in the B-cell lineage is induced by positive selection and antigen-receptor (BCR) signaling, i.e. encounter of cognate antigen. Self-renewal at this stage leads to clonal expansion and survival. Unlike stemness in AML and CML, which is determined by a developmental hierarchy, we propose that self-renewal in the B-cell lineage is transient and driven by environmental antigen and the ability of BCRs to bind with high affinity. In B-cell malignancies, positive B-cell selection events, resulting in Lgr5 surface expression, are mimicked by transforming oncogenes (e.g. BCR-ABL1, NRASG12D, MYD88L265P). Results: Combining flow cytometry and genetic approaches, we identified surface expression of the leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) as new biomarker of positively selected pre-B cells in the bone marrow. Conversely, conditional ablation of Lgr5 during earliest stages of B-cell development resulted in near-complete failure to develop a mature B cell pool (reduced by 2-3 log orders). Lgr5 is a Wnt target gene and an established cancer stem cell marker for epithelial cancers (e.g. colorectal cancer and mammary tumors), however, a role for Lgr5 in normal and malignant hematopoiesis is not known. Importantly, Lgr5 represents a previously unrecognized predictor of poor clinical outcome in children and adults with pre-B ALL, including worse overall survival and higher risks of drug-resistance and relapse. Limiting dilution transplant experiments showed that Lgr5-overexpression increased LIC-frequencies in NSG recipient mice. Inducible activation of Cre in Lgr5fl/fl mouse models for BCR-ABL1- or NRASG12D-driven B-ALL resulted in cell cycle arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG transplant recipients. Deletion of Lgr5 in pre-B ALL cells caused massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes. Phosphoproteomic analyses revealed increased levels of β-catenin S675-phosphorylation, which increases β-catenin transcriptional activity (Taurin 2006; Hino 2015). Inducible activation of a gain-of-function mutant of β-catenin revealed that pre-B ALL cells are extremely sensitive to β-catenin activation. Thus, Lgr5 enables positive selection and self-renewal of B-ALL cells by curbing β-catenin activity. Therapeutic implication: To assess Lgr5 surface expression on B-ALL as a target for antibody-drug conjugate (ADC), we treated refractory B-ALL PDX with the Lgr5-MMAE ADC. Single-agent treatment with Lgr5-MMAE significantly reduced B-ALL leukemia burden. Treatment with dexamethasone not only enforced persistent surface expression of Lgr5, but also potentiated efficacy of by Lgr5-MMAE. Conclusion: Unlike self-renewal in myeloid leukemia that is determined by a developmental hierarchy, our results here show that self-renewal in the B-cell lineage is transient and driven by the ability of BCRs to bind antigen with high affinity. Lgr5 is a biomarker of this selection event, critical for the initiation of B-ALL and other B-cell malignancies in transplant recipients. Given that positive B-cell selection events, resulting in Lgr5 surface expression are mimicked by transforming oncogenes, Lgr5 also represents a promising target for ADC therapy for instance Lgr5-MMAE (Genentech). Disclosures No relevant conflicts of interest to declare.