Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical, and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.

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.

Introduction: Multiple myeloma (MM) is the second most common blood cancer in the United States. Recent breakthroughs in immunotherapy have the potential to transform MM treatment. An immunotherapy target that shows considerable promise in myeloma is the B-cell maturation antigen (BCMA). BCMA is specifically expressed in myeloma cells and plasma cells, making it an ideal target in myeloma. Results from two early-phase clinical trials using anti-BCMA therapy, showed remarkable response in most patients. Although immunotherapy has been promising, recent findings suggest that patients can develop resistance to such therapies by lowering the levels of the target. Here we employ our innovative CRISPR-interference/activation (CRISPRi/a)-based functional genomics platform to identify mechanisms that regulate BCMA expression, which would enable us to design strategies to improve the efficacy of available BCMA immunotherapy agents. Methods: The CRISPRi/a platform utilizes a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor domain to silence genes (CRISPRi) or to activator domains to activate transcription (CRISPRa). The CRISPR machinery is targeted to specific genes using single guide-RNAs (sgRNA). We have engineered a panel of myeloma cell lines to express components of the CRISPRi system. Here we transduced CRISPRi-AMO1 cell line with sgRNAs targeting the human genome. The sgRNA expressing cells were stained with a fluorescent-tagged BCMA antibody and FACS sorted into cells expressing high and low levels of BCMA. The cells were processed for next generation sequencing to determine the frequency of sgRNA in each of these populations. To develop BCMA chimeric antigen receptor-T (CAR-T) cells, CD8+ T cells were transduced with BCMA CAR construct specifically recognizing BCMA. To examine the anti-myeloma activity of the BCMA CAR-T cells, CAR-T cells were co-cultured with MM cell lines at a ratio of 1:2 (Effector:Target) for 24hrs. The cells were analyzed by flow cytometry for expression of CD69, an activation marker on T cells and for apoptosis of cancer cells using propidium iodide. Results: Our FACS-based genome-wide CRISPRi screen identified several genes and pathways regulating BCMA cell surface expression. In addition to previously reported gamma-secretase complex and transcription factor POU2AF1 we identified genes involved in peroxisome biogenesis, subunits of the proteasome, transcription factors and a few druggable targets regulating cell surface expression of BCMA. We are currently validating the novel genes identified from our primary genome-wide screen in a panel of MM cell lines and developing MM cell lines expressing CRISPRa machinery to perform gain-of-function screens that will complement our CRISPRi screen. Furthermore, we have developed active CAR-T cells targeting BCMA and demonstrated its efficacy in MM cell lines expressing different levels of BCMA. We are currently testing the novel genes identified from our CRISPRi screen in combination with BCMA CAR-T cells to identify genes that alter sensitivity to BCMA immunotherapy. Conclusions: Our studies have identified several novel genes and pathways regulating BCMA expression including some druggable targets. Through these studies, we expect to uncover mechanisms regulating expression of BCMA and its impact on sensitivity to BCMA immunotherapy, and pinpoint potential combination therapy targets that pre-empt resistance to BCMA immunotherapy. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.