Identifying Factors in Multiple Myeloma Controlling Response to B-Cell Maturation Antigen (BCMA)-Targeted Immunotherapy Using CRISPR-Based Functional Genomics

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.