In Vivo Validation of Essential Genes for Myeloma Cells By CRISPR/Cas9 Studies in a Scaffold-Based System Engineered to Establish a "Humanized" Bone Marrow-like Microenvironment

The bone marrow (BM) microenvironment plays critical roles in the pathophysiology of multiple myeloma (MM), other hematologic malignancies and metastatic solid tumors, including the ability of nonmalignant cells of the BM microenvironment, such as bone marrow mesenchymal stromal cells (BMMSCs) to support tumor cell survival and proliferation. Our recent CRISPR/Cas9 genome-wide screens, using MM cell lines, have identified several candidate myeloma essential genes, which exhibit, when MM cells are co-cultured with BMMSCs, variable upregulation in the MM cell compartment, depending on the specific cell line. This raises the possibility that the functional dependence of MM cells on some of these genes may be altered in the context of in vivo interactions with BMMSCs, and potentially other elements of the BM milieu. While in vitro cocultures between tumor cells and BMMSCs provide insights into the mechanisms regulating tumor-microenvironment interactions, there is limited, if any, experience at the functional genomic level, on whether the molecular dependencies of MM cells, as identified from in vitro studies, are also relevant for the context of the in vivo microenvironment of the BM. In addition, several cytokines and growth factors present in the BM milieu have limited cross-reactivity between species, thus confounding the interpretation of results from in vivo xenografts of human tumor cells into the murine BM milieu. To address these limitations, we applied the CRISPR/Cas9 technology in the context of an in vivo model of subcutaneous bone marrow-like scaffold-based niches, which have been "humanized" with the use of human primary BMMSCs that have been induced to differentiate towards the osteogenic lineage, establishing in mice a "human BM"-like system. In this system, we inoculated MM.1S cells engineered to stably express the Cas9 nuclease and transduced with lentiviral particles for a pooled library of ~5000 sgRNAs covering a limited set of candidate essential genes; a larger group of control sgRNAs against genes with no significant role for essentiality in MM cell lines tested in our in vitro studies; and ~1,000 sgRNAs targeting sequences absent from the human genome. MM.1S-Cas9+ sgRNA+ cells were introduce into mice (n=10; 2 scaffolds per mouse) by direct intra-scaffold injections, and tumor cells were monitored serially until tumor growth was established and lead to eventual outgrowth of tumor cells from the scaffolds, at which point mice were sacrificed, and tumor cells were extracted from the scaffold for PCR and next-generation sequencing to quantify the proportion of different sgRNAs transduced into the human MM cells. We observed significant decrease in the read counts for sgRNAs for prominent MM selective-essential genes (including IRF4, CCND2, IKZF1, PRDM1), as well as additional candidate dependencies identified from our in vitro studies. In an additional cohort of mice harboring humanized scaffold-based BM niches and injected with our focused library of MM.1S-Cas9+ sgRNA+ cells, we observed that treatment with bortezomib, and subsequent monitoring until development of resistant tumor cells, was not associated with enrichment for sgRNAs for these plasma cell lineage-associated transcription factors, suggesting that their loss of function due to sgRNA-mediated gene editing does not lead to bortezomib resistance in this in vivo humanized system. Ongoing studies of our groups are extending these observations to additional larger scale screens, including efforts to validate candidate genes exhibiting more pronounced role as MM cell dependencies in the in vivo context compared to the in vitro setting.