Functional Interactions between Transcription Factors Involved in Myeloma Pathogenesis - Biological and Therapeutic Implications

The clinical success of combination therapies for multiple myeloma (MM) has led our group and others to perform pharmacological screens seeking to identify promising candidates for combination regimens. However, in our genome-scale CRISPR knockout (KO) studies, we have observed that the biological behavior of MM cells is driven by key transcription factors (TFs), including several that represent major preferential dependencies for MM cells compared to other blood cancers or to solid tumors. Most of these TFs are currently considered "undruggable", as they lack identifiable hydrophobic pockets that can be selectively engaged with small-molecule inhibitors and are not thalidomide derivative (IMID)-induced neo-substrates for CRBN. Therefore, pharmacological screens are inherently limited in their ability to probe the functional interactions of MM-related TFs. To bypass this limitation and interrogate in MM cells how key TFs may interact with each other or with other pathways, we performed combinatorial CRISPR-based gene editing studies, in which we examined how MM cell survival/proliferation is impacted by CRISPR KO of each gene individually vs. simultaneous KO of two genes. We applied a combinatorial CRISPR dual knockout (DKO) study in MM.1S or KMS-11 cells, which have been engineered to express 2 orthologous Cas9 nucleases (from S. pyogenes and S. aureus), to increase the efficiency and specificity of combinational KO. Synergy (or antagonism) of the KO of gene pairs was determined by comparing the average (+/-95% CIs) normalized log2-fold change (log2FC) of sgRNA readcounts for each gene pair vs. the sum of log2FC for the respective "singeletons" (pair of sgRNA for one gene and control sgRNA) vs. "double controls"; and focusing on genes pairs with concordant results for sgRNA pairs involving Sp.Cas9-Sa.Cas9 and vice versa. We performed a focused study on ~100 genes, including 45 TFs (including 20 TFs that represent MM-preferential dependencies); additional MM-preferential or broad-spectrum dependencies; tumor suppressors (e.g. TP53, PTEN); as well as TFs and other genes that are not major dependencies for MM cells in single KO studies. We also used this DKO system to study MM.1S cells implanted in "humanized" scaffold-based bone marrow-like model in NSG mice. Both in vitro and in vivo, synergistic combinations of gene KOs were heavily enriched for presence of at least one TF, most often one of the MM-preferentially essential TFs, including IRF4, POU2AF1, TCF3, and NF-kappaB pathway members. Many of these synergistic combinations had greater effect on MM cell survival/proliferation than the combined KO of IKZF1/IKZF3 (IKZF1/ZFP91 or IKZF3/ZFP91 did not result in synergy): these observations suggest that many MM TFs which are not CRBN neo-substrates are involved in synergistic DKO combinations with more potent anti-MM activity compared to all DKOs of the main TFs (IKZF1, IKZF3, ZFP91) degraded by IMIDs. This observation suggests that efforts to advance the therapeutic targeting of currently "undruggable" MM TFs (alone or paired) which do not interact with CRBN may have therapeutic implications that do not overlap with the effect of IMIDs. The results of our DKO studies exhibited a time-dependent effect: in early time-points, strong dependencies (e.g. IRF4) are highly recurrent partners in synergistic pairs, while later time-points uncover interactions between genes with limited, if any, individual roles as essential genes, including TFs (e.g. ZBP1) but also other pathways (the translation regulator ELL2, a gene proposed to be associated with increased risk for myelomagenesis). MM cell "dedifferentiation" (e.g. through suppression of plasma cell-specific TFs such as XBP1) has been proposed as potential mechanism for proteasome inhibitor resistance. However, in our study, loss of XBP1 or other MM-related TFs (alone or in combination with other genes) was not associated with significant resistance to MM.1S or KMS11 cell treated with clinically relevant pulses of bortezomib, suggesting that perturbation of key MM-related TFs can be therapeutically compatible with proteasome inhibition. More broadly, our DKO studies revealed critical interactions between TFs with central roles in MM biology and also others with previously underappreciated effects, pointing to combinatorial effects that may be exploited in the future through novel therapeutic strategies.