Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. >10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches.