P-362: ID2 acts as a novel tumor suppressor in MM by inhibition of TCF3 transcriptional activity

Introduction: Multiple myeloma (MM) is often sustained by chromosomal translocations that involve super-enhancers and transcription factors (TF), thus leading to a profound rewiring of transcriptional circuits. This generates MM specific non-oncogene addictions on TFs, including a strong dependency on the poorly characterized basic helix-loop-helix transcription factor (bHLH) TCF3 (E2A). Here we identify and describe Inhibitor of DNA binding 2 (ID2) as a potent tumor suppressor in MM thanks to its ability to prevent TCF3 activity.

Methods: We combined genome-wide unpublished CRISPRa and CRISPRi screen and RNA-seq of MM patients to identify deregulation of ID2-TCF3 axis in MM. We manipulated ID2 and TCF3 expression by inducible lentiviral platforms and performed both unbiased (RNA-seq, proteomics, ATAC-seq) and hypothesis driven (analysis of cell cycle, proliferation, EMSA assays) experiments to test its role in MM cell.

Results: We performed a genome-wide CRISPRa screen in three MM cell lines (AMO1, ANBL6 and JJN3), and identified the Inhibitor of DNA binding (ID) genes among the top hits inhibiting MM cell fitness. Importantly, the ID proteins (ID1, ID2, ID3, and ID4) can inhibit bHLH transcription factors, in particular E proteins like TCF3, by preventing their binding to DNA. We have therefore evaluated their expression in MM patients and found that ID2 is specifically and significantly downregulated in malignant plasma vs. normal plasma cells (p = 0.0059). Moreover, data from a genome-wide CRISPRi screen in 16 MM cell lines showed that only ID2 KO confers a proliferative advantage in a subset of MM cell lines (CSS score ≥ 0.5), mainly those with naturally higher ID2 expression.
Mechanistically, by co-immunoprecipitation and mass-spectrometry we found that ID2 preferentially binds to TCF3 in MM cells, and we used an EMSA assay to find that ID2 overexpression caused a loss of TCF3 DNA-binding activity. This led to transcriptomic changes that caused cell-cycle arrest in G1 and subsequent senescence, with a complete halt in proliferation in three MM cell lines. Importantly, this anti-myeloma effect was partially rescued by TCF3 overexpression. In summary, we hypothesize that the lower ID2 expression observed in MM cells confers a proliferative advantage by allowing higher TCF3 activity, supporting growth and proliferation.
Finally, we demonstrated that soluble factors secreted by bone marrow stromal cells induce further downregulation of ID2 expression at the transcriptional and protein levels, together with an increase in TCF3 binding motifs availability in open chromatin regions. Also in this context, overexpression of ID2 reduces TCF3 binding activity and prevents MM proliferation.

Conclusions: In conclusion, we identified a novel tumor suppressor role for ID2 in MM and propose a model where MM cells unleash the whole oncogenic/proliferative potential of TCF3 by deregulating its inhibitor with the support of the bone marrow milieu.