Transcriptional Deregulation Mediated By ID2-TCF3 Axis Supports MM Cell Growth and Proliferation in the Context of the Bone Marrow Milieu
Authors: Yao Yao , Perini Tommaso , Samur Mehmet K. , Szalat Raphael , Binder Moritz , Anderson Kenneth C. , Fulciniti Mariateresa , Munshi Nikhil C. 
Published: November 2021

Abstract
Multiple myeloma (MM) is a complex and heterogenous disease which is dependent on the surrounding microenvironment for growth and survival. In MM, dysregulation of transcriptional control is a major driver of tumor transformation and progression. To evaluate transcriptional programs activated in MM cells in the context of the bone marrow milieu, we have performed extensive transcriptomic analysis by RNA-seq and ATAC-seq using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines. We observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 cells significantly downregulated expression of Inhibitor of DNA Binding 2 (ID2), while footprint analysis of the open chromatin regions in MM cells upon interaction with BMSC revealed enrichment for binding motifs of the TCF family of transcription factors (E proteins). Inhibitors of DNA binding (ID) proteins control crucial transcriptional programs in B cell maturation via their heterodimerization with E proteins which are members of the basic helix-loop-helix (bHLH) class I family of transcription factors, repressing their DNA binding and therefore transcriptional activity.
We found that ID2 expression is significantly lower in primary CD138+ MM cells from patients compared to normal plasma cells (NPCs). Moreover, we have previously implicated the B-cell factor TCF3 as a novel MM dependency. Using MM cell lines and primary samples, we observed elevated enhancer activity at TCF3 locus in primary malignant plasma cells compared to NPCs, which resulted in significant upregulation of TCF3 expression in MM patients. We also showed that TCF3 is regulated by a large proximal enhancer that is bound by MYC and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. Genetic perturbation of TCF3 confirmed its critical role on MM cell growth and viability especially in IgH MYC translocated MM cell lines.
We here further explored the role of ID2-TCF3 axis and the hypothesis that lower expression of ID2 drives higher TCF3 activity in MM cells, which is further enhanced in presence of the bone marrow microenvironment.

Genetic modulation of ID2 significantly affected MM cell viability, with MM cells ectopically expressing ID2 displaying a cell growth arrest even in the presence of the supportive BM milieu. To define the mechanism of the observed oncosuppressive role of ID2 in MM, which is in line with preliminary observations in other hematological malignancy but in contrast with the pro-tumoral role described in solid tumors, we first performed immunoprecipitation of ID2 followed by mass spectrometry in 3 MM cell lines, and identified a very consistent and specific interaction with E proteins TCF3 and TCF12. Next, to explore the transcriptional programs dependent on ID2 we performed RNA-seq of 2 MM cell lines after ID2 overexpression. In line with our in vitro data, gene ontology and gene set enrichment analysis showed a significant downregulation of genes involved in E2F pathway, cell cycle progression and regulation of gene transcription. Interestingly, among the known TCF3 targets in B cells, only XBP-1 was significantly downregulated in MM cells after ID2 overexpression, suggesting the existence of a cell-specific TCF3 dependent transcriptional program in MM. Indeed, ATAC-seq experiments revealed ID2 overexpression led to a significant decrease of TCF3 binding motifs in open chromatin regions, confirming the relevance of ID2 in regulating TCF3 transcriptional activity in MM.
In conclusion, while both E and ID proteins have been implicated in malignant transformation, their role in supporting MM transcriptional deregulation and tumor growth in the context of the microenvironment is being defined. Here, we have identified ID2 as a major regulator of the TCF3 dependent transcriptional program in MM, whose downregulation is essential to maintain MM proliferation and to mediate the benefits induced by MM-stroma interaction.

Disclosures
Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.