Myeloma Cell Addiction to the Transcription Factor TCF11

BACKGROUND: TCF11/NRF1 is a transcription factor transiently and post-translationally activated in response to metabolic challenges and endoplasmic reticulum (ER) stress. However, the genome-wide compendium of genes regulated by TCF11 has not been defined and its role in tumour biology has not been explored. Since malignant plasma cells (PC) in multiple myeloma are under a constant state of ER stress due to immunoglobulin secretion, we hypothesised that the expression and functional role of TCF11 would be altered in myeloma compared to normal cells.

METHODS: We employed qPCR for mRNA quantitation, western blotting for protein expression analysis, constitutive and dox-inducible lentiviral shRNA-based knock-down of TCF11, DAPI staining and flow-cytometry for cell cycle analysis, RNA-seq for transcriptome analysis after TCF11knock-down, ChIP-seq against Flagx3-TCF11 in H929 myeloma cells, and a subcutaneous myeloma xenograft model in NSG mice following inducible TCF11 knock-down. Computational analysis of RNA-seq and ChIP-seq was performed using standard work-flows while cistrome-transcriptome integration was performed using the Binding and Expression Target Analysis package. Pathway enrichment analysis was performed using EnrichR and GSEA.

RESULTS: We found that at the protein level TCF11 is undetectable in erythroblasts, monocytes, B and T cells but expressed at low level in normal PC and at higher levels in myeloma cell lines (n=5) as well as primary myeloma PC (n=10). Therefore, myeloma cells and to a lesser extent normal PC constitutively express TCF11. Depletion of TCF11 using 2 validated shRNAs (either constitutive or inducible) was toxic to 5/5 and 2/2 myeloma cell lines tested in vitro and in vivo respectively, revealing an absolute requirement for TCF11 expression for myeloma cell survival.

Next we performed transcriptome analysis upon TCF11 depletion in 2 myeloma cell lines (H929 and U266) with divergent primary and secondary oncogenic events (CCND1 vs MMSET and MYC vs MYCL respectively). As well as its previously described role in the regulation of several metabolic processes, pathway enrichment analysis revealed in both cell lines a hitherto unknown function of TCF11 in cell cycle regulation. Indeed, inducible TCF11 depletion in myeloma cells resulted in a significant cell cycle arrest in G0/1 (n=5; p<0.01).

To investigate whether TCF11 directly regulates genes involved in cell cycle and proliferation, we generated and analysed the first genome-wide maps of TCF11 binding in H929 cells (n=2). Significantly enriched peaks (n=3829) bind in roughly equal proportions to promoters, introns and enhancers and are highly enriched for the previously described TCF11-preferred ARE motif.

Integrated transcriptome-cistrome maps revealed that 608 and 390 genes are directly activated or repressed by TCF11. Again, pathway enrichment analysis of TCF11 direct targets, as well as positive regulation of various metabolic processes (glycolysis, adipogenesis, unfolded protein response, oxidative phosphorylation), it also confirmed a direct role of TCF11 in the regulation of cell cycle and of the transcriptional programmes of MYC and E2F1/2. Low throughput ChIP-qPCR analysis validated TCF11 binding to the promoters of MYC and reciprocally, binding of MYC to the promoter of TCF11, thus establishing TCF11 as a novel co-operative factor of oncogenic MYC. Further, we confirmed that TCF11 binds to its own promoter and since TCF11 mRNA levels increase following proteasome inhibitor treatment, we conclude that TCF11 activates its own transcription.

Finally, we detected constitutively high protein levels of TCF11 in several other haematological cancer cell lines, i.e., (ALL: BV173; AML: HL60 and KG1 and Burkitt lymphoma: Ramos) and solid tumour cell lines (breast: MCF7 and MCF7TR; prostate: DU145 and PC3; ovarian: SKOV3 and OVC433). In all cases, depletion of TCF11 was toxic to the cells.

CONCLUSIONS: Myeloma cells are addicted to the constitutive expression of TCF11. As well as essential metabolic processes, TCF11 regulates cell cycle progression. At the transcriptional level, TCF11 regulated expression of MYC, E2F1/2 and of their respective transcriptional programmes in a direct co-operative fashion. These data establish a multifaceted role of TCF11 in tumour biology and link its function to cell cycle regulation in co-operation with oncogenic MYC in myeloma cells.