Non-Canonical Transcription Activator Activity of RNA-Binding Protein MBNL1 Uniquely Controls FLT3 expression in MLL-Rearranged Leukemias

Abstract

MLL-rearranged (MLL-r) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLL-r patients ultimately relapsed after an initial remission. So far, the molecular mechanism by which MLL-r leukemia maintains progression and prevents differentiation remains largely unclear. Observations in the clinic support aberrant expression and concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 that occur in leukemia, including the MLL-r subtype. To interrogate the novel MLL-r vulnerable genes regulating FLT3, we developed an algorithm so-called “context-dependent association analysis” (CDAA) to explore potential MLL-r dependent FLT3 regulators using the DepMap dataset. The RNA-binding protein MBNL1 was identified as the top candidate. MBNL1 has been recognized as an RNA-binding protein (RBP) involved in splicing, RNA export, and stability. However, our study suggested its non-canonical transcriptional activator function by uniquely controlling FLT3 expression in MLL-r leukemias. CRISPR/Cas9 disruption of the coding region of MBNL1 or CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines. However, the FLT3 gene splicing defect was not observed upon MBNL1 loss, suggesting MBNL1 could be affecting FLT3 expression through a transcriptional regulation mechanism. Genome-wide ChIP-seq assays performed in MLL-r SEM and MOLM13 cells identified only two reproducible MBNL1-bound peaks at a genome-wide scale, one of which was located in the distal enhancer of FLT3, ~170kb away from the FLT3 promoter. CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines without affecting enhancer-promoter looping. Using in vivo assay, SEM cells targeted with sgRNA against the MBNL1-bound site in the distal enhancer of FLT3 transplanted into NSG mice exhibited significant growth retardation. Furthermore, the impaired cell fitness crisis phenotype could be entirely rescued by overexpression of FLT3 cDNA. To identify the protein domains of MBNL1 required for its oncogenic function in MLL-r leukemia cells, an MBNL1 domain CRISPR screen was performed on the SEM cell line, stably expressing Cas9 and a pooled sgRNA library targeting the coding region of MBNL1. Essential domains observed from the screen included MBNL1's zinc finger domains. A biochemistry electrophoretic mobility shift assay (EMSA) was conducted using purified recombinant MBNL1 protein and a 207 bp DNA oligo synthesized based on the MBNL1 ChIP-seq peak at the FLT3 enhancer. The result clearly demonstrated that MBNL1 could directly bind to the enhancer DNA of FLT3 through its zinc fingers 1 and 2 domains, supporting its role as a novel transcriptional regulator of FLT3. Using state-of-art research tools, including genome editing, ChIP-seq, and EMSA assays, we have systematically interrogated an undocumented transcriptional regulation axis of MBNL1/FLT3 in MLL-r leukemias. This work will significantly advance our understanding of how transcription programs and novel factors reconstruct the regulatory network specifying MLL-r dependency, promoting the future development of alternative therapeutic targets for MLL-r leukemia in patients.