Uncovering the HOXA9 Translational Regulatory Complex That Promotes AML Leukemic Stem Cells

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.4106-4106
• Main Authors: Xie, Xueqin, Chavez, Florisela Herrejon, Baali, Ilyes, Chu, Eren L., Evans, Chiara, Yang, Xuejing, Wei, Wei, Luo, Hanzhi, Park, Sun Mi, Chang, Kathryn, Pierson, Aspen, Han, Grace, Batchelor, Emily, Morris, Quaid D., Vu, Ly, Nguyen, Diu T.T., Kharas, Michael G.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-211477

HOXA9 is a master transcription factor of hematopoiesis and essential for maintaining self-renewal of leukemia stem cells (LSCs). Although mechanisms associated with its transcriptional regulation are extensively studied, how Hoxa9 mRNA translation is controlled remain poorly understood. We have previously identified the RNA binding protein (RBP) MUSASHI-2 (MSI2) as a central regulator of myeloid LSCs by controlling translation of crucial transcription factors including Hoxa9 (Park et al. JCI, 2015; Nguyen et al. Nature Comm, 2020). However, the mechanisms by which HOXA9 translation is modulated and the role of MSI2 remain unknown. To characterize the HOXA9 3'UTR mRNA regulon, we used it as a bait to perform an RNA-pull down followed by mass spectrometry (MS) with control or MSI2 depleted cells. To determine the direct MSI2's protein interactors on HOXA9, we also performed MSI2 co-immunoprecipitation (co-IP) MS in control or MSI2 deficient AML cells. We identified 10 RBPs that directly bind to MSI2 and are dependent on MSI2 for their binding to the HOXA9-3'UTR mRNA. Among these, we focused on SYNCRIP, a known MSI2 interactor (Vu et al. Nat Gen, 2017), HNRNPC and MOV10, of which binding ability on HOXA9 3'UTR was most reduced and enhanced, respectively, upon MSI2 loss. Reciprocal co-IP confirmed the interactions between these RBPs with MSI2. We then reasoned that if these RBPs are in the same regulatory complex with MSI2, they might phenocopy MSI2 loss of function in LSCs. To understand how each selected factor impacts LSC function and gene control, we first assessed MSI2's global regulation by performing integrative omic studies including MSI2-HyperTRIBE, RNA-seq and proteomics in MLL-AF9 murine LSCs that have acutely deleted (68hrs) of Msi2. We found that MSI2 binding promotes translation of 264 genes while suppresses 8 genes; and only 10 genes affected at mRNA level. These data found that MSI2 mediates translation of the self-renewal program in LSCs. We then found that SYNCRIP is required for both leukemia initiation and maintenance in the MLL-AF9 driven AML model with the Syncrip conditional knockout mice. Serial transplantation assays of Syncrip deleted cells show a continued delay in leukemogenesis in vivo, suggesting SYNCRIP is required for functional LSC activity. To identify SYNCRIP's targets, we performed SYNCRIP-HyperTRIBE and iCLIP, and found that majority (~64%) of SYNCRIP targets overlapped with MSI2 targets and their binding sites are proximi