The RNA-binding protein Musashi2 is regulated by NOTCH1/KLF4 pathway and support tumor survival keeping CLL cells in proliferative niches during disease progression

Abstract

Post-transcriptional regulation is an essential mechanism for the cells to control gene regulation where the RNA-binding proteins (RBPs) orchestrate the fate of the RNA molecules. Because of its critical role, a dysregulation of the RBPs can lead to cancer. Specifically, in chronic lymphocytic leukemia (CLL) high levels of the RBP Musashi2 (MSI2) has been related with tumor cell survival and poor prognosis underlining a key role for MSI2 during disease evolution (Palacios et al., Leukemia, 2021). The increase in CLL-cells in patients stems from a small fraction of dividing CD5+ B cells. The growth of this proliferative fraction (PF) correlates directly with poor outcome, making the PF an important target for therapy. Interestingly, we observed higher MSI2 levels in CLL than healthy donors (HD) B cells, even higher in the PF. Downregulating MSI2 expression or blocking its function eliminates CLL-cells. These results allow us to propose that MSI2 itself, molecules that induce MSI2 expression or the downstream molecules that MSI2 regulates, could be responsible for the clinical course of CLL patients. Therefore, we studied the molecular mechanisms that induce MSI2 overexpression in CLL-B cells. Interestingly, in CLL-cells NOTCH1 suppresses the expression of the transcription factor Kruppel-like factor 4 (KLF4), a known negative regulator of MSI2 in adenocarcinoma. NOTCH1 is a transmembrane receptor that is cleaved upon engaging ligand with an intracellular domain migrating to the nucleolus and activating genes related to cell survival, as c-myc. As we observed for MSI2, higher NOTCH1 active forms were found in lymph nodes (LN) than peripheral blood (PB) of CLL-B cells. Because low levels of KLF4 were reported for CLL-B cells, we wondered whether MSI2 overexpression in CLL was due to alterations in the NOTCH1/KLF4 pathway. To answer this, we first determined at mRNA levels by quantitative PCR KLF4/MSI2 expression in CLL-cells and HD. Results showed that there was a negative correlation between KLF4/MSI2 (r=0.68; p=0.009). To further study the role of NOTCH1/KLF4/MSI2, poor outcome CLL patients (previously selected for activated NOTCH1), were treated with NOTCH1 inhibitor (gamma-secretase) and NOTCH1/KLF4/MSI2 levels were determined. NOTCH1 inhibition increased KLF4 levels (p= 0.01) and downregulates MSI2 and c-myc expression (p < 0.05). To confirm that the decrease expression of MSI2 it is because of the negative action of KLF4 on MSI2 promoter, chromatin immunoprecipitation using anti-KLF4 and PCR MSI2 promotor amplification was performed. Remarkably, treated CLL-cells exposed to NOTCH1 inhibitor immunoprecipitated a fragment corresponding to the MSI2-promoter, indicating that KLF4 effectively binds and negatively regulates MSI2 expression in CLL-cells. Results suggested that MSI2 overexpression in LN and in activating/dividing cells is due to the absence of KLF4 regulated by NOTCH1. Which are the molecules that MSI2 regulates in the active microenvironment it is now a relevant question to answer. Because MSI2 regulates different targets in a cell type-specific manner we studied MSI2´s role in activated/dividing CLL-cells. To do this, we analyzed the proteomes of MSI2 knock-down in activated (CpG-ODN+IL15) CLL-B cells (n=12) and compare with the control. Results showed that MSI2 knock-down significantly increased the levels of 12 proteins (p ≤ 0.01) consistent with MSI2 functioning as a mRNA inhibitor of a set of molecules involved in cell migration. To confirm these results, we selected and verified protein levels expression by flow cytometry of Fer (non-tyrosine-protein kinase receptor), VAV1 (guanine nucleotide exchange factors) and its active form (phosphorY174). Since Fer/VAV1 plays a role in the cytoskeleton regulation, we also knocked-down MSI2 in CLL-B cells from the same cohort and evaluated by microscopy cytoskeleton rearrangement of cells bound to fibronectin-coated slides, showing that MSi2 downregulation in activated cells present more elongated cells implying an active cytoskeleton. Because: 1-high MSI2 levels are associated to poor outcome, 2-there is more MSI2 in LN>PB, even more in dividing-cells and 3-in activated cells MSI2 inhibit cell migration, we hypothesize that MSI2 may be playing a role on keeping the cells in solid-tissues receiving surviving signals, favoring disease progression. Altogether, our results provide novel findings about the molecular mechanisms that regulates MSI2 expression, highlighting the role of NOTCH1/KLF4 pathway in this protein and the potential function of MSI2 inhibiting CLL-cell migration in an active microenvironment supporting CLL progression, which postulate new tools to modulate/prevent disease progression, potentially rendering CLL more manageable for patients.