The RNA-binding protein Musashi2 regulated by the NOTCH1/KLF4 pathway, modulates CLL cell migration and contributes to disease progression

Abstract

Post-transcriptional regulation is a crucial mechanism for cells to control gene expression, with RNA-binding proteins (RBPs) playing a key role in determining the fate of RNA molecules. Due to its significant function, any alteration in this process has been linked to various cancers. In chronic lymphocytic leukemia (CLL), we found that the RBP Musashi2(MSI2) promotes tumor cell survival and proliferation. CLL patients with elevated MSI2 levels experience shorter time to first treatment and reduced overall survival. This leads us to propose that MSI2, its regulators, or downstream targets could influence the progression of CLL in patients. Therefore, we studied the molecular mechanisms that induce MSI2 overexpression in CLL-B cells. In CLL-cells NOTCH1 suppresses the expression of the transcription factor Kruppel-likefactor 4(KLF4), a negative regulator of MSI2 in adenocarcinoma. Because low levels of KLF4were reported for CLL-B cells, we wondered whether MSI2 overexpression in CLL was due toalterations in NOTCH1/KLF4 pathway. To answer this, we determined mRNA expression levels of KLF4/MSI2 in CLL-cells(n=16) and HD(n=3). Results showed a negative correlation between KLF4/MSI2(p=0.0028). To further study the role of NOTCH1/KLF4/MSI2, poor outcome CLL patients’ cells were treated with NOTCH1 inhibitor(secretase) in-vitro andNOTCH1/KLF4/MSI2 levels were determined. NOTCH1 inhibition increased KLF4levels(p=0.01) and downregulates MSI2 expression(p<0.05). To confirm that KLF4 negatively regulates MSI2 by binding to MSI2-promoter, chromatin immunoprecipitation using anti-KLF4, and PCR MSI2-promoter amplification was performed in CLL-cells(n=6). Blocking NOTCH1immunoprecipitated a fragment of the MSI2-promoter, showing that KLF4 binds and negativelyregulates MSI2 expression in CLL-cells. Supporting this data, we observed higher levels of MSI2 in CLL-cells with NOTCH1-mutated (constitutive active NOTCH1) than NOTCH1-non-mutated, suggesting that NOTCH1 regulates MSI2 in CLL patients. Moreover, NOTCH1 activates survival genes, including oncoprotein c-MYC. Thus, weinvestigated the effects on c-MYC expression in CLL samples(n=13) in-vitro treated withNOTCH1 and MSI2 (Ro082750) inhibitors. The results show that combined inhibitors significantly reduce c-MYC expression(P≤0.001), suggesting a strategy to reduce tumor viability. Because MSI2 is highly expressed in dividing cells and regulates different targets in a cell type-specific manner, we studied MSI2´s role in activated/dividing CLL-cells. We analyzed theproteome in activated (CpG-ODN+IL15) CLL-B cells(n=12) treated with siRNA-MSI2(MSI2 low) and compare them to the control siRNA-CTR (MSI2 high). Results showed that the reduction ofMSI2 protein levels significantly increased the expression of 12 proteins(p≤0.01) involved in cell-migration and cytoskeleton rearrangement. Cytoskeleton rearrangements of cells bound to fibronectin-coated slides with high and low MSI2 were analyzed by microscopy. Results showed that high MSI2 levels inhibit cell-migration. Given that: 1-high MSI2 levels are associated with poor outcomes, 2-MSI2 is higher in lymph nodes than in peripheral blood, particularly in dividing cells, and 3-MSI2 inhibits cell-migration in activated cells, we hypothesize that MSI2 may help retain cells in solid tissues where they receive survival signals,promoting disease progression. Further studies will be done to confirm the hypothesis. These results reveal novel insights into the molecular mechanisms regulating MSI2 expression, emphasizing the role of NOTCH1/KLF4 pathway. Additionally, we highlight the potential function of MSI2 inhibiting CLL-cells migration within an active microenvironment support CLL progression.