The growth of CLL cells stems from a small fraction of dividing CD5+B cells. The size and rate of growth of this proliferative fraction (PF) correlates directly with poor outcome prognostic markers and inversely with time-to-first-treatment. Furthermore, since dividing cells upregulate DNA mutators (AID and APOBEC), the PF can acquire new DNA abnormalities that can lead to more lethal disease. Hence, cells of the PF are important targets for therapy.
By gene expression profile analysis, we found that Musashi 2 (MSI2) is highly expressed in the PF (CXCR4DimCD5Bright) compared with the resting fraction (RF) that expresses the reciprocal phenotype (CXCR4BrightCD5Dim). In normal cells, MSI2 binds to mRNA and blocks or enhances protein translation. MSI2 levels are higher in proliferating normal and malignant stem cells and during tumorigenesis. In CLL, high MSI2 mRNA levels associate with poor outcome. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we studied the biological role of MSI2 in CLL B cells.
We found that CLL B cells express higher levels of MSI2 protein than those of healthy donors (HD). MSI2 levels were higher in U-CLL than M-CLL, and M-CLL B cells expressed more than HD cells, consistent with MSI2 associating with poor prognosis. Within a CLL clone, MSI2 was higher in PF than RF. Also, microenvironment signals that induce B cell proliferation also increased MSI2 protein levels. Consistent with these observations in patients, in vitro experiments showed that dividing cells contain more MSI2 protein than undivided cells.
Next, we investigated the importance of MSI2 on CLL survival. In primary CLL cells, we knocked down MSI2 levels using MSI2 siRNA and co-cultured CLL cells with stromal cells (HS5) or stimulated them with CpG ODN + IL15, with or without HS5 cells. In the three conditions tested, knock-down of MSI2 resulted in a significant decrease in tumor-cell survival compared with the control (P = 0.0342, 0.0079, and 0.0274, respectively). In addition, down regulation of MSI2 led to an upregulation of cleaved caspase3 (1.3 fold change;P= 0.029), p27kip1 (1.10 fold change; P= 0.0026) and phospho p53 (1.16 fold change; P= 0.017) compared with siRNA control (siCTR). Collectively, the results highlight the importance of MSI2 in primary CLL-cell survival and suggest that these are mediated by caspase 3, p27kip1 and p53 signaling pathways.
Reported comparisons of the RNAs that MSI2 binds in different cell types suggest that MSI2 regulates both common and unique mRNA targets in a cell type-specific manner. Hence to study the role of MSI2 in CLL, we analyzed the proteomes of MSI2 knockdown B cells from 12 CLL patients and controls. This showed that MSI2 knock down significantly increased the levels of 12 proteins (P ≤ 0.01 and fold change ≤ -1.2 ≥ 1.2). Since Ingenuity Pathway Analysis of these proteins suggested an increase cellular movement, we confirmed protein levels by flow cytometry of proteins involved in migration: Fer (non-tyrosine-protein kinase receptor), VAV1 (guanine nucleotide exchange factors) and its active form (phosphorY174 VAV, pVAV). Since Fer and VAV1 play a role in actin cytoskeleton regulation, we next knocked down MSI2 in B cells from the same 12 CLL patients and evaluated by microscopy cytoskeleton rearrangement of cells bound to fibronectin-coated slides. We used an established method to classify round cells as having a non-modified cytoskeleton and elongated cells as having undergone cytoskeleton rearrangement. Also, we counted the number of cells with an accumulation of polarized actin and others with multiple protrusions. MSI2 downregulation increased the number of elongated, polarized and cells with multi protrusions.
Thus, MSI2 levels are higher in B cells from poor outcome patients and also in the dividing/divided cells of the PF before and after stimulation. Also, MSI's downregulation induces apoptosis of CLL cells and increases adhesion and migration. Therefore, we propose that MSI2 is a valuable target for therapeutic intervention since inhibiting its functions will likely abort clonal evolution and disease progression, making CLL an even more chronic and manageable condition.
Barrientos:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.
Asterisk with author names denotes non-ASH members.