CLL develops from a small fraction of dividing monoclonal CD5+ B cells. The size and rate of growth of this proliferative fraction (PF) correlates inversely with time-to-first-treatment and directly with poor outcome prognostic markers. Furthermore, since the dividing cells upregulate DNA mutators such as AID and APOBEC family members, the PF has a greater propensity for acquiring new DNA abnormalities that can lead to more lethal disease. Hence, cells of the PF are important targets for therapy for patients with worst outcome category.
The PF (CXCR4DimCD5Bright) differs by more than 1000 genes from the resting fraction (RF, CXCR4Bright CD5Dim); these genes relate to replication, migration, and regulation of gene expression. Some of these genes are also preferentially expressed in the PF of U-CLL cases. One such gene is Musashi 2 (MSI2).
MSI2 regulates gene expression by binding consensus sequences of mRNA and blocking protein translation. High MSI2 expression is involved in proliferation of normal and malignant stem cells, tumorigenesis, and poor outcome. In CLL, high MSI2 mRNA expression has been identified in patients with worse prognosis. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we report studies of the biological role of MSI2 in B-CLL cells and its possible association with B-cell proliferation and CLL disease progression.
First, we evaluated MSI2 protein levels by flow cytometry in CD19+CD5- and CD19+CD5+ cells from healthy donors (HDs; n=25) and in CD19+CD5+ from CLL patients (n=55). Higher MSI2 expression was observed in CLL than HD B cells, whereas no differences were found in CD19+CD5+ and CD19+CD5- cells from HDs. Also, MSI2 protein levels were higher in U-CLL than M-CLL, and M-CLL B cells express more MSI2 than HDs. Finally, MSI2 protein levels correlated with CD38, a CLL poor prognosis marker, suggesting MSI2 associates with poor prognosis in CLL.
Within the leukemic clone, we observed 25% more MSI2 in the PF than the Int (defined as CXCR4intCD5int) and 15% more in the Int than the RF (PF>Int >RF). The PF contains 40% more MSI2 than the RF, suggesting the highest amounts of MSI2 protein are in dividing and recently-divided cells.
Since CLL B cell proliferation occurs in the microenvironment of lymphoid organs, presumably delivered by external signals, we tested whether such signals could stimulate MSI2 expression. Results indicate that CD40L+IL4 and Toll-like 9 stimulation plus IL15 (TLR9+IL5) increase MSI2 synthesis in vitro 1.4 and 1.8 fold, respectively. The increases are associated with the appearance of phospho ERK and AKT. Also, inhibition of AKT signaling by a PI3K inhibitor decreases MSI2 levels, suggesting AKT is involved in MSI2 synthesis. In this regard, signals from the microenvironment inducing cell growth and proliferation promote MSI2 synthesis in B cells from CLL patients.
In addition, cells entering the cell cycle (Ki-67+ cells, those incorporating the thymidine analogue EdU, and cells in S, G2 and M cell cycle phases) express higher MSI2 levels than quiescent cells. Furthermore, dividing cells contain higher MSI2 levels than non-dividing cells as determined by CFSE dilution. These results suggest that cells entering the cell cycle or recently dividing have greater MSI2 expression.
Since high MSI2 levels associate with cell proliferation and its inhibition is said to promote apoptosis, we studied the effect of MSI2 downregulation in the CLL MEC1 cell line to determine if MSI2 is a potential therapeutic target for CLL. Our findings show that siRNAs decrease MSI2 mRNA (80%) and protein (40%) levels compared to negative controls. Downregulation of MSI2 in MEC1 led to cleaved caspase 3, TRAIL R1 and R2, FADD, TNFR1, P21, P27, phosho-p53, and decreased levels of inhibitors of apoptosis such as cIAP2 and survivin. Hence these data suggest downregulation of MSI2 in CLL cells could induce apoptosis.
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 cell line. Therefore, we propose that MSI2 is a valuable target for therapeutic intervention. Inhibiting its function and its role in cell proliferation will likely abort clonal evolution and disease progression, and make CLL an even more chronic and manageable condition.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.