Deregulated Bcl-2 Family Expression Drives Chronic Myeloid Leukemia Cancer Stem Cell Survival.

Introduction: A growing body of evidence suggests that a relatively rare subset of cells within a cancer subverts properties normally ascribed to stem cells in regenerating tissues, such as enhanced self-renewal and survival capacity. Recent studies suggest that these cancer stem cells (CSC) are resistant to treatments that target rapidly dividing cells. In blast crisis chronic myeloid leukemia (BC CML) and some forms of acute myelogenous leukemia (AML), research performed by ourselves and others indicates that CSC originate from the CD34+CD38+lineage- compartment of hematopoietic cells and can serially transplant blast crisis leukemia in immunodeficient mice. Despite abundant data indicating that Bcl-2 family proteins are involved in CML progression, the importance of these proteins in CSC survival remains to be elucidated. Clinical data have shown that CML stem cells become more resistant to therapies targeting BCR-ABL with progression to blast crisis. As BCR-ABL targeted therapy initiates apoptosis, these results suggest that CML CSC may become increasingly resistant to apoptosis with progression. Based on these findings and the results from our serial transplantation experiments, we hypothesized that CML CSC deregulate apoptosis pathways by differential expression of Bcl-2 family molecules and that these changes contribute to CSC ability to survive serial transplantation.

Methods: Quantitative FACS Aria analysis of Bcl-2 protein levels was compared in blast crisis (n=5) and chronic phase CML (n=3) patient samples. Mean fluorescence intensity (MFI) of Bcl-2 staining was compared between different hematopoietic populations within the patient samples. For gene expression analysis, cDNA was made from RNA isolated from sorted progenitor populations (CD34+CD38+Lin −). Isoform specific RT-PCR was used to determine expression levels of Bcl-2, Bcl-X, and Mcl-1 isoforms. Mcl-1 expression was confirmed using qPCR. In addition, preliminary experiments were performed (n=2) to determine if CSC engraftment could be reduced in vivo by targeted inhibition of Bcl-2 family molecules using Apogossypol, a clinically tested Bcl-2-family inhibitor. Briefly, immunocompromised neonatal mice were transplanted intrahepatically with luciferase GFP transduced granulocytic sarcomas from mice transplanted with BC CSC using our previously published methodology (Geron et al, Cancer Cell 2008). Transplanted mice were treated for 15 days with Apogossypol by oral gavage and engraftment was monitored by weekly bioluminescent imaging. Engraftment levels were determined by FACS analysis of human CD45+ expression in mouse livers on week 11 post-transplant.

Results: Comparing the MFI of Bcl-2 staining in the entire live mononuclear cell population, we detected no statistically significant difference in levels between the blast crisis and chronic phase samples. However, when we gated on separate cell populations, differences in the Bcl-2 MFI emerged. There was a statistically significant increase (P<0.03) in Bcl-2 MFI exclusively in the CD34+CD38+lineage- population of the blast crisis samples indicative of cell type and context specific deregulation of apoptosis in the CSC population. Further, we were interested in whether there were differences in Bcl-2 family expression at the transcriptional level. Notably, while we detected no difference in the levels of the isoforms of Bcl-2 and Bcl-X, splice isoform specific RT-PCR and qPCR revealed a decrease in the expression of the short isoform of Mcl-1, which encodes a pro-apoptotic protein, in serially transplanted BC CSC (CD34+CD38+). Together these results indicate that CML CSC may indeed deregulate the expression of several Bcl-2 family proteins. To test the therapeutic potential of inhibiting these deregulated apoptotic pathways in CML, we treated mice engrafted with CML CSC with Apogossypol, a broad-spectrum inhibitor of pro-survival Bcl-2 molecules. We saw a statistically significant decrease (P<0.05) in the number of CD45+ cells engrafted in the mouse liver after 3 weeks of Apogossypol treatment. Overall, our results suggest that the subversion of apoptosis plays an important role in allowing CML CSC to be serially transplanted and that apoptotic pathways may be a useful target for therapeutics aimed at inhibiting these cells.