Abstract

Abl kinase inhibitors are the preferred treatment for Chronic Myelogenous Leukemia (CML) and they are highly efficient at inducing remission but continued treatment is required as not all BCR/ABL positive cells are eradicated. Survival of tumor cells may be mediated by deregulation of apoptosis pathways as cells transformed by BCR/ABL are highly resistant to a variety of apoptotic stimuli. Resistance to drug-induced apoptosis in CD34+ CML patient cells and BCR/ABL+ cell lines is mediated by downstream signaling pathways activated by BCR/ABL. The signal transducer and activator of transcription 5 pathway which regulates the expression of the anti-apoptotic Bcl-x gene is constitutively activated in BCR/ABL-positive cells. Several lines of evidence led to the hypothesis that Bcl-x is a critical mediator in evasion of apoptosis required to sustain leukemia. In order to examine the in vivo role of Bcl-x in CML we generated a tetracycline-inducible transgenic mouse model in which BCR/ABL is expressed in hematopoietic stem cells and myeloid progenitor cells resulting in a CML-like phenotype, while at the same time the Bcl-x gene is excised. Efficient recombination and loss of Bcl-x expression in this model was verified by Three Primer PCR analysis in whole bone marrow and spleen. DNA from Gr-1+/Mac-1+ cells showed 80% recombination of the Bcl-x locus and Real-Time PCR confirmed reduction of Bcl-x expression in LSK cells (Lin,Sca-1+, c-Kit+) isolated from bone marrow. Induction of transgenic expression demonstrated increased mortality within the first 3 weeks in Bclx−/−;CML mice with 50% (8/16) of the animals succumbing to disease, whereas only 28.5% (4/14) of CML mice expired. Analysis of peripheral blood revealed increased neutrophila in the remaining Bclx−/−;CML mice compared to CML mice. Loss of Bcl-x exacerbated infiltration of myeloid cells into non-hematopoietic tissue leading to severe damage of kidneys in 100% (8/8) of the Bclx−/−;CML mice that lived for 3 months compared to 25% of CML mice. Furthermore, 37% of Bclx−/−;CML mice that survived longer than 3 months progressed to an accelerated disease characterized by up to 30-fold increase of LSK cells in the bone marrow compared to wild type mice. Massive infiltration of the liver with c-Kit+/Mac-1+ cells, characteristic of immature myeloid cells, accompanied this phenotype. Real-time PCR data revealed that Bcl-x expression decreases as cell differentiate from common myeloid progenitors (CMP) to granulocyte/monocyte progenitor cells (GMP). Thus, loss of Bcl-x may bias differentiation to the myeloid lineage leading to the higher neutrophil counts observed in Bclx−/−;CML mice. One characteristic of the inducible transgenic CML model is progression to B lymphoblastic leukemia in 30% of mice after 8 weeks; deletion of Bcl-x resulted in loss of this phenotype. The Bcl-x gene plays an important role in development of B cell lineage and it is possible that its deletion reduced the pool of cells available for transformation by BCR/ABL. In addition, the promoter/enhancer construct used to direct expression of transgenes in this animal model displays weak activity in immature B cells. An alternative explanation assumes an essential role of Bcl-x in the maintenance of a myeloid/B-cell bipotent progenitor cell. Taken together, our results suggest that Bcl-x is not required for the generation and maintenance of CML-like disease, and may in fact exacerbate the myeloproliferation, due to biased lineage allocation, however, it may be required for progression to lymphoid blast crisis.

Author notes

Disclosure: No relevant conflicts of interest to declare.