Loss of Bcl-x Does Not Ameliorate Chronic Myeloid Leukemia in an Inducible Murine Model System.

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.