Distinct Drug Responses of CD8⁺CD4⁺ and CD8⁺CD4⁻leukemic Cell Subsets in Mice with Notch1-Induced T-ALL

Abstract 1504

Leukemic cells are heterogeneous, so defining the differences of leukemic cell sub-populations in response to drug treatment may ultimately help design better strategies to maximize the efficacy of chemotherapeutic agents for patients. We have begun to study this issue with Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) in mice. The Notch1 signaling pathway plays an essential role in cell growth and differentiation, and activated mutations in the Notch1 gene are frequently observed in human T-ALL. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-ALL in mice. In our current study, we examined T-ALL cells, specifically the CD8 and CD4 double positive (CD8⁺CD4⁺) and CD8 single positive (CD8⁺CD4⁻) cell sub-populations, for their distinct sensitivities to drug treatment.

We first treated T-ALL mice with a cell-cycle nonspecific chemical drug, cyclophosphamide (CTX), or a mTORC1 inhibitor, rapamycin, when the overall T-ALL cell population rose to more than 10% in mouse peripheral blood. We found that CTX treatment effectively reduced the T-ALL cells and led to an increase in the ratio of CD8⁺CD4⁺ versus CD8⁺CD4⁻ T-ALL, indicating that CD8⁺CD4⁻ T-ALL cells were more sensitive to CTX treatment. In contrast, rapamycin had the opposite effect. When compared with mice treated with rapamycin or CTX alone, the combination of the two drugs led to a significant improvement for T-ALL bearing mice with respect to leukemia initiation, progress and overall survival. We then set out to investigate the mechanism of different sensitivities of CD8⁺CD4⁺ and CD8⁺CD4⁻ T-ALL cells upon CTX or rapamycin treatments. We found that CD8⁺CD4⁺ and CD8⁺CD4⁻T-ALL cell groups responded similarly to CTX treatment after 12 hours of treatment. However, CD8⁺CD4⁻T-ALL cells continued to undergo apoptosis 24 hours after CTX treatment. The expression of the anti-apoptosis gene Bcl-2 in CD8⁺CD4⁺ T-ALL cells was significantly higher than that in CD8⁺CD4⁻ T-ALL cells, while the expression of the pro-apoptosis genes Bax, p53 and Noxa in CD8⁺CD4⁺ T-ALL cells was lower than that of CD8⁺CD4⁻T-ALL cells after 24 hour CTX treatment. This suggests that CD8⁺CD4⁺ and CD8⁺CD4⁻ T-ALL cells activate apoptosis differently after CTX treatment. Interestingly, rapamycin treatment did not affect apoptosis in the same manner as CTX treatment, but instead, it arrested more T-ALL cells in the G0 phase. More importantly, only CD8⁺CD4⁺, but not CD8⁺CD4⁻T-ALL cells were sensitive to rapamycin treatment in arresting cell cycle at different time points. The expression levels of CDK2 and CDK4 were significantly lower in CD8⁺CD4⁺ T-ALL cells when compared with CD8⁺CD4⁻ T-ALL cells, while p27 expression in CD8⁺CD4⁺ T-ALL cells was higher than that of CD8⁺CD4⁻ T-ALL cells after rapamycin treatment for 3 or 10 days. These data provide a molecular basis for the distinct apoptotic and cell cycle arrest responses of CD8⁺CD4⁺ and CD8⁺CD4⁻ T-ALL cell subsets to CTX or rapamycin treatment.

Taken together, our study documents previously unappreciated, yet distinct properties of CD8⁺CD4⁺ and CD8⁺CD4⁻Notch1-induced T-ALL cells in the context of specific drug treatment and suggests a therapeutic potential of combining CTX and rapamycin treatment for T-ALL patients. This strategy may also help design better chemotherapeutical regimes for other types of leukemia and cancer.