The Order of Exposure to Cytotoxic Chemotherapy and the Proteasome Inhibitor Bortezomib Dictates the Extent of Reversal of Chemotherapy Resistance in T-ALL

While most children with T-cell acute lymphoblastic leukemia (T-ALL) are cured, the outcome for children who respond poorly to therapy or relapse is almost universally fatal. Our laboratory is interested in understanding the mechanisms contributing to therapy resistance and has developed a mutliparameter phosphoflow cytometry assay to study signal transduction events at a single cell level.

Nuclear Factor kappa B (NF-κB) is a family of transcription factors that plays an important role in cancer development by preventing apoptosis and facilitating tumor cell growth. Consistent with this, constitutive NF-κB activity has been reported in T-ALL, and has been suggested as a predictor of relapse. We hypothesized that the proteasome inhibitor Bortezomib, which blocks NF-κB activation, induces apoptosis in T-ALL. We further hypothesized that combining Bortezomib with conventional chemotherapeutic agents sensitizes T-ALL cells to apoptosis and overcomes chemotherapy resistance by altering the balance of downstream pro- and anti- apoptotic mediators.

A panel of T-ALL cell lines was cultured in the presence of vehicle alone, conventional cytotoxic drugs (Dexmethasone, Etoposide, Cyclophosphamide, Cytarabine), Bortezomib, or combinations of these. Cytotoxic effect was measured by trypan blue staining and flow cytometric detection of cleaved caspase-3. Multiparameter phosphoflow cytometry was used to measure expression levels of downstream apoptosis mediators. An advantage of multiparameter phosphoflow cytometry is the ability to gate on caspase 3-negative (i.e., chemotherapy resistant) cells, allowing us to interrogate potential mechanisms of resistance by comparing expression profiles of cell survival proteins before and after treatment.

T-ALL cells were exposed to cytotoxic chemotherapy or vehicle for 24 to 48 hours and subjected to phosphoflow cytometry. Analysis of caspase-3 negative cells indicated that chemotherapy resistant cells rewired their signaling networks by upregulating their MAPK, NF-κB, and AKT/PI3K/S6 signaling networks in response to genotoxic stress. This correlated with upregulation of the pro-survival protein survivin, suggesting a potential mechanism for chemotherapy resistance. To test whether Bortezomib could reverse this resistance, cells were exposed to subtoxic concentrations of Bortezomib alone or in combination with cytotoxic drugs. Combination therapy produced only a modest synergistic effect. We then wondered whether the sequence of administration of cytotoxic drug and Bortezomib could potentiate this effect. T-ALL cell lines were therefore 1) pretreated with Bortezomib for 24 hours followed by cytotoxic drug for an additional 24 hours, 2) pretreated with cytotoxic drug for 24 hours followed by addition of Bortezomib for an additional 24 hours, or 3) treated with both agents simultaneously. Surprisingly, the most pronounced cytotoxic synergy was observed when T-ALL cells were pretreated with cytotoxic drug followed by Bortezomib. While simultaneous or pretreatment with Bortezomib had no effect on upregulation of surivin in response to cytoxic chemotherapy, addition of Bortezomib post genotoxic stress resulted in markedly decreased levels of survivin.

We found that Bortezomib lowered the apoptotic threshold to conventional cytotoxic drugs and can reverse drug resistance in T-ALL cell lines. Interestingly, the order of exposure to cytotoxic chemotherapy and Bortezomib determines the extent of reversal of chemotherapy resistance. Our data is in agreement with studies in multiple myeloma and provides evidence supporting the use of Bortezomib in combination with conventional cytotoxic agents in the treatment of relapsed or refractory disease.

No relevant conflicts of interest to declare.