Aberrant MAPK and PI3K Signaling Contribute to Chemotherapy Resistance in T Cell Acute Lymphoblastic Leukemia by Altering the Balance of Apoptosis Mediators,

Relapse and induction failure due to chemotherapy resistance are major causes of mortality in T Cell Acute Lymphoblastic Leukemia (T-ALL), but the mechanisms of this resistance are incompletely understood. Pro-survival signals in the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathways are known to be hyperactive in T-ALL, and several mutations in the proteins governing these signaling pathways have been described. We hypothesized that remodeling of these signaling networks contribute to chemotherapy resistance by altering the balance of downstream pro- and anti-apoptosis mediators, such that survival is favored. We further hypothesized that inhibition of the PI3K pathway can reverse this effect, allowing conventional chemotherapy to more effectively induce apoptosis.

Human thymocytes, T-ALL cell lines, and primary T-ALL patient samples were cultured in the presence of vehicle alone, conventional cytotoxic drugs, targeted signal inhibitors, or combinations of these. Multiparameter phosphoflow cytometry was used to measure signaling network activities and expression levels of downstream apoptosis mediators at several time points. Cytotoxic effect was measured by trypan blue staining and flow cytometric detection of cleaved caspase-3.

In comparison to healthy thymocytes, all untreated T-ALL cell lines demonstrated augmented expression of pERK, pS6, Survivin, and Bcl2. Treatment with conventional cytotoxic drugs alone for 24 hours induced further increases in pERK, pS6, Survivin, and Bcl2 levels. Bim expression increased in accordance with that of cleaved caspase-3; PUMA expression was unchanged. Targeted inhibition of PI3K and mTOR with the dual inhibitor NVP-BEZ235 suppressed pS6 activation, and this corresponded with a decrease in Survivin and Bcl2 levels. PUMA, Bim, and cleaved Caspase-3 expression were minimally affected, and pERK levels remained stable or slightly increased. However, when NVP-BEZ235 was used in combination with conventional cytotoxic drugs, Bim and cleaved Caspase-3 were synergistically up-regulated, and this correlated with a decrease in viability by trypan blue staining. In comparison, the mTOR inhibitor rapamycin suppressed pS6 activity in all cell lines, but the cytotoxic effect in combination with conventional therapy was inferior to that of NVP-BEZ235. Conversely, the PI3K inhibitor PIK90 produced less effective pS6 suppression, but its cytotoxic effect in combination with conventional therapy was comparable to that of NVP-BEZ235.

T-ALL cells up-regulate MAPK and PI3K pathway activities in response to conventional chemotherapy. This correlates with augmented expression of the pro-survival factors Survivin and Bcl2, illustrating a potential mechanism for evasion of apoptosis in response to cytotoxic agents. This provides rationale for the use of a targeted PI3K pathway inhibitor in combination with conventional chemotherapy in the treatment of T-ALL. By inhibiting PI3K/mTOR signaling with the pharmacologic agent NVP-BEZ235, Survivin and Bcl2 are suppressed, allowing greater cytotoxicity in response to conventional chemotherapy. Although rapamycin effectively inhibits pS6 activity, activation of alternative PI3K targets may promote cell survival; targeting this pathway further upstream with agents such as NVP-BEZ235 or PIK90 may be required to effectively potentiate apoptosis. This data supports the need for further investigation of this complex signaling network in human T-ALL, and it provides evidence supporting the use of targeted therapy in combination with conventional cytotoxic agents in the treatment of relapsed or refractory disease.

No relevant conflicts of interest to declare.