Inhibition of Calcineurin/NFAT Signalling Breaks Resistance of Ph+ ALL to Imatinib

Abstract 2486

Approximately twenty percent of acute lymphoblastic leukemias (ALL) in adults are positive for the Philadelphia chromosome t(9;22) upon cytogenetic examination and express a Bcr-abl fusion protein with constitutive tyrosine kinase activity as part of their pathogenesis. While the majority of these leukemias initially respond well to tyrosine kinase inhibition (TKI) with imatinib or dasatinib, most of them develop TKI-resistance over time and exhibit progressive disease to which most patients eventually succumb. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT proteins get rephosphorylated and inactivated by a network of multiple NFAT kinases including GSK-3, CK-1 and DYRK. Several recent studies have demonstrated that Calcineurin/NFAT signalling is involved in the pathogenesis of a wide array of hematological malignancies including diffuse large B cell lymphoma, CLL as well as Burkitt and Burkitt-like lymphomas.

In this study, we investigated the role of the Ca/NFAT signalling pathway in the development of TKI-resistance in Ph+ ALL. We analyzed several Ph+ ALL cell lines (SD-1, TOM-1, SUP-B15), which had been shown previously to be resistant to imatinib, as well as primary leukemia samples from patients with TKI-resistant disease (n=3). NFAT expression and aberrant nuclear translocation was assessed by Western Blotting and the NFAT2 (NFATc1) mRNA level was confirmed by quantitative RT-PCR. The cells were subsequently grown under optimized cell culture conditions in the presence or absence of TKI (0–1 μM imatinib), and cell viability was assessed using propidium iodide staining and flow cytometry. Proliferation assays were performed after 48–72 h using a luminescence assay measuring the amount of cellular ATP.

All cell lines and primary leukemia samples showed marked expression and evidence of aberrant nuclear translocation of NFAT2 when analyzed by Western Blotting. Even maximum concentrations of imatinib had only minor effects on cellular proliferation documenting resistance to tyrosine kinase inhibition. To inhibit the calcineurin/NFAT signalling cascade as a potential therapeutic target, we treated the cells with the calcineurin inhibitor cyclosporin A (CsA) in addition to imatinib. The combined treatment showed a dramatic effect on ALL cell proliferation, resulting in almost complete cell death after 48 h. This effect could be reproduced using tacrolimus (FK506) instead of CsA indicating that it was specifically mediated through calcineurin inhibition and not by potential off target-activites of CsA.

In summary, our data provide strong evidence that Calcineurin/NFAT signalling contributes to the proliferation of TKI-resistant Ph+ ALL cells and that pharmacological inhibition of NFAT signalling can break treatment resistance to TKI. Targeting Ca/NFAT signalling in combination with Bcr-abl inhibition might therefore be a novel option in the treatment of Ph+ ALL.