Statin Treatment Prevents FLT3 Glycosylation and Overcomes Resistance to FLT3 Tyrosine Kinase Inhibitors

Abstract 1421

FLT3 is a receptor tyrosine kinase that is expressed on hematopoietic stem and progenitor cells where it functions in cell differentiation, survival and proliferation. It is also one of the most frequently mutated genes in acute myeloid leukemia (AML), and has thus become a target for modulation by the use of FLT3 tyrosine kinase inhibitors (TKI). Unfortunately, clinical resistance emanating via several different mechanisms has limited the potential benefit of FLT3 TKI. Some of these include FLT3 mutations that reduce drug binding, elevated FLT3 ligand (FL) levels that shift the dose-response curve and activation of parallel signaling pathways. These all pose major challenges to TKI effectiveness that must be overcome to improve patient outcomes. Since FLT3 is dependent upon N-linked glycosylation for its maturation and surface localization, we sought to determine whether statins might disrupt FLT3 signaling. Statins inhibit the mevalonate pathway and reduce levels of all ensuing end-products including dolichol, which transfers preassembled oligosaccharides onto nascent polypeptides. Here, we demonstrate by Western blotting that statins can indeed prevent complex FLT3 glycosylation, thus leading to loss of surface receptor expression. Immunofluorescence microscopy confirms a reduction in surface localization and a concomitant increase in intracellular FLT3/ITD accumulation. Interestingly, this aberrant localization was associated with increased STAT5 activation while inhibiting both MAP kinase and AKT phosphorylation. We have extended our previous findings that statins are cytotoxic to mutant FLT3 expressing cell lines to examine whether they are also able to overcome the resistance mechanisms discussed above. We show that the following mechanisms of resistance could be circumvented by fluvastatin. First, stimulation of BaF3 FLT3/ITD cells with exogenous FL at physiologic concentrations induces a three-fold increase in the IC₅₀ for inhibition of phosphorylated FLT3/ITD by the FLT3 TKI lestaurtinib. Pretreatment with fluvastatin for 24 h eliminated surface FLT3/ITD: FL interactions and restored the potency of lestaurtinib. Secondly, resistance to another clinical FLT3 TKI, sorafenib, caused by the FLT3/ITD N676K or D835Y mutations increased the IC₅₀ from 5 nM for BaF3/ITD cells to 30 nM and >50 nM, respectively. In contrast, both of these mutants remained just as sensitive to treatment with fluvastatin as were the BaF3/ITD cells (approx. IC₅₀ of 0.2 μM). A third mechanism of resistance that can be circumvented by fluvastatin is the over expression and/or activation of alternative pathways that can compensate for inhibited FLT3. A model of this is rescue of BaF3 FLT3/ITD cell lines from FLT3 TKI-mediated cytotoxicity by stimulation with IL-3. When this cell line is stimulated with IL-3, fluvastatin is able to inhibit activation of STAT5, AKT and MAP kinase caused by the IL-3 receptor, as well as FLT3/ITD signaling, leading to cell death. Fluvastatin was also effective in other cell lines expressing constitutively activated FLT3 (Molm-14, MV4;11, HB1119 and SEM-K2 cells) and in other cell lines in which glycosylated transmembrane receptors drive proliferation (mutant c-Kit in Kasumi cells) but not in cells that rely upon intracellular kinases (BCR-ABL in K562 cells). Importantly, fluvastatin also reduced FLT3 glycosylation in and was cytotoxic to primary AML patient samples harboring FLT3/ITD mutations at therapeutically achievable concentrations (1 μM). Finally, fluvastatin reduced engraftment of BaF3 FLT3/ITD cells transplanted in syngeneic Balb/c mice and prolonged their survival. These results demonstrate that statins, a class of drugs already FDA approved, might be useful, either alone or in combination with a FLT3 TKI, in the management of FLT3 AML cases including those resistant to FLT3 TKI.