MCL1 down‐regulation plays a critical role in mediating the higher anti‐leukaemic activity of the multi‐kinase inhibitor Sorafenib with respect to Dasatinib (2012)

Acute myeloid leukaemia (AML) is the most frequent acute leukaemia of adults. Mutations in the FMS-like tyrosine kinase-3 (FLT3) occur in 25–30% of AML patients, leading to internal tandem duplications in the juxtamembrane domain of the receptor (FLT3-ITD) (Stirewalt & Radich, 2003). Since the FLT3-ITD mutation dictates a particularly poor clinical outcome (Stirewalt & Radich, 2003), several specific FLT3 inhibitors have been developed and evaluated in clinical trials, but their overall clinical efficacy in AML to date must be considered as minor. A new class of pharmacological compounds with the potential to target FLT3-ITD is represented by the second generation of protein kinase inhibitors. Therefore, we have compared the anti-leukaemic activity of the multi-kinase inhibitors Dasatinib and Sorafenib in a panel of myeloid leukaemic cells and primary AML blasts with different FLT3 (FLT3wild-type and FLT3mutated) status. Dasatinib (BMS-354825), which is currently used in clinical trials for the treatment of chronic myeloid leukaemia, was chosen based on recent data suggesting that it might also display potential cytotoxic activity in AML (Guerrouahen et al, 2010), while Sorafenib (Nexavar) was chosen based on preliminary clinical studies in which it was found particularly active in FLT3-ITD positive patients (Metzelder et al, 2009). Dasatinib and Sorafenib (both from Selleck Chemicals, Houston, TX, USA) were comparatively analysed on five AML cell lines characterized by different FLT3 and TP53 status: FLT3wild-type/TP53wild-type (OCI), FLT3wild-type/TP53mutated (NB4), FLT3wild-type/TP53deleted (HL60), FLT3mutated/TP53wild-type (MOLM), FLT3mutated/TP53mutated (MV4-11). Results were evaluated by using analysis of variance with subsequent comparisons by Student's t-test and with the Mann–Whitney rank-sum test. Treatment with Sorafenib for 48 h exhibited a marked cytotoxicity, resulting in cell viability lower than 40% of the untreated cultures in all leukaemic cell lines (Fig 1A), as evaluated by Trypan blue dye exclusion (Zauli et al, 1992). As documented also by the 50% inhibitory concentration (IC50) values, Sorafenib showed maximal citotoxicity on FLT3mutated MV4-11 (IC50 < 1 μmol/l) and MOLM (IC50 < 1 μmol/l) followed by FLT3wild-type HL60 (IC50 5·1 μmol/l), OCI (IC50 5·3 μmol/l) and NB4 (IC50 8·8 μmol/l). On the other hand, Dasatinib showed an appreciable cytotoxicity only on MV4-11 (IC50 < 6·5 μmol/l) and to a much lesser extent on MOLM (IC50 16·6 μmol/l), while OCI (IC50 29·4 μmol/l), NB4 (IC50 24·7 μmol/l) and HL-60 (IC50 > 30 μmol/l) were completely resistant to Dasatinib. Thus, Sorafenib showed a significantly (P < 0·05) higher cytotoxicity with respect to Dasatinib in all cell lines. Both Sorafenib and Dasatinib induced accumulation in G1 phase of the cell cycle, with maximal cell cycle block in FLT3mutated MV4-11 and MOLM cells (Fig 1B). In addition, the degree of apoptosis was evaluated by annexin-V/propidium iodide (PI) staining and flow cytometry analysis performed as described (Secchiero et al, 2007). Sorafenib promoted apoptosis in all cell lines with maximal effects in MV4-11, MOLM and HL60 cells (Fig 1C), while Dasatinib induced apoptosis only in MV4-11 and MOLM cells (Fig 1C). We next investigated the molecular mechanisms underlying the disparity in cytotoxicity of Sorafenib versus Dasatinib in leukaemic cells. Dasatinib and Sorafenib inhibited at a comparable degree the phosphorylation levels of a variety of kinases (Fig S1A) and STAT transcription factor family members (Fig S1B). However, at variance to Dasatinib, Sorafenib potently inhibited phospho-ERK1/2, as confirmed by Western blot analysis in all leukaemic cell lines (Fig S1C). Data in solid tumours have shown that MCL1 is a potential down-stream target of Sorafenib (Inuzuka et al, 2011), while the ability of Dasatinib to modulate MCL1 is less clear (Nguyen et al, 2007). Strikingly, we found that Sorafenib potently down-regulated MCL1 in all leukaemic cell lines, while Dasatinib increased MCL1 expression in OCI and HL60 cells (Fig 2A). MCL1 and phospho-ERK1/2 down-regulation started 2 h after treatment with Sorafenib (Fig 2B), well before the onset of apoptosis, as evaluated by PARP cleavage and loss of cell viability (Fig 2B). In addition, the possibility that the down-regulation of MCL1 merely reflected a caspase-dependent degradation consequent to the induction of apoptosis by Sorafenib was ruled out in experiments performed with the pan-caspase inhibitor z-VAD (Calbiochem, La Jolla, CA, USA) and recombinant TRAIL, prepared as described (Campioni et al, 2005) (Fig 2C). While z-VAD completely abrogated the down-regulation of MCL1 induced by TRAIL, a death-inducing ligand known to induce apoptosis through the activation of caspases (Secchiero & Zauli, 2008), z-VAD did not affect the down-regulation of MCL1 induced by Sorafenib (Fig 2C). On the contrary, the pharmacological inhibitor of ERK1/2 pathway, PD98059 (Calbiochem), significantly down-modulated MCL1 protein levels in both untreated and Sorafenib-treated cultures (Fig 2D), suggesting that the strong inhibitory activity of phospho-ERK1/2 by Sorafenib probably has a role in the Sorafenib-mediated down-regulation of MCL1. PD98059 also induced a significant (P < 0·05) increment of Dasatinib-induced cytotoxicity (Fig 2D). In a final set of experiments, leukaemic cells were transfected with MCL1 siRNA before exposure to Sorafenib and Dasatinib (Fig 2E). While siRNA for MCL1 significantly (P < 0·05) increased apoptosis in untreated cultures as well as in cultures treated with Dasatinib, siRNA had minor effects on Sorafenib-mediated cytotoxicity. Overall, these data indicate the existence of a correlation between MCL1 down-regulation and leukaemic cytotoxicity. In this respect, the paradoxical induction of MCL1 in HL60 and OCI correlated with the higher resistance of these leukaemic cell lines in response to Dasatinib. It has been recently shown that FLT3-ITD can up-regulate MCL1 to promote survival of stem cells in AML (Yoshimoto et al, 2009) and thus it represents an axis for therapeutic interventions in the perspective to eradicate the leukaemic clone. In this respect, we have established for the first time that Sorafenib displays a higher cytotoxic activity against AML with respect to Dasatinib and that the different effects between these two multi-kinase inhibitors strongly correlates with their differential ability to down-regulated (Sorafenib) or not (Dasatinib) the anti-apoptotic Bcl-2 family member MCL1. This work was supported by grants from: the Italian Association for Cancer Research (AIRC) and Beneficientia Foundation (both to G.Z.), and by FIRB-MIUR Project (to P.S.). PS and EM collected, analysed, and interpreted the data, and wrote the manuscript. RV, FL contributed data and interpretation of results and wrote the manuscript. RV assisted in statistical analysis and writing of the manuscript. GZ interpreted the data and wrote the manuscript. All authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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