ALBERT

Description: Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with relapse rates approaching 40% in children. Aberrant activation of the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is implicated in promoting many cancer phenotypes, including AML. Additionally, the important role of STAT3 in microenvironment-mediated chemoresistance is well established. Therefore, STAT3 is an important target for the development of new agents. Like other transcription factors, the structure of the STAT3 protein does not easily lend itself to the development of a small molecule inhibitor that is both potent and specific. Several commercially available and academic STAT3 inhibitors have been reported, but none is yet suitable for broad clinical application. We have developed a novel class of naphthalene sulfonamide small molecule STAT3 inhibitors with efficacy in AML cell lines and primary samples (e.g. C188-9 [Redell, et al, 2011, Blood 117:5701-9]). Rational optimization steps have yielded a new lead compound, MM-206, with improved stability in cellular contexts. Our aim is to evaluate the activity of MM-206 in preclinical AML models that include the bone marrow stromal environment. Inhibition of STAT3 activity: MM-206 potently inhibited the STAT3 SH2 domain-phosphopeptide interaction, with IC50 1.2 μM by surface plasmon resonance assays. Further, MM-206 attenuated phosphorylation of G-CSF-induced STAT3 as measured by intracellular flow cytometry. AML cell lines were treated with increasing doses of MM-206 for 30 minutes, followed by stimulation with G-CSF to induce STAT3 phosphorylation (pY705). The IC50 values for inhibition of phosphorylation were 1-3 µM, indicating potent inhibitory activity in cells. Similarly, MM-206 inhibited G-CSF-induced PIM1 transcription in a dose dependent manner in AML cell lines. Anti-leukemia activity in vitro: MM-206 demonstrated dose-dependent induction of apoptosis in AML cell lines and pediatric AML samples, even in the presence of bone marrow stromal cells. Cell lines and patient samples were cultured alone or co-cultured with mOrange-transduced HS5 or HS27A stromal cells, then treated with increasing doses of MM-206 for 24 hours. Cells were stained with Annexin V-FITC and apoptosis in the mOrange-negative cells was quantified by FACS. EC50 values were consistent with the doses for inhibition of phosphorylation, ranging from 1 - 10 µM. The increase in EC50s in the stroma co-cultured AML cells, compared to AML cells cultured alone, was ~3-fold, indicating only moderate environment-derived protection against this compound. In complementary experiments, luciferase-transduced AML cell lines were cultured alone or with stromal cells, treated with MM-206 for 24 hours, then AML cell viability was assessed by luminescence. Again, IC50s were in the range of 3 µM for all cell lines tested. There was no difference in potency for cells on stroma in the luminescence assay. In contrast to the anti-AML activity, EC50s were 〉30 µM in cell line models of acute lymphoblastic leukemia, a disease not typically associated with STAT3-dependent survival. Anti-leukemia activity in vivo: We engrafted NSG mice with luciferase-expressing MV4-11 AML cells. After 2 weeks, mice were randomized to receive MM-206 (30 mg/kg) or vehicle, ip daily Monday-Friday, for 2 or 4 weeks. MM-206 treatment delayed disease progression, as evidenced by significantly lower luminescence values (p