A Novel Small Molecule STAT Inhibitor, BP-4-018, Demonstrates Significant Anti-Tumour Activity and Synergism With Bortezomib In Pre-Clinical In Vivo Models Of Multiple Myeloma

Abstract

Despite significant advances in the treatment of Multiple Myeloma (MM), it remains incurable and thus, novel therapeutic strategies are critically needed. Prior work has demonstrated that the transcription factor STAT3 is aberrantly activated in a number of human tumors and drives hallmark oncogenic processes such as proliferation, survival, angiogenesis and drug resistance. As such, this master regulator of malignant processes has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activation, including MM. In the present studies, we characterize a newly developed small molecule, BP-4-018. The inhibitory mechanism of BP-4-018 is based on its ability to function as a phosphotyrosine mimetic, and thus bind the STAT3 SH2 domain to inhibit STAT3 phosphorylation (pSTAT3), STAT3 dimerization and DNA binding. Based on the 3D crystal structure of STAT3, SH2 docking simulations and fluorescence polarization assays, BP-4-018 demonstrates high affinity binding to the SH2 domain of STAT3. Using a panel of molecularly heterogeneous human myeloma cell lines (HMCLs), BP-4-018 demonstrated potent and broad anti-tumor activity in MTT assays, with IC50 values in the low μM range (1.91 μM - 6.48 μM). These anti-MM effects are mediated through the induction of the apoptosis as evidence by Annexin V+ staining, PARP cleavage and activation of caspases 3 and 9. Using patient derived bone marrow (BM) cells, BP-4-018 was shown to retain its anti-MM activity against HMCLs cultured in the presence of a BM stroma, induce apoptosis in primary MM tumor cells, and importantly, displayed little to no toxicity against non-malignant BM cells. Using cell-based assays, we show that BP-4-018 inhibits basal and cytokine-induced pSTAT3, as well as STAT3 transcriptional activity in HMCLs that stably express a STAT3-driven luciferase reporter construct. Consistent with these effects, western blot analyses of BP-4-018-treated cells reveal decreased expression of STAT3 target genes including c-Myc, Mcl-1 and Bcl-xL. Interestingly, BP-4-018 did not demonstrate preferential inhibition of cytokine induced STAT3 phosphorylation over that of STATs 1 or 5 suggesting that this compound may in fact be a pan-STAT inhibitor. BP-4-018 does not however inhibit the phosphorylation of other proteins whose regulation is SH2-dependent including AKT, ERK, SRC and LYN. In vitro combinatorial studies revealed that BP-4-018 is synergistic in combination with dexamethasone (CI=0.62), melphalan (CI=0.94), lenalidomide (CI=0.82) and is highly synergistic with bortezomib (BTZ) (CI=0.37). To examine the potential mechanisms of combinatorial synergy with BTZ, we evaluated the effects of BTZ on STAT3 signaling in HMCLs. BTZ treatment alone augmented STAT3 transcriptional activity and led to the accumulation of Mcl-1 protein, which was reversed when cells were co-treated with BP-4-018. Furthermore, HMCLs expressing a dominant negative STAT3 mutant display increased sensitivity to BTZ. Collectively these data suggest that the combinatorial synergism with BTZ is at least in part related to BP-4-018-mediated inhibition of STAT3. Finally, we used a novel STAT3-driven luciferase xenograft model of MM to show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase activity in vivo, significantly delays tumor growth as a single agent, and in combination with BTZ, induces marked and in some animals complete tumor regression. Neither BP-4-018 alone or in combination elicited significant toxicities as assessed by weight loss, and more extensive pharmacodynamic, pharmacokinetic and toxicology studies of BP-4-018 are currently underway. Taken together, our results highlight the promising therapeutic potential of BP-4-018 and support the continued development of targeted therapies for MM with a focus on aberrant STAT signaling. Disclosures: No relevant conflicts of interest to declare.