Studies of BP-1-102, a Novel Direct Small-Molecule Inhibitor of Stat3 Demonstrates Substantial Anti-Myeloma Pre-Clinical Activity

Abstract Abstract 138 Stat3 is essential for transducing signals from extacellular stimuli, but also functions as a nuclear transcription factor required for regulating genes involved in proliferation, apoptosis, angiogenesis and invasion, in addition to genes encoding cytokines, chemokines and growth factors. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. In addition, activation of Stat3 is observed in MM bone marrow stroma cells (BMSCs), which is a major source of interleukin-6 (IL-6) that induces persistent activation of Stat3 in MM cells, thereby establishing a feedforward loop. Stat3 downstream target genes are critical to the dyregulated biological processes that promote MM cell growth, survival and induce chemoresistance, thus targeting JAK2/Stat3 signaling represents an important therapeutic target in MM. We describe here a novel, highly specific and potent small molecule inhibitor of Stat3 derived by molecular modeling of the phosphotyrosine (pY)-SH2 domain interactions in the Stat3:Stat3 dimerization, combined with in silico structural analysis of the Stat3 dimerization disruptor, S31-201. We present evidence from in vitro biochemical and biophysical studies that a structural analogs, BP-1-102 directly interacts with Stat3′s SH2 domain, with high affinity and disrupts the binding of Stat3 to a high affinity pY-peptide, GpYLPQTV-NH2, with IC50′s of 19 mM (BP-1-102) making this one of the most effective disruptors of Stat3 protein-phosphopeptide complexation events by a non-phosphorylated small molecule. We evaluated the activity of BP-1-102 in a series of pre-clinical models of MM. Analyses against a panel of 14 genetically diverse human myeloma cells lines (HMCLs) revealed that BP-1-102 suppressed myeloma cell growth in a dose-dependent fashion. After 72 h of in vitro exposure, the IC50 values ranged from 4.5–9.5 mM in responsive cell lines. Increased sensitivity to BP-1-102 was observed in HMCLs demonstrating a high IL-6/Stat3 gene expression signature. As expected, chemically optimized BP-1-102 demonstrated greater inhibitory potency then its parent compound, SF-1-66 (IC50 values 18–43.1 mM). BP-1-102 demonstrated a good therapeutic window failing to inhibit normal bone marrow (BM)-derived CD34 colony formation at doses up to 20 mM. Similar potent activity was observed against primary MM cells. Exposure of 6 patient derived BM mononuclear cells to BP-1-102 revealed selective activity against CD138+ MM cells (>50% in viable cells) in 4/6 patients treated with 10 mM BP-1-102, while minimal toxicity was observed against the CD138− cells. We confirmed activity of BP-1-102 against Stat3 in MM cells observing dose-dependent inhibition of IL-6 induced Tyr705Stat3 phosphophorylation in OPM2 cells presumably through the blockade of Stat3 binding to the pY motifs of pg130 component of the IL-6 receptor and the prevention of de novo phosphorylation. Further, the inhibitor blocked IL-6 stimulated nuclear translocation of Stat3. Treatment of JJN3 MM cells that demonstrate constitutive Tyr705Stat3 phosphorylation and thus harbor aberrant Stat3 activity induced marked apoptosis (annexin V+/PI-) that was preceded by rapid, pronounced and sustained inhibition of Stat3 phosphorylation. Interestingly, we also observed delayed inhibition of JAK2 and ERK1/2 phosphorylation that we speculate is a result of downregulation of an autocrine Stat3/IL-6 feedforward loop. On the otherhand, Akt phosphorylation was not suppressed consistent with lack of PI3k/Akt activation by IL-6. Finally, we observed downregulation of expression of Stat3 target genes, c-Myc and Mcl-1. Together the data are consistent with the targeted activity of BP-1-102 against Stat3 and suggest that Stat3 is a viable therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

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