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This file contains a detailed description of the synthesis as well as the characterization of lead compounds PG-S3-001, PG-S3-002, and PG-S3-003. Results pertaining to the interaction of PG-S3-001 with relevant kinases is also included; Table 1. Results of Kinome Screen for Kinases involved in Regulation of STAT3.

Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, concomitant assessment of drug-associated phenotypes has proven challenging. We have developed cellular target engagement by accumulation of mutant (CeTEAM) as a platform that can seamlessly evaluate drug-target interactions and phenotypic responses in a single multiparametric experiment. In the presence of binding ligand, accumulation of an initially unstable target protein acts as a biosensor that permits holistic assessment of drug pharmacology under physiological conditions. We demonstrate this proof-of-concept by uncoupling target binding from divergent cellular activities of MTH1 inhibitors, repurposing the R139C variant to dissect complex NUDT15-thiopurine interactions, and profiling the live-cell dynamics of DNA trapping by PARP inhibitors. Further, PARP1-derived drug biosensors facilitated multimodal ex vivo analysis of drug-target engagement and non-invasive tracking of drug binding in live animals. CeTEAM empowers real-time, comprehensive characterization of target engagement by bridging drug binding events and their biological consequences.

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.

Mutations in the BCR-ABL1 kinase domain are a well-established mechanism of tyrosine kinase inhibitor (TKI) resistance, but fail to explain many cases of clinical TKI failure. In the remaining patients, resistance occurs via activation of alternative signaling pathways that maintain survival despite BCR-ABL1 inhibition (BCR-ABL1-independent resistance). STAT3 mediates TKI resistance in chronic myeloid leukemia (CML) cells cultured in the presence of bone marrow-derived factors (Bewry et al., 2008; Traer et al., 2012; Nair et al., 2012), and also plays a critical role in survival of CML cells with BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). While targeting transcription factors is notoriously difficult, our combination of synthetic chemistry, in vitro reporter assays, and computational modeling has led to a low micromolar mechanism-based STAT3 inhibitor, which, in combination with TKIs, shows promise as a treatment for CML patients with BCR-ABL1-independent resistance. The original compound of the series, SF1-066 (10 µM; Fletcher et al., 2009), combines with TKIs to reduce survival of CML CD34+ cells exhibiting BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). To improve the potency and selectivity of SF1-066, we synthesized successive STAT3 inhibitor libraries and ranked candidates by structure-activity relationship using a luciferase-based reporter screen (Kraft et al. #2445, ASH 2012). This reporter assay quantifies STAT3 transcriptional activity in TKI-resistant AR230R cells, which grow in the continuous presence of imatinib (1.0 µM), lack BCR-ABL1 kinase domain mutations, and exhibit high levels of pSTAT3Y705, thereby enabling convenient, high-throughput screening for potency and selectivity in the context of endogenous STAT3 activation. Among three sequential STAT3 inhibitor libraries, BP5-087 emerged as the new lead compound. Fluorescence polarization assays verified that BP5-087 was 5-fold more effective than SF1-066 in outcompeting an SH2 peptide probe, and computational simulations predicted better overall binding of BP5-087 (-9.6 kcal/mol) versus SF1-066 (-7.6 kcal/mol) to the STAT3 SH2 interface. In AR230R cell growth assays, BP5-087 was effective at a 5-fold lower dose compared to SF1-066, with minimal effects on TKI-sensitive parental controls. Therefore, we tested BP5-087 in the context of primary TKI resistance. BP5-087 (1 µM) in combination with imatinib (2.5 µM) reduced colony formation and increased apoptosis of CD34+ cells from CML patients with BCR-ABL1-independent resistance. These cells have no BCR-ABL1 kinase domain mutations and undergo BCR-ABL1 kinase inhibition as detected by immunoblot analyses. In contrast, BP5-087 had no effect on CD34+ cells from newly diagnosed CML patients or normal individuals. Immunofluorescence demonstrated that dual treatment of TKI-resistant CML CD34+ cells resulted in reduced levels of nuclear pSTAT3Y705, consistent with an inhibitor of STAT3 dimerization. In more primitive CML stem cells, long term culture-initiating cell (LTC-IC) assays revealed that neither inhibitor alone had any effect on colony formation of primitive LTC-IC progenitors, whereas imatinib (2.5 µM) in combination with BP5-087 (1.0 µM) reduced LTC-IC colony formation by 66%. Consistent with this observation, immunofluorescence showed high levels of pSTAT3Y705 in primitive TKI-resistant CD34+CD38- cells when cultured in the presence but not absence of TKIs. To test the feasibility of BP5-087 for in vivo use, we treated mice orally with BP5-087 (25 mg/kg/day) for 4 weeks and observed no changes in body weight, peripheral blood cellularity, or bone marrow colony forming ability. Mass spectrometry confirmed that BP5-087 is orally bioavailable. In summary, BP5-087 is a systematically-derived, direct inhibitor of STAT3 that, in combination with TKIs, reduces survival of CML cells with BCR-ABL1-independent resistance. Further rounds of structure-activity optimization may reveal an inhibitor with a clinically-relevant effective concentration. Disclosures: Deininger: Bristol Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.