Abstract 2445

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) correlates with drug resistance and a poor prognosis in many cancers. STAT3 signaling is mediated by phosphorylation at tyrosine-705 (p STAT3Y705), dimerization, and nuclear transactivation. In chronic myeloid leukemia (CML), pSTAT3Y705 is demonstrable under two distinct resistance scenarios: (1) extrinsic resistance, in which BCR-ABL1 kinase-independent survival signals originating from the bone marrow (BM) microenvironment activate pSTAT3Y705 in a JAK2- or TYK2-dependent manner, and (2) intrinsic resistance, in which BCR-ABL1 kinase-independent signals activate pSTAT3Y705 in response to kinase inhibition. Based on these observations, we identified TKI-resistant CML as an excellent model for developing and optimizing pharmacologic STAT3 inhibitors.

Using K562 and AR230 CML cells that are resistant to 1 μM imatinib (K562R and AR230R; intrinsic resistance) and primary CML CD34+ progenitor cells exposed to BM stromal-derived conditioned medium (CM; extrinsic resistance), we examined the effects of direct pharmacologic inhibition of STAT3 in TKI-resistant CML. Here, we report the design and validation of next-generation STAT3 inhibitors identified through computational modeling and screening in AR230R CML cells expressing high levels of pSTAT3Y705.

We initially examined the effects of an established STAT3 inhibitor, S3I-201.1066 (SF1–066). K562R or AR230R cells were treated with 1 μM imatinib and/or 10 μM SF1–066, followed by culture in methylcellulose medium and scoring after 14–16 days. Combination treatment reduced the clonogenicity of K562R and AR230R cells to 31.4% (p<0.02) and 27.5% (p<0.004) of controls, respectively. In contrast, SF1–066 did not synergize with imatinib in parental K562 and AR230 cells that lack constitutive pSTAT3Y705 (p>0.05). Next, CD34+ cells from newly diagnosed CML patients (n=4) were cultured for 96 hours in the presence of CM and treated with 2.5 μM imatinib, 10 μM SF1–066 or both. Equal numbers of cells were then plated in colony forming assays. Imatinib combined with SF1–066 reduced colony formation to 54.8% (p<0.002) of controls treated with imatinib. Given that dual treatment reduces but does not completely suppress colony formation, we reasoned that some STAT3 signaling must persist in the presence of SF1–066. Thus, we developed next-generation STAT3 inhibitors with increased activity compared to SF1–066.

Computational modeling informed the design and synthesis of a second-generation, SF1–066-based library. We evaluated compounds with shared molecular functionalities using AR230R cells expressing a luciferase reporter containing sequential STAT3 sis-inducible elements (AR230R-SIE). At 10 μM, we found two inhibitors, BP2–047 and BP3–163, that reduced luminescence by 69.2% (p<3.6×10−6) and 59.7% (p<8.6×10−6), respectively, compared to controls (n=3). However, the compounds also reduced luminescence in AR230R cells expressing a scrambled luciferase reporter (AR230R-NEG). This screen provided information on the structure-activity relationships of the compounds and was used to generate 64 more candidate STAT3 inhibitors.

To account for the reduced luminescence in AR230R-NEG cells and analyze for more potent inhibitors, we performed the luciferase assay with candidate inhibitors at 5 μM. We identified BP5–087 and BP5–088, each with increased potency and STAT3 selectivity compared to SF1–066. These compounds decreased luminescence in AR230R-SIE cells by 36.1% (p<2.6×10−7) and 25.5% (p<6.1×10−10), respectively (n=3), with minimal effects on AR230R-NEG cells (p>0.05). Confirmation of STAT3 binding was obtained using fluorescence polarization assays, in which the EC50 values of BP5–087 and BP5–088 measured 8.5 μM and 4.6 μM, respectively. Studies with mouse and human liver microsomes also revealed that BP5–087 and BP5–088 exhibit enhanced metabolic stability compared to SF1–066. Interestingly, treatment with BP5–087 or BP5–088 (both at 1 and 5 μM) in CML CD34+ progenitors grown in CM showed increased cytoplasmic accumulation of pSTAT3Y705 compared to controls.

Thus, we identified BP5–087 and BP5–088 as two of the most potent small-molecule binders of STAT3 reported. These compounds are promising frontrunners toward new therapies for TKI-resistant CML and other diseases in which STAT3 activation drives malignant phenotypes.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.