STAT3 Inhibition Synergizes with BCR-ABL1 Inhibition to Overcome Kinase-Independent TKI Resistance in Chronic Myeloid Leukemia (CML)

Abstract 31

In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CML^CD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562^R and AR230^R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3^Y705 activation despite suppression of BCR-ABL1 kinase activity.

To investigate the role of pSTAT3^Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p<0.008) and increased apoptosis (17.9% increase, p<0.03) of parental K562 cells following imatinib exposure, thereby abolishing the protective effect of extrinsic BM-derived factors. In contrast, shSTAT3 had no effect on cells grown in regular medium. In intrinsic resistance, shSTAT3 reduced clonogenicity of K562^R cells by 58.3% (p<0.0004) and AR230^R cells by 62.2% (p<0.002) in the presence of 1.0 μM imatinib, with no effect on TKI-sensitive controls. A dnSTAT3 mutant lacking the C-terminal activation domain also impaired the clonogenicity of K562^R and AR230^R cells by 36.1% (p<0.003) and 50.0% (p<0.004), respectively. Thus, STAT3 plays a functional role in mediating intrinsic and extrinsic TKI resistance in BCR-ABL1-expressing cell lines.

Preliminary immunofluorescence data indicates that pSTAT3^Y705 levels are higher in drug-resistant CML CD34⁺CD38⁻ stem cells than in CD34⁺CD38⁺ progenitor cells, suggesting a potential role for pSTAT3^Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562^R cells by 68.6% (p<0.03) and AR230^R cells by 72.5% (p<0.0004), with no effect on TKI-sensitive controls. When CML^CD34+ progenitor cells from newly diagnosed patients (n=4) were cultured in the presence of HS-5 CM and treated with the SF1-066/imatinib combination, clonogenicity following drug exposure was reduced by 45.2% (p<0.002) compared to imatinib. Therefore, pharmacologic inhibition of STAT3 and BCR-ABL1 overcomes intrinsic and extrinsic TKI resistance in both CML cell lines and CML^CD34+ progenitor cells.

We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230^R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p<3.6×10⁻⁶) and 51.8% (p<8.6×10⁻⁶), respectively, with little effect on cells expressing a scrambled reporter. Selected inhibitors then provided scaffolds for the synthesis of focused libraries with enhanced potency and selectivity. Immunofluorescence data revealed that two of these inhibitors, BP5-087 and BP5-088, resulted in cytoplasmic accumulation of pSTAT3^Y705 in CML^CD34+ progenitors grown in HS-5 CM, consistent with a block of dimerization. Screening and validation of third-generation STAT3 inhibitors are underway and will be reported.

Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity.