In:
Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 854-854
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood.V122.21.854.854
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2013
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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