Direct Contact With Bone Marrow Stromal Cells Protects CML Progenitors From Imatinib Through Cytoplasmic Stabilization Of β-Catenin
Authors: Eiring Anna M. , Anderson David J. , Zhang Tian Y. , Kraft Ira L. , Reynolds Kimberly R. , Pomicter Anthony D. , Mason Clinton C. , O'Hare Thomas , Deininger Michael W. 
Published: October 2019

Abstract
A β-catenin expression signature is associated with primary resistance to tyrosine kinase inhibitors (TKIs) (McWeeney et al. 2010), but its role in TKI resistance is not completely understood. To assess the role of β-catenin in TKI resistance, we used shRNA targeting β-catenin (shβcat) in in vitro models of BCR-ABL1 kinase-independent resistance. To model resistance in the absence of bone marrow (BM)-derived factors, we used TKI-resistant K562R cells that are adapted for growth in the presence of imatinib, as well as primary CD34+ progenitors from CML patients who had failed treatment with two or more TKIs. These cells are cultured in regular medium (RM), proliferate in 1.0-2.5 μM imatinib, and exhibit β-catenin expression that is independent of BCR-ABL1 kinase activity. To model resistance mediated by the BM microenvironment, we cultured TKI-sensitive parental K562 cells and CD34+ progenitors from newly diagnosed CML patients in direct contact (DC) with HS-5 BM stromal cells. HS-5 co-culture increases β-catenin protein levels and clonogenic potential by >3-fold despite continued suppression of BCR-ABL1 kinase activity. All cells lack BCR-ABL1 kinase domain mutations and undergo TKI-mediated kinase inhibition as detected by immunoblot analyses.
CML cell lines and primary cells with BCR-ABL1-independent resistance were lentivirally transduced with shβcat or a scrambled control (shSCR), and knockdown was confirmed by immunoblot and/or qRT-PCR. In RM, shβcat reduced the clonogenicity of TKI-sensitive K562 cells and CD34+ cells from newly diagnosed CML patients by 49% and 39%, respectively, compared to shSCR controls. In TKI-resistant K562R cells and CD34+ cells from TKI-resistant CML patients, shβcat reduced clonogenicity by 60% and 50%, respectively, in the presence or absence of imatinib (0-10 μM), suggesting a role for β-catenin in the development or maintenance of TKI resistance. In contrast to cells grown in RM, clonogenicity of cell lines and patient samples cultured in HS-5 DC was unaffected by shβcat compared to imatinib alone. Immunoblot analyses revealed that β-catenin protein levels were fully restored in HS-5 DC, despite the continued presence of shβcat. qRT-PCR revealed that while cells in HS-5 DC have high amounts of β-catenin protein, the mRNA levels remained similar to shβcat-expressing cells cultured in RM, consistent with post-translational stabilization of β-catenin. Importantly, increased β-catenin was not observed when cells were cultured in HS-5 conditioned medium, indicating that stabilization requires DC with the bone marrow stroma. These data are consistent with a role for β-catenin in TKI resistance mediated by DC with the BM microenvironment, similar to a recent report (Zhang et al., 2013).
To understand nuclear versus cytoplasmic distribution following HS-5 DC, CD34+ cells from newly diagnosed CML patients were cultured in RM or HS-5 DC for 36 hours and analyzed for β-catenin localization by immunfluorescence. As expected, cells cultured in RM had low levels of β-catenin in the nucleus and cytoplasm that decreased upon treatment with imatinib (2.5 μM). In contrast, cells cultured in HS-5 DC had a marked increase of β-catenin that was unaffected by treatment with imatinib. While detectable in the nucleus, the majority of β-catenin protein was localized in the cytoplasm and at the cell membrane, consistent with its role in cell-cell junctions. Accordingly, in CD34+ cells from newly diagnosed CML patients, an N-cadherin blocking antibody impaired the clonogenic potential of cells cultured in HS-5 DC, with no significant effect on cells grown in RM. Affymetrix Human Gene 1.0 ST arrays revealed high levels of genes encoding the CDH2 and CDH13 cadherins, which may be involved in N-cadherin-mediated β-catenin stabilization, even in the presence of shβcat. Preliminary data also suggests that HS-5 DC reduces luciferase reporter activity from a construct harboring sequential β-catenin binding elements (pGF1-Lef/Tcf-eFGP-luc), further supporting a role for cytoplasmic β-catenin in TKI resistance. These data demonstrate a critical role for β-catenin in BCR-ABL1 kinase-independent TKI resistance, and suggest new strategies for targeting TKI resistance in the absence of BCR-ABL1 kinase domain mutations.

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.