β-Catenin is required for intrinsic but not extrinsic BCR-ABL1 kinase-independent resistance to tyrosine kinase inhibitors in chronic myeloid leukemia

Abstract

Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin has a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.

Figure 1. β-catenin protein expression is upregulated in CML cells under conditions of TKI resistance

a–c. Immunoblot analyses revealed increased levels of β-catenin protein in TKI-resistant K562^R (a, n=6), AR230^R (b, n=4), and CML CD34⁺ cells from TKI-resistant patients lacking BCR-ABL1 kinase domain mutations (c, n=3), all of which exhibit BCR-ABL1 kinase-independent TKI resistance (left). β-catenin protein levels are also maintained in parental K562^S (a, n=3) and AR230^S (b, n=3) cells, as well as CML CD34⁺ cells from newly diagnosed patients (c, n=3), when cultured in direct contact (DC) with HS-5 bone marrow (BM) stromal cells in the presence of 2.5 μM imatinib (right), but not when cultured in HS-5 conditioned medium (CM) (middle).

Figure 2. shβcat reduces survival of CML cells with intrinsic BCR-ABL1 kinase-independent TKI resistance in the presence of imatinib

a. Immunoblot analysis reveals near complete reduction (>90%) of β-catenin protein levels upon lentiviral delivery of shβcat into K562^R (left, n=3) and AR230^R (right, n=3) cells. b–e. shβcat results in reduced in vitro growth (b) and increased apoptosis (c) in the presence of 1 μM imatinib, as well as reduced colony formation (d–e) in semisolid medium in K562^R (left, n=3) and AR230^R (right, n=4) cells. f. shβcat also reduced colony formation of CML CD34⁺ cells from patients exhibiting clinical resistance to multiple TKIs but harboring no BCR-ABL1 kinase domain mutations both with and without imatinib (n=3). g–h. Nucleocytoplasmic fractionation and/or luciferase reporter assays revealed enhanced β-catenin transcriptional activity in intrinsically TKI resistant K562^R (g, n=3) and AR230^R (h, n=3) cell lines compared to parental TKI-sensitive controls. Bars represent standard error of the mean (SEM). *p<0.05.

Figure 3. shRNA-mediated β-catenin knockdown is abolished at the protein but not mRNA level when K562^S cells are co-cultured in direct contact (DC) with HS-5 BM stromal cells

a. Immunoblot analysis demonstrates restored β-catenin protein levels in K562^S-shβcat-expressing cells under conditions of co-culture in DC with HS-5 BM stromal cells (right, n=5). qRT-PCR confirmed maintenance of the knockdown at the mRNA level (left, n=2). Bars represent SEM and qRT-PCR data were normalized to expression of GUS. *p<0.05. b. shβcat increased apoptosis (left, n=3) and reduced colony formation (right, n=3) of K562^S cells cultured in regular medium (RM) or HS-5 conditioned medium (CM), but these effects were reduced or abolished when cultured in DC with HS-5 BM stromal cells. Bars represent SEM. *p<0.05. c. Immunoblot analysis demonstrates restored β-catenin protein levels in K562^S-shβcat-expressing cells under conditions of co-culture in DC with HS-23 and HS-27a BM stromal cells, which are closely related to the HS-5 cells used in these studies (n=2).

Figure 4. The effects of HS-5 co-culture are confirmed in CD34⁺ cells from newly diagnosed CML patients, and treatment of cells with antibodies blocking N-cadherin or H-cadherin reduces survival under conditions of direct contact

a. Immunoblot analysis revealed restored β-catenin protein expression in shβcat-expressing CML CD34⁺ cells cultured in HS-5 DC (left, n=3). qRT-PCR confirmed β-catenin knockdown at the mRNA level (right, n=3). Immunoblots were normalized to α-tubulin and qRT-PCR was normalized to GUS. b. Consistent with restored β-catenin protein levels, HS-5 DC also restored colony forming ability of shβcat-expressing CML CD34⁺ cells under scenarios of DC in the presence of imatinib (n=4). c. Addition of an N-cadherin blocking antibody to co-cultures restored the effects of imatinib and shβcat on colony formation of CML CD34⁺ cells grown in HS-5 DC (n=3). d,e. Addition of antibodies blocking N-cadherin (n=9), H-cadherin (n=8), or both (n=6), to HS-5 co-cultures restored the effects of imatinib on CML CD34⁺ cells cultured under HS-5 DC in the presence of imatinib (d), but did not have an effect on β-catenin protein levels as demonstrated by immunoblot analyses (e). Bars represent SEM. *p<0.05.

Figure 5. WNT5A has no effect on survival of CML CD34⁺ cells cultured in HS-5 DC

a–b. shRNA-mediated knockdown of human WNT5A in HS-5 BM stromal cells was confirmed by both qRT-PCR (a, n=3) and immunoblot (b, n=2) analyses in the presence but not absence of doxycycline (0.1 μg/mL). As expected, the non-silencing control (NSC) did not induce a knockdown, whereas shWNT5A induced ~60% knockdown at both the mRNA and protein level. c. Co-culture of CML CD34⁺ cells with shWNT5A-expressing HS-5 cells in the presence of doxycycline had no effect on colony forming ability in the presence of imatinib (n=3). Bars represent SEM. *p<0.05.

Figure 6. Nucleocytoplasmic fractionation, luciferase reporter activity, and immunofluorescence reveal minimal nuclear β-catenin transcriptional activity under conditions of extrinsic TKI resistance

a,b. Nucleocytoplasmic fractionation (a, n=3) and immunofluorescence staining with a pan-β-catenin antibody (b, n=3) confirms that β-catenin is located primarily within the cytoplasm of CD34⁺ cells from newly diagnosed CML patients, when cultured in RM or HS-5 DC, and when sorted for the CD34⁺38⁻ or CD34⁺38⁺ fractions. c,d. Luciferase reporter assays revealed that transcriptional activity is only minimally increased by HS-5 DC (b, n=5), and that addition of N-cadherin (n=3) or H-cadherin (n=3) blocking antibodies to HS-5 co-cultures results in a slight increase of luciferase reporter activity, while at the same time reducing colony forming ability (see Figure 4). d. Collectively, these data suggest that β-catenin transcriptional activity under conditions of DC does not correlate with survival. Bars represent SEM. *p<0.05.

Figure 7. HS-5 direct contact is sufficient to enhance survival of K562^S cells in the presence of imatinib, despite efficient β-catenin knockdown at 4 h and 8 h of co-culture

a,b. K562^S cells were infected with either shSCR or shβcat and subject to culture in RM or HS-5 DC. A high dose of imatinib (5 μM) was used to push the system during short time points. At the indicated times, all cells were harvested and the GFP⁺ cells were isolated by FACS. Immunoblot analysis revealed that β-catenin stabilization takes at least 24 hours to occur and is not due to contamination by HS-5 stromal cells (a). Colony assays revealed that, despite efficient β-catenin knockdown at 4 h and 8 h of HS-5 co-culture, HS-5 DC still protected cells from the effects of short-term treatment with imatinib (b). Thus, while β-catenin protein is stabilized by HS-5 DC, it is not required for direct contact-mediated protection against TKI treatment. Bars represent SEM. *p<0.05.