BTK acts as a modulator of the response to imatinib in chronic myeloid leukemia

Abstract

The use of tyrosine kinase inhibitors, such as imatinib, against the chronic myeloid leukemia (CML)-causing kinase BCR::ABL1 has become the model for successful targeted therapy. Nevertheless, drug resistance remains a clinical problem. Analysis of genome-wide expression and genetic aberrations of an in vitro imatinib-resistant CML cell line revealed downregulation of Bruton's tyrosine kinase (BTK), predominantly associated with B cell malignancies, and a novel BTK kinase domain variant in imatinib resistance. This raised the question of the role of BTK in imatinib-resistant CML. In the present study, BTK downregulation and the presence of the BTK variant c.1699_1700delinsAG p.(Glu567Arg) were confirmed in imatinib resistance in vitro. Similarly, BTK inhibition or small interfering RNA-mediated BTK knockdown reduced imatinib susceptibility by 84 and 71%, respectively. BTK overexpression was detrimental to CML cells, as proliferation was significantly reduced by 20.5% under imatinib treatment. In addition, BTK rescue in imatinib-resistant cells restored imatinib sensitivity. The presence of the BTK p.(Glu567Arg) variant increased cell numbers (57%) and proliferation (37%) under imatinib exposure. These data demonstrate that BTK is important for the development of imatinib resistance in CML: Its presence increased drug response, while its absence promotes imatinib resistance. Moreover, the BTK p.(Glu567Arg) variant abrogates imatinib sensitivity. These findings demonstrate a context-dependent role for BTK as an oncogene in B cell malignancies, but as a tumor suppressor in other neoplasms.

Keywords: Bruton's tyrosine kinase; chronic myeloid leukemia; drug resistance; ibrutinib; imatinib; tyrosine kinase inhibitor.

Figure 1.

BTK downregulation and gain of BTK variant p.(Glu567Arg)/E567R in imatinib resistance. (A) BTK mRNA expression in imatinib resistance analyzed by RT-qPCR and normalized to GAPDH, TBP and HPRT1 as housekeeping genes and treatment-naïve K-562 cells. n=3. (B) Immunoblotting of BTK protein levels in imatinib-resistant cells compared with GAPDH. n=3. (C) BTK mRNA expression in response to treatment with 2 µM imatinib for 0 to 24 h analyzed by RT-qPCR and normalized to GAPDH, TBP and HPRT1 and 0 h. n=3. (D) VAF of the BTK dinucleotide variant c.1699_1700delinsAG p.(Glu567Arg)/E567R in imatinib-resistant cell lines (0.5 and 2 µM imatinib) compared with treatment naïve K-562 cells obtained from exome and in-depth sequencing. Error bars indicate standard deviation. *P<0.05, **P<0.01, ***P<0.001 compared with (A) treatment-naïve and (C) 0 h. IM, imatinib; IM-R, imatinib resistance; VAF, Variant allele frequencies; BTK, Bruton's tyrosine kinase; RT-qPCR, reverse transcription-quantitative PCR; HPRT1, Hypoxanthine-guanine phosphoribosyltransferase.

Figure 2.

Inhibition of BTK by ibrutinib hampers susceptibility to low dose imatinib. Cell viability of K-562 cells after exposure to (A) imatinib (0–2,000 nM), (B) ibrutinib (0–1,000 nM), as well as to (C) 100 or (D) 2,000 nM imatinib, respectively, in a dose-dependent combination with ibrutinib for 48 h. Data were normalized to NTC. n=3. Error bars indicate standard deviation. *P<0.05, **P<0.01, ***P<0.001 compared with the respective cell line at 0 nM. IM, imatinib; IM-R, imatinib resistance; BTK, Bruton's tyrosine kinase; NTC, no treatment controls.

Figure 3.

Knockdown of BTK is beneficial for CML cells and promotes imatinib resistance. (A) BTK mRNA expression 24–72 h after small interfering RNA-mediated BTK knockdown measured by RT-qPCR normalized to GAPDH, TBP and HPRT1 as housekeeping genes and negative control-transfected cells (NC). n=3. Cellular fitness after BTK knockdown of (B) K-562 cells and (C) under exposure to 2 µM imatinib analyzed by cell numbers and proliferation rates. Data were normalized to NC. n=3. Error bars indicate standard deviation. *P<0.05, ***P<0.001 compared with 0 h or NC groups using (A) ANOVA with subsequent Dunnett's test or (B) student's t-tests. IM, imatinib; BTK, Bruton's tyrosine kinase; RT-qPCR, reverse transcription-quantitative PCR; HPRT1, Hypoxanthine-guanine phosphoribosyltransferase; NC, negative control.

Figure 4.

BTK modulation alters imatinib susceptibility in treatment naïve and imatinib-resistant CML cells. (A and B) BTK overexpression in treatment naïve K-562 cells. (A) BTK protein level after stable transfection of a BTK-encoding plasmid into K-562 cells analyzed by immunoblotting and compared to GAPDH. (B) Cellular fitness after BTK transfection into K-562 cells under exposure to 2 µM imatinib. (C and D) Restoration of BTK expression in imatinib-resistant K-562 cells. (C) BTK protein level after BTK rescue by plasmid transfection of imatinib-resistant cells analyzed by immunoblotting and compared to GAPDH. (D) Cell numbers, proliferation and cell viability after restoration of BTK expression after 48 h of imatinib treatment. n=3. Data were normalized to NC-transfected cells. Error bars indicate standard deviation. *P<0.05, **P<0.01, ***P<0.001 compared with NC using student's t-tests. IM, imatinib; NC, negative control; BTK, Bruton's tyrosine kinase; pBTK, transfection with an BTK-encoding plasmid.

Figure 5.

Presence of BTK p.(Glu567Lys)/E567K and p.(Glu567Arg)/E567R, but not p.(Glu567Gly)/E567G promotes imatinib resistance. (A) BTK mRNA and protein levels after stable transfection of BTK WT, the variants p.(Glu567Lys)/E567K, p.(Glu567Gly)/E567G and p.(Glu567Arg)/E567R and the NC into treatment naïve K-562 cells analyzed by immunoblotting compared to GAPDH (top) and by RT-qPCR normalized to GAPDH, TBP and HPRT1 (bottom). n=3. Presence of the variants in the stable transfected cell lines was confirmed using Sanger sequencing. (B) Cell fitness after overexpression of BTK WT or the variants E567K, E567G or E567R after exposure to 100 nM ibrutinib. n=3. Data were normalized to WT. (C) Cell number, proliferation and cell viability of BTK variant cell lines after treatment with 2 µM imatinib compared to WT cells. n=3. Data were normalized to WT. Error bars indicate standard deviation. *P<0.05, **P<0.01, ***P<0.001 compared with WT. WT, wild-type; BTK, Bruton's tyrosine kinase; NC, empty vector-control; RT-qPCR, reverse transcription-quantitative PCR; HPRT1, Hypoxanthine-guanine phosphoribosyltransferase; NC, negative control.