Long-Term Exposure to Imatinib Mesylate Downregulates Hippo Pathway and Activates YAP in a Model of Chronic Myelogenous Leukemia

Abstract

Despite the success of tyrosine kinase inhibitor (TKI) therapy in chronic myelogenous leukemia (CML), leukemic stem/progenitor cells remain detectable even in the state of deep molecular remission. Mechanisms that allow them to persist despite continued kinase inhibition remain unclear. We have previously shown that prolonged exposure to imatinib mesylate (IM) results in dysregulation of Akt/Erk 1/2 signaling, upregulation of miR-181a, enhanced adhesiveness, and resistance to high IM. To characterize the molecular basis and reversibility of those effects, we applied gene and protein expression analysis, quantitative phosphoproteomics, and direct miR-181a inhibition to our cellular model of CML cells subjected to prolonged exposure to IM. Those cells demonstrated upregulation of pluripotency markers (SOX2, SALL4) and adhesion receptors (CD44, VLA-4, CXCR4), as well as downregulation of Hippo signaling and upregulation of transcription coactivator YAP. Furthermore, inhibition of miR-181a using a microRNA sponge inhibitor resulted in decreased transcription of SOX2 and SALL4, decreased activation of YAP, and increased sensitivity to IM. Our findings indicate that long-term exposure to IM results in dysregulation of stem cell renewal-regulatory Hippo/YAP signaling, acquisition of expression of stem cell markers and that experimental interference with YAP activity may help to restore chemosensitivity to TKI.

Overexpression of Tpl2 is linked to imatinib resistance and activation of MEK‐ERK and NF‐κB pathways in a model of chronic myeloid leukemia

“…The human CML K562 cell line and its IM-resistant counterpart, clone K562-STI-R, were established and grown as described previously (Chorzalska et al, 2017). K562 and K562-STI-R cell lines are routinely tested for the presence of the hematopoietic cell marker CD45 by FACS and for the presence of Bcr-Abl1 oncogene by immunoblotting.…”

“…Total RNA from CD34+ cells cultured in the presence of 5 lM IM was purified using an RNeasy Plus Mini Kit (Qiagen Hilden, Germany). RT/PCR was performed as described (Chorzalska et al, 2017). The following primers were designed for transcript quantification: MAP3K8: For: 5 0 ATGGAGTAC-ATGAGCACTGGA3 0 and Rev: 5 0 GCTGGCTCTTC ACTTGCATAAAG3 0 ; NFKB1: For: 5 0 AACAGAGA-GGATTTCGTTTCCG3 0 and Rev: 5 0 TTTGACCT-GAGGGTAAGACTTCT3 0 ; ACTB: For: 5 0 CATG TACGTTGCTATCCAGGC3 0 and Rev: 5 0 CTCCTT AATGTCACGCACGAT3 0 , S18 rRNA: For: 5 0 GGG CGGAGATATGCTCATGTG3 0 and Rev: 5 0 TCTG GGATCTTGTACTGTCGT3 0 .…”

“…K562 or K562-STI-R cells in a logarithmic growth phase were subjected to phosphopeptide enrichment and proteomic analysis as described previously (Chorzalska et al, 2017). Five independently revived and expanded cellular clones per cell line were harvested, lysed, and used to prepare tryptic digested peptides.…”

“…MS/MS spectra were searched against a human-specific database (UniProt; downloaded 2/1/2013) using MASCOT v. 2.4 (Matrix Science, Ltd, London, UK). Peptide assignments from the database search were filtered down to 1% false discovery rate (FDR) by a logistic spectral score, as previously described (Chorzalska et al, 2017;Elias and Gygi, 2007;Yu et al, 2009). To validate the position of phosphorylation sites, the Ascore algorithm (Beausoleil et al, 2006) was applied and the reported phosphorylation site position reflected the top Ascore prediction.…”

“…The role of Bcr-Abl1 signaling in the regulation of these pathways in CML stem cells remains unclear, and the effect of long-term TKI exposure on these cascades is not understood. Notably, recent reports indicate that prolonged treatment with TKI supports stem cell-like characteristics of primitive, leukemia-initiating CML stem cells (Charaf et al, 2017;Chorzalska et al, 2017).…”