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.

Keywords: Hippo pathway; YAP; chemoresistance; imatinib mesylate; leukemic stem cells; tyrosine kinase inhibitor.

FIG. 1.

NOTCH1, STAT3, and CD44 expression in hematopoietic progenitor cells obtained from CML patients. (A) Affymetrix Human Genome U219 Array results for NOTCH1, STAT3, CD44 expression were analyzed. Analyses were carried out on CD34⁺ cells obtained from the bone marrow of CML patients at diagnosis (n = 6, pre-IM) and 1 week after the initiation of therapy (n = 6, post-IM; top three panels), and at diagnosis (n = 9, pre-IM), and 4 weeks after initiation of therapy (n = 9, post-IM; lower three panels). The following probe sets were evaluated using the deposited GEO GSE12211 and GSE33075 databases (GPL571 and GPL570 platforms, respectively). The status of expression of probes 218902_at for NOTCH1, 208991_at for STAT3, and 204489_s_at for CD44 is presented as interquartile range and median of normalized, log-transformed raw data. P-values are denoted on the graphs. (B) Hematopoietic progenitor cells were obtained from CML patients (n = 3) before initiation of TKI therapy. NOTCH1, STAT3, and CD44 expression was determined on CML CD34⁺ cells cultured with or without 5 μM IM for 1 week. Normalized expression relative to ACTB expressed as fold change, is presented. Samples were cultured and run in triplicate **P < 0.01. CML, chronic myelogenous leukemia; IM, imatinib mesylate.

FIG. 2.

Stem cell markers and sensitivity to IM in K562 and IM-resistant K562-STI-R cells. (A) Expression of NANOG, POU5F1, SOX2, CITED2, SALL4, and TAL1 was determined in K562 or IM-resistant K562-STI-R cells. Data represent normalized expression relative to ACTB, S18, and GAPDH. Samples were run in triplicate. Representative data from three independent analyses are shown. ***P < 0.001 (B) Expression of CD45, CD44, CXCR4, VLA-4, CD24, NOTCH1, and CD34 was determined by FACS in K562 and K562-STI-R cells. 3D histograms and a bar graph summary of the quantitative results are presented. Representative data from three independent analyses are shown. ***P < 0.001 (C) Coexpression of CD44 and CD24 by FACS was determined for K562 and K562-STI-R cells. Analyses were performed in quadruplicate. Representative data are shown. (D) FACS analyses were used to determine the percentage of apoptotic cells in a population of K562 and K562-STI-R cells subjected to incremental concentrations of IM for 24 h. The percent Annexin V-FITC-positive cells was determined. Representative data from three independent analyses are shown *P < 0.05.

FIG. 2.

Stem cell markers and sensitivity to IM in K562 and IM-resistant K562-STI-R cells. (A) Expression of NANOG, POU5F1, SOX2, CITED2, SALL4, and TAL1 was determined in K562 or IM-resistant K562-STI-R cells. Data represent normalized expression relative to ACTB, S18, and GAPDH. Samples were run in triplicate. Representative data from three independent analyses are shown. ***P < 0.001 (B) Expression of CD45, CD44, CXCR4, VLA-4, CD24, NOTCH1, and CD34 was determined by FACS in K562 and K562-STI-R cells. 3D histograms and a bar graph summary of the quantitative results are presented. Representative data from three independent analyses are shown. ***P < 0.001 (C) Coexpression of CD44 and CD24 by FACS was determined for K562 and K562-STI-R cells. Analyses were performed in quadruplicate. Representative data are shown. (D) FACS analyses were used to determine the percentage of apoptotic cells in a population of K562 and K562-STI-R cells subjected to incremental concentrations of IM for 24 h. The percent Annexin V-FITC-positive cells was determined. Representative data from three independent analyses are shown *P < 0.05.

FIG. 3.

Effect of long-term exposure to IM on the Hippo pathway and YAP. (A) Volcano plots showing fold change versus q-value of phosphopeptide peak areas were generated. Of the 4,807 unique phosphopeptides identified, 36.9% peptides (red in inset pie chart) showed a statistically significant (q < 0.05) difference and an absolute fold change greater than 2 comparing the K562-STI-R cells to control K562 cells. Within this group of significantly different peptides, 88.3% showed lower abundance in the K562-STI-R cells. (B) The five most highly scored pathways identified in K562-STI-R cells reflect the enrichment analysis of gene IDs corresponding to the proteins for which identified phosphopeptides showed statistically a significant (q < 0.05) fold change >2. (C) This schematic representation of Hippo/YAP cross-talk shows phosphosites that were significantly different in K562-STI-R cells versus control K562 cells. (D) Western blot analysis was performed on K562 and K562-STI-R whole cell lysates. Levels of Sav1, Lats1, Mob, and YAP along with phosphorylation of YAP on Ser127 and 397 were evaluated. Data from three independent, replicate cultures of each cell line representing three independent experiments are shown. (E) RT-PCR was performed to quantify AREG, BIRC5, and CTGF expression in K562 and IM-resistant K562-STI-R cells. Data are presented as normalized expression relative to ACTB. Samples were run in triplicate. Representative data from three independent analyses are shown *P < 0.05.

FIG. 3.

Effect of long-term exposure to IM on the Hippo pathway and YAP. (A) Volcano plots showing fold change versus q-value of phosphopeptide peak areas were generated. Of the 4,807 unique phosphopeptides identified, 36.9% peptides (red in inset pie chart) showed a statistically significant (q < 0.05) difference and an absolute fold change greater than 2 comparing the K562-STI-R cells to control K562 cells. Within this group of significantly different peptides, 88.3% showed lower abundance in the K562-STI-R cells. (B) The five most highly scored pathways identified in K562-STI-R cells reflect the enrichment analysis of gene IDs corresponding to the proteins for which identified phosphopeptides showed statistically a significant (q < 0.05) fold change >2. (C) This schematic representation of Hippo/YAP cross-talk shows phosphosites that were significantly different in K562-STI-R cells versus control K562 cells. (D) Western blot analysis was performed on K562 and K562-STI-R whole cell lysates. Levels of Sav1, Lats1, Mob, and YAP along with phosphorylation of YAP on Ser127 and 397 were evaluated. Data from three independent, replicate cultures of each cell line representing three independent experiments are shown. (E) RT-PCR was performed to quantify AREG, BIRC5, and CTGF expression in K562 and IM-resistant K562-STI-R cells. Data are presented as normalized expression relative to ACTB. Samples were run in triplicate. Representative data from three independent analyses are shown *P < 0.05.

FIG. 4.

The effect of IM exposure on expression of YAP1 and WWTR1 (TAZ) (A) Affymetrix Human Genome U219 Array data were used to examine YAP1 and WWTR1 (TAZ) transcript expression. CD34⁺ cells obtained from the bone marrow of CML patients at diagnosis (n = 6, pre-IM) and 1 week after the initiation of therapy (n = 6, post-IM), and at diagnosis (n = 9, pre-IM) and 4 weeks after the initiation of therapy (n = 9, post-IM). The following probe sets were evaluated in GEO GSE12211 and GSE33075 213342_at for YAP1 and 202134_s_at for WWTR (TAZ)1, expression of the probes is presented as interquartile range and median of normalized, log-transformed raw data. P-values are denoted on the graphs. (B) YAP and WWTR1 expression in CML CD34⁺ cells cultured in the presence of 5 μM IM was determined. CML CD34⁺ cells were obtained from the bone marrow of three CML patients prior initiation of TKI therapy. Normalized expression relative to ACTB expressed as fold change, is presented. Samples were cultured and run in triplicate. Representative data from three independent analyses are shown **P < 0.01.

FIG. 5.

Effect of long-term exposure to IM on levels of miR-181a. Taqman Human MicroRNA A (Card v2.0) Array data were used to examine miR-181a expression in unselected CML patients from the STOP-IM group (n = 7) and 7 healthy volunteers (n = 7). The hsa-miR-181a-4373117 probe set data were extracted from the GEO GSE75392 (platform GPL13987) data set. The geometric mean of normalized, log-transformed raw data is presented. P-value is denoted on the graph.

FIG. 6.

The effects of an miR-181a sponge inhibitor on IM-resistant K562-STI-R cells. (A) Schematic representation of the miR-181a sponge and the control sponge. The central mismatch at positions 11–14 (in which the nucleotide at position 14 was deleted) in each miRNA-binding site is shown in blue. The scrambled sequence for the control sponge is shown in red. Overhangs compatible with the restriction endonuclease SanDI are shown in underlined, bold font. (B) RT-PCR analyses of the levels of miR-181a in K562-STI-R cells electroporated with the control vector or that encoding the miR-181a sponge inhibitor. Expression relative to RU19 and RU6B is shown as fold difference. Triplicate analyses representative of data from three independent experiments are shown. ***P < 0.001 (C) FACS analyses of the percentage of apoptotic, Annexin V-FITC-positive K562-STI-R cells electroporated with the control vector or the miR-181a sponge inhibitor. Data represent four independent experiments. (D) K562-STI-R cells electroporated with the control vector or the miR-181a sponge inhibitor were cultured for 24 h. Cell count was determined. Data from three independent experiments are presented.

FIG. 7.

Effect of miR-181a on SOX2 and SALL4 expression, YAP activity, and sensitivity to IM. (A) Analysis of expression of POU5F1, SOX2, CITED2, and SALL4 in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB, S18, and GAPDH is presented as fold difference. Triplicate analyses representing three independent experiments are shown. *P < 0.05 (B) Analyses of CD45, CD44, CXCR4, VLA-4, CD24, NOTCH1, and CD34 expression by FACS in K562-STI-R expressing the control sponge or the miR-181a sponge inhibitor. Representative data from three independent analyses are shown. (C) Western blot analysis was performed on the whole cell lysates prepared from K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Levels of Lats1, Mob, and YAP along with phosphorylation of YAP on Ser127 and 397 were evaluated. Data from two independent electroporation reactions using control (ctrl sp: 1, 2) and miR-181a sponge (miR-181a sp: 1, 2) are presented. The results are representative of three independent experiments. Immunofluorescent analysis was performed to examine the cellular distribution of YAP (D) and F-actin (E). K562-STI-R cells were electroporated with GFP-tagged control sponge or miR-181a sponge. Anti-YAP followed by secondary antibody conjugated with AlexaFluor 555 and phalloidin conjugated with AlexaFluor 555 were used to visualize YAP and F-Actin, respectively (red), and DAPI (blue) was used to stain the nucleus. Confocal images were acquired with a Nikon C1si confocal. Z series sections were collected at 0.2 μm with a 60 × PlanApo objective. (F) RT-PCR analysis of AREG, BIRC5, and CTGF expression in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB and GAPDH is presented as fold difference. The triplicate analyses are representative of data from three independent experiments. *P < 0.05 (G) FACS analyses of the percentage of apoptotic cells in a population of K562-STI-R cells expressing control or miR-181a sponge inhibitor subjected to the incremental concentrations of IM. Cells 16 h after electroporation with vectors encoding the control sponge or the miR-181a sponge inhibitor were cultured for 24 h in the presence of IM. The percent of Annexin V-FITC-positive cells was determined. Representative data from three independent analyses are shown *P < 0.05.

FIG. 7.

Effect of miR-181a on SOX2 and SALL4 expression, YAP activity, and sensitivity to IM. (A) Analysis of expression of POU5F1, SOX2, CITED2, and SALL4 in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB, S18, and GAPDH is presented as fold difference. Triplicate analyses representing three independent experiments are shown. *P < 0.05 (B) Analyses of CD45, CD44, CXCR4, VLA-4, CD24, NOTCH1, and CD34 expression by FACS in K562-STI-R expressing the control sponge or the miR-181a sponge inhibitor. Representative data from three independent analyses are shown. (C) Western blot analysis was performed on the whole cell lysates prepared from K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Levels of Lats1, Mob, and YAP along with phosphorylation of YAP on Ser127 and 397 were evaluated. Data from two independent electroporation reactions using control (ctrl sp: 1, 2) and miR-181a sponge (miR-181a sp: 1, 2) are presented. The results are representative of three independent experiments. Immunofluorescent analysis was performed to examine the cellular distribution of YAP (D) and F-actin (E). K562-STI-R cells were electroporated with GFP-tagged control sponge or miR-181a sponge. Anti-YAP followed by secondary antibody conjugated with AlexaFluor 555 and phalloidin conjugated with AlexaFluor 555 were used to visualize YAP and F-Actin, respectively (red), and DAPI (blue) was used to stain the nucleus. Confocal images were acquired with a Nikon C1si confocal. Z series sections were collected at 0.2 μm with a 60 × PlanApo objective. (F) RT-PCR analysis of AREG, BIRC5, and CTGF expression in K562-STI-R cells expressing the control sponge or the miR-181a sponge inhibitor. Normalized expression relative to ACTB and GAPDH is presented as fold difference. The triplicate analyses are representative of data from three independent experiments. *P < 0.05 (G) FACS analyses of the percentage of apoptotic cells in a population of K562-STI-R cells expressing control or miR-181a sponge inhibitor subjected to the incremental concentrations of IM. Cells 16 h after electroporation with vectors encoding the control sponge or the miR-181a sponge inhibitor were cultured for 24 h in the presence of IM. The percent of Annexin V-FITC-positive cells was determined. Representative data from three independent analyses are shown *P < 0.05.