BIRC6 mRNA, protein, and BIRC6 phosphopeptide are higher in imatinib resistant MYL-R cells.

<p>(A) Sequence and position of the BIRC6 phosphopeptide. (B) Short-term ponatinib treatment of MYL-R cells reduced BIRC6 phosphopeptide. The BIRC6 phosphopeptide was isolated from cell lysates of MYL, MYL-R, and MYL-R cells treated with 10 nM ponatinib or 0.1% DMSO for 1 hour. Label-free quantification of mass spectral data was done using MaxQuant and normalized to MYL. (C and D) Short-term ponatinib treatment suppressed Bcr-Abl and Lyn signaling in MYL-R cells. Total BIRC6 and Mcl-1 proteins were not affected. Immunoblot analyses of the same lysates were performed to validate changes in Bcr-Abl and Lyn observed from the MIB/MS data. (E and F) BIRC6 mRNA and protein were elevated in MYL-R cells compared to MYL and K562 cells. QRT-PCR and immunoblot analyses were performed as described in Materials and Methods on parental MYL, MYL-R, and K562 CML cell lines to examine BIRC6 expression. * Represents <i>p < 0</i>.<i>05</i>. Data are representative of three independent experiments.</p

CDK9 regulates BIRC6 mRNA levels.

<p>(A) Inhibition of CDK9 with dinaciclib, HY-16462, and flavopiridol significantly reduced BIRC6 and Mcl-1 mRNA levels in a dose-dependent manner. MYL-R cells were treated with dinaciclib (10 or 100 nM), HY-16462 (250 nM or 1 μM), and flavopiridol (250 nM or 1 μM) for 24 hours and BIRC6 and Mcl-1 mRNA measured by QRT-PCR. (B) Immunoblot analyses of lysates from the same conditions in (A) above had no substantial effect on BIRC6 protein. By contrast, Mcl-1 protein levels were reduced in a dose-dependent manner except with 10 nM dinaciclib that showed a substantial increase in Mcl-1 protein. (C and D) shRNA knockdown of CDK9 in MYL-R cells and immunoblot analysis recapitulated the data obtained in (B) above. The data presented here are representative of three independent experiments. * Represents <i>p < 0</i>.<i>05</i>.</p

Lyn knockdown reduced the half-life of BIRC6 protein in MYL-R cells.

<p>(A) MYL-R cells were infected with lentiviral particles containing anti-Lyn shRNA constructs (shLyn-01 and -04), and Lyn knockdown confirmed by immunoblot analysis. (B) Lyn knockdown in MYL-R cells reduced the half-life of BIRC6 4-fold (~24 hrs in shCtrl cells to ~ 6 hrs in Lyn knockdown cells). Lyn knockdown MYL-R cells were incubated with 50 μg/mL cycloheximide (CHX) in a time-course manner and BIRC6 protein determined by immunoblotting. (C) BIRC6 half-life in MYL cells was ~24 hours. MYL cells were treated with CHX as in (B) and BIRC6 protein determined by immunoblotting. (D) GraphPad^™ Prism was used to plot change in BIRC6 stability over time upon Lyn-knockdown in MYL-R cells as described in (B). The BIRC6 stability data presented here are representative of two independent experiments.</p

BIRC6 mediates drug resistance in MYL-R cells independently of Mcl-1.

<p>(A) Anti-BIRC6 shRNAs were used to knock down BIRC6 in MYL-R cells. Immunoblot analyses showed that shBIRC6-58, -59, and -61 yielded efficient knockdown without affecting Mcl-1. (B) MYL-R cells were resistant to imatinib (IC50 ~3.0 μM) as compared to MYL cells (IC50 ~0.2 μM), and (C) BIRC6 knockdown sensitized MYL-R cells to imatinib (IC50 ~0.2 μM). MYL, MYL-R, and BIRC6 knockdown MYL-R cells were cultured in triplicate in 96-well plates with increasing concentrations of imatinib for 72 hours, and cell viability was assessed by MTS assay. (D) Treatment of BIRC6 knockdown MYL-R cells with imatinib showed elevation in caspase-3/7 activity. BIRC6 knockdown MYL-R cells were treated with 1 μM imatinib for 24 hours. Parental MYL-R cells were treated with DMSO or 1 nM dasatinib or 1 μM imatinib. Caspase-3/7 activity was measured using a fluorogenic substrate as described in Materials and Methods. (E) BIRC6 knockdown or imatinib treatment in MYL-R cells did not affect total Lyn or total Mcl-1 protein levels. BIRC6 knockdown MYL-R cells were treated with DMSO or 300nM or 1 μM imatinib (IM) for 24 hours, and immunoblot analyses used to measure total Lyn and total Mcl-1 proteins. (F) Knockdown of BIRC6 in MYL-R cells increased sensitivity to gemcitabine. BIRC6 knockdown MYL-R cells were cultured as described in (C) with increasing concentrations of gemcitabine for 72 hours and cell viability determined by MTS assay. The data presented in this figure are representative of at least three independent experiments, and * represents <i>p < 0</i>.<i>05</i>.</p

Lyn regulates caspase-mediated degradation of BIRC6 in MYL-R cells.

<p>(A) Caspase inhibitors, but not proteasome inhibitors inhibited ponatinib-mediated BIRC6 degradation. MYL-R cells were incubated with 10 nM ponatinib or 0.1% DMSO and/or a pan-caspase inhibitor (Z-VAD-FMK 10 or 20 μM) and/or the proteasome inhibitor MG132 (300 nM) for 24 hours and BIRC6 protein examined by immunoblotting. Inhibition of Lyn (or Src family kinases) was validated by the loss in phospho-Src family (Y416). (B) Inhibition of Lyn in MYL-R cells caused a 30% reduction in BIRC6 protein and induced PARP cleavage as determined by immunoblot analyses. MYL-R cells were incubated with 5 nM ponatinib or 0.1% DMSO for 24 hours and BIRC6 protein measured. Induction of apoptosis was demonstrated by PARP cleavage. (C) Inhibition of Lyn in MYL-R cells caused cytochrome c release from mitochondria. MYL-R cells were treated with 5 nM ponatinib or 0.1% DMSO for 24 hours and mitochondria enriched using the Cytochrome C Releasing Apoptosis Assay Kit. Cytochrome c release was measured by immunoblotting cytosolic and mitochondrial fractions. Erk2 and Hsp60 were used as cytosolic and mitochondrial markers respectively. The data presented here are representative of three independent experiments.</p

Combined MIB/MS and phosphopeptide enrichment strategy for studying proteome dynamics in CML cells.

<p>MIB/MS was used to study kinome dynamics in MYL, MYL-R, and MYL-R cells treated with ponatinib (10 nM, 1 hr.). In parallel, phosphoproteomics was used to study global phosphorylation differences from the same cells. Identification of peptides was accomplished by LC-MS/MS and label-free quantification (LFQ) of mass spectral data was performed using MaxQuant and the integrated ANDROMEDA search engine [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177871#pone.0177871.ref061" target="_blank">61</a>].</p

Lyn kinase regulates BIRC6 expression.

<p>(A) Treatment of MYL-R cells with dasatinib significantly increased BIRC6 and Mcl-1 mRNA levels as determined by QRT-PCR. MYL-R cells were treated with dasatanib (1 nM or 5 nM, 24 hours) and BIRC6 and Mcl-1 mRNA levels measured by QRT-PCR. (B) Immunoblotting of lysates from the same cells showed that dasatinib treatment reduced BIRC6 protein in MYL-R cells in a dose-dependent manner. (C) Ponatinib treatment of MYL-R cells recapitulated BIRC6 protein reduction observed with dasatinib. MYL-R cells were treated for 24 hours with increasing concentrations of ponatinib and immunoblot analyses used to measure BIRC6, Mcl-1, Lyn, and phospho-Src family (Y416) protein levels. Whereas BIRC6, phospho-Src family (Y416), and Mcl-1 were reduced in a dose-dependent manner, total Lyn was increased. (D) Whereas shRNA knockdown of Lyn in MYL-R cells substantially reduced BIRC6 protein, but not Mcl-1, (E) both BIRC6 and Mcl-1 mRNA levels were significantly increased by the more efficient Lyn knockdown shRNA constructs (shLyn-05 and -06). MYL-R cells were infected with lentiviral particles containing shRNA directed against Lyn. Upon selection of stably transduced cells, BIRC6 and Mcl-1 protein and mRNA levels were measured by immunoblotting and QRT-PCR. (F) Lyn knockdown in MYL-R cells was sufficient to increase caspase-3/7 activity. Data was normalized to shCtrl. (G) Ponatinib treatment of MYL-R cells significantly increased caspase-3/7 activity. MYL-R cells were treated with 1 nM, 5 nM, or 10 nM ponatinib or 0.1% DMSO for 24 hours and caspase-3/7 activity measured. The data are representative of three independent experiments, and * represents <i>p < 0</i>.<i>05</i>.</p