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Comparative Study
. 2015 Aug;27(8):1666-75.
doi: 10.1016/j.cellsig.2015.03.014. Epub 2015 Mar 26.

GW5074 and PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn function as drivers of retinoic acid-induced maturation

Holly A Jensen  1 , Rodica P Bunaciu  2 , Jeffrey D Varner  1 , Andrew Yen  2
Affiliations
Comparative Study

GW5074 and PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn function as drivers of retinoic acid-induced maturation

Holly A Jensen et al. Cell Signal. 2015 Aug.

Abstract

The multivariate nature of cancer necessitates multi-targeted therapy, and kinase inhibitors account for a vast majority of approved cancer therapeutics. While acute promyelocytic leukemia (APL) patients are highly responsive to retinoic acid (RA) therapy, kinase inhibitors have been gaining momentum as co-treatments with RA for non-APL acute myeloid leukemia (AML) differentiation therapies, especially as a means to treat relapsed or refractory AML patients. In this study GW5074 (a c-Raf inhibitor) and PP2 (a Src-family kinase inhibitor) enhanced RA-induced maturation of t(15;17)-negative myeloblastic leukemia cells and rescued response in RA-resistant cells. PD98059 (a MEK inhibitor) and Akti-1/2 (an Akt inhibitor) were less effective, but did tend to promote maturation-uncoupled G1/G0 arrest, while wortmannin (a PI3K inhibitor) did not enhance differentiation surface marker expression or growth arrest. PD98059 and Akti-1/2 did not enhance differentiation markers and have potential, antagonistic off-targets effects on the aryl hydrocarbon receptor (AhR), but neither could the AhR agonist 6-formylindolo(3,2-b)carbazole (FICZ) rescue differentiation events in the RA-resistant cells. GW5074 rescued early CD38 expression in RA-resistant cells exhibiting an early block in differentiation before CD38 expression, while for RA-resistant cells with differentiation blocked later, PP2 rescued the later differentiation marker CD11b; but surprisingly, the combination of the two was not synergistic. Kinases c-Raf, Src-family kinases Lyn and Fgr, and PI3K display highly correlated signaling changes during RA treatment, while activation of traditional downstream targets (Akt, MEK/ERK), and even the surface marker CD38, were poorly correlated with c-Raf or Lyn during differentiation. This suggests that an interrelated kinase module involving c-Raf, PI3K, Lyn and perhaps Fgr functions in a nontraditional way during RA-induced maturation or during rescue of RA induction therapy using inhibitor co-treatment in RA-resistant leukemia cells.

Keywords: Inhibitors; Leukemia; Lyn; Resistance; Retinoic acid; c-Raf.

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Figures

Fig. 1
Fig. 1
Phenotypic markers during RA and kinase inhibitor co-treatment in HL-60. Wild-type (WT), R38+ and R38−HL-60 cells were treated for 48 h with 1 μM RA, or RA combined with 2 μM PD98059 (PD), 2 μM GW5074 (GW), 1 μM wortmannin (Wo), or 1 μM Akti-1/2 (Akti) and analyzed by flow cytometry for (A) CD38 expression, (B) CD11b expression, or (C) G1/G0 cell cycle arrest. Error bars represent standard error. Asterisks for p-values of treatment group means compared to control indicate whether p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**) or p < 0.05 (*). (A) CD38 is maximally expressed in WT and R38+ HL-60 and not inhibited by any kinase inhibitor treatment. GW5074 significantly increase CD38 expression in R38−cells. (B) CD11b is increased by RA and enhanced by GW5074 in WT HL-60 cells, while all other co-treatments reduced the RA-induced CD11b expression. GW5074 can significantly increase CD11b expression in R38+ and R38−. (C) GW5074 can enhance RA-induced G1/G0 arrest in WT HL-60 and rescue G1/G0 arrest in R38+ and R38−. Akti-1/2 and PD98059 also tended to increase G1/G0 arrest.
Fig. 2
Fig. 2
Signaling network of factors investigated in this study and hierarchical clustering. (A) Simplified diagram of the interactions and functional relationships between the proteins investigated in this study, curated from literature: Bunaciu and Yen 2013 Mol Can; Bunaciu et al. submitted; Yim et al. 2004 Biochem Biophys Res Commun; Congleton et al. 2012 Leukemia; Congleton et al. 2014 Cell Signal; Bunda et al. 2014 Oncogene; Dhillon et al. 2007 Oncogene; Steelman et al. 2011 Aging; Zimmermann and Moelling 1999. The targets affected by the inhibitors used in this study are indicated. (B) Repeat Western blot data was quantified using ImageJ and subject to hierarchical clustering analysis using the Pearson correlation coefficient as a distance metric and an average linkage method (see Section 2). Data includes both cytoplasmic and nuclear signaling factor expression and phosphorylation for wild-type (Supplemental figs. S2–7), R38+ (Supplemental figs. S2–7) and R38− (Supplemental figs. S2–7) treated with 1 μM RA or RA combined with 2 μM PD98059, 2 μM GW5074, 1 μM wortmannin, 1 μM Akti-1/2 or 10 μM PP2. Distances between clusters (1 — Pearson correlation coefficient) are indicated on the x-axis.
Fig. 3
Fig. 3
Phenotypic markers during combined GW5074, PP2 and RA treatment. R38+ and R38− HL-60 cells were treated for 48 h with RA, or RA combined with GW5074 (GW) and/or PP2 and analyzed by flow cytometry for (A) CD38 and CD11b expressions and (B) G1/G0 cell cycle arrest. PP2-treated R38+ and R38− HL-60 cells have been reported previously [25] but are recapitulated here for clear comparison. Error bars represent standard error. Asterisks for p-values of treatment group means compared to control indicate whether p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**) or p < 0.05 (*). (A) GW5074-induced CD38 expression in higher than PP2 but GW5074-induced CD11b is reduced in R38+ and R38− cells. Combined PP2 + GW5074 with or without RA enhances markers even more in R38+ but actually decreases marker expression compared to GW5074 alone in R38−. (B) GW5074 does not appear to greatly increase the PP2-induced G1/G0 arrest in either RA-resistant HL-60 cell line.
Fig. 4
Fig. 4
Quantified signaling factor expression during combined GW5074, PP2 and RA treatment and correlation analysis. 48 h Western blot data (at least three repeats) of total lysates were quantified using ImageJ and fold change to respective R38+ or R38− control calculated. Error bars represent standard error. p-value analysis cannot be performed on quantified blot data, which is nonlinear. Instead a representative blot is displayed beneath each graph. GAPDH loading controls were performed to ensure even loading (not shown). (A) Signaling factors for c-Raf and Src-family kinase events and activated ERK were assessed for R38+ or R38− cell treated with PP2, PP2 + RA (reported previously in [25]), GW5074 (GW), GW + RA, PP2 + GW and PP2 + GW + RA. Since quantified data is estimated from immunoblot images in which signal detection may or may not have been in the linear range, p-value analysis is not applicable to quantified blot data. (B) Signaling factors for CD38-associated factors c-Cbl, Vav1 and Slp76, the p47phox protein. (C) Pearson correlation coefficient matrix between MAPK and Src-family kinase signaling factor events from Fig. 4A and phenotypic results from Fig. 3A, i.e. for R38+ and R38− across treatments with PP2, PP2 + RA, GW5074, GW5074 + RA, PP2 + GW5074 and PP2 + GW5074 + RA. Colorbar: white (1) indicates positive correlation, mid-gray (0) indicates no correlation, black (−1) indicates negative correlation. (D) Hierarchical clustering analysis of data presented in Fig. 4A using the Pearson correlation coefficient as a distance metric and an average linkage method (see Section 2). Distances between clusters (1 — Pearson correlation coefficient) are indicated on the x-axis.
Fig. 5
Fig. 5
Treatment of wild-type, R38+ and R38− HL-60 with the AhR agonist FICZ. Wild-type (WT), R38+ and R38− were treated for 48 h with RA or RA and the AhR agonist FICZ and analyzed by flow cytometry for (A) CD38 expression, (B) CD11b expression, or (C) G1/G0 cell cycle arrest. Asterisks for p-values of treatment group means compared to control indicate whether p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**) or p < 0.05 (*). FICZ treatment does not significantly rescues (A) CD38 expression, (B) CD11b expression or (C) G1/G0 cell cycle arrest in either RA-resistant cell line.
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