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

•Multi-targeted therapies are needed for correcting signaling aberrations in cancers.•Kinase inhibitors rescue retinoic acid maturation therapy in resistant leukemia.•Lyn and Raf inhibitors enhance differentiation; downstream target inhibitors do not.•Downstream factors like ERK and Akt are uncoupled from RA-induced differentiation.•A Lyn/c-Raf module exhibits nontraditional function and ambiguous phosphorylation.

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.