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

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.

Synthesis and Antiproliferative Effect of Ethyl 4‐[4‐(4‐Substituted Piperidin‐1‐yl)]benzylpyrrolo[1,2‐a]quinoxalinecarboxylate Derivatives on Human Leukemia Cells

“…Thus, we recently published four series of new pyrrolo[1,2‐ a ]quinoxaline derivatives (Figure ) endowed with interesting activity toward human leukemia cells . These antileukemia pyrroloquinoxaline compounds have been previously reported as new structural analogues of derivative A6730, a reference Akt inhibitor that exhibits antiproliferative activity against various human leukemia cell lines …”

“…Taking into account the best results obtained in the previous series (A–B, Figure ), we decided to refer to the JG564 and JG572 pyrrolo[1,2‐ a ]quinoxaline pharmacophores as a template for the design of new compounds 1 a – o , in which the pyrrolo[1,2‐ a ]quinoxaline ring is replaced in various positions by an ethyl ester functionality (Figure ). In addition, further modulations on the piperidine core were also considered, such as the introduction of new substituted heterocyclic moieties (benzimidazolone, fluorobenzimidazole, and pyridinyltriazole substituents), in analogy to previously described antileukemia compounds . The antiproliferative activity of the resulting derivatives ( 1 a – o ) was then evaluated in vitro against various myeloid (U937, HL60, K562) or lymphoid (Jurkat, U266) leukemia cell lines.…”

“…[15][16][17][18] These antileukemia pyrroloquinoxaline compounds have been previously reported as new structural analogues of derivative A6730, ar eference Akt inhibitor that exhibits antiproliferative activity against various human leukemia cell lines. [15][16][17][18][19][20] In this context, and by considering the biological activities of previousp yrrolo[1,2-a]quinoxalines on human leukemia cells, we undertook and investigatedt he synthesis of an ew series of ethyl 4-[4-(4-substituted piperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxalinecarboxylate derivatives. Taking into account the best results obtained in the previouss eries (A-B, Figure 1), we decided to refer to the JG564 and JG572 pyrrolo[1,2-a]quinoxalinep harmacophores [15][16][17][18] as at emplate for the design of new compounds 1a-o,i nw hich the pyrrolo[1,2-a]quinoxaline ring is replaced in various positions by an ethyl ester functionality ( Figure 1).…”

“…In addition, further modulations on the piperidine core werea lso considered, such as the introductiono f new substituted heterocyclic moieties( benzimidazolone, fluorobenzimidazole, and pyridinyltriazole substituents), in analogy to previously described antileukemia compounds. [15][16][17][18][19][20] The antiproliferative activity of the resulting derivatives (1a-o)w as then evaluated in vitro against variousm yeloid (U937, HL60, K562) or lymphoid (Jurkat, U266) leukemia cell lines. To determine their respective cytotoxicity,d erivatives 1a-o were also tested on activated human peripheral blood mononuclear cells (PBMCs).…”

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program

“…While the makeup of the assembled the connectivity of the signal integration and phenotypic programs driven by Trigger and cRaf-pS621 from literature (Table 1). We estimated the parameters for the signal initiation, and phenotype modules from steady-state and dynamic measurements of transcription factor and phenotypic marker expression following the addition of ATRA [28][29][30][31] . However, the bulk of the model parameters were taken from literature 32 and were not estimated in this study (see materials and methods).…”