The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells

Abstract

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3in acute myeloid leukemia cells

“…45 Moreover, the SFK inhibitor PP2 has recently been shown to rescue inducible differentiation in emergent ATRA-resistant myeloblastic leukemia cells, confirming the important role of SFKs in this process. 46 The detailed molecular mechanisms whereby erlotinib exerts differentiation-inducing effects remain largely elusive. However, it appears plausible that this activity involves the inhibition of p38 MAPK and/or SFKs, as (1) several distinct EGFR-targeting agents are known to interfere with the enzymatic functions of these kinases, 47,48 and (2) phosphorylates serine and threonine (rather than tyrosine) residues and hence is unlikely to be directly inhibited by erlotinib or gefitinib.…”

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

“…Two retinoic acid (RA)-resistant HL-60 sublines (R38+ and R38−) were isolated as described previously [25]. Cell cultures were maintained in 1640 RPMI medium (Invitrogen, Carlsbad, CA) supplemented with heat-inactivated 5% fetal bovine serum (FBS; Hyclone, Logan, UT) and 1% antibiotic/antimycotic (Invitrogen) and grown at 37 °C in a 5% CO 2 humidified environment.…”

“…c-Raf propels RA-induced differentiation [12,19] but induced phosphorylation at the c-Raf activating sites S338 and Y340/Y341 cannot be detected in RA-treated HL-60 [20,21]. Instead, phosphorylation occurs at the S259 putative inhibitory site [22], the S621 putative stability site [23] and the S289/296/301 c-Raf sites [24,25]. The S289/296/301c-Raf sites are targets of ERK, but whether these sites are inhibitory [26] or activating [27] remains unclear (see Discussion).…”

“…Both fail to upregulate CD11b expression, G1/G0 arrest and signaling factor expression/activation after RA treatment [25]. However, one RA-resistant cell line retains RA-inducible CD38 expression (R38+ HL-60) while the other has lost this marker as well (R38− HL-60).…”

“…However, one RA-resistant cell line retains RA-inducible CD38 expression (R38+ HL-60) while the other has lost this marker as well (R38− HL-60). The SFK inhibitor PP2, which enhances RA-induced differentiation in wild-type HL-60 and NB4 cells [24,39], rescues differentiation in both R38+ and R38− RA-resistant HL-60 cells [25]. Using the kinase inhibitors PD98059, GW5074, wortmannin, and Akti-1/2, we found that the c-Raf inhibitor GW5074 also emerges as an augmenter of RA-induced differentiation in wild-type and RA-resistant HL-60 cells, and had a similar effect to PP2 as reported previously.…”