Roscovitine enhances all-trans retinoic acid (ATRA)-induced nuclear enrichment of an ensemble of activated signaling molecules and augments ATRA-induced myeloid cell differentiation

Abstract

Although ATRA represents a successful differentiation therapy for APL, it is largely ineffective for non-APL AMLs. Hence combination therapies using an agent targeting ATRA-regulated molecules that drive cell differentiation/arrest are of interest. Using the HL-60 human non-APL AML model where ATRA causes nuclear enrichment of c-Raf that drives differentiation/G0-arrest, we now observe that roscovitine enhanced nuclear enrichment of certain traditionally cytoplasmic signaling molecules and enhanced differentiation and cell cycle arrest. Roscovitine upregulated ATRA-induced nuclear c-Raf phosphorylation at S259 and S289/296/301. Nuclear c-Raf interacted with RB protein and specifically with pS608RB, the hinge region phosphorylation controlling E2F binding and cell cycle progression. ATRA-induced loss of pS608RB with cell cycle arrest was associated with loss of RB-sequestered c-Raf, thereby coupling cell cycle arrest and increased availability of c-Raf to promote differentiation. Part of this mechanism reflects promoting cell cycle arrest via ATRAinduced upregulation of the p27 Kip1 CDKI. Roscovitine also enhanced the ATRAinduced nuclear enrichment of other signaling molecules traditionally perceived as cytoplasmic promoters of proliferation, but now known to promote differentiation; in particular: SFKs, Lyn, Fgr; adaptor proteins, c-Cbl, SLP-76; a guanine exchange factor, Vav1; and a transcription factor, IRF-1. Akin to c-Raf, Lyn bound to RB, specifically to pS608RB. Lyn-pS608RB association was greatly diminished by ATRA and essentially lost in ATRA plus roscovitine treated cells. Interestingly Lyn-KD enhanced such ATRAinduced nuclear signaling and differentiation and made roscovitine more effective. ATRA thus mobilized traditionally cytoplasmic signaling molecules to the nucleus where they drove differentiation which were further enhanced by roscovitine.

Role for Fgr and Numb in retinoic acid-induced differentiation and G0 arrest of non-APL AML cells

“…Arguably the prototype is Raf. RA-induced signalsome activation is associated with nuclear enrichment of Raf [ 17 , 20 ]. The nuclear Raf binds the NFATc3 transcription factor on the promoter of CXCR5 downstream of a non-canonical RARE [ 16 ].…”

“…The proposed paradigm also offers a rationalization for why very diverse agents have been serendipitously found to enhance RA action. For example, arsenic trioxide (a poison that generates oxidative stress with widespread toxicological effects) [ 68 ], Bosutinib (originally introduced as an SFK inhibitor) [ 69 ], Roscovitine (originally introduced as a CDK inhibitor) [ 20 , 45 ], and RRD-251 (originally introduced as a small molecule targeting Raf interactions) [ 19 ] all enhance the effects of RA. These agents have in common that they also have unanticipated abilities to target and stimulate signalsome components.…”

“…Raf is also implicated in putative Raf/Cdk2/RARα and Raf/GSK-3/RARα axes during RA-induced differentiation, suggesting that it also acts in other capacities to propel differentiation induced by RA [ 18 , 19 ]. We have found, too, that other signalosome components also become enriched in the nucleus by RA, in particular Lyn, Fgr, Slp-76, Cbl and Vav [ 20 ]. The signalosome ergo appears to generate signaling targeting RAREs that enables RA-induced transcriptional activation for driving cell differentiation.…”

Kinase Inhibitors and Interferons as Other Myeloid Differentiation Inducers in Leukemia Therapy

“…c-Raf phosphorylates transcription factors required for ATRA-induced differentiation [9]. Recently, Rashid et al [10, 11] used the t(15;17)-negative HL-60 human myeloblastic leukemia model and found that a CDK inhibitor, roscovitine, enhanced differentiation. Roscovitine also enhanced the ATRA-induced nuclear enrichment of several signaling molecules, including pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src family kinases (SFKs), Lyn Fgr, adaptor proteins c-Cbl and SLP-76, guanine exchange factor Vav1, and transcription factor IRF-1, which are known to play important roles in not only proliferation but also differentiation [10].…”

“…Roscovitine also enhanced the ATRA-induced nuclear enrichment of several signaling molecules, including pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src family kinases (SFKs), Lyn Fgr, adaptor proteins c-Cbl and SLP-76, guanine exchange factor Vav1, and transcription factor IRF-1, which are known to play important roles in not only proliferation but also differentiation [10]. They further revealed that c-Raf or Lyn formed a complex with retinoblastoma (RB), and that the c-Raf- or Lyn-retinoblastoma association was greatly diminished by ATRA and lost in ATRA and roscovitine co-treated cells [11]. Furthermore, Lyn knockdown enhanced ATRA-induced differentiation markers, such as CD11b, G1/G0 arrest, and ROS production [10].…”