Roscovitine enhances all-<i>trans</i> retinoic acid (ATRA)-induced nuclear enrichment of an ensemble of activated signaling molecules and augments ATRA-induced myeloid cell differentiation
Asif Rashid, Xin Duan, Feng Gao, Mengsu Yang, Andrew Yen
Abstract
// Asif Rashid 1 , 2 , Xin Duan 3 , Feng Gao 3 , Mengsu Yang 1 and Andrew Yen 2 1 Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, People’s Republic of China 2 Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA 3 The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China Correspondence to: Mengsu Yang, email: [email protected] Andrew Yen, email: [email protected] Keywords: ATRA; roscovitine; APL; HL-60; Lyn Received: December 14, 2019     Accepted: February 08, 2020     Published: March 24, 2020 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 ATRA-induced upregulation of the p27 Kip1 CDKI. Roscovitine also enhanced the ATRA-induced 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 ATRA-induced 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.