title: RRD-251 enhances all-trans retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells.
abstract: All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest of HL-60 cells. Responding to an RA-induced cytosolic signaling machine, c-Raf translocates to the nucleus, providing propulsion for RA-induced differentiation. This novel mechanism is not understood, but presumably reflects c-Raf binding with nuclear gene regulatory proteins. RRD-251 is a small molecule that prevents the interaction of c-Raf and RB, the retinoblastoma tumor suppressor protein. The involvement of c-Raf and RB in RA-induced differentiation motivates interest in the effects of combined RA and RRD-251 treatment on leukemic cell differentiation. We demonstrate that RRD-251 enhances RA-induced differentiation. Mechanistically, we find that nuclear translocated c-Raf associates with pS608 RB. RA causes loss of pS608 RB, where cells with hypophosphorylated S608 RB are G0/G1 restricted. Corroborating the pS608 RB hypophosphorylation, RB sequestration of E2F increased with concomitant loss of cdc6 expression, which is known to be driven by E2F. Hypophosphorylation of S608 RB releases c-Raf from RB sequestration to bind other nuclear targets. Release of c-Raf from RB sequestration results in enhanced association with GSK-3 which is phosphorylated at its S21/9 inhibitory sites. c-Raf binding to GSK-3 is associated with dissociation of GSK-3 and RARα, thereby relieving RARα of GSK-3 inhibition. RRD-251 amplifies each of these RA-induced events. Consistent with the posited enhancement of RARα transcriptional activity by RRD-251, RRD-251 increases the RARE-driven CD38 expression per cell. The RA/c-Raf/GSK-3/RARα axis emerges as a novel differentiation regulatory mechanism susceptible to RRD-251, suggesting enhancing RA-effects with RRD-251 in therapy.
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Wallace
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Harrison T
authors.initials: HT
authors.lastname: Supnick
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Rodica P
authors.initials: RP
authors.lastname: Bunaciu
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Yen
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
chemical_list: C556907:RRD 251; D014212:Tretinoin; D013890:Thiourea
keywords: GSK-3; RRD-251; c-Raf; retinoblastoma protein (RB); all-trans retinoic acid (RA)