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 invol… Show more
“…Motivated by the enhancement of RA-induced differentiation in HL-60 cells [ 23 ], we sought to determine if co-treating with RRD-251 had a similar impact on D3-induced differentiation. Like RA, D3 induces phenotypic changes in growth arrest, inducible oxidative metabolism, and the surface markers CD38 and CD11b, as well as CD14, a marker specific to the monocyte lineage.…”
Section: Resultsmentioning
confidence: 99%
“…RA-induced RB-E2F complexes begin to appear at 48 hours and are inhibited by phosphorylation of RB at serine 608 [ 22 ]. This same phosphorylation site shows a transient interaction with c-Raf early in the differentiation time course, which concomitantly acts as a reservoir, releasing nuclear c-Raf to interact with other partners during later differentiation events as RB hypophosphorylates [ 23 ].…”
Section: Introductionmentioning
confidence: 99%
“…Recent studies indicate that inhibition of glycogen synthase kinase-3 (GSK-3) can induce differentiation in AML cells on its own [ 24 , 25 ] and enhance RA and D3 differentiation therapies via respective hyperactivation of RARα and VDR transcriptional activity [ 23 , 26 ]. Interestingly, the mechanisms of transcriptional hyperactivation differ.…”
Section: Introductionmentioning
confidence: 99%
“…Initially synthesized by Chellappan and colleagues, RRD-251, a selective disrupter of the interaction between c-Raf and RB, enhances RA-induced myeloid differentiation of HL-60 cells through a novel c-Raf/GSK-3/RARα axis [ 23 , 28 ]. Dissociation of c-Raf and RB leads to increased E2F sequestration by RB and G 1 / 0 arrest, while simultaneously increasing associations of c-Raf with other nuclear partners including GSK-3.…”
Differentiation therapy is used in cancer treatment. Epidemiologic studies showed that higher vitamin D levels are associated with reduced cancer risks. However, the therapeutic doses needed for differentiation are accompanied by hypercalcemia and intolerable pathological sequelae. In the present work we evaluated if RRD-251, a small-molecule, can enhance vitamin D3-induced differentiation of leukemic cells, in the hope of decreasing the needed vitamin D3-dose.We demonstrate that RRD-251 enhances vitamin D3-induced differentiation of leukemic cells, the enrichment of the c-Raf kinase in the nucleus, the binding of nuclear c-Raf to GSK-3, increased phosphorylation of GSK-3 ser 21/9 inhibitory sites, and the binding of GSK-3 to VDR, where GSK-3 inhibition is known to enhance transcriptional activation by VDR. Enhancement of D3-induced p-GSK-3 ser 21/9 by RRD-251 was associated with enhanced Akt-GSK-3 binding, Akt being a known GSK-3 inhibitor, and diminished Erk1/2 binding. Diminishing Erk interaction with GSK-3 was associated with enhanced interaction with Vav1, a known driver of myeloid differentiation. This is redolent of an ATRA/c-Raf/GSK-3/RARα axis we previously reported, although the phosphorylation effects to enhance transcriptional activation on RARα vs VDR diverge. Taken together this indicates potential therapeutic significance for a c-Raf/GSK-3/VDR or RARα axis for leukemic myelo-monocytic differentiation.
“…Motivated by the enhancement of RA-induced differentiation in HL-60 cells [ 23 ], we sought to determine if co-treating with RRD-251 had a similar impact on D3-induced differentiation. Like RA, D3 induces phenotypic changes in growth arrest, inducible oxidative metabolism, and the surface markers CD38 and CD11b, as well as CD14, a marker specific to the monocyte lineage.…”
Section: Resultsmentioning
confidence: 99%
“…RA-induced RB-E2F complexes begin to appear at 48 hours and are inhibited by phosphorylation of RB at serine 608 [ 22 ]. This same phosphorylation site shows a transient interaction with c-Raf early in the differentiation time course, which concomitantly acts as a reservoir, releasing nuclear c-Raf to interact with other partners during later differentiation events as RB hypophosphorylates [ 23 ].…”
Section: Introductionmentioning
confidence: 99%
“…Recent studies indicate that inhibition of glycogen synthase kinase-3 (GSK-3) can induce differentiation in AML cells on its own [ 24 , 25 ] and enhance RA and D3 differentiation therapies via respective hyperactivation of RARα and VDR transcriptional activity [ 23 , 26 ]. Interestingly, the mechanisms of transcriptional hyperactivation differ.…”
Section: Introductionmentioning
confidence: 99%
“…Initially synthesized by Chellappan and colleagues, RRD-251, a selective disrupter of the interaction between c-Raf and RB, enhances RA-induced myeloid differentiation of HL-60 cells through a novel c-Raf/GSK-3/RARα axis [ 23 , 28 ]. Dissociation of c-Raf and RB leads to increased E2F sequestration by RB and G 1 / 0 arrest, while simultaneously increasing associations of c-Raf with other nuclear partners including GSK-3.…”
Differentiation therapy is used in cancer treatment. Epidemiologic studies showed that higher vitamin D levels are associated with reduced cancer risks. However, the therapeutic doses needed for differentiation are accompanied by hypercalcemia and intolerable pathological sequelae. In the present work we evaluated if RRD-251, a small-molecule, can enhance vitamin D3-induced differentiation of leukemic cells, in the hope of decreasing the needed vitamin D3-dose.We demonstrate that RRD-251 enhances vitamin D3-induced differentiation of leukemic cells, the enrichment of the c-Raf kinase in the nucleus, the binding of nuclear c-Raf to GSK-3, increased phosphorylation of GSK-3 ser 21/9 inhibitory sites, and the binding of GSK-3 to VDR, where GSK-3 inhibition is known to enhance transcriptional activation by VDR. Enhancement of D3-induced p-GSK-3 ser 21/9 by RRD-251 was associated with enhanced Akt-GSK-3 binding, Akt being a known GSK-3 inhibitor, and diminished Erk1/2 binding. Diminishing Erk interaction with GSK-3 was associated with enhanced interaction with Vav1, a known driver of myeloid differentiation. This is redolent of an ATRA/c-Raf/GSK-3/RARα axis we previously reported, although the phosphorylation effects to enhance transcriptional activation on RARα vs VDR diverge. Taken together this indicates potential therapeutic significance for a c-Raf/GSK-3/VDR or RARα axis for leukemic myelo-monocytic differentiation.
“…Such molecular mechanisms could underly aspects of cellular memory associated to consecutive ATRA treatments. Figure 9 This schematic diagram shows the hypothetical principal pathways in the ATRA–induced signaling that results in cell differentiation in the HL-60 myeloid leukemia model 17 , 67 – 71 . It is based on modules and feedback loops.…”
In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
