All-trans Retinoic Acid (ATRA) Induces Activation and Nuclear Translocation of an Ensemble of Novel Signaling Molecules and Causes Leukemia Cell Differentiation


Student thesis: Doctoral Thesis

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Awarding Institution
Award date27 Jun 2019


All-trans retinoic acid (ATRA) is a cancer differentiation therapy agent successful in treatment of acute promyelocytic leukemia (APL) but not for acute myeloid leukemia (AML) in general. ATRA causes the nuclear enrichment of c-Raf, which forces the question whether it has functional significance for ATRA-induced differentiation; and in particular what nuclear molecules it targets and whether those are rationalizable for causing differentiation with cell cycle G1/0 arrest. Here, we reported that in HL-60 cells, a non-APL AML, ATRA induced the enhanced expression of activated c-Raf in the nucleus and regulated its interaction with several novel partners; i.e. Cdk2, RB, SMARCD1. ATRA treatment diminished the amount of Cdk2 bound to c-Raf and enhanced Cdk2 interaction with retinoic acid receptor α (RARα). c-Raf also bound to RB, specifically its pS608 form (pS608RB) and ATRA induced S608 RB hypophosphorylation putatively drives G1/G0 cell cycle arrest. ATRA enhances nuclear c-Raf expression and at the same time releases nuclear c-Raf from known cell cycle drivers. ATRA thus switches c-Raf from pro-proliferation partners to putative pro-differentiation partners.

ATRA based differentiation therapy has been successful in treating t(15;17) positive AML patients, but t(15;17) negative are largely unresponsive, which motivates interest in combination therapies using ATRA plus other agents to increase therapeutic response. Pursuing the suggestion of Cdk2 regulatory involvement in ATRA-induced differentiation, we found that the putative cyclin dependent kinase inhibitor, roscovitine, enhanced the ATRA-induced expression of an ensemble of signaling molecules and promotes cell differentiation. Using the t(15; 17) negative HL-60 human myeloblastic leukemia model show that the roscovitine modulates members of MAPK signaling pathway and Src family kinases (SFKs) and enhances ATRA-induced differentiation. A prominent differentiation driver in this ensemble of signalsome is c-Raf. Roscovitine enhanced ATRA-induced c-Raf and Mek1/2 phosphorylation. We explored the role of key signaling molecule using the shRNA knockdown and found that loss of Lyn facilitated differentiation and enhanced ATRA-induced up-regulation of key signalsome molecules. ATRA plus roscovitine induced differentiation characterized by enhanced p47 phox and CD11b expression, G1/G0 cell cycle arrest and enhanced ROS production. Roscovitine thus has effects beyond its original presentation as a CDK inhibitor. Our data also suggested a mechanism for coupling cell cycle arrest and differentiation, we reported that roscovitine regulates novel nuclear pathways formed in part by various canonically cytoplasmic signaling molecules now enriched in the nucleus by ATRA. Roscovitine enhanced the ATRA-induced nuclear enrichment of c-Raf, pS259-c-Raf, pS289/296/301-c-Raf and the Lyn and Fgr SFKs. RB specifically, pS608 RB, interacted with nuclear c-Raf, and Lyn. ATRA plus roscovitine co-treatment diminished the amount of RB bound to c-Raf and Lyn and enhanced the availability of freed c-Raf and Lyn in nucleus. Expression of several adaptor proteins, c-Cbl, SLP-76, a guanine exchange factor, Vav1, and the transcription factor, IRF-1 were likewise found in the nucleus, with enhancement by roscovitine. Interestingly, like Lyn, Vav likewise binds RB. We showed that combination therapy caused decreased phosphorylation (hypo-phosphorylation) of S608 RB and Cdk2 expression, which correlated with induced differentiation and G1/G0 arrest via p27 Kip1/cyclin E1/Cdk2/RB axis. We also explored the modulation of novel nuclear molecules by Lyn knockdown and showed that roscovitine affects the ATRA-inducible nuclear components in these cells, which facilitated myeloid differentiation.

At its heart, this work addresses the basic biological question of how a fundamental developmental regulator act. The results motivate a novel paradigm that augments the historically dominant one of retinoic acid response element (RARE) predicated transcriptional regulation. Significantly ATRA induces enrichment of c-Raf in the nucleus where it regulates the interaction of c-Raf with several targets that have prominent roles in control of cell cycle and differentiation. There are new nuclear signaling pathways that use traditionally cytoplasmic signaling molecules now imported to the nucleus by ATRA. One is a novel nuclear Raf/Cdk2/RARα axis. These novel nuclear pathways illuminate novel targets for therapeutic intervention in cancer differentiation therapy. One drug that targets such these is roscovitine which show here to promote differentiation and growth arrest of leukemic cells. The holistic picture is that directed there by ATRA, c-Raf in the nucleus switches from pro-proliferation targets to pro-differentiation targets. c-Raf in the nucleus ergo drives differentiation and ATRA promotes this by driving it from cytoplasm to nucleus and by freeing it from arguably pro-proliferation targets where it might be sequestered to go to target driving differentiation.

    Research areas

  • All-trans retinoic acid, ATRA, Acute promyelocytic leukemia, APL, Acute myeloid leukemia, AML, c-Raf, Cyclin-dependent kinase-2, SMARCD1, RB, Src family kinase, SFK, Roscovitine, Lyn