Abstract 2031: RRD-251 enhances retinoic acid induced differentiation of myeloblastic leukemic cells

Abstract

Abstract All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest in HL-60 cells. This is driven by sustained activation of the MAPK pathway, where Raf-1 (MAPKKK) acts as a critical signaling molecule. RRD-251 is a small molecule that binds to the Rb protein and prevents its interaction with Raf-1. Hyperphosphorylation of Rb leads to a conformational change, inhibiting its ability to sequester the E2F family of transcription factors. To determine the effect of RRD-251 on RA-induced differentiation, cells were treated with RA and/or RRD-251 and then analyzed for proliferation, expression of key markers of differentiation, cell cycle arrest, and modifications in protein levels. RRD-251 by itself significantly inhibited proliferation, but cells treated with both RA and RRD-251 showed even further reduction in cellular growth. Similarly, both RA and RRD-251 induce cell cycle arrest, but cells treated with both show higher levels of G0/G1 than singly treated cells. Expression of CD11b is used a late classical marker for differentiation in HL-60. RRD-251 does not induce expression of CD11b, but cultures treated with RRD-251 and RA have nearly double the amount of cells expressing CD11b as compared to RA treated cells alone 72 hours after treatment. Additionally, we had wanted to see the relationship between specific Rb phosphorylation, the cell cycle, and treatment of RA and RRD-251. At 72 hours after treatment, RA induces the formation of a population of cells with no phosphorylation at serine 608 of Rb, which was 99.9% in G0/G1. The appearance of two populations for Rb phosphorylation was unique to serine 608 out of the four phospho-sites that we studied: serine 608, serine 780, serine 795, serine 807/811. Cells treated with both RA and RRD-251 had more cells in the no-phosphorylation population at 72 hours, as well as the appearance of this population at 48 hours. Finally, we observed the changes in protein amounts and localizations by western blotting. RA is known to cause the phosphorylation of Raf-1 at serine 621 and its translocation to the nucleus at 48 hours. This was observed on our blots and that cells treated with both RA and RRD-251 had increased amounts of total nuclear Raf-1 and of pS621 Raf-1 at 48 and 72 hours. The results reported here further support the usefulness of RRD-251 as an anti-cancer drug, from reducing tumor size and preventing angiogenesis in drug-resistant melanoma, to enhancing retinoic acid-induced differentiation in myeloblastic leukemic cells. Citation Format: Aaron S. Wallace, Wendy M. Geil, Andrew Yen. RRD-251 enhances retinoic acid induced differentiation of myeloblastic leukemic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2031. doi:10.1158/1538-7445.AM2015-2031