Effects of Rigosertib (RIGO) Alone or in Combination with Azacitidine or Vorinostat on Epigenetic Reprogramming of CD34+ Cells in the Myelodysplastic Syndrome

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.

RIG in combination with AZA or VOR (AZA/RIG, RIG/AZA, VOR/RIG or RIG/VOR) demonstrated differential effects on the association of RNA polymerase II (Pol II) with active histone marks (H3K4me3 and H3K4me2) in MDS-L and BW-90 cells. An overall decrease in association of Pol II/H3K4me2 was observed with the combinations (AZA/RIG, VOR/RIG or vice versa) compared to control in MDS-L and BW-90, 10-33% (ANOVA, p=0.0006), 9-20% (ANOVA, p=0.0004), respectively. Significant differences were observed in association of H3K4me3/Pol II in BW-90 cells (7-30%; ANOVA, p<0.0001). Similarly, in BM from a pt with MDS after 1 cycle of RIG and AZA treatment demonstrated a decrease in association of Pol II with H3K4me2 (67%) and H3K4me3 (28%).

Treatment of MDS-L cells with RIG alone or in combination with AZA failed to induce expansion of CD34⁺ cells, but the RIG/VOR combination induced 1.9-fold expansion of CD34⁺ cells (ANOVA p=0.002). Individually AZA or VOR treatment led to an enhanced expansion of CD34⁺ cells, 3.8 and 4.0 fold, respectively (ANOVA, p=0.004). RIG alone or with AZA yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSPCs) (ANOVA, p=0.006). However, a marked decrease in ALDH activity was observed in AZA or VOR or RIG/VOR, that was inversely proportion to the expansion of CD34⁺ cells.

In a single MDS pt treated with RIG/AZA, expansion of primitive HPSCs expressing low levels of CD34 was observed. A highly expressed subpopulation of HPSCs, which co-existed prior to treatment of the pt and disappeared upon treatment, suggests either an induced cell death or commitment of HSPCs towards improved lineage differentiation in conjunction with clinical response.

In order to evaluate effects of RIG on expansion of CD34⁺ cells, we examined the pluripotent genes (SOX2, OCT4 NANOG and ZIC3) in the BM from MDS pts, prior and after RIG/AZA treatment. RNA expression levels of SOX2, OCT4, NANOG and ZIC3 were elevated post treatment (≥2 fold). In MDS-L cells the pluripotency genes were highly upregulated in the presence of RIG or RIG/AZA or RIG/VOR (1.7-34 folds). These findings indicate that expression of pluripotency genes is a consequence of epigenetic reprogramming that favors expansion of more primitive HSPCs in a pt, while increase in ALDH activity was inversely proportional to the enhanced expansion of CD34⁺ cells in MDS-L suggesting a more primitive state of HSPC.

The epigenetic events modulated by RIG in combination with either AZA or VOR led to global histone PTMs, differential Pol II association with active histone marks, epigenetic reprogramming of pluripotency genes and expansion of primitive HSPC with downregulation of the PI3K/AKT pathway and cell cycle checkpoint proteins. Collectively, these studies indicate that epigenetic effects of RIG on chromatin alterations lead to HSPC reprogramming that may lead to improved hematopoietic function and response in the clinical setting. These preclinical models suggest potential novel clinical strategies to improve outcomes for patients with higher-risk MDS.