Synergistic Effect of DHODH Inhibitor PTC299 with DNA Demethylating Agent Decitabine in Myelodysplastic Syndromes

Background:Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine synthesis, the conversion of DHO to orotate. DHODH inhibition has been described recently as a new approach for treating acute myeloid leukemia (AML) by inducing differentiation of diverse AML subclasses. PTC299 represents a novel potent DHODH inhibitor and recently clinical development of PTC299 as a potential treatment option for acute leukemia was initiated. Here, we explored the efficacy of DHODH inhibitor PTC299 for MDS.

Methods:Anti-MDS efficacy of PTC299 was studied using human MDS cell lines and primary MDS cells in vitro. PTC299 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometric analysis and RNA-sequencing (RNA-seq). Gene expression levels were analyzed by quantitative PCR (qPCR).

Results:PTC299 inhibited proliferation of AML cell lines and induced their differentiation. As previously reported in other DHODH inhibitors, upregulation of CD11b was observed after PTC299 treatment in both HL-60 and THP-1 cells. In addition, PTC299 inhibited the proliferation of MDS cell lines, MDSL and SKM-1 cells, with EC50s of 12.6 nM in MDSL cells and 19.7 nM in SKM-1 cells. The inhibitory effect was reversed by the exogenous addition of 100 µM uridine, which bypasses the requirement for de novo pyrimidine synthesis by feeding into the salvage pathway, thereby negating the need for DHODH. Because the basal expression levels of CD11b are high in MDS cells, we examined the expression levels of CD38. Both cell lines showed dose-dependent upregulation of CD38 after PTC299 treatment.

To investigate the possible synergism between PTC299 and decitabine, we treated MDSL and SKM-1 cells with increasing concentrations of PTC299 and decitabine as single agents or in combination. After 3 days of culture, cells were analyzed by MTS assays. PTC299 and decitabine exerted a enhanced cytotoxic effect on MDSL and SKM-1 cells. Similar results were obtained with primary MDS samples. In Annexin/PI assays, the percentage of apoptotic cells was increased by combination of PTC299 with decitabine in both cell lines.

Mechanistically, treatment with PTC299 induced an intra-S-phase arrestfollowed by entry intoapoptotic cell death. It has also been reported that the expression of p53 is increased in response to the intra-S-phase arrest. To understand the genome-wide effects and target genes of PTC299 and the combination with decitabine, we performed RNA-seq of MDSL and SKM-1 cells treated with PTC299, decitabine, or the combination of both agents versus DMSO-treated cells. Gene set enrichment analysis (GSEA) using our RNA-seq data confirmed that MYC target gene sets were negatively enriched in both PTC299-, decitabine- and combination- treated cells. KEGG pathway enrichment analysis revealed activation of genes associated with apoptosis in both cell lines. To better elucidate a synergistic effect of PTC299 and decitabine, we performed qPCR of CDKN1A, which is a major target of p53 activity. The mRNA expression levels of CDKN1A were upregulated after treatment with PTC299, which was further enhanced by the combination with decitabine.

Conclusions:Our result indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells in vitro and acts in at least an additive and possibly synergistic manner with decitabine in this process. This combination therapy could be a new therapeutic option for the treatment of MDS.