Targeting of Hematologic Malignancies with PTC299, A Novel Potent Inhibitor of Dihydroorotate Dehydrogenase with Favorable Pharmaceutical Properties.

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin.

Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes. While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe. Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S. pombe pre-messenger RNAs (pre-mRNAs). Second, as in extracts from mammalian cells, ESE function in S. pombe is compromised by mutations and increased distance from the 3'-splice site. Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast. Finally, we have identified five natural purine-rich elements from S. pombe that promote splicing of our reporter pre-mRNAs. Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution.

Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo.

The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.