Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. METHODS: The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. RESULTS: EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. CONCLUSIONS: Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Comparative genomics of pneumocystis species suggests the absence of genes for myo-inositol synthesis and reliance on inositol transport and metabolism.

UNLABELLED: In the context of deciphering the metabolic strategies of the obligate pathogenic fungi in the genus Pneumocystis, the genomes of three species (P. carinii, P. murina, and P. jirovecii) were compared among themselves and with the free-living, phylogenetically related fission yeast (Schizosaccharomyces pombe). The underrepresentation of amino acid metabolism pathways compared to those in S. pombe, as well as the incomplete steroid biosynthesis pathway, were confirmed for P. carinii and P. jirovecii and extended to P. murina. All three Pneumocystis species showed overrepresentation of the inositol phosphate metabolism pathway compared to that in the fission yeast. In addition to those known in S. pombe, four genes, encoding inositol-polyphosphate multikinase (EC 2.7.1.151), inositol-pentakisphosphate 2-kinase (EC 2.7.1.158), phosphoinositide 5-phosphatase (EC 3.1.3.36), and inositol-1,4-bisphosphate 1-phosphatase (EC 3.1.3.57), were identified in the two rodent Pneumocystis genomes, P. carinii and P. murina. The P. jirovecii genome appeared to contain three of these genes but lacked phosphoinositide 5-phosphatase. Notably, two genes encoding enzymes essential for myo-inositol synthesis, inositol-1-phosphate synthase (INO1) and inositol monophosphatase (INM1), were absent from all three genomes, suggesting that Pneumocystis species are inositol auxotrophs. In keeping with the need to acquire exogenous inositol, two genes with products homologous to fungal inositol transporters, ITR1 and ITR2, were identified in P. carinii and P. murina, while P. jirovecii contained only the ITR1 homolog. The ITR and inositol metabolism genes in P. murina and P. carinii were expressed during fulminant infection as determined by reverse transcriptase real-time PCR of cDNA from infected lung tissue. Supplementation of in vitro culture with inositol yielded significant improvement of the viability of P. carinii for days 7 through 14. IMPORTANCE: Microbes in the genus Pneumocystis are obligate pathogenic fungi that reside in mammalian lungs and cause Pneumocystis pneumonia in hosts with weakened immune systems. These fungal infections are not responsive to standard antifungal therapy. A long-term in vitro culture system is not available for these fungi, impeding the study of their biology and genetics and new drug development. Given that all genomes of the Pneumocystis species analyzed lack the genes for inositol synthesis and contain inositol transporters, Pneumocystis fungi, like S. pombe, appear to be inositol auxotrophs. Inositol is important for the pathogenesis, virulence, and mating processes in Candida albicans and Cryptococcus neoformans, suggesting similar importance within the Pneumocystis species as well. This is the first report to (i) characterize genes in the inositol phosphate metabolism and transport pathways in Pneumocystis species and (ii) identify inositol as a supplement that improved the viability of P. carinii in in vitro culture.

Sequence and structure of the linear mitochondrial genome of Pneumocystis carinii.

With the exception of a few genes, most of the mitochondrial (mt) genome of Pneumocystis carinii has not previously been sequenced. Shotgun sequences generated as a result of the Pneumocystis Genome Project (PGP) were assembled with the gap4 assembly program into a 23-kb contig. Annotation of the mt genome identified 4 open reading frames and 20 tRNAs in addition to 17 other genes: ATP synthase, subunits 6, 8, and 9; cytochrome c oxidase, subunits 1, 2, and 3; NADH dehydrogenase, subunits 1, 2, 3, 4, 4L, 5, and 6; apocytochrome b; RNase P RNA gene; and the mitochondrial large and small ribosomal RNA subunits. A 24-bp unit that repeated from one to five times was identified interior to the ends of the mt genome. Migration of the genome on CHEF gels was consistent with that of linear DNA and digestion with BAL31 showed a concomitant reduction in size of the genome, a characteristic of linear DNA. Together with the identification of terminal repeats similar to those found in other linear fungal mt genomes and the inability to join the ends by PCR, these data provide strong evidence that the mt genome of P. carinii is linear.