Screening and characterization of antimalarial resistance related genetic structural variations in P. falciparum

The emergence of parasite resistance to antimalarials has been impeding the efforts in malaria elimination. Continuous surveillance of treatment failures from the clinical population informs the efficacy of the currently used antimalarial treatment and provide essential clues in deciphering the molecular mechanism of resistance. Screening of resistance biomarkers from clinical isolates has undermined the identification of large genetic variants, mainly due to technical challenges imposed by low quantity of genetic material and low complexity Plasmodium genome, making the analysis prone to bias. This study screened large genetic structural variations from 413 clinical samples collected in Greater Mekong Subregions, the epicentre of antimalarial treatment failures. Samples were obtained in collaboration with Tracking Resistance to Artemisinin Collaboration (TRAC) studies. Large structural variants were screened using an optimized microarray based Comparative Genomic Hybridization (aCGH) technique. We identified Copy Number Amplification in pfmdr1, pfgch1, and pfpm2, previously reported in association with mainly Mefloquine, Sulfadoxine-Pyrimethamine, and Piperaquine resistance, respectively. In addition, we also identified novel CNV in pfvit and pfyhm2 within the same locus, known for function in iron detoxification and mitochondrial electron transport chain, respectively. Four variant genes: pfpm2, pfvit, pfcyp19b, and pfk13, were chosen for biological and phenotypical characterization using an in vitro parasite model. The overall work suggested the CYP19B as a potential downstream effector of K13 C580Y allele as well as PMII and K13 probable compensatory/synergistic relationship in Ring stage; the stage had been shown to exhibit reduced susceptibility to Artemisinin treatment. Collectively, this work presented an additional potential molecular mechanism to the current notion of antimalarial resistance.Doctor of Philosoph

Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum

Resistance of the human malaria parasites, Plasmodium falciparum, to artemisinins is now fully established in Southeast Asia and is gradually emerging in Sub-Saharan Africa. Although nonsynonymous SNPs in the pfk13 Kelch-repeat propeller (KREP) domain are clearly associated with artemisinin resistance, their functional relevance requires cooperation with other genetic factors/alterations of the P. falciparum genome, collectively referred to as genetic background. Here we provide experimental evidence that P. falciparum cyclophilin 19B (PfCYP19B) may represent one putative factor in this genetic background, contributing to artemisinin resistance via its increased expression. We show that overexpression of PfCYP19B in vitro drives limited but significant resistance to not only artemisinin but also piperaquine, an important partner drug in artemisinin-based combination therapies. We showed that PfCYP19B acts as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P). Curiously, artemisinin and piperaquine affect eIF2α-P in an inverse direction that in both cases can be modulated by PfCYP19B towards resistance. Here we also provide evidence that the upregulation of PfCYP19B in the drug-resistant parasites appears to be maintained by a short tandem repeat (SRT) sequence polymorphism in the gene's promoter region. These results support a model that artemisinin (and other drugs) resistance mechanisms are complex genetic traits being contributed to by altered expression of multiple genes driven by genetic polymorphism at their promoter regions

Pulsed SILAC-based proteomic analysis unveils hypoxia- and serum starvation-induced de novo protein synthesis with PHD finger protein 14 (PHF14) as a hypoxia sensitive epigenetic regulator in cell cycle progression

Hypoxia is an environmental cue that is associated with multiple tumorigenic processes such as immunosuppression, angiogenesis, cancer invasion, metastasis, drug resistance, and poor clinical outcomes. When facing hypoxic stress, cells initiate several adaptive responses such as cell cycle arrest to reduce excessive oxygen consumption and co-activation of oncogenic factors. In order to identify the critical novel proteins for hypoxia responses, we used pulsed-SILAC method to trace the active cellular translation events in A431 cells. Proteomic discovery data and biochemical assays showed that cancer cells selectively activate key glycolytic enzymes and novel ER-stress markers, while protein synthesis is severely suppressed. Interestingly, deprivation of oxygen affected the expression of various epigenetic regulators such as histone demethylases and NuRD (nucleosome remodeling and deacetylase) complex in A431 cells. In addition, we identified PHF14 (the plant homeodomain finger-14) as a novel hypoxia-sensitive epigenetic regulator that plays a key role in cell cycle progress and protein synthesis. Hypoxia-mediated inhibition of PHF14 was associated with increase of key cell cycle inhibitors, p14ARF, p15INK4b, and p16INK4a, which are responsible for G1-S phase transition and decrease of AKT-mTOR-4E-BP1/pS6K signaling pathway, a master regulator of protein synthesis, in response to environmental cues. Analysis of TCGA colon cancer (n=461) and skin cancer (n=470) datasets revealed a positive correlation between PHF14 expression and protein translation initiation factors, eIF4E, eIF4B, and RPS6. Significance of PHF14 gene was further demonstrated by in vivo mouse xenograft model using PHF14 KD cell lines.Ministry of Education (MOE)National Medical Research Council (NMRC)Published versionThis study was supported by the Singapore Ministry of Education (MOE2014-T2-2-043, MOE2016-T2-2-018 and MOE2016-T3-1-003) and the National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016)