Drug screening in human physiologic medium identifies uric acid as an inhibitor of rigosertib efficacy.

The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.

CRISPR-Cas9-mediated somatic correction of a one-base deletion in the Ugt1a gene ameliorates hyperbilirubinemia in Crigler-Najjar syndrome mice.

(AAV)-mediated episomal gene replacement therapy for monogenic liver disorders is currently limited in pediatric settings due to the loss of vector DNA, associated with hepatocyte duplication during liver growth. Genome editing is a promising strategy leading to a permanent and specific genome modification that is transmitted to daughter cells upon proliferation. Using genome targeting, we previously rescued neonatal lethality in mice with Crigler-Najjar syndrome. This rare monogenic disease is characterized by severe neonatal unconjugated hyperbilirubinemia, neurological damage, and death. Here, using the CRISPR-Staphylococcus aureus Cas9 (SaCas9) platform, we edited the disease-causing mutation present in the Ugt1a locus of these mice. Newborn mice were treated with two AAV8 vectors: one expressing the SaCas9 and single guide RNA, and the other carrying the Ugt1a homology regions with the corrected sequence, while maintained in a temporary phototherapy setting rescuing mortality. We observed a 50% plasma bilirubin reduction that remained stable for up to 6 months. We then tested different Cas9:donor vector ratios, with a 1:5 ratio showing the greatest efficacy in lowering plasma bilirubin, with partial lethality rescue when more severe, lethal conditions were applied. In conclusion, we reduced plasma bilirubin to safe levels and partially rescued neonatal lethality by correcting the mutant Ugt1a1 gene of a Crigler-Najjar mouse model.

ASCT2 is the primary serine transporter in cancer cells.

The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic for serine and therefore reliant on the uptake of exogenous serine. Importantly, however, the transporter(s) that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (coded for by the gene SLC1A5) as the primary serine transporter in cancer cells. ASCT2 is well-known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that ERα promotes serine uptake by directly activating SLC1A5 transcription. Together, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target in serine metabolism.

Drug screening in human physiologic medium identifies uric acid as an inhibitor of rigosertib efficacy.

The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism waste product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. Structural modelling studies suggest that uric acid interacts with the tubulin-rigosertib complex and may act as an uncompetitive inhibitor of rigosertib. These results offer a possible explanation for the failure of rigosertib in clinical trials and demonstrate the utility of physiological media to achieve in vitro results that better represent human therapeutic responses.

Differential genetic hitchhiking around mutant pfcrt alleles in the Indian Plasmodium falciparum population.

OBJECTIVES: To study the origin and spread of the chloroquine-resistant Plasmodium falciparum population in the Indian subcontinent. METHODS: Fourteen microsatellites spanning a ∼120 kb region, flanking the P. falciparum chloroquine resistance transporter (pfcrt) gene, were analysed in 185 parasite isolates. RESULTS: The Indian P. falciparum population exhibited a selective valley of reduced genetic variation in the flanking microsatellites of the mutant pfcrt alleles (up to ±29 kb) as compared with the wild-type allele. This valley is much narrower than the ±200 kb valley reported from African and South-East Asian countries. The majority of the isolates showed asymmetry in the selective valley, where upstream microsatellites showed less genetic variation than the downstream microsatellites. Regional variation in the width and symmetry of the selective valley was noticed, which seems to be related to the number of pfcrt alleles present in the parasite population of a region. Forty-six different microsatellite haplotypes were observed among the P. falciparum isolates containing mutant pfcrt alleles. Parasite populations from different regions of mainland India shared microsatellite haplotypes between them, but they shared none with the isolates from the Andaman and Nicobar Islands, and vice versa. Indian isolates shared microsatellite haplotypes with the isolates from Papua New Guinea and Thailand. CONCLUSIONS: With regard to chloroquine there is regional variation in the selection pressure on the P. falciparum population in India. These findings will help the regional implementation of drug policy in India's malaria control programme.

Transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor-resistant pancreatic ductal adenocarcinoma.

Metabolic reprogramming, due in part to the overexpression of metabolic enzymes, is a key hallmark of cancer cells. Lactate dehydrogenase (LDHA), a metabolic enzyme that catalyzes the interconversion of lactate and pyruvate, is overexpressed in a wide variety of cancer types, including pancreatic ductal adenocarcinoma (PDAC). Furthermore, the genetic or pharmacological inhibition of LDHA suppresses cancer growth, demonstrating a cancer-promoting role for this enzyme. Therefore, several pharmacological LDHA inhibitors are being developed and tested as potential anti-cancer therapeutic agents. Because cancer cells are known to rapidly adapt and become resistant to anti-cancer therapies, in this study, we modeled the adaptation of cancer cells to LDHA inhibition. Using PDAC as a model system, we studied the molecular aspects of cells resistant to the competitive LDHA inhibitor sodium oxamate. We performed unbiased RNA-sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and metabolomics analyses of parental and oxamate-resistant PDAC cells treated with and without oxamate to identify the transcriptional, chromatin, and metabolic landscapes of these cells. We found that oxamate-resistant PDAC cells were significantly different from parental cells at the levels of mRNA expression, chromatin accessibility, and metabolites. Additionally, an integrative analysis combining the RNA-seq and ATAC-seq datasets identified a subset of differentially expressed mRNAs that directly correlated with changes in chromatin accessibility. Finally, functional analysis of differentially expressed metabolic genes in parental and oxamate-resistant PDAC cells treated with and without oxamate, together with an integrative analysis of RNA-seq and metabolomics data, revealed changes in metabolic enzymes that might explain the changes in metabolite levels observed in these cells. Collectively, these studies identify the transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor resistance in PDAC cells. Future functional studies related to these changes remain necessary to reveal the direct roles played by these changes in the development of LDHA inhibitor resistance and uncover approaches for more effective use of LDHA inhibitors in cancer therapy.

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

A major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

PSPH promotes melanoma growth and metastasis by metabolic deregulation-mediated transcriptional activation of NR4A1.

Metabolic deregulation, a hallmark of cancer, fuels cancer cell growth and metastasis. Here, we show that phosphoserine phosphatase (PSPH), an enzyme of the serine metabolism pathway, is upregulated in patient-derived melanoma samples. PSPH knockdown using short hairpin RNAs (shRNAs) blocks melanoma tumor growth and metastasis in both cell culture and mice. To elucidate the mechanism underlying PSPH action, we evaluated PSPH shRNA-expressing melanoma cells using global metabolomics and targeted mRNA expression profiling. Metabolomics analysis showed an increase in 2-hydroxyglutarate (2-HG) levels in PSPH knockdown cells. 2-HG inhibits the TET family of DNA demethylases and the Jumonji family of histone demethylases (KDM and JMJD), which is known to impact gene expression. Consistent with these data, PSPH knockdown in melanoma cells showed reduced DNA 5-hydroxymethylcytosine (5hmC) and increased histone H3K4me3 modifications. 2-HG treatment also inhibited melanoma growth. The nCounter PanCancer Pathways Panel-based mRNA expression profiling revealed attenuation of a number of cancer-promoting pathways upon PSPH knockdown. In particular, PSPH was necessary for nuclear receptor NR4A1 expression. Ectopic NR4A1 expression partly rescued the growth of melanoma cells expressing PSPH shRNA. Collectively, these results link PSPH to the facilitation of melanoma growth and metastasis through suppression of 2-HG and thus activation of pro-oncogenic gene expression.

Bilirubin-Induced Oxidative Stress Leads to DNA Damage in the Cerebellum of Hyperbilirubinemic Neonatal Mice and Activates DNA Double-Strand Break Repair Pathways in Human Cells.

Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free bilirubin (Bf), as determined by γH2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia.