The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies

One-carbon metabolism in cancer

Cells require one-carbon units for nucleotide synthesis, methylation and reductive metabolism, and these pathways support the high proliferative rate of cancer cells. As such, anti-folates, drugs that target one-carbon metabolism, have long been used in the treatment of cancer. Amino acids, such as serine are a major one-carbon source, and cancer cells are particularly susceptible to deprivation of one-carbon units by serine restriction or inhibition of de novo serine synthesis. Recent work has also begun to decipher the specific pathways and sub-cellular compartments that are important for one-carbon metabolism in cancer cells. In this review we summarise the historical understanding of one-carbon metabolism in cancer, describe the recent findings regarding the generation and usage of one-carbon units and explore possible future therapeutics that could exploit the dependency of cancer cells on one-carbon metabolism

A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, ratelimiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use a quantitative high-throughput screen to identify small molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.Damon Runyon Cancer Research Foundation (Sally Gordon Fellowship DRG-112-12)United States. Dept. of Defense. Breast Cancer Research Program (Postdoctoral Fellowship BC120208)American Society for Radiation Oncology (Resident Seed Grant RA-2011-1)European Molecular Biology Organization (Long-Term Fellowship)National Institutes of Health (U.S.) (R03 DA034602-01A1, R01 CA129105, R01 CA103866, and R37 AI047389)United States. Department of Defense (W81XWH-14-PRCRP-IA)Alexander and Margaret Stewart Trus

PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD + regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD + /NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. Keyword: PIK3CA; 2OG; OGDH; TCA cycle; glycolysisDamon Runyon Cancer Research Foundation (HHMI Fellowship

Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery

One of the promises of nanoparticle (NP) carriers is the reformulation of promising therapeutics that have failed clinical development due to pharmacologic challenges. However, current nanomedicine research has been focused on the delivery of established and novel therapeutics. Here we demonstrate proof of the principle of using NPs to revive the clinical potential of abandoned compounds using wortmannin (Wtmn) as a model drug. Wtmn is a potent inhibitor of phosphatidylinositol 3′ kinase-related kinases but failed clinical translation due to drug-delivery challenges. We engineered a NP formulation of Wtmn and demonstrated that NP Wtmn has higher solubility and lower toxicity compared with Wtmn. To establish the clinical translation potential of NP Wtmn, we evaluated the therapeutic as a radiosensitizer in vitro and in vivo. NP Wtmn was found to be a potent radiosensitizer and was significantly more effective than the commonly used radiosensitizer cisplatin in vitro in three cancer cell lines. The mechanism of action of NP Wtmn radiosensitization was found to be through the inhibition of DNA-dependent protein kinase phosphorylation. Finally, NP Wtmn was shown to be an effective radiosensitizer in vivo using two murine xenograft models of cancer. Our results demonstrate that NP drug-delivery systems can promote the readoption of abandoned drugs such as Wtmn by overcoming drug-delivery challenges

Functional genomics reveals serine synthesis is essential in PHGDH-amplified breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation[superscript 1, 2]. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes[superscript 3]. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.Susan G. Komen Breast Cancer Foundation (Fellowship)Life Sciences Research Foundation (Fellowship)W. M. Keck FoundationDavid H. Koch Cancer Research FundAlexander and Margaret Stewart TrustNational Institutes of Health (U.S.) (Grant CA103866

Association Rate Constants of Ras-Effector Interactions Are Evolutionarily Conserved

Evolutionary conservation of protein interaction properties has been shown to be a valuable indication for functional importance. Here we use homology interface modeling of 10 Ras-effector complexes by selecting ortholog proteins from 12 organisms representing the major eukaryotic branches, except plants. We find that with increasing divergence time the sequence similarity decreases with respect to the human protein, but the affinities and association rate constants are conserved as predicted by the protein design algorithm, FoldX. In parallel we have done computer simulations on a minimal network based on Ras-effector interactions, and our results indicate that in the absence of negative feedback, changes in kinetics that result in similar binding constants have strong consequences on network behavior. This, together with the previous results, suggests an important biological role, not only for equilibrium binding constants but also for kinetics in signaling processes involving Ras-effector interactions. Our findings are important to take into consideration in system biology approaches and simulations of biological networks