Cancer cells exhibit several unique metabolic phenotypes that are critical for cell growth and proliferation. Specifically, they over-express the M2 isoform of the tightly regulated enzyme pyruvate kinase (PKM2), which controls glycolytic flux, and they are highly dependent on de novo biosynthesis of serine and glycine. Here we describe a novel rheostat-like mechanistic relationship between PKM2 activity and serine biosynthesis. We show that serine can bind to and activate human PKM2 and that following serine deprivation, PKM2 activity in cells is reduced. This reduction in PKM2 activity shifts cells to a fuel-efficient mode where more pyruvate is diverted to the mitochondria and more glucose derived carbon is channelled into serine biosynthesis to support cell proliferation.

Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the and activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models. This study included preclinical activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. PQR309 had antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. .

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (Nterminal SH2) domains of p85α, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85α. The mutant proteins are still able to bind to the catalytic subunits p110α and p110β. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110α, and p110α is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85α and p110α while preserving the stabilizing interaction between p85α iSH2 and the adapter-binding domain of p110α.oncogenic transformation | target of rapamycin T he phosphoinositide 3-kinase (PI3K) signaling pathway is deregulated in most human cancers by differential gene expression, amplification, or mutation. Of particular interest are mutations that occur in the catalytic subunit p110α of class I PI3K, because they confer a strong gain of function upon the enzyme, resulting in enhanced catalytic activity, constitutive signaling, and oncogenicity in vitro and in vivo (1-8). There have also been early reports of cancer-specific mutations in p85α, a regulatory subunit of class I PI3K (9-14). Such mutations gained high significance by recent comprehensive genomic analyses of glioblastomas (15,16). Approximately 9% of these tumors harbor a mutation in p85α. The mutations cluster in the inter-SH2 (iSH2) domain of p85α, involving residues that interact with the C2 domain of the catalytic subunit p110α (15,17). The iSH2-C2 domain interaction has an inhibitory effect on enzyme activity, and the mutations in the iSH2 domain of p85α could weaken this interaction and release the inhibition of PI3K activity (15,(17)(18)(19). A similar mechanism has been proposed for the gain-of-function mutations in the helical domain of p110α that alleviate an inhibitory interaction with the N-terminal SH2 domain (nSH2) of p85α (20).We have studied mutations in p85α (referred to as p85). Most of these were identified in a genomic characterization of glioblastoma (15) and map to the iSH2 domain of p85; one was an engineered mutation that maps to the nSH2 domain of p85. These mutations show oncogenic potency in cell culture and elevated levels of downstream signaling and operate through the p110α isoform of the catalytic subunit of class I PI3K. Our observations extend recent studies of the p85α mutants using different cell systems (17,19) by providing quantitative data on the oncogenic potency of the mutations and by presenting evidence that suggests a unique role of p110α for the p85 mutation-induced gain of function in PI3K activity. ResultsCancer-Derived Mutations of p85 Induce Oncogenic Transform...

The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.

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Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague−Dawley mice, maximum concentration (C max ) in plasma and brain was reached after 30 min, with a half-life (t 1/2 ) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

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