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Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition.Significance: Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. Cancer Res; 78(5); 1358-70. ©2017 AACR.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

Inhibition of both the de novo (DNP) and salvage (NSP) pathways of nucleoside synthesis has been demonstrated to impair leukemia cells. We endeavored to determine whether this approach would be efficacious in glioblastoma. To diminish nucleoside biosynthesis, we utilized compound DI-39, which selectively targets NSP, in combination with thymidine (dT), which selectively targets DNP. We employed in vitro and ex vivo models to determine the effects of pretreatment with dT + DI-39 on brain tumor stem cells (BTSC). Here, we demonstrate that this combinatorial therapy elicits a differential response across a spectrum of human patient-derived glioblastoma cultures. As determined by apoptotic markers, most cultures were relatively resistant to treatment, although a subset was highly sensitive. Sensitivity was unrelated to S-phase delay and to DNA damage induced by treatment. Bioinformatics analysis indicated that response across cultures was associated with the transcription factor PAX3 (associated with resistance) and with canonical pathways, including the nucleotide excision repair pathway, PTEN (associated with resistance), PI3K/AKT (associated with sensitivity), and ErbB2-ErbB3. Our in vitro assays demonstrated that, in sensitive cultures, clonal sphere formation was reduced upon removal from pretreatment. In contrast, in a resistant culture, clonal sphere formation was slightly increased upon removal from pretreatment. Moreover, in an intracranial xenograft model, pretreatment of a sensitive culture caused significantly smaller and fewer tumors. In a resistant culture, tumors were equivalent irrespective of pretreatment. These results indicate that, in the subset of sensitive glioblastoma, BTSCs are targeted by inhibition of pyrimidine synthesis. Mol Cancer Ther; 15(6); 1271-8. ©2016 AACR.

We discovered recently that the central metabolite α-ketoglutarate (α-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

Patient tumor tissue processing is an important step in the generation of clinically relevant specimens for in vitro and in vivo studies. Proper disassociation and tissue sample cleanup is a multistep, time-consuming process that ultimately effects the generation of patient derived xenografts and neurosphere cultures. Here we describe a detailed protocol on how to process and disassociate patient glioma tissue and subsequent steps on orthotopic implantation and in vitro generation of neurospheres.

When it comes to biobanking and working with different types of laboratory specimens, it is important to understand potential biohazards to ensure safety of the operator and laboratory personnel. Biological safety levels (BSL) are a series of designations used to inform laboratory personnel about the level of biohazardous risks in a laboratory setting. There are a total of four levels ranked in order of increasing risk as stipulated by the Center of Disease Control and Prevention (CDC) (Biosafety in microbiological and biomedical laboratories, 5th edn. HHS publication no. (CDC) 21-1112. https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf . Accessed 2 Jan 2016, 2009). We will address the main distinctions between these levels including briefly introducing hazards characteristics that classify biohazardous agents, as well as define the essentials in meeting safety requirements.

The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low Pi concentrations. Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced. The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant. The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.

Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg2+, the Km values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The Ksi values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole carbon and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by EDTA-lysozyme treatment. The R. meliloti acid phosphatase activity of crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence of 5 mM choline, betaine, trimethylammonium or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. Among several phosphoesters tested only pyridoxal-5-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) of crude extracts obtained from bacteria grown in the presence of serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected with alpha-naphthylphosphate or pyridoxal-5-phosphate were used as substrates. In conclusion, although the choline metabolites are capable of increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.

In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.

The inhibition by atropine of cholinesterase from Pseudomonas aeruginosa has been studied in parallel with the membrane bound acetylcholinesterase from rat red cells. Acetylcholinesterase of rat red cells, like other animal cholinesterases, was competitively inhibited while the cholinesterase from Pseudomonas aeruginosa was partially non competitively inhibited by atropine. These results clearly indicated a different behavior of cholinesterase from Pseudomonas aeruginosa in comparison with the enzyme of Pseudomonas fluorescens and other animal cholinesterases.

Choline, acetylcholine and betaine used as a sole carbon source, effectuate in Ps. aeruginosa an acid phosphatase activity in addition to a cholinesterase activity. Induction of both enzyme activities was repressed by succinate or glucose. Cyclic AMP failed to relieve the repression produced by these compounds. Substrates not related to choline and used as a sole source of carbon, were inefficient to produce induction of both enzymes. The in-vitro action of choline, acetylcholine and betaine on Ps. aeruginosa acid phosphatase and cholinesterase has also been studied. To perform these studies periplasmic extracts obtained by EDTA-lysozyme treatment of the cells grown on choline or betaine as sole source of carbon, were used. Acid phosphatase activity was competitively inhibited by betaine, whereas the inhibition produced by choline and acetylcholine showed competitive and noncompetitive components. Cholinesterase activity was noncompetitively inhibited by betaine. At low acetylthiocholine concentration choline was an inhibitor of cholinesterase, whereas at high substrate concentration choline raised the hydrolysis rate of acetylthiocholine. These findings allow the conclusion that acid phosphatase and cholinesterase are specifically induced by choline and its metabolites derivatives. Kinetic results led us to postulate that acid phosphatase and cholinesterase contain a similar allosteric site. This site would either be of an anionic nature or show affinity to a methyl group or display both characteristics.

Different compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.

In this work the action of the following compounds upon Ps. aeruginosa acid phosphatase has been studied: 1) alkylammonium compounds; 2) aminoalcohols and aminoacids with different substituents (-H, -CH2OH and -CH3) attached to the nitrogen atom; 3) alcohols analogous to some compounds of the above series, but without the amino group. It was found that the enzyme inhibition was more effective with N-trimethylated compounds than with the triethylated ones. The degree of inhibition depended on the number of methyl groups bound to the nitrogen atom. Taking into account the choline and betaine series the hydroxyl derivatives showed more affinity for the enzyme than the carboxylated ones. In each series the Ki values increased with the decrease of methyl groups bound to the nitrogen atom. The presence of a positively charged nitrogen atom in the molecule of the effector was essential. These results enable us to confirm that in the molecule of Ps. aeruginosa acid phosphatase there exists an anionic site with one subsite with affinity for methyl groups.

Choline used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km = 3 microM) and another of low affinity (Km = 400 microM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na+ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.