Development of a biomarker to monitor target engagement after treatment with dihydroorotate dehydrogenase inhibitors.

Dihydroorotate dehydrogenase (DHODH) catalyzes a key step in pyrimidine biosynthesis and has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that toxicity could result from on-target DHODH inhibition in those species. We describe here the use of dihydroorotate (DHO) as a biomarker of DHODH inhibition. Treatment of mammalian cells with DSM265 or the mammalian DHODH inhibitor teriflunomide led to increases in DHO where the extent of biomarker buildup correlated with both dose and inhibitor potency on DHODH. Treatment of mice with leflunomide (teriflunomide prodrug) caused a large dose-dependent buildup of DHO in blood (up to 16-fold) and urine (up to 5,400-fold) that was not observed for mice treated with DSM265. Unbound plasma teriflunomide levels reached 20-85-fold above the mouse DHODH IC50, while free DSM265 levels were only 1.6-4.2-fold above, barely achieving âˆ¼ IC90 concentrations, suggesting that unbound DSM265 plasma levels are not sufficient to block the pathway in vivo. Thus, any toxicity associated with DSM265 treatment in mice is likely caused by off-target mechanisms. The identification of a robust biomarker for mammalian DHODH inhibition represents an important advance to generally monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease.

Glutaminase Activity of L-Asparaginase Contributes to Durable Preclinical Activity against Acute Lymphoblastic Leukemia.

We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.

Assessment of l-Asparaginase Pharmacodynamics in Mouse Models of Cancer.

l-asparaginase (ASNase) is a metabolism-targeted anti-neoplastic agent used to treat acute lymphoblastic leukemia (ALL). ASNase's anticancer activity results from the enzymatic depletion of asparagine (Asn) and glutamine (Gln), which are converted to aspartic acid (Asp) and glutamic acid (Glu), respectively, in the blood. Unfortunately, accurate assessment of the in vivo pharmacodynamics (PD) of ASNase is challenging because of the following reasons: (i) ASNase is resilient to deactivation; (ii) ASNase catalytic efficiency is very high; and (iii) the PD markers Asn and Gln are depleted ex vivo in blood samples containing ASNase. To address those issues and facilitate longitudinal studies in individual mice for ASNase PD studies, we present here a new LC-MS/MS bioanalytical method that incorporates rapid quenching of ASNase for measurement of Asn, Asp, Gln, and Glu in just 10 µL of whole blood, with limits of detection (s:n ≥ 10:1) estimated to be 2.3, 3.5, 0.8, and 0.5 µM, respectively. We tested the suitability of the method in a 5-day, longitudinal PD study in mice and found the method to be simple to perform with sufficient accuracy and precision for whole blood measurements. Overall, the method increases the density of data that can be acquired from a single animal and will facilitate optimization of novel ASNase treatment regimens and/or the development of new ASNase variants with desired kinetic properties.

RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells.

Among the 3 GTPases in the DIRAS family, DIRAS3/ARHI is the best characterized. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26-kDa GTPase that shares 60% homology to RAS and RAP. DIRAS3 is downregulated in many tumor types, including ovarian cancer, where re-expression inhibits cancer cell growth, reduces motility, promotes tumor dormancy and induces macroautophagy/autophagy. Previously, we demonstrated that DIRAS3 is required for autophagy in human cells. Diras3 has been lost from the mouse genome during evolutionary re-arrangement, but murine cells can still undergo autophagy. We have tested whether DIRAS1 and DIRAS2, which are homologs found in both human and murine cells, could serve as surrogates to DIRAS3 in the murine genome affecting autophagy and cancer cell growth. Similar to DIRAS3, these 2 GTPases share 40-50% homology to RAS and RAP, but differ from DIRAS3 primarily in the lengths of their N-terminal extensions. We found that DIRAS1 and DIRAS2 are downregulated in ovarian cancer and are associated with decreased disease-free and overall survival. Re-expression of these genes suppressed growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death. Mechanistically, DIRAS1 and DIRAS2 induce and regulate autophagy by inhibition of the AKT1-MTOR and RAS-MAPK signaling pathways and modulating nuclear localization of the autophagy-related transcription factors FOXO3/FOXO3A and TFEB. Taken together, these data suggest that DIRAS1 and DIRAS2 likely serve as surrogates in the murine genome for DIRAS3, and may function as a backup system to fine-tune autophagy in humans.

Hyperthermia sensitizes Rhizopus oryzae to posaconazole and itraconazole action through apoptosis.

The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 µg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 µg/ml) and 42°C (0.25 to 1 µg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4',6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.

Evaluation of the clinical relevance of body composition parameters in patients with cancer metastatic to the liver treated with hepatic arterial infusion chemotherapy.

The association between body composition parameters and toxicity from hepatic arterial infusion (HAI) chemotherapy regimens has not been analyzed. We assessed data from patients with advanced cancer and liver metastases treated on a clinical trial of a regimen of HAI oxaliplatin combined with systemic 5-fluorouracil/leucovorin and bevacizumab. Correlations between patient characteristics, response, and toxicity and body composition data taken from CT images were analyzed. Forty-eight of 57 patients (mean age 56 yr; 60% women) had available CT scans. The most common diagnosis was colorectal cancer (22/48, 46%); 30/48 patients (63%) had body mass index (BMI) ≥25 kg/m(2). Twenty (42%) of 48 patients were sarcopenic. Grade 3-4 adverse events did not differ among patients with and without sarcopenia or according to BMI. The median survival (95% C]) was 167 (128-206) days for sarcopenic and 280 (214-346) days for nonsarcopenic patients (P = 0.271). Among patients treated at the maximum tolerated dose, the median survival was 103 days for sarcopenic and 312 days for nonsarcopenic patients (P = 0.173). Sarcopenia was present in 30% (6/20) of patients with reduction in tumor size posttreatment, and in 52% (14/27) of patients with increased tumor size (P = 0.171). In conclusion, body composition was not significantly associated with toxicities or survival in our small sample.