Microbiota- and Radiotherapy-Induced Gastrointestinal Side-Effects (MARS) Study: A Large Pilot Study of the Microbiome in Acute and Late-Radiation Enteropathy.

PURPOSE: Radiotherapy is important in managing pelvic cancers. However, radiation enteropathy may occur and can be dose limiting. The gut microbiota may contribute to the pathogenesis of radiation enteropathy. We hypothesized that the microbiome differs between patients with and without radiation enteropathy.Experimental Design: Three cohorts of patients (n = 134) were recruited. The early cohort (n = 32) was followed sequentially up to 12 months post-radiotherapy to assess early radiation enteropathy. Linear mixed models were used to assess microbiota dynamics. The late cohort (n = 87) was assessed cross-sectionally to assess late radiation enteropathy. The colonoscopy cohort compared the intestinal mucosa microenvironment in patients with radiation enteropathy (cases, n = 9) with healthy controls (controls, n = 6). Fecal samples were obtained from all cohorts. In the colonoscopy cohort, intestinal mucosa samples were taken. Metataxonomics (16S rRNA gene) and imputed metataxonomics (Piphillin) were used to characterize the microbiome. Clinician- and patient-reported outcomes were used for clinical characterization. RESULTS: In the acute cohort, we observed a trend for higher preradiotherapy diversity in patients with no self-reported symptoms (P = 0.09). Dynamically, diversity decreased less over time in patients with rising radiation enteropathy (P = 0.05). A consistent association between low bacterial diversity and late radiation enteropathy was also observed, albeit nonsignificantly. Higher counts of Clostridium IV, Roseburia, and Phascolarctobacterium significantly associated with radiation enteropathy. Homeostatic intestinal mucosa cytokines related to microbiota regulation and intestinal wall maintenance were significantly reduced in radiation enteropathy [IL7 (P = 0.05), IL12/IL23p40 (P = 0.03), IL15 (P = 0.05), and IL16 (P = 0.009)]. IL15 inversely correlated with counts of Roseburia and Propionibacterium. CONCLUSIONS: The microbiota presents opportunities to predict, prevent, or treat radiation enteropathy. We report the largest clinical study to date into associations of the microbiota with acute and late radiation enteropathy. An altered microbiota associates with early and late radiation enteropathy, with clinical implications for risk assessment, prevention, and treatment of radiation-induced side-effects.See related commentary by Lam et al., p. 6280.

Application of meso scale technology for the measurement of phosphoproteins in human tumor xenografts.

In this age of molecularly targeted drug discovery, robust techniques are required to measure pharmacodynamic (PD) responses in tumors so that drug exposures can be associated with their effects on molecular biomarkers and efficacy. Our aim was to develop a rapid screen to monitor PD responses within xenografted human tumors as an important step towards a clinically applicable technology. Currently there are various methods available to measure PD end points, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction, gene expression profiling, and western blotting. These may require relatively large samples of tumor, surrogate tissue, or peripheral blood lymphocytes with subsequent analyses taking several days. The phosphoinositide 3-kinase (PI3-kinase) pathway is frequently deregulated in cancer and is also important in diabetes and autoimmune conditions. In this paper, optimization of the Meso Scale Discovery (MSD) (Gaithersburg, MD) platform to quantify changes in phospho-AKT and phospho-glycogen synthase kinase-3beta in response to a PI3-kinase inhibitor, LY294002, is described, initially in vitro and then within xenografted solid tumors. This method is highly practical with high throughput since large number of samples can be run simultaneously in 96-well format. The assays are robust (coefficient of variation for phospho-AKT 13.4%) and offer significant advantages (in terms of speed and quantitation) over western blots. This optimized procedure can be used for both in vitro and in vivo analysis, unlike an established fixed-cell ELISA with a time-resolved fluorescent end point.

Evaluation of by disubstituted acridone derivatives as telomerase inhibitors: the importance of G-quadruplex binding.

The synthesis and evaluation of a group of 2,6-, 2,7- and 3,6-bis-aminoalkylamido acridones are reported, which show a similar level of activity against telomerase in vitro compared to their acridine counterparts. Computer modelling and calculations of relative binding energies suggest an equivalent binding mode to human intramolecular G-quadruplex DNA, but with significantly reduced affinity, as a result of the limited delocalisation of the acridone chromophore compared to the acridine system. Thermal melting studies on acridone and acridine quadruplex complexes using a FRET approach support these predictions. Long-term cell proliferation studies at sub-cytotoxic doses with two representative acridones using the SKOV3 cell line, show that neither compound produces growth arrest, in contrast with the effects produced by the tri-substituted acridine compound BRACO-19. It is concluded that telomerase inhibitory activity is a necessary though by itself insufficient property in order for cellular growth arrest to occur at sub-toxic concentrations, and that tight quadruplex binding is also required.

Trisubstituted acridine derivatives as potent and selective telomerase inhibitors.

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity.

The telomerase complex is responsible for telomere maintenance and represents a promising cancer therapeutic target. We describe herein the antitelomerase and antitumor properties of a small-molecule compound designed by computer modeling to interact with and stabilize human G-quadruplex DNA, a structure that may form with telomeric DNA, thereby inhibiting access to telomerase. The 3,6,9-trisubstituted acridine 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis(3-pyrrolodinopropionamido) acridine (BRACO19) represents one of the most potent cell-free inhibitors of human telomerase yet described (50% inhibitory concentration of 115 +/- 18 nM). Moreover, in contrast to G-quadruplex interactive agents described previously, BRACO19 did not cause nonspecific acute cytotoxicity at similar concentrations to those required to completely inhibit telomerase activity. There exists a 90-fold differential (mean 50% inhibitory concentration for acute cell kill across seven human tumor cell lines of 10.6 +/- 0.7 microM). The exposure of 21NT human breast cancer cells, which possess relatively short telomeres, to nonacute cytotoxic concentrations of BRACO19 (2 microM) resulted in a marked reduction in cell growth after only 15 days. This was concomitant with a reduction in intracellular telomerase activity and onset of senescence as indicated by an increase in the number of beta-galactosidase positive-staining cells. Intraperitoneal administration of nontoxic doses of BRACO19 (2 mg/kg) to mice bearing advanced stage A431 human vulval carcinoma subcutaneous xenografts and previously treated with paclitaxel induced a significant increase in antitumor effect compared with that observed with paclitaxel alone. BRACO19 thus represents the first of a "second generation" of G-quadruplex-mediated telomerase/telomere-interactive compounds. It possesses nanomolar potency against telomerase but low nonspecific cytotoxicity, growth inhibitory effects, and induction of senescence in a human breast cancer cell line and, moreover, significant antitumor activity in vivo when administered post paclitaxel to mice bearing a human tumor xenograft carcinoma.

Antitumor polycyclic acridines. 8.(1) Synthesis and telomerase-inhibitory activity of methylated pentacyclic acridinium salts.

Two short routes to novel methylated pentacyclic quinoacridinium salts have been devised. New compounds display telomerase-inhibitory potency (<1 microM) in the TRAP assay. 3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (12d, RHPS4, NSC 714187) has a higher selectivity for triplex and quadruplex DNA structures than the 3,6,8,11,13-pentamethyl analogue (12c, RHPS3, NSC 714186) and a low overall growth-inhibitory activity in the NCI 60 cell panel (mean GI(50) 13.18 microM); in addition, the activity profile of 12d does not COMPARE with agents of the topoisomerase II class. Compound 12d is soluble in water, stable in the pH range of 5-9, efficiently transported into tumor cells, and is currently the lead structure for further elaboration in this new class of telomerase inhibitor.