Martina Proctor

Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TGs actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing. Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

Neutralizing IL-23 is superior to blocking IL-17 in suppressing intestinal inflammation in a spontaneous murine colitis model.

Background:IL-23/TH17 inflammatory responses are regarded as central to the pathogenesis of inflammatory bowel disease, but clinically IL-17A antibodies have shown low efficacy and increased infections in Crohns disease. Hence, we decided to closely examine the role of the IL-23/TH17 axis in 3 models of colitis. Methods:IL-17A−/− and IL-17Ra−/− T cells were transferred into Rag1−/− and RaW mice to assess the role of IL-17A-IL-17Ra signaling in T cells during colitis. In Winnie mice with spontaneous colitis due to an epithelial defect, we studied the progression of colitis in the absence of IL-17A and the efficacy of neutralizing antibodies against the IL-17A or IL-23p19 cytokines. Results:In transfer colitis models, IL-17A-deficient T cells failed to ameliorate disease, and IL-17Ra-deficient T cells were more colitogenic than wild-type T cells. In Winnie mice with an epithelial defect and spontaneous TH17-dominated inflammation, genetic deficiency of IL-17A did not suppress initiation of colitis but limited colitis progression. Furthermore, inhibition of IL-17A by monoclonal antibodies did not reduce colitis severity. In contrast, neutralizing IL-23 using an anti-p19 antibody significantly alleviated both emerging and established colitis, downregulating TH17 proinflammatory cytokine expression and diminishing neutrophil infiltration. Conclusions:Our results support clinical studies showing that IL-17 neutralization is not therapeutic but that targeting IL-23 suppresses intestinal inflammation. Effects of IL-23 distinct from its effects on maturation of IL-17A-producing lymphocytes may underlie the protection from inflammatory bowel disease conveyed by hypomorphic IL-23 receptor polymorphisms and contribute to the efficacy of IL-23 neutralizing antibodies in inflammatory bowel disease.

Inhibition of Aurora A and Aurora B Is Required for the Sensitivity of HPV-Driven Cervical Cancers to Aurora Kinase Inhibitors

The activity and efficacy of Aurora inhibitors have been reported in a wide range of cancer types. The most prominent Aurora inhibitor is alisertib, an investigational Aurora inhibitor that has been the subject of more than 30 clinical trials. Alisertib has inhibitory activity against both Aurora A and B, although it is considered to be primarily an Aurora A inhibitor in vivo. Here, we show that alisertib inhibits both Aurora A and B in vivo in preclinical models of HPV-driven cervical cancer, and that it is the inhibition of Aurora A and B that provides the selectivity and efficacy of this drug in vivo in this disease setting. We also present formal evidence that alisertib requires progression through mitosis for its efficacy, and that it is unlikely to combine with drugs that promote a G2 DNA damage checkpoint response. This work demonstrates that inhibition of Aurora A and B is required for effective control of HPV-driven cancers by Aurora kinase inhibitors. Mol Cancer Ther; 16(9); 1934–41. ©2017 AACR.

Mechanism of action of the third generation benzopyrans and evaluation of their broad anti-cancer activity in vitro and in vivo

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

Endogenous replication stress marks melanomas sensitive to CHK1 inhibitors in vivo

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo. Here, we have investigated the molecular basis of this activity. Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo. The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines. Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo. In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i. Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivo. Clin Cancer Res; 24(12); 2901–12. ©2018 AACR.