Gut Toxicity Research Articles

Editorial introductions

Editorial introductions

Toll-like receptors in the pathogenesis of chemotherapy-induced gastrointestinal toxicity.

Intestinal mucositis represents a common complication and dose-limiting toxicity of cancer chemotherapy. So far chemotherapy-induced intestinal mucositis remains poorly treatable resulting in significant morbidity and reduced quality of life in cancer patients. This review discusses recent insights into the pathophysiology of chemotherapy-induced intestinal mucositis. Novel mechanisms linking gut microbiota, host innate immunity and anticancer drug metabolism are highlighted. Gut microbiota may affect xenobiotic metabolism by direct and indirect mechanisms, critically modulating gut toxicity of chemotherapy drugs. Composition and metabolic function of the gut microbiome as well as innate immune responses of the intestinal mucosa are severely altered during chemotherapy. Commensal-mediated innate immune signaling via Toll-like receptors (TLRs) ambiguously shapes chemotherapy-induced genotoxic damage in the gastrointestinal tract. TLR2 may accelerate host detoxification by activating the multidrug transporter ATP-binding cassette 1 (ABCB1)/MDR1 P-glycoprotein to efflux harmful drugs, thus controlling the severity of cancer therapy-induced mucosal damage in the gastrointestinal tract. In contrast, selective chemotherapy drugs may drive LPS hyperresponsiveness via TLR4, which exacerbates mucosal injury through aberrant cytokine storms. Broad-spectrum antibiotic treatment does not seem to represent a valid therapeutic option, as drastic reduction in global gut microbiota may enhance risk of gastrointestinal toxicity and reduce efficacy of some chemotherapy drugs, at least in murine models. Several variables (environment, metabolism, dysbiosis, infections and/or genetics) influence the outcome of mucosal TLR signaling during cancer treatment. Differences in innate immune responses also reflect chemotherapy drug-specific effects. Future studies must investigate in more detail whether manipulating the delicate balance between gut microbiota and host immune responses by either monotherapy or combinations of different TLR agonists and antagonists may be indeed useful to limit the toxic side-effects of complex chemotherapy regimens, accelerate mucosal tissue regeneration and improve the anticancer treatment response.

A novel in vitro platform for the study of SN38-induced mucosal damage and the development of Toll-like receptor 4-targeted therapeutic options.

Tight junction and epithelial barrier disruption is a common trait of many gastrointestinal pathologies, including chemotherapy-induced gut toxicity. Currently, there are no validated invitro models suitable for the study of chemotherapy-induced mucosal damage that allow paralleled functional and structural analyses of tight junction integrity. We therefore aimed to determine if a transparent, polyester membrane insert supports a polarized T84 monolayer with the phenotypically normal tight junctions. T84 cells (passage 5-15) were seeded into either 0.6 cm(2), 0.4 µm pore mixed-cellulose transwell hanging inserts or 1.12 cm(2), 0.4 µm pore polyester transwell inserts at varying densities. Transepithelial electrical resistance was measured daily to assess barrier formation. Immunofluoresence for key tight junction proteins (occludin, zonular occludens-1, claudin-1) and transmission electron microscopy were performed to assess tight junction integrity, organelle distribution, and polarity. Reverse transcription-polymerase chain reaction was performed to determine expression of toll-like receptor 4 (TLR4). Liquid chromatography was also conducted to assess SN38 degradation in this model. Polyester membrane inserts support a polarized T84 phenotype with functional tight junctions invitro. Transmission electron microscopy indicated polarity, with apico-laterally located tight junctions. Immunofluorescence showed membranous staining for all tight junction proteins. No internalization was evident. T84 cells expressed TLR4, although this was significantly lower than levels seen in HT29 cells (P = .0377). SN38 underwent more rapid degradation in the presence of cells (-76.04 ± 1.86%) compared to blank membrane (-48.39 ± 4.01%), indicating metabolic processes. Polyester membrane inserts provide a novel platform for paralleled functional and structural analysis of tight junction integrity in T84 monolayers. T84 cells exhibit the unique ability to metabolize SN38 as well as expressing TLR4, making this an excellent platform to study clinically relevant therapeutic interventions for SN38-induced mucosal damage by targeting TLR4.

Peritransplant Serum Albumin Decline Predicts Subsequent Severe Acute Graft-versus-Host Disease after Mucotoxic Myeloablative Conditioning

Conditioning-related gut toxicity can result in a protein-losing enteropathy manifesting as a decline in serum albumin in the peritransplant period. Inspired by the pathogenesis of acute graft-versus-host disease (aGVHD), we hypothesized that the magnitude of decline in serum albumin from the day of conditioning initiation until its nadir in the first 2 weeks after hematopoietic cell transplantation HCT (DeltaAlb) predicts the risk for subsequent severe aGVHD. We reviewed the medical records of all 88 patients with acute myeloid leukemia or myelodysplastic syndrome who underwent highly mucotoxic myeloablative (busulfan/cyclophosphamide or cyclophosphamide/total body irradiation) allogeneic HCT from a matched related donor (MRD) or matched unrelated donor (MUD) at our institution between January 1, 2012 and January 1, 2015. Severe aGVHD was associated with MUD (47% versus 14% with MRD; P = .001) and DeltaAlb, which was significantly greater among patients who developed versus did not develop severe aGVHD (1.2 ± .5 versus .8 ± .4 g/dL, respectively; P < .001). In multivariate analysis DeltaAlb remained a significant predictor of severe aGVHD (odds ratio, 5.68; 95% CI, 1.65 to 19.64; P = .006; area under the ROC curve, .74; 95% CI, .63 to .86; P < .001). The best cutoff for DeltaAlb to predict severe aGVHD was .9, with a sensitivity, specificity, and overall classification accuracy of 77%, 66%, and 69%, respectively. The model was validated using the bootstrap technique, with no significant change in its performance. These results were not generalizable to a cohort of 30 patients who received less mucotoxic myeloablative or reduced-intensity conditioning. In conclusion, with mucotoxic myeloablative HCT, each .1-g/dL increase in DeltaAlb was associated with an approximately 23% increase in the odds of developing severe aGVHD. As an early biomarker of gut damage, DeltaAlb can be incorporated in composite risk models for aGVHD prediction, with hopes for ultimately allowing for individualized GVHD prophylaxis and potential intervention according to the predicted risk.

Chemotherapy Modulates Intestinal Immune Gene Expression Including Surfactant Protein-D and Deleted in Malignant Brain Tumors 1 in Piglets

Background: Information about chemotherapy-induced intestinal gene expression may provide insight into the mechanisms underlying gut toxicity and help identify biomarkers and targets for intervention. Methods: We analyzed jejunal tissue from piglets subjected to two different, clinically relevant chemotherapy regimens: (1) busulfan plus cyclophosphamide (BUCY) and (2) doxorubicin (DOX). Results: Gene expression analysis identified 1,328 differentially expressed genes in the BUCY piglets and 594 in the DOX piglets, compared to controls. Similar changes in expression were found for 137 genes across the BUCY and DOX piglets. Selected genes of potential biological significance with a similar change in expression across the treatments were controlled by real-time polymerase chain reaction. Key innate defense molecules, including surfactant protein-D and deleted in malignant brain tumors 1, were among the upregulated genes for both treatments. Conclusion: In the developing intestine, chemotherapy increases the expression of genes related to innate immune functions involved in surveillance, protection, and homeostasis of mucosal surfaces.

Abstract B150: Identifying tolerable schedules for targeted anti-cancer agents by applying ordered logistic regression modeling to empirical pathology scores in mice

Abstract Even targeted oncology agents may have significant toxicity, either target-mediated or otherwise. Some targets are simultaneously drivers of cancer yet essential for normal turnover in certain tissues. We sought to develop a predictive model for gut toxicity, to enable model-based selection of the optimal dose and schedule in mice. Mice were treated under several schedules (twice daily, once every one, two or three days), at various doses (6.25-100 mg/kg) of the inhibitor AZ17, and for various durations. Target inhibition is expected to disregulate normal turnover of the gut lining. Epithelial histopathology findings collected from individual animals at various timepoints after the final dose were manually scored on an empirical five-point scale. An ordered logistic regression model was fit to all animals individually across all regimens simultaneously, to describe the probability of observing toxicity at a particular level on the five-point scale as a function of dose and time. It has recently been applied to clinical data, but this is a novel approach in the pre-clinical space. This model-based approach allows integration of data collected at various timepoints and from different studies. It differs from the standard PK/PD approach in that it attempts to predict not the toxicity score itself (which can only be an integer), but the probability of observing each score on the scale. It allows for recovery once dosing stops. The predicted probabilities will thus vary with dose, schedule, duration of treatment and time since last dose. We illustrate with a study of 30 mice, showing that the model was able to capture a number of essential features of any such model: (i) the steep nature of the dose-response relationship; (ii) robustness to outliers (e.g., when a single animal experienced severe tox at a generally tolerated dose); (iii) robustness to non-exposure-driven tox (e.g., finding in vehicle-treated animal). Since staining/reading of pathology slides is not resource intensive, this approach is suitable even for lead-optimization projects. Such data could be collected at the end of a standard tolerability study, in which body-weight is the typical endpoint. It could also be collected from tumor-bearing animals, either a pharmacodynamic or efficacy study. Both applications conform with the 3R's imperative to ‘reduce, refine, replace’ use of animals. Since the model, by design, makes no assumptions about mechanism, it could easily be extended to assess synergistic tox when combined with chemotherapy. Citation Format: Francis Gibbons, Shenghua Wen, Prasad Nadella. Identifying tolerable schedules for targeted anti-cancer agents by applying ordered logistic regression modeling to empirical pathology scores in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B150.

Chemotherapy-induced gut toxicity and pain: involvement of TLRs.

Chemotherapy-induced gut toxicity is associated with significant pain, and pain influences gut function. Toll-like receptors (TLRs) that regulate gut homeostasis are activated by tissue damage and microbes, and their altered expression following chemotherapy may change cellular responses. This study examined the interaction between chemotherapy-induced gut toxicity and pain and related these to gut TLR and glial fibrillary acidic protein (GFAP) expression. Female tumor bearing Dark Agouti rats received irinotecan (175 mg/kg, n = 34) or vehicle (n = 5) and were assessed over 120 h for gut toxicity (diarrhea, weight loss), pain (facial), and GFAP, TLR2, 4, 5, and 9 gut expression. Irinotecan caused diarrhea (72 % of animals grade ≥ 1), weight loss (11.1 ± 6.6 %, P < 0.0001), and pain (5 (0-5), P < 0.0001) all peaking at 72 h. Higher pain scores were observed in rats with diarrhea versus those without: median (range) of 2.0 (0-5) versus 0 (0-5), P = 0.01. Irinotecan also caused a decrease in TLR4 and 5, and an increase in GFAP expression in jejuna crypt at 96 and 120 h (all P < 0.05); with lower TLR4 expression associated with lower pain (P = 0.012). The association between gut toxicity and pain suggests these toxicities are linked, possibly via TLR-mediated inflammatory pathways. Further, as TLR4 and 5 expression was absent during recovery in the jejuna and GFAP expression was increased in the jejuna, this implies expression of these may be critical in the healing phase following chemotherapy. Detailed studies of gut TLRs and GFAP are now warranted.

Mycotoxin mechanisms of action and health impact: ‘in vitro’ or ‘in vivo’ tests, that is the question

The aim of this paper is to present examples ofin vitro andin vivo tests for mycotoxin mechanisms of action and evaluation of health effects, with a focus on the gut environment and toxicity testing.In vivo investigations may provide information on the net effects of mycotoxins in whole animals, whereasin vitro models represent effective tools to perform simplified experiments under uniform and well-controlled conditions and a suitable alternative toin vivo animal testing providing insights not achievable with animal studies. The main limits ofin vitro models are the lack of interactions with other cells and extracellular factors, lack of hormonal or immunological influences, and lack or different levels ofin vitro expression of genes involved in the overall response to mycotoxins. The translation ofin vitro data into meaningfulin vivo effects remains an unsolved problem. The main issues to be considered are the mycotoxin concentration range in accordance with levels encountered in realistic situations, the identification of reliable biomarkers of mycotoxin toxicity, the measurement of the chronic toxicity, the evaluation of single- or multi-toxin challenge. The gastrointestinal wall is the first barrier preventing the entry of undesirable substances. The intestinal epithelium can be exposed to high concentrations of mycotoxins upon ingestion of contaminated food and the amount of mycotoxin consumed via food does not always reflect the amount available to exert toxic actions in a target organ.In vitro digestion models in combination with intestinal epithelial cells are powerful tools to screen and predict thein vivo bioavailability and digestibility of mycotoxins in contaminated food and correctly estimate health effects. In conclusion,in vitro andin vivo tests are complementary approaches for providing a more accurate picture of the health impact of mycotoxins and improved understanding and evaluation of relevant dietary exposure and risk scenarios.

In vivo studies of the PARP inhibitor, AZD-2281, in combination with fractionated radiotherapy: An exploration of the therapeutic ratio

Background and purposePre-clinical data have shown that PARP inhibitors (PARPi) may increase the efficacy of radiotherapy in prostate cancer. However, it is uncertain as to whether PARPi lead to clonogenic kill when combined with radiotherapy (RT). Material and methodsWe tested the PARP inhibitor AZD-2281 as a radiosensitizing agent under oxic and hypoxic conditions for clonogenic survival in vitro and in vivo using the human prostate cancer cell line, 22Rv1. In addition, the effects of PARPi+RT on normal tissue were investigated using a crypt clonogenic assay. ResultsAZD-2281 inhibited cellular PARP activity under both oxic and hypoxic conditions. The addition of AZD-2281 radiosensitized 22Rv1 cells under oxia, acute hypoxia and chronic hypoxia in vitro. The combination of AZD-2281 with fractionated radiotherapy resulted in a significant growth delay and clonogenic kill in vivo. No increased gut toxicity was observed using this combined PARPi+radiotherapy regimen. ConclusionsThis is the first preclinical study to demonstrate direct clonogenic kill in vivo by the addition of AZD-2281 to radiotherapy. As we did not observe gut toxicity, the use of PARPi in the context of prostate cancer radiotherapy warrants further investigation in clinical trials.

Bovine Colostrum Modulates Myeloablative Chemotherapy–Induced Gut Toxicity in Piglets1–3

Intensive chemotherapy frequently results in gut toxicity, indicated by oral and intestinal mucositis, resulting in poor treatment outcomes and increased mortality. There are no effective preventive strategies against gut toxicity and the role of diet is unknown. We hypothesized that the severity of chemotherapy-induced gut toxicity in early life is diet-dependent, and that intake of bovine colostrum (BC) provides better gut protection than an artificial milk replacer (MR). A total of 37 3-d-old pigs received for 6 d either intravenous saline control or myeloablative treatment with busulfan and cyclophosphamide, and were fed either BC or MR, resulting in the following 4 treatments (n = 8-10/group): bovine colostrum plus saline control (Ctr-BC), milk replacer plus saline control (Ctr-MR), bovine colostrum plus busulfan and cyclophosphamide chemotherapy (BUCY-BC), and milk replacer plus busulfan and cyclophosphamide chemotherapy (BUCY-MR). The gut was collected for analysis 11 d after the start of chemotherapy. Relative to the control groups, both busulfan and cyclophosphamide chemotherapy (BUCY) groups showed signs of gut toxicity, with oral ulcers, reduced intestinal dimensions, and hematologic toxicity. Diet type did not affect mucosal structure on day 11, but BUCY-BC pigs had less vomiting than BUCY-MR pigs (1 of 10 vs. 10 of 10, P < 0.05). Markers of intestinal function were higher (up to 20-fold greater galactose absorption and 2-3-fold greater brush border enzyme activity, all P < 0.05), and tissue inflammatory cytokine concentrations and serum liver enzyme values were lower in BUCY-BC than in BUCY-MR pigs (30-50% reductions in interleukin 6 and 8, aminotransferase, and bilirubin concentrations, P < 0.05). Gut colonization was not significantly affected except that BUCY pigs had lower microbial diversity with a higher abundance of Lactobacilli. BC may reduce gut toxicity during myeloablative chemotherapy in piglets by preserving intestinal function and reducing inflammation. Whether similar effects occur in children remains to be tested.

Abstract B13: Hypoxia-induced chemokine activation and lymphnode metastases in primary cervix xenografts

Abstract Aim: To examine the role of the chemokine pathway in cervix cancer in the regulation of hypoxia-induced metastases and bone marrow derived myeloid cells (BMDCs) in the tumor microenvironment. Methods: Cervix cancer biopsies from patients in clinical studies at PMH were used for implantation into the cervix of NRG mice. Relative CXCR4 expression by qRT-PCR in biopsies from 117 patients with cervical cancer and 17 primary, orthotopic, cervical cancer xenografts was conducted. Mice bearing orthotopic xenografts were exposed to cyclic hypoxia (12x 10min cycles of breathing 7%O2 or air; 4hr/day for 2 wks) during their growth in combination with AMD3100 (Plerixafor), administered by osmotic pumps (5mg/kg/day) implanted subcutaneously for 3 weeks. Primary tumor size and para-aortic lymph node metastases were assessedfor metastases (H&amp;E staining). Mice received 15x30Gy (2Gy daily 5x/wk) targeted radiotherapy with cisplatin (4mg/kg/wk)(RTCTx) with or without AMD3100. The mice were monitored weekly for tumor growth by CT scanning. Immunohistochemistry was used to analyse levels of hypoxia (EF5/CA-9), blood (CD31) and lymphatic (LYVE-1) vessels, and CXCR4/CXCL12 in the orthotopic tumors and involved lymphnodes. Cd11b staining was used to assess neutrophil infiltration into the tumors. The GI toxicity of AMD3100 with RTCTx was assessed by gut crypt assay. Results: The (early passage) primary orthotopic cervical xenografts used in this study invade and metastasize to lymph nodes in a manner similar to cervical cancer in patients. The microenvironmental features (vascularity, stroma content, hypoxia, IFP) of the xenografts match the original biopsy. The variability in baseline CXCR4 gene expression (by qRT-PCR) in our xenograft models is also similar to that seen in the patient biopsies. Preliminary data also shows comparable CXCL12 and CD11b+ BMDC infiltration in a patient biopsy and in primary xenografts. Induction of tumor hypoxia by exposing the animals to a low oxygen environment increased CXCL12 and CXCR4 gene and protein expression and resulted in a significant increase in the number of lymph node metastases. Treatment with the CXCR4 antagonist AMD3100 reversed these effects. Tumor-bearing animals treated with targeted RTCTx combined with AMD3100 demonstrated significantly enhanced growth delay compared to RTCTx alone and demonstrated reduced metastases at the end of the study. There was no evidence that AMD3100 enhanced the gut toxicity of the RTCTx treatment. Conclusions: Our results suggest that CXCR4 plays an important role in metastasis and tumor growth, under hypoxic conditions, in human cervical cancer. Targeting the CXCR4-CXCL12 axis, with chemokine specific inhibitors,may serve as a novel and attractive therapeutic approach in cervix cancer.These studies provide a strong focus on developing and optimizing new targeted therapeutic approaches aimed at mitigating the adverse consequences of altered chemokine/BMDC signaling and translating these to high-impact phase I/II clinical trials. Citation Format: Naz Chaudary, Salomeh Jelveh, Patricia Lindsay, Melania Pintilie, Michael Milosevic, Richard P. Hill. Hypoxia-induced chemokine activation and lymphnode metastases in primary cervix xenografts. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B13. doi:10.1158/1538-7445.CHTME14-B13

Selectively Targeting Mutated NOTCH1 with a Folate-Thapsigargin Derivative

Gain-of-function mutations in NOTCH1, encoding a trans-membrane receptor involved in a transcriptional activation complex, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Thus, targeting NOTCH1 represents one promising therapeutic opportunity in T-ALL. The implementation of NOTCH1 pathway antagonists, however, poses several challenges secondary to concerns about inhibiting normal Notch receptors, leading to gut toxicity and secondary malignancies.We recently reported the results of a gene-expression based, NOTCH1 chemical genomic loss-of-function screen and a cell-based screen for cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA (Sarco-endoplasmic reticulum Ca2+ ATPases) emerged at the intersection of these complementary approaches. We discovered that SERCA inhibition impairs the maturation of mutated NOTCH1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human T-ALL cells. A small-molecule SERCA inhibitor, thapsigargin, a sesquiterpene-g-lactone, had on-target activity in mouse models of human T-ALL and also interfered with Notch signaling in Drosophila. Remarkably, thapsigargin preferentially inhibited mutated NOTCH1 receptors. This selectivity provides a therapeutic window not observed with other Notch pathway inhibitors, such as gamma-secretase inhibitors, that are equipotent inhibitors of mutated and wild-type receptors.While thapsigargin is a highly potent tool compound, in its native form, it is unlikely to have direct clinical application because of its global effects on intracellular Ca2+. Aberrant expression of folate receptors (FR) on the surface of cancer cells, including leukemia, has previously been reported, and efforts have been undertaken to leverage folate receptor-mediated endocytosis to deliver cancer therapeutics in vivo. We therefore developed a thapsigargin derivative conjugated with folic acid (JQ-FT) for preclinical testing in T-ALL.The FR gene family includes four members: FOLR1-4. The high affinity of FOLR1 and FOLR2 for folate binding, their endocytic capacity and their restricted expression pattern have prompted evaluation of folate-drug conjugates as a therapeutic approach in cancer. Analysis of gene-expression profiling suggested that FOLR2 is more highly and consistently expressed compared to FOLR1 in human T-ALL cell lines. We confirmed this finding by qRT-PCR across a panel of T-ALL cell lines and three primary patient samples. Surface expression of FOLR1 and FOLR2 measured by flow cytometry paralleled the transcriptional findings. We next validated the dependency of the uptake of a folate-FITC probe in T-ALL cells on active endocytosis via FR. First, we demonstrated that treatment of T-ALL cells with increasing concentrations of folate-FITC corresponds to an increase in measurable fluorescent signal by flow cytometry. The uptake is specific for FR as overexpression of FOLR1 or FOLR2 leads to increased, and folic acid competition decreased, fluorescent signal. Moreover, active endocytosis is supported by data demonstrating that folate-FITC fluorescence is not eliminated with an acidic wash step and that cold temperature does diminish internalization of the chemical probe.Next we demonstrated that increasing concentrations of JQ-FT impede the growth of T-ALL cells and induce apoptosis as measured by the activation of caspase 2. Moreover, the overexpression of ICN1, the intracellular active form of NOTCH1, partially rescues the small-molecule anti-proliferative effect indicating that the JQ-FT retains the on target activity similar to the underivatized thapsigargin. As observed with thapsigargin treatment of T-ALL cells, JQ-FT treatment decreased the levels of the intracellular active form of NOTCH1, ICN1, and the furin-processed transmembrane form of NOTCH1, while the full length precursor accumulated.In summary, these pre-clinical studies confirm the selective uptake of a folate-conjugated thapsigargin by an FR-mediated mechanism with the suppression of mutated NOTCH1 maturation and support the ongoing testing of this approach in mouse models of NOTCH1-mutated T-ALL. If successful, this approach would enhance the therapeutic window of thapsigargin as a NOTCH1 inhibitor providing dual selectivity: leukemia over normal cell and NOTCH1 mutated over wild-type receptors. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-40: Inducible in vivo silencing of Brd4 identifies potential toxicities of sustained BET protein inhibition

Abstract BET family proteins are novel therapeutic targets for cancer and inflammation and represent the first epigenetic readers against which small-molecule inhibitors have been developed. First generation BET inhibitors have shown some therapeutic efficacy in pre-clinical models, however the consequences of disrupting BET protein function in normal tissues are largely unknown. Using an inducible and reversible transgenic RNAi mouse model targeting the BET family protein Brd4, we show that acute suppression of Brd4 in adult animals has immediate and dramatic effects in multiple tissues. Brd4-depleted mice displayed epidermal hyperplasia, alopecia, and decreased cellular diversity and stem cell depletion in the small intestine. To test whether the loss of intestinal stem cells induced by Brd4 suppression would have consequences in combination with agents that induce acute gut toxicity, we subjected mice to a sub-lethal irradiation. Whereas control mice showed intestinal recovery 4-5 days following irradiation, Brd4-depleted mice showed dramatic and continued crypt-villus atrophy, implying that potent BET protein inhibition in combination with cytotoxic chemotherapy may show significantly enhanced toxicity in the clinic. These findings provide important insight into Brd4 function in normal tissues and importantly, predict several potential outcomes associated with potent and sustained inhibition of BET protein function. Citation Format: Jessica Bolden, Nilgun Tasdemir, Lukas Dow, Johan H. van Es, John E. Wilkinson, Zhen Zhao, Hans Clevers, Scott W. Lowe. Inducible in vivo silencing of Brd4 identifies potential toxicities of sustained BET protein inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-40. doi:10.1158/1538-7445.AM2014-LB-40

Abstract 2990: Notch1 monoclonal antibody inhibits tumor growth and modulates angiogenesis

Abstract AVEO Oncology has developed a series of inducible mouse models of cancer, which through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding was consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit the Notch1 receptor. Characterization of monoclonal antibodies targeting Notch1 through cell-based and biochemical studies demonstrated that the Notch1 monoclonal antibody bound with high affinity and high specificity to the Notch1 receptor, prevented ligand-mediated activation of the target receptor, and specifically repressed Notch-dependent signaling with high potency. Significantly, specific inhibition by the Notch1 monoclonal antibody did not result in the dose-limiting gut toxicity observed with Notch1 mAb resulted in tumor inhibition that was accompanied by increased vascular density, inhibition of Notch target gene expression, and increased expression of angiogenesis-related genes, consistent with expected changes resulting from Notch1/Dll4 pathway inhibition. In addition, several tumor models treated in combination with Notch1 mAb and an investigational VEGF pathway inhibitor, tivozanib, demonstrated increased tumor inhibition compared to either agent alone. Interestingly, combination treatment resulted in decreased vascular density and downregulated expression of angiogenesis genes, similar to treatment with VEGF inhibitor alone. Collectively, our work suggests that combination therapy of Notch1 antibodies with VEGF inhibitors should be evaluated as a potential viable therapeutic option for VEGF-resistant tumors. Citation Format: Heidi Okamura, Theresa Proia, Alisa Bell, Qing Liu, Zakir Siddiquee, Jie Lin, Jeno Gyuris. Notch1 monoclonal antibody inhibits tumor growth and modulates angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2014-2990

Matrix metalloproteinases and gut toxicity following cytotoxic cancer therapy.

Chemotherapy is an effective anticancer treatment; however, it induces mucositis in a wide range of patients. This condition is characterized by pain and ulceration, vomiting, bloating and diarrhea, depending on the area of the alimentary tract affected. Although treatment is available for a small subset of patients suffering from mucositis, the majority rely on pain relief as their only treatment option. Much progress has been made in recent years into understanding the pathobiology underlying the development of mucositis and matrix metalloproteinases (MMPs) have been implicated as being key mediators. The purpose of this review was to evaluate recent literature implicating MMPs in mucositis. MMPs are well known for their roles in induction of inflammation and contribution to tissue injury. Recent literature provides a role for MMPs in mucositis development possibly through inflammatory pathways, alterations in extracellular matrix composition, adhesion molecules and tight junctions. Better understanding of the precise roles of MMPs is now required in order to target appropriate treatment strategies.

Near fatal 5-FU gut toxicity post surgery – remarkable effect of high-dose sucralfate

Background:The objective of this review article and case report was to investigate the effectiveness of high-dose sucralfate on severe life-threatening 5-fluorouracil (5-FU) gut toxicity, with reference to, but not limited to dihydropyrimidine dehydrogenase (DPD) deficiency.Methods:A search was conducted on PubMed from 1950 to July 2013 for original studies on 5-FU gut toxicity and sucralfate. Studies were limited to human trials and English language and all articles included in this study were assessed with the application of predetermined selection criteria. Each article was then reviewed independently by two reviewers. A case report from our own centre was included in this review.Results:From 33 results, 6 manuscripts were identified including 4 randomized controlled trial. One trial evaluated the use of sucralfate to alleviate stomatitis in patients with 5-FU-based chemotherapy. The other three trials evaluated the role of sucralfate in radiation toxicity. There was one case report which showed gastroscopy confirmed normalization of severe dysplastic erosive gastroduodenitis attributed to hepatic arterial infusion of 5-FU following a 2-month course of sucralfate and cimetidine and one case series showing clinical and sigmoidoscopically demonstrated improvement in ulcerative colitis in majority of patients receiving sucralfate enemas. There was no current literature specifically focussed on the role of sucralfate in 5-FU gut toxicity. Our case report describes the clinical course and successful treatment with sucralfate of a patient with Pseudomyxoma peritonei (PMP) who experienced 5-FU gut toxicity resulting in life-threatening bleeding due to presumed DPD deficiency post intraperitoneal 5-FU administration.Conclusion:This review article showed a lack of literature concerning the use of sucralfate in 5-FU gut toxicity. In our patient’s case, sucralfate had a crucial role in the management of near fatal 5-FU gut toxicity, and further evaluation is required.

Gastrointestinal Mucositis: The Role of MMP-Tight Junction Interactions in Tissue Injury

Chemotherapy for cancer causes significant gut toxicity known as mucositis. The pathogenesis of mucositis is ill defined. Recent clinical research guidelines have highlighted epithelial junctional complexes as emerging targets within mucositis research. Given the robust biological evidence linking tight junctions and matrix metalloproteinases, key mediators of mucositis, tight junction proteins have received significant attention. Despite this, the link between tight junctions, matrix metalloproteinases and mucositis development is yet to be established. This critical review therefore aims to describe the role of matrix metalloproteinases in mucositis, and how matrix metalloproteinase-dependent tight junction disruption may contribute to the pathobiology of mucositis.

TLR4/PKC‐mediated tight junction modulation: A clinical marker of chemotherapy‐induced gut toxicity?

Chemotherapy-induced gut toxicity is a major clinical and economic burden to oncology practice. The mechanisms responsible for its development are ill defined, hampering the development of therapeutic interventions. In light of newly published research foci and clinical practice guidelines in supportive care in cancer, there has been renewed interest in the role tight junctions play in the pathobiology of chemotherapy-induced gut toxicity. Several preclinical studies have identified molecular defects in intestinal tight junctions following chemotherapy. Despite these findings, the mechanisms responsible for chemotherapy-induced tight junction disruption remain unclear. Recent research has highlighted roles for toll-like receptor 4 and protein kinase C signalling in the regulation of tight junctions. This critical review therefore aims to provide evidence linking toll-like receptor 4 expression, protein kinase C activation and tight junction disruption and their relationship to clinical toxicity.

Irinotecan disrupts tight junction proteins within the gut

Chemotherapy for cancer causes significant gut toxicity, leading to severe clinical manifestations and an increased economic burden. Despite much research, many of the underlying mechanisms remain poorly understood hindering effective treatment options. Recently there has been renewed interest in the role tight junctions play in the pathogenesis of chemotherapy-induced gut toxicity. To delineate the underlying mechanisms of chemotherapy-induced gut toxicity, this study aimed to quantify the molecular changes in key tight junction proteins, ZO-1, claudin-1, and occludin, using a well-established preclinical model of gut toxicity. Female tumor-bearing dark agouti rats received irinotecan or vehicle control and were assessed for validated parameters of gut toxicity including diarrhea and weight loss. Rats were killed at 6, 24, 48, 72, 96, and 120 h post-chemotherapy. Tight junction protein and mRNA expression in the small and large intestines were assessed using semi-quantitative immunohistochemistry and RT-PCR. Significant changes in protein expression of tight junction proteins were seen in both the jejunum and colon, correlating with key histological changes and clinical features. mRNA levels of claudin-1 were significantly decreased early after irinotecan in the small and large intestines. ZO-1 and occludin mRNA levels remained stable across the time-course of gut toxicity. Findings strongly suggest irinotecan causes tight junction defects which lead to mucosal barrier dysfunction and the development of diarrhea. Detailed research is now warranted to investigate posttranslational regulation of tight junction proteins to delineate the underlying pathophysiology of gut toxicity and identify future therapeutic targets.

A Phase II Study Of The Efficacy and Safety Of An Intensified Schedule Of Azacitidine (AZA) In Intermediate-2 and High Risk MDS Patients

BackgroundAlthough azacitidine (AZA) improves survival over conventional treatments in higher risk MDS (Lancet Oncol, 2009), median overall survival (OS) with AZA is only about 2 years, the CR+PR rate about 30%, and further improvements are needed. Gene hypomethylation appears to be a major mechanism of action of AZA but, with the typical 7 days of administration every 28 days, reversal of gene hypomethylation is seen at the end of each cycle (Braiteh F, Clin Cancer Res 2008), suggesting that increasing the number of treatment days could improve AZA results. This trial tested the hypothesis that an AZA regimen with more days of drug administration (ie using the standard daily dose of 75 mg/m2, but during 5 days every 14 days ) could increase the response rate, and that this improvement could translate into better OS. MethodsPatients (pts) aged 18-75 years with ECOG performance status (PS) of 0–2 and no major comorbidities preventing administration of an intensified regimen of AZA, with IPSS int-2 or high MDS, CMML with WBC < 13,000/mm3 and marrow blasts > 10% , and AML with 20-30% marrow blasts (ie EU label for AZA) who had received no prior treatment for their MDS/AML except ESAs could be included. Treatment consisted of AZA 75mg/m2/d for 5 days every 14 days for 4 cycles (AZA-14, cycles 1-4). Patients achieving CR or PR then received 4 cycles of AZA 75mg/m2/d during 5 days every 21 days (AZA-21, cycles 5 to 8) followed by classical cycles of AZA 75mg/m2/d for 7 days every 28 days, to be continued until progression/relapse or toxicity arose. This schedule corresponded to a 20% increase in the number of days of AZA during the first 8 weeks of treatment. Patients not obtaining CR or PR after the initial 4 cycles of AZA-14 received 4 additional cycles of AZA 14 (cycles 5 to 8). Patients not obtaining CR, PR or HI after 8 cycles of AZA-14 were taken off-study. The primary endpoint was response after 4 and 8 cycles (IWG 2006 criteria). We present here the 1st interim analysis of the trial based on the first 22 patients/26 inclusions. Median [IQR] are reported unless specified. ResultsOne patient was excluded for consent withdrawal. 21 patients (M/F: 14/7, median age 66) were enrolled between 2011 and 2013, including 2 RCMD, 2 RAEB1, 10 RAEB2 and 5 AML (with 20 to 30% marrow blasts). Karyotype (IPSS) was favorable in 9 pts, intermediate in 4 patients and unfavorable in 8 pts. Median marrow blast was 13% (range 9.5-15), baseline platelet count was 71 G/l (37-204) including 33% with platelet<50 G/L, baseline Hb level was 9.6 g/dl (8.9-10.2), and baseline ANC was 1.2 G/l (0.6-2.3), including 38% with less than 0.8 G/L. IPSS was int-2 in 13 patients and high in 8 patients. With a median follow-up of 8.6 months, 184 cycles were administered (median 8 /patient, including 13 patients who received 6 or more cycles). Cycle 2 was performed at a median of 14 (14-14) days after cycle 1 and had to be delayed beyond d15 in only 1 patient, due to pleural effusion. Cycles 3 and 4, scheduled at d28 and d42, were slightly delayed in many pts (and were administered at a median of d34 and d50 from AZA onset). 1 pt terminated the study before cycle 4 due to CNS bleeding. 6 pts had died and 24 grade 3-4 SAEs were reported in 7 pts, including transient bilirubin increase in 2 pts, transient renal failure in 1pt, gut toxicity in 2 pts and CNS bleeding in 1 pt.20 patients were evaluable for response after 4 cycles: 1 achieved CR, 5 PR , 5 marrow CR, , 8 stable disease without HI, and 1 progression (overall response rate ORR 55%). After 8 cycles, 1 PR patient at 4 cycles achieved CR and 5 additional responses were observed (3 marrow CR and 2 CR) leading to an ORR of 14/20 (70%). With a median follow up of 8.6 months, 1year overall survival was 69%. ConclusionIn this population of relatively “fit” Higher risk MDS, an intensified schedule of AZA seems feasible without obvious increase in toxicity compared to the classical 7 day schedule of azacitidine. Cycles were delayed in a limited proportion of patients and no extra toxicities were observed. Both the ORR of 70% and the 69% overall survival at 1 year are encouraging. Disclosures:Guerci-Bresler:Novartis: Honoraria; BMS: Honoraria; Celgene: Honoraria; Amgen: Honoraria. Fenaux:CELGENE: Research Funding.