Kristin E. Burke

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin–proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

Clostridium difficile Infection: A Worldwide Disease

Clostridium difficile, an anaerobic toxigenic bacterium, causes a severe infectious colitis that leads to significant morbidity and mortality worldwide. Both enhanced bacterial toxins and diminished host immune response contribute to symptomatic disease. C. difficile has been a well-established pathogen in North America and Europe for decades, but is just emerging in Asia. This article reviews the epidemiology, microbiology, pathophysiology, and clinical management of C. difficile. Prompt recognition of C. difficile is necessary to implement appropriate infection control practices.

Exclusive antagonism of the α4β7 integrin by vedolizumab confirms the gut-selectivity of this pathway in primates†

Background: Biological therapies that antagonize specific molecules have demonstrated efficacy in inflammatory bowel diseases, but infections resulting from systemic immunosuppression underscore the need for safer therapies. The objective of this investigation was to determine if antagonism of the &agr;4&bgr;7 integrin would exclusively yield gut‐selective antiinflammatory activity in primates. Methods: A series of experiments were conducted to investigate potential intra‐ and extraintestinal effects in healthy nonhuman primates dosed repeatedly with the &agr;4&bgr;7‐exclusive antagonist vedolizumab (former versions: MLN0002, MLN02, LDP‐02) for 4, 13, and 26 weeks. Results: No adverse clinical effects of vedolizumab were observed in healthy cynomolgus monkeys up to the highest doses tested (100 mg/kg). Histomorphologic analyses indicated a reduction in the frequency of leukocytes in gastrointestinal tissue, but not other organs. A significant (P < 0.05) decrease in the frequency of &bgr;Symbol lymphocytes in gastrointestinal tissues corresponded to a significant (P < 0.05) increase in &agr;4&bgr;Symbol memory helper T lymphocytes in peripheral blood. This elevation was specific to &agr;4&bgr;Symbol memory helper T lymphocytes; levels of other leukocyte subsets remained unaffected. Systemic opportunistic infections were not observed, and vedolizumab did not inhibit adaptive or innate immune responses systemically. Symbol. No caption available. Symbol. No caption available. Symbol. No caption available. Conclusions: These data demonstrate that blocking the &agr;4&bgr;7 integrin exclusively yields gut‐selective antiinflammatory activity in primates. (Inflamm Bowel Dis 2012;)

Fecal Transplantation for Recurrent Clostridium difficile Infection in Older Adults: A Review

Recurrent Clostridium difficile infection (CDI) is a common nosocomial infection that has a large effect on morbidity and quality of life in older adults in hospitals and long‐term care facilities. Because antibiotics are often unsuccessful in curing this disease, fecal transplantation has emerged as a second‐line therapy for treatment of recurrent CDI. A comprehensive literature search of PubMed, Embase, and Web of Science regarding fecal transplantation for CDI was performed to further evaluate the efficacy and side effects of this promising therapy in older adults. Data were extracted from 10 published articles from 1984 to the present that met inclusion criteria, including nine open‐label reports and one randomized controlled trial. Baseline characteristics and outcomes of individuals undergoing fecal transplantation and effects of fecal transplantation on the fecal microflora were reviewed. Methods of fecal transplantation and donor selection were reviewed. Fecal transplantation was performed in 115 individuals aged 60 to 101, with a female predominance. CDI cure was achieved in 103 (89.6%) individuals over a follow‐up period of 2 months to 5 years (mean 5.9 months). There was no significant difference in cure rate between older and younger participants in included studies. Most failed transplantation occurred in individuals infected with the aggressive NAP1/027 strain of C. difficile. Microbiological studies of fecal biodiversity before and after fecal transplantation demonstrated greater bacterial diversity and shift in flora species to resemble donor flora after transplantation that correlated with clinical remission. Fecal transplantation provides a safe and durable cure for older adults with recurrent CDI.

Phase I Study of the Investigational NEDD8-Activating Enzyme Inhibitor Pevonedistat (TAK-924/MLN4924) in Patients with Advanced Solid Tumors.

Purpose: To determine the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of the investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) and to investigate pevonedistat pharmacokinetics and pharmacodynamics in patients with advanced nonhematologic malignancies. Experimental Design: Pevonedistat was administered via 60-minute intravenous infusion on days 1 to 5 (schedule A, n = 12), or days 1, 3, and 5 (schedules B, n = 17, and C, n = 19) of 21-day cycles. Schedule B included oral dexamethasone 8 mg before each pevonedistat dose. Dose escalation proceeded using a Bayesian continual reassessment method. Tumor response was assessed by RECIST 1.0. Results: Schedule A MTD was 50 mg/m2; based on the severity of observed hepatotoxicity, this schedule was discontinued. Schedules B and C MTDs were 50 and 67 mg/m2, respectively. DLTs on both these schedules included hyperbilirubinemia and elevated aspartate aminotransferase. There were no grade ≥3 treatment-related serious adverse events reported on schedules B or C. Twenty-three (74%) evaluable patients on schedules B and C had stable disease. Intermittent dexamethasone use did not significantly influence pevonedistat pharmacokinetics. NAE inhibition by pevonedistat was demonstrated in multiple tumor types via IHC detection of pevonedistat-NEDD8 adduct and accumulation of Cullin-RING ligase substrates CDT1 and NRF2 in tumor biopsies. Conclusions: Pevonedistat was generally well tolerated on a day 1, 3, 5 schedule every 3 weeks with an MTD between 50 mg/m2 and 67 mg/m2. DLTs were predominantly hepatic enzyme elevations. Pharmacodynamic studies demonstrated that pevonedistat inhibited NAE in tumors. Clinical trials are ongoing. Clin Cancer Res; 22(4); 847–57. ©2015 AACR.

Applications of Pathology-Assisted Image Analysis of Immunohistochemistry-Based Biomarkers in Oncology

Immunohistochemistry-based biomarkers are commonly used to understand target inhibition in key cancer pathways in preclinical models and clinical studies. Automated slide-scanning and advanced high-throughput image analysis software technologies have evolved into a routine methodology for quantitative analysis of immunohistochemistry-based biomarkers. Alongside the traditional pathology H-score based on physical slides, the pathology world is welcoming digital pathology and advanced quantitative image analysis, which have enabled tissue- and cellular-level analysis. An automated workflow was implemented that includes automated staining, slide-scanning, and image analysis methodologies to explore biomarkers involved in 2 cancer targets: Aurora A and NEDD8-activating enzyme (NAE). The 2 workflows highlight the evolution of our immunohistochemistry laboratory and the different needs and requirements of each biological assay. Skin biopsies obtained from MLN8237 (Aurora A inhibitor) phase 1 clinical trials were evaluated for mitotic and apoptotic index, while mitotic index and defects in chromosome alignment and spindles were assessed in tumor biopsies to demonstrate Aurora A inhibition. Additionally, in both preclinical xenograft models and an acute myeloid leukemia phase 1 trial of the NAE inhibitor MLN4924, development of a novel image algorithm enabled measurement of downstream pathway modulation upon NAE inhibition. In the highlighted studies, developing a biomarker strategy based on automated image analysis solutions enabled project teams to confirm target and pathway inhibition and understand downstream outcomes of target inhibition with increased throughput and quantitative accuracy. These case studies demonstrate a strategy that combines a pathologist’s expertise with automated image analysis to support oncology drug discovery and development programs.

Modifiable Environmental Factors in Inflammatory Bowel Disease

Purpose of ReviewEnvironmental factors may influence predisposition to develop inflammatory bowel diseases (Crohn’s disease, ulcerative colitis) or alter its natural history by modification of both the host immune response and intestinal microbial composition. The purpose of this review is to translate such evidence into clinical practice by a focus on interventional studies that have modified such environmental influences to improve disease outcomes.Recent FindingsSeveral environmental influences have been identified in the recent literature including tobacco use, diet, antibiotics, vitamin D deficiency, stress, appendectomy, and oral contraceptive use. Some risk factors have similar influences on both Crohn’s disease and ulcerative colitis while others are disease-specific or have divergent effects.SummaryEmerging epidemiologic evidence has confirmed the association of many of these factors with incident disease using prospective data. In addition, laboratory data has supported their mechanistic plausibility and relevance to intestinal inflammation.

Indications for Mode of Delivery in Pregnant Women with Inflammatory Bowel Disease

Background: Reasons for the increased incidence of cesarean delivery among women with inflammatory bowel disease remain unclear. We assessed cesarean delivery incidence and factors influencing mode of delivery in women with inflammatory bowel disease. Methods: We performed a 10-year retrospective cohort study of nulliparous women who delivered a singleton infant at our institution. We compared the risk of each mode of delivery in women with Crohns disease and ulcerative colitis with women without inflammatory bowel disease. We assessed mode of delivery indications for patients with inflammatory bowel disease and whether cesarean deliveries were planned. Results: The overall incidence of cesarean delivery among women with Crohns disease (24/59; 40.7%) was similar to that among women without inflammatory bowel disease (7868/21,805; 36.1%) (risk ratio 1.1 [95% confidence interval, 0.83, 1.5]; P = 0.46), but was increased in the subgroups with active and inactive perianal disease (risk ratio 2.3; P < 0.01). Women with ulcerative colitis had a 1.8-fold increased relative risk of cesarean delivery (41/65; 63.1%) (95% confidence interval, 1.5, 2.1; P < 0.01), with highest incidence in patients with ileal pouch-anal anastomosis. Forty-nine percent of ulcerative colitis and 66.7% of Crohns disease cesarean deliveries were unplanned, with only 1 unplanned delivery performed for active inflammatory bowel disease. Most unplanned deliveries were for arrest of descent/dilation and nonreassuring fetal heart tracings. Seventy-five percent of planned cesarean deliveries were for inflammatory bowel disease–related indications. Conclusions: Women with ulcerative colitis and perianal Crohns disease have an increased incidence of cesarean delivery. At least half of cesarean deliveries are unplanned.

Genetic Markers Predict Primary Nonresponse and Durable Response to Anti–Tumor Necrosis Factor Therapy in Ulcerative Colitis

Background Despite a high nonresponse rate, predictors of response to anti-tumor necrosis factor (anti-TNF) therapy in ulcerative colitis (UC) remain limited. We aim to determine clinical and genetic predictors of primary nonresponse (PNR) and durable response (DR) to anti-TNF therapy in a large prospective UC cohort. Methods Using the Illumina Immunochip, candidate polymorphisms associated with clinical outcomes of PNR and DR were separately evaluated and combined into weighted genetic risk scores. Combined genetic and clinical multivariable models for PNR and DR were compared with clinical predictive models using area under the receiver operating characteristic (AUROC) curves. Models were internally (DR) or externally (PNR) validated. Multivariable logistic regression was utilized to assess the association of genetic risk scores with infliximab levels and antibodies. Results Of 231 patients, 28 (12%) experienced PNR and 120 (52%) experienced DR. There was no significant difference in clinical features between primary nonresponders and responders. Eight alleles were associated with PNR. A combined clinical-genetic model (AUROC, 0.87) more accurately predicted PNR compared with a clinical-only model (AUROC, 0.57; P < 0.0001). In an external cohort of 131 patients, increasing tertiles of PNR genetic risk score correlated with increased risk of PNR (P = 0.052). Twelve candidate loci were associated with DR. Genetic risk score quartiles for DR demonstrated a strong dose-response relationship in predicting treatment duration. Genetic risk scores for PNR and DR were not associated with infliximab levels or antibody formation. Conclusion Genetic polymorphisms enhance prediction of PNR and DR to anti-TNF therapy in patients with UC.

Abstract A41: Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin

Background: Immunohistochemistry provides visual context to protein modulation and is relied upon to elucidate mechanism of action, marker translocation and pharmacodynamics (PD) in drug discovery. Markers of the PI3K-MTOR pathway have been monitored in this way to aid in the clinical development of various inhibitors targeting this pathway. Here, assays were developed using preclinical xenograft PD studies to explore the feasibility of automated staining of pAKT 473, pS6, p4EBP1 and pRb. In preparation for clinical assessment of these markers in human skin samples; these markers were also examined in normal mouse skin. p4EBP1 was selected to examine the reproducibility of these automated assays and to explore the correlation between pathway inhibition in PD xenograft tissues and murine skin tissues. The automated staining technique was coupled with whole slide scanning and digital image analysis for quantitative comparison. Methods: Assays were chosen for evaluation based on known biology. FFPE PTEN-null breast cancer primary human tumor xenografts with high, medium and low biomarker expression, with companion skin samples, were selected for feasibility and reproducibility testing. Whole slide scanning and reproducibility analysis was executed using Aperio Scanscope XT and Spectrum software. Statistical analysis methods of mixed effect linear models and two sample t-tests were applied. Xenograft and skin samples from NCI-H1048 tumor bearing Nude mice treated with MTOR (MLN0128)and PI3Kα (MLN1117) investigational inhibitors were immunohistochemically stained for p4EBP1. Definiens object-based imaging software was used to isolate and analyze viable tumor area and exclude necrotic and stromal components in xenografts. A customized Definiens® algorithm was used on skin to identify the epithelial layer for analysis. Results: After evaluation, baseline biomarker expression in the xenograft and skin samples determined marker selection for future steps. Baseline p4EBP1 levels were high in both xenograft and skin. The reproducibility testing yielded consistent results in both xenograft and skin, validating autostaining use for PD studies. Phosphomarker p4EBP1 expression was inhibited after MLN0128 administration, with increased reduction after combination with MLN1117. Development of a novel analysis solution using Definiens software for skin samples permitted the area of interest to be restricted to the epithelial layer and excluded other epidermal features with similar morphometric characteristics. The p4EBP1 modulation was concordant between skin and tumor though more pronounced in skin, indicating that skin is a suitable surrogate tissue for determining modulation of this PI3K-MTOR pathway marker. Conclusions: Autostaining platforms provide reproducible results and stain consistently for PI3K-MTOR phosphomarkers on both xenografts and mouse skin samples. Utilizing p4EBP1, target effect was observed after MTOR and PI3Kα administration. This development and evaluation of the phosphobiomarker using automated staining technology and advanced image analysis supports preclinical PD assessment and combination clinical trials as well as influenced timepoint selection for clinical assessment. Citation Format: Anna Kreshock, Natalia Iartchouk, Yueying Cao, Jeffrey Szwaya, Feng Gao, Christopher Simpson, Evan Luongo, Kristine Burke, Esha Gangolli, Keisuke Kuida, Rachael Brake, Stephen Tirrell, Vaishali Shinde. Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A41.