James S. Hardwick

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

γ-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Phase II Trial of Vorinostat in Recurrent Glioblastoma Multiforme: A North Central Cancer Treatment Group Study

PURPOSE Vorinostat, a histone deacetylase inhibitor, represents a rational therapeutic target in glioblastoma multiforme (GBM). PATIENTS AND METHODS Patients with recurrent GBM who had received one or fewer chemotherapy regimens for progressive disease were eligible. Vorinostat was administered at a dose of 200 mg orally twice a day for 14 days, followed by a 7-day rest period. RESULTS A total of 66 patients were treated. Grade 3 or worse nonhematologic toxicity occurred in 26% of patients and consisted mainly of fatigue (17%), dehydration (6%), and hypernatremia (5%); grade 3 or worse hematologic toxicity occurred in 26% of patients and consisted mainly of thrombocytopenia (22%). Pharmacokinetic analysis showed lower vorinostat maximum concentration and area under the curve (0 to 24 hours) values in patients treated with enzyme-inducing anticonvulsants, although this did not reach statistical significance. The trial met the prospectively defined primary efficacy end point, with nine of the first 52 patients being progression-free at 6 months. Median overall survival from study entry was 5.7 months (range, 0.7 to 28+ months). Immunohistochemical analysis performed in paired baseline and post-vorinostat treatment samples in a separate surgical subgroup of five patients with recurrent GBM showed post treatment increase in acetylation of histones H2B and H4 (four of five patients) and of histone H3 (three of five patients). Microarray RNA analysis in the same samples showed changes in genes regulated by vorinostat, such as upregulation of E-cadherin (P = .02). CONCLUSION Vorinostat monotherapy is well tolerated in patients with recurrent GBM and has modest single-agent activity. Histone acetylation analysis and RNA expression profiling indicate that vorinostat in this dose and schedule affects target pathways in GBM. Additional testing of vorinostat in combination regimens is warranted.

Single-Dose Fosaprepitant for the Prevention of Chemotherapy-Induced Nausea and Vomiting Associated With Cisplatin Therapy: Randomized, Double-Blind Study Protocol—EASE

PURPOSE Addition of aprepitant, a neurokinin-1 receptor antagonist (NK1RA), to an ondansetron and dexamethasone regimen improves prevention of chemotherapy-induced nausea/vomiting (CINV), particularly during the delayed phase (DP; 25 to 120 hours). Therefore, recommended antiemetic regimens include multiple-day NK1RA administration. Preliminary data suggested that single-dose aprepitant before chemotherapy could provide CINV protection throughout the overall risk phase (OP; 0 to 120 hours). This study compared a 3-day oral aprepitant schedule to a regimen containing a single dose of the intravenous NK1RA fosaprepitant. PATIENTS AND METHODS A randomized, double-blind, active-control design was used to test whether fosaprepitant is noninferior to aprepitant. Patients receiving cisplatin ≥ 70 mg/m(2) for the first time received ondansetron and dexamethasone with a standard aprepitant regimen (125 mg on day 1, 80 mg on day 2, 80 mg on day 3) or a single-dose fosaprepitant regimen (150 mg on day 1). The primary end point was complete response (CR; no vomiting, no rescue medication) during OP. Secondary end points were CR during DP and no vomiting during OP. Accrual of 1,113 evaluable patients per treatment arm was planned to confirm noninferiority with expected CR of 67.7% and noninferiority margin of minus 7 percentage points. RESULTS A total of 2,322 patients were randomly assigned, and 2,247 were evaluable for efficacy. Antiemetic protection with aprepitant and fosaprepitant was equivalent within predefined bounds for noninferiority. Both regimens were well tolerated, although more frequent infusion site pain/erythema/thrombophlebitis was seen with fosaprepitant relative to aprepitant (2.7% v 0.3%, respectively). CONCLUSION Given with ondansetron and dexamethasone, single-dose intravenous fosaprepitant (150 mg) was noninferior to standard 3-day oral aprepitant in preventing CINV during OP and DP.

Development of vorinostat: Current applications and future perspectives for cancer therapy

Vorinostat is a potent histone deacetylase inhibitor that blocks the catalytic site of these enzymes. A large number of cellular proteins are modified post-translationally by acetylation, leading to altered structure and/or function. Many of these proteins, such as core nucleosomal histones and transcription factors, function in key cellular processes and signal transduction pathways that regulate cell growth, migration, and differentiation. At concentrations that are non-toxic to normal cells, vorinostat dramatically alters cellular acetylation patterns and causes growth arrest and death and in a wide range of transformed cells, both in vitro and in animal tumor models. Vorinostat has shown promising clinical activity against hematologic and solid tumors at doses that have been well tolerated by patients. Recent non-clinical experiments that explored the effects of vorinostat in combination with other chemotherapeutic agents have begun to illuminate potential mechanisms of action for this histone deacetylase inhibitor and are providing guidance for new avenues of clinical investigation.

Inhibition of NOTCH signaling by gamma secretase inhibitor engages the RB pathway and elicits cell cycle exit in T-cell acute lymphoblastic leukemia cells.

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.

Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett’s esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPα, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine–cytokine receptor interactions and TGF-β. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

A Phase I Pharmacokinetic and Pharmacodynamic Study of Dalotuzumab (MK-0646), an Anti-Insulin-like Growth Factor-1 Receptor Monoclonal Antibody, in Patients with Advanced Solid Tumors

Purpose: Insulin-like growth factor-1 receptor (IGF-1R) mediates cellular processes in cancer and has been proposed as a therapeutic target. Dalotuzumab (MK-0646) is a humanized IgG1 monoclonal antibody that binds to IGF-1R preventing receptor activation. This study was designed to evaluate the safety and tolerability of dalotuzumab, determine the pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and identify a recommended phase II dose. Experimental Design: Patients with tumors expressing IGF-1R protein were allocated to dose-escalating cohorts of three or more patients each and received intravenous dalotuzumab weekly, every 2 or 3 weeks. Plasma was collected for PK analysis. Paired baseline and on-treatment skin and tumor biopsy samples were collected for PD analyses. Results: Eighty patients with chemotherapy-refractory solid tumors were enrolled. One dose-limiting toxicity was noted, but a maximum-tolerated dose was not identified. Grade 1 to 3 hyperglycemia, responsive to metformin, occurred in 15 (19%) patients. At dose levels or more than 5 mg/kg, dalotuzumab mean terminal half-life was 95 hours or more, mean Cmin was more than 25 μg/mL, clearance was constant, and serum exposures were approximately dose proportional. Decreases in tumor IGF-1R, downstream receptor signaling, and Ki67 expression were observed. 18F-Fluorodeoxy-glucose positron emission tomography metabolic responses occurred in three patients. One patient with Ewings sarcoma showed a mixed radiologic response. The recommended phase II doses were 10, 20, and 30 mg/kg for the weekly, every other week, and every third week schedules, respectively. Conclusions: Dalotuzumab was generally well-tolerated, exhibited dose-proportional PK, inhibited IGF-1R pathway signaling and cell proliferation in treated tumors, and showed clinical activity. The low clearance rate and long terminal half-life support more extended dosing intervals. Clin Cancer Res; 17(19); 6304–12. ©2011 AACR.

Phase I trial of vorinostat combined with bortezomib for the treatment of relapsing and/or refractory multiple myeloma.

UNLABELLED Preclinical studies have shown that targeted combination therapy consisting of vorinostat and bortezomib has antitumor activity in multiple myeloma (MM). We examined this drug combination in advanced relapsing and/or refractory MM patients (n = 34). Although the maximum tolerated dose was not reached, the study found this combination regimen generally well tolerated and clinically active in relapsed and/or refractory MM patients. BACKGROUND Development of targeted therapies for MM has improved response rates and increased patient survival, but ultimately the disease becomes refractory and progresses. Vorinostat combined with bortezomib has demonstrated synergistic antiproliferative and proapoptotic activity in preclinical models of MM. The objectives of this study were to determine the maximum tolerated dose for vorinostat with bortezomib in patients with advanced MM and to evaluate the clinical benefit of this new drug combination. PATIENTS AND METHODS Patients ≥ 18 years old with relapsed and/or refractory MM were enrolled into escalating dose cohorts of vorinostat and bortezomib combination therapy. Thirty-four patients were enrolled and were evaluable for safety and efficacy analyses. RESULTS All patients reported adverse events, 89% of which were mild to moderate in severity. Thirteen patients experienced 29 serious adverse events, 12 (41%) of which were considered drug-related. The maximum tolerated dose was not reached. Partial responses were observed in 9 (27%) patients. Minimal responses were observed in 2 additional patients (6%), and another 20 patients (59%) experienced disease stabilization. CONCLUSION Vorinostat with bortezomib is generally well-tolerated and has clinical activity in patients with relapsed and/or refractory MM. Response rates were similar in patients previously exposed to bortezomib and patients who were naive to bortezomib therapy.

Downregulation of MHC-I expression is prevalent but reversible in Merkel cell carcinoma.

Paulson and colleagues report that 84% of Merkel cell carcinoma (MCC) tumors downregulated MHC-I expression, and MCC patients treated with intralesional IFNs had increased MHC-I expression on their tumor cells, thus promoting the use of immune-stimulating therapies for MCC. Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P < 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC. Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR.