Purpose DNX-2401 (Delta-24-RGD; tasadenoturev) is a tumor-selective, replication-competent oncolytic adenovirus. Preclinical studies demonstrated antiglioma efficacy, but the effects and mechanisms of action have not been evaluated in patients. Methods A phase I, dose-escalation, biologic-end-point clinical trial of DNX-2401 was conducted in 37 patients with recurrent malignant glioma. Patients received a single intratumoral injection of DNX-2401 into biopsy-confirmed recurrent tumor to evaluate safety and response across eight dose levels (group A). To investigate the mechanism of action, a second group of patients (group B) underwent intratumoral injection through a permanently implanted catheter, followed 14 days later by en bloc resection to acquire post-treatment specimens. Results In group A (n = 25), 20% of patients survived > 3 years from treatment, and three patients had a ≥ 95% reduction in the enhancing tumor (12%), with all three of these dramatic responses resulting in > 3 years of progression-free survival from the time of treatment. Analyses of post-treatment surgical specimens (group B, n = 12) showed that DNX-2401 replicates and spreads within the tumor, documenting direct virus-induced oncolysis in patients. In addition to radiographic signs of inflammation, histopathologic examination of immune markers in post-treatment specimens showed tumor infiltration by CD8 and T-bet cells, and transmembrane immunoglobulin mucin-3 downregulation after treatment. Analyses of patient-derived cell lines for damage-associated molecular patterns revealed induction of immunogenic cell death in tumor cells after DNX-2401 administration. Conclusion Treatment with DNX-2401 resulted in dramatic responses with long-term survival in recurrent high-grade gliomas that are probably due to direct oncolytic effects of the virus followed by elicitation of an immune-mediated antiglioma response.

Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser 139 (;-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration-and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of ;-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of ;-H2AX -positive cells increased with concentrations of gemcitabine up to 0.1 Mmol/L, and ;-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser 1981 was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure.

Nucleoside analogs are structurally similar antimetabolites that have a broad range of action and are clinically active in both solid tumors and hematological malignancies. Many of these agents are incorporated into DNA by polymerases during normal DNA synthesis, an action that blocks further extension of the nascent strand and causes stalling of replication forks. The molecular mechanisms that sense stalled replication forks activate cell cycle checkpoints and DNA repair processes, which may contribute to drug resistance. When replication forks are not stabilized by these molecules or when subsequent DNA repair processes are overwhelmed, apoptosis is initiated either by these same DNA damage sensors or by alternative mechanisms. Recently, strategies aimed at targeting DNA damage checkpoints or DNA repair processes have demonstrated effectiveness in sensitizing cells to nucleoside analogs, thus offering a means to elude drug resistance. In addition to their DNA synthesisdirected actions many nucleoside analogs trigger apoptosis by unique mechanisms, such as causing epigenetic modifications or by direct activation of the apoptosome. A review of the cellular and molecular responses to clinically relevant agents provides an understanding of the mechanisms that cause apoptosis and may provide rationale for the development of novel therapeutic strategies.

Chronic lymphocytic leukemia (CLL) is characterized by cells that exhibit dysfunctional apoptosis. Here, we show that deacetylase inhibition led to the E2F1-and myc-mediated transcriptional activation of the microRNA miR106b in primary CLL cells. Induction of miR106b was associated with a down-regulation in the levels of the E3-ubiquitin ligase Itch. Decreases in Itch protein levels were associated with a reciprocal accumulation of its proapoptotic substrate, TAp73

SummaryIn this open-label, multicentre, phase 1 study a fully human anti-CD40 antagonist monoclonal antibody, lucatumumab, was evaluated in patients with relapsed/refractory multiple myeloma (MM). The primary objective was to determine the maximum tolerated dose (MTD) based on dose-limiting toxicities (DLTs). Secondary objectives included safety, pharmacokinetics, pharmacodynamics and antimyeloma activity. Twenty-eight patients, enrolled using a standard '3 + 3' dose escalation, received one or two (n = 3) cycles of lucatumumab 1Á0, 3Á0, 4Á5 or 6Á0 mg/kg once weekly for 4 weeks. Common lucatumumab-related adverse events were reversible, mild-to-moderate infusion reactions. Severe adverse events were anaemia, chills, hypercalcaemia and pyrexia (7% each). DLTs included grade 4 thrombocytopenia, grade 3 increased alanine aminotransferase and grade 4 increased lipase (n = 1 each). The MTD was 4Á5 mg/kg. At doses ! 3Á0 mg/ kg, sustained receptor occupancy ( ! 87%), observed throughout weekly infusions up to 5 weeks after the last infusion, correlated with an estimated half-life of 4-19 d. Twelve patients (43%) had stable disease, and one patient (4%) maintained a partial response for ! 8 months. These findings indicate that single-agent lucatumumab was well tolerated up to 4Á5 mg/kg with modest clinical activity in relapsed/refractory MM, warranting further study as a combination therapy.

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Purpose The double strand breaks elicited by sapacitabine, a clinically active nucleoside analog prodrug are repaired by Rad51 and the homologous recombination repair (HR) pathway which could potentially limit its toxicity. We investigated the mechanism by which HDAC inhibitors targeted Rad51 and HR to sensitize AML cells to sapacitabine. Experimental Design Chromatin immunoprecipitation identified the role of HDACs in silencing microRNA-182 in AML. Immunoblotting, gene expression, overexpression or inhibition of miR-182 and luciferase assays established that miR-182 directly targeted Rad51. HR reporter assays, apoptotic assays and colony forming assays established that the miR-182, as well as the HDAC inhibition-mediated decreases in Rad51 inhibited HR repair and sensitized cells to sapacitabine. Results The gene repressors, HDAC1 and HDAC2, became recruited to the promoter of miR-182 to silence its expression in AML. HDAC inhibition induced miR-182 in AML cell lines and primary AML blasts. miR-182 targeted Rad51 protein both in luciferase assays and in AML cells. Overexpression of miR-182, as well as HDAC inhibition-mediated induction of miR-182 were linked to time- and dose-dependent decreases in the levels of Rad51, an inhibition of HR, increased levels of residual damage and decreased survival after exposure to double strand damage inducing agents. Conclusions Our findings define the mechanism by which HDAC inhibition induces miR-182 to target Rad51 and highlights a novel pharmacological strategy that compromises the ability of AML cells to conduct HR, thereby sensitizing AML cells to DNA damaging agents that activate HR as a repair and potential resistance mechanism.

; San Diego, CA: abstract 3702. SummaryDespite advancements in the treatment of non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL), patients continue to relapse and thus a need for new targeted therapies remains. The CD40 receptor is highly expressed on neoplastic B cells and activation leads to enhanced proliferation and survival. Lucatumumab (HCD122) is a fully human antagonistic CD40 monoclonal antibody. A phase IA/II study was designed to determine the maximum tolerated dose (MTD) and activity of lucatumumab in patients with relapsed/refractory lymphoma. Determination of the MTD was the primary objective of the phase IA dose escalation portion and clinical response was the primary objective of the phase II dose expansion portion. Patients received escalating doses of lucatumumab administered intravenously once weekly for 4 weeks of an 8-week cycle. MTD was determined at 4 mg/kg of lucatumumab. A total of 111 patients with NHL (n = 74) and HL (n = 37) were enrolled. Responses were observed across various lymphoma subtypes. The overall response rate by computed tomography among patients with follicular lymphoma (FL) and marginal zone lymphoma of mucosa-associated lymphatic tissue (MZL/MALT) was 33Á3% and 42Á9%, respectively. Lucatumumab demonstrates modest activity in relapsed/refractory patients with advanced lymphoma, suggesting that targeting of CD40 warrants further investigation.