Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×1010 (the first 4 patients) or 5×1010 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).

Phase I Study of DNX-2401 (Delta-24-RGD) Oncolytic Adenovirus: Replication and Immunotherapeutic Effects in Recurrent Malignant Glioma.

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.

HDAC Inhibition Induces MicroRNA-182, which Targets RAD51 and Impairs HR Repair to Sensitize Cells to Sapacitabine in Acute Myelogenous Leukemia.

PURPOSE: The double-strand breaks elicited by sapacitabine, a clinically active nucleoside analogue prodrug, are repaired by RAD51 and the homologous recombination repair (HR) pathway, which could potentially limit its toxicity. We investigated the mechanism by which histone deacetylase (HDAC) inhibitors targeted RAD51 and HR to sensitize acute myelogenous leukemia (AML) cells to sapacitabine. EXPERIMENTAL DESIGN: Chromatin immunoprecipitation identified the role of HDACs in silencing miR-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 pharmacologic 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. Clin Cancer Res; 22(14); 3537-49. ©2016 AACR.

Phase IA/II, multicentre, open-label study of the CD40 antagonistic monoclonal antibody lucatumumab in adult patients with advanced non-Hodgkin or Hodgkin lymphoma.

Despite 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 33·3% and 42·9%, respectively. Lucatumumab demonstrates modest activity in relapsed/refractory patients with advanced lymphoma, suggesting that targeting of CD40 warrants further investigation.

A phase 1 study of lucatumumab, a fully human anti-CD40 antagonist monoclonal antibody administered intravenously to patients with relapsed or refractory multiple myeloma.

In 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 1·0, 3·0, 4·5 or 6·0 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 4·5 mg/kg. At doses ≥3·0 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 4·5 mg/kg with modest clinical activity in relapsed/refractory MM, warranting further study as a combination therapy.

Killing of chronic lymphocytic leukemia by the combination of fludarabine and oxaliplatin is dependent on the activity of XPF endonuclease.

PURPOSE: Chronic lymphocytic leukemia (CLL) resistant to fludarabine-containing treatments responds to oxaliplatin-based therapy that contains fludarabine. We postulated that a mechanism for this activity is the incorporation of fludarabine into DNA during nucleotide excision repair (NER) stimulated by oxaliplatin adducts. EXPERIMENTAL DESIGN: We analyzed CLL cell viability, DNA damage, and signaling pathways in response to treatment by fludarabine, oxaliplatin, or the combination. The dependency of the combination on oxaliplatin-induced DNA repair was investigated using siRNA in CLL cells or cell line models of NER deficiency. RESULTS: Synergistic apoptotic killing was observed in CLL cells after exposure to the combination in vitro. Oxaliplatin induced DNA synthesis in CLL cells, which was inhibited by fludarabine and was eliminated by knockdown of XPF, the NER 5'-endonuclease. Wild-type Chinese hamster ovarian cells showed synergistic killing after combination treatment, whereas only additive killing was observed in cells lacking XPF. Inhibition of repair by fludarabine in CLL cells was accompanied by DNA single-strand break formation. CLL cells initiated both intrinsic and extrinsic apoptotic pathways as evidenced by the loss of mitochondrial outer membrane potential and partial inhibition of cell death upon incubation with FasL antibody. CONCLUSIONS: The synergistic cell killing is caused by a mechanistic interaction that requires the initiation of XPF-dependent excision repair in response to oxaliplatin adducts, and the inhibition of that process by fludarabine incorporation into the repair patch. This combination strategy may be useful against other malignancies.

Specific activation of microRNA106b enables the p73 apoptotic response in chronic lymphocytic leukemia by targeting the ubiquitin ligase Itch for degradation.

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 (p73), and induction of p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein. This event was accompanied by mitochondrial dysfunction, processing of caspase-9, and apoptosis of CLL cells. Ectopic expression of miR106b in CLL cells demonstrated that Itch was a direct target of miR106b such that miR106b-induced decreases in Itch resulted in an accumulation of p73. Thus, our results identify a novel regulatory mechanism wherein microRNA regulate cell survival by mediating the posttranscriptional down-regulation of an ubiquitin ligase, leading to the induction of a proapoptotic regulator in malignant cells. Silencing of miRNA expression in CLL may selectively suppress proapoptotic pathways, providing such tumors with a survival advantage. Consequently, chemotherapeutic drugs that activate miR106b could initiate a p53-independent mechanism that targets CLL cells.

ATM and the Mre11-Rad50-Nbs1 complex respond to nucleoside analogue-induced stalled replication forks and contribute to drug resistance.

The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser(1981)-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA during replication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser(139) (gamma-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-beta-d-arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using gamma-H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by forming nuclear foci that colocalize with gamma-H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential.

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation.

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) (gamma-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 gamma-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 gamma-H2AX-positive cells increased with concentrations of gemcitabine up to 0.1 micromol/L, and gamma-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. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation.