Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RARa. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXRa), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RARa to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identi®ed in a pediatric case of APL. RT ± PCR, cloning, and sequence studies identi®ed NPM as the RARa partner on chromosome 5, and both NPM-RARa and RARa-NPM fusion mRNAs were expressed in this patient. We further explored the e ects of the NPM-RARa chimeric protein on the subcellular localization of PML, RXRa, NPM, and PLZF using immuno¯uorescent confocal microscopy. While PML remained localized to its normal 10 ± 20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXRa, and NPM-RARa in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RARa partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.

Oncolytic viruses that selectively replicate in cancer cells have been described for over 50 years. Despite the observation by several groups that multimutated herpes simplex type 1 vectors are oncolytic in a variety of murine tumor models, the oncolytic potential of ICP0 null mutants has not been described. This study characterizes a novel second-generation oncolytic herpes simplex type 1 vector null for the ICP0 gene. We tested three mutant viruses and found that all were selectively cytotoxic in a variety of human and murine tumor cells in vitro. Furthermore, we provide evidence of a mechanistic link between ICP0's function in interferon signaling pathways and the observed oncolytic capacity of ICP0 mutants. Using an immunocompetent murine model of breast adenocarcinoma we demonstrate that the ICP0 mutant KM100 completely eradicates tumors in approximately 80% of treated animals and significantly increases survival. Our data suggest that active viral replication is necessary for effective tumor regression. In addition, we characterized the potential of KM100 as an anti-tumor vaccine since cured mice were found to elicit a robust anti-tumor immune response and were refractory to subsequent tumor growth upon rechallenge.

Oncolytic activity of the MG1 strain of the Maraba vesiculovirus has proven efficacy in numerous preclinical cancer models, and relied not only on a direct cytotoxicity but also on the induction of both innate and adaptive antitumor immunity. To further expand tumor-specific T-cell effector and long-lasting memory compartments, we introduced the MG1 virus in a prime-boost cancer vaccine strategy. To this aim, a replication-incompetent adenoviral [Ad] vector together with the oncolytic MG1 have each been armed with a transgene expressing a same tumor antigen. Immune priming with the Ad vaccine subsequently boosted with the MG1 vaccine mounted tumor-specific responses of remarkable magnitude, which significantly prolonged survival in various murine cancer models. Based on these promising results, we validated the safety profile of the Ad:MG1 oncolytic vaccination strategy in nonhuman primates and initiated clinical investigations in cancer patients. Two clinical trials are currently under way (NCT02285816; NCT02879760). The present review will recapitulate the discoveries that led to the development of MG1 oncolytic vaccines from bench to bedside.

We have investigated the therapeutic potential of a prototypic melanoma vaccine based on recombinant adenovirus expressing human dopachrome tautomerase in the B16F10 murine melanoma model. We found that in the presence of a tumor, the magnitude of T-cell immunity evoked by the vaccine was significantly reduced. This impairment was compounded by defects in cytokine production and degranulation within the tumor-infiltrating lymphocytes (TILs). We showed that the combination of vaccination with high-dose cyclophosphamide was able to skew the response toward the target antigen and enhanced both the quantity and quality of antigen-specific CD8 þ and CD4 þ T-cell responses in tumor-bearing mice, which resulted in the inhibition of tumor growth. Furthermore, when tumor-specific antigens were targeted by the vaccine, the combination therapy could actually produce tumor regression, which appeared to result from the high frequency of antigen-specific T cells. These data show that recombinant adenovirus vaccines are compatible with conventional high-dose chemotherapy and that the combined treatment results in improved therapeutic outcomes relative to either agent individually.

e14637 Background: OVs display oncolytic activity and boost adaptive cell immunity. MG1MA3 is a Maraba virus modified to express tumour antigen MAGE-A3. MG1MA3, both alone and after immune priming with a MAGE-A3 modified adenovirus (AdMA3) may trigger anti-tumour T-cell responses. Methods: N = 41 patients (pts) with MAGE-A3 expressing solid tumours were evaluated in 3 groups (A) Dose escalation of MG1MA3 iv d1+4, q8w (n = 9); (B) Single fixed dose AdMA3 1e10 pfu IM d1 (n = 6); (C) AdMA3 priming d(-14) followed by dose escalated MG1MA3 (d1+4, q8w) (n = 25). Arm A and C had a 3+3 design. Pre + post treatment blood and tissue biopsies were evaluated for viral and immune markers. Endpoints included MTD/MFD, RP2DL, safety, tolerability, pharmacokinetics, viral delivery and replication. Results: Dose limiting toxicities (hypoxia/dyspnea, vomiting, headache) occurred in 4 pts (2 each Arm A +C). RP2DL for arm C was AdMA3 1e10 pfu IM d(-14) then MG1MA3 1e11 pfu iv d1+4. Common treatment related toxicities on Arm C occurring hours to a few days after MG1MA3 included diarrhea, nausea, vomiting, anorexia, chills, fatigue, fever, flu-like symptoms, hypophosphatemia, headache, and hypotension. Preliminary tumour gene expression results reveal induction of pro-inflammatory genes, including chemokines (CCL2, CCL5, CX3CL1, CXCL10), acute phase response proteins (IL-6, TNF), antigen presenting cell (APC) activation markers (CD80, HLA-A, HLA-B), markers of APC and Natural Killer cell infiltration (CD56, CD68, TLR3), as well as a co-incident decrease in the suppressive cytokine TGF-β. MG1MA3 replication was observed in some pts, inferred by detection of circulating genomes on days 4, 8 and 15 after clearance of the input dose. Induction of anti-tumour immune responses (CD8 T cells and antibodies vs MAGE-A3) was demonstrated in 3 of 6 Arm C pts evaluated to date. In one patient, over 1% of circulating CD8 T cells were directed against MAGE-A3. Conclusions: AdMA3 prime followed by MG1MA3 OV boost is feasible with a defined RP2DL, and capable of inducing potent anti-tumour immune response. Alternate schedules will be evaluated. Clinical trial information: NCT02285816.