Background: The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. Methods: By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. Results: We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. Conclusions: Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

Adoptive cell therapy (ACT) using ex vivo expanded tumor infiltrating lymphocytes (TIL) has shown great promise as a treatment for metastatic melanoma and has the potential to deliver durable responses in other solid tumors. Clonal neoantigens, which are derived from mutations occurring very early in the tumor development, are present in all cancer cells within a patient and therefore could be the optimal targets for TIL-based therapies. Recently it was shown that the number of clonal neoantigens within a tumor is associated with improved clinical outcomes following checkpoint inhibition in patients with non-small cell lung cancer (NSCLC) and melanoma. An approach that targets multiple clonal neoantigens with specific T cells has the potential to demonstrate high specificity and efficacy whilst mitigating the risk of immune escape. Achilles Therapeutics is developing a personalized ACT product, ATL001, to target clonal neoantigens, which are identified using tumor exome sequencing and the PELEUS™ bioinformatics platform. Clonal neoantigen reactive T cells (cNeTs) are then manufactured from TIL using the VELOS™ manufacturing process. Two Phase I/IIa clinical trials of ATL001 are ongoing in patients with advanced NSCLC and metastatic or recurrent melanoma. In common with the development of other ACT products, the key to characterizing and improving cNeT products relies on evaluating a diverse set of exploratory endpoints in early clinical trials, including understanding the procedural, clinical and biological factors that influence cNeT manufacturing rate and product reactivity; monitoring the expansion, persistence and phenotype of the infused cells in vivo and identifying potential biomarkers of clinical activity or safety of cNeTs in treated patients. These insights may suggest further improvements to cNeT product development in ensuing iterations. The evaluation of these endpoints requires the collection of a rich longitudinal dataset that traces each patient's journey from tissue procurement and cNeT manufacture, to final product infusion and follow up. The data collected will include clinical and disease characteristics, tumor microenvironment insights from exome sequencing and immunohistochemistry of procured tumor, and metrics from the VELOS™ manufacturing process, along with a comprehensive immune-monitoring programme comprising immuno-sequencing, immunophenotyping, bespoke ctDNA panels and reactivity assays at specified timepoints, all to be evaluated against clinical outcomes data. The amalgamation of diverse streams of data requires the development of robust processes and systems for data collection, processing and storage. Furthermore, the evaluation of multiple exploratory endpoints will require integration and modelling of baseline covariates, time-series immune-monitoring and efficacy data, all of which will be described Citation Format: Michael Epstein, Rebecca Pike, Emma Leire, Jen Middleton, Megan Wileman, Lylia Ouboussad, Leah Manning, Theres Oakes, Eva Pekle, Amy Baker, Mark Brown, Daisy Melandri, Pablo Becker, Anabel Ramirez, Natasa Hadjistephanou, Samra Turaljic, Mariam Jamal-Hanjani, Martin Forster, Iraj Ali, Jane Robertson, Karl Peggs, Sergio Quezada. Characterization of a novel clonal neoantigen reactive T cell (cNeT) product through a comprehensive translational research program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1508.

The Suppressor APC Domain Containing 2 (SAPCD2) gene, also known by its aliases p42.3 and c9orf140, encodes a protein with an approximate molecular weight of 42.3 kDa. It was initially recognized as a cell cycle-associated protein involved in mitotic progression. Since the initial discovery of this gene, emerging evidence has suggested that its functions extend beyond that of regulating cell cycle progression to include modulation of planar polarization of cell progenitors and determination of cell fate throughout embryonic development. The underlying mechanisms driving such functions have been partially elucidated. However, the detailed mechanisms of action remain to be further characterized. The expression level of SAPCD2 is high throughout embryogenesis but is generally absent in healthy postnatal tissues, with restored expression in adult tissues being associated with various disease states. The pathological consequences of its aberrant expression have been investigated, most notably in the development of several types of cancers. The role of SAPCD2 in tumorigenesis has been supported by in vitro, in vivo, and retrospective clinical investigations and the mechanisms underlying its oncogenic function have been partially revealed. The potential of SAPCD2 as a diagnostic marker and therapeutic target of cancers have also been explored and have shown great promise. However, many questions pertaining to its oncogenic mechanisms as well as its value as a diagnostic marker and therapeutic target remain to be answered. In addition to its function as an oncogene, an involvement of SAPCD2 in other pathological processes such as inflammation has also been implicated and provides additional directions that warrant future investigation. This article reviews the current understanding of the normal cellular functions of SAPCD2 and the relevance of SAPCD2 in disease development with a primary focus on tumorigenesis. The mechanisms that regulate p43.2 expression, including the potential role of microRNAs in regulating its expression, are also reviewed. To the best of our knowledge, we are the first to comprehensively review the published findings regarding the physiological and pathological functions of this gene.

Pediatric neuroblastoma is the most common extra‐cranial malignancy in infants and children. Neuroblastoma is a neuroendocrine cancer arising from sympathetic neuronal precursors that circumvent the differentiation process into mature neurons and glia. The poor differentiation status of neuroblastoma cells allows them to retain features of early embryonic stem cells. SAPCD2 is a newly identified cell cycle regulator involved in mitotic progression and spindle assembly. Although the mechanism by which SAPCD2 exerts its effect is poorly understood, its function appears to be critical in early embryonic development where it regulates mitotic spindle orientation, planar polarization in cell progenitors, and tissue segmentation. More relevant to this study, a recent investigation has revealed that SAPCD2 plays a role in symmetric planar divisions during early embryonic segmentation by interacting with the Gαi‐LGN‐NuMA ternary spindle assembly complex, which appears to be critically important for modulating differentiation of neuroepithelial cells. The expression of SAPCD2 is generally exclusive to embryonic tissues except in the case of malignancies where it is dramatically overexpressed compared to normal tissues. Its overexpression has been observed in several cancers such as in the lung and liver, but its relevance to neuroblastoma has not been reported in the current literature. The objective of this study is to investigate whether SAPCD2 expression levels in human neuroblastoma tissues are correlated with disease progression and if so, to further characterize its role in regulating neuroblastoma cell survival in vitro. The correlation of SAPCD2 with neuroblastoma patient survival was investigated using two data sets obtained from the public R2: Genomics database ( http://r2.amc.nl). The patients were divided into two groups based on SAPCD2 mRNA level (high or low), and the difference in patient survival between the two groups was compared using Kaplan‐Meier analysis. To further investigate the potential oncogenic role of SAPCD2 in vitro, the neuroblastoma cell line BE(2)‐C was transfected with siRNAs against SAPCD2 mRNA to determine the effect of SAPCD2 knockdown on neuroblastoma cell survival and proliferation. Our results demonstrate a statistically significant difference in the survival probabilities of neuroblastoma patients within the two SAPCD2 expression groups, with patients possessing high SAPCD2 mRNA levels showing significantly reduced survival compared to patients with low SAPCD2 mRNA level in both datasets. In in vitro experimental investigations, siRNA‐mediated knockdown of SAPCD2 caused a dramatic reduction in neuroblastoma cell viability after five days and a dramatic reduction in colony formation capacity after 14 days. These results strongly suggest that SAPCD2 functions as an oncogene in neuroblastoma and elevated SAPCD2 expression in neuroblastoma cells promote poor patient prognosis. Future elucidation of the role of SAPCD2 in contributing to the oncogenic transformation of neuroblastoma may ...

BackgroundAdoptive transfer of ex-vivo expanded tumor-infiltrating lymphocytes (TIL) has shown promise in the clinic. However, the non-specific expansion of TIL and the lack of understanding of the active component of TIL has resulted in poor correlation between clinical response and dose as well as poor understanding of response and resistance mechanisms. The VELOSTM manufacturing process generates a precision and personalized treatment modality by targeting clonal neoantigens with the incorporation of an antigen-specific expansion step to enrich the product for these specificities. Achilles has developed a second generation manufacturing process (VELOSTM Process 2) to boost the neoantigen-reactive cell dose while maintaining key qualitative features associated with function. Here we report the in-depth characterization of clonal neoantigen-reactive T cells (cNeT) products expanded using the two VELOSTM processes.MethodsMatched tumors and peripheral blood from patients undergoing routine surgery were obtained from patients with primary NSCLC or metastatic melanoma (NCT03517917). TIL were expanded from tumor fragments and peptide pools corresponding to the clonal mutations identified using the PELEUSTM bioinformatics platform were synthesized. cNeT were expanded by co-culture of TIL with peptide-pulsed autologous dendritic cells, with an optimized cytokine cocktail and co-stimulation for Process 2. Neoantigen reactivity was assessed using our proprietary potency assay with peptide pool re-challenge followed by intracellular cytokine staining. Single peptide reactivities were identified using ELISPOT and flow cytometric analysis for in-depth phenotyping of cNeT was performed.ResultsCD3+ T cells displayed higher fold expansion in Process 2 (median 77.4) compared to Process 1 (median 3.8)(n=5). Both processes showed similar CD3+ T cell content (median Process 1=91.3%, Process 2=96.9% n=5) and contained both CD4+ and CD8+ T cells showing reactivity to clonal neoantigens. Proportion of cells responding to neoantigen re-challenge was similar across both processes (median Process 1=19.9% and Process 2=18.2%) leading to higher reactive dose when coupled with higher T cell doses in Process 2. Phenotypically T cells were predominantly effector memory for both processes and Process 2 had lower frequencies of terminally differentiated T cells.ConclusionsAchilles’ proprietary potency assay enables the optimization of new processes that deliver high cNeT doses to patients by detecting the active drug component. We have generated proof of concept data that supports the transfer of the VELOSTM Process 2 to clinical manufacture for two first-in-human studies for the treatment of solid cancers.Ethics ApprovalThe samples for the study were collected under an ethically approved protocol (NCT03517917)