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Accurate assessment of treatment response and residual disease is indispensable for the evaluation of cancer treatment efficacy. However, performing tissue biopsies for longitudinal follow-up poses a major challenge in the management of solid tumours like neuroblastoma. In the present study, we evaluated whether circulating miRNAs are suitable to monitor neuroblastoma tumour burden and whether treatment-induced changes of miRNA abundance in the tumour are detectable in serum. We performed small RNA sequencing on longitudinally collected serum samples from mice carrying orthotopic neuroblastoma xenografts that were exposed to treatment with idasanutlin or temsirolimus. We identified 57 serum miRNAs to be differentially expressed upon xenograft tumour manifestation, out of which 21 were also found specifically expressed in the serum of human high-risk neuroblastoma patients. The murine serum levels of these 57 miRNAs correlated with tumour tissue expression and tumour volume, suggesting potential utility for monitoring tumour burden. In addition, we describe serum miRNAs that dynamically respond to p53 activation following treatment of engrafted mice with idasanutlin. We identified idasanutlin-induced serum miRNA expression changes upon one day and 11 days of treatment. By limiting to miRNAs with a tumour-related induction, we put forward hsa-miR-34a-5p as a potential pharmacodynamic biomarker of p53 activation in serum.

This study seeks to develop a systemic immunomodulatory nanoparticle (immuno-NP) therapy that harnesses proinflammatory innate immune cells to remodel tumor microenvironment (TME) immunity in humanized mouse models of high-risk neuroblastoma (NB). Currently, survival for high-risk metastatic NB remains ~50% at 5 years, even with chemotherapy given at maximal tolerated dosing, which itself imparts debilitating long-term sequelae in >90% of survivors. The success of immunotherapies for leukemias and lymphomas has not translated to pediatric solid tumors, which typically present as immunosuppressed “cold” tumors with microenvironments that limit antigen presentation and sustained activation of cytotoxic CD8+ T cells. To date, immune checkpoint inhibitor and chimeric antigen receptor (CAR) T approaches have primarily focused on augmenting CD8+ T cells that are suppressed by the “cold” TME of high-risk solid tumors. However, it has now been demonstrated that proinflammatory cytokines secreted by innate antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, can restimulate anti-tumor T cell responses by remodeling the TME from “cold” to “hot”. Here, we use a novel engineering approach to design and systemically deliver lipid-based immuno-NPs coencapsulating synergistic innate immune agonists of the stimulator of interferon genes (STNG) and Toll-like receptor 4 (TLR4) pathways to target APCs that are enriched in the TME perivascular region. APCs taking up NPs synergistically produce Type I interferons, priming and recruiting CD8+ T cells for clearance. We hypothesized that treatment of humanized mice bearing human NB xenografts with immuno-NPs will drive APC-mediated “cold”-to-“hot” TME remodeling, leading to sustained recruitment and activation of CD8+ T cells. Humanized NOD scid gamma (NSG) mice bearing orthotopic CMYC-expressing metastatic SH-SY5Y cell xenografts were treated with immuno-NPs and tumor burden assessed via bioluminescent imaging just 3 days afterwards indicated a significant 47% reduction in primary tumor mass and 53% reduction in liver metastasis. Confocal imaging of both the primary tumor and metastatic sites indicated clear immuno-NP deposition surrounding the CD31+ vascular tumor endothelium. Flow cytometry analysis of primary tumors indicated a significant 7.8-fold increase in infiltrating CD8+ T cells in mice that received immuno-NPs compared to control mice that received empty NPs. Subsequent survival experiments demonstrated that immuno-NPs significantly increased survival by driving proinflammatory cytokine production in the TME and also combined effectively with PD1 blockade for enhanced CD8+ T cell activation and function. In conclusion, these findings demonstrated the feasibility of our engineering strategy as a safer, more effective therapy for high-risk NB by confirming that immuno-NPs deposit avidly in NB primary tumors and even sites of metastasis upon systemic delivery, bolster TME APC function and CD8+ T cell-mediated clearance, and drive protective long-term anti-tumor immunity. Citation Format: Prabhani Atukorale, Jason Shohet, Christina Lusi, Kim Wigglesworth, Griffin Kane, Esteban Rozen. Engineering immunomodulatory nanoparticles for "cold"-to-"hot" tumor microenvironment remodeling for treatment of high-risk neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B47.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed a siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of NB cell lines, those with MYCN amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of its known target histone H3, and upregulation of p53 pathway in MYCN-amplified NB cells with wild-type TP53. Both wild-type and p53-mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. Citation Format: Dominik Bogen, Jun S. Wei, David O. Azorsa, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Jonathan M. Keller, Lesley A. Mathews Griner, Marc Ferrer, Young K. Song, Hongling Liao, Arnulfo Mendoza, Berkley E. Gryder, Sivasish Sindri, Jianbin He, Xinyu Wen, Xinyu Wen, Shile Zhang, John F. Shern, Marielle E. Yohe, Sabine Taschner-Mandl, Jason Shohet, Craig J. Thomas, Scott E. Martin, Peter F. Ambros, Javed Khan. Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B31.

Identification and Characterization of Rp5: A Novel Gene Differentially Expressed in Germinal Center B-Cells