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Activating mutations in the RAS/MAPK pathway are observed in relapsed neuroblastoma. Preclinical studies indicate that these tumors have an increased sensitivity to inhibitors of the RAS/MAPK pathway, such as MEK inhibitors. MEK inhibitors do not induce durable responses as single agents, indicating a need to identify synergistic combinations of targeted agents to provide therapeutic benefit. We previously showed preclinical therapeutic synergy between a MEK inhibitor, trametinib, and a monoclonal antibody specific for IGF1R, ganitumab in RAS-mutated rhabdomyosarcoma. Neuroblastoma cells, like rhabdomyosarcoma cells, are sensitive to the inhibition of the RAS/MAPK and IGF1R/AKT/mTOR pathways. We hypothesized that the combination of trametinib and ganitumab would be effective in RAS-mutated neuroblastoma. In this study, trametinib and ganitumab synergistically suppressed neuroblastoma cell proliferation and induced apoptosis in cell culture. We also observed a delay in tumor initiation and prolongation of survival in heterotopic and orthotopic xenograft models treated with trametinib and ganitumab. However, the growth of both primary and metastatic tumors was observed in animals receiving the combination of trametinib and ganitumab. Therefore, more preclinical work is necessary before testing this combination in patients with relapsed or refractory RAS-mutated neuroblastoma.

Introduction: Cancer stem cells (CSCs), a group of tumor-initiating and tumor-maintaining cells, may be major players in the treatment resistance and recurrence distinctive of chordoma. Characterizing CSCs is crucial to better targeting this subpopulation., Methods: Using flow cytometry, six chordoma cell lines were evaluated for CSC composition. In vitro, cell lines were stained for B7H6, HER2, MICA-B, ULBP1, EGFR, and PD-L1 surface markers. Eighteen resected chordomas were stained using a multispectral immunofluorescence (mIF) antibody panel to identify CSCs in vivo. HALO software was used for quantitative CSC density and spatial analysis., Results: In vitro, chordoma CSCs express more B7H6, MICA-B, and ULBP1, assessed by percent positivity and mean fluorescence intensity (MFI), as compared to non-CSCs in all cell lines. PD- L1 percent positivity is increased by >20% in CSCs compared to non-CSCs in all cell lines except CH22. In vivo, CSCs comprise 1.39% of chordoma cells and most are PD-L1+ (75.18%). A spatial analysis suggests that chordoma CSCs cluster at an average distance of 71.51 mm (SD 73.40 mm) from stroma., Discussion: To our knowledge, this study is the first to identify individual chordoma CSCs and describe their surface phenotypes using in vitro and in vivo methods. PD-L1 is overexpressed on CSCs in chordoma human cell lines and operative tumor samples. Similarly, potential immunotherapeutic targets on CSCs, including B7H6, MICA-B, ULBP1, EGFR, and HER2 are overexpressed across cell lines. Targeting these markers may have a preferential role in combating CSCs, an aggressive subpopulation likely consequential to chordoma's high recurrence rate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lopez, Fabian, Padget, Robbins, Kowalczyk, Lassoued, Pastor, Allen, Gallia, Gulley, Hodge and London.)

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

There is strong evidence that chemotherapy can induce tumor necrosis which can be exploited for the targeted delivery of immuno-oncology agents into the tumor microenvironment (TME). We hypothesized that docetaxel, a chemotherapeutic agent that induces necrosis, in combination with the bifunctional molecule NHS-IL-12 (M9241), which delivers recombinant IL-12 through specific targeting of necrotic regions in the tumor, would provide a significant antitumor benefit in the poorly inflamed murine tumor model, EMT6 (breast), and in the moderately immune-infiltrated tumor model, MC38 (colorectal). Docetaxel, as monotherapy or in combination with NHS-IL-12, promoted tumor necrosis, leading to the improved accumulation and retention of NHS-IL-12 in the TME. Significant antitumor activity and prolonged survival were observed in cohorts receiving docetaxel and NHS-IL-12 combination therapy in both the MC38 and EMT6 murine models. The therapeutic effects were associated with increased tumor infiltrating lymphocytes and were dependent on CD8 + T cells. Transcriptomics of the TME of mice receiving the combination therapy revealed the upregulation of genes involving crosstalk between innate and adaptive immunity factors, as well as the downregulation of signatures of myeloid cells. In addition, docetaxel and NHS-IL-12 combination therapy effectively controlled tumor growth of PD-L1 wild-type and PD-L1 knockout MC38 in vivo, implying this combination could be applied in immune checkpoint refractory tumors, and/or tumors regardless of PD-L1 status. The data presented herein provide the rationale for the design of clinical studies employing this combination or similar combinations of agents., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: While radiation and chemotherapy are primarily purposed for their cytotoxic effects, a growing body of preclinical and clinical evidence demonstrates an immunogenic potential for these standard therapies. Accordingly, we sought to characterize the immunogenic potential of radiation and cisplatin in human tumor models of HPV-associated malignancies. These studies may inform rational combination immuno-oncology (IO) strategies to be employed in the clinic on the backbone of standard of care, and in so doing exploit the immunogenic potential of standard of care to improve durable responses in HPV-associated malignancies., Methods: Retroviral transduction with HPV16 E7 established a novel HPV-associated sinonasal squamous cell carcinoma (SNSCC) cell line. Three established HPV16-positive cell lines were also studied (cervical carcinoma and head and neck squamous cell carcinoma). Following determination of sensitivities to standard therapies using MTT assays, flow cytometry was used to characterize induction of immunogenic cell stress following sublethal exposure to radiation or cisplatin, and the functional consequence of this induction was determined using impedance-based real time cell analysis cytotoxicity assays employing HPV16 E7-specific cytotoxic lymphocytes (CTLs) with or without N803 (IL-15/IL-15-Rα superagonist) or exogenous death receptor ligands. In vitro observations were translated using an in vivo xenograft NSG mouse model of human cervical carcinoma evaluating cisplatin in combination with CTL adoptive cell transfer., Results: We showed that subpopulations surviving clinically relevant doses of radiation or cisplatin therapy were more susceptible to CTL-mediated lysis in four of four tumor models of HPV-associated malignancies, serving as a model for HPV therapeutic vaccine or T-cell receptor adoptive cell transfer. This increased killing was further amplified by IL-15 agonism employing N803. We further characterized that radiation or cisplatin induced immunogenic cell stress in three of three cell lines, and consequently demonstrated that upregulated surface expression of Fas and TRAIL-R2 death receptors at least in part mediated enhanced CTL-mediated lysis. In vivo, cisplatin-induced immunogenic cell stress synergistically potentiated CTL-mediated tumor control in a human model of HPV-associated malignancy., Conclusion: Standard of care radiation or cisplatin therapy induced immunogenic cell stress in preclinical models of HPV-associated malignancies, presenting an opportunity poised for exploitation by employing IO strategies in combination with standard of care., Competing Interests: Competing interests: NRL receives research funding from Merck Sharp & Dohme, holds stock in Navigen Pharmaceuticals and was a consultant for Cooltech, none of which are relevant to the present manuscript. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Innovative strategies to re-establish the immune-mediated destruction of malignant cells is paramount to the success of anti-cancer therapy. Accumulating evidence suggests that radiotherapy and select chemotherapeutic drugs and small molecule inhibitors induce immunogenic cell stress on tumors that results in improved immune recognition and targeting of the malignant cells. Through immunogenic cell death, which entails the release of antigens and danger signals, and immunogenic modulation, wherein the phenotype of stressed cells is altered to become more susceptible to immune attack, radiotherapies, chemotherapies, and small-molecule inhibitors exert immune-mediated anti-tumor responses. In this review, we discuss the mechanisms of immunogenic cell death and immunogenic modulation and their relevance in the anti-tumor activity of radiotherapies, chemotherapies, and small-molecule inhibitors. Our aim is to feature the immunological aspects of conventional and targeted cancer therapies and highlight how these therapies may be compatible with emerging immunotherapy approaches.

Background: Chordoma is a rare, invasive, and devastating bone malignancy of residual notochord tissue that arises at the skull base, sacrum, or spine. In order to maximize immunotherapeutic approaches as a potential treatment strategy in chordoma it is important to fully characterize the tumor immune microenvironment (TIME). Multispectral immunofluorescence (MIF) allows for comprehensive evaluation of tumor compartments, molecular co-expression, and immune cell spatial relationships. Here we implement MIF to define the myeloid, T cell, and natural killer (NK) cell compartments in an effort to guide rational design of immunotherapeutic strategies for chordoma., Methods: Chordoma tumor tissue from 57 patients was evaluated using MIF. Three panels were validated to assess myeloid cell, T cell, and NK cell populations. Slides were stained using an automated system and HALO software objective analysis was utilized for quantitative immune cell density and spatial comparisons between tumor and stroma compartments., Results: Chordoma TIME analysis revealed macrophage infiltration of the tumor parenchyma at a significantly higher density than stroma. In contrast, helper T cells, cytotoxic T cells, and T regulatory cells were significantly more abundant in stroma versus tumor. T cell compartment infiltration more commonly demonstrated a tumor parenchymal exclusion pattern, most markedly among cytotoxic T cells. NK cells were sparsely found within the chordoma TIME and few were in an activated state. No immune composition differences were seen in chordomas originating from diverse anatomic sites or between those resected at primary versus advanced disease stage., Conclusion: This is the first comprehensive evaluation of the chordoma TIME including myeloid, T cell, and NK cell appraisal using MIF. Our findings demonstrate that myeloid cells significantly infiltrate chordoma tumor parenchyma while T cells tend to be tumor parenchymal excluded with high stromal infiltration. On average, myeloid cells are found nearer to target tumor cells than T cells, potentially resulting in restriction of T effector cell function. This study suggests that future immunotherapy combinations for chordoma should be aimed at decreasing myeloid cell suppressive function while enhancing cytotoxic T cell and NK cell killing., Competing Interests: NL receives research funding from Merck Sharp & Dohme, LLC regarding HPV related sinonasal carcinomas, holds stock in Navigen Pharmaceuticals and was a consultant for Cooltech Inc., none of which are relevant to the present manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lopez, Robbins, Kowalczyk, Lassoued, Gulley, Miettinen, Gallia, Allen, Hodge and London.)

Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS., (©2020 American Association for Cancer Research.)