Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells.

Purpose: Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. This study delineates how osteosarcoma cells educate the lung microenvironment during metastatic progression. Experimental design: Using single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence. We evaluated the ability of nintedanib to impair metastatic colonization and prevent osteosarcoma-induced education of the lung microenvironment in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Results: Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially-differentiated epithelial intermediates. Inhibition of fibrotic pathways with nintedanib prevented metastatic progression in multiple murine and human xenograft models. Conclusions: Our work demonstrates that osteosarcoma cells co-opt fibrosis to promote metastatic outgrowth. When harmonized with data from adult epithelial cancers, our results support a generalized model wherein aberrant mesenchymal-epithelial interactions are critical for promoting lung metastasis. Adult epithelial carcinomas induce fibrotic pathways in normal lung fibroblasts, whereas osteosarcoma, a pediatric mesenchymal tumor, exhibits fibrotic reprogramming in response to the aberrant wound-healing behaviors of an otherwise normal lung epithelium, which are induced by tumor cell interactions. Statement of translational relevance: Therapies that block metastasis have the potential to save the majority of lives lost due to solid tumors. Disseminated tumor cells must educate the foreign, inhospitable microenvironments they encounter within secondary organs to facilitate metastatic colonization. Our study elucidated that disseminated osteosarcoma cells survive within the lung by co-opting and amplifying the lung's endogenous wound healing response program. More broadly, our results support a model wherein mesenchymal-epithelial cooperation is a key driver of lung metastasis. Osteosarcoma, a pediatric mesenchymal tumor, undergoes lung epithelial induced fibrotic activation while also transforming normal lung epithelial cells towards a fibrosis promoting phenotype. Conversely, adult epithelial carcinomas activate fibrotic signaling in normal lung mesenchymal fibroblasts. Our data implicates fibrosis and abnormal wound healing as key drivers of lung metastasis across multiple tumor types that can be targeted therapeutically to disrupt metastasis progression.

Host-derived growth factors drive ERK phosphorylation and MCL1 expression to promote osteosarcoma cell survival during metastatic lung colonization.

PURPOSE: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. METHODS: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. RESULTS: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. CONCLUSION: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.

Osteosarcoma Multi-Omics Landscape and Subtypes.

Osteosarcoma (OS) is the most common primary bone malignancy that exhibits remarkable histologic diversity and genetic heterogeneity. The complex nature of osteosarcoma has confounded precise molecular categorization, prognosis, and prediction for this disease. In this study, we performed a comprehensive multiplatform analysis on 86 osteosarcoma tumors, including somatic copy-number alteration, gene expression and methylation, and identified three molecularly distinct and clinically relevant subtypes of osteosarcoma. The subgrouping criteria was validated on another cohort of osteosarcoma tumors. Previously unappreciated osteosarcoma-type-specific changes in specific genes' copy number, expression and methylation were revealed based on the subgrouping. The subgrouping and novel gene signatures provide insights into refining osteosarcoma therapy and relationships to other types of cancer.

Post-zygotic rescue of meiotic errors causes brain mosaicism and focal epilepsy.

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development.

Oncolytic virus-driven immune remodeling revealed in mouse medulloblastomas at single cell resolution.

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma.

Structurally Complex Osteosarcoma Genomes Exhibit Limited Heterogeneity within Individual Tumors and across Evolutionary Time.

Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNA) are the genetic drivers of disease. Models around genomic instability conflict-it is unclear whether osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single-cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single-cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single-cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little subclonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic timepoints (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. Significance: Chromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications for diagnosis, biomarker assessment, mechanisms of treatment resistance, and represents a conceptual advance in our understanding of intratumoral heterogeneity and tumor evolution.

Osteosarcoma tumors maintain intra-tumoral transcriptional heterogeneity during bone and lung colonization.

BACKGROUND: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. RESULTS: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. CONCLUSIONS: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma.

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ T cells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

Redundant Signaling as the Predominant Mechanism for Resistance to Antibodies Targeting the Type-I Insulin-Like Growth Factor Receptor in Cells Derived from Childhood Sarcoma.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.

Osteosarcoma.

Osteosarcoma is the most common primary malignant tumour of the bone. Osteosarcoma incidence is bimodal, peaking at 18 and 60 years of age, and is slightly more common in males. The key pathophysiological mechanism involves several possible genetic drivers of disease linked to bone formation, causing malignant progression and metastasis. While there have been significant improvements in the outcome of patients with localized disease, with event-free survival outcomes exceeding 60%, in patients with metastatic disease, event-free survival outcomes remain poor at less than 30%. The suspicion of osteosarcoma based on radiographs still requires pathological evaluation of a bone biopsy specimen for definitive diagnosis and CT imaging of the chest should be performed to identify lung nodules. So far, population-based screening and surveillance strategies have not been implemented due to the rarity of osteosarcoma and the lack of reliable markers. Current screening focuses only on groups at high risk such as patients with genetic cancer predisposition syndromes. Management of osteosarcoma requires a multidisciplinary team of paediatric and medical oncologists, orthopaedic and general surgeons, pathologists, radiologists and specialist nurses. Survivors of osteosarcoma require specialized medical follow-up, as curative treatment consisting of chemotherapy and surgery has long-term adverse effects, which also affect the quality of life of patients. The development of osteosarcoma model systems and related research as well as the evaluation of new treatment approaches are ongoing to improve disease outcomes, especially for patients with metastases.

An international working group consensus report for the prioritization of molecular biomarkers for Ewing sarcoma.

The advent of dose intensified interval compressed therapy has improved event-free survival for patients with localized Ewing sarcoma (EwS) to 78% at 5 years. However, nearly a quarter of patients with localized tumors and 60-80% of patients with metastatic tumors suffer relapse and die of disease. In addition, those who survive are often left with debilitating late effects. Clinical features aside from stage have proven inadequate to meaningfully classify patients for risk-stratified therapy. Therefore, there is a critical need to develop approaches to risk stratify patients with EwS based on molecular features. Over the past decade, new technology has enabled the study of multiple molecular biomarkers in EwS. Preliminary evidence requiring validation supports copy number changes, and loss of function mutations in tumor suppressor genes as biomarkers of outcome in EwS. Initial studies of circulating tumor DNA demonstrated that diagnostic ctDNA burden and ctDNA clearance during induction are also associated with outcome. In addition, fusion partner should be a pre-requisite for enrollment on EwS clinical trials, and the fusion type and structure require further study to determine prognostic impact. These emerging biomarkers represent a new horizon in our understanding of disease risk and will enable future efforts to develop risk-adapted treatment.

Lurbinectedin Inhibits the EWS-WT1 Transcription Factor in Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric sarcoma with poor overall survival. This tumor is absolutely dependent on the continued expression and activity of its defining molecular lesion, the EWS-WT1 transcription factor. Unfortunately, the therapeutic targeting of transcription factors is challenging, and there is a critical need to identify compounds that inhibit EWS-WT1. Here we show that the compound lurbinectedin inhibits EWS-WT1 by redistributing the protein within the nucleus to the nucleolus. This nucleolar redistribution interferes with the activity of EWS-WT1 to reverse the expression of over 70% of the transcriptome. In addition, the compound blocks the expression of the EWS-WT1 fusion protein to inhibit cell proliferation at the lowest GI50 ever reported for this compound in any cell type. The effects occur at concentrations that are easily achievable in the clinic and translate to the in vivo setting to cause tumor regressions in multiple mice in a xenograft and PDX model of DSRCT. Importantly, this mechanism of nucleolar redistribution is also seen with wild-type EWSR1 and the related fusion protein EWS-FLI1. This provides evidence for a "class effect" for the more than 18 tumors driven by EWSR1 fusion proteins. More importantly, the data establish lurbinectedin as a promising clinical candidate for DSRCT.

Characterizing the metabolic role of STAT3 in canine osteosarcoma.

Signal transducer and activator of transcription 3 (STAT3) dysregulation has been characterized in canine OS, with previous data suggesting that constitutive STAT3 activation contributes to survival and proliferation in OS cell lines in vitro. Recently, the contribution of STAT3 to tumour metabolism has been described across several tumour histologies, and understanding the metabolic implications of STAT3 loss may elucidate novel therapeutic approaches with synergistic activity. The objective of this work was to characterize metabolic benchmarks associated with STAT3 loss in canine OS. STAT3 expression and activation was evaluated using western blotting in canine OS cell lines OSCA8 and Abrams. STAT3 was deleted from these OS cell lines using CRISPR-Cas9, and the effects on proliferation, invasion and metabolism (respirometry, intracellular lactate) were determined. Loss of STAT3 was associated with decreased basal and compensatory glycolysis in canine OS cell lines, without modulation of cellular proliferation. Loss of STAT3 also resulted in diminished invasive capacity in vitro. Interestingly, the absence of STAT3 did not impact sensitivity to doxorubicin in vitro. Our data demonstrate that loss of STAT3 modulates features of aerobic glycolysis in canine OS impacting capacities for cellular invasions, suggesting a role for this transcription factor in metastasis.

Clinical outcomes and efficacy of stereotactic body radiation therapy in children, adolescents, and young adults with metastatic solid tumors.

OBJECTIVE: The objective of this study is to report disease outcomes and toxicity with the use of stereotactic body radiation therapy (SBRT) in the treatment of pediatric metastatic disease. METHODS: All pediatric and adolescent young adult (AYA) patients' who received SBRT were included between the years 2000 and 2020. Study endpoints included local control (LC), progression-free survival (PFS), overall survival (OS), cumulative incidence (CI) of death or local failure and toxicity. The end points with respect to survival and LC were calculated using the Kaplan-Meier estimate. The cumulative incidence of local failure was calculated using death as a competing risk. RESULTS: 16 patients with 36 lesions irradiated met inclusion criteria and formed the study cohort. The median OS and PFS for the entire cohort were 17 months and 15.7 months, respectively. The 1 year OS for the entire cohort was 75%. The 6- and 12 month local control was 85 and 78%, respectively. There were no local failures in irradiated lesions for patients who received a BED10≥100 Gy. Patients who were treated with SBRT who had ≤5 metastatic lesions at first recurrence had a superior 1 year OS of 100 vs 50% for those with >5 lesions. One patient (6.3%) experienced a Grade 3 central nervous system toxicity. CONCLUSION: LC was excellent with SBRT delivered to metastatic disease, particularly for lesions receiving a BED10≥100 Gy. High-grade toxicity was rare in our patient population. Patients with five or fewer metastatic sites have a significantly better OS compared to >5 sites. ADVANCES IN KNOWLEDGE: This study demonstrates that SBRT is safe and efficacious in the treatment of pediatric oligometastatic disease.

Targeted Therapy in a Young Adult With a Novel Epithelioid Tumor Driven by a PRRC2B-ALK Fusion.

This case report describes an 18-year-old woman with an unusual epithelioid tumor of the omentum with a novel PRRC2B-ALK fusion. Although the atypical pathologic features raised significant diagnostic challenges, expression of CD30 on tumor cells and detection of an ALK rearrangement provided critical information for selecting targeted therapy in a patient not suitable for surgical resection. Despite an initially promising therapeutic response, the patient died. The efficacy of treatment was confirmed by the lack of viable tumor cells at autopsy. This case highlights the role of timely targeted therapy in patients with rare tumors and novel actionable molecular targets.

Endogenous retrovirus envelope as a tumor-associated immunotherapeutic target in murine osteosarcoma.

Osteosarcoma remains one of the deadliest cancers in pediatrics and young adults. We administered two types of immunotherapies, oncolytic virotherapy and immune checkpoint inhibition, to two murine osteosarcoma models and observed divergent results. Mice bearing F420 showed no response, whereas those with K7M2 showed prolonged survival in response to combination therapy. K7M2 had higher expression of immune-related genes and higher baseline immune cell infiltrates, but there were no significant differences in tumor mutational burden or predicted MHC class I binding of nonsynonymous mutations. Instead, we found several mouse endogenous retrovirus sequences highly expressed in K7M2 compared with F420. T cell tetramer staining for one of them, gp70, was detected in mice with K7M2 but not F420, suggesting that endogenous retrovirus proteins are targets for the anti-tumor immune reaction. Given prior observations of endogenous retrovirus expression in human osteosarcomas, our findings may be translatable to human disease.

Charting a path for prioritization of novel agents for clinical trials in osteosarcoma: A report from the Children's Oncology Group New Agents for Osteosarcoma Task Force.

Osteosarcoma is the most common bone tumor in children and young adults. Metastatic and relapsed disease confer poor prognosis, and there have been no improvements in outcomes for several decades. The disease's biological complexity, lack of drugs developed specifically for osteosarcoma, imperfect preclinical models, and limits of existing clinical trial designs have contributed to lack of progress. The Children's Oncology Group Bone Tumor Committee established the New Agents for Osteosarcoma Task Force to identify and prioritize agents for inclusion in clinical trials. The group identified multitargeted tyrosine kinase inhibitors, immunotherapies targeting B7-H3, CD47-SIRPα inhibitors, telaglenastat, and epigenetic modifiers as the top agents of interest. Only multitargeted tyrosine kinase inhibitors met all criteria for frontline evaluation and have already been incorporated into an upcoming phase III study concept. The task force will continue to reassess identified agents of interest as new data become available and evaluate novel agents using this method.