Genomic regions with reduced chromatin activity.

Gene alteration frequency of PREX1.

Clinical and Histological Characteristics of Patient Specimens

Differential expression of RNA-seq between the different groups.

Differential chromatin activity shapes transcriptional profile.

Effects of PREX1 knockdown on localized PDX-derived TCCC-OS94 cells.

Abstract Osteosarcoma is the most common primary malignant bone tumor affecting the pediatric population with a high potential to metastasize. However, insights into the molecular features enabling its metastatic potential are limited. We mapped the active chromatin landscapes of osteosarcoma tumors by integrating histone H3 lysine–acetylated chromatin state (n = 13), chromatin accessibility profiles (n = 11), and gene expression (n = 13) to understand the differences in their active chromatin profiles and their impact on molecular mechanisms driving the malignant phenotypes. Primary osteosarcoma tumors from patients with metastasis (primary met) have a distinct active chromatin landscape compared with those without metastasis (localized). This difference shapes the transcriptional profile of osteosarcoma. We identified novel candidate genes, including PPP1R1B, PREX1, and IGF2BP1, that exhibit increased chromatin activity in primary met. Loss of PREX1 in primary met osteosarcoma cells significantly diminishes osteosarcoma proliferation, invasion, migration, and colony formation capacity. Differential chromatin activity in primary met is associated with genes regulating cytoskeleton organization, cellular adhesion, and extracellular matrix, suggesting their role in facilitating osteosarcoma metastasis. Chromatin profiling of tumors from metastatic lung lesions shows increased chromatin activity in genes involved in cell migration and Wnt pathway. These data demonstrate that metastatic potential is intrinsically present in primary met tumors, with cellular chromatin profiles further adapting for successful dissemination, migration, and colonization at the distal site. Implications: Our study demonstrates that metastatic potential is intrinsic to primary metastatic osteosarcoma tumors, with chromatin profiles further adapting for successful dissemination, migration, and colonization at the distal metastatic site.

Hierarchical clustering of signal intensity (tags per million) of top 20% most variable regions by median absolute deviation (n = 31,690) of all H3K27ac enriched peaks.

Accurate nuclei segmentation in microscopy whole slide images (WSIs) remains challenging due to variability in staining, imaging conditions, and tissue morphology. We propose CellGenNet, a knowledge distillation framework for robust cross-tissue cell segmentation under limited supervision. CellGenNet adopts a student-teacher architecture, where a capacity teacher is trained on sparse annotations and generates soft pseudo-labels for unlabeled regions. The student is optimized using a joint objective that integrates ground-truth labels, teacher-derived probabilistic targets, and a hybrid loss function combining binary cross-entropy and Tversky loss, enabling asymmetric penalties to mitigate class imbalance and better preserve minority nuclear structures. Consistency regularization and layerwise dropout further stabilize feature representations and promote reliable feature transfer. Experiments across diverse cancer tissue WSIs show that CellGenNet improves segmentation accuracy and generalization over supervised and semi-supervised baselines, supporting scalable and reproducible histopathology analysis.

Cancer continues to be a significant global health issue, influenced by genetic mutations and external factors like carcinogenic exposure, lifestyle choices, and chronic inflammation. The myelocytomatosis (MYC) oncogene family, including c-MYC, MYCN, and MYCL, is essential in the development, progression, and metastasis of various cancers such as breast, colorectal, osteosarcoma, and neuroblastoma. Beyond its well-known roles in cell growth and metabolism, MYC significantly shapes the tumor immune microenvironment (TIME) by altering immune cell dynamics, antigen presentation, and checkpoint expression. It contributes to immune evasion by upregulating checkpoints such as programmed death-ligand 1 (PD-L1) and cluster of differentiation (CD)47, suppressing antigen-presenting major histocompatibility complex (MHC) molecules, and promoting the recruitment of suppressive immune cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). While direct targeting of MYC has proven challenging, recent advances in therapeutic strategies, including MYC-MYC-associated factor X (MAX) dimerization inhibitors, bromodomain and extra terminal domain (BET) and cyclin dependent kinase (CDK) inhibitors, synthetic lethality approaches, and epigenetic modulators, have shown promising results in preclinical and early clinical settings. This review discusses MYC's comprehensive impact on TIME and examines the promising therapeutic strategies of MYC inhibition in enhancing the effectiveness of immunotherapies, supported by recent preclinical and clinical findings.

Osteosarcoma (OS) is the most common primary malignant bone tumor affecting the pediatric population with high potential to metastasize to distal sites, most commonly the lung. Insights into defining molecular features contributing to metastatic potential are lacking. We have mapped the active chromatin landscapes of OS tumors by integrating histone H3 lysine acetylated chromatin (H3K27ac) profiles (n=13), chromatin accessibility profiles (n=11) and gene expression (n=13) to understand the differences in their active chromatin profiles and its impact on molecular mechanisms driving the malignant phenotypes. Primary OS tumors from patients with metastasis (primary met) have a distinct active chromatin landscape compared to primary tumors from patients without metastatic disease (localized). The difference in chromatin activity shapes the transcriptional profile of OS. We identified novel candidate genes involved in OS pathogenesis and metastasis, including PPP1R1B, PREX1 and IGF2BP1, which exhibit increased chromatin activity in primary met along with higher transcript levels. Overall, differential chromatin activity in primary met occurs in proximity of genes regulating actin cytoskeleton organization, cellular adhesion, and extracellular matrix suggestive of their role in facilitating OS metastasis. Furthermore, chromatin profiling of tumors from metastatic lung lesions noted increases in chromatin activity in genes involved in cell migration and key intracellular signaling cascades, including the Wnt pathway. Thus, this data demonstrates that metastatic potential is intrinsically present in primary metastatic tumors and the cellular chromatin profiles further adapt to allow for successful dissemination, migration, and colonization at the distal metastatic site.

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Approximately 25-30% of these tumors carry amplification of chromosome 8q24, which harbors the oncogene c-Myc, and correlates with a poor prognosis in patients with OS. To understand the mechanisms that underlie the ability of Myc to alter both the tumor and its surrounding tumor immune microenvironment (TiME), we generated and molecularly characterized an osteoblast-specific Cre-Lox-Stop-Lox;(LSL)-c-MycT58A;p53f/+ knockin genetically engineered mouse model (GEMM). Phenotypically, the Myc knockin-GEMM had rapid tumor development with a high incidence of metastasis. Myc-dependent gene signature in our murine model demonstrated significant homology to the human Myc-amplified OS.Interestingly, we noticed a significant reduction in the osteoclast (OCL) cell population in the Myc knockin OS tumor compared to the p53-driven. We found the expression of RANK was significantly downregulated in the Myc knockin tumor compared to the Non-knockin p53 heterozygous tumors. The RANK/RANKL pathway is vital in OCL maturation and bone modeling/remodeling. To understand the involvement of Myc in RANK regulation, we used murine-derived OS cell lines and transiently knocked down of Myc expression using siRNA. We observed a significant upregulation in RANK expression after Myc knockdown. To decipher the molecular mechanism behind the Myc-dependent regulation of RANK expression in OS, we looked into the Myc-mediated microRNAs. Myc regulates the expression of several microRNAs, including the polycistronic miR-17-92 cluster. The expression of miR17-5p and Mir20a-5p was significantly higher in the GEMM tumor tissue samples isolated from the Myc knockin compared to the p53-driven. Further, we validated the Myc-dependent regulation of miR-17-5p/20a-5p expression using transient knockdown of Myc in mouse Myc knockin-derived cell lines. To examine the role of miR17-5p/20a-5p on the RANK regulation, we performed both gain and loss-of-function studies using microRNA-17/20a mimics and inhibitors. After the treatment with miR-17-5p/20a-5p inhibitors, the expression of RANK was significantly upregulated whereas in the case of miR17/20a mimics reversed these effects and led to a downregulation of RANK expression. We established that miR-17-5p/20a-5p is causally responsible for at least part of the mechanism by which Myc regulates the RANK expression in OS.We concluded that the Myc-regulated miR17/20a modulates the RANK expression that is involved in the OCL cell population regulation and function in the OS. Citation Format: Bikesh Kumar Nirala, Lyazat Kurenbekova, Tajhal Patel, Ryan Lane Shuck, Atreyi Dasgupta, Nino Carlo Rainusso, Jason T. Yustein. Myc-regulated miR17, 20a modulate RANK expression in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6713.

Background: Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Two-thirds of OS patients are cured through chemotherapy and tumor resection. However, survival rates have plateaued over the past 30 years, especially for patients with relapsed and/or metastatic disease, with overall long-term outcomes of less than 30%. Transducin beta-like protein 1 (TBL1) protects beta-catenin from nuclear degradation and mediates Wnt targeted transcription by forming a TBL1-beta-catenin complex. Overexpression of TBL1 and nuclear beta-catenin are positively correlated to adverse clinicopathological features and poor prognosis for osteosarcoma patients. Functional experiments reveal that down-regulation of TBL1 and nuclear beta-catenin activity inhibit proliferation, migration, and invasion of osteosarcoma cells. The objective of this study was to determine TBL1 levels within a panel of OS patient-derived xenograft (PDX) tumors. Methods: TBL1 and beta-catenin protein levels were measured via western blot and immunohistochemistry in OS samples. We examined protein levels in OS PDX cell lines collected at distinct clinical stages of metastatic OS and OS primary tumor samples. TBL1, total beta-catenin, and activated beta-catenin levels were measured by immunohistochemical (IHC) staining in tissue microarray (TMA) derived from patient derived xenograft (PDX) tumor and clinical samples. Each tumor sample were formalin fixed, paraffin embedded, and spotted in triplicate on the TMA. Results: Beta-catenin and TBL1 protein were detected in OS samples, and we found higher TBL1 protein levels in cells derived from advanced disease stages. Assessment of TBL1 protein was expanded to OS tissue microarray where TBL1, total beta-catenin, and active beta-catenin were independently stained in a TMA. TBL1, total beta-catenin, and activated beta-catenin were present in 100% of OS PDX cores. Finally, a majority of OS cores were shown to have high expression for both TBL1 and active beta-catenin. Conclusion: Elevated TBL1 levels have been associated with metastatic disease and poor survival. We have measured TBL1 and activated beta-catenin protein levels in a variety of osteosarcoma samples and identified a large percentage of osteosarcoma that co-express both proteins. These results indicate that metastatic OS represents a TBL1 enriched patient population and is a candidate for therapeutics that target TBL1. Citation Format: Kimberly R. Holloway, Taku Yamamichi, Bikesh Nirala, Nino Rainusso, Jason T. Yustein, Stephen Horrigan. TBL1, a multifunctional transcriptional regulator, is highly expressed in osteosarcoma and correlates with activated beta catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6166.

Osteosarcoma (OS) is a heterogeneous, highly metastatic bone malignancy in children and adolescents. Despite advancements in multimodal treatment strategies, the prognosis for patients with metastatic or recurrent disease has not improved significantly in the last four decades. OS is a highly heterogeneous tumor; its genetic background and the mechanism of oncogenesis are not well defined. Unfortunately, no effective molecular targeted therapy is currently available for this disease. Understanding osteosarcoma's tumor microenvironment (TME) has recently gained much interest among scientists hoping to provide valuable insights into tumor heterogeneity, progression, metastasis, and the identification of novel therapeutic avenues. Here, we review the current understanding of the TME of OS, including different cellular and noncellular components, their crosstalk with OS tumor cells, and their involvement in tumor progression and metastasis. We also highlight past/current clinical trials targeting the TME of OS for effective therapies and potential future therapeutic strategies with negligible adverse effects.

Osteosarcoma (OS) is the predominant primary bone tumor in the pediatric and adolescent populations. It has high metastatic potential, with the lungs being the most common site of metastasis. In contrast to many other sarcomas, OS lacks conserved translocations or genetic mutations; instead, it has heterogeneous abnormalities, including somatic DNA copy number alteration, ploidy, chromosomal amplification, and chromosomal loss and gain. Unfortunately, clinical outcomes have not significantly improved in over 30 years. Currently, no effective molecularly targeted therapies are available for this disease. Several genomic studies showed inactivation in the tumor suppressor genes, including p53, RB, and ATRX, and hyperactivation of the tumor promoter genes, including MYC and MDM2, in OS. Alterations in the major signaling pathways, including the PI3K/AKT/mTOR, JAK/STAT, Wnt/β-catenin, NOTCH, Hedgehog/Gli, TGF-β, RTKs, RANK/RANKL, and NF-κB signaling pathways, have been identified in OS development and metastasis. Although OS treatment is currently based on surgical excision and systematic multiagent therapies, several potential targeted therapies are in development. This review focuses on the major signaling pathways of OS, and we propose a biological rationale to consider novel and targeted therapies in the future.

Ewing Sarcoma (EwS) is the second most common malignant bone tumor in adolescents and young adults. The single-most powerful predictor of outcome in EwS is presence of metastatic burden at the time of diagnosis. Patients with metastatic Ewing Sarcoma have an abysmal 5-year survival rate of 10%-25%, which has not changed over the past 30-40 years. Thus, unraveling underlying mechanisms of EwS metastasis are imperative for developing effective therapeutic measures. Investigations towards this goal are limited by the lack of reliable genetically engineered mouse models and specialized metastatic models. Using two established cell lines, A673 and TC71, we generated lung specific metastatic cell lines by serial orthotopic intra-tibial injection followed by isolation of cells from lung metastases. The lung metastatic lines generated exhibit distinct differential molecular signatures from the parental cells when analyzed using a multi-omics approach. These signatures overlapped with EwS patient primary bone and metastatic lung specimens supporting the clinical relevance of these preclinical models of EwS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Intra-Tibial injection in NSG mice Basic Protocol 2: Development and characterization of lung metastatic cell line.

Osteosarcoma (OS) is the most common primary bone tumor of childhood. Approximately 20%-30% of OSs carry amplification of chromosome 8q24, which harbors the oncogene c-MYC and correlates with a poor prognosis. To understand the mechanisms that underlie the ability of MYC to alter both the tumor and its surrounding tumor microenvironment (TME), we generated and molecularly characterized an osteoblast-specific Cre-Lox-Stop-Lox-c-MycT58A p53fl/+ knockin genetically engineered mouse model (GEMM). Phenotypically, the Myc-knockin GEMM had rapid tumor development with a high incidence of metastasis. MYC-dependent gene signatures in our murine model demonstrated significant homology to the human hyperactivated MYC OS. We established that hyperactivation of MYC led to an immune-depleted TME in OS demonstrated by the reduced number of leukocytes, particularly macrophages. MYC hyperactivation led to the downregulation of macrophage colony-stimulating factor 1, through increased microRNA 17/20a expression, causing a reduction of macrophage population in the TME of OS. Furthermore, we developed cell lines from the GEMM tumors, including a degradation tag-MYC model system, which validated our MYC-dependent findings both in vitro and in vivo. Our studies utilized innovative and clinically relevant models to identify a potentially novel molecular mechanism through which MYC regulates the profile and function of the OS immune landscape.

Osteosarcoma (OS) is the most common bone tumor in pediatric patients, particularly in adolescents and young adults. Currently, no effective molecular targeted therapies are available for the OS, however, many OS patients possess genetically defined somatic DNA copy number alterations, including amplification of chromosome 8q24, which harbors the oncogene Myc, and correlates with a worse prognosis. To better understand the molecular pathogenesis, and identify targeted therapy for OS, a robust and reliable preclinical model is needed both for the primary and metastatic disease. We have developed a novel conditional, osteoblast-specific Myc knock-in murine model to understand the mechanisms that underlie the ability of Myc to drive the development and progression of OS and utilize it for the identification of Myc-dependent intrinsic and extrinsic molecular vulnerabilities. A tissue-specific Myc-in genetically engineered murine model was generated by crossing Col2.3-Cre; TP53Flox/+ mice with LSL-MycT58A mice and characterized integrating RNA-seq and Mass Spectrometry. Syngeneic murine OS cell lines were also generated and characterized. The molecular characterization of the GEMM model was compared with high and low Myc expressing human tumors using the OS-TARGET dataset. Differential gene expression in Myc-driven and non-driven-tumor tissue samples were quantified by RNA sequencing. The immune landscape of OS was evaluated using SymphonyFACS and IHC staining. Murine OS tumor type was confirmed by H&E staining. OS tumor was highly aggressive and metastatic in more than 60% of the mice. Myc expression was significantly higher both at the transcriptional and protein levels in the Myc-in tissue/cells compared to the non-Myc-driven model. Expression of 1479 genes (out of 13781 genes) were significantly altered in which 543 were upregulated and 936 were downregulated at transcriptional levels in Myc-in tumor tissue samples as compared to the low-Myc-driven OS tumor model analyzed by RNA sequencing. Myc-target-genes, splicing factor-related genes, E2F, G2M check-point, and DNA repair associated genes were significantly upregulated whereas the immune-related genes, oxidative phosphorylation, adipogenesis, and E2M transition genes were significantly downregulated when compared between two groups, which correlates to differential gene ontologies noted in high Myc human tumors. The total immune cell population, specifically the myeloid and macrophage populations were significantly diminished in the Myc-in tumor models, which could contribute to the poor prognosis in OS.Our murine model closely resembles human OS and will provide new opportunities for dissecting the molecular pathogenesis, identification of novel therapeutic strategies, and pre-clinical modeling for direct translational applications and targeting of Myc-driven OS. Citation Format: Bikesh K. Nirala, Lyazat Kurenbekova, Ryan L. Shuck, Tajhal Patel, Kimal Rajapakshe, Jason T. Yustein. Development and characterization of a c-Myc-driven preclinical mouse model of osteosarcoma to investigate the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1668.

Background: Osteosarcoma (OS) is the most common bone malignancy in childhood and adolescence. Though the prognosis of OS patients has been improved by multimodal therapies, patients with metastatic disease still have a very poor prognosis. We previously reported a novel TBL1/β-catenin inhibitor, Tegavivint, suppresses OS primary tumor and lung metastasis using patient-derived cell lines. Wnt/β-catenin signaling is reported to influence intratumoral T cell infiltration and using murine syngeneic OS models we investigated the effects of Tegavivint on improving immune checkpoint inhibition and the OS tumor immune microenvironment. Methods: We used murine OS cell lines F331 and F420 for in vitro and in vivo studies. We performed cytotoxicity studies using the CCK-8 assay and western blots to investigate downstream β-catenin signaling perturbations secondary to Tegavivint. Intratibial injections were performed into C57BL/6 mice using both murine cell lines. Tegavivint and anti-PD-1 antibody was administered as monotherapy or in combination. After treatment, we compared the tumor volume in the control, monotherapy (Tegavivint or PD-1 only) and combination therapy groups (Tegavivint and PD-1). We assessed T cell tumor infiltration using immunohistochemistry and flow cytometry. Results: In vitro, Tegavivint suppresses the growth of F331 and F420 cell lines with IC50s of 9.52nM and 49.4nM, respectively after 72hours of treatment. In vivo, anti-PD1 monotherapy did not show any significant anti-tumor activity, but Tegavivint monotherapy significantly suppressed the growth of murine tumors (p<0.05) and the combination therapy further improved overall anti-tumor activity. IHC staining showed an increase in CD8-positive T cells in Tegavivint monotherapy and combination therapy treated tumors compared with the control tumors. Furthermore, flow cytometry validated the increase in CD8-positive T cell infiltration and also showed an increase in intratumoral NK cells in Tegavivint treated tumors. Conclusions: These results indicate that TBL1 inhibition, via Tegavivint, inhibits nuclear β-catenin activity and alters the tumor immune microenvironment and enhances immune checkpoint blockade in OS. Future directions include more comprehensive profiling and molecular analysis of the tumor and immune microenvironment, and evaluation of additional immune modulatory approaches for the treatment of OS. Citation Format: Kengo Nakahata, Bikesh K. Nirala, Ryan L. Shuck, Lyazat Kurenbekova, Jason T. Yustein. Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6046.