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Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using Eu II -based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and Eu II . Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated Eu II/III -containing probes. We studied three different Eu II/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time ( T 1 ) and 19 F signal of solutions containing different ratios of Eu II - and Eu III -containing complexes was plotted against the percentage of Eu II -containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.

Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft-tissue sarcoma. Multimodal treatment, including surgery and traditional chemotherapy with radiotherapy, has contributed to improvements in overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here, we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models in vitro and in vivo resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small-molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant antitumor and antimetastatic potential in several primary and metastatic in vivo models, including a relapsed RMS patient-derived xenograft model. Transcriptomic analysis of PAK4-targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of antitumor immune response signatures. This PAK4-targeting gene signature showed prognostic significance for patients with sarcoma. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS.Significance:These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced rhabdomyosarcoma.

Detail description of some of the methods mentioned in main text.

Contains supplementary figures and tables with corresponding legends. Figure S1: PAK expression and signaling in RMS. Figure S2: Generation of PAK4 knock down stable clones and phenotypic effects on tumorigenicity. Figure S3: Knockdown of PAK4 inhibits tumor growth in vivo. Figure S4: Phenotypic effects of small molecule inhibitors of PAK4. Figure S5: Pharmacological inhibition of PAK4 in vitro attenuating invasion and migration of cells. Figure S6: Target validation and body weight measurement upon treatment. Figure S7: Molecular analysis of PAK4 targeted proteome. Figure S8: Transcriptomic Analysis of KPT-9274 treated RMS tumors indicating immune modulation. Supplementary table 1: Sequence of primers. Supplementary table 2: Patient annotations.

Osteosarcoma (OS) is the most common bone tumor in pediatrics. After resection, allografts or metal endoprostheses reconstruct bone voids, and systemic chemotherapy is used to prevent recurrence. This urges the development of novel treatment options for the regeneration of bone after excision. We utilized a previously developed biomimetic, biodegradable magnesium-doped hydroxyapatite/type I collagen composite material (MHA/Coll) to promote bone regeneration in the presence of chemotherapy. We also performed experiments to determine if human mesenchymal stem cells (hMSCs) seeded on MHA/Coll scaffold migrate less toward OS cells, suggesting that hMSCs will not contribute to tumor growth and therefore the potential of oncologic safety in vitro. Also, hMSCs seeded on MHA/Coll had increased expression of osteogenic genes ( BGLAP, SPP1, ALP) compared to hMSCs in the 2D condition, even when exposed to chemotherapeutics. This is the first study to demonstrate that a highly osteogenic scaffold can potentially be oncologically safe because hMSCs on MHA/Coll tend to differentiate and lose the ability to migrate toward tumor cells. Therefore, hMSCs on MHA/Coll could potentially be utilized for bone regeneration after OS excision.

Ponatinib (Pon) is a multi-tyrosine kinase inhibitor that demonstrated high efficiency for treating cancer. However, severe side effects caused by Pon off-targeting effects prevent its extensive use. Using our understanding into the mechanisms by which Pon is transported by bovine serum albumin in the blood, we have successfully encapsulated Pon into a biomimetic nanoparticle (NP). This lipid NP (i.e., "leukosomes") incorporates membrane proteins purified from activated leukocytes that enable immune evasion, and enhanced targeting of inflamed endothelium NPs have been characterized for their size, charge, and encapsulation efficiency. Membrane proteins enriched on the NP surface enabled modulation of Pon release. These NP formulations showed promising dose-response results on two different murine osteosarcoma cell lines, F420 and RF379. Our results indicate that our fabrication method is reproducible, nonuser-dependent, efficient in loading Pon, and applicable toward repurposing numerous therapeutic agents previously shelved due to toxicity profiles.

Non-small cell lung cancer (NSCLC) comprises the majority (~85%) of all lung tumors, with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being the most frequently diagnosed histological subtypes. Multi-modal omics profiling has been carried out in NSCLC, but no studies have yet reported a unique metabolite-related gene signature and altered metabolic pathways associated with LUAD and LUSC.We integrated transcriptomics and metabolomics to analyze 30 human lung tumors and adjacent noncancerous tissues. Differential co-expression was used to identify modules of metabolites that were altered between normal and tumor.We identified unique metabolite-related gene signatures specific for LUAD and LUSC and key pathways aberrantly regulated at both transcriptional and metabolic levels. Differential co-expression analysis revealed that loss of coherence between metabolites in tumors is a major characteristic in both LUAD and LUSC. We identified one metabolic onco-module gained in LUAD, characterized by nine metabolites and 57 metabolic genes. Multi-omics integrative analysis revealed a 28 metabolic gene signature associated with poor survival in LUAD, with six metabolite-related genes as individual prognostic markers.We demonstrated the clinical utility of this integrated metabolic gene signature in LUAD by using it to guide repurposing of AZD-6482, a PI3Kβ inhibitor which significantly inhibited three genes from the 28-gene signature. Overall, we have integrated metabolomics and transcriptomics analyses to show that LUAD and LUSC have distinct profiles, inferred gene signatures with prognostic value for patient survival, and identified therapeutic targets and repurposed drugs for potential use in NSCLC treatment.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC–derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a–regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

Chimeric antigen receptor (CAR) T-cell therapy offers promise to improve outcomes for pediatric patients with recurrent/refractory osteosarcoma (OS), a ‘poor-prognosis’ cancer. However, early-phase clinical studies have shown limited activity despite potent antitumor activity in preclinical xenograft models, highlighting the need to develop better preclinical models. B7-H3 has emerged as a promising immunotherapeutic target for pediatric solid tumors including OS, and we and other investigators are actively exploring B7-H3-CAR T cell therapies in early-phase clinical studies. Our goal was to develop a murine immunocompetent OS model (F331) for the realistic pre-clinical evaluation of B7-H3-CAR T cells and to explore additional genetic modifications to improve their effector function. We generated murine B7-H3-CAR T cells expressing a 2nd generation B7-H3-CD28.ζ CAR by retroviral transduction. In vitro, B7-H3-CAR T cells recognized B7-H3-positive F331 cells as judged by IFNγ production and cytolytic activity in contrast to B7-H3-negative tumor cells; Control (ctrl) CAR T-cells recognizing an irrelevant antigen (SP6) did not recognize or kill B7-H3-positive tumor cells confirming specificity. Since lung is the primary OS metastasis site, we focused on the intravenous (i.v.) disseminated lung F331 OS metastasis model. In this model, a single i.v. infusion of 5x106 CD8+ B7-H3-CAR T cells demonstrated significant antitumor activity compared to CD8+ ctrl CAR T cells, resulting in a significant survival advantage (median: 70 vs 38 days respectively). Unfortunately, tumors invariably recurred. We found limited B7-H3-CAR T cell expansion and persistence, even though recurring tumors still expressed B7-H3, excluding tumor antigen escape mechanism. We therefore embarked on a comprehensive screen to knock out (KO) known negative regulators of T-cell function, starting with Regnase-1 (Reg-1). Reg-1 is known to have RNase activity and to regulate activation of immune cells. KO of Reg-1 in CD8+ B7-H3-CAR T cells improved their expansion post-infusion in spleen and lungs as judged by flow cytometric analysis without systemic toxicities based on mice body weight assessment. This resulted in a significant improvement in overall survival in comparison to control KO B7-H3-CAR T cells at a cell dose (1x106) at which ctrl-KO CD8+ B7-H3-CAR T cells were ineffective. Reg1-KO ctrl CAR T cells had no therapeutic benefit, excluding nonspecific effects. In summary, we have established an immune-competent OS model to evaluate the effector function of B7-H3-CAR T cells and have demonstrated the advantage given by 2nd genetic modifications to enhance their antitumor activity. We are currently using this model to define mechanisms of immune resistance with the goal of further enhancing OS-redirected CAR T-cell therapy. Citation Format: Adeleye O. Adeshakin, Peipei Zhou, Jean-Yves Métais, Phuong Nguyen, Scott Perry, Heather Sheppard, Xiang Sun, Trevor Cunningham, Hao Shi, Jessica Wagner, Jason T. Yustein, Christopher DeRenzo, Giedre Krenciute, Hongbo Chi, Stephen Gottschalk. B7-H3-CAR T-cell therapy in immune-competent osteosarcoma models: Regnase-1 KO overcomes limited CAR T-cell expansion [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 1778.

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 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.

Simple Summary Osteosarcoma is the most common cancer of the bone for children and adolescents. Although chemotherapy regimens have been widely available and effective for many patients for decades, there have been few reliable options available when these standard regimens fail to adequately cure the disease. This suggests internal resistance mechanisms that allow the tumor cells to survive. Here, we review many of the recent studies that have provided new insights into understanding and overcoming chemo- and radioresistance in osteosarcoma. Abstract Osteosarcoma, the most common bone malignancy of childhood, has been a challenge to treat and cure. Standard chemotherapy regimens work well for many patients, but there remain minimal options for patients with progressive or resistant disease, as clinical trials over recent decades have failed to significantly improve survival. A better understanding of therapy resistance is necessary to improve current treatments and design new strategies for future treatment options. In this review, we discuss known mechanisms and recent scientific advancements regarding osteosarcoma and its patterns of resistance against chemotherapy, radiation, and other newly-introduced therapeutics.

The identification of mechanisms to promote memory T cells (T(MEM)) has important implications for vaccination and anti-cancer immunotherapy(1–4). Using a CRISPR-based screen for negative regulators of T(MEM) generation in vivo(5), we discovered multiple components of the mammalian canonical Brg1/Brg-associated factor (cBAF)(6,7). Several components of the cBAF complex are essential for the differentiation of activated CD8(+) T cells into T effector (T(EFF)) cells, and their loss promotes T(MEM) formation in vivo. During the first division of activated CD8(+) T cells, cBAF and c-Myc(8) frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high c-Myc and high cBAF display a cell fate trajectory towards T(EFF) cells, while those with low c-Myc and low cBAF preferentially differentiate towards T(MEM) cells. The cBAF complex and c-Myc physically interact to establish the chromatin landscape in activated CD8(+) T cells. Treatment of naïve CD8(+) T cells with a putative cBAF inhibitor during the first 48 hours of activation, prior to the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a solid tumor model. Our results establish cBAF as a negative determinant of T(MEM) cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.

Osteosarcoma (OS) is the most common primary pediatric malignancy of the bone having poor prognosis and long-term survival rates of less than 30% in patients with metastasis. MicroRNA-509 was reported to be downregulated in OS. We and others previously published that miR-509-3p can strongly attenuate cellular migration/invasion and sensitize ovarian cancer to cisplatin. Here, we show that overexpression of miR-509-3p inhibited migration of primary OS cell lines U2OS, HOS, and SaOS2 as well as metastatic derivatives 143B and LM7. miR-509-3p overexpression also inhibited proliferation and invasion of HOS and 143B cells and sensitized cells to cisplatin. Luciferase reporter assays using 3′-UTRs of predicted miR-509-3p targets associated with metastatic phenotypes revealed ARHGAP1 could be one of the downstream effectors of miR-509-3p in HOS. To find the global impact of miR-509-3p overexpression and cisplatin treatment we performed Reverse Phase Protein Analysis (RPPA). AXL, which has been reported to play a critical role in cisplatin resistance and confirmed as direct target of miR-509-3p was downregulated upon miR-509-3p treatment and further down-regulated upon miR-509-3p + cisplatin treatment. We propose that the miR-509-3p/AXL and miR-509-3p/ARHGAP1 axes have the potential to uncover new druggable targets for the treatment of drug resistant metastatic osteosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of about 65-70%. Thus, additional molecular insights and representative models are critical for further identifying and evaluating new treatment modalities. Using MyoD-Cre mediated introduction of mutant K-RasG12D and perturbations in p53 we have developed a novel genetically engineered mouse model (GEMM) for RMS. Specifically, we directly crossed mice expressing MyoD promoter-regulated Cre-recombinase with germline p53Flox or Lox-Stop-Lox (LSL) knock-in alleles expressing oncogenic p53R172H and/or K-RasG12D mutants. The anatomic sites of primary RMS development observed in these mice recapitulated human disease, with the most frequent sites of tumor growth seen in the head, neck, extremities, and abdomen.We have confirmed RMS histology and diagnosis through hematoxylin and eosin (H&E) staining, as well as positive immunohistochemistry (IHC) staining for desmin, myogenin, and phosphotungstic acid hematoxylin (PTAH). We established cell lines from several of the GEMM tumors with the ability to engraft and develop tumors in immunocompetent mice with similar histological and staining features as the primary tumors. Furthermore, injection of syngeneic RMS lines via tail vein had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Specifically, we noted alterations in gene ontologies including immune response, metabolism and mRNA processing. Finally, pre-clinical use of these murine RMS lines demonstrated similar therapeutic responsiveness to relevant chemotherapy and targeted therapies as human cell line models.Summary StatementWe have developed a new conditional genetically engineered mouse model of rhabdomyosarcoma (RMS) with homologous molecular signature to human RMS that provides valuable pre-clinical models for evaluating novel therapies.

Simple Summary Osteosarcoma (OS) is the most common bone tumor in the pediatric population, and long-term survival occurs in less than a third of the population with metastatic or recurrent tumors. Secreted frizzled-related protein 2 (SFRP2) promotes metastatic OS cell migration and tumor angiogenesis. This study aimed to assess the role of antagonizing SFRP2 with a humanized monoclonal antibody to SFRP2 (hSFRP2 mAb) in OS metastases in vivo and the role of SFRP2 in T-cells. Our results demonstrate that hSFRP2 mAb treatment inhibits metastases in two metastatic models of OS and can overcome resistance to a PD-1 monoclonal antibody. hSFRP2 mAb treatment restores T-cell proliferation and, in T-cells, inhibits NFATc3, CD38 and PD-1 expression. We conclude that SFRP2-targeted immunotherapy reduces the growth of metastatic osteosarcoma, not only through a direct antitumor and antiangiogenic effect but also by impacting the immune system. Abstract Secreted frizzled-related protein 2 (SFRP2) promotes the migration/invasion of metastatic osteosarcoma (OS) cells and tube formation by endothelial cells. However, its function on T-cells is unknown. We hypothesized that blocking SFRP2 with a humanized monoclonal antibody (hSFRP2 mAb) can restore immunity by reducing CD38 and PD-1 levels, ultimately overcoming resistance to PD-1 inhibitors. Treating two metastatic murine OS cell lines in vivo, RF420 and RF577, with hSFRP2 mAb alone led to a significant reduction in the number of lung metastases, compared to IgG1 control treatment. While PD-1 mAb alone had minimal effect, hSFRP2 mAb combination with PD-1 mAb had an additive antimetastatic effect. This effect was accompanied by lower SFRP2 levels in serum, lower CD38 levels in tumor-infiltrating lymphocytes and T-cells, and lower PD-1 levels in T-cells. In vitro data confirmed that SFRP2 promotes NFATc3, CD38 and PD-1 expression in T-cells, while hSFRP2 mAb treatment counteracts these effects and increases NAD+ levels. hSFRP2 mAb treatment further rescued the suppression of T-cell proliferation by tumor cells in a co-culture model. Finally, hSFRP2 mAb induced apoptosis in RF420 and RF577 OS cells but not in T-cells. Thus, hSFRP2 mAb therapy could potentially overcome PD-1 inhibitor resistance in metastatic osteosarcoma.

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8(+) T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8(+) T cells induced HIF-1α/HIF-2α–dependent differentiation of tissue-resident memory–like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8(+) TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of ∼65-70%. Thus, additional molecular insights and representative models are critical for identifying and evaluating new treatment modalities. Using MyoD-Cre-mediated introduction of mutant K-RasG12D and perturbations in p53, we developed a novel genetically engineered mouse model (GEMM) for RMS. The anatomic sites of primary RMS development recapitulated human disease, including tumors in the head, neck, extremities and abdomen. We confirmed RMS histology and diagnosis through Hematoxylin and Eosin staining, and positive immunohistochemical staining for desmin, myogenin, and phosphotungstic acid-Hematoxylin. Cell lines from GEMM tumors were established with the ability to engraft in immunocompetent mice with comparable histological and staining features as the primary tumors. Tail vein injection of cell lines had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Finally, pre-clinical use of these murine RMS lines showed similar therapeutic responsiveness to chemotherapy and targeted therapies as human RMS cell lines. ispartof: DISEASE MODELS & MECHANISMS vol:15 issue:2 ispartof: location:England status: published

The introduction of targeted anticancer drugs has revolutionized treatment. Multi-tyrosine kinase inhibitors (TKIs) are potential therapeutics targeting specific signaling pathways that contribute to cancer progression, including sarcomas. Osteosarcoma is the most common pediatric tumor with a worldwide incidence of 3.4 cases/million annually but without effective treatment. Of the TKIs, Ponatinib demonstrated potent anti-tumor activity; however, it received an FDA black box warning for potential side effects. New treatment and delivery systems must be identified to use Ponatinib clinically. Our laboratory developed novel biomimetic nanoparticles (NPs) synthesized from activated leukocytes called Leukosomes. Leukosomes maintain leukocyte tropism towards inflamed endothelium and can encapsulate and effectively release Ponatinib. We aimed to validate Leukosome technology’s therapeutic potential to specifically target and inhibit primary murine osteosarcoma growth and reduce detrimental side effects. In a 3D sarcosphere model, we observed effective penetration and internalization of NPs in both murine (RF379, 577) and human patient derived xenograft (PDX94, PDX202) osteosarcoma cell lines. Leukosome exhibited 20% increased targeting versus control liposomes. Cell viability decreased 20% after treatment with Ponatinib IC50. Leukosome-Ponatinib IC50 also induced complete inhibition of murine sarcosphere formation and ~60-80% reduction of cell viability. We developed an osteosarcoma orthotopic mouse model by intratibial injection of murine F420 osteosarcoma cells. After 3 weeks, tumor was detected and the mice were injected with Ponatinib NPs. Leukosome showed increased tumor targeting and penetration 1 and 3 hours post-NP injection. Intravenous tail injection of Ponatinib was lethal due to drug-related toxic effects, while intraperitoneal injection of Ponatinib was well tolerated. Conversely, intravenous injection of Ponatinib-loaded NPs did not show toxic effects. In vivo efficacy studies demonstrated that 3 weeks of Ponatinib treatment (2 treatments/week) inhibited tumor growth and increased survival. Similar results were also observed after Leukosome Ponatinib treatment, despite 10 times less Ponatinib in NPs than in free drug. Overall, these results show that leukocyte-derived membrane proteins enhance the accumulation of Ponatinib at the tumor site and surrounding inflamed tissue. While limited Ponatinib encapsulation in NPs remains an open challenge, this formulation underlies the possibility of reducing drug dosage and side effects. Thus, suggest Leukosome’s translational potential as a new targeted drug delivery approach with better outcomes and fewer complications for osteosarcoma patients. Citation Format: Federica Giordano, Stefania Lenna, Riccardo Rampado, Gherardo Baudo, Matteo Massaro, Ashley Rivera, Enrica De Rosa, Jason T. Yustein, Francesca Taraballi. Ponatinib loaded leukocyte-based nanoparticles: A new platform for treating 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 307.

Introduction: Ewing Sarcoma (EwS) is the second most common bone tumor in children. At diagnosis, approximately 20-25% of patients with EwS have metastatic disease. While systemic chemotherapy, along with surgery and/or radiotherapy, has significantly increased the survival rate of patients with localized disease, metastatic Ewing sarcoma (EwS) has an extremely poor overall survival with a 5-year survival rate of approximately 20-30%. This prognosis has not changed in the last several decades. Thus, there is an imperative need to focus particularly on mechanisms of metastatic growth and progression and to identify and target the underlying intrinsic and extrinsic molecular features. Unfortunately, presently there is no reliable engineered mouse model to functionally study EwS development and metastasis. Using two independent EwS cell lines, we have developed highly aggressive, lung-specific metastatic models that show mechanistic overlap with human patient tumor data and are valuable resources to further study and target metastatic EwS. Approach: Using a serial transplantation approach, we generated lung specific metastatic lines by repeated intra-tibial injection of two commonly used EwS cell lines (A673 and TC71), allowing metastatic foci to form, followed by dissociation and subsequent re-injection into the tibia. We repeated this 5 times to generate highly aggressive lung-specific metastatic cell lines. As a proof of principle, when equal numbers of GFP+ parental cells and mCherry+ lung metastatic cells are injected into the tibia in NSG mice they form primary tumors with red and green cells, while the metastatic lesions in lungs are almost exclusively mCherry+. A multi-omics approach including glycoproteomics, secretome, and transcriptomic analysis were then used to characterize these cells and compare them with patient tumor samples. Results: Integration of focused proteomics and glycoproteomics data revealed upregulation in AXL, MEK6, STAT1, and STAT3 proteins and phosphoproteins in metastatic lesions. Focused RNA profiling with Nanostring technologies using formalin-fixed paraffin-embedded (FFPE) slides of patient tumor samples revealed increased expression of several other upstream genes that signal through STAT1, including IL-6, IKK-β, TRAF-6, and Myd88. In addition, upregulation in factors involved in tumor invasive front such as Furin, TIMP-2, MMP-2, and MMP-14 were also noted. We are currently in the process of further validating some of the key regulators using larger set of patient derived tumors and by targeting them in orthotopic mouse models. Citation Format: Atreyi Dasgupta, Lyazat Kurenbekova, Tajhal Patel, Jason T. Yustein. Development and characterization of Ewing Sarcoma lung metastasis model [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 6324.

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 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.

Osteosarcoma (OS) is the most prevalent bone tumor in pediatric patients. Regimens of neoadjuvant chemotherapy have improved survival of OS patients greatly, however the 5-year survival rate for localized OS is 75% with a 30-50% recurrence rate. We sought to identify genes which could predict chemo-response and survival in localized OS. The TARGET OS RNA-seq dataset was utilized to identify genes and pathways associated with localized patient relapse and survival. We identified 478 differentially expressed genes with a 1.5 FC and FDR < 0.05 common to overall survival and relapse We further performed string analysis to generate a protein-protein interaction network followed by hub analysis with Cytohubba using betweenness centrality and radiality measures. Combining the top 10 hub genes from these two methods resulted in a total of 13 genes: MYOM2, VEGFA, VCAM1, EGFR, MUC1, IHH, GLI1, GPC3, IGF2, GRIA1, GNG12, GNGT1 and C3. These 13 genes were used to stratify localized patients in the TARGET dataset into high-risk and low-risk tertiles. The low-risk group had 100% overall survival while the high-risk group had 44% 5-year survival (p=2e-4). We also found a significant correlation between the 13 genes and time to death in localized patients (p=0.04). Additionally, there was a significant difference in expression of the 13 genes between alive and deceased patients (p=2e-5) and patients who relapsed (p=1.5e-4). Overall, these data suggest that these 13 genes could predict relapse and overall survival in OS patients with localized disease in the TARGET cohort. We performed Weighted Gene Correlation Network Analysis (WGCNA) on the 478 overlapping genes and identified five modules, with our 13 genes split across these modules. All modules were also significantly correlated with vital status suggesting that the genes in our signature represent distinct sub-groups with possibly separate mechanisms. Over-representation analysis was performed for each module and while each module did have distinct pathways, there were 65 pathways which overlapped between 3 of the modules. Of particular interest was Hedgehog signaling, with 2 of our 13 genes, IHH and GLI1, key to Hedgehog signaling, and a Hedgehog pathway inhibitor, Gant-58, scored high in reversing the 478 gene signature as determined using Connectivity Map (Broad Institute). We tested Gant-58 against two PDX OS models. Gant-58 did not inhibit a non-relapsed, chemo sensitive localized PDX-derived cell line, but showed potent activity towards a recurrent localized PDX with elevated IHH and GLI1 expression (p Citation Format: Tajhal D. Patel, Kshiti Dholakia, Tanmay R. Gandhi, Rupa S. Kanchi, Sandra L. Grimm, Chenlian Fu, Jason T. Yustein, Cristian Coarfa. Identification of a 13 gene signature to predict survival in localized 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 6047.

Background: Recapitulation of the full spectrum of genomic changes driving patient tumors have resulted in increased use of patient-derived xenograft (PDX) models in studies of basic cancer biology and preclinical drug development. Given the translational potential of PDXs and limited availability of pediatric cancer models, we established a PDX program to expand the existing collection of pediatric PDXs in the community and enable pre- and post-clinical studies. Methods: PDX generation requests were integrated into clinical workflows to maximize identification of eligible patients for informed consent and tissue collection at Memorial Sloan Kettering Cancer Center. Methodologies for tissue procurement and cryopreservation were optimized to facilitate implantation into host immunodeficient mice and enable multi-institutional tissue exchange for model building. A bioinformatics pipeline was established to allow molecular validation of engrafted PDXs using a next-generation targeted gene panel (MSK-IMPACT) evaluating concordance based on acquired mutations, copy number alterations and clonal structure. Results: Between November 2016 - October 2021, 379 PDX models were developed (265 distinct models) representing 69 discrete diagnoses. Sarcoma represents the most common model type (50 discrete osteosarcoma, 20 desmoplastic small round cell tumor, 14 Ewing sarcoma, 24 rhabdomyosarcoma, 2 CIC/DUX4 and 2 BCOR-rearranged sarcoma) followed by neuroblastoma (n=35), leukemia (n=44), and Wilms tumor (n=15). While the majority of PDXs were established from recurrent or metastatic tissue, 7 paired diagnostic/pre-therapy and post-therapy or relapse models were generated. Genomic characterization of PDXs demonstrate excellent concordance and recapitulation of single nucleotide variants (90%), structural (88%) and copy number variants (94%) between patient tumor and matched PDX. Discrepancies between matched patient/PDX pairs are due to sub-clonal heterogeneity in source tumors with clonal outgrowth in the PDX. Analysis of serial PDX passages also demonstrate stable recapitulation of the genomic profile. Establishment of a diverse PDX collection allowed preclinical evaluation of 10 targeted agents across a spectrum of pediatric tumors and provided the preclinical rationale for 3 investigator-initiated pediatric clinical trials. Conclusions: Investment in the development of a phenotypically diverse and biologically faithful collection of pediatric PDX models enables the goals of precision medicine. Optimization of PDX workflows and methods has also enabled the development of a pediatric PDX consortium (PROXC - Pediatric Research in Oncology Xenografting Consortium) to further support the development of pre- and post-clinical studies for pediatric cancer. Citation Format: Filemon S. Dela Cruz, Joseph G. McCarter, Daoqi You, Nancy Bouvier, Xinyi Wang, Kristina C. Guillan, Armaan H. Siddiquee, Katie B. Souto, Hongyan Li, Teng Gao, Dominik Glodzik, Daniel Diolaiti, Neerav N. Shukla, Joachim Silber, Umeshkumar K. Bhanot, Faruk Erdem Kombak, Diego F. Coutinho, Shanita Li, Juan E. Arango Ossa, Juan S. Medina-Martinez, Michael V. Ortiz, Emily K. Slotkin, Michael D. Kinnaman, Sameer F. Sait, Tara J. O'Donohue, Marissa Mattar, Maximiliano Meneses, Michael P. LaQuaglia, Todd E. Heaton, Justin T. Gerstle, Nicola Fabbri, Chelsey M. Burke, Irene M. Rodriquez-Sanchez, Christine A. Iacobuzio-Donahue, Julia L. Glade Bender, Ryan D. Roberts, Jason T. Yustein, Nino C. Rainusso, Brian D. Crompton, Elizabeth Stewart, Alejandro Sweet-Cordero, Leanne C. Sayles, Andrika D. Thomas, Michael H. Roehrl, Elisa de Stanchina, Elli Papaemmanuil, Andrew L. Kung. Development of a patient-derived xenograft (PDX) modeling program to enable pediatric precision medicine [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 704.

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.

The complexes described here serve as contrast agents for magnetic resonance imaging thermometry. The complexes differentially enhance contrast between 275 and 325 K. The basis of the temperature response of the fluorinated contrast complex is the modulation of water exchange caused by trifluoromethyl groups that can be chemically controlled.

Oncolytic virotherapy has been tested in numerous early phase clinical studies. However, the antitumor activity of oncolytic viruses thus far has been limited. Numerous strategies are being explored to enhance their antitumor activity by activating the adaptive arm of the immune system. We reasoned that it might also be possible to engineer oncolytic viruses to redirect tumor-associated macrophages to tumor cells for therapeutic benefit. We engineered an oncolytic vaccinia virus (VV) to disrupt the CD47/SIRPα interaction by expressing a chimeric molecule that consists of the ectodomain of SIRPα and the Fc domain of IgG4 (SIRPα-Fc-VV). SIRPα-Fc-VV readily replicated in tumor cells and redirected M1 as well as M2 macrophages to tumor cells in vitro. In contrast, control VVs that either encoded YFP (YFP-VV) or SIRPα (SIRPα-VV) did not. In vivo, SIRPα-Fc-VV had greater antitumor activity than YFP-VV and SIRPα-VV in an immune competent osteosarcoma model resulting in a significant survival advantage. Pretreatment with cytoxan further augmented the antitumor activity of SIRPα-Fc-VV. Thus, arming oncolytic viruses with SIRPα-Fc may present a promising strategy to enhance their antitumor activity for the virotherapy of solid tumors.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, β-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-ɣ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.

Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft-tissue sarcoma. Multimodal treatment, including surgery and traditional chemotherapy with radiotherapy, has contributed to improvements in overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here, we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models in vitro and in vivo resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small-molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant antitumor and antimetastatic potential in several primary and metastatic in vivo models, including a relapsed RMS patient-derived xenograft model. Transcriptomic analysis of PAK4-targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of antitumor immune response signatures. This PAK4-targeting gene signature showed prognostic significance for patients with sarcoma. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS. Significance: These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced rhabdomyosarcoma.

Ewing Sarcoma (ES) is a highly aggressive bone tumor with peak incidence in the adolescent population. It has a high propensity to metastasize, which is associated with dismal survival rates of approximately 25%. To further understand mechanisms of metastasis we investigated microRNA regulatory networks in ES. Our studies focused on miR-130b due to our analysis that enhanced expression of this microRNA has clinical relevance in multiple sarcomas, including ES. Our studies provide insights into a novel positive feedback network involving the direct regulation of miR-130b and activation of downstream signaling events contributing toward sarcoma metastasis. Specifically, we demonstrated miR-130b induces proliferation, invasion, and migration in vitro and increased metastatic potential in vivo. Using microarray analysis of ES cells with differential miR-130b expression we identified alterations in downstream signaling cascades including activation of the CDC42 pathway. We identified ARHGAP1, which is a negative regulator of CDC42, as a novel, direct target of miR-130b. In turn, downstream activation of PAK1 activated the JNK and AP-1 cascades and downstream transcriptional targets including IL-8, MMP1 and CCND1. Furthermore, chromatin immunoprecipitation of endogenous AP-1 in ES cells demonstrated direct binding to an upstream consensus binding site within the miR-130b promoter. Finally, small molecule inhibition of PAK1 blocked miR-130b activation of JNK and downstream AP-1 target genes, including primary miR-130b transcripts, and miR-130b oncogenic properties, thus identifying PAK1 as a novel therapeutic target for ES. Taken together, our findings identify and characterize a novel, targetable miR-130b regulatory network that promotes ES metastasis.

Ewing sarcoma (EwS) is an aggressive pediatric tumor treated with intensive cytotoxic chemotherapies. Overall survival for metastatic or relapsed disease is only 20-30%. Metformin has long been an attractive therapeutic option for EwS, but hypoxia limits its efficacy. Through a systematic integration of drug combination screening, bioinformatics analyses, functional and in vivo studies, and correlation with clinical outcome, we identified another known drug, imatinib that could augment the in vivo anti-tumor capacity of metformin by attenuating tumor hypoxic response. This drug combination regimen widely suppressed multiple dominant mechanisms in EwS genesis, growth, and metastasis, including key EWS-FLI1 downstream targets that converge into the PI3K/AKT/mTOR signaling pathway. In addition, the combination significantly enhanced inhibition on tumor cell proliferation by standard EwS chemotherapy drugs, including cyclophosphamide and ifosfamide. This suggests a potential clinical benefit of the metformin/imatinib combination by allowing the reduction in dose intensity of standard chemotherapy without compromising survival outcome and represents a potential faster track application for EwS patients.

Non-protein coding RNAs have emerged as a regulator of cell signaling and cancer progression through regulation of cell proliferation, metastatic burden, and cancer stem cell capacity. A subtype of non-protein coding RNA is long non-protein coding RNA (lncRNA). Besides their aforementioned roles in cancer cell biology, dysregulation of lncRNAs contribute to resistance to therapeutic treatments. A couple of important therapeutic classes are chemotherapy and targeted/hormone therapies. This review highlights the variety of malignancies affected by lncRNA dysregulation and the underlying mechanism causing therapeutic resistance.

We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4 mg/kg intravenously every 3 days for 3 weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40 days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p

Introduction: Osteosarcoma is the most common bone tumor in children and adolescents. Many patients have disease progression through standard chemotherapy regimens of methotrexate, cisplatin, and doxorubicin. Presently, there are no salvage therapies that have added significant survival benefits, leaving minimal options for relapsed and resistant disease. Additionally, there are no clinically significant biomarkers for this disease that would predict treatment failures. N-acetylgalactosaminyltransferase 14 (GALNT14) is an enzyme that initiates O-linked glycosylation to outer membrane-bound and extracellular proteins. Using transcriptomic analyses, we identified GALNT14 overexpression correlated with poor tumor necrosis and survival outcomes. Therefore, we hypothesize that GALNT14 contributes to metastatic and chemoresistant phenotypes in pediatric osteosarcoma. Methods: Transcriptomic data from the Therapeutically Applicable Research for Generating Effective Treatments (TARGET) database and RNA-sequencing of institutional patient-derived xenografts (PDXs) revealed that overexpression of GALNT14 correlated to poor necrosis rates. Gene Set Enrichment Analysis (GSEA) also identified upregulation of glycosylation pathways. Aberrant expression of GALNT14 was confirmed using quantitative polymerase chain reaction (qPCR) and Western blot analyses in various osteosarcoma cell lines. Transient GALNT14 knockdown and overexpression were then analyzed for effects on proliferation, invasion, migration, and chemotherapy response in vitro. Kaplan-Meier curves demonstrating overall survival (OS) and event-free survival (EFS) were established using data from the TARGET database. Results: GALNT14 expression was higher in both commercial and PDX cell lines that were known to be more prone for chemoresistance and invasiveness. The highest quartile of patients based on GALNT14 overexpression had 40% OS and 25% EFS, compared to 80% (p=0.002) and 75% (p Summary: GALNT14 shows promise as a predictive marker of chemoresistance and metastasis for pediatric osteosarcoma. Future functional genomic studies through gain and loss-of-function assays will evaluate the role of GALNT14 in osteosarcoma chemoresistance and metastatic potential. Citation Format: Zachary D. Prudowsky, Tajhal Patel, Kimal Rajapakshe, Christian Coarfa, Ryan Shuck, Nino Rainusso, Jason Yustein. The role of GALNT14 in chemoresistant and metastatic osteosarcoma [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 405.

Osteosarcoma (OS) is the most common pediatric bone tumor with a worldwide incidence of 3.4 cases per million people annually. Unfortunately, current treatments are still not sufficient to eradicate OS, due to its ability to resist the upfront standard chemotherapy. Therefore, it is essential to identify and effectively deliver novel therapeutic regimens. Multi-tyrosine kinase inhibitors have been explored as new therapeutics for different sarcomas. Among many, Ponatinib (Pon) demonstrated very potent anti-tumor activity, however, it received a black box warning from the FDA due to significant cardiovascular side effects. Recently, our lab developed novel biomimetic nanoparticles (NPs) called Leukosomes (Leuko) capable of encapsulating and effectively releasing Pon. These NPs are synthesized from activated leukocytes, maintaining leukocytes' tropism towards inflamed endothelium. Leveraging on this technology, we aim to validate the therapeutic potential of Leuko in 3D OS tumor model (sarcospheres) and their ability to target primary murine OS model while concomitantly reducing the detrimental side effects. Given its biological relevance and ability to better recapitulate the tumor structure, a 3D tumor model was chosen to reproduce key properties, such as diffusion limitations and the cellular network of solid tumors that have significant impacts on cancer drug efficacy.In the sarcospheres model, we observed efficient penetration and internalization of NPs in both, murine (RF379, 577) and human PDX derived (PDX94, pPDX202) OS cell lines, where Leuko exhibited a 20% increase in targeting vs the control Liposome (Lipo). This difference was not detected in the control 2D model. Moreover, our data demonstrated a 2-fold increase in the Leuko cytotoxic effect in 3D compared to 2D systems. Murine OS cell viability was 20% lower in sarcospheres after treatment with the IC50 for Pon. In addition, NPs induced complete inhibition of murine sarcosphere formation in the extreme limiting dilution assay (ELDA). These findings were also confirmed using the PDX derived OS cell lines. Subsequently, an in vivo intratibial syngeneic orthotopic mouse model was utilized to determine the targeting and biodistribution of the NPs. Leuko showed an increased targeting and penetration in the tumor 1 and 3h post NPs injection as anticipated by the 3D in vitro studies. By exploiting the inflammatory conditions within the tumor, which increases the Leuko accumulation, these preliminary results advocate the translational potential of this innovative formulation as a new targeted drug delivery approach for OS. This promising platform could improve the clinical therapeutic approaches and lead to improved outcomes and reduced side effects for OS patients. Citation Format: Federica Giordano, Stefania Lenna, Riccardo Rampado, April Ewing, Gherardo Baudo, Matteo Massaro, Assaf Zinger, Enrica De Rosa, Jason T Yustein, Francesca Taraballi. Ponatinib loaded leukocyte-based nanoparticles for osteosarcoma treatment in sarcosphere tumor model [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 311.

Magnetic resonance imaging (MRI) using redox-active, EuII-containing complexes is one of the most promising techniques for noninvasively imaging hypoxia in vivo. In this technique, positive (T1-weighted) contrast enhancement persists in areas of relatively low oxidizing ability, such as hypoxic tissue. Herein, we describe a fluorinated, EuII-containing complex in which the redox-active metal is caged by intramolecular interactions. The position of the fluorine atoms enables temperature-responsive contrast enhancement in the reduced form of the contrast agent and detection of the oxidized contrast agent via MRI in vivo. Positive contrast is observed in 1H-MRI with Eu in the +2 oxidation state, and chemical exchange saturation transfer and 19F-MRI signal are observed with Eu in the +3 oxidation state. Contrast enhancement is controlled by the redox state of Eu, and modulated by the fluorous interactions that cage a bound water molecule reduce relaxivity in a temperature-dependent fashion. Together, these advancements constitute the first report of in vivo, redox-responsive imaging using 19F-MRI.