Sensitization of osteosarcoma to irradiation by targeting nuclear FGFR1.

Over the past 25 years, chemotherapy regimens for osteosarcoma have failed to improve the 65-70% long-term survival rate. Radiation therapy is generally ineffective except for palliative care. We here investigated whether osteosarcoma can be sensitized to radiation therapy targeting specific molecules in osteosarcoma. Large-scale RNA sequencing analysis in osteosarcoma tissues and cell lines revealed that FGFR1 is the most frequently expressed receptor tyrosine kinase in osteosarcoma. Nuclear FGFR1 (nFGFR1) was observed by subcellular localization assays. The functional studies using a FGFR1IIIb antibody or small molecule FGFR1 inhibitors showed that nFGFR1, but not membrane-bound FGFR1, induces G2 cell-cycle checkpoint adaptation, cell survival and polyploidy following irradiation in osteosarcoma cells. Further, the activation of nFGFR1 induces Histone H3 phosphorylation at Ser 10 and c-jun/c-fos expression to contribute cell survival rendering radiation resistance. Furthermore, an in vivo mouse study revealed that radiation resistance can be reversed by the inhibition of nFGFR1. Our findings provide insights into the potential role of nFGFR1 to radiation resistance. Thus, we propose nFGFR1 could be a potential therapeutic target or a biomarker to determine which patients might benefit from radiation therapy.

SMARCA4 biology in alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

Interleukin-4 Receptor Inhibition Targeting Metastasis Independent of Macrophages.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma occurring in children and carries a dismal prognosis when metastatic disease is detected. Our previous work has suggested the cytokine receptor IL4Rα may play a role in contributing to metastasis in the alveolar subtype of rhabdomyosarcoma (aRMS), and thus could present a therapeutic target. The IL4 signaling axis has been characterized in various adult cancers as well; however, pediatric trials often follow similar adult trials and the role of the IL4Rα receptor has not been explored in the context of a mediator of metastasis in adult disease. Here, we demonstrate that the impact of IL4Rα blockade in an orthotopic allograft model of aRMS is not mediated by a macrophage response. We further examine the effect of IL4 blockade in adult colon, breast, and prostate cancers and find that inhibition of IL4Rα signaling modulates in vitro cell viability of HCT-116 colon carcinoma cells; however, this finding did not translate to an autocrine-related in vivo difference in tumor burden or lung metastasis. Our results suggest that if humanized IL4 mouse host strains are not available (or not ideal due to the need for immunosuppressing the host innate immune response for xenograft systems), then genetically-engineered mice and mouse allograft studies may be the best indicator of therapeutic targeting efficacy.

N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection.

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

Defining the Extracellular Matrix of Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades-underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

In vitro benchmarking of NF-κB inhibitors.

Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds.

Preclinical testing of the glycogen synthase kinase-3ß inhibitor tideglusib for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3ß is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3ß inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo. In this study, we tested the irreversible GSK3ß-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3ß inhibitors may not be a viable treatment for aRMS or eRMS.

NFκB signaling in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (aRMS) is a pediatric soft tissue cancer commonly associated with a chromosomal translocation that leads to the expression of a Pax3:Foxo1 or Pax7:Foxo1 fusion protein, the developmental underpinnings of which may give clues to its therapeutic approaches. In aRMS, the NFκB-YY1-miR-29 regulatory circuit is dysregulated, resulting in repression of miR-29 and loss of the associated tumor suppressor activity. To further elucidate the role of NFκB in aRMS, we first tested 55 unique sarcoma cell lines and primary cell cultures in a large-scale chemical screen targeting diverse molecular pathways. We found that pharmacological inhibition of NFκB activity resulted in decreased cell proliferation of many of the aRMS tumor cultures. Surprisingly, mice that were orthotopically allografted with aRMS tumor cells exhibited no difference in tumor growth when administered an NFκB inhibitor, compared to control. Furthermore, inhibition of NFκB by genetically ablating its activating kinase inhibitor, IKKß, by conditional deletion in a mouse model harboring the Pax3:Foxo1 chimeric oncogene failed to abrogate spontaneous tumor growth. Genetically engineered mice with conditionally deleted IKKß exhibited a paradoxical decrease in tumor latency compared with those with active NFκB. However, using a synthetic-lethal approach, primary cell cultures derived from tumors with inactivated NFκB showed sensitivity to the BCL-2 inhibitor navitoclax. When used in combination with an NFκB inhibitor, navitoclax was synergistic in decreasing the growth of both human and IKKß wild-type mouse aRMS cells, indicating that inactivation of NFκB alone may not be sufficient for reducing tumor growth, but, when combined with another targeted therapeutic, may be clinically beneficial.

EphB4/EphrinB2 therapeutics in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma affecting children and is often diagnosed with concurrent metastases. Unfortunately, few effective therapies have been discovered that improve the long-term survival rate for children with metastatic disease. Here we determined effectiveness of targeting the receptor tyrosine kinase, EphB4, in both alveolar and embryonal RMS either directly through the inhibitory antibody, VasG3, or indirectly by blocking both forward and reverse signaling of EphB4 binding to EphrinB2, cognate ligand of EphB4. Clinically, EphB4 expression in eRMS was correlated with longer survival. Experimentally, inhibition of EphB4 with VasG3 in both aRMS and eRMS orthotopic xenograft and allograft models failed to alter tumor progression. Inhibition of EphB4 forward signaling using soluble EphB4 protein fused with murine serum albumin failed to affect eRMS model tumor progression, but did moderately slow progression in murine aRMS. We conclude that inhibition of EphB4 signaling with these agents is not a viable monotherapy for rhabdomyosarcoma.

Combined targeting of SET and tyrosine kinases provides an effective therapeutic approach in human T-cell acute lymphoblastic leukemia.

Recent evidence suggests that inhibition of protein phosphatase 2A (PP2A) tumor suppressor activity via the SET oncoprotein contributes to the pathogenesis of various cancers. Here we demonstrate that both SET and c-MYC expression are frequently elevated in T-ALL cell lines and primary samples compared to healthy T cells. Treatment of T-ALL cells with the SET antagonist OP449 restored the activity of PP2A and reduced SET interaction with the PP2A catalytic subunit, resulting in a decrease in cell viability and c-MYC expression in a dose-dependent manner. Since a tight balance between phosphatases and kinases is required for the growth of both normal and malignant cells, we sought to identify a kinase inhibitor that would synergize with SET antagonism. We tested various T-ALL cell lines against a small-molecule inhibitor screen of 66 compounds targeting two-thirds of the tyrosine kinome and found that combined treatment of T-ALL cells with dovitinib, an orally active multi-targeted small-molecule receptor tyrosine kinase inhibitor, and OP449 synergistically reduced the viability of all tested T-ALL cell lines. Mechanistically, combined treatment with OP449 and dovitinib decreased total and phospho c-MYC levels and reduced ERK1/2, AKT, and p70S6 kinase activity in both NOTCH-dependent and independent T-ALL cell lines. Overall, these results suggest that combined targeting of tyrosine kinases and activation of serine/threonine phosphatases may offer novel therapeutic strategies for the treatment of T-ALL.

The Stone Cold Truth: The Effect of Concrete Encasement on the Rate and Pattern of Soft Tissue Decomposition.

This study provides a descriptive analysis of taphonomic changes observed in the soft tissue of ten pigs (Sus scrofa) after being encased in Quickrete (®) concrete and excavated at monthly or bimonthly intervals over the course of 2 years. The best method of subject excavation was investigated. Rate and pattern of decomposition were compared to a nonencased control subject. Results demonstrate subjects interred in concrete decomposed significantly slower than the control subject (p < 0.01), the difference being observable after 1 month. After 1 year, the encased subject was in the early stage of decomposition with purging fluids and intact organs present, versus complete skeletonization of the control subject. Concrete subjects also display a unique decomposition pattern, exhibiting a chemically burned outer layer of skin and a common separation of the dermal and epidermal layers. Results suggest using traditional methods to estimate postmortem interval on concrete subjects may result in underestimation.