Cell Sarcoma Research Articles

Can a signature molecular-profile define disparate survival in BRAF-positive Gliosarcoma and identify novel targets for therapeutic intervention?

Gliosarcoma (GS) has a low incidence but is aggressively invasive, with poor-survival. Even though GS is recognized as a different subgroup from glioblastoma (GB), there is no molecular panel available to define its clinical outcome. The objective was to identify the molecular imprint of GS in terms of expression of human telomerase reverse transcriptase (hTERT), high mobility group A1 (HMGA-1), kinesin superfamily protein-14 (KIF-14), epidermal growth factor receptor (EGFR) markers with reference to disparate prognosis and identify plausible targets for intervention. We retrieved 9-GS samples from a cohort of 57-GB patients during a 36 months study period and compared them with 10 molecularly typed GB-samples and 15 controls. Conventional-immunohistochemistry (IHC) was used for histopathology of GS and immunofluorescence-IHC was performed for quantification of identified marker-panel. Statistical tools for non-parametric data were used for inferring results. GS was confirmed by reticulin-staining and positivity for glial fibrillary acidic protein, Vimentin, smooth muscle actin. Immune-reactivity for BRAF-V600Ewas present in both glial and sarcomatous cells and negative expression of isocitrate dehydrogenase, ATRX, TP53.Comparison between GS, GB, and control tissues showed that the expression of markers reached significance (P < 0.0001), without the influence of confounders. Significant correlation of EGFR was found with hTERT (r = 0.77), HMGA-1 (r = 0.72), KIF-14 (r = 0.82) suggesting that their combined analysis can define prognosis. To establish the diagnostic accuracy (threshold ≥80% specificity), AUC for EGFR was 0.78 (>3.95), KIF-14 0.97 (>7.45), hTERT 0.63 (>23.86), and HMGA-1 0.53 (>15.45). This is the first evidence-based investigation presenting differential expression of proliferation and stemness markers hTERT, HMGA-1, KIF-14 in-correlation with EGFR, indicating a plausible-association between survival and disease-progression in individual GS-cases. It can serve as a model for further studies in this glioma-subgroup and the designing of a target panel for personalized treatment.

CLEAR CELL HEPATOCELLULAR CARCINOMA, A CASE REPORT

Clear Cell Hepatocellular Carcinoma (CCHCC) represents 2.2 to 6.7% of all Hepatocellular Carcinomas (HCC), affects mostly women and is frequently associated with liver cirrhosis, viral infections (HBV, HCV), aflatoxins, hemochromatosis, oral contraceptives, obesity and type 2 diabetes mellitus. The most frequent manifestation is a solitary tumor with a pseudocapsule, which is more frequent than in other subtypes of HCC. Histologically, CCHCC can be observed as cells with an empty appearance with abundant cytoplasm, vacuolated and foamy due to the accumulation of glycogen and fat, constituting more than 50% of the total cells. Differential diagnosis with liver metastases can be difficult, so immunohistochemistry is an important diagnostic tool. 69-year-old female with a history of hepatitis C virus infection in 2018 receiving direct-acting antiviral treatment for 12 weeks with sustained viral response-12, Child Pugh B liver cirrhosis is documented. 2 years later, the follow-up ultrasound reports liver injury cystic and alpha-fetoprotein at 84.27 ng / ml, so a triphasic tomography was performed, observing liver lesion in segment VII of 35 × 27 × 31 mm suggestive of hepatocellular carcinoma with atypical characteristics, no tumors were reported in another abdominal site, as there was no conclusive radiological criterion for hepatocellular carcinoma, a liver lesion biopsy was performed with a histological report of moderately differentiated clear cell carcinoma and immunohistochemistry with Hepatocyte antigen positive, Carcino-embryonic antigen negative, internal Arginase 1 positive, Glypican 3 positive and Internal renal carcinoma antigen negative, concluding diagnosis of clear cells hepatocellular carcinoma T1B, N0, M0, therefore the patient was referred for transarterial chemoembolization of the lesion. The importance of the current report is to identify histopathological characteristics and establish the usefulness of Immunohistochemistry to make a differential diagnosis with other tumors that can metastasize to and be confused with a primary CCHCC of the liver. CHCC is a rare subtype of HCC with a more favorable prognosis than other forms of hepatocellular carcinoma, the histological differential diagnosis through immunohistochemistry should be performed with renal cell carcinoma, adrenal cortical carcinoma, clear cell sarcoma, angiolipomas, pulmonary and neuroendocrine clear cell variant, which can metastasize to the liver and be confused. The immunohistochemical study was decisive for the treatment and favorable prognosis of the patient. The authors declare that there is no conflict of interest.

CIC-DUX4 sarcoma: A case report and review of the literature

CIC-DUX4 sarcoma is highly aggressive and rapidly develops lethal metastatic disease and chemoresistance. Its histology is similar to that of Ewing sarcoma and other small round cell sarcomas. Correlation with clinical data, radiological findings, pathological results (including immunohistochemistry and fluorescence in situ hybridization), and/or molecular techniques is necessary. We present the case of a 44-year-old woman who was initially diagnosed as having high-grade undifferentiated round cell sarcoma confirmed to be a CIC-DUX sarcoma by next-generation sequencing.

Early Complications after Limb Salvage Procedures: Prospective Cross Sectional Study

ABSTRACT Introduction: Sarcomas are tumors that are mesenchymal in origin involving bone and soft tissues occurring in all age groups. Giant cell tumor, although benign, is locally aggressive and can also destroy the bone. Amputation was once considered primary treatment but now with advancements in diagnostic imaging modalities, surgical procedures and adjuvant therapies, limb salvage surgeries have become the preferred treatment modality. The primary objective of our study was to determine the incidence of early complications occurring within the first thirty days in patients who underwent limb salvage surgery at our hospital. The secondary objective was to assess the association of these complications with patient-related factors, surgical modality, tumor characteristics and neoadjuvant therapy. Methodology: All the patients with bone or soft tissue sarcoma and aggressive giant cell tumors of the extremities who underwent wide excision and limb salvage surgery from February 2020 to December 2021 were included in this study. Data were collected prospectively on follow-up visits in the clinic for one month. Data were compiled and analyzed using SPSS version 22. A p-value of &lt;0.05 was considered significant throughout the study. Results: A total of 60 patients were included in this study. The mean age of the patients was 39.90 years. Bone and soft tissue tumors were found in 35 and 25 patients respectively. A total of 13 patients experienced postoperative complications within 30 days of surgery. The association of these postoperative complications with gender distribution, prior comorbid conditions, type of tumor, anatomical location of the tumor and neoadjuvant therapy was found to be statistically insignificant. Conclusion: The most frequently occurring postoperative complications are wound-related and the incidence of these complications is independent of the proposed risk factors. Research registry number: Researchregistry4695. Highlights

Targeting CD99 Compromises the Oncogenic Effects of the Chimera EWS-FLI1 by Inducing Reexpression of Zyxin and Inhibition of GLI1 Activity.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

The Study on the Effect and Mechanism of Crocin against Human Ewing Sarcoma

Ewing sarcoma is the second most common primary malignant bone tumor in pediatrics after osteosarcoma, which mainly occurs in children and adolescents aged 5-20 y. Crocin is the main active ingredient extracted from Crocus sativus and has significant inhibitory effect on many cancer cells. Therefore, this study observed the effect of crocin on human Ewing sarcoma and its specific mechanism and provided molecular theoretical basis for clinical application. We cultured Human Ewing sarcoma cells lines SK- ES-1 and RD-ES in vitro and treated them with 0, 2.5, 5 and 10 mmol/l crocin solution. And we used cell counting kit-8 assay to detect cell viability and adopted flow cytometry to detect cell apoptosis and cell cycle changes, used western-blot to detect the expression of apoptotic pathway-related proteins. At the same time, we also combined vincristine and crocin to treat the cells, to detect cell viability and apoptosis, and to observe for synergistic effect. We established the xenografts model of Ewing’s sarcoma in nude mice and observe the inhibitory effect of crocin and vincristine on xenografts. Crocin could not only inhibit the growth of human Ewing sarcoma cells SK-ES-1 and RD-ES in a dose-dependent manner, but also inhibit the growth of xenografts in nude mice and increase cell apoptosis and block cell cycle progression. The results of mechanism analysis showed that crocin and vincristine significantly increased the degradation of poly adenosine diphosphate-ribose polymerase and caspase-3, and increased the levels of anti-apoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra-large and p21. And crocin could increase the effect of vincristine on proliferation inhibition and apoptosis increase of human Ewing sarcoma. Crocin could inhibit the growth of human Ewing sarcoma either alone or in combination with vincristine. This effect was mediated by increasing apoptosis-related pathways.

SPARC-mediated long-term retention of nab-paclitaxel in pediatric sarcomas

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein overexpressed by several cancers. Because SPARC shows high binding affinity to albumin, we reasoned that pediatric sarcoma xenografts expressing SPARC would show enhanced uptake and accumulation of nanoparticle albumin-bound (nab)-paclitaxel, a potent anticancer drug formulation. We first evaluated the expression of SPARC in patient-derived xenografts (PDXs) of Ewing sarcoma, rhabdomyosarcoma and osteosarcoma, finding variable SPARC gene expression that correlated well with SPARC protein measured by immunoblotting. We revealed that the activity of the fusion gene chimera EWSR1-FLI1, the genetic driver of Ewing sarcoma, leads to lower expression of the gene SPARC in these tumors, likely due to enriched acetylation marks of the histone H3 lysine 27 at regions including the SPARC promoter and potential enhancers. Then, we used SPARC-edited Ewing sarcoma cells (A673 line) to demonstrate that SPARC knocked down (KD) cells accumulated significantly less amount of nab-paclitaxel in vitro than SPARC wild type (WT) cells. In vivo, SPARC KD and SPARC WT subcutaneous xenografts in mice achieved similar maximum intratumoral concentrations of nab-paclitaxel, though drug clearance from SPARC WT tumors was significantly slower. We confirmed such SPARC-mediated long-term intratumoral accumulation of nab-paclitaxel in Ewing sarcoma PDX with high expression of SPARC, which accumulated significantly more nab-paclitaxel than SPARC-low PDX. SPARC-high PDX responded better to nab-paclitaxel than SPARC-low tumors, although these results should be taken cautiously, given that the PDXs were established from different patients that could have specific determinants predisposing response to paclitaxel. In addition, SPARC KD Ewing sarcoma xenografts responded better to soluble docetaxel and paclitaxel than to nab-paclitaxel, while SPARC WT ones showed similar response to soluble and albumin-carried drugs. Overall, our results show that pediatric sarcomas expressing SPARC accumulate nab-paclitaxel for longer periods of time, which could have clinical implications for chemotherapy efficacy.

Ovarian Carcinosarcoma with Yolk Sac Tumor in a Post-Menopausal Female: A Case Report and Review of Literature

Yolk sac tumor is the second most common subtype of ovarian germ cell tumors and is rare in postmenopausalwomen. The few cases in literature have found that in this age group, yolk sac tumors more commonly present as a mixed component, combined with epithelial tumors. We report a case of a 60-year-old female who presented with an enlarging abdominopelvic mass. Imaging pointed to an ovarian new growth. Total abdominal hysterectomy with bilateral salpingo-oophorectomy revealed a tumor with three populations composed of carcinomatous, sarcomatous, and germ cell components, which was ultimately diagnosed as an ovarian carcinosarcoma with concurrent yolk sac tumor based on histomorphology and immunohistochemical staining. This report also discusses the proposed pathogenesis, treatment, and prognosis of this uncommon entity.

FZD10-targeted α-radioimmunotherapy with 225 Ac-labeled OTSA101 achieves complete remission in a synovial sarcoma model.

Synovial sarcomas are rare tumors arising in adolescents and young adults. The prognosis for advanced disease is poor, with an overall survival of 12‐18 months. Frizzled homolog 10 (FZD10) is overexpressed in most synovial sarcomas, making it a promising therapeutic target. The results of a phase 1 trial of β‐radioimmunotherapy (RIT) with the 90Y‐labeled anti‐FZD10 antibody OTSA101 revealed a need for improved efficacy. The present study evaluated the potential of α‐RIT with OTSA101 labeled with the α‐emitter 225Ac. Competitive inhibition and cell binding assays showed that specific binding of 225Ac‐labeled OTSA101 to SYO‐1 synovial sarcoma cells was comparable to that of the imaging agent 111In‐labeled OTSA101. Biodistribution studies showed high uptake in SYO‐1 tumors and low uptake in normal organs, except for blood. Dosimetric studies showed that the biologically effective dose (BED) of 225Ac‐labeled OTSA101 for tumors was 7.8 Bd higher than that of 90Y‐labeled OTSA101. 90Y‐ and 225Ac‐labeled OTSA101 decreased tumor volume and prolonged survival. 225Ac‐labeled OTSA101 achieved a complete response in 60% of mice, and no recurrence was observed. 225Ac‐labeled OTSA101 induced a larger amount of necrosis and apoptosis than 90Y‐labeled OTSA101, although the cell proliferation decrease was comparable. The BED for normal organs and tissues was tolerable; no treatment‐related mortality or obvious toxicity, except for temporary body weight loss, was observed. 225Ac‐labeled OTSA101 provided a high BED for tumors and achieved a 60% complete response in the synovial sarcoma mouse model SYO‐1. RIT with 225Ac‐labeled OTSA101 is a promising therapeutic option for synovial sarcoma.

SHED-Dependent Oncogenic Signaling of the PEAK3 Pseudo-Kinase.

Simple SummaryThe human kinome is composed of about 50 pseudo-kinases with unclear function, because they are predicted to be catalytically inactive; however, they are shown to play an important role in cancer, similar to active kinases. Understanding how these pseudo-kinases promote tumor formation despite their catalytic inactivity is a great challenge, which may lead to innovative anti-cancer therapies. The PEAK1 and 2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism via a conserved split helical dimerization (SHED) module. In this study, we uncovered a similar SHED-dependent oncogenic activity for PEAK3, a recently discovered new member of this family. We also show that this new signaling mechanism may be implicated in acute myeloid leukemia.The PEAK1 and Pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signaling mechanism remains unclear. In this study, we found that, although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, the PEAK3 protein is strongly expressed in human hematopoietic cells and is upregulated in acute myeloid leukemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signaling, independent of growth-factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signaling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2, which promoted PEAK3 tyrosine phosphorylation, its association with GRB2 and ASAP1, and AKT signaling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signaling proteins to promote oncogenesis.

Combination of tetrapyridylporphyrins and arene ruthenium(II) complexes to treat synovial sarcoma by photodynamic therapy

Four tetrapyridylporphyrin and four dipyridylporphyrin arene ruthenium complexes have been synthesized and characterized. In these complexes, the porphyrin core is either metal-free or occupied by zinc, and the arene ligand of the arene ruthenium units are either the standard methyl-isopropyl-benzene ([Formula: see text]cymene) or the less common phenylpropanol (PhPrOH) derivative. The porphyrin derivatives are coordinated to four arene ruthenium units or only two, in accordance with the number of pyridyl substituents at the periphery of the porphyrins, 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (TPyP) and 5,15-diphenyl-10,20-di(pyridin-4-yl)porphyrin (DPhDPyP). All eight complexes were evaluated as anticancer agents on synovial sarcoma cells, in the presence and absence of light, suggesting that both the arene ligand and the porphyrin core substituent can play a crucial role in fine-tuning the photodynamic activity of such organometallic photosensitizers.

Enhancement of Radiosensitivity by DNA Hypomethylating Drugs through Apoptosis and Autophagy in Human Sarcoma Cells

The targeting of DNA methylation in cancer using DNA hypomethylating drugs has been well known to sensitize cancer cells to chemotherapy and immunotherapy by affecting multiple pathways. Herein, we investigated the combinational effects of DNA hypomethylating drugs and ionizing radiation (IR) in human sarcoma cell lines both in vitro and in vivo. Clonogenic assays were performed to determine the radiosensitizing properties of two DNA hypomethylating drugs on sarcoma cell lines we tested in this study with multiple doses of IR. We analyzed the effects of 5-aza-dC or SGI-110, as DNA hypomethylating drugs, in combination with IR in vitro on the proliferation, apoptosis, caspase-3/7 activity, migration/invasion, and Western blotting using apoptosis- or autophagy-related factors. To confirm the combined effect of DNA hypomethylating drugs and IR in our in vitro experiment, we generated the sarcoma cells in nude mouse xenograft models. Here, we found that the combination of DNA hypomethylating drugs and IR improved anticancer effects by inhibiting cell proliferation and by promoting synergistic cell death that is associated with both apoptosis and autophagy in vitro and in vivo. Our data demonstrated that the combination effects of DNA hypomethylating drugs with radiation exhibited greater cellular effects than the use of a single agent treatment, thus suggesting that the combination of DNA hypomethylating drugs and radiation may become a new radiotherapy to improve therapeutic efficacy for cancer treatment.

Altertoxin II, a Highly Effective and Specific Compound against Ewing Sarcoma.

Simple SummaryEwing sarcoma is a cancer of the bone and soft tissues that affects children and adolescents. Unfortunately, only 20–30% of patients with metastatic Ewing sarcoma survive, necessitating the need to identify new, more effective therapies. We screened natural product extracts from plants and fungal cultures to identify compounds with selective cytotoxic activity against Ewing sarcoma cells, which led to the identification of altertoxin II as a compound with highly selective activity against Ewing sarcoma cells. Mechanism of action studies showed that altertoxin II selectively induces DNA damage in Ewing sarcoma cells, but does not bind to DNA. Additionally, we found that altertoxin II has antitumor activity in a mouse model of Ewing sarcoma, suggesting it will be useful as a lead compound to help identify new molecular targets for the development of new Ewing-sarcoma-specific therapies.A screening program designed to identify natural products with selective cytotoxic effects against cell lines representing different types of pediatric solid tumors led to the identification of altertoxin II as a highly potent and selective cytotoxin against Ewing sarcoma cell lines. Altertoxin II, but not the related compounds altertoxin I and alteichin, was highly effective against every Ewing sarcoma cell line tested, with an average 25-fold selectivity for these cells as compared to cells representing other pediatric and adult cancers. Mechanism of action studies revealed that altertoxin II causes DNA double-strand breaks, a rapid DNA damage response, and cell cycle accumulation in the S phase. Our studies also demonstrate that the potent effects of altertoxin II are partially dependent on the progression through the cell cycle, because the G1 arrest initiated by a CDK4/6 inhibitor decreased antiproliferative potency more than 10 times. Importantly, the cell-type-selective DNA-damaging effects of altertoxin II in Ewing sarcoma cells occur independently of its ability to bind directly to DNA. Ultimately, we found that altertoxin II has a dose-dependent in vivo antitumor efficacy against a Ewing sarcoma xenograft, suggesting that it has potential as a therapeutic drug lead and will be useful to identify novel targets for Ewing-sarcoma-specific therapies.

Endosialin/CD248 may be a potential therapeutic target to prevent the invasion and metastasis in osteosarcoma.

Endosialin/CD248/tumor endothelial marker 1 is classified as a C-type lectin-like transmembrane receptor, found on the plasma membrane of activated mesenchymal cells, which binds to fibronectin. Although endosialin is expressed at high levels in stem-like cells of sarcomas, its role has not been fully uncovered. The present study aimed to determine whether endosialin expression is associated with tumor progression and metastasis, and whether endosialin has the potential to act as a novel therapeutic target in osteosarcoma (OS) using MORAb-004/ontuxizumab, a humanized monoclonal antibody, which targets the type C lectin domain of endosialin. The results demonstrated that endosialin was highly expressed in OSs with metastatic disease. Furthermore, MORAb-004 had no cytostatic effect on OS cells in vitro and did not change the expression of stem cells and differentiation markers; however, it inhibited migration of OS cells. Taken together, these results suggest that endosialin may play a role in migration, and may be involved in the metastatic process of OSs. Furthermore, MORAb-004 reduces the motility of OS cells, and suppresses invasion and the development of metastatic lesions.

Abstract P087: Targeting LSD1 protein scaffolding function in FET-rearranged sarcomas with SP-2577

Abstract The RNA-binding proteins, FUS, EWSR1, and TAF15 (FET protein family) are frequently fused to transcription factors, and the resulting oncogenic fusion proteins characterize various sarcomas. These fusion proteins act as aberrant transcription factors and promote oncogenic gene expression profiles. FET family rearranged sarcomas include Ewing Sarcoma, desmoplastic round cell tumors, myxoid liposarcoma, and clear cell sarcoma, amongst others. A growing body of work suggests that, independently of Lysine Specific Demethylase 1 (LSD1) demethylase activity, LSD1’s association with FET family oncogenic transcription factors is critical for FET-rearranged sarcoma development and progression. Extensive ongoing work aims to characterize the functional relationship of LSD1 and EWS/FLI, as well as the anti-tumor effect of disrupting that interaction. SP-2577 is an oral, first-in-class, small molecule with reversible, noncompetitive inhibition of LSD1 demethylase activity (IC50: 25–50 nM) and, importantly, also disrupts LSD1 protein-protein interactions. In vitro and in vivo data demonstrate SP-2577, or analogs, reverses EWS/ETS-mediated transcriptional regulation at both up- and downregulation target genes, leading to significant tumor growth inhibition in Ewing Sarcoma mouse xenograft studies. In this study, we set out to expand our understanding of SP-2577’s efficacy in diverse FET-rearranged malignancies including desmoplastic small round cell tumor (DSCRT: EWSR1/WT1), myxoid liposarcoma (ML: FUS/DDIT3), and clear cell sarcoma (CCS: EWS/ATF1). To examine whether these additional FET-rearranged sarcomas are sensitive to LSD1 scaffolding inhibition, we measured viability of Ewing sarcoma cells A673, TC32, TTC-446, and SK-N-MC, clear cell sarcoma cell lines SU-CCS-1, DTC-1, myxoid liposarcoma cell lines DL-221, 402-91, and 1765-92, and DSCRT patient derived xenograft organoids from SJDSRCT046151, X0069, SJDSRCT046155 after 96 hours of treatment with the clinical compound SP-2577, a first generation compound SP-2509, an inactive control, SP2513, and, a TCP-scaffold based, irreversible inhibitor of LSD1, OG-L002. While SP-2513 and OG-L002 had no activity against these cells SP-2509 and SP-2577 demonstrated potent activity across cell lines in vitro. Preliminary in vivo assessment of SP-2577 efficacy in DSCRT PDX SJDSRCT046151 resulted in a significant delay in time to event (p&amp;lt;0.02). Together, these data demonstrate that the unique scaffolding inhibition function of SP-2577 is essential for reducing cell viability through LSD1 targeting. Currently, we are elucidating changes to the transcriptome and proteome following SP-2577 treatment, in vitro. These data, in combination with results from the ongoing dose expansion phase 1 trial of SP-2577 in select sarcoma patients, may offer further mechanistic insight into the dependency of FET-rearranged sarcomas on LSD1 scaffolding functions and potentially lead to identifying biomarkers of sensitivity and response. Citation Format: Galen Rask, Emily R. Theisen, Aundrietta D. Duncan, Daniela Y. Santiesteban. Targeting LSD1 protein scaffolding function in FET-rearranged sarcomas with SP-2577 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P087.

Abstract P222: Combinations of receptor tyrosine kinase inhibitors targeting the tumor and stromal cells of complex spheroids from the National Cancer Institute’s Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/)

Abstract Tumor stroma is critical in shaping the tumor microenvironment and promoting tumor growth and metastasis. Six stroma-targeted kinase inhibitors (cediranib, vandetanib, axitinib, pazopanib, cabozantinib, and lenvatinib) were tested both as single agents and in combination with five tumor cell-targeted kinase inhibitors (erlotinib, gefitinib, osimertinib, rociletinib, and erdafitinib)using a complex spheroid cell culture model. Complex spheroids, containing 60% tumor cells, 25% endothelial cells (HUVEC) and 15% mesenchymal stem cells (MSC), serve as a cell culture model of human solid tumors incorporating both malignant and stromal cells. Seventeen tumor cell lines were grown as complex spheroids, including varied sarcoma and non-small cell lung cancer types. Drug sensitivities of the HUVEC and MSC were also evaluated, both individually as monolayers and mixed as monolayers and spheroids. Overall, the HUVEC and MSC were more sensitive to the VEGFR inhibitors than to the EGFR inhibitors. Growth as complex spheroids reduced the sensitivity of HUVEC and MSC to several kinase inhibitors compared to each cell type alone or in mixed monolayer culture. A significant correlation was observed between the EGFR mRNA expression in patient-derived tumor lines and their sensitivity to the EGFR inhibitors. In combination regimens, Erlotinib was most effective when combined with the VEGFR inhibitors pazopanib, vandetanib and cediranib, which produced more than one log of cell killing at concentrations less than the clinical Cmax of each drug. Osimertinib and rociletinib, which irreversibly target EGFR variants, were more cytotoxic towards complex spheroids of NCI-H1975 NSCLC (EGFR L858R, T790M) than NCI-H522 NSCLC (EGFR wildtype). Combinations of the mutant-selective EGFR inhibitors with a VEGFR inhibitor (especially, vandetanib, lenvatinib or cediranib) increased the cytotoxicity in complex spheroids containing EGFR mutant and wildtype tumor lines. Highly effective combinations included erdafitinib with vandetanib and erlotinib with cediranib. The combination of cediranib and erlotinib was also evaluated in nine patient-derived xenograft (PDX) models and resulted in two partial responses in a penile squamous carcinoma and uterine sarcoma, while the remainder showed stable, slowed, or progressive disease. Using &amp;gt;1 log of cell kill at drug concentrations less than the mouse Cmax concentration for each drug as a predictor for in vivo responsiveness, the complex spheroid assay was 88% accurate in predicting response or progressive disease in the PDX models. This criteria for predicting in vivo activity from in vitro model results will be further explored. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Thomas S. Dexheimer, Julie Laudeman, Thomas Silvers, Rene Delosh, Russell Reinhart, Chad Ogle, Eric Jones, Nathan P. Coussens, Beverly A. Teicher, Naoko Takebe, Alice Chen, James H. Doroshow. Combinations of receptor tyrosine kinase inhibitors targeting the tumor and stromal cells of complex spheroids from the National Cancer Institute’s Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov/) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P222.

Abstract P210: MDM2 gene amplification as a predictive biomarker for the MDM2 inhibitor milademetan

Abstract MDM2 is an E3 ubiquitin ligase that plays a critical role in the degradation of the tumor suppressor p53. Milademetan (RAIN-32) is an orally available, small molecule inhibitor of MDM2 that disrupts the MDM2-p53 protein complex thereby restoring p53 expression and activity. Milademetan has demonstrated anti-tumor activity in de-differentiated liposarcoma, a tumor type characterized by MDM2 gene amplification (amp) and WT TP53, in the phase I study of monotherapy milademetan (NCT01877382). Here we investigate the use of MDM2 gene amplification as a predictive biomarker for the selection of patients with advanced cancers who might benefit from milademetan. In this study we evaluated milademetan in MDM2 gene amp cancer models from a variety of tumor types using both in vitro assays, including 2D viability assays and organoid models, as well as in vivo patient-derived xenografts. We employed a mutual exclusivity analysis between MDM2 copy number (CN) and TP53 mutation status using publicly available next generation sequencing data to derive an optimal MDM2 CN threshold for patient selection and present clinical data from patients with MDM2 amp tumors treated with milademetan in the phase 1 study (NCT01877382). Three cell lines, CCFSTTG1 (astrocytoma), DKMG (glioblastoma), and SJSA1 (osteosarcoma), with MDM2 amp were identified and demonstrated inhibition of cell proliferation by milademetan with GI50 &amp;lt;100 nM. Evidence of MDM2 target engagement and activation of p53 was demonstrated through induction of p21 and PUMA, transcriptional targets of p53. Four patient-derived organoid models from diverse cancers (2 lung adenocarcinoma, 1 cholangiocarcinoma, and 1 renall cell carcinoma) were identified with MDM2 amp and WT TP53 and demonstrated differential selectivity compared to non-MDM2 amp models also with WT TP53 (head and neck scquamous cell carcinoma, endometrial carcinoma). Xenograft models from tumor types including gastric, lung adenocarcinoma and osteosarcoma with MDM2 amp showed dose-dependent anti-tumor activity to milademetan in vivo. Induction of MIC-1, a p53 target gene, was observed in vivo following milademetan dosing. An MDM2 CN threshold of ≥ 12 was derived using mutual exclusivity analysis using the AACR Genie dataset across solid tumor types. Using TCGA Pan-Cancer Atlas dataset, we identified 1.1% of cancers that met the criteria of MDM2 CN ≥ 12 and WT TP53. Finally, 3 patients (breast cancer, synovial sarcoma, and small cell lung cancer) with MDM2 CN ≥ 12 were previously enrolled on the U101 study of milademetan, Schedule D (3 days on 11 days off), which was determined to be the dose schedule for future clinical trials, and all 3 patients experienced tumor reduction with 2 patients demonstrating a partial response and 1 confirmed partial response. Milademetan shows evidence of preclinical and clinical anti-tumor activity in genetically selected tumors using MDM2 amp and WT TP53 as selection criteria. A basket study evaluating milademetan in solid tumors with MDM2 CN ≥ 12 and WT TP53 (MANTRA-2) is planned. Citation Format: Vijaya G. Tirunagaru, Mrinal M. Gounder, Prasanna R. Kumar, David S. Hong, Robert C. Doebele. MDM2 gene amplification as a predictive biomarker for the MDM2 inhibitor milademetan [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P210.

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P233.

Abstract P051: Identification of an orally bioavailable dual Cyclin K glue degrader - CDK12/13 inhibitor

Abstract CDK12 and CDK13 regulate gene expression through translation and splicing of mRNA. Impaired activity of CDK12 has been shown to reduce the expression of a range of genes involved in DNA homologous repair leading to the induction of a ‘BRCAness’ phenotype in cells. CDK12 inhibitors have the potential in many different indications, including overcoming emergent resistance to PARP inhibition, enhancing the efficacy of PARP inhibition in patients with BRCA1/2 wt status [1] and extending the efficacy of immune checkpoint inhibition through immunogenic cell death [2]. Recently, certain classes of CDK12 inhibitors have also demonstrated ‘glue degrader’ properties [1]: causing proteosomal degradation of the obligate co-factor for CDK12 and 13, Cyclin K. Such glue-degraders have the capability of enhancing cellular potency and specificity over classical kinase inhibitors. Taking as a starting point the selective CDK7 inhibitor samuraciclib, we have taken a rational design approach to develop inhibitors that preferentially inhibit CDK12. By utilising differences in the active site between CDK7 and CDK12 we were able to increase potency against CDK12 in biochemical assays by &amp;gt; 100 fold, with no significant inhibition of kinases outside the CDK class. Cellular potency of these molecules was evaluated in the Ewing’s sarcoma cell line A673, which is known to be sensitive to CDK12 inhibition [4]. Remarkably the potency of the molecules to induce cell death was greater than their biochemical potency against CDK12 by &amp;gt; 10 fold. Analysis of Cyclin K levels in cells demonstrated that the molecules lead to Cyclin K degradation. Furthermore, cellular potency was reduced by the presence of the neddylation inhibitor MLN4924 confirming that the compounds enhanced potency was mediated by glue degradation of Cyclin K. The molecules were found to cause potent inhibition of DNA homologous repair genes including BRCA1, FANCF and ERCC4 and lead to the accumulation of DNA strand breaks as assessed by the marker γ-H2AX. In the BRCA wt ovarian cancer cell line, OV-90, strong synergy was observed in the ability to induce cell death with a range of PARP inhibitors. In A673 tumour bearing mice, dosing with the compounds was found to cause significant reduction in the levels of cyclin K in tumours, which was sustained for 24 hr. Additionally, assessment of CDK12 protein in these tumours also showed a reduction, suggesting greater turnover of CDK12 protein in the absence of Cyclin K. Repeat dose studies at doses that lead to Cyclin K degradation in tumours demonstrated that the compounds were well tolerated, with no weight loss in mice. Two molecules, CT7439 and CT7510, were found to have oral bioavailability in mice that indicate that they would be suitable for once-daily oral administration in humans and are being progressed through regulatory toxicity studies. [1] Johnson, et al, (2016) Cell Rep. 17, 2367 [2] Li, et al, (2020) Cancer Lett. 495, 12 [3] Słabicki, et al, (2020) Nature 585, 293 [4] Iniguez, et al, (2018) Cancer Cell 33, 202 Citation Format: Edward K. Ainscow, Adrian Campbell, Michael Cripps, Robert Workman, Stuart Thomson, Kam Chohan, Damien Crepin, Mahiro Sunose, Jamie Patient, Jane Kendrew, Ash Bahl. Identification of an orally bioavailable dual Cyclin K glue degrader - CDK12/13 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P051.

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety.

Simple SummaryThe prevalence of TEM-1 in the vasculature and the stroma of solid tumors and in malignant cells of sarcomas suggests that targeting TEM-1 could have therapeutic benefit. In this context, an anti-TEM-1 companion diagnostic may assist in the personalized medicine approach, whereby TEM-1 expression is exploited as a biomarker to select patients that would most benefit from a treatment directed toward the TEM-1 antigen. In our previous works, we have selected 1C1m-Fc, a fusion protein antibody, radiolabeled it with 177Lu and demonstrated that [177Lu]Lu-1C1m-Fc has interesting therapeutic performance. To define a suitable radiopharmaceutical companion for theranostic applications, 64Cu was chosen to radiolabel the fusion protein antibody. The aim of this work was thus to determine if [64Cu]Cu-1C1m-Fc can be considered for TEM-1 PET imaging and to predict the dosimetry of the [177Lu]Lu-1C1m-Fc companion therapy.1C1m-Fc, a promising anti-TEM-1 DOTA conjugate, was labeled with 64Cu to target cancer cells for PET imaging and predicting the efficacy and safety of a previously studied [177Lu]Lu-1C1m-Fc companion therapy. DOTA-conjugated 1C1m-Fc was characterized by mass spectrometry, thin layer chromatography and immunoreactivity assessment. PET/CT and biodistribution studies were performed in human neuroblastoma xenografted mice. Absorbed doses were assessed from biodistribution results and extrapolated to 177Lu based on the [64Cu]Cu-1C1m-Fc data. The immunoreactivity was ≥ 70% after 48 h of incubation in serum, and the specificity of [64Cu]Cu-1C1m-Fc for the target was validated. High-resolution PET/CT images were obtained, with the best tumor-to-organ ratios reached at 24 or 48 h and correlated with results of the biodistribution study. Healthy organs receiving the highest doses were the liver, the kidneys and the uterus. [64Cu]Cu-1C1m-Fc could be of interest to give an indication of 177Lu dosimetry for parenchymal organs. In the uterus and the tumor, characterized by specific TEM-1 expression, the 177Lu-extrapolated absorbed doses are overestimated because of the lack of later measurement time points. Nevertheless, 1C1m-Fc radiolabeled with 64Cu for imaging would appear as an interesting radionuclide companion for therapeutic application with [177Lu]Lu-1C1m-Fc.