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Supplementary Figure 1 - PDF file 39K, S1P and C1P activate MAPK and AKT in human RMS cell lines in G proteincoupled receptor-dependent manner

Evidence suggests that bioactive lipids may regulate pathophysiologic functions such as cancer cell metastasis. Therefore, we determined that the bioactive lipid chemoattractants sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) strongly enhanced the in vitro motility and adhesion of human rhabdomyosarcoma (RMS) cells. Importantly, this effect was observed at physiologic concentrations for both bioactive lipids, which are present in biologic fluids, and were much stronger than the effects observed in response to known RMS prometastatic factors such as stromal derived factors-1 (SDF-1/CXCL12) or hepatocyte growth factor/scatter factor (HGF/SF). We also present novel evidence that the levels of S1P and C1P were increased in several organs after γ-irradiation or chemotherapy, which indicates an unwanted prometastatic environment related to treatment. Critically, we found that the metastasis of RMS cells in response to S1P can be effectively inhibited in vivo with the S1P-specific binder NOX-S93 that is based on a high-affinity Spiegelmer. These data indicate that bioactive lipids play a vital role in dissemination of RMS and contribute to the unwanted side effects of radio/chemotherapy by creating a prometastatic microenvironment. Mol Cancer Res; 11(7); 793–807. ©2013 AACR.

Supplementary Figure Legends - PDF files 72K, Supplementary Figure Legends

Supplementary Figure 3 - PDF file 66K, S1P and C1P levels create a pro-metastatic microenvironment in organs after administration of vincristine

Supplementary Figure 2 - PDF file 174KB, High levels of S1P and C1P in CM from irradiated organs create a pro-metastatic microenvironment

Supplementary Tables S1-S3, Figures S1-S4

Purpose:Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES.Experimental Design:Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy.Results:Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed.Conclusions:Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

Supplementary Figure from M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression

Osteosarcoma is the most common malignancy of the bone, yet the survival for patients with osteosarcoma is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In patients with osteosarcoma, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy-number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Methyl RNA immunoprecipitation sequencing analysis and functional studies showed that ALKBH5 mediates its protumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key protumorigenic genes, consequently driving unchecked cell-cycle progression, incessant replication, and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4–based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth.Significance:RNA demethylase ALKBH5 upregulates USP22 and RNF40 to inhibit histone H2A ubiquitination and induces expression of key replication and DNA repair–associated genes, driving osteosarcoma progression.

Supplementary Data from M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression

The high rate of negative clinical trials and failed drug development programs calls into question the use of preclinical testing as currently practiced. An important issue for the in vitro testing of agents that have advanced into the clinic is the use of clinically irrelevant concentrations in reports making claims for anticancer activity, as illustrated by publications for sorafenib, vorinostat, and metformin. For sorafenib, high protein binding leads to a dichotomy between concentrations active in the 10% serum conditions commonly used for in vitro testing and concentrations active in plasma. Failure to recognize this distinction leads to inappropriate claims of activity for sorafenib based on the micromolar concentrations commonly used for in vitro testing in low serum conditions. For vorinostat and metformin, results using in vitro concentrations higher than those achievable in patients are reported despite the availability of publications describing human pharmacokinetic data for each agent. We encourage journal editors and reviewers to pay greater attention to clinically relevant concentrations when considering reports that include in vitro testing of agents for which human pharmacokinetic data are available. Steps taken to more carefully scrutinize activity claims based on in vitro results can help direct researchers away from clinically irrelevant lines of research and toward lines of research that are more likely to lead to positive clinical trials and to improved treatments for patients with cancer. Clin Cancer Res; 19(11); 2828–33. ©2013 AACR.

Supplemental Materials for Smith and Houghton (A Proposal Regarding Reporting of in Vitro Testing Results): References to justify specific statements made in the manuscript

Purpose: microRNAs have been shown to be involved in different human cancers. We therefore have performed expression profiles on a panel of pediatric tumors to identify cancer-specific microRNAs. We also investigated if microRNAs are coregulated with their host gene.Experimental Design: We performed parallel microRNAs and mRNA expression profiling on 57 tumor xenografts and cell lines representing 10 different pediatric solid tumors using microarrays. For those microRNAs that map to their host mRNA, we calculated correlations between them.Results: We found that the majority of cancer types clustered together based on their global microRNA expression profiles by unsupervised hierarchical clustering. Fourteen microRNAs were significantly differentially expressed between rhabdomyosarcoma and neuroblastoma, and 8 of them were validated in independent patient tumor samples. Exploration of the expression of microRNAs in relationship with their host genes showed that the expression for 43 of 68 (63%) microRNAs located inside known coding genes was significantly correlated with that of their host genes. Among these 43 microRNAs, 5 of 7 microRNAs in the OncomiR-1 cluster correlated significantly with their host gene MIRHG1 (P < 0.01). In addition, high expression of MIRHG1 was significantly associated with high stage and MYCN amplification in neuroblastoma tumors, and the expression level of MIRHG1 could predict the outcome of neuroblastoma patients independently from the current neuroblastoma risk-stratification in two independent patient cohorts.Conclusion: Pediatric cancers express cancer-specific microRNAs. The high expression of the OncomiR-1 host gene MIRHG1 correlates with poor outcome for patients with neuroblastoma, indicating important oncogenic functions of this microRNA cluster in neuroblastoma biology. (Clin Cancer Res 2009;15(17):5560–8)

Supplementary Data from microRNA Profiling Identifies Cancer-Specific and Prognostic Signatures in Pediatric Malignancies

STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (–NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated. Cancer Res; 74(8); 2316–27. ©2014 AACR.

Supplementary Methods, Tables 1-4, Figure 1 from Credentialing Preclinical Pediatric Xenograft Models Using Gene Expression and Tissue Microarray Analysis

PDF file - 282K, H&E-stained organ tissue samples and TUNEL Stain.

PDF file - 172K, EPR spectra from organ tissue samples obtained from HO-3867-treated mice.

PDF file - 146K, Methods for HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.

PDF file - 77K, Immunoblot analysis for pSTAT3, and Bcl-2 using 2 different mice tissue.

PDF file - 220K, Representative Annexin V flow cytomety plots of CHO, H9c2 and A2780 cells.

PDF file - 122K, Illustration of the GFP-Histone 2B fusion protein (pBOS H2BGFP) vector.

Human tumor xenografts have been used extensively for rapid screening of the efficacy of anticancer drugs for the past 35 years. The selection of appropriate xenograft models for drug testing has been largely empirical and has not incorporated a similarity to the tumor type of origin at the molecular level. This study is the first comprehensive analysis of the transcriptome of a large set of pediatric xenografts, which are currently used for preclinical drug testing. Suitable models representing the tumor type of origin were identified. It was found that the characteristic expression patterns of the primary tumors were maintained in the corresponding xenografts for the majority of samples. Because a prerequisite for developing rationally designed drugs is that the target is expressed at the protein level, we developed tissue arrays from these xenografts and corroborated that high mRNA levels yielded high protein levels for two tested genes. The web database and availability of tissue arrays will allow for the rapid confirmation of the expression of potential targets at both the mRNA and the protein level for molecularly targeted agents. The database will facilitate the identification of tumor markers predictive of response to tested agents as well as the discovery of new molecular targets. [Cancer Res 2007;67(1):32–40]

PDF file - 156K, Effect of H-4073 in both cancer and normal cells. Phosphokinase assay array using CHO cells.

PDF file - 177K, Effect of DAPs on SKOV3 ovarian cancer and hOSE cells.

PDF file - 116K, Viability of SKOV3, hOSE, and H9c2 cells treated with H-4073 or HO-3867.

PDF file - 95K, Results of Tukey's post-hoc test and Molecular weights of the pure DAP compounds.

PDF file - 692K, (A) H-4073 Cell uptake in A2780 and CHO cell lines; (B) Ion chromatogram of pure HO-3867 in A2780 cells; (C) Ion chromatogram of pure HO-3867 in CHO cells; (D) Extracted ion chromatograms - H-4073; (E)Extracted ion chromatograms - H-4073.

Osteosarcoma is the most common malignancy of the bone, yet the survival for patients with osteosarcoma is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In patients with osteosarcoma, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy-number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Methyl RNA immunoprecipitation sequencing analysis and functional studies showed that ALKBH5 mediates its protumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key protumorigenic genes, consequently driving unchecked cell-cycle progression, incessant replication, and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4–based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth. Significance: RNA demethylase ALKBH5 upregulates USP22 and RNF40 to inhibit histone H2A ubiquitination and induces expression of key replication and DNA repair–associated genes, driving osteosarcoma progression.

Copanlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor, with activity against all four PI3K class I isoforms (PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ). Whole-genome and RNA sequencing data have revealed several PI3K aberrations in osteosarcoma tumor samples. The in vivo anticancer effects of copanlisib were assessed in a panel of six osteosarcoma models. Copanlisib induced prolonged event-free survival in five of six osteosarcoma models; however, all models demonstrated progressive disease suggesting minimal activity. While copanlisib did not result in tumor regression, more data are needed to fully explore the role of the PI3K pathway in the pathogenesis of osteosarcoma.

The centromere DNA-kinetochore complex is the specialized chromatin structure that mediates chromosome attachment to microtubules and is required for high-fidelity chromosome transmission. CENP-A is the centromere-specific histone H3 variant that epigenetically determines centromere position on the chromosome to ensure kinetochore assembly. Despite its importance, the precise mechanism of maintenance of CENP-A, i.e., the maintenance of centromere identity, remains obscure. In this study, we found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required for CENP-A deposition at centromeres by binding to centromeric RNA. We determined that the CENP-A binding domain of EWSR1 is the SYGQ2 region within the prion-like domain, which plays an important role in phase separation for chromatin remodeling. Our data suggest that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA. This is a novel mechanism to maintain the position of the centromere on the chromosome i.e., centromere identify. As EWSR1-FLI1 is the oncogenic fusion protein in Ewing sarcoma and as it inhibits EWSR1 function as a dominant negative mutant, it contributes to chromosome instability in Ewing sarcoma, which may lead to therapy resistance and metastasis. Citation Format: Risa Kitagawa, Argentina Becker, Peter Houghton, Katsumi Kitagawa. EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB034.

Background Adrenocortical cancer (ACC) is a rare endocrine malignancy frequently diagnosed at an advanced stage when prognosis is dismal. Mitotane, the only approved therapy for locally advanced or metastatic disease, is minimally effective, and recent clinical trials with multi-kinase inhibitors or immunotherapies have been disappointing. OR-449 is a potent, selective, and orally bioavailable small molecule antagonist to steroidogenic factor-1 (SF-1 or NR5A1), a nuclear receptor and a transcription factor essential for the growth and development of the adrenal gland. SF-1 was recently described as a candidate "master transcription factor" in adult ACC (1). We previously reported that OR-449, dosed orally at 30 mg/kg for 28 days, completely blocked the growth of SJ-ACC3, a pediatric ACC patient-derived tumor xenograft (PDX), in immunocompromised mice. To help identify biomarkers for following the action of OR-449 in patient tumors, we evaluated a second pediatric ACC PDX, SW1939, which has high SF-1 expression and responded to OR-449 (10, 30 or 100 mg/kg) with inhibition of tumor growth and improved event-free survival over 24 days of dosing (an event is a 4-fold increase in tumor volume). Methods We first measured circulating steroids, steroid precursors and steroid sulfates in SW1939 tumor-bearing mice with or without OR-449 treatment using liquid chromatography/tandem mass spectrometry. Second, we investigated common pathways of gene regulation by OR-449 in SW1939 and SJ-ACC3 tumors by RNAseq after seven days of dosing (100 mg/kg or 30 mg/kg, respectively). Results Cortisol, DHEAS, steroid precursors (11-deoxycortisol, 17OH-progesterone, 17OH-pregnenolone) and sulfated Δ5 steroid precursors (pregnenolone sulfate, 17OH-pregnenolone sulfate, androstenediol-sulfate) were elevated in serum from SW1939 tumor-bearing mice compared to CD-1 non tumor-bearing mice, consistent with tumor-derived origin. Moreover, DHEAS, pregnenolone sulfate, 17OH-pregnenolone sulfate and androstenediol-sulfate were significantly suppressed (12-fold, 7.6-fold, 10.6-fold and 4.4-fold, respectively) following administration of OR-449 (100 mg/kg/day for 24 days) in SW1939 tumor-bearing mice. Final tumor weights were ∼2-fold lower than controls, indicating that circulating steroid reduction was disproportionately greater than the change in tumor volume. Global gene expression was significantly altered following 7-day exposure to OR-449 in both tumors. Significantly, PAPSS2 and SULT2A1 within the sulfation pathway were down-regulated in both SW1939 (2.3-fold and 1.2-fold, respectively) and SJ-ACC3 (1.6-fold and 5-fold, respectively). Conclusions OR-449 reduces steroid production, alters gene expression, and suppresses growth of two PDX models of ACC in mice. Tumor gene expression and steroid regulation by OR-449 are currently under investigation in other pediatric and adult ACC PDX models to evaluate these endpoints as potential biomarkers of SF-1 receptor engagement for initial clinical trials of OR-449 in ACC. Reference: (1) Corces et al., Science. 2018 Oct 26;362(6413): eaav1898; Reddy et al., Sci Adv. 2021 Nov 26;7(48): eabf6123. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma characterized by an impaired myogenic differentiation despite the expression of myogenic master genes MYOD and MYOG. Therefore, the restoration of differentiation is considered an anti-cancer therapy. SKP2 is an oncogenic E3-ubiquitin ligase that promotes cell proliferation by targeting the CDKi p21Cip1 and p27Kip1. Previous works showed that SKP2 overexpression is induced by the fusion oncoprotein PAX3-FOXO1 expressed in fusion positive (FP)-RMS cells, and promotes tumor cell proliferation through p27kip1 degradation. However, the role of SKP2 in fusion negative (FN)-RMS cells, devoid of any fusion gene, remains unclear. We report here that SKP2 transcript and protein levels are up-regulated in RMS patients and cell lines compared to normal tissue. Accordingly, we observed increased acetylation of H3K27 histone mark in RMS patients and cell lines compared to myoblasts and muscle tissue. We then show that in RMS cell lines SKP2 expression is induced by MYOD, which binds two SKP2 regulatory regions, an intronic and a distal enhancers, identified by Hi-C and 3C experiments. SKP2 knockdown in FN-RMS cells leads to p21Cip1 and p27Kip1 protein levels up-regulation coupled with G1/S cell cycle arrest. Rescue experiments showed that SKP2 promotes cell proliferation directly targeting p27Kip1. Moreover, SKP2 binds and promotes degradation of p57Kip2 and its silencing restores myogenic differentiation associated to MYOG and de novo MyHC expression in FN-RMS cells. SKP2 depletion also induces cell senescence and prevents anchorage-independent growth and stemness in vitro, and tumor growth in vivo. In turn, SKP2 forced expression partially rescued the anti-cancer effects preventing the increase of p21Cip1, p27Kip1, p57Kip2 and MYOG, promoting re-entry into cell cycle, inhibiting human myoblasts cell differentiation and restoring the tumorigenic potential in FN-RMS. Since neddylation is an essential step for the activity of SKP2, we used MLN4924, an inhibitor of the Nedd8 Activating Enzyme (NAE), under clinical investigation, to resume SKP2 knockdown features. MLN4924 induces p21Cip1 and p27Kip2 expression, promotes senescence and apoptosis, and hampers cell growth in vitro and in vivo both in FP- and FN-RMS. These results unveil an unprecedented role for SKP2 in governing both proliferation and myogenic differentiation in RMS, suggesting that targeting SKP2 functions through MLN4924 treatment might have clinical relevance in FP- and FN-RMS. The study has been founded by AIRC and 5xmille 2021/Ministero della Salute to RR. Citation Format: Silvia Pomella, Matteo Cassandri, Doris Phelps, Clara Perrone, Michele Pezzella, Marco Wachtel, Benjamin Sunkel, Antonella Cardinale, Zoe Walters, Cristina Cossetti, Sonia Rodriguez, Nadia Carlesso, Janet Shipley, Lucio Miele, Beat Schafer, Enrico Velardi, Peter Houghton, Berkley Gryder, Benjamin Stanton, Concetta Quintarelli, Biagio De Angelis, Franco Locatelli, Rossella Rota. A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo [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 668.

Introduction: B7-H3 (encoded by the CD276 gene) is suggested to act as an immune checkpoint molecule and is highly expressed in some pediatric solid tumors. Monoclonal antibodies targeting B7-H3 (8H9 and MGA271) are well-tolerated but have demonstrated limited success in clinical trials. DS-7300a is a B7-H3 targeting ADC with a payload of DXd (an exatecan derivative that inhibits DNA topoisomerase I). DS-7300a has a drug-to-antibody ratio of 4 and it has shown promising early clinical activity in adults with advanced solid cancers (Johnson, Annals of Oncology 2021; 32:S583-S585). Methods: Xenograft models for several pediatric cancer types were selected for preclinical testing of DS-7300a based on the high rates of B7-H3 expression in these cancers. Models were dosed at 10 mg/kg administered intravenously every other week for two doses (Q2wk x 2). Different experimental designs were used for efficacy testing. For rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, and other pediatric solid tumors a single mouse trial (SMT) design with 40 models was used. A cohort design (N=10) was used for efficacy testing in 6 osteosarcoma models, 3 patient-derived orthotopic xenograft glioblastoma (PDOX) models, and 3 PDOX ependymoma models. For neuroblastoma models, 10 models (2 mice per model) were tested. To evaluate treatment efficacy, objective response measures based on changes in relative tumor volume were used (Houghton, Pediatr Blood Cancer 2007;49:928-940). Results: As a single agent, DS-7300a induced a statistically significant prolongation of survival in 2 of 3 orthotopic glioblastoma and 1 of 3 ependymal tumor models. DS-7300a demonstrated high efficacy (maintained complete response, complete response, and partial response) in most non-CNS models tested: 17 of 21 rhabdomyosarcomas, 5 of 7 osteosarcomas, 7 of 10 neuroblastomas and 2 of 2 Wilms tumors. For Ewing sarcoma, most models were classified as progressive disease (9 of 15). For osteosarcoma models, the log cell kill per dose values ranged from 0.95 to 2.76 indicating high activity of the agent in these models. The activity for DS-7300a followed the general pattern of protein and RNA expression levels for B7-H3/CD276 in the models, with Ewing sarcoma models showing lower expression compared to the other models. Conclusions: DS-7300a shows tumor-regressing anticancer activity across a wide range of pediatric solid tumor models. The maintained complete remissions observed for osteosarcoma models are noteworthy, as this level of response is uncommon for these models and as osteosarcoma shows the highest B7-H3 expression among pediatric cancers. The high level of preclinical activity observed for DS-7300a combined with the promising early clinical activity observed for adult patients provide strong rationale for studying DS-7300a in children with B7-H3 expressing solid tumors. Citation Format: Richard Gorlick, E. Anders Kolb, Yifei Wang, Peter Houghton, Raushan Kurmasheva, Yael Mosse, John Maris, Matthew Tsang, David Groff, Kateryna Krytska, Xiao-Nan Li, Yuchen Du, Jun Hasegawa, Nanae Izumi, Steven Neuhauser, Anuj Srivastava, Tim Stearns, Vivek Philip, Emily L. Jocoy, Jeff Chuang, Carol J. Bult, Beverly Teicher, Malcolm Smith. Evaluation of the in vivo efficacy of the B7-H3 targeting antibody-drug conjugate (ADC) DS7300a: A report fro the Pediatric Preclinical In Vivo Resting (PIVOT) program [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 LB061.