Supplementary Table 1. Cell line and growth media information.

Preclinical translational research has increasingly utilized patient-derived xenograft (PDX) models for mechanistic and experimental therapeutic studies. However, most existing models have been developed from adult cancer types. Here, we describe the establishment of a PDX program to expand the availability of pediatric-specific PDXs for preclinical research and enable studies of pediatric cancer histologies, including ultra-rare diseases. Processes for PDX generation were integrated into established clinical workflows to facilitate universal model generation. Methodologies for tissue procurement, processing, and cryopreservation were optimized to enable intra- and inter-institutional PDX model generation. Over a 6-year span, 388 PDX tumor models representing >40 diagnoses were generated, including ultra-rare tumors and longitudinal models established from pre-therapy, post-therapy, and relapse tumors from the same patient. Genomic characterization of these PDXs demonstrated excellent concordance and recapitulation of molecular alterations of the source tumor. Successful PDX generation was enhanced from relapsed samples, was higher in sarcomas compared to other solid tumor types, and was a negative prognosticator for clinical outcome. The utility of the broad portfolio of molecularly annotated models for validating cross-histology biomarker-driven therapeutic strategies was established by demonstrating anti-tumor activity of a MAT2A inhibitor in MTAP-deficient PDXs. Universal model creation also allowed for experimental validation of therapeutic hypotheses on a patient-specific basis, as highlighted by the characterization of a RAF1 fusion (EPB41L2::RAF1) in an osteosarcoma PDX. Overall, development of a diverse collection of pediatric PDX models enables hypothesis-driven and cross-histology studies that expand our understanding of cancer biology and aid ongoing drug prioritization efforts in rare tumors.

Trastuzumab deruxtecan (T-DXd) is an ERBB2/HER2-targeting antibody-drug conjugate (ADC) with efficacy across adult cancers exhibiting variable HER2 expression. Prior studies demonstrating HER2 expression in osteosarcoma motivated a clinical trial of T-DXd in pediatric and adolescent/young adults with osteosarcoma, but the trial was terminated early because of inactivity. We evaluated the activity of T-DXd using osteosarcoma patient-derived xenograft (PDX) models and found a 22% objective response rate despite no detectable HER2 expression across PDXs tested. To further assess non-HER2-mediated activity, we evaluated the activity of T-DXd across 31 pediatric cancer cell lines and found osteosarcoma to be amongst the most resistant to T-DXd, as well as unconjugated deruxtecan, providing a potential explanation for the negative results observed in the clinical trial of T-DXd in osteosarcoma. T-DXd evaluation in PDX models representing pediatric histologies with greater intrinsic sensitivity to deruxtecan, including pediatric renal tumors and desmoplastic small round cell tumor, revealed both HER2-enhanced activity as well as substantial non-HER2-mediated activity, as evidenced by equipotent activity using an isotype-matched control ADC. Together, these results underscore translational opportunities for ADC therapeutics in tumor histologies with high sensitivity to the payload and in which enhanced tumor delivery may be mediated by antibody-targeted mechanisms as well as macromolecular characteristics of ADCs (e.g., enhanced permeability and retention effect) and tumor microenvironmental factors (e.g., proteolytic payload release). Our findings challenge the role of HER2 as a biomarker predictive of T-DXd response in pediatric cancers and support further biomarker-agnostic clinical development of T-DXd in desmoplastic small round cell tumor and pediatric renal tumors.

Supplementary Figure 2 depicts whole genome sequencing findings

Supplementary Figure 4 shows data related to FGFR4 inhibition in DSRCT and control models

Supplementary Table 1 shows point mutations found in DSRCT samples

Supplementary Figure 3 provides a breakdown of the structural variant types found via whole genome sequencing in DSRCT samples

Supplementary figures and tables

Supplementary Table 2 shows the raw data further described in Figure 3

Introduction: Malignant rhabdoid (MRT) and Wilms tumor (WT) comprise more than 5% of all pediatric cancers. Despite intensive multimodality therapy, outcomes remain dismal for a subset of patients with aggressive or high-risk molecular features. Characteristic of most pediatric cancers, MRT and WT demonstrate relatively low frequencies of somatic mutations compared to adult tumors and generally lack therapeutically targetable genetic alterations. Hence, we applied a systems biology approach to identify and evaluate non-genetically encoded vulnerabilities in MRT and WT. Methods: MetaVIPER analysis was performed to computationally infer protein activity from MRT and WT whole transcriptomic data available in the TARGET database. Expanded metaVIPER analysis of TARGET and TCGA cohorts demonstrated XPO1 as having consistently high inferred activity in MRT and WT. Functional in vitro studies using a selective inhibitor of XPO1, selinexor, were performed on a panel of MRT and WT cell lines to evaluate the effects of XPO1 inhibition on proliferation, cell cycle transition and apoptosis induction. In vivo validation of anti-tumor activity following XPO1 inhibition were performed in cell line-derived (CDX) and patient-derived xenograft (PDX) models of MRT and WT. Results: MetaVIPER analysis identified consistent high inferred activity of XPO1 in MRT and WT compared to other tumor types. MRT and WT cell lines demonstrated in vitro sensitivity to selinexor treatment resulting in cell cycle arrest and apoptosis induction. Furthermore, protein expression analysis showed increased nuclear sequestration of tumor suppressors proteins following treatment with selinexor. In vivo treatment of panel of MRT and WT CDX and PDX models with selinexor and a next-generation XPO1 inhibitor, eltanexor, resulted in significant abrogation of tumor growth with associated decreases in inferred XPO1 activity. Pharmacodynamic analysis of treated PDX tumors show decreased levels of XPO1, RB1-pSer780, and increased p53, p27 and p21 protein levels. Based on promising preclinical data, we describe a case report of a child with relapsed and progressive Wilms tumor who experienced a sustained complete remission on maintenance selinexor therapy. Conclusion: XPO1 represents a non-genetically encoded vulnerability in MRT and WT. Promising preclinical activity in MRT and WT models has provided the preclinical rationale for evaluation of XPO1 inhibition in an investigator-initiated clinical trial of Selinexor in pediatric MRT and WT. Citation Format: Diego F. Coutinho, Chelsey Burke, Prabhjot Mundi, Michael V. Ortiz, Kelly L. Vallance, Matthew Long, Nestor Rosales, Glorymar Ibanez, Lianna J. Marks, Daniel Diolaiti, Andoyo Ndengu, Daoqi You, Armaan Siddiquee, Ervin S. Gaviria, Allison R. Rainey, Andrea Califano, Andrew L. Kung, Filemon S. Dela Cruz. Targeting of the nuclear export protein XPO1 represents a non-genetically encoded vulnerability in malignant rhabdoid and Wilms tumors [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 1810.

Decrease in DNA dioxygenase activity generated by TET2 gene family is crucial in myelodysplastic syndromes (MDS). The general downregulation of 5-hydroxymethylcytosine (5-hmC) argues for a role of DNA demethylation in MDS beyond TET2 mutations, which albeit frequent, do not convey any prognostic significance. We investigated TETs expression to identify factors which can modulate the impact of mutations and thus 5-hmC levels on clinical phenotypes and prognosis of MDS patients. DNA/RNA-sequencing and 5-hmC data were collected from 1665 patients with MDS and 91 controls. Irrespective of mutations, a significant fraction of MDS patients exhibited lower TET2 expression, whereas 5-hmC levels were not uniformly decreased. In searching for factors explaining compensatory mechanisms, we discovered that TET3 was upregulated in MDS and inversely correlated with TET2 expression in wild-type cases. Although TET2 was reduced across all age groups, TET3 levels were increased in a likely feedback mechanism induced by TET2 dysfunction. This inverse relationship of TET2 and TET3 expression also corresponded to the expression of L-2-hydroxyglutarate dehydrogenase, involved in agonist/antagonist substrate metabolism. Importantly, elevated TET3 levels influenced the clinical phenotype of TET2 deficiency whereby the lack of compensation by TET3 (low TET3 expression) was associated with poor outcomes of TET2 mutant carriers.

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

Background: Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young chil-dren comprising ,-,5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations. Methods: Comparative protein activity analysis of MRTs (n = 68) and WTs (n = 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. In vitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. In vivo anti-tumor activity was as-sessed in patient-derived xenograft (PDX) models of MRTs and WTs. Findings: metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with in vitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. In vivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sus-tained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor. Conclusions: We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in ge-nomically quiescent pediatric cancers. These results have provided pre -clinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activ-ity as a potential predictive biomarker for response. Funding: This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

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.

Wilms’ tumor is the most common childhood kidney cancer. Two distinct histological subtypes of Wilms’ tumor have been described: tumors lacking anaplasia (the favorable subtype) and tumors displaying anaplastic features (the unfavorable subtype). Children with favorable disease generally have a very good prognosis, while those with anaplasia are oftentimes refractory to standard treatments and suffer poor outcomes. MYCN dysregulation has been associated with a number of pediatric cancers including the anaplastic subtype of Wilms’ tumor. In this context, we undertook a functional genomics approach to uncover novel therapeutic strategies for those patients with anaplastic Wilms’ tumor. Genomic analysis and in vitro experimentation demonstrate that Wilms’ tumor cell growth can be reduced by modulating MYCN overexpression via BRD4 inhibition. We observed a time dependent reduction of MYCN and MYC protein levels upon BRD4 inhibition in Wilms’ tumor cell lines which led to increased cell death and suppressed proliferation. We suggest that AZD5153, a novel dual-BRD4 inhibitor, can reduce MYCN levels and should be further explored for its therapeutic potential against Wilms’ tumor.

Abstract Introduction Natural orange juice is a phenolic compounds rich beverage, whose disposal on market and food catering has increased with the expansion of the concept of healthy food, raising concerns about the effect of storage on total antioxidant capacity (TAC), and scientific evidence on this is scarce. Regarding TAC, is well established that phenolic compounds account more to it than ascorbic acid [1]. Our aim was to evaluate the concentration of total phenolic compounds (CTPC) in fresh squeezed orange juice and analyse the influence of storage conditions (temperature and light) during 48 h on CTPC. Materials and methods Fresh oranges (Citrus sinensis, variety “Valencia Late”, Portugal) of the same calibre and producer were squeezed aliquoted into 3 glasses stored in different conditions: (1) Room temperature (20 °C) and exposed to sunlight; (2) Room temperature (20 °C) fully wrapped in aluminium; and (3) Refrigerated (1 °C). The procedure was performed according to the methodology described by Keskin-Šašić et al. [2]. CTPC was quantified by Folin-Ciocalteu spectrophotometric method, using gallic acid as standard, at 0, 2.5, 4, 8 and 48 h. (a) Samples preparation: 1 mL of orange juice was diluted from each vial in water, to a volume of 25 mL. Part of solution was centrifuged at 5300 rpm for 20’. The supernatant solution was used to analyse. (b) Determination of CTPC: approximately 0.2 mL of each sample was transferred to test tubes containing 1.0 mL of a dilution of Folin–Ciocalteu reagent in water (1:10). After 10’, 0.8 mL of a sodium carbonate solution (7.5% m/v) was added to the sample. The test tubes were allowed to stand at room temperature for 30’, and then absorbance was measure at 743 nm. CTPC was expressed as equivalents of gallic acid (EGA) in mg/100 ml of squeezed orange juice. Samples’ CTPC was determined from a standard curve of gallic acid varying between 0.2 and 4 mg/L. Results CTPC of recently squeezed fresh oranges was 30.1 mg EGA/100 mL (basal). After 48 h, CTPC decreased in all samples: 38.5% in sample 1 (18.5 mg EGA/100 mL), 28.6% in sample 2 (21.8 mg EGA/100 mL), and 27.6% in sample 3 (21.5 mg EGA/100 mL). The higher variation in CTPC occurred for sample 1, stored at room temperature and exposed to sunlight. Discussion and conclusions: Our results suggest that exposure to sunlight is the variable that most influences the decrease CTPC of the fresh orange juice after 48 h. More studies on the influence of these variables are needed to confirm these results and to increase knowledge about storage of fruit fresh juices, to ensure the nutritional quality of this product. Squeezed orange juice is widely present on markets and coffee shops, usually refrigerated but not protected from sunlight, which means that the storage conditions would need to be reconsidered.

Limited clinical data are available regarding the utility of multikinase inhibition in neuroblastoma. Repotrectinib (TPX-0005) is a multikinase inhibitor that targets ALK, TRK, JAK2/STAT, and Src/FAK, which have all been implicated in the pathogenesis of neuroblastoma. We evaluated the preclinical activity of repotrectinib monotherapy and in combination with chemotherapy as a potential therapeutic approach for relapsed/refractory neuroblastoma. In vitro sensitivity to repotrectinib, ensartinib, and cytotoxic chemotherapy was evaluated in neuroblastoma cell lines. In vivo antitumor effect of repotrectinib monotherapy, and in combination with chemotherapy, was evaluated using a genotypically diverse cohort of patient-derived xenograft (PDX) models of neuroblastoma. Repotrectinib had comparable cytotoxic activity across cell lines irrespective of ALK mutational status. Combination with chemotherapy demonstrated increased antiproliferative activity across several cell lines. Repotrectinib monotherapy had notable antitumor activity and prolonged event-free survival compared with vehicle and ensartinib in PDX models (P < 0.05). Repotrectinib plus chemotherapy was superior to chemotherapy alone in ALK-mutant and ALK wild-type PDX models. These results demonstrate that repotrectinib has antitumor activity in genotypically diverse neuroblastoma models, and that combination of a multikinase inhibitor with chemotherapy may be a promising treatment paradigm for translation to the clinic.

Despite all knowledge acquired regarding the mutational profile of pediatric myelodysplastic syndrome (MDS), the somatic genomic landscape underlying that disease remains unclear. We evaluated the presence of somatic variants in 37 genes related to myeloid malignancies through targeted NGS in 20 Brazilian patients with refractory cytopenia of childhood (RCC). Only 15% (3/20) of patients showed at least one somatic driver mutation – all in genes coding to regulators of cell signaling (TP53 and CBLB) or epigenetics (ASXL1 and DNMT3A). Interestingly, those variants were identified in patients with no detected clonal chromosomal abnormalities.