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HER2 is expressed in many pediatric solid tumors and is a target for innovative immune therapies including CAR-T cells and antibody–drug conjugates (ADC). We evaluated the preclinical efficacy of trastuzumab deruxtecan (T-DXd, DS-8201a), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient- and cell line–derived xenograft (PDX/CDX) models. HER2 mRNA expression was determined using RNA-seq and protein expression via IHC across multiple pediatric tumor PDX models. Osteosarcoma (OS), malignant rhabdoid tumor (MRT), and Wilms tumor (WT) models with varying HER2 expression were tested using 10 mice per group. Additional histologies such as Ewing sarcoma (EWS), rhabdomyosarcoma (RMS), neuroblastoma (NB), and brain tumors were evaluated using single mouse testing (SMT) experiments. T-DXd or vehicle control was administered intravenously to mice harboring established flank tumors at a dose of 5 mg/kg on day 1. Event-free survival (EFS) and objective response were compared between treatment and control groups. HER2 mRNA expression was observed across histologies, with the highest expression in WT (median = 22 FPKM), followed by MRT, OS, and EWS. The relationship between HER2 protein and mRNA expression was inconsistent. T-DXd significantly prolonged EFS in 6/7 OS, 2/2 MRT, and 3/3 WT PDX models. Complete response (CR) or maintained CR (MCR) were observed for 4/5 WT and MRT models, whereas stable disease was the best response among OS models. SMT experiments also demonstrated activity across multiple solid tumors. Clinical trials assessing the efficacy of a HER2-directed ADC in pediatric patients with HER2-expressing tumors should be considered.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody–drug conjugated (ADC) with a pyrrolobenzodiazepine dimer toxin targeting DLL3, in preclinical models of human neuroblastoma.We evaluated DLL3 expression in RNA-sequencing datasets and performed IHC on neuroblastoma patient-derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays. We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared antitumor activity with expression levels.DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose–response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3 of 11 and stable disease in another 3 of 11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for antitumor activity.Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.Significance:GD2-directed antibody therapy is standard of care for high-risk neuroblastoma; therapy is toxic, and relapses often occur. DLL3, an inhibitory Notch ligand, is overexpressed in several neuronal cancers. A DLL3-targeting ADC showed objective activity only in neuroblastoma models with high DLL3 expression. These data provide vigilance about clinical development of DLL3 immunotherapies for neuroblastoma.

Supplementary Figures 1-7 with legends included

Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Furthermore, we tested BT1769, an MT1-MMP–targeted Bicycle toxin conjugate, in osteosarcoma patient-derived xenograft models. The results showed that BT1769 had encouraging antitumor activity, high affinity for its target, and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.

Supplementary Figure from Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

Supplementary Data from Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

Purpose:Vincristine combined with camptothecin derivatives showed synergy in preclinical pediatric cancer models, and the combinations are effective in treatment of childhood solid tumors. We determined whether the synergy between vincristine and irinotecan extends to eribulin, another microtubule inhibitor.Experimental Design:Vincristine or eribulin, alone or combined with irinotecan, was studied in 12 xenograft models. Tumor regression and time to event were used to assess antitumor activity. Pharmacodynamic studies and RNA sequencing (RNA-seq) were conducted 24 and 144 hours after single-agent or combination treatment. Effects on vascular development were studied in Matrigel plugs implanted in mice. The interaction between binary combinations was examined in vitro.Results:Eribulin combined with irinotecan was more effective than vincristine–irinotecan in 6 of 12 models. Pharmacodynamic markers induced by eribulin (phospho-histone H3) and irinotecan (γ-H2A.X) were abrogated in combination-treated tumors. The predominant RNA-seq signature in combination-treated tumors was activation of the TP53 pathway with increased nuclear TP53. Massive apoptosis was observed 24 hours only after treatment with the eribulin combination. In vitro, neither combination showed interaction using combination index analysis. Eribulin alone and the combination caused alterations in developing vasculature.Conclusions:The eribulin combination is very active in these xenograft models, but not synergistic in vitro. The combination reduced pharmacodynamic markers indicative of single-agent mechanisms but in tumors, dramatically activated the TP53 pathway. Although a mechanism for in vivo synergy requires further study, it is possible that eribulin-induced inhibition of microtubule dynamics enhances irinotecan-induced nuclear accumulation of TP53, leading to rapid cell death.

Purpose:Checkpoint kinase 1 (CHK1) inhibitors potentiate the DNA-damaging effects of cytotoxic therapies and/or promote elevated levels of replication stress, leading to tumor cell death. Prexasertib (LY2606368) is a CHK1 small-molecule inhibitor under clinical evaluation in multiple adult and pediatric cancers. In this study, prexasertib was tested in a large panel of preclinical models of pediatric solid malignancies alone or in combination with chemotherapy.Experimental Design:DNA damage and changes in cell signaling following in vitro prexasertib treatment in pediatric sarcoma cell lines were analyzed by Western blot and high content imaging. Antitumor activity of prexasertib as a single agent or in combination with different chemotherapies was explored in cell line–derived (CDX) and patient-derived xenograft (PDX) mouse models representing nine different pediatric cancer histologies.Results:Pediatric sarcoma cell lines were highly sensitive to prexasertib treatment in vitro, resulting in activation of the DNA damage response. Two PDX models of desmoplastic small round cell tumor and one malignant rhabdoid tumor CDX model responded to prexasertib with complete regression. Prexasertib monotherapy also elicited robust responses in mouse models of rhabdomyosarcoma. Concurrent administration with chemotherapy was sufficient to overcome innate resistance or prevent acquired resistance to prexasertib in preclinical models of neuroblastoma, osteosarcoma, and Ewing sarcoma, or alveolar rhabdomyosarcoma, respectively.Conclusions:Prexasertib has significant antitumor effects as a monotherapy or in combination with chemotherapy in multiple preclinical models of pediatric cancer. These findings support further investigation of prexasertib in pediatric malignancies.

Supplementary Tables and Figures

Purpose:OBI-3424 is a highly selective prodrug that is converted by aldo-keto reductase family 1 member C3 (AKR1C3) to a potent DNA-alkylating agent. OBI-3424 has entered clinical testing for hepatocellular carcinoma and castrate-resistant prostate cancer, and it represents a potentially novel treatment for acute lymphoblastic leukemia (ALL).Experimental Design:We assessed AKR1C3 expression by RNA-Seq and immunoblotting, and evaluated the in vitro cytotoxicity of OBI-3424. We investigated the pharmacokinetics of OBI-3424 in mice and nonhuman primates, and assessed the in vivo efficacy of OBI-3424 against a large panel of patient-derived xenografts (PDX).Results:AKR1C3 mRNA expression was significantly higher in primary T-lineage ALL (T-ALL; n = 264) than B-lineage ALL (B-ALL; n = 1,740; P < 0.0001), and OBI-3424 exerted potent cytotoxicity against T-ALL cell lines and PDXs. In vivo, OBI-3424 significantly prolonged the event-free survival (EFS) of nine of nine ALL PDXs by 17.1–77.8 days (treated/control values 2.5–14.0), and disease regression was observed in eight of nine PDXs. A significant reduction (P < 0.0001) in bone marrow infiltration at day 28 was observed in four of six evaluable T-ALL PDXs. The importance of AKR1C3 in the in vivo response to OBI-3424 was verified using a B-ALL PDX that had been lentivirally transduced to stably overexpress AKR1C3. OBI-3424 combined with nelarabine resulted in prolongation of mouse EFS compared with each single agent alone in two T-ALL PDXs.Conclusions:OBI-3424 exerted profound in vivo efficacy against T-ALL PDXs derived predominantly from aggressive and fatal disease, and therefore may represent a novel treatment for aggressive and chemoresistant T-ALL in an AKR1C3 biomarker-driven clinical trial.

Supplementary Methods, Supplementary Tables 1-6, Supplementary Figures 1-9

Purpose:Patients with relapsed pediatric solid malignancies have few therapeutic options, and many of these patients die of their disease. B7-H3 is an immune checkpoint protein encoded by the CD276 gene that is overexpressed in many pediatric cancers. Here, we investigate the activity of the B7-H3–targeting antibody–drug conjugate (ADC) m276-SL-PBD in pediatric solid malignancy patient-derived (PDX) and cell line–derived xenograft (CDX) models.Experimental Design:B7-H3 expression was quantified by RNA sequencing and by IHC on pediatric PDX microarrays. We tested the safety and efficacy of m276-SL-PBD in two stages. Randomized trials of m276-SL-PBD of 0.5 mg/kg on days 1, 8, and 15 compared with vehicle were performed in PDX or CDX models of Ewing sarcoma (N = 3), rhabdomyosarcoma (N = 4), Wilms tumors (N = 2), osteosarcoma (N = 5), and neuroblastoma (N = 12). We then performed a single mouse trial in 47 PDX or CDX models using a single 0.5 m/kg dose of m276-SL-PBD.Results:The vast majority of PDX and CDX samples studied showed intense membranous B7-H3 expression (median H-score 177, SD 52). In the randomized trials, m276-SL-PBD showed a 92.3% response rate, with 61.5% of models showing a maintained complete response (MCR). These data were confirmed in the single mouse trial with an overall response rate of 91.5% and MCR rate of 64.4%. Treatment-related mortality rate was 5.5% with late weight loss observed in a subset of models dosed once a week for 3 weeks.Conclusions:m276-SL-PBD has significant antitumor activity across a broad panel of pediatric solid tumor PDX models.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL subtype with high rates of relapse and poor patient outcome. Activating mutations affecting components of the JAK-STAT signaling pathway occur in the majority of Ph-like ALL cases. The use of JAK inhibitors represents a potential treatment option for Ph-like ALL, although we and others have shown that CRLF2-rearranged Ph-like ALL responds poorly to single-agent JAK inhibitors in the preclinical setting. Therefore, the aim of this study was to identify effective combination treatments against CRLF2-rearranged Ph-like ALL, and to elucidate the underlying mechanisms of synergy. We carried out a series of high-throughput combination drug screenings and found that ruxolitinib exerted synergy with standard-of-care drugs used in the treatment of ALL. In addition, we investigated the molecular effects of ruxolitinib on Ph-like ALL by combining mass spectrometry phosphoproteomics with gene expression analysis. Based on these findings, we conducted preclinical in vivo drug testing and demonstrated that ruxolitinib enhanced the in vivo efficacy of an induction-type regimen consisting of vincristine, dexamethasone, and L-asparaginase in 2/3 CRLF2-rearranged Ph-like ALL xenografts. Overall, our findings support evaluating the addition of ruxolitinib to conventional induction regimens for the treatment of CRLF2-rearranged Ph-like ALL.

Membrane protein leucine–rich repeat containing 15 (LRRC15) is known to be expressed in several solid tumors including osteosarcoma. ABBV-085, an antibody–drug conjugate against LRRC15, conjugated to monomethyl auristatin E (MMAE), was studied in osteosarcoma patient-derived xenografts (PDXs) by the Pediatric Preclinical Testing Consortium (PPTC). LRRC15 expression data were obtained from PPTC RNA-sequencing data for the PDX models. The TARGET database was mined for LRRC15 expression in human osteosarcoma. Protein expression was confirmed via IHC in three PDX models. Seven osteosarcoma PDX models (OS1, OS9, OS33, OS34, OS42, OS55, and OS60) with varying LRRC15 gene expression were studied. ABBV-085 was administered at 3 mg/kg (OS33), 6 mg/kg (all seven PDXs), and 12 mg/kg (OS60) weekly for 4 consecutive weeks via intraperitoneal injection. Control cohorts included vehicle and an isotype MMAE-linked antibody. Tumor volumes and responses were reported using PPTC statistical analysis. OS1, OS33, OS42, OS55, and OS60 had high LRRC15 expression while OS9 and OS34 had low LRRC15 expression. ABBV-085 inhibited tumor growth in six of seven PDX models as compared with vehicle control and significantly improved event-free survival in five of seven models as compared with isotype controls. Two models showed maintained complete responses while all others showed progressive disease. Response correlated with LRRC15 expression. ABBV-085’s antitumor activity against osteosarcoma PDX suggests LRRC15 may be a rational target for pursuing clinical trials in patients with this disease.

Purpose: Patients with relapsed pediatric solid malignancies have few therapeutic options, and many of these patients die of their disease. B7-H3 is an immune checkpoint protein encoded by the CD276 gene that is overexpressed in many pediatric cancers. Here, we investigate the activity of the B7-H3–targeting antibody–drug conjugate (ADC) m276-SL-PBD in pediatric solid malignancy patient-derived (PDX) and cell line–derived xenograft (CDX) models. Experimental Design: B7-H3 expression was quantified by RNA sequencing and by IHC on pediatric PDX microarrays. We tested the safety and efficacy of m276-SL-PBD in two stages. Randomized trials of m276-SL-PBD of 0.5 mg/kg on days 1, 8, and 15 compared with vehicle were performed in PDX or CDX models of Ewing sarcoma (N = 3), rhabdomyosarcoma (N = 4), Wilms tumors (N = 2), osteosarcoma (N = 5), and neuroblastoma (N = 12). We then performed a single mouse trial in 47 PDX or CDX models using a single 0.5 m/kg dose of m276-SL-PBD. Results: The vast majority of PDX and CDX samples studied showed intense membranous B7-H3 expression (median H-score 177, SD 52). In the randomized trials, m276-SL-PBD showed a 92.3% response rate, with 61.5% of models showing a maintained complete response (MCR). These data were confirmed in the single mouse trial with an overall response rate of 91.5% and MCR rate of 64.4%. Treatment-related mortality rate was 5.5% with late weight loss observed in a subset of models dosed once a week for 3 weeks. Conclusions: m276-SL-PBD has significant antitumor activity across a broad panel of pediatric solid tumor PDX models.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody–drug conjugated (ADC) with a pyrrolobenzodiazepine dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA-sequencing datasets and performed IHC on neuroblastoma patient-derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays. We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared antitumor activity with expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose–response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3 of 11 and stable disease in another 3 of 11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for antitumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection. Significance: GD2-directed antibody therapy is standard of care for high-risk neuroblastoma; therapy is toxic, and relapses often occur. DLL3, an inhibitory Notch ligand, is overexpressed in several neuronal cancers. A DLL3-targeting ADC showed objective activity only in neuroblastoma models with high DLL3 expression. These data provide vigilance about clinical development of DLL3 immunotherapies for neuroblastoma.

OBJECTIVE: To test associations between grades 3 or 4 (severe) intraventricular hemorrhage (IVH) and single nucleotide polymorphisms (SNPs) associated with coagulation, inflammation, angiogenesis, and organ development in an exploratory study. STUDY DESIGN: Extremely low-birthweight (ELBW) infants enrolled in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network’s (NRN) Cytokines Study were included if they had cranial ultrasound (CUS) and genotyping data available in the NRN Anonymized DNA Repository and Database. Associations between SNPs and IVH severity were tested with multivariable logistic regression analysis. RESULT: One hundred thirty-nine infants with severe IVH and 687 infants with grade 1 or 0 IVH were included. One thousand two hundred seventy-nine SNPs were genotyped. Thirteen were preliminarily associated with severe IVH including five related to central nervous system (CNS) neuronal and neurovascular development. CONCLUSION: Genetic variants for CNS neuronal and neurovascular development may be associated with severe IVH in premature infants.

Background: Youth with Duchenne and Becker muscular dystrophy (DBMD) experience challenges in attaining adult roles, which may impact quality of life. New interventions and treatments may facilitate adult role attainment through improved function. Historical data on adult role attainment is important to assess the impact of new interventions on teens and young adults with DBMD. This study assesses medical knowledge, independence and employment, and relationships among adolescents and young adults with DBMD. Methods: This study uses data from a 2013 Muscular Dystrophy Surveillance, Tracking, and Research Network (MD STARnet) survey on adult transition. Males with DBMD aged 16–30 years were included. Results: Sixty-five of 258 eligible males participated; we report results on 60 participants with an MD STARnet case definition of DMD or BMD. Individuals with BMD reported higher rates than those with DMD of frequently staying home without supervision (50% BMD; 14% DMD), independently performing daily physical needs (93% BMD; 7% DMD) and being employed full or part time (33% BMD; 4% DMD). Most participants understood medication and physical therapy goals; less than half indicated being often or always responsible for scheduling DMBD-related management and refilling medications. Most had not been in a romantic relationship but reported desiring such relationships. Conclusions: Our data reinforce the impact of DMD (and to a lesser extent, BMD) on transition to adult roles. These results provide an important historical comparator for teen and adult patients who are trying new interventions and therapies. Such data are important for assessing the quality-of-life impact of new treatments and to inform support and training programs for people with DBMD as they transition to new adult roles and responsibilities.

Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Furthermore, we tested BT1769, an MT1-MMP–targeted Bicycle toxin conjugate, in osteosarcoma patient-derived xenograft models. The results showed that BT1769 had encouraging antitumor activity, high affinity for its target, and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.

SP-2577(Seclidemstat), an inhibitor of lysine-specific demthylase KDM1A (LSD1) that is overexpressed in pediatric sarcomas, was evaluated against pediatric sarcoma xenografts. SP-2577 (100 mg/kg/day × 28 days) statistically significantly (p < .05) inhibited growth of three of eight Ewing sarcoma (EwS), four of five rhabdomyosarcoma (RMS), and four of six osteosarcoma (OS) xenografts. The increase in EFS T/C was modest (

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited genetic disorder characterized by recurrent and chronic open wounds with significant morbidity, impaired quality of life, and early mortality. RDEB patients demonstrate reduction or structural alteration type VII collagen (C7) owing to mutations in the gene COL7A1, the main component of anchoring fibrils (AF) necessary to maintain epidermal-dermal cohesion. While over 700 alterations in COL7A1 have been reported to cause dystrophic epidermolysis bullosa (DEB), which may be inherited in an autosomal dominant (DDEB) or autosomal recessive pattern (RDEB), the incidence and prevalence of RDEB is not well defined. To date, the widely estimated incidence (0.2-6.65 per million births) and prevalence (3.5-20.4 per million people) of RDEB has been primarily characterized by limited analyses of clinical databases or registries. Methods Using a genetic modelling approach, we use whole exome and genome sequencing data to estimate the allele frequency of pathogenic variants. Through the ClinVar and NCBI database of human genome variants and phenotypes, DEB Register, and analyzing premature COL7A1 termination variants we built a model to predict the pathogenicity of previously unclassified variants. We applied the model to publicly available sequences from the Exome Aggregation Consortium (ExAC) and Genome Aggregation Database (gnomAD) and identified variants which were classified as pathogenic for RDEB from which we estimate disease incidence and prevalence. Results Genetic modelling applied to the whole exome and genome sequencing data resulted in the identification of predicted RDEB pathogenic alleles, from which our estimate of the incidence of RDEB is 95 per million live births, 30 times the 3.05 per million live birth incidence estimated by the National Epidermolysis Bullosa Registry (NEBR). Using a simulation approach, we estimate a mean of approximately 3,850 patients in the US who may benefit from COL7A1-mediated treatments in the US. Conclusion We conclude that genetic allele frequency estimation may enhance the underdiagnosis of rare genetic diseases generally, and RDEB specifically, which may improve incidence and prevalence estimates of patients who may benefit from treatment.

The Pediatric Preclinical Testing Program (PPTP) previously reported the activity of the EZH2 inhibitor tazemetostat (EPZ6438) against xenograft models of rhabdoid tumors. Here, we determined whether an inhibitor of EZH2 enhanced the effect of standard of care chemotherapeutic agents: irinotecan, vincristine, and cyclophosphamide. EPZ011989 significantly prolonged time to event in all the six rhabdoid models studied but did not induce tumor regression. The addition of EPZ011989 to standard of care agents significantly improved time to event in at least one model for each of the agents studied, although this effect was observed in only a minority of the combination testing experiments.

Eltrombopag is a small molecule, thrombopoietin receptor agonist approved for the treatment of patients with aplastic anemia and chronic immune thrombocytopenia. It is also a polyvalent cation chelator and inhibits leukemia cell proliferation via reduction of intracellular iron. The in vivo efficacy of eltrombopag was tested against a panel of six Pediatric Preclinical Testing Consortium osteosarcoma xenografts at doses of 5 mg/kg/day (moderate dose) and 50 mg/kg/day (high dose). Eltrombopag, at moderate doses, failed to significantly improve event-free survival (EFS) in 6/6 models. At high doses, eltrombopag significantly prolonged EFS in 2/2 models, though the effect size was small. All models tested demonstrated progressive disease. While eltrombopag did not meaningfully inhibit osteosarcoma growth, it also did not stimulate tumor growth, suggesting it may be safely investigated as a supportive care agent to enhance platelet recovery post chemotherapy.

Background VTP-50469 is a potent inhibitor of the menin-MLL1 interaction and is implicated in signaling downstream of EWSR1-FLI1. Procedure VTP-50469 was evaluated against seven Ewing sarcoma (EwS) xenograft models and in vitro against EwS cell lines. Results VTP-50469 showed limited antitumor activity, statistically significantly slowing tumor progression in four tumor models but with no evidence of tumor regression. In vitro, the IC50 concentration was 10 nM for the mixed lineage leukemia (MLL)-rearranged leukemia cell line MV4;11, but > 3 μM for EwS cell lines. Conclusions In contrast to its high level of activity against MLL1-rearranged leukemia xenografts, VTP-50469 shows little activity against EwS models.

Background Regorafenib is a small molecule multikinase inhibitor that inhibits multiple kinases including BRAF, KIT, PDGFRB, RAF, RET, and VEGFR1-3. Procedures The in vivo anticancer effects of regorafenib were assessed in a panel of six osteosarcoma models, three rhabdomyosarcoma models, and one Ewing sarcoma model. Results Regorafenib induced modest inhibition of tumor growth in the models evaluated. Conclusion The overall pattern of response to regorafenib appears similar to that of the kinase inhibitor sorafenib, with pronounced slowing of tumor growth in some models, limited to the period of agent administration, being the primary treatment effect.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL subtype with high rates of relapse and poor patient outcome. Activating mutations affecting components of the JAK-STAT signaling pathway occur in the majority of Ph-like ALL cases. The use of JAK inhibitors represents a potential treatment option for Ph-like ALL, although we and others have shown that CRLF2-rearranged Ph-like ALL responds poorly to single-agent JAK inhibitors in the preclinical setting. Therefore, the aim of this study was to identify effective combination treatments against CRLF2-rearranged Ph-like ALL, and to elucidate the underlying mechanisms of synergy. We carried out a series of high-throughput combination drug screenings and found that ruxolitinib exerted synergy with standard-of-care drugs used in the treatment of ALL. In addition, we investigated the molecular effects of ruxolitinib on Ph-like ALL by combining mass spectrometry phosphoproteomics with gene expression analysis. Based on these findings, we conducted preclinical in vivo drug testing and demonstrated that ruxolitinib enhanced the in vivo efficacy of an induction-type regimen consisting of vincristine, dexamethasone, and L-asparaginase in 2/3 CRLF2-rearranged Ph-like ALL xenografts. Overall, our findings support evaluating the addition of ruxolitinib to conventional induction regimens for the treatment of CRLF2-rearranged Ph-like ALL.

The pediatric preclinical testing program previously demonstrated activity of eribulin in osteosarcoma patient-derived xenograft (PDX) models. The phase 2 trial in patients with relapsed osteosarcoma failed to meet response endpoints. Eribulin was evaluated in the original and an expanded set of PDX models and tested at multiple dose levels and schedules to evaluate dose-response. Maximal response was observed at the highest dose, consistent with prior results. The alternative schedule generated similar responses. We demonstrate steep dose-response for eribulin in osteosarcoma PDX models, implying that any deviation from achievement of effective concentrations may have a significant impact on activity.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited genetic disorder characterized by recurrent and chronic open wounds with significant morbidity, impaired quality of life, and early mortality. RDEB patients demonstrate reduction or structural alteration type VII collagen (C7) owing to mutations in the geneUsing a genetic modelling approach, we use whole exome and genome sequencing data to estimate the allele frequency of pathogenic variants. Through the ClinVar and NCBI database of human genome variants and phenotypes, DEB Register, and analyzing premature COL7A1 termination variants we built a model to predict the pathogenicity of previously unclassified variants. We applied the model to publicly available sequences from the Exome Aggregation Consortium (ExAC) and Genome Aggregation Database (gnomAD) and identified variants which were classified as pathogenic for RDEB from which we estimate disease incidence and prevalence.Genetic modelling applied to the whole exome and genome sequencing data resulted in the identification of predicted RDEB pathogenic alleles, from which our estimate of the incidence of RDEB is 95 per million live births, 30 times the 3.05 per million live birth incidence estimated by the National Epidermolysis Bullosa Registry (NEBR). Using a simulation approach, we estimate a mean of approximately 3,850 patients in the US who may benefit fromWe conclude that genetic allele frequency estimation may enhance the underdiagnosis of rare genetic diseases generally, and RDEB specifically, which may improve incidence and prevalence estimates of patients who may benefit from treatment.

Aim To determine if genetic variation associated with decreased dopamine neurotransmission predicts a decrease in motor development in a convenience cohort study of infants born extremely-low-birthweight (ELBW). Method Four hundred and ninety-eight infants born ELBW had genome-wide genotyping and a neurodevelopmental evaluation at 18 to 22 months of age, corrected for preterm birth. A polygenic risk score (PRS) was created to combine into one predictor variable the hypothesized influences on motor development of alleles at seven independent single nucleotide polymorphisms previously associated with relative decreases in both dopamine neurotransmission and motor learning, by summing the number of alleles present in each infant (range=0-14). The motor development outcome was the Psychomotor Development Index (PDI) of the Bayley Scales of Infant Development, Second Edition. The linear regression models were adjusted for seven clinical and four genetic ancestry covariates. The mean PRS of infants with cerebral palsy (CP) was compared to those without CP. Results PRS was inversely related to PDI (p=0.011). Each 1-point increase in PRS resulted in an average decrease in PDI of 1.37 points. Patients with CP did not have a greater mean PRS than those without (p=0.67), both with and without adjustment for covariates. Interpretation Genetic variation that favors a decrease in dopamine neurotransmission predisposes to a decrease in motor development in infants born ELBW, but not to the diagnosis of CP. What this paper adds Genetic variation in dopamine neurotransmission was associated with a decrease in motor development in infants born at an extremely-low-birthweight. It does not predispose to the diagnosis of cerebral palsy.

Introduction WEE1 is a serine kinase central to the G2 checkpoint. Inhibition of WEE1 can lead to cell death by permitting cell-cycle progression despite unrepaired DNA damage. AZD1775 is a WEE1 inhibitor that is in clinical development for children and adults with cancer. Methods AZD1775 was tested using a dose of 120 mg/kg administered orally for days 1 to 5. Irinotecan was administered intraperitoneally at a dose of 2.5 mg/kg for days 1 to 5 (one hour after AZD1775 when used in combination). AZD1775 and irinotecan were studied alone and in combination in neuroblastoma (n = 3), osteosarcoma (n = 4), and Wilms tumor (n = 3) xenografts. Results AZD1775 as a single agent showed little activity. Irinotecan induced objective responses in two neuroblastoma lines (PRs), and two Wilms tumor models (CR and PR). The combination of AZD1775 + irinotecan-induced objective responses in two neuroblastoma lines (PR and CR) and all three Wilms tumor lines (CR and 2 PRs). The objective response measure improved compared with single-agent treatment for one neuroblastoma (PR to CR), two osteosarcoma (PD1 to PD2), and one Wilms tumor (PD2 to PR) xenograft lines. Of note, the combination yielded CR (n = 1) and PR (n = 2) in all the Wilms tumor lines. The event-free survival was significantly longer for the combination compared with single-agent irinotecan in all models tested. The magnitude of the increase was greatest in osteosarcoma and Wilms tumor xenografts. Conclusions AZD1775 potentiates the effects of irinotecan across most of the xenograft lines tested, with effect size appearing to vary across tumor panels.

Purpose: Vincristine combined with camptothecin derivatives showed synergy in preclinical pediatric cancer models, and the combinations are effective in treatment of childhood solid tumors. We determined whether the synergy between vincristine and irinotecan extends to eribulin, another microtubule inhibitor. Experimental Design: Vincristine or eribulin, alone or combined with irinotecan, was studied in 12 xenograft models. Tumor regression and time to event were used to assess antitumor activity. Pharmacodynamic studies and RNA sequencing (RNA-seq) were conducted 24 and 144 hours after single-agent or combination treatment. Effects on vascular development were studied in Matrigel plugs implanted in mice. The interaction between binary combinations was examined in vitro. Results: Eribulin combined with irinotecan was more effective than vincristine–irinotecan in 6 of 12 models. Pharmacodynamic markers induced by eribulin (phospho-histone H3) and irinotecan (γ-H2A.X) were abrogated in combination-treated tumors. The predominant RNA-seq signature in combination-treated tumors was activation of the TP53 pathway with increased nuclear TP53. Massive apoptosis was observed 24 hours only after treatment with the eribulin combination. In vitro, neither combination showed interaction using combination index analysis. Eribulin alone and the combination caused alterations in developing vasculature. Conclusions: The eribulin combination is very active in these xenograft models, but not synergistic in vitro. The combination reduced pharmacodynamic markers indicative of single-agent mechanisms but in tumors, dramatically activated the TP53 pathway. Although a mechanism for in vivo synergy requires further study, it is possible that eribulin-induced inhibition of microtubule dynamics enhances irinotecan-induced nuclear accumulation of TP53, leading to rapid cell death.

The prognosis for children diagnosed with high-risk acute lymphoblastic leukemia (ALL) remains sub-optimal, and more potent and less toxic treatments are urgently needed. We investigated the efficacy of a novel nicotinamide phosphoribosyltransferase inhibitor, OT-82, against a panel of patient-derived xenografts (PDXs) established from high-risk and poor outcome pediatric ALL cases. OT-82 was well-tolerated and demonstrated impressive single agent in vivo efficacy, achieving significant leukemia growth delay in 95% (20/21) and disease regression in 86% (18/21) of PDXs. In addition, OT-82 enhanced the efficacy of the established drugs cytarabine and dasatinib and, as a single agent, showed similar efficacy as an induction-type regimen combining three drugs used to treat pediatric ALL. OT-82 exerted its anti-leukemic action by depleting NAD(+) and ATP, inhibiting the NAD(+)-requiring DNA damage repair enzyme PARP-1, increasing mitochondrial ROS levels and inducing DNA damage, culminating in apoptosis induction. OT-82 sensitivity was associated with the occurrence of mutations in major DNA damage response genes, while OT-82 resistance was characterized by high expression levels of CD38. In conclusion, our study provides evidence that OT-82, as a single agent, and in combination with established drugs, is a promising new therapeutic strategy for a broad spectrum of high-risk pediatric ALL for which improved therapies are urgently needed.

Purpose: OBI-3424 is a highly selective prodrug that is converted by aldo-keto reductase family 1 member C3 (AKR1C3) to a potent DNA-alkylating agent. OBI-3424 has entered clinical testing for hepatocellular carcinoma and castrate-resistant prostate cancer, and it represents a potentially novel treatment for acute lymphoblastic leukemia (ALL). Experimental Design: We assessed AKR1C3 expression by RNA-Seq and immunoblotting, and evaluated the in vitro cytotoxicity of OBI-3424. We investigated the pharmacokinetics of OBI-3424 in mice and nonhuman primates, and assessed the in vivo efficacy of OBI-3424 against a large panel of patient-derived xenografts (PDX). Results: AKR1C3 mRNA expression was significantly higher in primary T-lineage ALL (T-ALL; n = 264) than B-lineage ALL (B-ALL; n = 1,740; P < 0.0001), and OBI-3424 exerted potent cytotoxicity against T-ALL cell lines and PDXs. In vivo, OBI-3424 significantly prolonged the event-free survival (EFS) of nine of nine ALL PDXs by 17.1–77.8 days (treated/control values 2.5–14.0), and disease regression was observed in eight of nine PDXs. A significant reduction (P < 0.0001) in bone marrow infiltration at day 28 was observed in four of six evaluable T-ALL PDXs. The importance of AKR1C3 in the in vivo response to OBI-3424 was verified using a B-ALL PDX that had been lentivirally transduced to stably overexpress AKR1C3. OBI-3424 combined with nelarabine resulted in prolongation of mouse EFS compared with each single agent alone in two T-ALL PDXs. Conclusions: OBI-3424 exerted profound in vivo efficacy against T-ALL PDXs derived predominantly from aggressive and fatal disease, and therefore may represent a novel treatment for aggressive and chemoresistant T-ALL in an AKR1C3 biomarker-driven clinical trial.

Introduction: Toxin-conjugated agents targeting cell-surface antigens (e.g. antibody-drug conjugates) show promising clinical activity for some solid tumors, but none yet for OS. There is an urgent need to identify high-confidence OS cell-surface antigens and evaluate the antitumor activity of toxin conjugates that can deliver cytotoxic payloads to OS tumors expressing these targets. Methods: To identify cell-surface proteins that are highly expressed in OS but not in normal tissues, we prepared surface protein extracts from 4 standard, 4 patient-derived OS cell lines, and 10 OS PDX tissues. Quantitative mass spectrometry (MS) was used to profile the surface proteins. We included the proteomics data of OS and 39 types of normal human tissues via a public MS database (ProteomicsDB). We compared RNA-sequencing data from 101 OS patient samples via the NCI TARGET project with the RNA-sequencing data of 54 types of normal human tissue (GTEx, 17382 samples). We integrated the MS and RNA sequencing datasets to prioritize candidate surface targets by differential and absolute expression at both protein and mRNA levels. The cell-surface expression of the targets was further validated by IHC and flow cytometry with OS cell lines and tumor tissue microarray. BT1769, which consists of a bicyclic peptide targeting the tumor antigen MT1-MMP, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker was tested in 6 OS PDX models using standard PPTC methods at 3 mg/kg given IV weekly x 4. Ewing sarcoma (EWS) models lacking MT1-MMP expression were also tested. Results: MS identified 1156 surface proteins as highly expressed in OS cell lines/PDX tissues. 129 proteins overexpressed in OS were identified from the public MS database. RNA sequencing data identified 195 overexpressed plasma membrane-associated genes in OS compared to normal tissues. The targets identified by both proteomics and transcriptomic data analysis were cross-referenced with existing toxin-conjugated agents. Three potential targets, MT1-MMP, CD276, and MRC2 were validated and MT1-MMP was prioritized because it had the highest absolute expression level in OS. BT1769 significantly inhibited tumor growth in 6/6 OS PDX models when compared to vehicle, with complete response maintained at 6 weeks in 3/6 models. Minimal activity was observed in 2/2 EWS models. Correlation analysis between the MT1-MMP gene expression level, IHC staining intensity and drug response are underway and will be presented. Conclusion: Integrated proteomic and transcriptomic OS surfaceome profiling identified high-confidence OS cell-surface antigens as therapeutic targets. MT1-MMP was highly expressed in most OS patient samples and PDX models, and BT1769 was highly active against select OS PDX models. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana N. Lazcano Segura, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Eric J. Earley, Stephen W. Erickson, Edward A. Kolb, Malcolm A. Smith, Peter Houghton, Raushan Kurmasheva, Tara Gelb, Philip Huxley, Johanna Lahdenranta, Gemma Mudd, Richard Gorlick. Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB008.

Introduction: CD47 is a cell surface marker that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells to negatively regulate phagocytosis. This interaction elicits a “don't eat me” signal preventing phagocytosis by immune cells, enabling cell survival. CD47 is expressed on tumor cells, including acute leukemia cells. AO-176 is a highly differentiated monoclonal antibody that targets CD47, exerts potent in vivo activity in mouse xenograft models, has an established safety profile in cynomolgus monkeys, and is being evaluated in clinical trials. Given the broad expression of CD47 in malignant hematopoietic cells, it was of interest for the Pediatric Preclinical Testing Consortium to evaluate AO-176 in vivo against patient-derived xenograft (PDX) models of pediatric ALL. Methods: CD47 mRNA and protein expression were quantified by RNAseq (https://pedcbioportal.org) and flow cytometry (relative fluorescence intensity, RFI), respectively. AO-176 was tested in vivo against 3 T-ALL (ALL-8, ALL-30, ALL-121) and one early T-cell precursor (ETP) ALL (ETP-2) PDXs in NSG mice at 25 mg/kg intraperitoneally once per week for 4 weeks. Engraftment was evaluated by enumerating the proportion of human CD45+ cells in the peripheral blood (%huCD45+) at weekly intervals. Events were defined as the %huCD45+ ≥ 25%. At Day 28 post-treatment initiation or event (whichever occurred first), 3 mice/group were humanely killed to assess leukemia infiltration of hematolymphoid organs. Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C, and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: CD47 mRNA expression was significantly higher in a panel of T-lineage ALL PDXs (n=25; mean + SD FPKM, 149 + 57.9) compared with B-ALL PDXs (n=65; 65.3 + 18.7 FPKM) (P Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Eric J. Earley, Stephen W. Erickson, Beverly A. Teicher, Victoria Sung, Malcolm A. Smith. The differentiated CD47 monoclonal antibody AO-176 exhibits significant in vivo activity against xenograft models of pediatric acute lymphoblastic leukemia (ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB171.

Introduction: ROR1 is an orphan-receptor tyrosine kinase-like surface antigen that is expressed primarily during development but is not expressed on normal adult tissues. ROR1 is expressed on multiple adult hematologic and solid cancers, making it a target for antibody-based therapies. UC-961 (cirmtuzumab), a humanized IgG1 monoclonal antibody, binds with high affinity to a specific extracellular epitope of human ROR1 and rapidly internalizes and traffics to lysosomes. VLS-101 is an ADC developed by conjugating UC-961 to a cleavable linker with an MMAE payload. VLS-211 was generated by conjugating UC-961 with a PNU payload. VLS-101 is in clinical development for adults with hematologic and solid cancers and has received FDA Fast Track and Orphan Drug designation for the treatment of patients with mantle cell lymphoma. To better define the utility of ROR1 as an immuno-oncology target for pediatric cancers, we evaluated VLS-101 and VLS-211 against pediatric preclinical models selected based on ROR1 expression. Methods: Models were selected based on RNA-seq transcript levels for ROR1 with subsequent confirmation by flow cytometry for acute lymphoblastic leukemia (ALL) models. For the ALL models, UC-961 (5 mg/kg), VLS-101 (2.5 and 5.0 mg/kg), and VLS-211 (0.25 and 0.5 mg/kg) were each administered weekly x 3. For the Ewing sarcoma (EWS) models, the dose of VLS-211 was 0.5 and 1 mg/kg. Four models were treated using a twice-weekly or every 4-day treatment schedule while 2 models were treated weekly. Results: Elevated ROR1 gene expression among PPTC models was most notable among the ALL and EWS panels. Leukemia cell surface expression of ROR1 correlated with transcript levels. ALL models with elevated ROR1 expression [TCF3-HLF (1), TCF3-PBX1 (2), ETV6-RUNX1 (2), and BCR-ABL1 (1)] as well as a non-expressing model were tested against the ROR1 ADCs. Activity of each ADC was ROR1 expression-level dependent, and for VLS-211 dose-dependency was noted. At the highest doses of each ADC studied, 2 of 6 (VLS-101) and 3 of 6 (VLS-211) ROR1-expressing models showed objective responses. For most models, median time to event was longer for VLS-211 versus VLS-101. Five EWS models were studied, and expression-level dependent activity was observed for both ADCs. VLS-101 induced tumor regressions in 2 of 5 models, while VLS211 induced regressions in 3 of 5 models. Time to event was generally longer for VLS-211 compared to VLS-101. No objective responses were observed to UC-961 for the ALL and EWS models. Conclusions: VLS-101 and VLS-211 showed expression-level dependent activity against ALL and EWS models. These results support ROR1 as a relevant immuno-oncology target for the subset of B-ALL and Ewing sarcoma cases with elevated ROR1 expression. Further research is required to identify the optimal payload for ROR1 ADCs for use against pediatric cancers. Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Brian J. Lannutti, Katti A. Jessen, Eric J. Earley, Stephen W. Erickson, Malcolm A. Smith, Raushan Kurmasheva, Peter J. Houghton. Evaluation of ROR1-targeted antibody-drug conjugates against ROR1-expressing pediatric preclinical models - a report from the pediatric preclinical testing consortium (PPTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3038.

Introduction: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains poor. Spleen Tyrosine Kinase (SYK), a cytosolic nonreceptor tyrosine kinase, is primarily expressed in the hematopoietic lineage and is essential for B-cell receptor signaling. It is associated with malignant transformation, cancer cell proliferation, and is a prominent tyrosine-phosphorylated protein in B-lineage pediatric ALL (Dolai et al, Cancer Res 76:2766-77, 2016). Fms Related Receptor Tyrosine Kinase 3 (FLT3) is a Class III receptor tyrosine kinase that regulates hematopoiesis. While uncommon in ALL, activating FLT3 mutations and internal tandem duplications are associated with poor outcome. TAK-659 is a dual SYK/FLT3 reversible inhibitor currently undergoing clinical trials against B-cell lymphoma, acute myeloid leukemia and solid tumors. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium to test TAK-659 in vivo against its patient-derived xenograft (PDX) models of pediatric ALL. Methods: SYK and FLT3 mRNA expression in ALL PDXs was quantified by RNAseq (https://pedcbioportal.org) and 8 B-lineage pediatric ALL PDXs (3 B-cell precursor, BCP; 4 mixed-lineage leukemia-rearranged, MLLr; one Philadelphia Chromosome-like, Ph-like) were selected for testing. Engraftment was evaluated by enumerating the proportion of human CD45+ cells (%huCD45+) in the peripheral (PB) blood at weekly intervals. TAK-659 was tested at 60 mg/kg by oral gavage daily for 21 days. Events were defined as %huCD45+ ≥ 25%. At Day 28 post-treatment initiation or event (whichever occurred first), 4 mice/group were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: The in vivo efficacy of TAK-659 was evaluated against B-lineage PDXs as FLT3 and SYK mRNA expression was higher in the PDXs from this lineage compared with PDXs from T-ALL. TAK-659 was well tolerated and significantly prolonged the time to event in 6/8 PDXs (T-C 0-25.5 days, T/C 1.0-2.2). Only one MLLr-ALL obtained an objective response (partial response) with all other models exhibiting progressive disease. The minimum mean %huCD45+ was significantly reduced in 5/8 PDXs in TAK-659 treated mice compared to control. Despite significant reductions in %huCD45+ in the PB for 5/8 PDXs, TAK-659 did not significantly reduce the spleen or BM infiltration of any PDXs. Conclusion: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric B-ALL PDXs representative of diverse disease subtypes. (Supported by NCI Grants CA199000 and CA199922) Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Eric J. Earley, Stephen W. Erickson, Beverly A. Teicher, Malcolm A. Smith. Pediatric preclinical testing consortium evaluation of the dual SYK/FLT3 inhibitor TAK-659 in xenograft models of pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3039.

Background. 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). Copanlisib was approved for use by the FDA in the treatment of refractory follicular lymphoma in 2017. Preclinical data have suggested that deregulation of the PI3K pathway is a key driver of multiple malignancies. The relevance of this pathway in osteosarcoma tumorigenesis remains a subject of debate, however, whole genome and RNA sequencing data have revealed several PI3K aberrations in osteosarcoma tumor samples. The in vivo effects of copanlisib were studied in the Pediatric Preclinical Testing Consortium osteosarcoma xenograft models. Methods. The in vivo anticancer effects of copanlisib were assessed in a panel of 6 osteosarcoma models (OS-2, OS-9, OS-31, OS-33, OS-36, and OS-60). Copanlisib was administered by oral gavage at a dose of 10 mg/kg/day, two days on and five days off, repeated weekly for 4 weeks. Time to event and tumor volume responses were defined and analyzed utilizing standard PPTC statistical methods. Results. Copanlisib was well tolerated in the models with minimal weight loss and no treatment related mortality as compared to controls. Copanlisib induced prolonged event-free survival (EFS) in 5/6 osteosarcoma models (p Citation Format: Douglas Harrison, Jonathan Gill, Pooja Hingorani, Michael Roth, Wendong Zhang, Beverly Teicher, Eric J. Earley, Stephen W. Erickson, Gregory Gatto, Raushan T. Kurmasheva, Peter J. Houghton, Malcolm A. Smith, Edward A. Kolb, Richard Gorlick. Evaluation of the pan-class I phosphoinositide 3-kinase (PI3K) inhibitor copanlisib in the Pediatric Preclinical Testing Consortium osteosarcoma in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB252.

10037 Background: B7-H3 (encoded by the CD276 gene) is an immunoregulatory molecule that is widely expressed in pediatric embryonal tumors and sarcomas, while expression is limited for normal tissues. Among PPTC models, osteosarcoma models showed highest CD276 transcript levels followed by Wilms tumor, neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Protein expression by IHC generally followed expression at the RNA level, with high expression by IHC observed across a range of models. MGC018 is a duocarmycin-based humanized ADC targeting B7-H3 that shows selective cytotoxicity for B7-H3 expressing cancer cells, robust in vivo activity against a range of adult cancer preclinical models, a favorable pharmacokinetic and safety profile in cynomolgus monkeys, and has entered clinical testing for adults with cancer. Herein we report the in vivo antitumor activity of MGC018 against preclinical models of pediatric solid tumors. Methods: MGC018 was tested at 6 mg/kg administered intraperitoneally (IP) on Day 1. SYD988, an anti-CD20 ADC with the same linker and payload as MGC018, was used as a control arm at 6 mg/kg IP on Day 1. Osteosarcoma models, due to their slower growth kinetics and lower rates of tumor regression, were tested with n = 10 mice per arm, while the sarcoma and neuroblastoma models were tested with n of 1 or n of 2 designs, respectively. Activity was evaluated using response criteria mirroring clinical criteria (progressive disease (PD), stable disease (SD), partial response (PR), complete response (CR), and maintained CR (MCR)], by minimum relative tumor volume across all models, and by event-free survival (EFS) comparisons between treatment groups. Results: For neuroblastoma models (n = 9), 3 and 2 neuroblastoma models showed objective responses (PR/CR/MCR) or SD, respectively, to MGC018, while none showed an objective response or SD to SYD988. For osteosarcoma, results are mature for 1 of 5 models, with this model showing an MCR response to MGC018 and a PD response to SYD988. For Ewing sarcoma (n = 5), a PR and MCR were observed to MGC018 with the remaining models showing PD; for SYD988 a single CR was observed with the remaining 4 models showing PD. For rhabdomyosarcoma (n = 3) 2 MCR and 1 PD were noted for MGC018, while only PD was noted for SYD988. EFS duration exceeding 100 days to a single dose of MGC018 was observed for 2 rhabdomyosarcoma, 1 Ewing sarcoma, and 1 neuroblastoma model. Evaluation of the relationship between B7-H3 expression and response to MGC018 will be performed when results from all studies are completed. Conclusions: B7-H3 is an important target for immuno-oncology agents for childhood cancers. Our results provide proof-of-principle for the ability of MGC018 to produce profound antitumor activity in select pediatric solid tumor models in a B7-H3 specific manner.

CD4+ T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4+ T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4+ T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5 mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4+ T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4+ T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4+ T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4+ T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4+ T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene.