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Subcutaneous patient-derived xenografts (PDXs) are an important tool for childhood cancer research. Here, we describe a resource of 68 early passage PDXs established from 65 pediatric solid tumor patients. Through genomic profiling of paired PDXs and patient tumors (PTs), we observe low mutational similarity in about 30% of the PT/PDX pairs. Clonal analysis in these pairs show an aggressive PT minor subclone seeds the major clone in the PDX. We show evidence that this subclone is more immunogenic and is likely suppressed by immune responses in the PT. These results suggest interplay between intratumoral heterogeneity and antitumor immunity may underlie the genetic disparity between PTs and PDXs. We further show that PDXs generally recapitulate PTs in copy number and transcriptomic profiles. Finally, we report a gene fusion LRPAP1-PDGFRA. In summary, we report a childhood cancer PDX resource and our study highlights the role of immune constraints on tumor evolution. Subcutaneous patient-derived xenografts are a common tool in cancer research. Here, the authors compare 65 paired early passage xenografts to their original paediatric tumour and show clonal evolution determines seeding of the xenograft.

Patient-derived xenograft (PDX) models are widely used in cancer research. Genomic and transcriptomic profiling of PDXs are inevitably contaminated by sequencing reads originated from mouse cells. Here, we examine the impact of mouse read contamination on RNA sequencing (RNAseq), Whole Exome Sequencing (WES), and Whole Genome Sequencing (WGS) data of 21 PDXs. We also systematically benchmark the performance of 12 computational protocols for removing mouse reads from PDXs. We find that mouse read contamination increases expression of immune and stromal related genes, and inflates the number of somatic mutations. However, detection of gene fusions and copy number alterations is minimally affected by mouse read contamination. Using gold standard datasets, we find that pseudo-alignment protocols often demonstrate better prediction performance and computing efficiency. The best performing tool is a relatively new tool Xengsort. Our results emphasize the importance of removing mouse reads from PDXs and the need to adopt new tools in PDX genomic studies.

B-cell lymphoma extra large (BCL-X L ) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-X L inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-X L proteolysis-targeting chimera (PROTAC), that targets BCL-X L to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-X L -dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-X L . The first BCL-X L -degrading PROTAC achieves safer and more potent antitumor activity than dual BCL-X L and BCL-2 inhibitor navitoclax because of reduced dose-limiting platelet toxicity and high target specificity.

BackgroundMetastatic castration-resistant prostate cancer (mCRPC) and Ewing sarcoma (EWS) are diseases for which immune therapies could potentially provide benefit. STEAP1 (Six Transmembrane Epithelial Antigen of the Prostate 1) is a cell surface protein with elevated expression in mCRPC 1 and EWS.2 MethodsWe designed AMG 509, a novel, half-life extended, STEAP1 x CD3 XmAb® 2+1 bispecific antibody to induce T cell-mediated cytotoxicity against STEAP1-expressing cancer cells. AMG 509 contains two identical anti-STEAP1 Fab domains, an anti-CD3 scFv domain, and an effectorless Fc domain that extends serum half-life. We characterized STEAP1 expression in normal and tumor tissues by immunohistochemistry, and we assessed the pharmacological properties of AMG 509 including binding, T cell-mediated redirected lysis, and in vivo antitumor activity.ResultsWe detected high STEAP1 surface expression on 80% of primary prostate tumors (n=88), 89% of mCRPC lesions (n=114), including 84% of mCRPC bone metastases (n=31), and 63% of EWS samples (n=35). In contrast, in normal tissues (n=72), low STEAP1 expression was detected in only six other tissues, including the normal prostate. AMG 509 bound to recombinant human CD3ε with a KD of 27.6 nM, and it bound specifically to 293T cells transfected with human STEAP1 with an EC50 of 3.8 nM. AMG 509 triggered potent T cell-redirected lysis of STEAP1-positive cancer cells, with a median EC50 of 37 pM across 19 cancer cell lines that endogenously express various levels of STEAP1. AMG 509-mediated cytotoxicity was specific, as it showed no activity against prostate cancer cells in which STEAP1 was knocked out. AMG 509 was 65-fold more potent in inducing the redirected lysis of prostate cancer cells in vitro than an XmAb® molecule with a single anti-STEAP1 Fab domain. AMG 509 had greater cytotoxic activity against high STEAP1-expressing cancer cells than against low STEAP1-expressing cancer cells, and it had minimal activity against normal cells. This preferential killing of high STEAP1-expressing cells is likely driven by the avidity conferred by the dual STEAP1-binding domains, a feature that may help reduce off-target effects in the clinic. In vivo, AMG 509 induced robust anti-tumor activity in prostate cancer and EWS mouse xenograft models, with concomitant CD8+ T-cell activation and expansion in tumors.ConclusionsAMG 509 is a specific, first-in-class T cell-recruiting antibody with avidity-driven activity against STEAP1-positive malignancies. AMG 509 is currently being evaluated for safety, pharmacokinetics, and efficacy in a phase 1, first-in-human study in patients with mCRPC (NCT04221542).AcknowledgementsThe authors acknowledge Micah Robinson, PhD of Amgen Inc. for medical writing support.Trial RegistrationClinicalTrials. gov Identifier: NCT04221542Ethics ApprovalAll animal experimental protocols were approved by an Institutional Animal Care and Use Committee (IACUC protocol number 2015-01243) and were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) (Amgen) or the standards of the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (IACUC protocol number 15015x) in a facility certified with an Office of Laboratory Animal Welfare (OLAW) (UTHSA).ReferencesGrunewald TGP, Ranft A, Esposito I, Silva-Buttkus P da, Aichler M, Baumhoer D, Schaefer KL, Ottaviano L, Poremba C, Jundt G, Jurgens H, Dirksen U, Richter GHS, Burdach S. High STEAP1 expression is associated with improved outcome of Ewing’s sarcoma patients. Ann Oncol 2012; 23:2185–2190.Hubert RS, Vivanco I, Chen E, Rastegar S, Leong K, Mitchell SC, Madraswala R, Zhou Y, Kuo J, Raitano AB, Jakobvits A, Saffran SC, Afar DE. STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA 1999;96:14523–14528.

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5′ end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.

B-cell lymphoma extra large (BCL-X ) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-X inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-X proteolysis-targeting chimera (PROTAC), that targets BCL-X to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-X -dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-X .

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5’ end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2- F1025Y,R1128G) whose product generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2- F1025Y,R1128G allele is associated with a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that over-express the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2. DNA-strand separation during transcription and replication creates topological problems that are resolved by topoisomerases. These enzymes nick DNA strands to allow strand passage and then reseal the broken DNA to restore its integrity. Topoisomerase II (Top2) nicks complementary DNA strands to create double-strand break (DSBs) intermediates that can be stabilized by chemotherapeutic drugs and are toxic if not repaired. We identified a mutant form of yeast Top2 that forms stabilized cleavage intermediates in the absence of drugs. Over- expression of the mutant Top2 was associated with a unique mutation signature in which small (1-4 bp), unique segments of DNA were duplicated. These de novo duplications required the nonhomologous end-joining pathway of DSB repair, and their Top2-dependence has clinical and evolutionary implications.