ABSTRACT Measuring the activity of the tumor suppressor p53 in living systems is essential for understanding its dysregulation in cancer and other conditions, such as aging and diabetes. Zebrafish ( Danio rerio ) are a powerful vertebrate model that enable such studies, due to the evolutionary conservation of p53 structure and function. However, p53 activity in zebrafish has mainly been assessed using pharmacological methods that induce DNA damage or have off-target effects, making it difficult to isolate p53-specific responses from broader stress responses. Here, by using biophysical assays, molecular dynamics, and molecular assays, we show that sulanemadlin, a stapled peptide inhibitor of MDM2, binds to zebrafish Mdm2 and transcriptionally activates downstream targets of p53, including cdkn1a , isoform Δ113p53 , and Mdm2 . No effect on gene expression was observed in embryos treated with a point-modified control peptide or in embryos carrying a mutation that renders p53 transcriptionally inactive. RNA sequencing further confirmed upregulation of p53 signaling and downregulation of DNA replication pathways, while an acridine orange assay showed no detectable increases in apoptosis. In contrast, the tested small molecule Mdm2 inhibitors exhibit reduced binding affinity to zebrafish Mdm2 due to an amino acid variation in the zebrafish Mdm2 binding pocket. By overcoming a species-specific barrier in p53-MDM2 binding, the stapled peptide sulanemadlin is the first pharmacological tool to specifically activate p53 in zebrafish without inducing measurable apoptosis, enabling direct in vivo studies of p53 regulation in cancer and other disease contexts.

ABSTRACT:Myeloid leukemia of Down syndrome (ML-DS) is a distinct form of pediatric acute myeloid leukemia (AML) that responds to reduced-intensity chemotherapy, as compared with non-DS AML that requires intensive chemotherapy and often stem cell transplant. While most patients with ML-DS have a favorable prognosis, outcomes for those with refractory or relapsed disease are dismal. Children's Oncology Group study AAML1531 introduced the use of measurable residual disease by multiparameter flow cytometry at the end of the first course of induction therapy (EOI-1 MRD) for risk stratification of treatment intensity. Of 280 patients with ML-DS who were enrolled, 41 were classified as high risk (HR) due to positive EOI-1 MRD, and treated with intensified chemotherapy similar to that used for pediatric non-DS AML. Treatment intensification did not improve the 2-year event-free survival compared with patients who were MRD-positive treated with reduced-intensity therapy in the predecessor study AAML0431 (80.5% ± 12.4% vs 76%; P = .247) or overall survival (80.5% ± 12.4% vs 76.2% ± 18.6%; P = .819), but significantly increased the frequency of febrile neutropenia and sepsis events. While stratification of treatment intensity based on MRD was not beneficial, molecular markers of relapse risk proposed by the Japan Children's Cancer Group for ML-DS (alterations of CDKN2A, ZBTB7A, JAK2, TP53) proved prognostic. Relapse risk was 50% in patients who were HR from AAML1531 with any high-risk molecular marker compared with 6.7% in those without. Similar relapse results were obtained in the MRD-negative AAML1531 group, suggesting molecular risk markers can predict outcome and thus be used to stratify therapy in ML-DS. This trial was registered at www.clinicaltrials.gov as #NCT02521493.

Larval zebrafish drug efficacy studies for patients with no available mouse PDX. A to C, Patient information, alongside larval zebrafish tumor cell numbers for each therapy for rhabdomyosarcoma zccs170 (A), gastrointestinal stromal tumor zccs15 (B), and Ewing sarcoma zccs276 (C). For larval zebrafish data, error bars represent mean ± SEM, each colored dot represents an individual larva, and a threshold is based on SEM of 1 dpi. Statistical analysis was conducted using a one-way ANOVA with the Dunnett multiple comparisons test, comparing the mean of each treatment group with control (4 dpi), P values: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001. D, Representative image of a five microns sectioned control larval zebrafish xenograft for patient zccs170. Deep Red–labeled patient cells present in the YS 4 dpi and labeled with DAPI to confirm viability. White circle in the images point to zccs170 tumor cells. Images were taken with 10× and 20× magnification. The scale bar is 100 µm. dpi, days post-injection.

Single agent and combination doses established in the larval zebrafish PDX at 35°C for 3 to 7 days post fertilization zebrafish for 72-hour treatment.

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with a highly penetrant cancer spectrum characterized by germline TP53 mutations. We characterized the first LFS zebrafish hotspot mutants, tp53 R217H and R242H (human R248H and R273H), and found these mutants exhibit partial-to-no activation of p53 target genes, have defective cell-cycle checkpoints, and display partial-to-full resistance to apoptosis, although the R217H mutation has hypomorphic characteristics. Spontaneous tumor development histologically resembling human sarcomas was observed as early as 6 months. tp53 R242H mutants had a higher lifetime tumor incidence compared to tp53 null and R217H mutants, suggesting it is a more aggressive mutation. We observed mutation-specific tumor phenotypes across tp53 mutants with associated diverse transcriptomic and DNA methylome profiles in tp53 mutant larvae, impacting metabolism, cell signalling, and biomacromolecule synthesis and degradation. These tp53 zebrafish mutants demonstrate fidelity to their human counterparts and provide new insights into underlying tumorigenesis mechanisms and kinetics that suggest metabolic rewiring and cellular signalling changes occur prior to tumor initiation, which will guide targeted therapeutics for LFS.

Genomics has transformed the diagnostic landscape of pediatric malignancies by identifying and integrating actionable features that refine diagnosis, classification, and treatment. Yet, translating precision oncology data into effective therapies for hard-to-cure childhood, adolescent, and young adult malignancies remains a significant challenge. We present the case for combining proteomics with patient-derived xenograft models to identify personalized treatment for an adolescent with primary and metastatic spindle epithelial tumor with thymus-like elements (SETTLE). Within two weeks of biopsy, proteomics identified elevated SHMT2 as a target for therapy with the anti-depressant sertraline. Drug response was confirmed within two months using a personalized chicken chorioallantoic membrane model of the patient’s SETTLE tumor. Following failure of cytotoxic chemotherapy and second-line therapy, the patient received sertraline treatment and showed decreased tumor growth rates, albeit with clinically progressive disease. We demonstrate that proteomics and fast-track xenograft models provide supportive pre-clinical data in a clinically meaningful timeframe to impact clinical practice. By this, we show that proteome-guided and functional precision oncology are feasible and valuable complements to the current genome-driven precision oncology practices.

Despite advances in precision medicine, 30% of high-risk pediatric cancers lack an actionable molecular target, hindering effective treatment and affecting survival outcomes. Although mouse patient-derived xenograft (PDX) models offer additional insights into clinical drug responses, delivering findings from these models within a clinically actionable time frame remains challenging. This international collaboration between two national precision medicine programs demonstrates proof-of-principle that individualized larval zebrafish PDXs can robustly and rapidly assess clinical responses in high-risk pediatric cancers. Retrospective zebrafish PDX testing was performed on tumor samples from 10 pediatric patients with high-risk cancers. Drug responses in zebrafish models were correlated with clinical responses for each patient and directly compared with responses in cognate mouse PDX models. Responses to conventional and targeted therapies, administered as single agents or in combinations, were assessed. Zebrafish PDXs were successfully established from all 10 patients and provided robust drug response data in every case, including from three patients whose tumor samples could not be engrafted in mice. Remarkably, zebrafish models accurately recapitulated patient responses for 11 of 12 treatment regimens. These findings highlight the potential of larval zebrafish PDX models to provide real-time, clinically relevant drug response data, supporting their potential use in prospective precision medicine studies.Significance: This proof-of-principle study is the first to compare drug responses in larval zebrafish and mouse PDX models with patient outcomes in pediatric precision oncology, showing high concordance. Results highlight the potential of zebrafish PDX models to predict drug responses in high-risk cancers more accurately, rapidly, and cost-effectively in prospective studies.

Consistency in drug response between patients and their cognate larval zebrafish and mouse PDX models. Evaluable response to therapy was compared for each patient and their cognate larval zebrafish PDX and mouse PDX models. Patient and mouse responses are indicated as OR criteria. Larval zebrafish PDX models are indicated as response (inhibition of cell growth), SD (cell number maintained), or PD (cell growth in the presence of drug). Maintained CR, CR, and PR for patient and mouse are considered equivalent to R (response) in the larval zebrafish model. The concordance of response for each model type with patient response is summarized in the legends on the right. ***For zccs373 (nonresponder to triple combination), mouse PDX responded to dual combination. MCR, maintained complete response; NT, not treated; zf, zebrafish.

Purpose: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. Methods: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. Results: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. Conclusion: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients. (c) 2024 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines.

Abstract Translation of precision oncology data into feasible precision therapies for hard-to-cure childhood, adolescent and young adult malignancies remains a significant challenge. Identifying therapeutic targets at the protein and pathway level and demonstrating treatment response in personalized models hold great promise, particularly for combination therapies, but may be considered complex and time consuming. Here, we present the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how proteomics combined with rapid patient-derived models can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. Elevated SHMT2 levels were validated by immunohistochemistry and compared to levels across AYA tumors. Based on the suitability for an innovative therapy trial, we prioritized single-agent sertraline, a commercially available anti-depressant medication that inhibits SHMT2, and confirmed a positive drug response in both chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Retrospective expansion in a murine xenograft enabled metabolic tracing on isolated SETTLE- patient-derived xenograft cells using 13C6-glucose confirming SHMT2 activity and response to in vitro treatment. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial of sertraline was initiated by the patient but stopped after 8 weeks after evidence of progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Combining sertraline with the common antibiotic trimethoprim, resulted in enhanced growth inhibition of SETTLE cells in the larval zebrafish xenografts. Significance: Overall, we demonstrate that proteomics and personalized xenograft models may provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact clinical practice. Citation Format: Georgina D. Barnabas, Tariq A. Bhat, Verena Goebeler, Pascal Leclair, Nadine Azzam, Nicole Melong, Jason N. Berman, Jennifer A. Chan, Donna L. Senger, Seth Parker, Christopher A. Maxwell, Gregor S. Reid, Jonathan Bush, Caron Strahlendorf, Rebecca Deyell, C James Lim, Philipp F. Lange. Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 937.

e23195 Background: Pediatric patients with relapsed or refractory malignancy have few options. The quality of life (QoL) impact for enrolling in a phase I or II trial compared to those not participating is unknown. This information is invaluable to the consenting process as well as to clinical trial design. The primary aim was to determine if those enrolled on a phase I or II trial had lower scores on the Symptom Screening in Pediatrics Tool (SSPedi) total score compared to those not enrolled on a trial. Methods: This Canadian multi-site study had nine sites open for accrual. SSPedi was calculated based on patient reports for 8-18 years and parent-proxy reports for all children. The total score is the sum of 15 items’ 4 Likert scale scores, score ranges from 0 to 60 (worst possible). Scores from baseline, 4 weeks and 8 weeks of those enrolled on an early phase trial were compared to those who did not enroll in such trials. Results: Of the 54 49 patients, 21 (43%) patients were enrolled on an early phase trial and 28 (57%) patients were not enrolled in a study. For those enrolled on the study at enrollment time, 4 weeks and 8 weeks time points, the total SSPedi score for the participant mean was 11.1 (SD 8.1), 9.2 (SD 6.3) and 10.1 (8.3) respectively compared to those not enrolled on a study with mean scores at the same time points of 13.8 (SD 8.6), 13.5 (SD 9.4), and 15.4 (SD 12.6). Conclusions: These results suggest that it is feasible to evaluate patients enrolled and not enrolled on early phase trials, and to compare their symptom experience. Further efforts will focus on more recruitment and using the PedsQL 3.0 Acute Cancer Module to further define differences in QoL. The results suggest that those enrolled in an early phase trial had an improved quality of life compared to those non-enrolled. This research is supported through granting from the C17 Council and Kindred Foundation. [Table: see text]

Background . Myeloid leukemia of Down syndrome (ML-DS) is a distinct form of pediatric AML that is treated with DS-specific reduced intensity chemotherapy with a resulting favorable prognosis (5-year EFS 89.9%). In contrast, non-responders and patients with relapsed ML-DS have a dismal outcome. The aim of Children's Oncology Group (COG) study AAML1531 was introduce risk stratification of chemotherapy intensity for patients with ML-DS based on measurable residual disease by multi-parameter flow cytometry at the end of the first course of induction (EOI-1 MRD), which is used to risk stratify treatment intensity for non-DS pediatric AML patients and was prognostic in the preceding ML-DS trial, AAML0431. Methods . AAML1531 enrolled 280 patients with ML-DS between November 2015 and April 2022. All patients received the same first course of induction therapy (Induction I): daunorubicin, cytarabine, 6-thioguanine (DAT). Those with EOI-1 MRD <0.05% were classified as Standard Risk (SR, Arm A) and treated with reduced-intensity chemotherapy based on the historical control, AAML0431, but with elimination of the second induction course of AAML0431 therapy, which consisted of high-dose cytarabine/asparaginase, to reduce infectious events. Patients with EOI-1 MRD >0.05% were classified as High Risk (HR, Arm B) and had their subsequent treatment intensified to a level consistent with that of pediatric non-DS AML (Induction II: mitoxantrone/high-dose cytarabine; Intensification I: cytarabine/etoposide; Intensification II: high-dose cytarabine/asparaginase) with the aim of reducing the number of relapse events. Cytogenetic results were centrally reviewed and MRD was measured by multidimensional flow cytometry in a reference laboratory (Hematologics, Inc., Seattle, WA). Results.We previously reported outcomes for the SR group (n=114) (Hitzler et al. Blood 2021). We now report outcomes of patients in the ML-DS HR group (n=41). Efficacy: Intensification of chemotherapy for EOI-1 MRD-positive patients did not significantly improve the 2-year EFS compared to that of the AAML0431 EOI-1 MRD-positive cohort (AAML1531: 80.5 + 12.4% vs. AAML0431: 76%, p=0.247). OS also did not differ significantly (AAML1531: 80.5 + 12.4% vs. AAML0431: 76.2 + 18.6%, p=0.819). There were 7 relapses and 1 death as first event. Of the 7 patients with relapse, 6 did not survive (2-year-OS 14.3 + 26.5% after relapse). Adverse events: Febrile neutropenia (FN) occurred in 25.4% of all AAML1531 patients during the common Induction I phase. During intensified post-induction therapy on the HR arm, the course-specific proportions of FN were 31.7% of 41 patients (Induction II), 27.5% of 40 patients (Intensification I) and 26.3% of 38 patients (Intensification II). The corresponding proportions on the reduced-intensity SR arm were significantly lower than on the HR arm: 3.7% of 108 patients (Induction II, p<0.001), 6.9% of 101 patients (Induction III, no corresponding HR course), 6.1% of 98 patients (Intensification I, p=0.001) and 8.6% of 93 patients (Intensification II, p=0.008). Sepsis grade 3 or greater was reported in 9.8% of 41 patients (Induction II, p=0.005) treated on HR arm compared to none treated on the SR (Ind II, n=108), and in 3.3% in the common Induction phase (Induction I). Conclusions. Intensification of chemotherapy for patients with ML-DS with positive EOI-1 MRD neither improved EFS nor OS and resulted in more FN and a greater number of sepsis events. While EOI-1 flow cytometric MRD detected ML-DS patients whose outcomes were poorer than those without MRD (Taub et al. Blood 2017), intensification of chemotherapy was not beneficial to the MRD-positive group. Overall, results of AAML1531 demonstrate that stratification of treatment intensity according to flow cytometric EOI-1 MRD did not did not improve outcomes for patients with ML-DS. Alternative approaches such as mutational profiling of ML-DS blasts should be evaluated with regard to prognostication, risk stratification and identification of targets for novel agents to improve the overall outcome for this disease.

Abstract Personalized medicine is revolutionizing cancer detection, characterization, and treatment, however, 30% of children with high-risk cancers lack an actionable molecular target and need alternate approaches to identify personalized treatment recommendations. While mouse patient-derived xenografts (PDXs) are widely regarded as the gold standard for preclinical drug response prediction, they remain highly resource-intensive, challenging to establish, and often have establishment times outside clinically relevant timeframes. The larval zebrafish model has gained prominence as a promising tool for personalized medicine, however, drug treatment responses in zebrafish and mouse PDX models, and in their cognate patients, have not previously been compared. Here, we present data from an international collaboration between two national child cancer precision medicine programs: Canada’s PRecision Oncology For Young peopLE (PROFYLE) and Australia’s Zero Childhood Cancer (ZERO), which, for the first time, directly compares single agent and combination treatment responses using individualized models in larval zebrafish, mouse, and short-term culture, with patient clinical responses. Samples were collected from ten high-risk childhood/adolescent cancer patients (aged 1.5-15 years) diagnosed with various tumor types and enrolled on ZERO for molecular profiling (whole genome and whole transcriptome sequencing analysis), and in vitro high throughput drug screening (HTS). Mouse PDX drug testing was guided by prior molecular sequencing findings, single agent HTS, and treatments received by each patient. Samples from these patients underwent retrospective zebrafish PDX testing. Labeled tumor cells were engrafted into the zebrafish larvae yolk sac at 48 hours, treated for 3 days from 72 hours by immersion, followed by ex vivo tumor cell quantification for drug response evaluation. Larval zebrafish models were successfully established for all ten patients, including three for whom a mouse PDX model was not able to be developed. Additionally, samples from three of the ten patients underwent a secondary in vitro screen of single drugs and drug combinations, for comparison with responses in larval zebrafish and mouse PDXs. Remarkably, a high degree of concordance was observed between evaluable patient responses and responses observed in preclinical models developed from the patient, with 10/11 zebrafish, 7/8 mouse and 3/3 short-term cultures recapitulating responses in patients. The larval zebrafish workflow was less than one week from engraftment to completion, comparable to direct HTS from patient samples, and far quicker than establishing mouse PDX models. These findings represent the first pediatric precision oncology study to demonstrate consistent and clinically informative drug responses across multiple modalities in successfully predicting drug responses in high-risk child cancer patients, and suggest the feasibility of the larval zebrafish PDX as an efficient preclinical tool for patient-specific therapeutic decision-making. Citation Format: Nadine Azzam, Jamie I. Fletcher, Nicole Melong, Loretta Lau, Emmy M. Dolman, Jie Mao, Gabor Tax, Roxanne Cadiz, Lissandra Tuzi, Alvin Kamili, Biljana Dumevska, Jinhan Xie, Jennifer A. Chan, Donna L. Senger, Stephanie A. Grover, David Malkin, Michelle Haber, Jason N. Berman. High-risk pediatric cancer models in zebrafish, mouse and short-term culture predict individual patient responses to therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B007.

Mutations in the DNAJC21 gene were recently described in Shwachman-Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.

Inherited bone marrow failure syndromes (IBMFS) are a clinically diverse group of rare genetic disorders characterized by cytopenia of one or more hematopoietic lineages. IBMFS account for 10-15% of marrow aplasia and >50% of chronic pediatric bone marrow failure disorders. Definitive treatment for all IBMFS currently requires allogeneic hematopoietic stem cell transplantation. However, post-transplant complications such as organ toxicity and engraftment failure are common in this population. A thorough understanding of the underlying disease biology would enable the development of targeted therapeutic interventions that could rescue marrow failure, and potentially prevent progression to myeloid malignancies. We hypothesized that in addition to the transcriptional dysregulation, known to underpin IBMFS, perturbations in metabolism are essential for the transition of hematopoietic cells from a state of hypo-proliferation in IBMFS to one of hyperproliferation in leukemia. Given the rarity of IBMFS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Zebrafish (Danio rerio) are ideal given their highly conserved and rapid hematopoiesis and access to early blood progenitors. Here, we used loss-of-function zebrafish mutants to study two IBMFS subtypes with increased propensity for malignancies, namely, DNAJC21-mutant Shwachman-Diamond syndrome (SDS) and PARN-mutant dyskeratosis congenita (DC). We recently showed that in dnajc21-/- embryos, poor DNA damage responses caused by nucleotide deficiency impedes cell cycle progression, contributing to neutropenia. Treatment of dnajc21-/- embryos with 100 mM uridine or thymidine nucleoside relieved the cell cycle block and restored neutrophil counts (Ketharnathan et al. Leukemia, in press). We extended these findings to our parn-/- zebrafish that also present with neutropenia and anemia at 48 hours post-fertilization (hpf). Reduced telomerase activity and shortened telomeres are inherent features of DC. Zebrafish telomere lengths are strikingly similar to that of humans (5-15 kb in zebrafish versus 20-150 kb in mice). Preliminary analysis revealed reduced telomerase activity in parn-/- whole kidney marrows (WKMs, human bone marrow equivalent) by 12 months of age. Thymidine treatment has been shown to support telomere elongation in human cells. Hence, we treated parn-/- embryos with thymidine (100 mM from 3 to 48 hpf). We found that thymidine treatment rescued neutropenia, but only partially improved erythrocyte counts, suggesting differences in underlying mechanisms. In addition to nucleotide imbalance, metabolomic analyses of dnajc21-/- embryos and WKMs identified deficiencies in vitamin B6 (pyridoxine) and its active form, pyridoxal 5-phosphate. We are currently evaluating the effectiveness of exogenous pyridoxal 5-phosphate supplementation for rescuing cytopenia in the dnajc21-/- mutants. In parn-/- WKMs, we identified several metabolic processes that are dysregulated at the transcriptional level: linoleic acid metabolism, fatty acid biosynthesis and glycine, serine and threonine metabolism were downregulated whereas, cholesterol biosynthesis, arachidonic acid metabolism, cysteine and methionine metabolism were upregulated. Importantly, squalene epoxidase (zebrafish sqlea), the second rate-limiting enzyme in the cholesterol biosynthesis pathway and a marker that is upregulated in various cancers, was elevated in the parn-/- mutants. We are currently investigating the effects of altered cholesterol metabolism on hematopoietic differentiation, and the potential for SQLE inhibitors such as terbinafine for rescuing cytopenia in parn-mutant DC. In summary, our zebrafish models of SDS and DC serve as promising in vivo platforms for revealing disease mechanisms and preclinical screening of targeted therapies.

Infant leukemia (IL) is an aggressive form of leukemia that occurs in children less than one year of age. It is characterized by high white blood cell counts, hepatosplenomegaly, central nervous system involvement and skin infiltration. Compared to leukemia in older children, IL has increased relapse rates and higher treatment-related morbidities. Fusions involving the KMT2A gene, termed ‘KMT2A-r‘ are seen in 70-80% of infant acute lymphoblastic leukemia and in 50% of infant acute myeloid leukemia patients. However, KMT2A-r alone is not sufficient to induce rapid-onset leukemia, suggesting the presence of additional cooperative mutations. Heterozygous germline mutations in the KMT2C gene were found to be enriched in IL patients with KMT2A-r. Further, the neonatal microenvironment strongly influences KMT2A-r-driven leukemia phenotypes. A highly conserved hematopoietic system and access to early embryonic progenitors make the zebrafish (Danio rerio) an ideal preclinical model for IL. We established a loss-of-function kmt2ca zebrafish mutant using CRISPR-Cas9 mutagenesis. Characterization of hematopoiesis by in situ hybridization showed reduced primitive and definitive erythropoiesis and myelopoiesis at 24- and 48-hours post-fertilization (hpf), respectively, in kmt2ca-/- embryos compared to wildtype. While we observed no changes in hematopoietic stem and progenitor cell specification, cebpa+ myeloid progenitors were upregulated in kmt2ca-/- embryos. Similarly, in adult kmt2ca-/- fish, we observed a significant expansion of myeloid cells accompanied by reduced erythrocytes and lymphocytes. Our findings suggest that kmt2ca loss inhibits hematopoietic differentiation in zebrafish. To model the co-operativity between KMT2C mutations and the KMT2A-MLLT1 fusion, we expressed the human KMT2A-MLLT1 fusion under the zebrafish lmo2 promoter, which drives expression in early hematopoietic progenitors. Using in situ hybridization, we found that zebrafish harboring both the kmt2ca-/- mutation and the KMT2A-MLLT1 fusion exhibited severely reduced expression of hbbe3+ erythrocytes and lcp1+ leukocytes, compared with zebrafish harboring kmt2ca mutation alone, fusion alone or wildtype. KMT2A-r fusions are known to disrupt hematopoietic differentiation via overexpression of HOX genes, regulated by the MEN1-MEIS1 complex. Moreover, enforced co-overexpression of Hoxa9 and Meis1 in mice promotes leukemia with short latency. At 24 hpf, using quantitative PCR, we observed an upregulation of meis1b and hoxa9a genes in kmt2ca-/-:KMT2A-MLLT1 embryos compared with zebrafish harboring kmt2ca mutation alone, fusion alone or wildtype. The inherent overexpression of these pro-leukemia genes may accelerate leukemia development, a phenotype currently under investigation. In conclusion, our preliminary findings suggest that kmt2ca mutations cooperate with the KMT2A-MLLT1 fusion to activate leukemogenic pathways. This in turn may inhibit hematopoietic differentiation and promote the expansion of immature myeloid progenitors, potentially resulting in rapid-onset leukemia. Our zebrafish mutant and transgenic lines thus represent high fidelity in vivo models of IL that can be leveraged for therapeutic screening.

Increased access to high-throughput DNA sequencing platforms has transformed the diagnostic landscape of pediatric malignancies by identifying and integrating actionable genomic or transcriptional features that refine diagnosis, classification, and treatment. Yet less than 10% of treated patients show a positive response and translating precision oncology data into feasible and effective therapies for hard-to-cure childhood, adolescent, and young adult malignancies remains a significant challenge. Combining the identification of therapeutic targets at the protein and pathway levels with demonstration of treatment response in personalized models holds great promise. Here we present the case for combining proteomics with patient-derived xenograft (PDX) models to identify personalized treatment options that were not apparent at genomic and transcriptomic levels. Proteome analysis with immunohistochemistry (IHC) validation of formalin-fixed paraffin-embedded sections from an adolescent with primary and metastatic spindle epithelial tumor with thymus-like elements (SETTLE) was completed within two weeks of biopsy. The results identified an elevated protein level of SHMT2 as a possible target for therapy with the commercially available anti-depressant sertraline. Within 2 months and ahead of a molecular tumor board, we confirmed a positive drug response in a personalized chick chorioallantoic membrane (CAM) model of the SETTLE tumor (CAM-PDX). Following the failure of cytotoxic chemotherapy and second-line therapy, a treatment of sertraline was initiated for the patient. After 3 months of sertraline treatment the patient showed decreased tumor growth rates, albeit with clinically progressive disease. Significance: Overall, we demonstrate that proteomics and fast-track personalized xenograft models can provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact the clinical practice. By this we show that proteome-guided and functional precision oncology are feasible and valuable complements to the current genome- driven precision oncology practices. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Data and/or materials used for this research were made available by the PRecision Oncology For Young peopLE (PROFYLE) program. PROFYLE has been supported by funds from many funders including Alberta Cancer Foundation, Alberta Childrens Hospital Foundation, BC Cancer Foundation, BC Childrens Hospital Foundation, Childhood Cancer Canada, Kids Cancer Care Foundation and Terry Fox Research Institute. This work was supported by the BC Childrens Hospital Foundation through the Better Responses through Avatars and Evidence (BRAvE) Initiative. Salary support was provided by the Michael Cuccione Foundation MCF (C.J.L., G.S.D.R., C.A.M., P.F.L., V.G.), the Canada Research Chairs Program (CRC-RS 950-230867, P.F.L.), the Canadian Institutes of Health Research (C.A.M., P.F.L.), the Michael Smith Foundation for Health Research Scholar Program (16442, P.F.L.), MITACS (T.A.B., G.B.) and the University of British Columbia (E.K.E.). Project support for J.A.C. and D.L.S. was provided by The Alberta Cancer Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Children and Womens Research Ethics Board of the University of British Columbia gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes To maintain the patients and familys privacy in this study of a single case the raw data is not made available at this point. Release of the raw data as part of an aggregated patient cohort is in preparation.