Children's Cancer Institute Australia

BackgroundOutcomes for patients with esophageal squamous cell carcinoma (ESCC) remain poor, partly due to treatment resistance, particularly to DNA-damaging therapies. Poly(ADP‑ribose) polymerase 1 (PARP1) plays a critical role in repairing single‑strand DNA breaks (SSBs). Unrepaired SSBs can be converted into double‑strand breaks (DSBs) during DNA replication, potentially leading to cell death. Genomic amplification of the distal portion of chromosome 3q (3q26-q29) is a frequent copy‑number alteration in ESCC, which harbors genes encoding several oncoproteins. However, whether long noncoding RNAs (lncRNAs) from this region contribute to ESCC pathogenesis and treatment resistance remains poorly understood.MethodsIn situ hybridization and qPCR were used to assess RNA expression. Protein PARylation was evaluated by immunoprecipitation followed by Western blotting. Cellular phenotypes were quantified using the cell counting kit-8, Annexin V/Propidium iodide staining, and clonogenic assays. DNA damage was monitored by immunofluorescence staining for phosphorylated histone H2AX (γH2AX) and p53-binding protein 1 (53BP1) and by comet assays. RNA-protein interactions were assessed through RNA pulldown coupled with mass spectrometry and RNA immunoprecipitation. Chromatin fractionation and detergent pre-extraction immunofluorescence were conducted to examine PARP1 chromatin association. ESCC growth and responses to treatments were evaluated using xenograft models.ResultsThe lncRNA LINC00885, hereafter referred to as PARylator, was the most upregulated lncRNA encoded within the 3q26-q29 amplicon in ESCC. PARylator was predominantly nuclear and interacted with PARP1. Knockdown of PARylator increased γH2AX and 53BP1 foci and comet tail moment, triggered apoptosis, reduced clonogenicity, sensitized ESCC cells to cisplatin and ionizing radiation. In vivo, PARylator knockdown impaired tumor growth and increased cisplatin sensitivity in ESCC xenografts. Mechanistically, PARylator promoted PARP1 recruitment to chromatin and catalytic activation, thereby increasing PARP1 auto-PARylation and enhancing the PARylation of X-ray repair cross-complementing 1 (XRCC1). PARylator was further upregulated in response to DNA damage.ConclusionsThe DNA damage-responsive, 3q26-q29 amplicon-encoded lncRNA PARylator promotes PARP1‑mediated PARylation and SSB repair, thereby limiting DSB accumulation and supporting ESCC cell survival and resistance to DNA-damaging therapies. Targeting PARylator, alone or in combination with DNA-damaging agents, may represent a novel avenue for ESCC treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40164-025-00739-z.

The acquisition of a chemoresistant state underlies poor prognosis in many cancers, including neuroblastoma. We previously demonstrated that heterogeneity in apoptosis induction through c-Jun amino-terminal kinase (JNK) promotes a form of nongenetic chemoresistance in neuroblastoma observable at both patient and single-cell levels. As the maintenance of this JNK-impaired state in the relapse setting is a substantial barrier to the efficacy of many standard-of-care chemotherapy drugs, we combined a mechanistic, mathematical model of JNK activation with a pediatric-focused drug screen and identified approved oncology drugs capable of inducing apoptosis in a JNK-independent manner. Functional genomics further revealed that synergy between these JNK-independent drugs and standard-of-care chemotherapies emerged from differential utilization of apoptotic network components, rather than from their direct mechanistic targets. Efficacy studies with patient-derived xenograft models also confirmed that including a JNK-independent drug within existing chemotherapy backbones significantly improved response in the relapse setting, where new approaches are urgently needed.

High-grade gliomas(HGG) are highly aggressive tumors,which arepredominately fatal for adults and pediatric patients. Identifyingcancer-selective therapeutic targets remains a critical unmet need.The overexpression of endoplasmic reticulum (ER) chaperones in variouscancers is well documented. Moreover, tumor cells exhibit an atypicalsurface expression of ER chaperones, suggesting the potential forselective targeting. Our study examined the differences in the mRNA,total protein, and surface expression levels of seven key ER chaperones,compared with those in non-neoplastic samples. Notably, a poor correlationwas found between mRNA, protein, and surface protein levels, underscoringthe limitations of transcriptomics alone in target discovery. We alsohighlight the limitations of surfaceome studies which exclude noncanonicalmembrane proteins, such as ectopically expressed ER chaperones, whichoften escape detection by conventional bioinformatic pipelines. Forthe first time, this study advances our understanding of the surfaceexpression of ER chaperones in both adult and pediatric HGG. Our findingshighlight the importance of surfaceome analysis in the discovery ofcancer selective targets against this devastating disease.

Previous research shows that families find concepts underpinning childhood cancer precision medicine challenging to understand. We developed three animated videos and then evaluated these videos' acceptability, accessibility, emotional impact, and impact on participants' understanding of precision medicine. Eligible participants included adolescents and young adults with cancer (12-25years) and parents of a child (< 25years) with cancer who recently enrolled in a precision medicine trial. Participants evaluated the videos using a questionnaire containing validated and purpose-designed quantitative and qualitative items. Twenty-three parents and five patients participated. Over 70% of participants rated each video as 'just right' in length, pace, and amount of information. Almost all felt the videos provided useful information about the precision medicine process (nparents = 19/20, 95%; npatients = 4/4, 100%) and would recommend the videos to other families (nparents = 18/20, 90%; npatients = 4/4, 100%). After watching the videos, parents' perceived understanding of key precision medicine concepts increased (p ≤ 0.011 for all items, e.g. possible risks and discomforts). Similarly, watching the videos increased parents' self-rated knowledge of all precision medicine-related terms that were initially poorly understood (p ≤ 0.016 for terms including 'precision medicine' and 'germline variant'). Qualitatively, participants reported valuing the videos and provided constructive feedback to improve the concision and quality of the videos. Parents and patients responded positively to the videos about precision medicine for childhood cancer and felt they would benefit other families. The videos are a potentially useful complement to written consent information and clinical discussions to support understanding of complex precision medicine concepts.

Aurora kinase A (AURKA) is an established oncogenic factorandtherapeutic target in neuroblastoma due to its roles in mitosis andstability of the MYCN protein. We previously identified SK2188 asa highly potent, selective, and fast-acting AURKA degrader capableof inducing MYCN destabilization. However, SK2188 showed low systemicexposure and rapid clearance in mice, necessitating further chemicaloptimization. Here, we describe our structure–activity optimizationefforts involving linker rigidification and incorporation of alternativecereblon- and AURKA-recruiting ligands, leading to two optimized PROTACs,SK4454 and SK5527. Both compounds retained rapid and selective AURKAdegradation with improved pharmacokinetic properties. Importantly,single intravenous administration of either degrader efficiently reducedAURKA levels in vivo in a neuroblastoma xenograft mouse model. Moreover,MDR1-mediated PROTAC efflux was identified as a key intrinsic mechanismlimiting in vitro potency. These results establish SK4454 and SK5527as advanced AURKA degraders with improved pharmacokinetic properties,warranting further preclinical evaluation.

Abstract Diffuse midline glioma (DMG) is a universally fatal high-grade glioma. Its immunologically cold tumor immune microenvironment (TIME) presents a major barrier to durable anti-tumor responses. Dordaviprone (ONC201) is a brain-penetrant DRD2 antagonist and ClpP agonist that disrupts mitochondrial complex II (CII) activity; currently under clinical evaluation for DMG. In melanoma, CII inhibition has been shown to enhance MHC presentation and T cell–mediated killing. Here, we investigated the immunomodulatory effects of dordaviprone in DMG mouse models, patient tumor tissues, and blood samples. Immunocompetent DMG-PPK mice (PdgfraD842V, Trp53DN, H3f3aK27M mutations) were treated with dordaviprone (125 mg/kg, twice weekly) for 1 week (acute) or 3 weeks (chronic) and compared to naïve, sham (orthotopic surgery without tumor engraftment), and vehicle-treated controls. Murine tumors were analyzed by single-cell multiome (scRNA-seq and scATAC-seq) and spatial transcriptomics. Murine blood and bone marrow (BM) and human blood samples were profiled by scRNA-seq and flow cytometry. In models, dordaviprone promoted microglial activation and macrophage infiltration within the TIME. Spatial transcriptomics identified increased expression of the MHC-I subunit B2m in tumor cells and tumor-infiltrating Cd8+ T cells following dordaviprone treatment. Flow cytometry of murine and human peripheral blood confirmed CD4+ and CD8+ lymphopenia at baseline. Dordaviprone increased circulating lymphocytes in models (log2 fold change=1.03; patient data pending), reversing DMG-induced CD3+ T cell sequestration in the BM. Immunohistochemistry confirmed elevated levels of B2M (p=0.0281) and tumor-infiltrating lymphocytes (TILs), including CD45+ (p=0.0275) and CD3+ T cells (p=0.0103) in dordaviprone-treated mice. Despite enhanced TIL recruitment, dordaviprone did not improve survival (n=10), likely due to persistent immunosuppressive myeloid activity, i.e. Havcr2 (TIM-3), Cd74, Cd274 (PD-L1). This study demonstrates that DMG tumors contribute to systemic lymphopenia, partially reversed by dordaviprone. While supporting its immunomodulatory effects, including enhanced antigen presentation and TIL recruitment, our findings highlight future combination strategies to leverage dordaviprone-induced immunoreactive TIME.

Many copy number variants (CNVs) are implicated in neurodevelopmental disability, but exhibit incomplete penetrance. The definition of penetrance is often unclear. In published literature, penetrance typically includes the background risk of disease, while clinicians tend to exclude risks unrelated to the genetic variant. We propose a more clinically relevant definition of penetrance and develop a new formula for this. These changes are applied to existing data sources to produce updated penetrance estimates. Our findings indicate that most CNVs studied have significantly lower penetrance than previously published. Eleven CNVs, previously described as low-penetrant, are recalculated as having a penetrance close to 0% for intellectual disability. These include 1q21.1 proximal duplications [RBM8A], 2q11.2 deletions [TMEM127], 2q13 proximal deletions and duplications [NPHP1], 6q16 duplications [SIM1], 13q12 deletions [CRYL1], 15q11.2 duplications [NIPA1, NIPA2], 15q13.3 duplications [CHRNA7], 16p12.2 duplications [CDR2], 16p13.11 duplications [MYH11] and Xp22.3 duplications [SHOX]. Previous estimates of CNV penetrance, which ranged from 10-40% have been recalculated as 1-10%. In conclusion, many previously published estimates of CNV penetrance are inflated. Re-evaluation of existing data reveals lower and more accurate penetrance estimates for intellectual disability. This has important implications for diagnosis, genetic counselling, and prenatal reporting of recurrent CNVs.

Abstract Germline CHEK2 pathogenic/likely pathogenic variants (GPV CHEK2 ) are associated with an increased risk of adult-onset malignancies such as breast cancer. However, their role in pediatric-onset cancer is unclear. Pediatric Precision Medicine Programs have reported GPV CHEK2 in some cases, suggesting a role in pediatric tumour development. Aims: To investigate the contribution of germline pathogenic CHEK2 variants (GPVCHEK2) to genetic cancer risk in children and explore their potential role in pediatric tumorigenesis. Methods: Patients under 21 years old with childhood cancer were enrolled in ZERO Childhood Cancer Program (2018-2025). We retrospectively analyzed clinical information, including demographics, diagnoses, treatment, and outcome data, obtained from medical records, and paired tumor-germline data from participants with GPV CHEK2 . Results: In total, 22 patients (0.94% of participants) harbored a GPV CHEK2 . Comparison of the rates of GPV CHEK2 between the ZERO cohort and gnomAD (Genome Aggregation Database) demonstrated a signal of statistical enrichment in the ZERO cohort. GPV CHEK2 were approximately 1.9 times more often in the ZERO cohort compared to gnomAD (q value (corrected p value) of 0.03; p value 0.003). Within the ZERO GPV CHEK2 cohort, there were 24 tumours and 26 episodes of paired tumor-germline profiling data (22 primary, 2 secondary and 2 relapsed cancers). The median age at diagnosis was 8 years old, 59% male, and a broad tumor spectrum including 6 hematological malignancies, and 18 non-hematological tumours (7 CNS tumours and 11 Solid tumours). We identified 10 distinct single nucleotide GPV CHEK2 ( no copy number changes were detected). Four patients had a second GPV in a cancer predisposition gene in addition to CHEK2. The CHEK2 c.1100del founder variant was seen in 64% of patients, with three patients harboring rare or novel variants. Half of them (55%) had high-risk tumors, 41% had non-high-risk tumors including 23% with rare tumors. Ploidy across the tumor samples ranged between 1.64 to 3.35 with a median of 2. Tumor Mutational Burden ranged between 0.08 to 48.46 mutation/Megabases (median 0.52mut/Mb). No homologous recombination deficiency (HRD) mutational signature was observed in the tumor samples. Genome Wide Loss of Heterozygosity across the samples ranged from 0% to 41.84%, with a median of 8.69%. Somatic second hits in CHEK2 were identified in two cases, both involving copy number variants. Additionally, each tumour also harbored an independent somatic driver in a different cancer gene deemed sufficient for tumorigenesis. Conclusions Germline pathogenic variants in CHEK2 are observed in children with diverse cancer types, without a clearly defined tumor spectrum. While no biomarkers of HRD were identified, some tumors exhibited biallelic inactivation of CHEK2 via a second somatic event on the wildtype allele. These findings suggest that GPV CHEK2 may contribute to genomic instability, enabling additional somatic alterations to occur and potentially cooperating with somatic drivers in tumorigenesis. Citation Format: Laura Raiti, Ann Altekoester, Sam El-Kamand, Gabi Ryan, Kathy Tucker, Meera Warby, Eliza Courtney, Paulette Barahona, Loretta Lau, Toby Trahair, Glenn Marshall, David Ziegler, Michelle Haber, Vanessa Tyrrell, Mark Cowley, Chelsea Mayoh, Mark Pinese, Dong-Anh Khuong-Quang, Noemi Fuentes-Bolanos. The biology of childhood tumors with germline CHEK2 loss of function variants [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A044.

Abstract Introduction: Circulating tumor DNA (ctDNA) is the most frequently utilized liquid biopsy. High sensitivity is critical for minimal residual disease detection to inform treatment response, risk stratification and molecular relapse. Whole-genome-based, tumor-informed strategy is increasingly being used in ctDNA detection in adult cancers but not in pediatric cancers. Methods: We designed a personalized ctDNA assay which utilized paired tumor-normal whole genome sequencing data of individual patient to design a panel of ∼400 patient-specific tumor biomarkers. 83 patients with high-risk solid tumors from the ZERO Childhood Cancer Precision Medicine Program in Australia who had 2 or more serial liquid biopsy samples collected were selected for analysis. Results: The cohort consisted of 54 sarcoma, 16 neuroblastoma and 13 other solid tumors. 303 liquid biopsy samples were analyzed for plasma ctDNA, ranging from 2 - 7 samples per patient. The detection limit was between 10^-4 and 10^-5 ctDNA fraction for 90% of the samples and between 10^-3 and 10^-4 for the remaining 10%. 73 (88%) patients had at least one blood sample with detectable ctDNA. ctDNA detection rate was 96% in newly diagnosed disease, with ctDNA fraction ranging from 0.01 to 99% (median 36%), 88% in off-treatment relapse (median 3.6%; range: 0.011 – 77%) and 85% in refractory/progressive disease (PD) (median 0.8%; range: 0.37 – 81%). For patients with complete response (CR), 89% had undetectable ctDNA. In the remaining 11% with CR and detectable ctDNA, majority (71%) experienced relapse. ctDNA levels were significantly associated with disease burden and treatment response. Patients with metastatic disease at enrolment had significantly higher ctDNA fraction (median 8.2%; range: 0 – 99%) than those with localized disease (median 0.06%; range: 0 – 42%) (P&amp;lt;0.05). The median ctDNA fraction corresponding to CR was 0% (range: 0 – 4.0%), 0.0095% (range: 0 – 4.7%) for partial response, 0.022% (range: 0 – 23%) for stable disease and 5.6% (range: 0 – 82%) for PD (P&amp;lt;0.0001). Most importantly, ctDNA predicted clinical outcomes. Patients with early clearance of ctDNA between 60 and 120 days from start of treatment, compared with those with detectable ctDNA, had significantly better PFS (5-yr PFS 47% vs 21%; P&amp;lt;0.01) and OS (5-yr OS 54% vs 37%; P&amp;lt;0.05). Patients with undetectable ctDNA at the end of treatment (+/-42 days from completing treatment), compared with those with persistence ctDNA, also had significantly better PFS (5-yr PFS 50% vs 0%; P&amp;lt;0.0001) and OS (5-yr OS 66% vs 17%; P&amp;lt;0.001). Conclusion: This ultrasensitive personalized ctDNA assay with a detection limit between 10^-4 and 10^-5 ctDNA fraction is highly correlative of disease burden and treatment response. Early clearance of ctDNA and persistence of ctDNA at end of the treatment both correlated with clinical outcome. The assay will be evaluated prospectively to determine its clinical utility in complementing treatment response evaluation, detecting early recurrence and predicting outcome in pediatric cancers. Citation Format: Loretta M.S. Lau, Rob Salomon, Wenhan Chen, Charles Shale, Mojgan Toumari, Wenyan Li, Jingwei Chan, Sajad Razavi-Bazaz,, Louise Cui, Sam El-Kamand, Marie Wong, Edwin Cuppen, Michelle Haber, Vanessa Tyrrell, Paul G Ekert, David S Ziegler, Peter Priestley, Mark J Cowley. Whole-genome-based, tumor-informed circulating tumor DNA detection correlates with treatment response and survival of pediatric solid tumor [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A001-PR009.