10029 Background: Several aggressive pediatric and young adult cancers are characterized by loss of INI1 expression, including malignant rhabdoid tumor, ATRT, epithelioid sarcoma and poorly differentiated chordoma. These cancers have a poor prognosis and limited effective treatments for relapsed or refractory disease. Prior data nominated immune checkpoint inhibition as a potential strategy for INI1-deficient tumors. Methods: In this phase II multicenter trial of nivolumab and ipilimumab, patients 6 months to 40 years with relapsed or refractory INI1-deficient cancers were enrolled in two strata: extracranial solid tumors (Stratum 1) and CNS tumors (Stratum 2). The primary endpoint was objective response ≥partial response (PR) by RECIST v1.1 (Stratum 1) or RANO (Stratum 2). Secondary endpoints included progression-free survival, overall survival and 12-month disease control rate. Patients received nivolumab 3mg/kg and ipilimumab 1mg/kg every 3 weeks for 4 cycles followed by nivolumab 3mg/kg every 2 weeks for up to 1 year. A Simon’s two-stage design targeted a response rate >25%, with each stratum assessed separately. Interim analysis at Stage 1 required ≥1 of 10 patients to have response ≥PR to proceed to Stage 2; at Stage 2, ≥3 of 20 patients with response ≥PR for evidence of efficacy. Evaluable patients received study treatment and were assessed for response or had progressive disease. Results: Thirty patients from six centers enrolled across both Strata from 8/2020 to 5/2025 and 27 were evaluable for the primary endpoint. Three inevaluable Stratum 1 patients were replaced per protocol. In Stratum 1, 1 patient (with poorly-differentiated chordoma) of 20 evaluable patients had ≥PR (ORR: 5%; 95% CI: 0.1-25%). In Stratum 1 (n=20), the 3-month EFS±SE and OS±SE were 20%±9% and 60%±11%, respectively, and the proportion of patients who were progression-free at 12-months was 5% (95%CI=0.1%-25%). Stratum 2 closed early with 7 patients enrolled, too few for Stage 1 interim analysis. No Stratum 2 patients had ≥PR [ORR: 0% (n=7)]. Patients received a median of 2 cycles of study treatment. The most common ≥ grade 3 treatment-related adverse events were decreased lymphocyte count (n=3), increased AST (n=3) anemia (n=2), and pneumonitis (n=2). Conclusions: The study did not meet the predetermined threshold for efficacy for Stratum 1. Immune checkpoint inhibitor therapy could warrant further exploration in poorly differentiated chordoma. Clinical trial information: NCT04416568 . Patient characteristics (n=27 evaluable patients). Median (Range) Age at enrollment (years) 7.1 (0.6-37.6) Lines of prior therapy 2 (1-7) n (%) Age group <18 yrs 24 (89%) ≥18 yrs 3 (11%) Sex Male 15 (56%) Female 12 (44%) Cancer diagnosis Stratum 1 20 (74%) Malignant Rhabdoid Tumor 12 (60%) Poorly Differentiated Chordoma 3 (15%) Epithelioid Sarcoma 2 (10%) Other 3 (15%) Stratum 2 7 (26%) Atypical Teratoid Rhabdoid Tumor 7 (100%)

Although most pediatric acute myeloid leukemia (pAML) patients achieve complete remission with standard-of-care chemotherapy, overall outcomes are poor, and 40% will eventually relapse. Improved methods for risk assessment at diagnosis and alternative therapies are needed to improve outcomes for these patients. Toward these objectives, we characterized the clonal composition of pAMLs, identifying subclones that expand or transform between diagnosis and relapse. We further showed that the abundance of these expanding and transforming subclones in diagnostic samples is predictive of patient outcomes and, similarly, predicts response to chemotherapy and targeted therapies in patient samples and patient-derived xenograft models. Moreover, gene expression programs previously associated with pAML chemoresistance are recurrently elevated in these predictive subclones. Consequently, we propose a novel strategy for improving pAML risk prediction at both diagnosis and during therapy that combines the detection of outcome-predictive tumor subclones in pAML blood or bone marrow with cytogenetic biomarkers and residual disease assessment. Critically, we showed that this combination dramatically improved risk prediction, including for patients who achieve complete remission after chemotherapy. Moreover, through our analyses of outcome-predictive pAML subclones, we identified potential personalized targeted therapies for pAML patients based on the composition of their tumors.

Rhabdoid tumours (RT) are malignancies of the central nervous system, kidneys, liver and soft tissues that most commonly affect very young children with survival rates below 30% in high-risk cohorts. Treatment entails surgery, intensive chemotherapy and radiotherapy, associated with substantial short- and long-term toxicities. There is an unmet need to develop targeted therapies for RT to improve patient outcomes and mitigate the toxicities of current therapy. Detailed research followed by a workshop had the objective of enabling the development of targeted therapeutics for RT. Given the inherent commonality of their biology (i.e. biallelic inactivation of SMARCB1 or more rarely SMARCA4) the therapeutic approach should be similar for intra-cranial and extra-cranial tumours. DDB1–CUL4-associated factor 5 is a promising target, and the development of small molecule binders/degraders is a priority. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) degraders may have greater therapeutic potential than inhibitors. Fibroblast growth factor receptor and platelet-derived growth factor receptor inhibitors may have value in subgroups. Mouse double minute 2 homologue (MDM2) is a priority target for novel therapeutic development and combination trials. Combinations of EZH2, MDM2 inhibitors and selective inhibitors of nuclear export should be evaluated robustly preclinically and drive early clinical studies.

ABSTRACT Background Hepatotoxicity is a common treatment‐related complication in pediatric acute lymphoblastic leukemia (ALL). Excess adiposity at diagnosis has been identified as a risk factor, but the impact of weight change during therapy has not been examined. Methods We included patients aged 2–20 years newly diagnosed with ALL between 2005 and 2021. Hepatotoxicity was defined as: (1) transaminitis (ALT/AST > 10× the upper limit of normal), or (2) conjugated hyperbilirubinemia (cbili; > 3.0 mg/dL during induction/> 2.0 mg/dL post‐induction). Youden's J was used to identify optimal BMI z‐score thresholds. Cox proportional hazards regression was used to assess associations between hepatotoxicity during treatment and diagnostic BMI and post‐induction hepatotoxicity and weight change during induction. Associations were adjusted for age, race, ethnicity, and treatment risk stratification. Patients were censored at qualifying event or 1200 days post‐diagnosis. Results Among 1070 patients (median age 6 years), 45.8% developed transaminitis and 7.6% cbili. Optimal BMI z‐score cut points were −1.5 and 1.5 for transaminitis and 1.5 for cbili. A BMI z‐score ≤ −1.5 was associated with reduced transaminitis (HR = 0.58, 95% CI: 0.39–0.86; p = 0.006), while a z‐score ≥ 1.5 was associated with increased transaminitis (HR = 1.32, 95% CI: 1.06–1.65; p = 0.01). A BMI z‐score ≥ 1.5 was also associated with increased cbili (HR = 1.97, 95% CI: 1.23–3.18; p = 0.01). Weight change was not associated with transaminitis, but weight loss during induction was associated with an increase in hazard of post‐induction cbili (HR = 1.08, 95% CI: 1.04–1.11; p < 0.0001). Conclusion Diagnostic BMI z‐score and weight loss during induction were associated with increased hepatotoxicity risk, highlighting potential targets for improved risk stratification and hepatoprotective interventions.

Treatment for childhood acute lymphoblastic leukemia (ALL) can result in hepatotoxicity. Despite being a common complication of ALL therapy, mechanisms and biomarkers of treatment-associated hepatotoxicity (TAH) are not well described. We conducted lipidomic profiling to identify plasma lipids associated with TAH in children receiving ALL therapy utilizing a nested case–control framework. TAH was defined as (1) transaminitis: ALT/AST ≥ CTCAE grade 3, and/or (2) conjugated hyperbilirubinemia: > 3.0 mg/dL during induction therapy or > 2.0 mg/dL post induction. A total of 90 patients (45 matched pairs) treated at Texas Children’s Hospital between 2012 and 2021 were selected for lipidomic profiling, with controls matched to cases based on the availability of samples collected at similar time points in therapy. Lipidomic profiling quantified 1056 lipids, with 751 retained after quality control. Associations with TAH were evaluated using multivariable conditional logistic regression controlling for age, diagnostic BMI z-score, race/ethnicity, and induction intensity. The cohort was 55

BACKGROUND:Hypoglycemia is a rarely reported complication of asparaginase (ASP) therapy in children with acute lymphoblastic leukemia/lymphoma (ALL/LLy). We sought to identify risk factors and outcomes among patients with ASP-induced hypoglycemia (AIH) at our institution. METHODS:Retrospective cohort study using electronic medical records to identify all patients who received ASP and had diagnosis of hypoglycemia between June 1, 2017 and June 30, 2022. Demographic and clinically relevant data were collected. RESULTS:A total of 672 patients received ASP, with 8% having AIH-defined by a measured low blood glucose level within 14 days of ASP administration and other causes of hypoglycemia excluded. Median age at ALL/LLy diagnosis was 4.4 years (inter-quartile range [IQR]: 2.5-7.7) that was younger than a comparison cohort of patients without AIH (median 6.7 years, p value 0.01). Median BMI z-score was 0.50 (IQR: -0.46 to 0.95). Initial hypoglycemia event was during Induction therapy in 71%, with a median of 11 days (IQR: 6-15) from ASP to hypoglycemia diagnosis. Median duration of the hypoglycemia episode was 11 days (IQR: 7-19). Recurrent hypoglycemia with subsequent ASP doses occurred in 84% of patients, with a median duration of 14 days (IQR: 8-21). Overall survival of the AIH cohort was 80% (85% if limited to patients with newly diagnosed ALL/LLy), with 3-year median follow-up. In univariate analysis, hypoglycemia severity was not associated with age, sex, ethnicity, or weight. CONCLUSION:AIH is relatively common with no clear risk factors besides younger age. It can recur and become more severe with longer duration. AIH screening and management should be implemented.

Treatment-associated hepatotoxicity (TAH) is a common complication of pediatric acute lymphoblastic leukemia (ALL) treatment, but genetic risk factors remain poorly understood. We evaluated the SOD2 rs4880 variant in 544 children with ALL at Texas Children's Hospital. After adjusting for demographic and clinical covariates, the rs4880 C allele was associated with an increased odds of conjugated hyperbilirubinemia and/or Grade 4 transaminitis in Hispanic children (adjusted odds ratio [aOR] = 2.73, 95% confidence interval [CI]: 1.72-4.47; p = 3.5 × 10-5), but decreased odds in non-Hispanic children (aOR = 0.55, 95% CI: 0.32-0.94, p = 0.03). These findings highlight potential ethnicity-specific differences in genetic contributors to TAH risk and highlight the need for mechanistic studies.

ABSTRACT:Hepatotoxicity is a well-documented complication of induction chemotherapy for acute lymphoblastic leukemia (ALL), but our understanding of its biological mechanisms is limited. We identified 314 patients with ALL (aged 1-19 years) treated at Texas Children's Hospital (2008-2019) with diagnostic bone marrow plasma available for metabolomic profiling: 234 for discovery and 80 for replication. Hepatotoxicity during induction was defined as follows: (1) transaminitis: grade ≥3 aspartate aminotransferase or alanine aminotransferase or (2) conjugated hyperbilirubinemia: conjugated bilirubin (c.bili) >3 mg/dL. Untargeted profiling detected 519 metabolites. Adjusted odds ratios (aORs) for each metabolite were calculated with logistic regression, accounting for sex, age, body mass index, race/ethnicity, and treatment intensity. The population was 56% Latino, 57% male, 92% B-ALL, 25% overweight/obese, and 57% National Cancer Institute standard-risk at a median age of 5 years. Transaminitis was observed in 34% of the discovery and 24% of the replication cohort. Seven instances of c.bili >3 mg/dL were observed in the discovery cohort, with none in the replication cohort. Furthermore, 12 metabolites were associated with transaminitis (P < .05) in the discovery cohort, including 2 that replicated (P < .05): 1,2-dipalmitoyl-glycerophosphocholine (GPC) (aOR combined = 1.88 [95% confidence interval [CI], 1.26-2.79], P = .002) and 1-(1-enyl-palmitoyl)-2-palmitoleoyl-GPC (aOR combined = 1.56 [95% CI: 1.17-2.09], P = .003). In the discovery cohort, 34 metabolites were associated with c.bili >3 mg/dL, including the top association of 1,2-dipalmitoyl-GPC (aOR = 5.76 [95% CI: 2.20-23.16], P = .002). We observed and replicated associations between phosphatidylcholine metabolites at ALL diagnosis and hepatotoxicity during induction therapy, suggesting a potential role for lipid dysregulation in the development of hepatotoxicity.

Most pediatric acute myeloid leukemia (pAML) patients achieve complete remission after chemotherapy, yet relapse is common, with nearly 40% ultimately dying of the disease. Prognosis is currently assessed using cytogenetic biomarkers and measurable residual disease after the first chemotherapy cycle, with the highest risk patients referred for stem cell transplantation (SCT) at first remission. Because aggressive therapies such as SCT are highly toxic, yet cures after relapse are rare, accurate early risk prediction is essential for improving outcomes. To address this need, we analyzed paired diagnosis-relapse samples from 33 pAML patients at single-cell resolution and identified chemoresistant cell populations whose abundance at diagnosis significantly improved risk prediction. Incorporating the detection of these cell populations into our risk model revealed a previously unrecognized patient subgroup with a 5-year event-free survival rate below 40%. Although this subgroup represents only 20% of pAML cases, it accounted for half of the deaths among patients who do not receive SCT at first remission. Moreover, molecular characterization of these chemoresistant cell populations uncovered potential therapeutic targets and candidate interventions relevant to most high-risk patients, paving the way for more effective targeted treatments for high-risk pAML patients.

Pediatric Blood & CancerEarly View e31657 COMMENTARY Acute Leukemia in the Crosshairs: First-in-Class Menin Inhibitor Approval for Adults and Children Joanna S. Yi, Corresponding Author Joanna S. Yi [email protected] orcid.org/0000-0002-0914-8728 Department of Pediatrics, Baylor College of Medicine/Texas Children's Hospital, Houston, Texas, USA Correspondence: Joanna S. Yi ([email protected])Search for more papers by this authorBranko Cuglievan, Branko Cuglievan Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USASearch for more papers by this author Joanna S. Yi, Corresponding Author Joanna S. Yi [email protected] orcid.org/0000-0002-0914-8728 Department of Pediatrics, Baylor College of Medicine/Texas Children's Hospital, Houston, Texas, USA Correspondence: Joanna S. Yi ([email protected])Search for more papers by this authorBranko Cuglievan, Branko Cuglievan Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USASearch for more papers by this author First published: 18 March 2025 https://doi.org/10.1002/pbc.31657Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookxLinkedInRedditWechat No abstract is available for this article. References 1B. Cuglievan, H. Kantarjian, J. E. 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The past 25 years of clinical trials have produced few improvements in pediatric AML (pAML) outcomes. This is acutely evident in patients with t(16;21)(p11;q22), yielding FUS::ERG. Patients with FUS::ERG-positive AML relapse quickly and do not respond to transplantation. Major histocompatibility complex (MHC) class I & II receptors and costimulatory molecules are absent at diagnosis in FUS::ERG-positive AML, mirroring the phenotype and outcomes of post-transplant relapse. We show that this is driven by overexpression of EZH2, in vitro and in multiple clinical cohorts. While FUS::ERG AML is the most extreme example, this phenotype is shared by lethal CBFA2T3::GLIS2-driven AML, and patients with RUNX1::RUNX1T1 have significantly worse outcomes when EZH2 overexpression co-occurs. The FDA-approved EZH2 inhibitor tazemetostat reverses this phenotype, re-establishes MHC presentation, and elicits immune effector cell-mediated elimination. EZH2 inhibitors may provide the first targeted therapeutic frontline option for AML patients with FUS::ERG, with the potential for broader frontline immunostimulatory benefits.

ABSTRACT:FLAG-IDA (fludarabine, cytarabine, granulocyte colony-stimulating factor, idarubicin) with venetoclax shows promise as frontline pediatric acute myeloid leukemia therapy. In 12 patients treated at MD Anderson, most achieved remission with good early survival outcomes, and many proceeded to transplant. Common toxicities included cytopenias, comparable to previous regimens.

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [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 4752.

Chromosomal translocations involving the mixed-lineage leukemia (MLL) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative MLL-rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose MLL oncoproteins promote lineage-switching events through dynamic chromatin binding at lineage-specific target genes, and may support resistance to menin inhibitors through similar changes in chromatin occupancy. The effects of chromosomal translocations involving the mixed-lineage leukemia (MLL) locus on gene expression regulation remain to be explored. Here, the authors find that MLL oncoproteins support lineage-switching events through dynamic chromatin binding.