The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions

Steliarova-Foucher E., Colombet M., Ries L.A., Moreno F., Dolya A., Bray F., Hesseling P., Shin H.Y., Stiller C.A., Bouzbid S., Hamdi-Cherif M., Hablas A., Chirpaz E., Buziba N., Chesumbai G., et. al.

Cancer is a major cause of death in children worldwide, and the recorded incidence tends to increase with time. Internationally comparable data on childhood cancer incidence in the past two decades are scarce. This study aimed to provide internationally comparable local data on the incidence of childhood cancer to promote research of causes and implementation of childhood cancer control.This population-based registry study, devised by the International Agency for Research on Cancer in collaboration with the International Association of Cancer Registries, collected data on all malignancies and non-malignant neoplasms of the CNS diagnosed before age 20 years in populations covered by high-quality cancer registries with complete data for 2001-10. Incidence rates per million person-years for the 0-14 years and 0-19 years age groups were age-adjusted using the world standard population to provide age-standardised incidence rates (WSRs), using the age-specific incidence rates (ASR) for individual age groups (0-4 years, 5-9 years, 10-14 years, and 15-19 years). All rates were reported for 19 geographical areas or ethnicities by sex, age group, and cancer type. The regional WSRs for children aged 0-14 years were compared with comparable data obtained in the 1980s.Of 532 invited cancer registries, 153 registries from 62 countries, departments, and territories met quality standards, and contributed data for the entire decade of 2001-10. 385 509 incident cases in children aged 0-19 years occurring in 2·64 billion person-years were included. The overall WSR was 140·6 per million person-years in children aged 0-14 years (based on 284 649 cases), and the most common cancers were leukaemia (WSR 46·4), followed by CNS tumours (WSR 28·2), and lymphomas (WSR 15·2). In children aged 15-19 years (based on 100 860 cases), the ASR was 185·3 per million person-years, the most common being lymphomas (ASR 41·8) and the group of epithelial tumours and melanoma (ASR 39·5). Incidence varied considerably between and within the described regions, and by cancer type, sex, age, and racial and ethnic group. Since the 1980s, the global WSR of registered cancers in children aged 0-14 years has increased from 124·0 (95% CI 123·3-124·7) to 140·6 (140·1-141·1) per million person-years.This unique global source of childhood cancer incidence will be used for aetiological research and to inform public health policy, potentially contributing towards attaining several targets of the Sustainable Development Goals. The observed geographical, racial and ethnic, age, sex, and temporal variations require constant monitoring and research.International Agency for Research on Cancer and the Union for International Cancer Control.

Li J., Riedt T., Goossens S., Carrillo García C., Szczepanski S., Brandes M., Pieters T., Dobrosch L., Gütgemann I., Farla N., Radaelli E., Hulpiau P., Mallela N., Fröhlich H., La Starza R., et. al.

Key Points Zeb2 controls stem cell pool size and lineage fidelity. Zeb2 deletion promotes a myeloproliferative phenotype resembling the early stage of primary myelofibrosis.

Liang K., Volk A.G., Haug J.S., Marshall S.A., Woodfin A.R., Bartom E.T., Gilmore J.M., Florens L., Washburn M.P., Sullivan K.D., Espinosa J.M., Cannova J., Zhang J., Smith E.R., Crispino J.D., et. al.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Zhang Y., Zhou H., Zhou J., Sun W.

Göllner S., Oellerich T., Agrawal-Singh S., Schenk T., Klein H., Rohde C., Pabst C., Sauer T., Lerdrup M., Tavor S., Stölzel F., Herold S., Ehninger G., Köhler G., Pan K., et. al.

Proteasomal degradation of EZH2 in AML patients in response to therapy triggers the expression of stem cell markers and has been identified as an epigenetic pathway leading to acquired drug resistance. Treatments aimed to restore EZH2 expression in relapsed AML patients have shown clinical efficacy and constitute a viable approach to re-sensitize tumors to chemotherapy. In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

Guryanova O.A., Shank K., Spitzer B., Luciani L., Koche R.P., Garrett-Bakelman F.E., Ganzel C., Durham B.H., Mohanty A., Hoermann G., Rivera S.A., Chramiec A.G., Pronier E., Bastian L., Keller M.D., et. al.

AML cells carrying R882 mutations in DNMT3A fail to sense and repair DNA damage induced by standard-dose chemotherapy as a result of impaired chromatin remodeling Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3AR882), have been observed in AML1,2,3 and in individuals with clonal hematopoiesis in the absence of leukemic transformation4,5. Patients with DNMT3AR882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy6,7, suggesting that DNMT3AR882 cells persist and drive relapse8. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3ITD) and the nucleophosmin gene (Npm1c) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3AR882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3AR882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3AR882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.

Faber Z.J., Chen X., Gedman A.L., Boggs K., Cheng J., Ma J., Radtke I., Chao J., Walsh M.P., Song G., Andersson A.K., Dang J., Dong L., Liu Y., Huether R., et. al.

Lars Bullinger, Jinghui Zhang, Jeffery Klco, James Downing and colleagues report a detailed genomic analysis of pediatric and adult core-binding factor acute myeloid leukemias (CBF-AMLs). They identify recurrent mutations in CCND2, MGA, DHX15 and ZBTB7A and highlight dramatic differences in the landscape of cooperating mutations between different CBF-AML subtypes. Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.

Ramamurthy R., Hughes M., Morris V., Bolouri H., Gerbing R.B., Wang Y., Loken M.R., Raimondi S.C., Hirsch B.A., Gamis A.S., Oehler V.G., Alonzo T.A., Meshinchi S.

Aberrant expression of microRNA-155 (miR-155) has been implicated in acute myeloid leukemia (AML) and associated with clinical outcome.We evaluated miR-155 expression in 198 children with normal karyotype AML (NK-AML) enrolled in Children's Oncology Group (COG) AML trial AAML0531 and correlated miR-155 expression levels with disease characteristics and clinical outcome. Patients were divided into quartiles (Q1-Q4) based on miR-155 expression level, and disease characteristics were then evaluated and correlated with miR-155 expression.MiR-155 expression varied over 4-log10-fold range relative to its expression in normal marrow with a median expression level of 0.825 (range 0.043-25.630) for the entire study cohort. Increasing miR-155 expression was highly associated with the presence of FLT3/ITD mutations (P < 0.001) and high-risk disease (P < 0.001) and inversely associated with standard-risk (P = 0.008) and low-risk disease (P = 0.041). Patients with highest miR-155 expression had a complete remission (CR) rate of 46% compared with 82% in low expressers (P < 0.001) with a correspondingly lower event-free (EFS) and overall survival (OS) (P < 0.001 and P = 0.002, respectively). In a multivariate model that included molecular risk factors, high miR-155 expression remained a significant independent predictor of OS (P = 0.022) and EFS (0.019).High miR-155 expression is an adverse prognostic factor in pediatric NK-AML patients. Specifically, high miR-155 expression not only correlates with FLT3/ITD mutation status and high-risk disease but it is also an independent predictor of worse EFS and OS.

Fooladinezhad H., Khanahmad H., Ganjalikhani-Hakemi M., Doosti A.

Li S., Garrett-Bakelman F.E., Chung S.S., Sanders M.A., Hricik T., Rapaport F., Patel J., Dillon R., Vijay P., Brown A.L., Perl A.E., Cannon J., Bullinger L., Luger S., Becker M., et. al.

Genome-wide methylome sequencing of serial samples obtained from patients with acute myeloid leukemia reveals that epigenetic alleles and genetic alleles follow independent courses during disease evolution. Genetic heterogeneity contributes to clinical outcome and progression of most tumors, but little is known about allelic diversity for epigenetic compartments, and almost no data exist for acute myeloid leukemia (AML). We examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epialleles), somatic mutations, and transcriptomes of AML patient samples at serial time points. We observed that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning followed different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression showed increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics likely to affect the biological and clinical features of tumors.

Maxson J.E., Ries R.E., Wang Y., Gerbing R.B., Kolb E.A., Thompson S.L., Guidry Auvil J.M., Marra M.A., Ma Y., Zong Z., Mungall A.J., Moore R., Long W., Gesuwan P., Davidsen T.M., et. al.

Papaemmanuil E., Gerstung M., Bullinger L., Gaidzik V.I., Paschka P., Roberts N.D., Potter N.E., Heuser M., Thol F., Bolli N., Gundem G., Van Loo P., Martincorena I., Ganly P., Mudie L., et. al.

BACKGROUND Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice. METHODS We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes. RESULTS We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials. CONCLUSIONS The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).

Mayakonda A., Koeffler H.P.

AbstractMutation Annotation Format (MAF) has become a standard file format for storing somatic/germline variants derived from sequencing of large cohort of cancer samples. MAF files contain a list of all variants detected in a sample along with various annotations associated with the putative variant. MAF file forms the basis for many downstream analyses and provides complete landscape of the cohort. Here we introduce maftools–an R package that provides rich source of functions for performing various analyses, visualizations and summarization of MAF files. Maftools uses data.table library for faster processing/summarization and ggplot2 for generating rich and publication quality visualizations. Maftools also takes advantages of S4 class system for better data representation, with easy to use and flexible functions.Availability and Implementationmaftools is implemented as an R package available at https://github.com/PoisonAlien/maftoolsContactcsiamt@nus.edu.sg

Farrar J.E., Schuback H.L., Ries R.E., Wai D., Hampton O.A., Trevino L.R., Alonzo T.A., Guidry Auvil J.M., Davidsen T.M., Gesuwan P., Hermida L., Muzny D.M., Dewal N., Rustagi N., Lewis L.R., et. al.

Abstract The genomic and clinical information used to develop and implement therapeutic approaches for acute myelogenous leukemia (AML) originated primarily from adult patients and has been generalized to patients with pediatric AML. However, age-specific molecular alterations are becoming more evident and may signify the need to age-stratify treatment regimens. The NCI/COG TARGET-AML initiative used whole exome capture sequencing (WXS) to interrogate the genomic landscape of matched trios representing specimens collected upon diagnosis, remission, and relapse from 20 cases of de novo childhood AML. One hundred forty-five somatic variants at diagnosis (median 6 mutations/patient) and 149 variants at relapse (median 6.5 mutations) were identified and verified by orthogonal methodologies. Recurrent somatic variants [in (greater than or equal to) 2 patients] were identified for 10 genes (FLT3, NRAS, PTPN11, WT1, TET2, DHX15, DHX30, KIT, ETV6, KRAS), with variable persistence at relapse. The variant allele fraction (VAF), used to measure the prevalence of somatic mutations, varied widely at diagnosis. Mutations that persisted from diagnosis to relapse had a significantly higher diagnostic VAF compared with those that resolved at relapse (median VAF 0.43 vs. 0.24, P &lt; 0.001). Further analysis revealed that 90% of the diagnostic variants with VAF &gt;0.4 persisted to relapse compared with 28% with VAF &lt;0.2 (P &lt; 0.001). This study demonstrates significant variability in the mutational profile and clonal evolution of pediatric AML from diagnosis to relapse. Furthermore, mutations with high VAF at diagnosis, representing variants shared across a leukemic clonal structure, may constrain the genomic landscape at relapse and help to define key pathways for therapeutic targeting. Cancer Res; 76(8); 2197–205. ©2016 AACR.

Chun H.E., Lim E.L., Heravi-Moussavi A., Saberi S., Mungall K.L., Bilenky M., Carles A., Tse K., Shlafman I., Zhu K., Qian J.Q., Palmquist D.L., He A., Long W., Goya R., et. al.

Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.

Mendoza-Castrejon J., Yang W., Denby E., Muthukumar R., Casey E.B., Patel R.M., Tasian S.K., Magee J.A.

ABSTRACTInfant leukemias arise as B-cell acute lymphocytic (B-ALL) or acute myeloid leukemia (AML). The majority are driven by chromosomal rearrangements of theMLL/ KMT2Agene (MLLr) and arise in utero, implying a fetal cell of origin. Fetal and neonatal hematopoietic progenitors have unique transcriptomes and epigenomes, raising the question of whether MLL fusion proteins activate distinct target gene profiles during these early stages of life. Here, we use a transgenic mouse model of MLL::ENL-driven leukemia to identifySkida1as a target gene that is more highly induced in fetal and neonatal progenitors than in adult progenitors.SKIDA1is highly expressed in humanMLLr leukemias and the protein associates with the Polycomb Repressive Complex 2 (PRC2). We show thatSkida1is dispensable for normal hematopoiesis, but it promotes B-cell priming and maintains MLL::ENL-expressing hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) during neonatal development. Conditional deletion ofSkida1has no effect on normal HSC function, yet it impairs B-cell production from neonatal MLL::ENL-expressing HSCs while leaving myeloid leukemogenesis unaffected. Temporally-restricted targets of MLL fusion proteins, such as SKIDA1, can therefore tune cell fates at different ages, potentially influencing the types MLLr leukemias that arise at different ages.

Hoff F.W., Huang Y., Welkie R.L., Swords R.T., Traer E., Stein E.M., Lin T.L., Patel P.A., Collins R.H., Baer M.R., Duong V.H., Blum W.G., Arellano M.L., Stock W., Odenike O., et. al.

Boudia F., Baille M., Babin L., Aid Z., Robert E., Riviere J., Galant K., Alonso-Pérez V., Anselmi L., Arkoun B., Abermil N., Marzac C., Bertuccio S.N., Regnault de Premesnil A., Lopez C.K., et. al.

Abstract Pediatric acute myeloid leukemia frequently harbors fusion oncogenes associated with poor prognosis, including KMT2A, NUP98, and GLIS2 rearrangements. Although murine models have demonstrated their leukemogenic activities, the steps from a normal human cell to leukemic blasts remain unclear. Here, we precisely reproduced the inversion of chromosome 16 resulting in the ETO2::GLIS2 fusion in human induced pluripotent stem cells (iPSCs). iPSC-derived ETO2::GLIS2-expressing hematopoietic cells showed differentiation alterations in vitro and efficiently induced in vivo development of leukemia that closely phenocopied human acute megakaryoblastic leukemia (AMKL), reflected by flow cytometry and single-cell transcriptomes. Comparison of iPS-derived cells with patient-derived cells revealed altered chromatin accessibility at early and later bona fide leukemia stages, with aberrantly higher accessibility and expression of the osteogenic homeobox factor DLX3 that preceded increased accessibility to ETS factors. DLX3 overexpression in normal CD34+ cells increased accessibility to ETS motifs and reduced accessibility to GATA motifs. A DLX3 transcriptional module was globally enriched in both ETO2::GLIS2 AMKL and some aggressive pediatric osteosarcoma. Importantly, DLX3 knockout abrogated leukemia initiation in this ETO2::GLIS2 iPSC model. Collectively, the characterization of a novel human iPSC-derived AMKL model revealed that hijacking of the osteogenic homeobox transcription factor DLX3 is an essential early step in chromatin changes and leukemogenesis driven by the ETO2::GLIS2 fusion oncogene.

Decker M., Huang B.J., Ware T., Boone C., Tang M., Ybarra J., Ballapuram A.C., Taran K.A., Chen P., Amendáriz M., Leung C.J., Harris M., Tjoa K., Hongo H., Abelson S., et. al.

SUMMARYNormal and oncogenic Ras proteins are functionally dependent on one or more lipid modifications1,2. Whereas K-Ras4b farnesylation is sufficient for stable association with the plasma membrane, farnesylated H-Ras, K-Ras4a, and N-Ras traffic to the Golgi where they must undergo palmitoylation before regulated translocation to cell membranes. N-Ras palmitoylation by the DHHC family of palmitoyl acyl transferases (PATs) and depalmitoylation by ABHD17 serine hydrolases is a dynamic process that is essential for the growth of acute myeloid leukemias (AMLs) harboring oncogenicNRASmutations3–6. Here, we have tested whether co-targeting ABHD17 enzymes and Ras signal output would cooperatively inhibit the proliferation and survival ofNRAS-mutant AMLs while sparing normal tissues that retain K-Ras4b function. We show that ABD778, a potent and selective ABHD17 inhibitor within vivoactivity, selectively reduces the growth ofNRAS-mutant AML cellsin vitroand is synergistic with the allosteric MEK inhibitor PD0325901 (PD901)7,8. Similarly, ABD778 and PD901 significantly extended the survival of recipient mice transplanted with three independent primary mouse AMLs harboring an oncogenicNrasG12Ddriver mutation. Resistant leukemias that emerged during continuous drug treatment acquired by-pass mutations that confer adaptive drug resistance and increase mitogen activated protein kinase (MAPK) signal output. ABD778 augmented the anti-leukemia activity of the pan-PI3 kinase inhibitor pictilisib9, the K/N-RasG12Cinhibitor sotorasib10, and the FLT3 inhibitor gilteritinib11. Co-treatment with ABD778 and gilteritinib restored drug sensitivity in a patient-derived xenograft model of adaptive resistance to FLT3 inhibition. These data validate the palmitoylation cycle as a promising therapeutic target in AML and support exploring it in otherNRAS-mutant cancers.

Zhong L., Luo J., Dong J., Yang X., Wang X.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and the second most common in children. Despite the introduction of targeted therapies, AML survival rates have shown limited improvement, particularly among older patients. This study explored personalized treatment strategies for AML by proposing a novel subtyping method. Through unsupervised clustering based on the enrichment scores of 14 pathways related to metabolism, immunity, DNA repair, and oncogenic signaling, we identified three AML subtypes: DNA repair (DR), immune-enriched (ImE), and immune-deprived (ImD), consistent in four independent datasets. DR is marked by high expression of DNA repair and metabolic pathways, high stemness and proliferation potential, as well as high sensitivity to chemotherapy. ImD is characterized by low expression of immune and oncogenic pathways, favorable survival prognosis, low mutation rates of RUNX1 and TP53, high homeostasis, and low migration potential. ImE exhibits high enrichment of immune and oncogenic pathways, low stemness and proliferation capacity, low homeostasis, high migration potential, and low sensitivity to chemotherapy. Our pathway enrichment-based subtyping approach would offer a promising framework for understanding the molecular heterogeneity of AML and guiding personalized treatment of this disease.

Wang K., Saniei S., Poddar N., Autar S., Carcamo S., Sreenath M., Peplinski J.H., Ries R.E., Martinez I.G., Chao C., Mei A.H., Rahman N., Mekerishvili L., Quijada-Álamo M., Freed G., et. al.

SummaryAccumulating evidence links pediatric cancers to prenatal transformation events, yet the influence of the developmental stage on oncogenesis remains elusive. We investigated how hematopoietic stem cell developmental stages affect leukemic transformation, disease progression, and therapy response using a novel, humanized model of NUP98::NSD1-driven pediatric acute myeloid leukemia, that is particularly aggressive with WT1 co-mutations. Fetal-derived hematopoietic stem cells readily transform into leukemia, andWT1mutations further enhance stemness and alter lineage hierarchy. In contrast, stem cells from later developmental stages become progressively resistant to transformation. Single-cell analyses revealed that fetal-origin leukemia stem cells exhibit greater quiescence and reliance on oxidative phosphorylation than their postnatal counterparts. These differences drive distinct therapeutic responses, despite identical oncogenic mutations. In patients, onco-fetal transcriptional programs correlate with worse outcomes. By targeting key vulnerabilities of fetal-origin leukemia cells, we identified combination therapies that significantly reduce aggressiveness, highlighting the critical role of ontogeny in pediatric cancer treatment.

Yi J.S., Cuglievan B.

Yang K., Yang B., Zhou Y., Huang Q., Yin X.

Sperb N., Maksakova I.A., Escano L., Abraham L., MacPhee L., Cabantog A., Kim D., Yu M., Krowiorz K., Im J., Grasedieck S., Pochert N., Ruess C., Rösler R., Flibotte S., et. al.

Mendoza-Castrejon J., Yang W., Denby E., Wang H., Casey E.B., Muthukumar R., Patel R.M., Yoon J., Li Y., White J.M., Chen R., Batista L., Magee J.A.

ABSTRACTMLLrearrangements (MLLr) are the most common cause of congenital and infant leukemias.MLLr arise prior to birth and require few cooperating mutations for transformation, yet congenital leukemias are 10-fold less common than infant leukemias and >100-fold less common than childhood leukemias overall. This raises the question of whether mechanisms exist to suppress leukemic transformation during fetal life, thereby protecting the developing fetus from malignancy during a period of rapid hematopoietic progenitor expansion. Here, we use mouse models to show that fetal MLL::ENL exposure creates a heritable, leukemia-resistant state. MLL::ENL imposes a negative selective pressure on fetal hematopoietic progenitors. It leads to postnatal loss of self-renewal gene expression and enhanced myeloid differentiation that precludes transformation. These changes do not occur when MLL::ENL is induced shortly after birth, and transformation proceeds efficiently in this context. The fetal barrier to transformation is enforced by the histone methyltransferase MLL3. It can be overcome by cooperating mutations, such asNrasG12D, or through somatic or germline inactivation of MLL3. Heritable fetal protection against leukemic transformation may explain the low incidence of congenital leukemias in humans despite prenatalMLLrearrangement.

Qiu K., Liao X., Fang J., Zhou D.

Purpose: We aimed to develop a simple nomogram and online calculator that can identify the optimal subpopulation of pediatric acute myeloid leukemia (AML) patients who would benefit most from gemtuzumab ozogamicin (GO) therapy. Methods: Within the framework of the phase Ⅲ AAML0531 randomized trial for GO, the event-free survival (EFS) probability was calculated using a predictor-based nomogram to evaluate GO treatment impact on EFS in relation to baseline characteristics. Nomogram performance was assessed by the area under the receiver operating characteristic curve (AUC) and the calibration curve with 500 bootstrap resample validations. Decision curve analysis (DCA) was performed to evaluate the clinical utility of the nomogram. Results: A total of 705 patients were randomly assigned to two arms: the No-GO arm (n = 358) and the GO arm (n = 347). We performed a nomogram model for EFS among childhood AML. The AUC (C statistic) of the nomogram was 0.731 (95%CI: 0.614–0.762) in the development group and 0.700 (95% CI: 0.506–0.889) in the validation group. DCA showed that the model in the development and validation groups had a net benefit when the risk thresholds were 0–0.75 and 0–0.75, respectively. Notably, an intriguing observation emerged wherein pediatric patients with AML exhibited a favorable outcome in the GO arm when the predicted 5-year EFS probability fell below 60%, demonstrating a superior EFS compared to the No-GO Arm. Conclusions: We have developed a nomogram and online calculator that can be used to predict EFS among childhood AML based on trial AAML0531, and this might help deciding which patients can benefit from GO.

Cui B., Ai L., Lei M., Duan Y., Tang C., Zhang J., Gao Y., Li X., Zhu C., Zhang Y., Zhu X., Isobe T., Yang W., Gottgens B., Zhu P.

Abstract Pediatric acute myeloid leukemia (pAML) is a clonal disease with recurrent genetic alterations that affect epigenetic states. However, the implications of epigenetic dysregulation in disease progression remain unclear. Here, we interrogated single-cell and clonal level chromatin accessibility of bone marrow samples from 28 patients with pAML representing multiple subtypes using mitochondrial single-cell assay for transposase-accessible chromatin with sequencing, which revealed distinct differentiation hierarchies and abnormal chromatin accessibility in a subtype-specific manner. Innate immune signaling was commonly enhanced across subtypes and related to improved advantage of clonal competition and unfavorable prognosis, with further reinforcement in a relapse-associated leukemia stem cell–like population. We identified a panel of 31 innate immunity–related genes to improve the risk classification of patients with pAML. By comparing paired diagnosis and postchemotherapy relapse samples, we showed that primitive cells significantly reduced major histocompatibility complex class II signaling, suggesting an immune evasion mechanism to facilitate their expansion at relapse. Key regulators orchestrating cell cycle dysregulation were identified to contribute to pAML relapse in drug-resistant clones. Our work establishes the single-cell chromatin accessibility landscape at clonal resolution and reveals the critical involvement of epigenetic disruption, offering insights into classification and targeted therapies of patients with pAML.

Myeong J., Lee M., Lee B., Kim J.H., Nam Y., Choi Y., Kim J., Jeon S.Y., Shim H., Jung D., Shin Y., Jeong M., Oh B., Jung J., Kim C.S., et. al.

Bubb Q.R., Balood M., Seir G.E., Swartzrock L., Haslett E., Ho K., Xu P., Wiltz S.G., Sotillo E., Gruber T.A., Richards R.M., Mackall C.L., Czechowicz A.

Nguyen N.H., Rafiee R., Parcha P.K., Tagmount A., Rubnitz J., Ribeiro R.C., Cao X., Pounds S.B., Vulpe C.D., Lamba J.K.

Abstract Cytarabine, daunorubicin, and etoposide (ADE) have been the standard backbone of induction chemotherapy regimen for patients with pediatric acute myeloid leukemia (pAML) for &gt;5 decades. However, chemoresistance is still a major concern, and a significant proportion of pAML becomes resistant to ADE treatment and relapse, leading to poor survival. Therefore, there is a considerable need to identify mechanisms mediating drug resistance for overcoming chemoresistance. Herein, we performed synthetic lethal CRISPR/Cas9 screens using the ADE components to identify response markers. We further integrated significant markers in 3 independent pAML clinical cohorts treated with only an ADE regimen to identify drug response biomarkers with prognostic significance. We were able to identify several mediators that represent clinically and biologically significant marker genes for ADE treatment, such as BCL2, CLIP2, and VAV3, which are resistant markers to ADE, with high expression associated with poor outcomes in pAML treated with ADE, and GRPEL1, HCFC1, and TAF10, which are sensitive markers to ADE, with high expression showing beneficial outcomes. Notably, BCL2, CLIP2, and VAV3 knockdowns in their expression in AML cell lines sensitized the cells more to the ADE components, suggesting that these modulators should be further studied as potential therapeutic targets to overcome chemoresistance.