Stanley Pounds

Relapsed B-acute lymphoblastic leukemia (B-ALL) generally occurs due to the emergence of subclones resistant to therapy, leading to poor outcome. Here, we describe novel genomic alterations responsible for therapeutic resistance at a subtype level in a clinical trial (AALL1331) which randomized patients to blinatumomab and chemotherapy versus chemotherapy alone. We performed whole-genome and transcriptome sequencing on 288 patients (183 diagnosis/first relapse/germline trios and 105 additional patients with first-relapse samples) and analyzed 41 second-relapse samples. For ETV6::RUNX1, we showed that cases with gain of the fusion allele or with fusion transcript involving exon 5 of ETV6 and exon 4 of RUNX1 (E5-E4) had a higher risk of first relapse than cases with E5-E3. For the hyperdiploid subtype, we demonstrated that low modal chromosomal numbers (51-52) were more prone to first relapse than those with ≥53 chromosomes, with an additive effect of CREBBP mutations. We showed enrichment of IKZF1 alterations in the Philadelphia-like subtype and that NSD2 E1099K was commonly associated with relapse in TCF3::PBX1. Together, these new features accounted for 30% (n=55) of first relapses. Intriguingly, ~3% of relapses are related to secondary B-ALL or germline predisposition. We also compared genomic alterations at diagnosis and first relapse in relation to second relapse status in randomized patients, with TCF3::PBX1/NT5C2 linked to worse outcomes regardless of randomized arm. Together, our findings provide novel insights into the genomics of relapsed childhood B-ALL as well as in the context of blinatumomab therapy. This trial was registered at www.clinicaltrials.gov as # NCT0201853.

Background: Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML) and contributes to leukemogenesis, treatment response, and clinical heterogeneity. While genome-wide methylation studies have identified prognostic methylation signatures, the impact of DNA methylation within pharmacologic pathways and AML-relevant disease genes remains incompletely understood. We investigated the association of DNA methylation in genes of pharmacokinetic/pharmacodynamic (PK/PD) pathways of drugs used to treat AML and in myeloid leukemia-related genes with treatment outcomes in pediatric AML. Methods: DNA methylation profiles from 924 pediatric AML patients treated on Children’s Oncology Group trials (AAML1031, AAML0531, and AAML03P1—available publicly) were analyzed as a discovery cohort. A validation cohort included 159 patients treated on the AML02 trial. A total of 2296 variable CpG sites mapping to 65 PK/PD genes and 107 AML biology/leukemia stemness genes were evaluated. Associations between CpG methylation, gene expression, event-free survival (EFS), overall survival (OS), and measurable residual disease after induction I (MRD1) were assessed using Cox proportional hazards, logistic regression, and correlation analyses. Results: Twenty-three CpG sites in PK/PD genes and forty-two CpG sites in AML-related genes were significantly associated with at least one clinical endpoint after Bonferroni correction (p < 2.17 × 10⁻⁵). Hypermethylation of drug transporters ABCA3, ABCC1, and SLC22A1, as well as pharmacologically relevant genes MPO, NOS3, and CTPS1, was associated with inferior survival and/or increased MRD1 positivity. Among AML biology genes, methylation of ETV6, NOTCH1, RUNX1, KIT, MPL, DNMT3A, and DNMT3B demonstrated consistent associations with outcomes across discovery and validation cohorts. Several genes exhibited significant inverse correlations between DNA methylation and gene expression, including MPO, KIT, MPL, SPINK2, and DNMT3B, supporting functional epigenetic regulation. Notably, hypermethylation of ABCA3, MPO, and MPL was reproducibly associated with poor OS and EFS in both cohorts. Conclusions: DNA methylation of key pharmacologic and leukemia-related genes is associated with clinical outcomes in pediatric AML. These findings identify biologically and clinically relevant epigenetic biomarkers that may improve risk stratification and support the development of precision medicine approaches incorporating DNA methylation profiling and epigenetic therapies in pediatric AML.

The increasing availability of genomic and transcriptomic sequencing has uncovered diverse genomic alterations and distinct gene expression profiles driving hematologic diseases, yet a data integration and sharing platform dedicated to hematology remains lacking. We developed the American Society of Hematology (ASH) HematOmics Program (ASHOP, ashop.hematology.org), a resource for exploring somatic alterations and gene fusions, transcriptomic results, and clinical data from 5,960 patients spanning B-cell precursor and T-cell acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndromes, and chronic lymphocytic leukemia. Users can explore genomic alteration landscapes and co-mutation patterns via lollipop and matrix plots, and analyze significantly altered genes in user-defined sub-cohorts. Transcriptomes can be explored through interactive UMAPs, clustering, differential expression, and pathway enrichment. Genomic, transcriptomic features, and clinical outcomes can be correlated in a user-driven manner, or combined to precisely define study cohorts. We illustrate four use cases of ASHOP: (1) stratification of DUX4-rearranged B-cell leukemias into Early/Multipotent and Committed subgroups with distinct outcomes, (2) characterization of HOXA/HOXB expression patterns in acute myeloid leukemias, (3) correlating mutational burden with mismatch repair deficiency and mutational signatures, (4) investigation of TP53 alteration landscape. ASHOP is an open-access resource to inform genomic and transcriptomic data interpretation for hematologic malignancies, and will expand to support additional diseases and data modalities from the ASH community.

By integrating short-read whole-genome sequencing and RNA sequencing (RNA-seq) data with long-read RNA-seq, we dissect the complex genomic architecture of PAX5 intragenic tandem multiplication, revealing that these complex rearrangements result in in-frame transcripts that likely encode proteins with altered domains.

Background: Rapid advances in biotechnology provide researchers with the opportunity to integrate omics profiles (genomics, epigenomics, transcriptomics, proteomics, etc.) with multiple phenotypes or experimental conditions. In cancers such as acute myeloid leukemia (AML), where combination therapies are standard of care, identifying genetic drivers of drug resistance requires evaluating how genes are associated with multiple drug response phenotypes. Statistical analyses associating omics profiles with multiple phenotypes yield multiple significance values and rankings for each of many genes. There is a great need to consolidate these multiple rankings into a consensus ranking to prioritize specific genes for detailed follow-up wet-lab or clinical studies. Methods/Results: Here, we evaluate the well-known Fisher’s method, the sum of squared z-statistics (SSz), and the recently published cellMCD method as tools for gene prioritization. In simulation studies, cellMCD showed very similar or highly superior performance to the widely used Fisher’s and SSz methods. These advantages were also observed in an example application involving a CRISPR drug screen of an acute myeloid leukemia cell line. Conclusions: In summary, our results indicate that cellMCD should be more widely used for prioritizing discoveries from multiple omic association studies. These methods are available as an R package on github.

Relapse remains the leading cause of mortality in pediatric acute myeloid leukemia (AML), yet the genetic changes contributing to relapse remain incompletely defined. To address this gap, we performed whole-genome sequencing and targeted-capture sequencing on 39 diagnosis–relapse and 2 relapse–relapse pairs of pediatric AML. Mutational burden increased at relapse, largely reflecting spontaneous mutagenesis, whereas therapy-related signatures were rarely observed and only occasionally associated with pathogenic mutations. Although recurrently enriched mutations at relapse included those in FLT3 , WT1 , and TP53 , relapse-fated subclones were frequently marked only by non-pathogenic or non-coding variants. Longitudinal deep sequencing in eight patients showed rapid depletion of major clones after induction therapy, whereas subclones often displayed variable chemosensitivity. Relapse-specific mutations emerged only late or remained undetectable during remission, suggesting that clonal selection of pre-existing clones is the predominant mechanism of relapse. Transcriptome analysis of paired RNA sequencing data revealed no differentially expressed genes, but gene set enrichment analysis and CIBERSORT deconvolution in each pair uncovered heterogeneous trajectories to relapse. Although relapse is often attributed to the emergence of stem-like phenotypes, our data demonstrate that transcriptional evolution is more diverse: some cases acquired stem-like features, whereas others showed partial differentiation, which was confirmed by re-analysis of a public single cell RNA sequence dataset. These changes were largely constrained by baseline differentiation states at diagnosis. Together, our data indicate that pediatric AML relapse arises through selection of pre-existing clones with diverse trajectories, underscoring the need to target both stem-like and differentiated populations to achieve durable cures.

Leukemias with NUP98 rearrangements exhibit heterogeneous phenotypes such as acute myeloid leukemia, T-cell acute lymphoblastic leukemia (T-ALL), or myelodysplastic syndrome/neoplasms associated with fusion partners, whereas the mechanism responsible for this heterogeneity is poorly understood. Through genome-wide mutational and transcriptional analyses of 177 NUP98-rearranged leukemias, we show that cooperating alterations are associated with differentiation status even among leukemias sharing the same NUP98 fusions, such as NUP98::KDM5A acute megakaryocytic leukemia with RB1 loss or T-ALL with NOTCH1 mutations. CUT&RUN profiling of in vitro cord blood CD34+ cell (cbCD34) models of major NUP98 fusions revealed that NUP98-fusion oncoproteins (FOs) directly regulate differentiation-related genes contributing to the disease phenotypes, represented by NUP98::KDM5A binding to MEIS2 or GFI1B for megakaryocyte (MK) differentiation. In patient samples, NUP98-FO binding patterns are heterogeneous, potentially shaped by somatic mutations and differentiation status. Using cbCD34 models and CRISPR/Cas9 gene editing, we show that RB1 loss cooperates with NUP98::KDM5A by blocking terminal differentiation toward platelets and expanding MK-like cells, whereas WT1 frameshift mutations skew differentiation toward dormant lymphoid-myeloid primed progenitor cells and cycling granulocyte-monocyte progenitor cells, providing evidence for NUP98-rearranged leukemia phenotypes affected by cooperating alterations. NUP98::KDM5A cbCD34 models with RB1 or WT1 alterations have different sensitivities to menin inhibition, suggesting that cellular differentiation provides stage-specific menin dependencies and resistance mechanisms that can be leveraged for future treatment strategies for NUP98-rearranged leukemia.

Stress granules (SGs) are dynamic RNA-protein assemblies that form in response to cellular stress and must be efficiently disassembled to restore normal cell function. Valosin-containing protein (VCP), an enzyme implicated in neurodegenerative diseases, is essential for SG disassembly, but whether and how this process is coordinated with SG assembly remains unclear. Here, we identify the VCP cofactor, Alveolar soft part sarcoma locus (ASPL) as a key regulator linking SG assembly and disassembly. ASPL promotes SG assembly by facilitating biomolecular condensation of Ras guanosine triphosphatase–activating protein-binding protein (G3BP) and stabilizing its interactions with other SG proteins. ASPL also facilitates phosphorylation and activation of VCP by UNC-51-like kinases 1 and 2 (ULK1/2), enabling G3BP extraction and efficient SG disassembly. Pathogenic VCP mutations that disrupt ASPL binding impair SG disassembly, a defect rescued by phosphomimetic mutations or ASPL depletion. Our findings suggest that disruptions in the ASPL-VCP interaction uncouple SG assembly and disassembly, representing a potential mechanism underlying VCP-associated neurodegenerative diseases.

Supplementary Figure S1. T-ALL subtype classifying driver genes among individuals of European, Admixed American, and African ancestry. Supplementary Figure S2. T-ALL subtype classifying driver genes among patients with Early T-cell Precursor (ETP)-like subtype among individuals of European, Admixed American and African ancestry. Supplementary Figure S3. Distribution of 15 genomically defined T-ALL subtypes described by Pölönen and colleagues (16) among individuals of African and Non-African ancestry. Supplementary Figure S4. 5-year event free survival for NOTCH1 and FBXW7 by ancestral group. Supplementary Figure S5. Prevalence of (A) coding versus non-coding alterations and (B) types of pathogenic variants in NOTCH1 among individuals of African versus non-African ancestry. Supplementary Figure S6. Event free survival among all patients, patients of European, Admixed American, and African ancestry by NOTCH1 alterations (no mutation, favorable, unfavorable). Supplementary Figure S7. NOTCH1 coding mutation types (frameshift, missense, nonsense) on a lollipop plot in patients of European, African, and Admixed American Ancestry. Supplementary Figure S8. 5-year event free survival for genes with at least 5% frequency for each ancestral group (CDKN2A, PHF6, PTEN, DNM2). Supplementary Figure S9. 5-year event free survival for genes with at least 5% frequency for each ancestral group (LEF1, MYB, MYC, 6q loss). Supplementary Figure S10. 5-year event free survival for genes with at least 5% frequency for each ancestral group (WT1, USP7, IL7R, N/KRAS). Supplementary Figure S11. Distribution of genetic ancestral composition of individuals by categorically defined genetic ancestry.

T-cell Acute Lymphoblastic Leukemia subtype by Categorical Genetic Ancestry