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PURPOSE We determined the safety and efficacy of coadministration of CD19- and CD22-chimeric antigen receptor (CAR) T cells in patients with refractory disease or high-risk hematologic or isolated extramedullary relapse of B-acute lymphoblastic leukemia. PATIENTS AND METHODS This phase II trial enrolled 225 evaluable patients age ≤ 20 years between September 17, 2019, and December 31, 2021. We first conducted a safety run-in stage to determine the recommended dose. After interim analysis of the first 30 patients treated (27 at the recommended dose) showing that the treatment was safe and effective, the study enrolled additional patients according to the study design. RESULTS Complete remission was achieved in 99.0% of the 194 patients with refractory leukemia or hematologic relapse, all negative for minimal residual disease. Their overall 12-month event-free survival (EFS) was 73.5% (95% CI, 67.3 to 80.3). Relapse occurred in 43 patients (24 with CD19+/CD22+ relapse, 16 CD19–/CD22+, one CD19–/CD22–, and two unknown). Consolidative transplantation and persistent B-cell aplasia at 6 months were associated with favorable outcomes. The 12-month EFS was 85.0% (95% CI, 77.2 to 93.6) for the 78 patients treated with transplantation and 69.2% (95% CI, 60.8 to 78.8) for the 116 nontransplanted patients ( P = .03, time-dependent covariate Cox model). All 25 patients with persistent B-cell aplasia at 6 months remained in remission at 12 months. The 12-month EFS for the 20 patients with isolated testicular relapse was 95.0% (95% CI, 85.9 to 100), and for the 10 patients with isolated CNS relapse, it was 68.6% (95% CI, 44.5 to 100). Cytokine release syndrome developed in 198 (88.0%) patients, and CAR T-cell neurotoxicity in 47 (20.9%), resulting in three deaths. CONCLUSION CD19-/CD22-CAR T-cell therapy achieved relatively durable remission in children with relapsed or refractory B-acute lymphoblastic leukemia, including those with isolated or combined extramedullary relapse. [Media: see text]

Intrahepatic cholangiocarcinoma (iCCA) is characterized by its highly desmoplastic stroma. Myofibroblasts (MFs) are present both within the tumor mass (intratumoral MFs, iMFs) and at the tumor border (peritumoral MFs, pMFs). Using a spheroid-based coculture system, we show that the initial iCCA-pMF contact is growth suppressive to the tumor cells. However, prolonged iCCA-pMF interaction elicits significant tumor cell invasion and dissemination. We find that vascular cell adhesion molecule-1 (Vcam1) level is elevated in tumor cells in contact with pMFs but low in disseminated tumor cells both in vitro and in vivo. A gene regulatory network analysis of mouse and patient iCCA tumors and Vcam1 knockout (Vcam1KO) demonstrate a heavy involvement of Vcam1 in epithelial-to-mesenchymal transition. While Vcam1KO has only a limited impact on tumor cell growth in their monoculture, Vcam1KO spheroids exhibit instant dissemination and a severe growth defect when cocultured with pMFs. When transplanted into the liver, Vcam1KO iCCA cells show a similar increase in dissemination but a significant defect in establishing primary and metastatic tumors. Incomplete blocking of Vcam1 in vivo reduces the size but increase the number of metastatic lesions. Overall, our study shows a spatiotemporal regulation of iCCA growth and dissemination by pMFs in a Vcam1-dependent manner.

Of 1003 children with acute lymphoblastic leukaemia (ALL), 147 (14.7%) presented without peripheral blood blasts (PBB). While absence of PBB was not independently associated with survival outcomes when compared to those with PBB, patients without PBB had distinct genetic and clinical characteristics. Notably, we identified a novel genotype-phenotype relationship, in that the patients without PBB had a significantly higher incidence of hyperdiploid B-ALL, accounting for almost half of all patients without PBB (46.9% vs. 22.7%, p 0.001). Further, absence of PBB was associated with decreased rates of leukaemia involvement of the central nervous system (p 0.001).

Identification of specific leukemia subtypes is a key to successful risk-directed therapy in childhood acute lymphoblastic leukemia (ALL). Although RNA sequencing (RNA-seq) is the best approach to identify virtually all specific leukemia subtypes, the routine use of this method is too costly for patients in resource-limited countries. This study enrolled 295 patients with pediatric ALL from 2010 to 2020. Routine screening could identify major cytogenetic alterations in approximately 69% of B-cell ALL (B-ALL) cases by RT-PCR, DNA index, and multiplex ligation-dependent probe amplification. STIL-TAL1 was present in 33% of T-cell ALL (T-ALL) cases. The remaining samples were submitted for RNA-seq. More than 96% of B-ALL cases and 74% of T-ALL cases could be identified based on the current molecular classification using this sequential approach. Patients with Philadelphia chromosome-like ALL constituted only 2.4% of the entire cohort, a rate even lower than those with ZNF384-rearranged (4.8%), DUX4-rearranged (6%), and Philadelphia chromosome-positive (4.4%) ALL. Patients with ETV6-RUNX1, high hyperdiploidy, PAX5 alteration, and DUX4 rearrangement had favorable prognosis, whereas those with hypodiploid and KMT2A and MEF2D rearrangement ALL had unfavorable outcomes. With the use of multiplex ligation-dependent probe amplification, DNA index, and RT-PCR in B-ALL and RT-PCR in T-ALL followed by RNA-seq, childhood ALL can be better classified to improve clinical assessments.

Although acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, it is unknown how or which host immune factors influence the long-term remission of this cancer. To this end, we systematically evaluated the effects of T-cell immunity on Ph+ ALL therapy outcomes. Using a murine Arf−/−BCR-ABL1 B-cell ALL model, we showed that loss of T cells in the host drastically increased leukemia relapse after dasatinib or cytotoxic chemotherapy. Although ABL1 mutations emerged early during dasatinib treatment in both immunocompetent and immunocompromised hosts, T-cell immunity was essential for suppressing the outgrowth of drug-resistant leukemia. Bulk and single-cell transcriptome profiling of T cells during therapy pointed to the activation of type 1 immunity-related cytokine signaling being linked to long-term leukemia remission in mice. Consistent with these observations, interferon γ and interleukin 12 directly modulated dasatinib antileukemia efficacy in vivo. Finally, we evaluated peripheral blood immune cell composition in 102 children with ALL during chemotherapy and observed a significant association of T-cell abundance with treatment outcomes. Together, these results suggest that T-cell immunity plays pivotal roles in maintaining long-term remission of ALL, highlighting that the interplay between host immunity and drug resistance can be harnessed to improve ALL chemotherapy outcomes.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121GA) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

Glucocorticoids (GCs) are a mainstay of contemporary, multidrug chemotherapy in the treatment of childhood acute lymphoblastic leukemia (ALL), and resistance to GCs remains a major clinical concern. Resistance to GCs is predictive of ALL relapse and poor clinical outcome, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in resistance. To address this, we mapped the gene regulatory response to GCs in two ALL cell lines using functional genomics and high-throughput reporter assays and identified thousands of GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and a depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. By integrating our GC response maps with genetic and epigenetic datasets in primary ALL cells from patients, we further uncovered cis-regulatory disruptions at GC-responsive genes that impact GC resistance in childhood ALL. Overall, these data indicate that GCs initiate pervasive effects on the leukemia epigenome, and that alterations to the GC gene regulatory network contribute to GC resistance.

Background Adult survivors of childhood cancer are at increased risk of cardiac late effects. Methods Using whole-genome sequencing data from 1870 survivors of European ancestry in the St. Jude Lifetime Cohort (SJLIFE) study, genetic variants were examined for association with ejection fraction (EF) and clinically assessed cancer therapy–induced cardiac dysfunction (CCD). Statistically significant findings were validated in 301 SJLIFE survivors of African ancestry and 4020 survivors of European ancestry from the Childhood Cancer Survivor Study. All statistical tests were 2-sided. Results A variant near KCNK17 showed genome-wide significant association with EF (rs2815063-A: EF reduction = 1.6%; P = 2.1 × 10-8) in SJLIFE survivors of European ancestry, which replicated in SJLIFE survivors of African ancestry (EF reduction = 1.5%; P = .004). The rs2815063-A also showed a 1.80-fold (P = .008) risk of severe or disabling or life-threatening CCD and replicated in 4020 Childhood Cancer Survivor Study survivors of European ancestry (odds ratio = 1.40; P = .04). Notably, rs2815063-A was specifically associated among survivors exposed to doxorubicin only, with a stronger effect on EF (3.3% EF reduction) and CCD (2.97-fold). Whole blood DNA methylation data in 1651 SJLIFE survivors of European ancestry showed statistically significant correlation of rs2815063-A with dysregulation of KCNK17 enhancers (false discovery rate Conclusions Leveraging the 2 largest cohorts of childhood cancer survivors in North America and survivor-specific polygenomic functional data, we identified a novel risk locus for CCD, which showed specificity with doxorubicin-induced cardiac dysfunction and highlighted dysregulation of KCNK17 as the likely molecular mechanism underlying this genetic association.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.

Maintenance therapy (MT) with oral methotrexate (MTX) and 6-mercaptopurine (6-MP) is essential for the cure of acute lymphoblastic leukemia (ALL). MTX and 6-MP interfere with nucleotide synthesis and salvage pathways. The primary cytotoxic mechanism involves the incorporation of thioguanine nucleotides (TGNs) into DNA (as DNA-TG), which may be enhanced by the inhibition of de novo purine synthesis by other MTX/6-MP metabolites. Co-medication during MT is common. Although Pneumocystis jirovecii prophylaxis appears safe, the benefit of glucocorticosteroid/vincristine pulses in improving survival and of allopurinol to moderate 6-MP pharmacokinetics remains uncertain. Numerous genetic polymorphisms influence the pharmacology, efficacy, and toxicity (mainly myelosuppression and hepatotoxicity) of MTX and thiopurines. Thiopurine S-methyltransferase (encoded by TPMT) decreases TGNs but increases methylated 6-MP metabolites (MeMPs); similarly, nudix hydrolase 15 (encoded by NUDT15) also decreases TGNs available for DNA incorporation. Loss-of-function variants in both genes are currently used to guide MT, but do not fully explain the inter-patient variability in thiopurine toxicity. Because of the large inter-individual variations in MTX/6-MP bioavailability and metabolism, dose adjustments are traditionally guided by the degree of myelosuppression, but this does not accurately reflect treatment intensity. DNA-TG is a common downstream metabolite of MTX/6-MP combination chemotherapy, and a higher level of DNA-TG has been associated with a lower relapse hazard, leading to the development of the Thiopurine Enhanced ALL Maintenance (TEAM) strategy—the addition of low-dose (2.5–12.5 mg/m2/day) 6-thioguanine to the 6-MP/MTX backbone—that is currently being tested in a randomized ALLTogether1 trial (EudraCT: 2018-001795-38). Mutations in the thiopurine and MTX metabolism pathways, and in the mismatch repair genes have been identified in early ALL relapses, providing valuable insights to assist the development of strategies to detect imminent relapse, to facilitate relapse salvage therapy, and even to bring about changes in frontline ALL therapy to mitigate this relapse risk.

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remain incompletely understood. Here, using integrated whole genome and transcriptome sequencing of 124 iAMP21-ALL patients, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL according to patterns of copy number alteration and structural variation. This large dataset enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which are differentially expressed compared to non-iAMP21-ALL cases, and including multiple genes implicated in the pathogenesis of acute leukemia: CHAF1B, DYRK1A, ERG, HMGN1 and RUNX1. Using multimodal single cell genomic profiling, including single cell whole genome sequencing of two cases, we documented clonal heterogeneity and genomic evolution, formally demonstrating that acquisition of the iAMP21-chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

Advancing cure rates for high-risk acute lymphoblastic leukemia (ALL) has been limited by the lack of agents that effectively kill leukemic cells sparing normal hematopoietic tissue. Molecular glues direct the cellular ubiquitin ligase cellular machinery to target neosubstrates for protein degradation. We developed a novel Cereblon modulator, SJ6986 that exhibited potent and selective degradation of GSPT1 and GSPT2, and cytotoxic activity against childhood cancer cell lines. Here we report in vitro and in vivo testing of the activity of this agent in a panel of ALL cell lines and xenografts. SJ6986 exhibited similar cytotoxicity to the previously described GSPT1 degrader CC-90009 in a panel of leukemia cell lines in vitro, resulting in apoptosis and perturbation of cell cycle progression. SJ6986 was more effective than CC-90009 in suppressing leukemic cell growth in vivo, partly attributable to favorable pharmacokinetic properties, and did not significantly impair differentiation of human CD34+ cells ex vivo. Genome wide CRISPR/Cas9 screening of ALL cell lines treated with SJ6986 confirmed components of the CRL4CRBN complex, associated adaptors, regulators and effectors were integral in mediating the action of SJ6986. SJ6986 is a potent, selective, orally bioavailable GSPT1/2 degrader that shows broad antileukemic activity and has potential for clinical development.

Background Survivors of pediatric central nervous system (CNS) tumors treated with craniospinal irradiation (CSI) exhibit long-term cognitive difficulties. Goals of this study were to evaluate longitudinal effects of candidate and novel genetic variants on cognitive decline following CSI. Methods Intelligence quotient (IQ), working memory (WM), and processing speed (PS) were longitudinally collected from patients treated with CSI (n = 241). Genotype-by-time interactions were evaluated using mixed-effects linear regression to identify common variants (minor allele frequency > 1%) associated with cognitive performance change. Novel variants associated with cognitive decline (P Results Participants were mostly males (65%) diagnosed with embryonal tumors (98%) at a median age of 8.3 years. Overall, 1150 neurocognitive evaluations were obtained (median = 5, range: 2–10 per participant). One of the five loci previously associated with cognitive outcomes in pediatric CNS tumors survivors demonstrated significant time-dependent IQ declines (PPARA rs6008197, P = .004). Two variants associated with IQ in the general population were associated with declines in IQ after Bonferroni correction (rs9348721, P = 1.7 × 10−5; rs31771, P = 7.8 × 10−4). In genome-wide analyses, we identified novel loci associated with accelerated declines in IQ (rs116595313, meta-P = 9.4 × 10−9), WM (rs17774009, meta-P = 4.2 × 10−9), and PS (rs77467524, meta-P = 1.5 × 10−8; rs17630683, meta-P = 2.0 × 10−8; rs73249323, meta-P = 3.1 × 10−8). Conclusions Inherited genetic variants involved in baseline cognitive functioning and novel susceptibility loci jointly influence the degree of treatment-associated cognitive decline in pediatric CNS tumor survivors.

Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers. However, the mechanisms controlling BETi response and resistance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a combination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts in vivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combination treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique ex vivo and in vivo KMT2A-r PDX models.

Supplementary Data Legend, Supplementary Table (S7) and Supplementary Figures (S1-S6)

We evaluated clinical significance of recently identified subtypes of acute lymphoblastic leukemia (ALL) in 598 children treated with minimal residual disease (MRD)–directed therapy. Among the 16 B-cell ALL (B-ALL) and 8 T-cell ALL subtypes identified by next-generation sequencing, ETV6–RUNX1, high-hyperdiploid, and DUX4-rearranged B-ALL had the best 5-year event-free survival rates (95.0%–98.4%); TCF3–PBX1, PAX5-altered (PAX5alt), T-cell, early T-cell precursor (ETP), intrachromosomal amplification of chromosome 21 (iAMP21), and hypodiploid ALL intermediate rates (80.0%–88.2%); and BCR–ABL1, BCR–ABL1-like, ETV6–RUNX1-like, and KMT2A-rearranged ALL the worst rates (64.1%–76.2%). All but 3 of the 142 patients with day 8 blood MRD DUX4-rearranged, BCR–ABL1-like, and ZNF384-rearranged ALL, and achievement of day 42 MRD MEF2D-rearranged, and ETV6–RUNX1-like ALL. Thus, new subtypes including DUX4-rearranged, PAX5alt, BCR–ABL1-like, ETV6–RUNX1-like, MEF2D-rearranged, and ZNF384-rearranged ALL have important prognostic and therapeutic implications.Significance:Genomic analyses and MRD should be used together for risk-directed treatment of childhood ALL. Six recently described subtypes—DUX4-rearranged, PAX5alt, BCR–ABL1-like, ETV6–RUNX1-like, MEF2D-rearranged, and ZNF384-rearranged ALL—had prognostic and therapeutic significance with contemporary risk-directed treatment.See related commentary by Segers and Cools, p. 294.See related video from the AACR Annual Meeting 2021: https://vimeo.com/558556916

Supplementary Table S1-S5

Supplementary Table S6

Cell line IC50 values

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5′-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B. Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia.Significance:Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries.This article is highlighted in the In This Issue feature, p. 2659

This file contains Supplementary Tables 1-15 detailing clinical and genomic information about each patient analyzed in this study, along with the results of RNA-seq and gene set enrichment analyses.

This supplementary table describes metabolite levels and statistics from metabolite experiments

This file contains Supplementary Figures 1-23 with corresponding supplementary figure legends, as well as the legends for Supplementary Tables 1-15.

Supplementary materials and methods and supplementary figure legends.

RNAseq Gene Lists

S1. Schematic diagram of NSD2 isoform indicating domains, the location of amino acid E1099 within the SET domain, and the targets of the shRNAs. S2. NSD2 Overexpression in B-ALL Cell Lines Does Not Impart Clonal Advantage. S3. Overexpression of REIIBP in B-ALL Cell Lines Does Not Impart Clonal Advantage. S4. Knockdown of WT NSD2 in B-ALL Cell Lines Does Not Lead to Phenotypic Differences. S5. Knockdown of NSD2 in RS4;11 and RCH-ACV lines leads to increased chemosensitivity. S6. Sensitivity to 6-MP Upon Knockdown of NSD2 in RS4;11 is Not Depedent of Level of Knockdown. S7. Knockdown of NSD2 in WT B-ALL Cell Lines Does Not Lead to Increased Sensitivity. S8. NSD2 WT and EK Overexpression in B-ALL Cell Lines Leads to Distinct Genetic Signature Unique to Each Cell Line. S9. NSD2 WT and EK Overexpression in B-ALL Cell Lines Leads to Distinct Epigenetic Signature Resulting in Gene Expression Changes. S10. NSD2 Knockdown in Mutant B-ALL Cell Lines Leads to Distinct Genetic Signature Unique to Each Cell Line. S11. ATAC peaks significantly correlate with gene expression for RS4;11 and RCH-ACV cell lines.

Supplementary figure 1 and 2 describe protein purity and expression. Supplementary Figure 3 shows cell-based cytotoxic assays. Supplementary Figure 4 shows protein activity against several substrates. Supplementary Figure 5 describes levels of individual metabolites.