Aberrant activation of TAL1, a key oncogenic driver, defines a major subgroup comprising ~30% of childhood T-lineage acute lymphoblastic leukemias (T-ALLs). We and others have shown that somatic non-coding mutations within upstream and intronic cis-regulatory regions of TAL1 contribute to transformation by creating binding sites for MYB and other transcription factors. Here we investigated cis-regulatory mechanisms mediated by somatic mutations occurring in an intergenic region located 29 kilobase pairs downstream of the canonical TAL1 transcription initiation site, implicated in 6% of TAL1-expressing T-ALLs. These somatic variants include i) complex indels resulting in de novo MYB transcription factor binding sites (TFBSs) and ii) internal tandem duplications (ITDs) encompassing canonical MYB TFBSs. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed binding of the TAL1 core regulatory circuit (CRC) transcription factors MYB, GATA3, and RUNX1, resulting in enhancer activity mediated by sequences with the mutant allele. Strikingly, ChIP-seq peaks for the repressive H3K27me3 mark and the active H3K27ac mark co-existed across TAL1 regulatory sequences but enriched for different haplotypes. TAL1 transcription from the mutant haplotype initiated from a promoter located within exon 4 of the canonical TAL1 transcript, resulting in a short isoform normally expressed by hematopoietic stem cells (HSC). Interestingly, neither the isoform expression nor the enhancer activity could be predicted by the sequence-to-function deep learning artificial intelligence (AI) model AlphaGenome, emphasizing the importance of experimental validation. Our findings indicate that selection for cis-regulatory, non-coding variants leads to reactivation of enhancers normally active in HSC but silenced in differentiated lineages during normal hematopoietic cell development.

Despite extensive studies of the error profiles of SNVs, those of insertions/deletions (indels)/structural variants (SVs) remain elusive. Using ultra-deep sequencing, we show that the error rates of indel/SVs are >100-fold lower than those of SNVs, although repeat indels have high error rates of 1%. We validated this pattern in a cohort of 103 patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We analyzed repeat indels in 339 cancer driver genes and demonstrated that the number of repeat units is highly predictive of the error rate. We then analyzed minimal residual disease samples from 72 patients with relapsed B-ALL and demonstrated that our approach had positive detections in 61% of cases, outperforming clinical flow cytometry (51% detection). Overall, we established indel and SV error profiles in deep next-generation sequencing (NGS) data, enabling superior tumor detection at very low burdens, which has a significant impact on the clinical diagnosis and monitoring of human cancers and other diseases.

Despite high cure rates of >90% for newly diagnosed childhood B-ALL, outcomes remain poor for relapsed patients. Prior genomics studies have revealed 12 genes with mutations enriched at relapse. However, the resistance mechanisms for >50% of relapses remain elusive. Here we performed WGS and RNA sequencing on initial diagnostic, relapse, and remission specimens from 183 patients enrolled in the AALL1331 trial of the Children’s Oncology Group. In this cohort, 96 cases did not harbor a known resistance mutation. These cases were enriched in several common subtypes, including those with favorable outcomes (hyperdiploid ALL, ETV6::RUNX1, and DUX4) and those with less favorable outcomes (Ph-Like, iAMP21). By analyzing tumor mutation burdens coupled with ancestral drivers such as fusions, we uncovered 5 cases where the relapses were a novel clone unrelated to the clone at diagnosis, indicative of secondary B-ALL. In the remaining 178 truly relapsed cases, we discovered four mechanisms of therapy resistance. First, a collaborating mutation can be acquired and confer resistance in either a subtype-dependent manner (NSD2 in TCF3::PBX1 (P=1.2×10-6) or IKZF1 in Ph-Like (P=1.7×10-22) and DUX4 (P=0.02) subtypes), or in a pan-subtype manner (the previously reported NT5C2/PRPS1/PRPS2 genes). Second, amplification of the fusion allele of ETV6::RUNX1 can confer elevated resistance. In the AALL1331 cohort, 26% (out of 31) of relapsed ETV6::RUNX1 tumors harbor an amplification of the fusion allele, as compared to 13% of the original 31 diagnostic tumors (P=0.12). We also validated this trend in a recently published cohort of diagnostic samples enriched for eventual relapse (MP2PRT, Cheng et al, JCO 2024) at a frequency of 10% in patients experiencing a subsequent relapse and 8% in those who remained in long term remission. Third, fusion isoforms formed from different introns can confer elevated resistance. In AALL1331, 19% of ETV6::RUNX1 patients have isoform E5-E4 in their diagnostic and relapse tumors, compared with the expected frequency of 4% (P=0.007) if E5-E4 had comparable selection pressure as isoform E5-E3. In the MP2PRT cohort, E5-E4 is observed in 20% of those who went on to relapse as compared to 11% (P=0.01) of patients who did not relapse. Fourth, the total number of chromosomes in hyperdiploid ALL can confer elevated resistance. In AALL1331, 41% (out of 29) of hyperdiploid ALL cases had ≤52 chromosomes, as compared to 13% (out of 403) in the MP2PRT cohort (P=2×10-4). Concordantly, in the MP2PRT cohort, hyperdiploid ALL with ≤52 chromosomes had an eventual relapse rate of 43% compared to 26% for patients with 53 chromosomes, versus ∼17% when there are ≥54 chromosomes. Together, these mechanisms explained 72% of relapses in our cohort, suggesting risk prediction may benefit from incorporating these newly discovered mutations. Xiaotu Ma, Yuan Feng, Hanxia Li, Ying Shao, Heather Mulder, Pandurang Kolekar, Yanling Liu, Quang Tran, Zhikai Liang, Li Fan, Scott Foy, Ti-Cheng Chang, Wenana Chen, Stanley Pounds, Gang Wu, Charles Mullighan, William L. Carroll, Jinghui Zhang, John Easton, Patrick Brown, Mignon L. Loh. Diverse mechanisms of therapy resistance in relapsed childhood B-cell acute lymphoblastic leukemia (B-ALL): a report from the Children’s Oncology Group [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1205.

While the outcomes for pediatric acute lymphoblastic leukemia (ALL) have improved dramatically over the past 30 years, up to 20% of patients relapse and their prognosis is poor. Collaborators at St. Jude’s Research Hospital discovered that mutations in ZMIZ1 are highly enriched at relapse in a subset of patients. Subsequent studies showed that the transcript level is correlated to event-free survival. Thus, our objective is to study the function of ZMIZ1 in cancer survival under the selective pressures of chemotherapy. To accomplish this, we generated a panel of B-ALL cell lines overexpressing either wild-type (WT), or two different mutant forms of ZMIZ1 found in patients (A291-A305 deletion (Del), or A297T substitution (Sub)) and compared them to an empty vector (EV) control. We performed reverse transcription quantitative PCR (RT qPCR) to confirm mRNA expression, western blots to determine expression of ZMIZ1, as well as cell viability, apoptosis, and proliferation assays to assess functional impact. While full length transcripts were confirmed in all three lines, full length protein was only detected in the WT overexpressing line. All lines expressing the mutant forms of ZMIZ1 demonstrated a lower molecular weight product, suggesting post-translational modification. Phenotypically, we observed profound resistance to prednisolone, a cornerstone of ALL therapy, upon WT (IC50 >1e-1mM) expression compared to EV (IC50=3e-4mM), while cell lines expressing the Del or Sub showed no difference in drug sensitivity (IC50s=3e-4mM). No differences were seen with other commonly used chemotherapies. Since these experiments were done in cell lines with two WT alleles, we then used CRISPR-cas9 to knock out endogenous ZMIZ1. Allele specific sequencing for individual clones was confirmed using IDT’s rhAmpSeqTM CRISPR kit. ZMIZ1 complete knockouts (KO) showed no detectable protein expression and had increased sensitivity to prednisolone (IC50=3e-5mM) compared to the Rosa control (IC50=5.7e-5mM) in both cell viability and apoptosis assays. Subsequently, we reintroduced ZMIZ1 (WT and mutants) into KO cells and again only WT had detectable full length protein expression, but now all three lines showed the lower molecular weight product. Resistance to prednisolone was restored completely upon rescue with WT ZMIZ1 (IC50>1e-1mM) compared to the complete KO cells and Rosa. In contrast, mutant cell lines now showed modest resistance to prednisolone (IC50s 4.9e-5mM Del; 5.6e-5mM Sub) compared to KO. Collectively our results indicate a complex relationship between the products of WT and mutant alleles mediated in part by a post-translationally modified species involved in glucocorticoid resistance. A more thorough understanding of how mutations in ZMIZ1 affect the regulation and drug resistance is vital in determining the clinical significance and evaluating ZMIZ1 as a potential therapeutic target in patients. Caroline S. Greenstone, Jessica Krugman, Danielle W. Cohen, John Easton, Heather Mulder, Ying Shao, Xiaotu Ma, Nikki A. Evensen, William L. Carroll. The impact of aberrant ZMIZ1 protein expression in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7056.

The transcription factor (TF) TAL1 is a master regulator expressed at the early stages of normal thymocyte development. It is one of the major drivers in childhood T-lineage acute lymphoblastic leukemia (T-ALL) and is aberrantly activated in 30-40% of T-ALL. While somatic alterations in upstream and intronic cis-regulatory regions of TAL1 are known mechanisms of activation, the role of downstream enhancer alterations (DEAs) remains less understood. We investigated somatic alterations in an intergenic region 11Kb downstream of TAL1, identifying a complex indel that created a de novo MYB transcription factor binding site (TFBS), and a 2.4Kb internal tandem duplication (ITD) encompassing a MYB TFBS. Alterations in this region were found to be present in 2% of the samples in a recent T-ALL landscape study involving 1,335 cases. Using the patient-derived xenograft (PDX) mouse models of the two T-ALLs harboring the complex indel and ITD DEA, we performed multi-omic profiling, including H3K27ac, H3K4me1, H3K27me3 and MYB chromatin immunoprecipitation sequencing (ChIP-seq); assay for transposase-accessible chromatin using sequencing (ATAC-seq), Hi-C, RNA-seq and full-length transcriptome sequencing (Iso-seq). Enhancer activity by DEA was validated by presence of H3K27ac peaks and cis-activation of TAL1 which exhibits mono-allelic expression in both the primary tumors and PDX models. Involvement of MYB in enhancer activity is validated by MYB ChIP-seq peaks centered around the de novo and existing MYB TFBSs. Interestingly, we found that TAL1 activation via DEA led to preferential initiation of transcription from the promoter located at the exon 4, resulting in predominant expression of the TAL1-short isoform in cases with DEA. Indeed, TAL1-short expression accounted for 78% of the TAL1 expression in T-ALLs harboring DEA in contrast to the 37% frequency in other TAL1-activated T-ALLs (Wilcoxon two-sided test P=0.001). Furthermore, using the full-length transcripts generated by PacBio Iso-seq platform on the PDX samples, only TAL1-short transcripts were detected. By comparing Hi-C data generated from our PDX samples with those from Jurkat (TAL1+, with upstream enhancer region) and DND41 (TAL1-) cell lines, we identified a re-organization of chromatin looping structure showing distinct interaction between DEA region and TAL1 promoter at exon 4. This study provides a detailed view of the regulatory mechanism of TAL1 activation by DEA, indicating that preferential expression of TAL1-short isoform, protein product of which is known to bind more strongly to TAL1 E-protein partners than that of TAL1-long isoform, is active in initiating and maintaining the TAL1 core regulatory circuit in the transformation of pre-T cell progenitors in T-ALL. Nadezhda V. Terekhanova, Xiaolong Chen, Wentao Yang, Ying Shao, Li Dong, Bensheng Ju, Yu Liu, John Easton, A. Thomas Look, Jinghui Zhang. Mechanism of TAL1 activation by alterations in the downstream enhancer in childhood T-lineage acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4057.

Supplementary results, figures, and table notes

Supplementary Data from EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma

PDX targeted-sequencing tissue concordance

Supplementary Figures S1-S11

Background: Accurate detection of low frequency mutations is of critical importance in the study of genetic heterogeneity, such as on the detection of minimal residual diseases for leukemias. Our prior work has resulted in successful error suppression for substitutions (10−4-10−5). However, the error profiles of indels and structural variants (SVs) remain elusive. Results: In this work, we generated ultra-deep sequencing data using our previously established dilution models (COLO829) on known somatic indels (n=23) and SVs (n=17). We discovered that the error rate of indels (10−6) and SVs (<2 × 10−7) are 100- to >1000-fold lower than that of SNVs. This finding was fully recapitulated in our analysis of 347 indels and 1248 SVs discovered from a relapsed B-ALL cohort of 103 patients, although homopolymer indels can have high error rates (>1%). We then performed a comprehensive study of homopolymer indels in 361 cancer driver genes by using whole genome data from 1662 healthy donors from the SJLIFE cohort. Our data indicated that the number of repeating units are highly predictive relative to the error rate of homopolymer indels (R2=0.988, p=4.89 × 10−8). Utilizing these insights, we assayed end-of-induction remission samples from 72 B-cell lymphoblastic leukemia patients that relapsed by selecting ~5 somatic clonal SNV/Indel/SV markers, which confirmed that SVs and indels have >10-fold lower error rates than SNVs. Our next generation sequencing (NGS) approach had 44 positive detections (61%) and outperformed the current standard method of clinical flow cytometry (n=37; 51%) for detecting minimal residual disease. The NGS-based method detected 92% of designed markers for samples with MRD>0.3%, and this detection rate dropped to 27% for MRD between 0.1% and 0.01%, indicating the difficulty in recovering mutant molecules when their frequencies are very low. Conclusions: Overall, we established indel and SV error profiles in deep next generation sequencing data enabling superior tumor detection performance at very low burdens, with lower error rates than what is observed for SNVs. Our work will have a significant impact on the clinical diagnosis and monitoring of human cancers and beyond. Citation Format: Ying Shao, Quang Tran, Pandurang Kolekar, Yanling Liu, Andrea McBride, Tyler Jones, Heather Mulder, Lingyun Ji, Benjamin Huang, Soheil Meshinchi, Jeffery Klco, Jinghui Zhang, William Carroll, Mignon Loh, Patrick Brown, John Easton, Xiaotu Ma. Analysis of indel and structural variant error profiles in deep next generation sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB077.

dPDX and dRI-PDX leukemia-initiating cell frequencies

Additional file 9. TAD_decreased.csv Lost TAD activity due to NSD2 coordinates.

BACKGROUND:The NSD2 p.E1099K (EK) mutation is shown to be enriched in patients with relapsed acute lymphoblastic leukemia (ALL), indicating a role in clonal evolution and drug resistance. RESULTS:To uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we perform Hi-C on three B-ALL cell lines heterozygous for NSD2 EK. The NSD2 mutation leads to widespread remodeling of the 3D genome, most dramatically in terms of compartment changes with a strong bias towards A compartment shifts. Systematic integration of the Hi-C data with previously published ATAC-seq, RNA-seq, and ChIP-seq data show an expansion in H3K36me2 and a shrinkage in H3K27me3 within A compartments as well as increased gene expression and chromatin accessibility. These results suggest that NSD2 EK plays a prominent role in chromatin decompaction through enrichment of H3K36me2. In contrast, we identify few changes in intra-topologically associating domain activity. While compartment changes vary across cell lines, a common core of decompacting loci are shared, driving the expression of genes/pathways previously implicated in drug resistance. We further perform RNA sequencing on a cohort of matched diagnosis/relapse ALL patients harboring the relapse-specific NSD2 EK mutation. Changes in patient gene expression upon relapse significantly correlate with core compartment changes, further implicating the role of NSD2 EK in genome decompaction. CONCLUSIONS:In spite of cell-context-dependent changes mediated by EK, there appears to be a shared transcriptional program dependent on compartment shifts which could explain phenotypic differences across EK cell lines. This core program is an attractive target for therapeutic intervention.

Patient characteristics and patient genomic analysis (WES and SNP 6.0)

Targeted-sequencing analysis of PDX and PairTree predicted mutational population clusters

Background CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF. Results We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcript changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID-tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.

RNA-sequencing data from Patient 9 PDX

RNA-sequencing, pathway enrichment (GSEA) reports and GSVA results (including gene list HSC vs B) of PDX and paired patient samples

Leukemia-initiating cell frequencies of paired diagnosis and relapse patient samples

Abstract Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. Significance: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587