The Role of the JAK-STAT Pathway in Childhood B-Cell Acute Lymphoblastic Leukemia.

B-cell lymphoblastic leukemia is a hematologic neoplasm that poses a serious health concern in childhood. Genetic aberrations, such as mutations in the genes IL-7, IL7R, JAK1, JAK2, TLSP, CRLF2, and KTM2A or gene fusions involving BCR::ABL1, ETV6::RUNX1, and PAX5::JAK2, often correlate with the onset of this disease. These aberrations can lead to malfunction of the JAK-STAT signaling pathway, which is implicated in various important biological processes, including those related to immunology. Understanding the mechanisms underlying the malfunction of the JAK-STAT pathway holds potential for research on drugs targeting its components. Available drugs that interfere with the JAK-STAT pathway include fludarabine, ruxolitinib, and fedratinib.

RAG1 and RAG2 non-core regions are implicated in leukemogenesis and off-target V(D)J recombination in BCR-ABL1-driven B-cell lineage lymphoblastic leukemia.

The evolutionary conservation of non-core RAG regions suggests significant roles that might involve quantitative or qualitative alterations in RAG activity. Off-target V(D)J recombination contributes to lymphomagenesis and is exacerbated by RAG2' C-terminus absence in Tp53-/- mice thymic lymphomas. However, the genomic stability effects of non-core regions from both Rag1c/c and Rag2c/c in BCR-ABL1+ B-lymphoblastic leukemia (BCR-ABL1+ B-ALL), the characteristics, and mechanisms of non-core regions in suppressing off-target V(D)J recombination remain unclear. Here, we established three mouse models of BCR-ABL1+ B-ALL in mice expressing full-length RAG (Ragf/f), core RAG1 (Rag1c/c), and core RAG2 (Rag2c/c). The Ragc/c (Rag1c/c and Rag2c/c) leukemia cells exhibited greater malignant tumor characteristics compared to Ragf/f cells. Additionally, Ragc/c cells showed higher frequency of off-target V(D)J recombination and oncogenic mutations than Ragf/f. We also revealed decreased RAG cleavage accuracy in Ragc/c cells and a smaller recombinant size in Rag1c/c cells, which could potentially exacerbate off-target V(D)J recombination in Ragc/c cells. In conclusion, these findings indicate that the non-core RAG regions, particularly the non-core region of RAG1, play a significant role in preserving V(D)J recombination precision and genomic stability in BCR-ABL1+ B-ALL.

Economic Impact of Whole Genome Sequencing and Whole Transcriptome Sequencing Versus Routine Diagnostic Molecular Testing to Stratify Patients with B-Cell Acute Lymphoblastic Leukemia.

Whole genome and whole transcriptome sequencing (WGTS) can accurately distinguish B-cell acute lymphoblastic leukemia (B-ALL) genomic subtypes. However, whether this is economically viable remains unclear. This study compared the direct costs and molecular subtype classification yield using different testing strategies for WGTS in adolescent and young adult/adult patients with B-ALL. These approaches were: (1) combined BCR::ABL1 by fluorescence in situ hybridization (FISH) + WGTS for all patients; and (2) sequential BCR::ABL1 FISH + WGTS contingent on initial BCR::ABL1 FISH test outcome. The cost of routine diagnostic testing was estimated using Medicare or hospital fees, and the additional cost of WGTS was evaluated from the health care provider perspective using time-driven activity-based costing with resource identification elicited from experts. Molecular subtype classification yield data were derived from literature sources. Parameter uncertainty was assessed through deterministic sensitivity analysis; additional scenario analyses were performed. The total per patient cost of WGTS was $4319 (all costs reported in US dollars); consumables accounted for 74% of the overall cost, primarily driven by sequencing-related consumables. The incremental cost per additional patient categorized into molecular subtype was $8498 for combined BCR::ABL1 FISH + WGTS for all patients and $5656 for initial BCR::ABL1 FISH + WGTS for select patients compared with routine diagnostic testing. A reduction in the consumable costs of WGTS or an increase in the yield of molecular subtype classification is favorable.

Unraveling Copy Number Alterations in Pediatric B-Cell Acute Lymphoblastic Leukemia: Correlation with Induction Phase Remission Using MLPA.

OBJECTIVE: Acute Lymphoblastic Leukemia (ALL) is the most common malignancy occurring in children. Copy number alterations (CNA) like PAX5, CDKN2A/2B, PAR1 Region, ETV6, IKZF1, BTG1, and RB1 gene deletion are important genetic events that define and prognosticate B-cell ALL. Thus, this study aimed to evaluate associations of CNA with induction phase remission status in childhood B-cell ALL. METHODS: This study was observational with a cross-sectional design at the Dharmais Cancer Hospital, Harapan Kita Mother and Children Hospital, and Tangerang Regional Public Hospital. We evaluated 74 pediatric B-cell ALL cases with 1-18-year-olds. Genomic DNA was analyzed by Multiplex Ligation Dependent Probe Amplification Assay (MLPA). This study used the P335 ALL-IKZF1 panel kit, which contains several ALL-related genes. The patient's clinical and laboratory characteristics were collected from medical records from January to December 2019. RESULT: We observed gene copy number alteration in children with B-Cell ALL. PAX5 was the most commonly observed gene deletion, followed by CDKN21/2B, ETV6, IKZF1, BTG1, RB1, and PAR1 Region. Based on gene mutations, only the PAX5 had a significant association with the remission status of pediatric B-cell ALL (p-value <0.05; OR = 3.91). It showed that patients with PAX5 gene mutations have 3.9 times the risk of no remission and/or relapse compared to those without PAX5 gene mutations. CONCLUSION: Patients with mutations in the PAX5 gene have a higher chance of not achieving remission and/or experiencing relapse than those without such mutations. The MLPA method can be utilized for examining copy number alterations, which is valuable for achieving more precise stratification in diagnosis.. Further research is needed to expand upon this finding.

The Molecular and Biological Function of MEF2D in Leukemia.

Myocyte enhancer factor 2 (MEF2) is a key transcription factor (TF) in skeletal, cardiac, and neural tissue development and includes four isoforms: MEF2A, MEF2B, MEF2C, and MEF2D. These isoforms significantly affect embryonic development, nervous system regulation, muscle cell differentiation, B- and T-cell development, thymocyte selection, and effects on tumorigenesis and leukemia. This chapter describes the multifaceted roles of MEF2 family proteins, covering embryonic development, nervous system regulation, and muscle cell differentiation. It further elucidates the contribution of MEF2 to various blood and immune cell functions. Specifically, in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), MEF2D is aberrantly expressed and forms a fusion protein with BCL9, CSF1R, DAZAP1, HNRNPUL1, and SS18. These fusion proteins are closely related to the pathogenesis of leukemia. In addition, it specifically introduces the regulatory effect of MEF2D fusion protein on the proliferation and growth of B-cell acute lymphoblastic leukemia (B-ALL) cells. Finally, we detail the positive feedback loop between MEF2D and IRF8 that significantly promotes the progression of acute myeloid leukemia (AML) and the importance of the ZMYND8-BRD4 interaction in regulating the IRF8 and MYC transcriptional programs. The MEF2D-CEBPE axis is highlighted as a key transcriptional mechanism controlling the block of leukemic cell self-renewal and differentiation in AML. This chapter starts with the structure and function of MEF2 family proteins, specifically summarizing and analyzing the role of MEF2D in B-ALL and AML, mediating the complex molecular mechanisms of transcriptional regulation and exploring their implications for human health and disease.

The Diverse Roles of ETV6 Alterations in B-Lymphoblastic Leukemia and Other Hematopoietic Cancers.

Genetic alterations of the repressive ETS family transcription factor gene ETV6 are recurrent in several categories of hematopoietic malignancy, including subsets of B-cell and T-cell acute lymphoblastic leukemias (B-ALL and T-ALL), myeloid neoplasms, and mature B-cell lymphomas. ETV6 is essential for adult hematopoietic stem cells (HSCs), contributes to specific functions of some mature immune cells, and plays a key role in thrombopoiesis as demonstrated by familial ETV6 mutations associated with thrombocytopenia and predisposition to hematopoietic cancers, particularly B-ALL. ETV6 appears to have a tumor suppressor role in several hematopoietic lineages, as demonstrated by recurrent somatic loss-of-function (LoF) and putative dominant-negative alterations in leukemias and lymphomas. ETV6 rearrangements contribute to recurrent fusion oncogenes such as the B-ALL-associated transcription factor (TF) fusions ETV6::RUNX1 and PAX5::ETV6, rare drivers such as ETV6::NCOA6, and a spectrum of tyrosine kinase gene fusions encoding hyperactive signaling proteins that self-associate via the ETV6 N-terminal pointed domain. Another subset of recurrent rearrangements involving the ETV6 gene locus appear to function primarily to drive overexpression of the partner gene. This review surveys what is known about the biochemical and genome regulatory properties of ETV6 as well as our current understanding of how alterations in these functions contribute to hematopoietic and nonhematopoietic cancers.

Rare Drivers at Low Prevalence with High Cancer Effects in T-Cell and B-Cell Pediatric Acute Lymphoblastic Leukemia.

The genomic analyses of pediatric acute lymphoblastic leukemia (ALL) subtypes, particularly T-cell and B-cell lineages, have been pivotal in identifying potential therapeutic targets. Typical genomic analyses have directed attention toward the most commonly mutated genes. However, assessing the contribution of mutations to cancer phenotypes is crucial. Therefore, we estimated the cancer effects (scaled selection coefficients) for somatic substitutions in T-cell and B-cell cohorts, revealing key insights into mutation contributions. Cancer effects for well-known, frequently mutated genes like NRAS and KRAS in B-ALL were high, which underscores their importance as therapeutic targets. However, less frequently mutated genes IL7R, XBP1, and TOX also demonstrated high cancer effects, suggesting pivotal roles in the development of leukemia when present. In T-ALL, KRAS and NRAS are less frequently mutated than in B-ALL. However, their cancer effects when present are high in both subtypes. Mutations in PIK3R1 and RPL10 were not at high prevalence, yet exhibited some of the highest cancer effects in individual T-cell ALL patients. Even CDKN2A, with a low prevalence and relatively modest cancer effect, is potentially highly relevant for the epistatic effects that its mutated form exerts on other mutations. Prioritizing investigation into these moderately frequent but potentially high-impact targets not only presents novel personalized therapeutic opportunities but also enhances the understanding of disease mechanisms and advances precision therapeutics for pediatric ALL.

Enhancer engagement sustains oncogenic transformation and progression of B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. METHODS: We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages-onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. RESULTS: We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. CONCLUSIONS: Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL.

The impact of exosomes derived from B-cell acute lymphoblastic leukemia as a growth factor on bone marrow mesenchymal stromal cells.

Background The incidence of various types of cancers, including leukemia, is on the rise and many challenges in both drug resistance and complications related to chemotherapy appeared. Recently, the development and application of extracellular vesicles (EV) such as exosomes in the management of cancers, especially leukemia, holds great significance. In this article, we extracted exosomes from NALM6 cells and assessed their regulatory effects on proliferation and apoptosis in mesenchymal stem cells (MSCs). Method and result We first verified the exosomes using various techniques, including flow cytometry, transient electron microscopy, dynamic light scattering (DLS), and BCA protein assay. Then MTT analysis and flowcytometry (apoptosis and cell cycle assay) besides gene expressions were employed to determine the state of MSC proliferations. The results indicated that exosome-specific pan markers like CD9, CD63, and CD81 were present. Through DLS, we found out that the mean size of the exosomes was 89.68 nm. The protein content was determined to be 956.292 µg/ml. Analysis of MTT, flow cytometry (cell cycle and apoptosis assay), and RT-qPCR showed that in the dose of 50 µg/ml the proliferation of MSCs was increased significantly (p-value < 0.05). Conclusion All these data showed that exosomes use several signaling pathways to increase the MSCs' proliferation and drug resistance, ultimately leading to high mortalities and morbidities of acute lymphoblastic leukemia.

Single-cell transcriptomic analysis of the immune microenvironment in pediatric acute leukemia.

Relapse and treatment resistance pose significant challenges in the management of pediatric B cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The efficacy of immunotherapy in leukemia remains limited due to factors such as the immunosuppressive tumor microenvironment (TME) and lack of suitable immunotherapeutic targets. Thus, an in-depth characterization of the TME in pediatric leukemia is warranted to improve the efficacy of immunotherapy. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the TME of pediatric B-ALL and AML, focusing specifically on bone-marrow-derived T cells. Moreover, we investigated the transcriptome changes during the initiation, remission, and relapse stages of pediatric AML. Our findings revealed that specific functional expression programs correlated with fluctuations in various T cell subsets, which may be associated with AML progression and relapse. Furthermore, our analysis of cellular communication networks led to the identification of VISTA, CD244, and TIM3 as potential immunotherapeutic targets in pediatric AML. Finally, we detected elevated proportions of γδ T cells and associated functional genes in samples from pediatric patients diagnosed with B-ALL and AML, which could inform the development of novel therapeutic approaches, potentially focusing on γδ T cells.

Identification of a novel MEF2C::SS18L1 fusion in childhood acute B-lymphoblastic leukemia.

PURPOSE: Leukemia-associated fusion genes are closely related to the occurrence, development, diagnosis, and treatment of leukemia. DNA microarrays and second-generation sequencing have discovered multiple B-ALL fusion genes. We identified a novel MEF2C::SS18L1 fusion gene in a child diagnosed with B-ALL. This study investigates the oncogenicity and prognosis of this fusion gene in B-ALL. METHODS: A child with B-ALL who has a MEF2C::SS18L1 fusion is reported as a newly discovered case. Compared the breakpoints, structural domains, clinical phenotypes, and differential expression genes of MEF2C::SS18L1 and MEF2D::SS18.Using "ONCOFUSE" software, the carcinogenicity of MEF2C::SS18L1 is predicted. Using whole transcriptome sequencing, we analyze the breakpoints and the secondary structure of the fusion protein. Further, we compared the structures, differentially expressed genes, and clinical phenotypes of MEF2D and MEF2C fusion genes by DESeq, GO functional enrichment, and flow cytometry immunophenotyping analysis. RESULTS: Whole transcriptome sequencing identified a MEF2C::SS18L1 fusion transcript in a 3-year-old child with B-ALL. The MADS box, MEF structural domain, HJURP_C structural domain, and TAD I structural domain of MEF2C, and the QPGY structural domain of SS18L1, make up the fusion protein. "Oncofuse" found a 0.99 Bayesian probability that the fusion gene drives cancer. The breakpoint positions, fusion protein secondary structures, differentially expressed genes, and clinical characteristics of this patient were identical to those with MEF2D::SS18 fusion gene. CONCLUSION: We identified a novel MEF2C::SS18L1 fusion gene in childhood ALL, which shares similar structural and clinical characteristics with MEF2D::SS18. Further studies with more samples should be conducted in future.

Relationship Between Cytotoxic T-Lymphocyte-associated Antigen-4: Programmed Death-1 Genes Polymorphisms and Susceptibility to Pediatric B-Cell Acute Lymphoblastic Leukemia.

Programmed death-1 (PD1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) have a vital role in immune checkpoint pathways. Single nucleotide polymorphisms (SNPs) of PD1 and CTLA4 have been reported to be associated with susceptibility to certain autoimmune diseases and cancers. The potential association between SNPs in these immune checkpoint genes and risk of acute lymphoblastic leukemia (ALL) still unclear. The aim of this study is to clarify the effect of PD1 and CTLA4 SNPs on the risk of developing ALL and the prognosis of the disease. The study was performed on 100 pediatric B-ALL patients and 100 controls. The PD1 and CTLA4 SNPs were examined by RFLP technique. The study revealed that CTLA4 (rs11571316) was associated with high risk of B-ALL developments OR 1.492 (CI: 1157 to 1924) ( P =0.002). PD1 (rs36084323) GA genotype was significantly associated with protective effect against nonremission ( P =0.007). PD1 (rs36084323) A allele were associated with protective effect against relapse ( P =0.008). CTLA4 and PD1 genotypes did not have significant impact on B-ALL patients outcome. The current study displayed for the first time that genetic variations of the CTLA-4, was associated with susceptibility to B-ALL and that PD1 (rs36084323) GA genotype was significantly associated with protective effect against nonremission, while PD1 (rs36084323) A allele was associated with protective effect against relapse.

Identification of a circular RNA isoform of WASHC2A as a prognostic factor for high-risk paediatric B-ALL patients.

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is a serious disease for which a better understanding of prognostic factors and new therapeutic targets is needed. Circular RNAs (circRNAs) are promising markers due to their stability and differential expression patterns in various diseases. However, their role in pediatric B-ALL patients, particularly in risk stratification and relapse prediction, remains poorly understood. In this study, we comprehensively examined the circRNA landscape in pediatric B-ALL patients, focusing on both high-risk and standard-risk patients. Using advanced sequencing techniques and sophisticated bioinformatics tools, we identified thousands of circRNAs, including a novel circRNA derived from the WASHC2A gene, termed circWASHC2A. CircWASHC2A showed differential expression between high-risk and standard-risk patients and exhibited potential for predicting relapse in high-risk patients. Functional experiments highlighted a role for circWASHC2A in regulating cell cycle progression and mitochondrial respiratory activity in leukaemic cells. Transcriptomic analysis further supported these findings, suggesting the involvement of circWASHC2A in signalling pathways relevant to leukaemia pathogenesis. This study provides in-depth insights into the circRNA landscape of pediatric B-ALL patients and identifies circWASHC2A as a potential biomarker for risk stratification and relapse prediction, with significant implications for tailoring diagnostic and therapeutic strategies in this patient population.

Prognosis of pediatric BCP-ALL with IKZF1 deletions and impact of intensive chemotherapy: Results of SCCLG-2016 study.

BACKGROUND: IKZF1 deletion (IKZF1del) is associated with poor prognosis in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). But the prognosis of IKZF1del combined with other prognostic stratification factors remains unclear. Whether intensified treatment improves BCP-ALL prognosis has not been determined. METHODS: A retrospective analysis was performed on 1291 pediatric patients diagnosed with BCP-ALL and treated with the South China Children's Leukemia 2016 protocol. Patients were stratified based on IKZF1 status for comparison of characteristics and outcome. Additionally, IKZF1del patients were further divided based on chemotherapy intensity for outcome assessments. RESULTS: The BCP-ALL pediatric patients with IKZF1del in south China showed poorer early response. Notably, the DFS and OS for IKZF1del patients were markedly lower than IKZF1wt group (3-year DFS: 88.7% [95% CI: 83.4%-94.0%] vs. 93.5% [95% CI: 92.0%-94.9%], P = .021; 3-year OS: 90.7% [95% CI: 85.8% to 95.6%] vs. 96.1% [95% CI: 95% to 97.2%, P = .003]), with a concurrent increase in 3-year TRM (6.4% [95% CI: 2.3%-10.5%] vs. 2.9% [95% CI: 1.9%-3.8%], P = .025). However, the 3-year CIR was comparable between the two groups (5.7% [95% CI: 1.8%-9.5%] vs. 3.7% [95% CI: 2.6%-4.7%], P = .138). Subgroup analyses reveal no factor significantly influenced the prognosis of the IKZF1del cohort. Noteworthy, intensive chemotherapy improved DFS from 85.7% ± 4.1% to 94.1% ± 0.7% in IKZF1del group (P = .084). Particularly in BCR::ABL positive subgroup, the 3-year DFS was remarkably improved from 53.6% ± 20.1% with non-intensive chemotherapy to 100% with intensive chemotherapy (P = .026). CONCLUSIONS: Pediatric BCP-ALL patients with IKZF1del in South China manifest poor outcomes without independent prognostic significance. While no factor substantially alters the prognosis in the IKZF1del group. Intensified chemotherapy may reduce relapse rates and improve DFS in patients with IKZF1del subset, particularly in IKZFdel patients with BCR::ABL positive.

An artificial intelligence-assisted clinical framework to facilitate diagnostics and translational discovery in hematologic neoplasia.

BACKGROUND: The increasing volume and intricacy of sequencing data, along with other clinical and diagnostic data, like drug responses and measurable residual disease, creates challenges for efficient clinical comprehension and interpretation. Using paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) as a use case, we present an artificial intelligence (AI)-assisted clinical framework clinALL that integrates genomic and clinical data into a user-friendly interface to support routine diagnostics and reveal translational insights for hematologic neoplasia. METHODS: We performed targeted RNA sequencing in 1365 cases with haematological neoplasms, primarily paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) from the AIEOP-BFM ALL study. We carried out fluorescence in situ hybridization (FISH), karyotyping and arrayCGH as part of the routine diagnostics. The analysis results of these assays as well as additional clinical information were integrated into an interactive web interface using Bokeh, where the main graph is based on Uniform Manifold Approximation and Projection (UMAP) analysis of the gene expression data. At the backend of the clinALL, we built both shallow machine learning models and a deep neural network using Scikit-learn and PyTorch respectively. FINDINGS: By applying clinALL, 78% of undetermined patients under the current diagnostic protocol were stratified, and ambiguous cases were investigated. Translational insights were discovered, including IKZF1plus status dependent subpopulations of BCR::ABL1 positive patients, and a subpopulation within ETV6::RUNX1 positive patients that has a high relapse frequency. Our best machine learning models, LDA and PASNET-like neural network models, achieve F1 scores above 97% in predicting patients' subgroups. INTERPRETATION: An AI-assisted clinical framework that integrates both genomic and clinical data can take full advantage of the available data, improve point-of-care decision-making and reveal clinically relevant insights promptly. Such a lightweight and easily transferable framework works for both whole transcriptome data as well as the cost-effective targeted RNA-seq, enabling efficient and equitable delivery of personalized medicine in small clinics in developing countries. FUNDING: German Ministry of Education and Research (BMBF), German Research Foundation (DFG) and Foundation for Polish Science.