ABSTRACT
ABSTRACT: Distinct diagnostic entities within BCR::ABL1-positive acute lymphoblastic leukemia (ALL) are currently defined by the International Consensus Classification of myeloid neoplasms and acute leukemias (ICC): "lymphoid only", with BCR::ABL1 observed exclusively in lymphatic precursors, vs "multilineage", where BCR::ABL1 is also present in other hematopoietic lineages. Here, we analyzed transcriptomes of 327 BCR::ABL1-positive patients with ALL (age, 2-84 years; median, 46 years) and identified 2 main gene expression clusters reproducible across 4 independent patient cohorts. Fluorescence in situ hybridization analysis of fluorescence-activated cell-sorted hematopoietic compartments showed distinct BCR::ABL1 involvement in myeloid cells for these clusters (n = 18/18 vs n = 3/16 patients; P < .001), indicating that a multilineage or lymphoid BCR::ABL1 subtype can be inferred from gene expression. Further subclusters grouped samples according to cooperating genomic events (multilineage: HBS1L deletion or monosomy 7; lymphoid: IKZF1-/- or CDKN2A/PAX5 deletions/hyperdiploidy). A novel HSB1L transcript was highly specific for BCR::ABL1 multilineage cases independent of HBS1L genomic aberrations. Treatment on current German Multicenter Study Group for Adult ALL (GMALL) protocols resulted in comparable disease-free survival (DFS) for multilineage vs lymphoid cluster patients (3-year DFS: 70% vs 61%; P = .530; n = 91). However, the IKZF1-/- enriched lymphoid subcluster was associated with inferior DFS, whereas hyperdiploid cases showed a superior outcome. Thus, gene expression clusters define underlying developmental trajectories and distinct patterns of cooperating events in BCR::ABL1-positive ALL with prognostic relevance.
Subject(s)
Fusion Proteins, bcr-abl , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Humans , Middle Aged , Young Adult , Acute Disease , Chromosome Deletion , Fusion Proteins, bcr-abl/genetics , Genomics , In Situ Hybridization, Fluorescence , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.
Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , CD47 Antigen , Humans , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapyABSTRACT
Acute leukemias are the most common malignant diseases in childhood. The aims of this retrospective cohort study were to investigate the frequency of cytogenetic abnormalities in acute pediatric leukemia; the correlation between cytogenetic abnormalities and 5-year survival; and the correlation between cytogenetic abnormalities and clinical and laboratory features. We included 105 patients; acute lymphoblastic leukemia (ALL) had 80.9% patients, B-cell lineage ALL (B-ALL) 84.7% of them, and T-cell lineage (T-ALL) 15.3%. The overall 5-year survival for B-ALL was 85.9% and for T-ALL was 84.6%. The most common cytogenetic abnormalities in patients with B-ALL were t(12;21)(p13.2;q22.1); ETV6-RUNX1 with 22.2% and hyperdiploidy with 19.4%. Our survival analysis showed that t(12;21)(p13.2;q22.1); ETV6-RUNX1 and t(1;19)(q23;p13.3); TCF3-PBX1 had the best 5-year survival with 100% of patients surviving, whereas t(v;11q23.3); KMT2A rearranged had the worst 5-year survival of just 33.3% of patients surviving after 5 years. We found no difference in 5-year survival in B-ALL when comparing clinical features. Acute myelogenous leukemia had 20 patients with 70.6% 5-year survival. The most common cytogenetic abnormality in acute myelogenous leukemia was t(8;21)(q21;q22.1); RUNX1-RUNX1T1 (20%). In conclusion, this study showed the correlation of different cytogenetic abnormalities with 5-year survival in B-ALL patients. Such correlation was not found when comparing clinical features and 5-year survival of patients with B-ALL. This emphasized the significance of cytogenetic analysis in pediatric leukemia.
Subject(s)
Leukemia, Myeloid, Acute , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Child , Humans , Retrospective Studies , Core Binding Factor Alpha 2 Subunit/genetics , Chromosome Aberrations , Translocation, Genetic , Cytogenetic Analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Leukemia, Myeloid, Acute/geneticsABSTRACT
In contrast to B-cell precursor acute lymphoblastic leukemia (ALL), molecular subgroups are less well defined in T-lineage ALL. Comprehensive studies on molecular T-ALL subgroups have been predominantly performed in pediatric ALL patients. Currently, molecular characteristics are rarely considered for risk stratification. Herein, we present a homogenously treated cohort of 230 adult T-ALL patients characterized on transcriptome, and partly on DNA methylation and gene mutation level in correlation with clinical outcome. We identified nine molecular subgroups based on aberrant oncogene expression correlating to four distinct DNA methylation patterns. The subgroup distribution differed from reported pediatric T-ALL cohorts with higher frequencies of prognostic unfavorable subgroups like HOXA or LYL1/LMO2. A small subset (3%) of HOXA adult T-ALL patients revealed restricted expression of posterior HOX genes with aberrant activation of lncRNA HOTTIP. With respect to outcome, TLX1 (n = 44) and NKX2-1 (n = 4) had an exceptionally favorable 3-year overall survival (3y-OS) of 94%. Within thymic T-ALL, the non TLX1 patients had an inferior but still good prognosis. To our knowledge this is the largest cohort of adult T-ALL patients characterized by transcriptome sequencing with meaningful clinical follow-up. Risk classification based on molecular subgroups might emerge and contribute to improvements in outcome.
Subject(s)
DNA Methylation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Humans , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Adult , Male , Female , Prognosis , Middle Aged , Young Adult , Adolescent , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aged , Biomarkers, Tumor/genetics , Mutation , Follow-Up Studies , Survival Rate , Transcriptome , Homeodomain Proteins/geneticsABSTRACT
Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell-derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)-itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient-derived EV induce AREG shedding and restore chemoresistance in ADAM17-deficient cells. Confirming that ADAM17-containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.
Subject(s)
Antineoplastic Agents , Extracellular Vesicles , Ovarian Neoplasms , Humans , Female , ADAM Proteins/metabolism , ErbB Receptors , Extracellular Vesicles/metabolism , Ovarian Neoplasms/drug therapy , ADAM17 ProteinABSTRACT
Introduction: The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Methods: Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. Results: We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Discussion: Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.
Subject(s)
Burkitt Lymphoma , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Adult , Humans , Polymerase Chain Reaction , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Genes, Immunoglobulin , Burkitt Lymphoma/genetics , Bone Marrow/pathologyABSTRACT
FAT atypical cadherin 1 (FAT1), a transmembrane protein, is frequently mutated in various cancer types and has been described as context-dependent tumor suppressor or oncogene. The FAT1 gene is mutated in 12-16% of T-cell acute leukemia (T-ALL) and aberrantly expressed in about 54% of T-ALL cases contrasted with absent expression in normal T-cells. Here, we characterized FAT1 expression and profiled the methylation status from T-ALL patients. In our T-ALL cohort, 53% of patient samples were FAT1 positive (FAT1pos) compared to only 16% FAT1 positivity in early T-ALL patient samples. Aberrant expression of FAT1 was strongly associated with FAT1 promotor hypomethylation, yet a subset, mainly consisting of TLX1-driven T-ALL patient samples showed methylation-independent high FAT1 expression. Genes correlating with FAT1 expression revealed enrichment in WNT signaling genes representing the most enriched single pathway. FAT1 knockdown or knockout led to impaired proliferation and downregulation of WNT pathway target genes (CCND1, MYC, LEF1), while FAT1 overexpressing conveyed a proliferative advantage. To conclude, we characterized a subtype pattern of FAT1 gene expression in adult T-ALL patients correlating with promotor methylation status. FAT1 dependent proliferation and WNT signaling discloses an impact on deeper understanding of T-ALL leukemogenesis as a fundament for prospective therapeutic strategies.
Subject(s)
Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Adult , Humans , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Wnt Signaling Pathway , Cadherins/genetics , Cadherins/metabolism , Cell Proliferation/genetics , T-Lymphocytes/metabolism , Cell Line, TumorABSTRACT
Acute myeloid leukemia (AML) is characterized by complex molecular alterations and driver mutations. Elderly patients show increased frequencies of IDH mutations with high chemoresistance and relapse rates despite recent therapeutic advances. Besides being associated with global promoter hypermethylation, IDH1 mutation facilitated changes in 3D DNA-conformation by CTCF-anchor methylation and upregulated oncogene expression in glioma, correlating with poor prognosis. Here, we investigated the role of IDH1 p.R132H mutation in altering 3D DNA-architecture and subsequent oncogene activation in AML. Using public RNA-Seq data, we identified upregulation of tyrosine kinase PDGFRA in IDH1-mutant patients, correlating with poor prognosis. DNA methylation analysis identified CpG hypermethylation within a CTCF-anchor upstream of PDGFRA in IDH1-mutant patients. Increased PDGFRA expression, PDGFRA-CTCF methylation and decreased CTCF binding were confirmed in AML CRISPR cells with heterozygous IDH1 p.R132H mutation and upon exogenous 2-HG treatment. IDH1-mutant cells showed higher sensitivity to tyrosine kinase inhibitor dasatinib, which was supported by reduced blast count in a patient with refractory IDH1-mutant AML after dasatinib treatment. Our data illustrate that IDH1 p.R132H mutation leads to CTCF hypermethylation, disrupting DNA-looping and insulation of PDGFRA, resulting in PDGFRA upregulation in IDH1-mutant AML. Treatment with dasatinib may offer a novel treatment strategy for IDH1-mutant AML.
Subject(s)
Isocitrate Dehydrogenase , Leukemia, Myeloid, Acute , Humans , Aged , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Dasatinib , Mutation , Oncogenes , Leukemia, Myeloid, Acute/genetics , Carcinogenesis/geneticsABSTRACT
Upregulation of surface expressed sialoglycans on tumor cells is one of the mechanisms which promote tumor growth and progression. Specifically, the interactions of sialic acids with sialic acid-binding immunoglobulin-like lectins (Siglecs) on lymphoid or myeloid cells transmit inhibitory signals and lead to suppression of anti-tumor responses. Here, we show that neutrophils express among others Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. Importantly, the cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In vivo a short-term xenograft mouse model confirmed the improved therapeutic efficacy of EGFR antibodies against sialic acid depleted, by a sialyltransferase inhibitor, tumor cells compared to untreated cells. Our studies demonstrate that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.
Subject(s)
N-Acetylneuraminic Acid , Neoplasms , Humans , Mice , Animals , N-Acetylneuraminic Acid/metabolism , Neutrophils/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Antibodies , Sialic Acids/metabolism , ErbB Receptors , Tumor MicroenvironmentABSTRACT
Current classifications (World Health Organization-HAEM5/ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by transcriptome sequencing (RNA-Seq) is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning-based classifier using RNA-Seq gene expression data to allocate BCP-ALL samples to all 21 gene expression-defined molecular subtypes. Trained on n = 1869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n = 1018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High-confidence predictions were achieved in 83.7% of samples with 98.9% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel driver candidates in previously unassigned samples. Additional modules provided predictions of samples blast counts, patient's sex, and immunophenotype, allowing the imputation in cases where these information are missing. We established a novel RNA-Seq reference of human B-lymphopoiesis using 7 FACS-sorted progenitor stages from healthy bone marrow donors. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory. This identified shared proximity patterns of BCP-ALL subtypes to normal lymphopoiesis stages, extending immunophenotypic classifications with a novel framework for developmental comparisons of BCP-ALL. ALLCatchR enables RNA-Seq routine application for BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocation and novel insights into underlying developmental trajectories.
ABSTRACT
Identification of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements in acute lymphoblastic leukemia (ALL) patients at initial presentation are crucial for monitoring of minimal residual disease (MRD) during subsequent follow-up and thereby for appropriate risk-group stratification. Here we describe how RNA-Seq data can be generated and subsequently analyzed with ARResT/Interrogate to identify possible MRD markers. In addition to the procedures, possible pitfalls will be discussed. Similar strategies can be employed for other lymphoid malignancies, such as lymphoma and myeloma.
Subject(s)
Gene Rearrangement , Genes, T-Cell Receptor , Immunoglobulins , Neoplasm, Residual , Precursor Cell Lymphoblastic Leukemia-Lymphoma , RNA-Seq , Aftercare , Gene Rearrangement/genetics , Genes, T-Cell Receptor/genetics , Humans , Immunoglobulins/genetics , Neoplasm, Residual/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA-Seq/methods , Risk AssessmentABSTRACT
Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all-trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy.
Subject(s)
CD47 Antigen , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Humans , Immunoglobulin AABSTRACT
BACKGROUND: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogenous malignancy with poor prognosis in relapsed adult patients. The genetic basis for relapse in aneuploid subtypes such as near haploid (NH) and high hyperdiploid (HeH) BCP-ALL is only poorly understood. Pathogenic genetic alterations remain to be identified. To this end, we investigated the dynamics of genetic alterations in a matched initial diagnosis-relapse (ID-REL) BCP-ALL cohort. Here, we firstly report the identification of the novel genetic alteration CYB5Aalt, an alternative transcript of CYB5A, in two independent cohorts. METHODS: We identified CYB5alt in the RNAseq-analysis of a matched ID-REL BCP-ALL cohort with 50 patients and quantified its expression in various molecular BCP-ALL subtypes. Findings were validated in an independent cohort of 140 first diagnosis samples from adult BCP-ALL patients. Derived from patient material, the alternative open reading frame of CYB5Aalt was cloned (pCYB5Aalt) and pCYB5Aalt or the empty vector were stably overexpressed in NALM-6 cells. RNA sequencing was performed of pCYB5Aalt clones and empty vector controls followed by differential expression analysis, gene set enrichment analysis and complementing cell death and viability assays to determine functional implications of CYB5Aalt. RESULTS: RNAseq data analysis revealed non-canonical exon usage of CYB5Aalt starting from a previously undescribed transcription start site. CYB5Aalt expression was increased in relapsed BCP-ALL and its occurrence was specific towards the shared gene expression cluster of NH and HeH BCP-ALL in independent cohorts. Overexpression of pCYB5Aalt in NALM-6 cells induced a distinct transcriptional program compared to empty vector controls with downregulation of pathways related to reported functions of CYB5A wildtype. Interestingly, CYB5A wildtype expression was decreased in CYB5Aalt samples in silico and in vitro. Additionally, pCYB5Aalt NALM-6 elicited a more resistant drug response. CONCLUSIONS: Across all age groups, CYB5Aalt was the most frequent secondary genetic event in relapsed NH and HeH BCP-ALL. In addition to its high subgroup specificity, CYB5Aalt is a novel candidate to be potentially implicated in therapy resistance in NH and HeH BCP-ALL. This is underlined by overexpressing CYB5Aalt providing first evidence for a functional role in BCL2-mediated apoptosis.
Subject(s)
Cytochromes b5 , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Adult , Aneuploidy , Cytochromes b5/genetics , Humans , Mutation , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , RecurrenceABSTRACT
The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4-/- mice as prone to developing BCP-ALL with age. Irf4-/- preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4-/- leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.
Subject(s)
Burkitt Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Adult , Animals , Humans , Mice , B-Lymphocytes , Burkitt Lymphoma/pathology , Interleukin-7/genetics , Janus Kinase 3/genetics , Mutation/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Signal TransductionABSTRACT
BACKGROUND: Resistance to therapy and subsequent relapse remain major challenges in the clinical management of relapsed childhood acute lymphoblastic leukemia. As the bone marrow environment plays an important role in survival and chemotherapy resistance of leukemia cells by activating different signaling pathways, such as the VLA-4 and PI3K/Akt pathways, we studied the prognostic and biological impact of VLA-4 expression in leukemia cells from children with relapsed B-cell precursor acute lymphoblastic leukemia and its influence on the sensitivity of the leukemia cells to drugs. DESIGN AND METHODS: VLA-4 expression was quantified by real-time polymerase chain reaction in leukemia cells from 56 patients with relapsed acute lymphoblastic leukemia enrolled in the ALL-REZ BFM 2002 trial of the Berlin-Frankfurt-Münster study group. Gene expression changes related to VLA-4 expression were investigated by microarray-based mRNA profiling. The effect of VLA-4 signaling on proliferation and drug resistance was studied in co-cultures of leukemia and stromal cells. RESULTS: High expression of VLA-4 at first relapse was associated with adverse prognostic factors, poor molecular response to therapy and significantly worse probabilities of event-free and overall survival. VLA-4 expression was an independent prognostic parameter. Comparing gene expression profiles of leukemia cells with high versus low VLA-4 expression, we identified 27 differentially expressed genes primarily involved in the PI3K/Akt, ephrin and Rho GTPase pathways. Blocking of VLA-4 signaling in combination with cytarabine treatment abolished the growth supportive effect of stromal cells. CONCLUSIONS: Our results show that high VLA-4 expression is a marker of poor prognosis and a potential therapeutic target in children with relapsed acute lymphoblastic leukemia and confirm that cellular interactions and biological effects related to VLA-4 play a decisive role in the survival of leukemia cells and response to therapy. (ClinicalTrials.gov identifier: NCT00114348).
Subject(s)
Gene Expression Regulation, Leukemic , Integrin alpha4beta1/biosynthesis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Adolescent , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Asparaginase/administration & dosage , Child , Child, Preschool , Daunorubicin/administration & dosage , Disease-Free Survival , Drug Resistance, Neoplasm , Humans , Male , Phosphatidylinositol 3-Kinases/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Prednisone/administration & dosage , Proto-Oncogene Proteins c-akt/metabolism , Recurrence , Retrospective Studies , Signal Transduction/drug effects , Survival Rate , Vincristine/administration & dosageABSTRACT
High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Leukemia, Myeloid, Acute/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Reverse Genetics/methods , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adult , Animals , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/antagonists & inhibitors , Child , Female , Gene Silencing , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Male , Mice , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitors , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/genetics , Precision Medicine/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. However, the role and expression pattern of lncRNAs in molecular subtypes of B cell acute lymphoblastic leukemia (BCP-ALL) have not yet been investigated. Here, we assess to what extent lncRNA expression and DNA methylation is driving the progression of relapsed BCP-ALL subtypes and we determine if the expression and DNA methylation profile of lncRNAs correlates with established BCP-ALL subtypes. METHODS: We performed RNA sequencing and DNA methylation (Illumina Infinium microarray) of 40 diagnosis and 42 relapse samples from 45 BCP-ALL patients in a German cohort and quantified lncRNA expression. Unsupervised clustering was applied to ascertain and confirm that the lncRNA-based classification of the BCP-ALL molecular subtypes is present in both our cohort and an independent validation cohort of 47 patients. A differential expression and differential methylation analysis was applied to determine the subtype-specific, relapse-specific, and differentially methylated lncRNAs. Potential functions of subtype-specific lncRNAs were determined by using co-expression-based analysis on nearby (cis) and distally (trans) located protein-coding genes. RESULTS: Using an integrative Bioinformatics analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific and 942 relapse-specific lncRNAs and the methylation profile of three subtypes of BCP-ALL. The 1235 subtype-specific lncRNA signature represented a similar classification of the molecular subtypes of BCP-ALL in the independent validation cohort. We identified a strong correlation between the DUX4-specific lncRNAs and genes involved in the activation of TGF-ß and Hippo signaling pathways. Similarly, Ph-like-specific lncRNAs were correlated with genes involved in the activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we found 23 promoter methylated lncRNAs epigenetically facilitating their expression levels. CONCLUSION: Here, we describe a set of subtype-specific and relapse-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. The subtype-specific lncRNAs are reproducible and can effectively stratify BCP-ALL subtypes. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining which lncRNAs are involved in the pathogenesis of disease and are relevant for the stratification of BCP-ALL subtypes.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA, Long Noncoding/genetics , Transcriptome/genetics , Adult , Base Sequence/genetics , Berlin , Biomarkers, Tumor/genetics , Bone Marrow , Child , Cohort Studies , DNA Methylation/genetics , Epigenesis, Genetic , Female , Humans , Male , Metabolic Networks and Pathways/genetics , Promoter Regions, Genetic/genetics , RecurrenceABSTRACT
Recent efforts reclassified B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) into more refined subtypes. Nevertheless, outcomes of relapsed BCP-ALL remain unsatisfactory, particularly in adult patients where the molecular basis of relapse is still poorly understood. To elucidate the evolution of relapse in BCP-ALL, we established a comprehensive multi-omics dataset including DNA-sequencing, RNA-sequencing, DNA methylation array and proteome MASS-spec data from matched diagnosis and relapse samples of BCP-ALL patients (n = 50) including the subtypes DUX4, Ph-like and two aneuploid subtypes. Relapse-specific alterations were enriched for chromatin modifiers, nucleotide and steroid metabolism including the novel candidates FPGS, AGBL and ZNF483. The proteome expression analysis unraveled deregulation of metabolic pathways at relapse including the key proteins G6PD, TKT, GPI and PGD. Moreover, we identified a novel relapse-specific gene signature specific for DUX4 BCP-ALL patients highlighting chemotaxis and cytokine environment as a possible driver event at relapse. This study presents novel insights at distinct molecular levels of relapsed BCP-ALL based on a comprehensive multi-omics integrated data set including a valuable proteomics data set. The relapse specific aberrations reveal metabolic signatures on genomic and proteomic levels in BCP-ALL relapse. Furthermore, the chemokine expression signature in DUX4 relapse underscores the distinct status of DUX4-fusion BCP-ALL.
Subject(s)
Cytokines , Gene Expression Regulation, Leukemic , Neoplasm Proteins , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Adult , Child , Cytokines/genetics , Cytokines/metabolism , Female , Genomics , Humans , Male , Metabolic Networks and Pathways , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/classification , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , ProteomicsABSTRACT
Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup ('PAX5-plus', n = 19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis.