RESUMEN
T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease.
RESUMEN
ABSTRACT: Inotuzumab ozogamicin (InO) is an antibody-drug conjugate that delivers calicheamicin to CD22-expressing cells. In a retrospective cohort of InO-treated patients with B-cell acute lymphoblastic leukemia, we sought to understand the genomic determinants of the response and resistance to InO. Pre- and post-InO-treated patient samples were analyzed by whole genome, exome, and/or transcriptome sequencing. Acquired CD22 mutations were observed in 11% (3/27) of post-InO-relapsed tumor samples, but not in refractory samples (0/16). There were multiple CD22 mutations per sample and the mechanisms of CD22 escape included epitope loss (protein truncation and destabilization) and epitope alteration. Two CD22 mutant cases were post-InO hyper-mutators resulting from error-prone DNA damage repair (nonhomologous/alternative end-joining repair, or mismatch repair deficiency), suggesting that hypermutation drove escape from CD22-directed therapy. CD22-mutant relapses occurred after InO and subsequent hematopoietic stem cell transplantation (HSCT), suggesting that InO eliminated the predominant clones, leaving subclones with acquired CD22 mutations that conferred resistance to InO and subsequently expanded. Acquired loss-of-function mutations in TP53, ATM, and CDKN2A were observed, consistent with a compromise of the G1/S DNA damage checkpoint as a mechanism for evading InO-induced apoptosis. Genome-wide CRISPR/Cas9 screening of cell lines identified DNTT (terminal deoxynucleotidyl transferase) loss as a marker of InO resistance. In conclusion, genetic alterations modulating CD22 expression and DNA damage response influence InO efficacy. Our findings highlight the importance of defining the basis of CD22 escape and eradication of residual disease before HSCT. The identified mechanisms of escape from CD22-targeted therapy extend beyond antigen loss and provide opportunities to improve therapeutic approaches and overcome resistance. These trials were registered at www.ClinicalTrials.gov as NCT01134575, NCT01371630, and NCT03441061.
Asunto(s)
Resistencia a Antineoplásicos , Inotuzumab Ozogamicina , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Humanos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Resistencia a Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Femenino , Mutación , Masculino , Antineoplásicos Inmunológicos/uso terapéutico , Antineoplásicos Inmunológicos/farmacología , Adulto , Persona de Mediana Edad , Estudios Retrospectivos , AdolescenteRESUMEN
Inotuzumab ozogamicin (InO) is an antibody-drug conjugate that delivers calicheamicin to CD22-expressing cells. In a retrospective cohort of InO treated patients with B-cell acute lymphoblastic leukemia, we sought to understand the genomic determinants of response to InO. Acquired CD22 mutations were observed in 11% (3/27) of post-InO relapsed tumor samples. There were multiple CD22 mutations per sample and the mechanisms of CD22 escape included protein truncation, protein destabilization, and epitope alteration. Hypermutation by error-prone DNA damage repair (alternative end-joining, mismatch repair deficiency) drove CD22 escape. Acquired loss-of-function mutations in TP53 , ATM and CDKN2A were observed, suggesting compromise of the G1/S DNA damage checkpoint as a mechanism of evading InO-induced apoptosis. In conclusion, genetic alterations modulating CD22 expression and DNA damage response influence InO efficacy. The escape strategies within and beyond antigen loss to CD22-targeted therapy elucidated in this study provide insights into improving therapeutic approaches and overcoming resistance. KEY POINTS: We identified multiple mechanisms of CD22 antigen escape from inotuzumab ozogamicin, including protein truncation, protein destabilization, and epitope alteration.Hypermutation caused by error-prone DNA damage repair was a driver of CD22 mutation and escape.
RESUMEN
The bone marrow microenvironment (BME) drives drug resistance in acute lymphoblastic leukemia (ALL) through leukemic cell interactions with bone marrow (BM) niches, but the underlying mechanisms remain unclear. Here, we show that the interaction between ALL and mesenchymal stem cells (MSCs) through integrin ß1 induces an epithelial-mesenchymal transition (EMT)-like program in MSC-adherent ALL cells, resulting in drug resistance and enhanced survival. Moreover, single-cell RNA sequencing analysis of ALL-MSC co-culture identifies a hybrid cluster of MSC-adherent ALL cells expressing both B-ALL and MSC signature genes, orchestrated by a WNT/ß-catenin-mediated EMT-like program. Blockade of interaction between ß-catenin and CREB binding protein impairs the survival and drug resistance of MSC-adherent ALL cells in vitro and results in a reduction in leukemic burden in vivo. Targeting of this WNT/ß-catenin-mediated EMT-like program is a potential therapeutic approach to overcome cell extrinsically acquired drug resistance in ALL.
Asunto(s)
Transición Epitelial-Mesenquimal , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , beta Catenina , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Técnicas de Cocultivo , Resistencia a Medicamentos , Proliferación Celular , Microambiente TumoralRESUMEN
RAB3GAP1 is GTPase activating protein localized to the ER and Golgi compartments. In humans, mutations in RAB3GAP1 are the most common cause of Warburg Micro syndrome, a neurodevelopmental disorder associated with intellectual disability, microcephaly, and agenesis of the corpus callosum. We found that downregulation of RAB3GAP1 leads to a reduction in neurite outgrowth and complexity in human stem cell derived neurons. To further define the cellular function of RAB3GAP1, we sought to identify novel interacting proteins. We used a combination of mass spectrometry, co-immunoprecipitation and colocalization analysis and identified two novel interactors of RAB3GAP1: the axon elongation factor Dedicator of cytokinesis 7 (DOCK7) and the TATA modulatory factor 1 (TMF1) a modulator of Endoplasmic Reticulum (ER) to Golgi trafficking. To define the relationship between RAB3GAP1 and its two novel interactors, we analyzed their localization to different subcellular compartments in neuronal and non-neuronal cells with loss of RAB3GAP1. We find that RAB3GAP1 is important for the sub-cellular localization of TMF1 and DOCK7 across different compartments of the Golgi and endoplasmic reticulum. In addition, we find that loss of function mutations in RAB3GAP1 lead to dysregulation of pathways that are activated in response to the cellular stress like ATF6, MAPK, and PI3-AKT signaling. In summary, our findings suggest a novel role for RAB3GAP1 in neurite outgrowth that could encompass the regulation of proteins that control axon elongation, ER-Golgi trafficking, as well as pathways implicated in response to cellular stress.
Asunto(s)
Discapacidad Intelectual , Microcefalia , Humanos , Discapacidad Intelectual/genética , Microcefalia/genética , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismo , Retículo Endoplásmico/metabolismo , Neuronas/metabolismo , Axones/metabolismoRESUMEN
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 remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, 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 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the 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.
Asunto(s)
Cromosomas Humanos Par 21 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Niño , Cromosomas Humanos Par 21/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aberraciones Cromosómicas , Citogenética , Genómica , Factor 1 de Ensamblaje de la Cromatina/genéticaRESUMEN
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 ubiquitin ligase cellular machinery to target neosubstrates for protein degradation. We developed a novel cereblon modulator, SJ6986, that exhibits 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 that 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.
Asunto(s)
Antineoplásicos , Piperidonas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Niño , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Piperidonas/uso terapéutico , Isoindoles/uso terapéuticoRESUMEN
Autism spectrum disorders (ASD) are associated with defects in neuronal connectivity and are highly heritable. Genetic findings suggest that there is an overrepresentation of chromatin regulatory genes among the genes associated with ASD. ASH1 like histone lysine methyltransferase (ASH1L) was identified as a major risk factor for ASD. ASH1L methylates Histone H3 on Lysine 36, which is proposed to result primarily in transcriptional activation. However, how mutations in ASH1L lead to deficits in neuronal connectivity associated with ASD pathogenesis is not known. We report that ASH1L regulates neuronal morphogenesis by counteracting the catalytic activity of Polycomb Repressive complex 2 group (PRC2) in stem cell-derived human neurons. Depletion of ASH1L decreases neurite outgrowth and decreases expression of the gene encoding the neurotrophin receptor TrkB whose signaling pathway is linked to neuronal morphogenesis. The neuronal morphogenesis defect is overcome by inhibition of PRC2 activity, indicating that a balance between the Trithorax group protein ASH1L and PRC2 activity determines neuronal morphology. Thus, our work suggests that ASH1L may epigenetically regulate neuronal morphogenesis by modulating pathways like the BDNF-TrkB signaling pathway. Defects in neuronal morphogenesis could potentially impair the establishment of neuronal connections which could contribute to the neurodevelopmental pathogenesis associated with ASD in patients with ASH1L mutations.
Asunto(s)
Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina , Proteínas de Unión al ADN/genética , Epigénesis Genética/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Increased production, oligomerization and aggregation of amyloid-ß (Aß) peptides are hallmark pathologies of Alzheimer's disease (AD). Expressing familial AD mutations (amyloid precursor protein and/or presenilins mutations), the Aß-pathologies of AD has been recapitulated in animal models of AD. Very few primary cell culture-based models of AD are available and they exhibit very weak Aß-pathologies compared to what is seen in AD patients and animal models of AD. CNS stem/progenitor cells are present in both embryonic and adult brains. They can be isolated, grown as neurospheres and differentiated into neurons, astrocytes and oligodendrocytes. It is not yet known whether CNS stem/progenitor cells can support the production of Aß peptides in culture. In this report, we have established Aß-pathologies such as production, secretion, oligomerization and aggregation of Aß peptides utilizing neurosphere cultures to create a new cellular model of AD. These cultures were developed from E15 embryonic brains of transgenic mice carrying the Swedish mutations in humanized mouse APP cDNA and the exon-9 deleted human presenilin 1 cDNA both regulated by mouse prion protein gene (Prnp) promoter. Results demonstrated the expression of transgene transcripts, APPswe protein and its processed products only in transgene positive neurosphere cultures. These cultures generate and secrete both Aß40 and Aß42 peptides into culture medium at levels comparable to the Aß load in the brain of AD patients and animal models of AD, and produce pathogenic oligomers of Aß peptides. The Aß42/Aß40 ratio in the medium of transgene positive neurosphere cultures is higher than any known cellular models of AD. Conformation dependent immunocytochemistry demonstrated the possible presence of intracellular and extracellular aggregation of Aß peptides in neurosphere cultures, which are also seen in AD brain and animal models of AD. Collectively, our neurosphere cultures provide robust Aß-pathologies of AD better than existing cellular model of Alzheimer's disease.
RESUMEN
Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido [4',5':4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly (ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Neuroblastoma/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Neuroblastoma/enzimología , Neuroblastoma/metabolismoRESUMEN
BACKGROUND: Malaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue. RESULTS: We obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi. CONCLUSION: 3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.