RESUMEN
ABSTRACT: Biallelic mutation in the DNA-damage repair gene NBN is the genetic cause of Nijmegen breakage syndrome, which is associated with predisposition to lymphoid malignancies. Heterozygous carriers of germ line NBN variants may also be at risk for leukemia development, although this is much less characterized. By sequencing 4325 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL), we systematically examined the frequency of germ line NBN variants and identified 25 unique, putatively damaging NBN coding variants in 50 patients. Compared with the frequency of NBN variants in gnomAD noncancer controls (189 unique, putatively damaging NBN coding variants in 472 of 118 479 individuals), we found significant overrepresentation in pediatric B-ALL (P = .004; odds ratio, 1.8). Most B-ALL-risk variants were missense and cluster within the NBN N-terminal domains. Using 2 functional assays, we verified 14 of 25 variants with severe loss-of-function phenotypes and thus classified these as nonfunctional or partially functional. Finally, we found that germ line NBN variant carriers, all of whom were identified as heterozygous genotypes, showed similar survival outcomes relative to those with wild type status. Taken together, our findings provide novel insights into the genetic predisposition to B-ALL, and the impact of NBN variants on protein function and suggest that heterozygous NBN variant carriers may safely receive B-ALL therapy. These trials were registered at www.clinicaltrials.gov as #NCT01225874, NCT00075725, NCT00103285, NCI-T93-0101D, and NCT00137111.
Asunto(s)
Proteínas de Ciclo Celular , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genéticaRESUMEN
Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers. However, the mechanisms controlling BETi response and resistance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a combination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts in vivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combination treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique ex vivo and in vivo KMT2A-r PDX models.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Leucemia , Humanos , Glucógeno Sintasa Quinasa 3/metabolismo , Línea Celular Tumoral , Leucemia/tratamiento farmacológico , Leucemia/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismoRESUMEN
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.
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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
Familial aggregation of Hodgkin lymphoma (HL) has been demonstrated in large population studies, pointing to genetic predisposition to this hematological malignancy. To understand the genetic variants associated with the development of HL, we performed whole genome sequencing on 234 individuals with and without HL from 36 pedigrees that had 2 or more first-degree relatives with HL. Our pedigree selection criteria also required at least 1 affected individual aged <21 years, with the median age at diagnosis of 21.98 years (3-55 years). Family-based segregation analysis was performed for the identification of coding and noncoding variants using linkage and filtering approaches. Using our tiered variant prioritization algorithm, we identified 44 HL-risk variants in 28 pedigrees, of which 33 are coding and 11 are noncoding. The top 4 recurrent risk variants are a coding variant in KDR (rs56302315), a 5' untranslated region variant in KLHDC8B (rs387906223), a noncoding variant in an intron of PAX5 (rs147081110), and another noncoding variant in an intron of GATA3 (rs3824666). A newly identified splice variant in KDR (c.3849-2A>C) was observed for 1 pedigree, and high-confidence stop-gain variants affecting IRF7 (p.W238∗) and EEF2KMT (p.K116∗) were also observed. Multiple truncating variants in POLR1E were found in 3 independent pedigrees as well. Whereas KDR and KLHDC8B have previously been reported, PAX5, GATA3, IRF7, EEF2KMT, and POLR1E represent novel observations. Although there may be environmental factors influencing lymphomagenesis, we observed segregation of candidate germline variants likely to predispose HL in most of the pedigrees studied.
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Enfermedad de Hodgkin , Humanos , Adulto Joven , Adulto , Enfermedad de Hodgkin/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Codón sin Sentido , Secuenciación Completa del Genoma , Linaje , Proteínas de Ciclo Celular/genéticaRESUMEN
Trisomy 21, the genetic cause of Down syndrome (DS), is the most common congenital chromosomal anomaly. It is associated with a 20-fold increased risk of acute lymphoblastic leukemia (ALL) during childhood and results in distinctive leukemia biology. To comprehensively define the genomic landscape of DS-ALL, we performed whole-genome sequencing and whole-transcriptome sequencing (RNA-Seq) on 295 cases. Our integrated genomic analyses identified 15 molecular subtypes of DS-ALL, with marked enrichment of CRLF2-r, IGH::IGF2BP1, and C/EBP altered (C/EBPalt) subtypes compared with 2257 non-DS-ALL cases. We observed abnormal activation of the CEBPD, CEBPA, and CEBPE genes in 10.5% of DS-ALL cases via a variety of genomic mechanisms, including chromosomal rearrangements and noncoding mutations leading to enhancer hijacking. A total of 42.3% of C/EBP-activated DS-ALL also have concomitant FLT3 point mutations or insertions/deletions, compared with 4.1% in other subtypes. CEBPD overexpression enhanced the differentiation of mouse hematopoietic progenitor cells into pro-B cells in vitro, particularly in a DS genetic background. Notably, recombination-activating gene-mediated somatic genomic abnormalities were common in DS-ALL, accounting for a median of 27.5% of structural alterations, compared with 7.7% in non-DS-ALL. Unsupervised hierarchical clustering analyses of CRLF2-rearranged DS-ALL identified substantial heterogeneity within this group, with the BCR::ABL1-like subset linked to an inferior event-free survival, even after adjusting for known clinical risk factors. These results provide important insights into the biology of DS-ALL and point to opportunities for targeted therapy and treatment individualization.
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Síndrome de Down , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Ratones , Síndrome de Down/complicaciones , Síndrome de Down/genética , Mutación , Factores de Riesgo , Genómica , Aberraciones Cromosómicas , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
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.
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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
Although acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, it is unknown how or which host immune factors influence the long-term remission of this cancer. To this end, we systematically evaluated the effects of T-cell immunity on Ph+ ALL therapy outcomes. Using a murine Arf-/-BCR-ABL1 B-cell ALL model, we showed that loss of T cells in the host drastically increased leukemia relapse after dasatinib or cytotoxic chemotherapy. Although ABL1 mutations emerged early during dasatinib treatment in both immunocompetent and immunocompromised hosts, T-cell immunity was essential for suppressing the outgrowth of drug-resistant leukemia. Bulk and single-cell transcriptome profiling of T cells during therapy pointed to the activation of type 1 immunity-related cytokine signaling being linked to long-term leukemia remission in mice. Consistent with these observations, interferon γ and interleukin 12 directly modulated dasatinib antileukemia efficacy in vivo. Finally, we evaluated peripheral blood immune cell composition in 102 children with ALL during chemotherapy and observed a significant association of T-cell abundance with treatment outcomes. Together, these results suggest that T-cell immunity plays pivotal roles in maintaining long-term remission of ALL, highlighting that the interplay between host immunity and drug resistance can be harnessed to improve ALL chemotherapy outcomes.
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Interferón gamma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Dasatinib/farmacología , Dasatinib/uso terapéutico , Interleucina-12 , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Linfocitos TRESUMEN
Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.
RESUMEN
BACKGROUND: This study analyzed data from two consecutive protocols for children newly diagnosed with acute lymphoblastic leukemia (ALL) to determine the clinical impact of minimal/measurable residual disease (MRD) and recently identified tumor genetic subtypes. METHODS: Genetic subtypes were determined by sequential approaches including DNA indexing, reverse transcriptase-polymerase chain reaction, multiplex ligation-dependent probe amplification, and RNA-sequencing. MRD was assessed by flow cytometry. The Taiwan Pediatric Oncology Group TPOG-ALL-2013 study enrolled patients who received MRD-directed therapy. RESULTS: The 5-year event-free survival (EFS) and overall survival rates in the 2013 cohort were 77.8% and 86.9% compared to those of the 2002 cohort, which were 62.4% and 76.5%. Among patients treated with MRD-guided therapy, those with ETV6-RUNX1 fusion and high hyperdiploidy had the highest 5-year EFS (91.4% and 89.6%, respectively). The addition of dasatinib improved outcomes in patients with BCR-ABL1 ALL. Recently identified subtypes like DUX4-rearranged, ZNF384-rearranged, MEF2D-rearranged, and PAX5alt subtypes were frequently positive for MRD after remission induction, and these patients consequently received intensified chemotherapy. Treatment intensification according to the MRD improved the outcomes of patients presenting DUX4 rearrangements. In high-risk or very-high-risk subtypes, the TPOG-ALL-2013 regimen did not confer significant improvements compared to TPOG-ALL-2002, and the outcomes of BCR-ABL1-like, MEF2D-rearranged, and KMT2A-rearranged ALL subtypes (in addition to those of T-cell ALL) were not sufficiently good. Novel agents or approaches are needed to improve the outcomes for these patients. CONCLUSIONS: The TPOG-ALL-2013 study yielded outcomes superior to those of patients treated in the preceding TPOG-ALL-2002 study. This study provides important data to inform the design of future clinical trials in Taiwan. PLAIN LANGUAGE SUMMARY: MRD-directed therapy improved the outcomes for pediatric ALL, especially standard-risk patients. Genomic analyses and MRD might be used together for risk-directed therapy of childhood ALL. Our work provides important data to inform the design of future clinical trials in Taiwan.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Niño , Humanos , Neoplasia Residual/genética , Neoplasia Residual/diagnóstico , Pronóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Dasatinib/uso terapéutico , Inducción de RemisiónRESUMEN
Of 1003 children with acute lymphoblastic leukaemia (ALL), 147 (14.7%) presented without peripheral blood blasts (PBB). While absence of PBB was not independently associated with survival outcomes when compared to those with PBB, patients without PBB had distinct genetic and clinical characteristics. Notably, we identified a novel genotype-phenotype relationship, in that the patients without PBB had a significantly higher incidence of hyperdiploid B-ALL, accounting for almost half of all patients without PBB (46.9% vs. 22.7%, p < 0.001). Further, absence of PBB was associated with decreased rates of leukaemia involvement of the central nervous system (p < 0.001).
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , LeucocitosRESUMEN
BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigate whether the neonatal liver provides a protumorigenic niche to HB development. APPROACH AND RESULTS: HB development was compared between orthotopic transplantation models established in postnatal day 5 (P5) and 60 (P60) mice (P5Tx and P60Tx models). Single-cell RNA-sequencing (sc-RNAseq) was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. CONCLUSIONS: We found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver that express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout (KO) in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in tumors from patients with metastatic HB, there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with prognosis of patients with HB among pediatric cancers. We demonstrated that the neonatal liver provides a prometastatic niche to HB development through the Cxcl1/Cxcr2 axis.
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Hepatoblastoma , Neoplasias Hepáticas , Ratones , Animales , Hepatoblastoma/metabolismo , Quimiocina CXCL1/metabolismo , Receptores de Interleucina-8B/genética , Neoplasias Hepáticas/patología , ARNRESUMEN
CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.
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Quinasas Janus/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Receptores de Citocinas/genética , Animales , Línea Celular Tumoral , Descubrimiento de Drogas , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Quinasas Janus/metabolismo , Ratones Endogámicos NOD , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
To identify the prognostic factors that are useful to improve central nervous system (CNS) control in children with acute lymphoblastic leukemia (ALL), we analyzed the outcome of 7640 consecutive patients treated on Chinese Children's Cancer Group ALL-2015 protocol between 2015 and 2019. This protocol featured prephase dexamethasone treatment before conventional remission induction and subsequent risk-directed therapy, including 16 to 22 triple intrathecal treatments, without prophylactic cranial irradiation. The 5-year event-free survival was 80.3% (95% confidence interval [CI], 78.9-81.7), and overall survival 91.1% (95% CI, 90.1-92.1). The cumulative risk of isolated CNS relapse was 1.9% (95% CI, 1.5-2.3), and any CNS relapse 2.7% (95% CI, 2.2-3.2). The isolated CNS relapse rate was significantly lower in patients with B-cell ALL (B-ALL) than in those with T-cell ALL (T-ALL) (1.6%; 95% CI, 1.2-2.0 vs 4.6%; 95% CI, 2.9-6.3; P < .001). Independent risk factors for isolated CNS relapse included male sex (hazard ratio [HR], 1.8; 95% CI, 1.1-3.0; P = .03), the presence of BCR-ABL1 fusion (HR, 3.8; 95% CI, 2.0-7.3; P < .001) in B-ALL, and presenting leukocyte count ≥50×109/L (HR, 4.3; 95% CI, 1.5-12.2; P = .007) in T-ALL. Significantly lower isolated CNS relapse was associated with the use of total intravenous anesthesia during intrathecal therapy (HR, 0.2; 95% CI, 0.04-0.7; P = .02) and flow cytometry examination of diagnostic cerebrospinal fluid (CSF) (HR, 0.2; 95% CI, 0.06-0.6; P = .006) among patients with B-ALL. Prephase dexamethasone treatment, delayed intrathecal therapy, use of total intravenous anesthesia during intrathecal therapy, and flow cytometry examination of diagnostic CSF may improve CNS control in childhood ALL. This trial was registered with the Chinese Clinical Trial Registry (ChiCTR-IPR-14005706).
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Neoplasias del Sistema Nervioso Central , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Adolescente , Factores de Edad , Neoplasias del Sistema Nervioso Central/líquido cefalorraquídeo , Neoplasias del Sistema Nervioso Central/mortalidad , Neoplasias del Sistema Nervioso Central/terapia , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/líquido cefalorraquídeo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/líquido cefalorraquídeo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Factores de Riesgo , Factores Sexuales , Tasa de SupervivenciaRESUMEN
There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with wild-type ETV6 with potentially dominant-negative effects. Whole-transcriptome and whole-genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy-number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.
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Predisposición Genética a la Enfermedad , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Niño , Genes Dominantes , Genoma Humano , Mutación de Línea Germinal/genética , Humanos , Proteína ETS de Variante de Translocación 6RESUMEN
There is growing evidence for an inherited basis of susceptibility to childhood acute lymphoblastic leukemia. Genomewide association studies by us and others have identified non-coding acute lymphoblastic leukemia risk variants at the ARID5B gene locus, but the molecular mechanisms linking ARID5B to normal and malignant hematopoiesis remain largely unknown. Using a Vav1-driven transgenic mouse model, we characterized the role of Arid5b in hematopoiesis in vivo. Arid5b overexpression resulted in a dramatic reduction in the proportion of circulating B cells, immature, and mature Bcell fractions in the peripheral blood and the bone marrow, and also a decrease of follicular B cells in the spleen. There were significant defects in B-cell activation upon Arid5b overexpression in vitro with hyperactivation of B-cell receptor signaling at baseline. In addition, increased mitochondrial oxygen consumption rate of naïve or stimulated B cells of Arid5b-overexpressing mice was observed, compared to the rate of wild-type counterparts. Taken together, our results indicate that ARID5B may play an important role in B-cell development and function.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Factores de Transcripción , Animales , Ratones , Factores de Transcripción/genética , Proteínas de Unión al ADN/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Estudio de Asociación del Genoma Completo , HematopoyesisRESUMEN
BACKGROUND: Thiopurines are an important class of immunosuppressants despite their risk for hematopoietic toxicity and narrow therapeutic indices. Benign neutropenia related to an ACKR1 variant (rs2814778-CC) is common among persons of African ancestries. OBJECTIVE: To test whether rs2814778-CC was associated with azathioprine discontinuation attributed to hematopoietic toxicity and lower thiopurine dosing. DESIGN: Retrospective cohort study. SETTING: Two tertiary care centers. PATIENTS: Thiopurine users with White or Black race. MEASUREMENTS: Azathioprine discontinuation attributed to hematopoietic toxicity. Secondary outcomes included weight-adjusted final dose, leukocyte count, and change in leukocyte count. RESULTS: The rate of azathioprine discontinuation attributed to hematopoietic toxicity was 3.92 per 100 person-years among patients with the CC genotype (n = 101) and 1.34 per 100 person-years among those with the TT or TC genotype (n = 1365) (hazard ratio [HR] from competing-risk model, 2.92 [95% CI, 1.57 to 5.41]). The risk remained significant after adjustment for race (HR, 2.61 [CI, 1.01 to 6.71]). The risk associated with race alone (HR, 2.13 [CI, 1.21 to 3.75]) was abrogated by adjustment for genotype (HR, 1.13 [CI, 0.48 to 2.69]). Lower last leukocyte count and lower dosing were significant among patients with the CC genotype. Lower dosing was validated in an external cohort of 94 children of African ancestries prescribed the thiopurine 6-mercaptopurine (6-MP) for acute lymphoblastic leukemia. The CC genotype was independently associated with lower 6-MP dose intensity relative to the target daily dose of 75 mg/m2 (median, 0.83 [IQR, 0.70 to 0.94] for the CC genotype vs. 0.94 [IQR, 0.72 to 1.13] for the TT or TC genotype; P = 0.013). LIMITATIONS: Unmeasured confounding; data limited to tertiary centers. CONCLUSION: Patients with the CC genotype had higher risk for azathioprine discontinuation attributed to hematopoietic toxicity and lower thiopurine doses. Genotype was associated with those risks, even after adjustment for race. PRIMARY FUNDING SOURCE: National Institutes of Health.
Asunto(s)
Azatioprina , Mercaptopurina , Azatioprina/efectos adversos , Niño , Estudios de Cohortes , Genotipo , Humanos , Mercaptopurina/efectos adversos , Estudios RetrospectivosRESUMEN
As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.
Asunto(s)
Antimetabolitos/administración & dosificación , Antimetabolitos/toxicidad , Mercaptopurina/administración & dosificación , Mercaptopurina/toxicidad , Variantes Farmacogenómicas , Pirofosfatasas/genética , Alelos , Sustitución de Aminoácidos , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Estabilidad de Enzimas , Células HEK293 , Humanos , Mutación Missense , Medicina de Precisión , Conformación Proteica en Hélice alfa/genética , Pirofosfatasas/química , RiesgoRESUMEN
To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.
Asunto(s)
Farmacogenética , Alelos , Genotipo , Haplotipos , Humanos , Análisis de Secuencia de ADN/métodosRESUMEN
The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.