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1.
Sci Adv ; 8(50): eadd6403, 2022 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-36516256

RESUMEN

Blinatumomab is an efficacious immunotherapeutic agent in B cell acute lymphoblastic leukemia (B-ALL). However, the pharmacogenomic basis of leukemia response to blinatumomab is unclear. Using genome-wide CRISPR, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity, i.e., the loss of CD58 as a top driver for resistance, in addition to CD19. Screening 1639 transcription factor genes, we then identified PAX5 as the key activator of CD58. ALL with the PAX5 P80R mutation also expressed the lowest level of CD58 among 20 ALL molecular subtypes in 1988 patients. Genome editing confirmed the effects of this mutation on CD58 expression and blinatumomab sensitivity in B-ALL, with validation in patient leukemic blasts. We described a PAX5-driven enhancer at the CD58 locus, which was disrupted by PAX5 P80R, and the loss of CD58 abolished blinatumomab-induced T cell activation with global changes in transcriptomic/epigenomic program. In conclusion, we identified previously unidentified genetic mechanisms of blinatumomab resistance in B-ALL, suggesting strategies for genomics-guided treatment individualization.


Asunto(s)
Anticuerpos Biespecíficos , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Antígenos CD19/genética , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo
2.
Hum Mutat ; 43(11): 1557-1566, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36057977

RESUMEN

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/métodos
3.
Br J Haematol ; 199(2): 270-276, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35905175

RESUMEN

6-Mercaptopurine (6-MP) is widely used for the treatment of paediatric leukaemia and lymphoma. Recently, germline variants in the NUDT15 gene have been identified as one of the major genetic causes for 6-MP-associated adverse effects such as myelosuppression. Patients with hypomorphic NUDT15 variants accumulate excessive levels of DNA-incorporated thioguanine in white blood cells, resulting in severe myelosuppression. Although preclinical studies suggest that these variants may influence the protein stability of NUDT15, this has not been directly characterised in patients. In this study, we report the development of a series of novel monoclonal antibodies against NUDT15, using which we quantitatively assessed NUDT15 protein levels in 37 patients with acute lymphoblastic leukaemia treated with 6-MP, using sandwich enzyme-linked immunosorbent assay (ELISA). The NUDT15 genotype was highly correlated with its protein levels (p < 0.0001), with homozygous and compound heterozygous patients showing exceedingly low NUDT15 expression. There was a positive correlation between NUDT15 protein level and 6-MP tolerance (r = 0.631, p < 0.0001). In conclusion, our results point to low NUDT15 protein abundance as the biochemical basis for NUDT15-mediated 6-MP intolerance, thus providing a phenotypic readout of inherited NUDT15 deficiency.


Asunto(s)
Mercaptopurina , Pirofosfatasas , Niño , Humanos , Anticuerpos Monoclonales/uso terapéutico , Mercaptopurina/efectos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirofosfatasas/genética , Tioguanina/uso terapéutico
4.
Pediatr Hematol Oncol ; 39(3): 254-266, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34665987

RESUMEN

Inter-individual variance in 6-mercaptopurine (6-MP) dose intensity is common in patients with acute lymphoblastic leukemia (ALL). We aimed to evaluate the association of common variants of ABCC4, ITPA, NUDT15, and TPMT with 6-MP dose intensity and toxicity in pediatric ALL patients. In this cohort, 13.8% of patients were intolerant to 6-MP with actual dosage less than 50% of scheduled dose. Twenty percent of patients were found to be heterozygous or homozygous mutated with NUDT15. NUDT15 c.415C > T and the genotype-predicted NUDT15 activity were significantly associated with 6-MP intolerance. TPMT*3C variants were not common in this cohort (2.8%). NUDT15 polymorphisms and genotype predicted NUDT15 activity were significantly associated with 6-MP dose intensity and leukopenia episodes. Combination of ABCC4 and ITPA variants (ABCC4 c.912G > T and ITPA c.94C > A) also showed significant positive association with 6-MP intolerance in Chinese children with ALL. Further study on pharmacogenetic screening for ALL patients to avoid 6-MP induced toxicity is recommended.Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2021.1973628.


Asunto(s)
Antimetabolitos Antineoplásicos , Mercaptopurina , Leucemia-Linfoma Linfoblástico de Células Precursoras , Antimetabolitos Antineoplásicos/efectos adversos , Antimetabolitos Antineoplásicos/uso terapéutico , Niño , China , Humanos , Mercaptopurina/efectos adversos , Mercaptopurina/uso terapéutico , Metiltransferasas/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirofosfatasas/genética
5.
Pharmacogenet Genomics ; 32(2): 60-66, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-34412101

RESUMEN

Thiopurines [e.g. 6-mercaptopurine (6MP)] are essential for the cure of acute lymphoblastic leukemia (ALL) but can cause dose-limiting hematopoietic toxicity. Germline variants in drug-metabolizing enzyme genes TPMT and NUDT15 have been linked to the risk of thiopurine toxicity. However, the full spectrum of genetic polymorphism in these genes and their impact on the pharmacological effects of thiopurines remain unclear. Herein, we comprehensively sequenced the TPMT and NUDT15 genes in 685 children with ALL from the Children's Oncology Group AALL03N1 trial and evaluated their association with 6MP dose intensity. We identified 6 and 5 coding variants in TPMT and NUDT15 respectively, confirming the association at known pharmacogenetic variants. Importantly, we discovered a novel gain-of-function noncoding variants in TPMT associated with increased 6MP tolerance (rs12199316), with independent validation in 380 patients from the St. Jude Total Therapy XV protocol. Located adjacent to a regulatory DNA element, this intergenic variant was strongly associated TPMT transcription, with the variant allele linked to higher expression (P = 2.6 × 10-9). For NUDT15, one noncoding common variant, rs73189762, was identified as potentially related to 6MP intolerance. Collectively, we described pharmacogenetic variants in TPMT and NUDT15 associated with thiopurine sensitivity, providing further insights for implementing pharmacogenetics-based thiopurine individualization.


Asunto(s)
Variantes Farmacogenómicas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Niño , Humanos , Mercaptopurina/efectos adversos , Metiltransferasas/genética , Polimorfismo Genético/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirofosfatasas/genética
6.
J Cell Mol Med ; 25(22): 10521-10533, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34636169

RESUMEN

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.


Asunto(s)
5'-Nucleotidasa/genética , Resistencia a Antineoplásicos/genética , Mercaptopurina/farmacología , Mutación , Polimorfismo Genético , Pirofosfatasas/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Alelos , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Relación Dosis-Respuesta a Droga , Genotipo , Humanos
7.
Blood Adv ; 5(23): 5420-5428, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34662904

RESUMEN

The effect of genetic variation on second malignant neoplasms (SMNs) remains unclear. First, we identified the pathogenic germline variants in cancer-predisposing genes among 15 children with SMNs after childhood leukemia/lymphoma using whole-exome sequencing. Because the prevalence was low, we focused on the association between SMNs and NUDT15 in primary acute lymphoblastic leukemia (ALL) cases. NUDT15 is one of the 6-mercaptopurine (6-MP) metabolic genes, and its variants are common in East Asian individuals. The prevalence of NUDT15 hypomorphic variants was higher in patients with SMNs (n = 14; 42.9%) than in the general population in the gnomAD database (19.7%; P = .042). In the validation study with a cohort of 438 unselected patients with ALL, the cumulative incidence of SMNs was significantly higher among those with (3.0%; 95% confidence interval [CI], 0.6% to 9.4%) than among those without NUDT15 variants (0.3%; 95% CI, 0.0% to 1.5%; P = .045). The 6-MP dose administered to patients with ALL with a NUDT15 variant was higher than that given to those without SMNs (P = .045). The 6-MP-related mutational signature was observed in SMN specimens after 6-MP exposure. In cells exposed to 6-MP, a higher level of 6-MP induced DNA damage in NUDT15-knockdown induced pluripotent stem cells. Our study indicates that NUDT15 variants may confer a risk of SMNs after treatment with 6-MP in patients with ALL.


Asunto(s)
Neoplasias Primarias Secundarias , Leucemia-Linfoma Linfoblástico de Células Precursoras , Antimetabolitos Antineoplásicos/uso terapéutico , Niño , Humanos , Incidencia , Neoplasias Primarias Secundarias/epidemiología , Neoplasias Primarias Secundarias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirofosfatasas/genética , Pirofosfatasas/uso terapéutico
8.
Nat Commun ; 12(1): 4181, 2021 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-34234136

RESUMEN

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.


Asunto(s)
Aciclovir/farmacología , Antivirales/farmacología , Infecciones por Citomegalovirus/prevención & control , Ganciclovir/análogos & derivados , Pirofosfatasas/genética , Aciclovir/uso terapéutico , Adolescente , Adulto , Anciano , Animales , Profilaxis Antibiótica , Antivirales/uso terapéutico , Variación Biológica Poblacional/genética , Línea Celular , Niño , Preescolar , Cristalografía por Rayos X , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/virología , ADN Viral/sangre , ADN Viral/aislamiento & purificación , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Femenino , Ganciclovir/farmacología , Ganciclovir/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Interacciones Microbiota-Huesped/genética , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Muromegalovirus/aislamiento & purificación , Muromegalovirus/patogenicidad , Variantes Farmacogenómicas , Polimorfismo de Nucleótido Simple , Pirofosfatasas/metabolismo , Pirofosfatasas/ultraestructura , Resultado del Tratamiento , Adulto Joven
9.
J Biol Chem ; 296: 100568, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33753169

RESUMEN

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.


Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Mutación , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/genética , Tioguanina/química , Tioguanina/farmacología , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Pirofosfatasas/química
11.
Blood ; 137(3): 364-373, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-32693409

RESUMEN

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.


Asunto(s)
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 6
12.
Clin Pharmacol Ther ; 109(6): 1538-1545, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33124053

RESUMEN

6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide (TGN) metabolites. A recent genomewide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms nominally associated with erythrocyte TGN during 6-MP treatment (P < 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to interpatient variability in thiopurine drug disposition.


Asunto(s)
5'-Nucleotidasa/genética , Antimetabolitos Antineoplásicos/farmacocinética , Mercaptopurina/farmacocinética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adolescente , Alelos , Niño , Preescolar , Estudios de Cohortes , Eritrocitos/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Mutación de Línea Germinal , Humanos , Desequilibrio de Ligamiento , Masculino , Familia de Multigenes , Análisis Multivariante , Mutación Missense , Polimorfismo de Nucleótido Simple , Tioguanina/sangre , Adulto Joven
13.
Blood ; 136(10): 1161-1168, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32391884

RESUMEN

Methotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10-8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10-9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms.


Asunto(s)
Metotrexato/análogos & derivados , Recurrencia Local de Neoplasia/patología , Péptido Sintasas/genética , Ácido Poliglutámico/análogos & derivados , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Tetrahidrofolato Deshidrogenasa/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Recién Nacido , Masculino , Metotrexato/administración & dosificación , Metotrexato/metabolismo , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Ácido Poliglutámico/administración & dosificación , Ácido Poliglutámico/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pronóstico , Adulto Joven
14.
Mol Cancer Res ; 18(8): 1153-1165, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32332049

RESUMEN

The NSD2 p.E1099K (EK) mutation is observed in 10% of acute lymphoblastic leukemia (ALL) samples with enrichment at relapse indicating a role in clonal evolution and drug resistance. To discover mechanisms that mediate clonal expansion, we engineered B-precursor ALL (B-ALL) cell lines (Reh, 697) to overexpress wildtype (WT) and EK NSD2, but observed no differences in proliferation, clonal growth, or chemosensitivity. To address whether NSD2 EK acts collaboratively with other pathways, we used short hairpin RNAs to knockdown expression of NSD2 in B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM). Knockdown resulted in decreased proliferation in all lines, decreased clonal growth in RCH-ACV, and increased sensitivity to cytotoxic chemotherapeutic agents, although the pattern of drug sensitivity varied among cell lines implying that the oncogenic properties of NSD2 mutations are likely cell context specific and rely on cooperative pathways. Knockdown of both Type II and REIIBP EK isoforms had a greater impact than knockdown of Type II alone, suggesting that both SET containing EK isoforms contribute to phenotypic changes driving relapse. Furthermore, in vivo models using both cell lines and patient samples revealed dramatically enhanced proliferation of NSD2 EK compared with WT and reduced sensitivity to 6-mercaptopurine in the relapse sample relative to diagnosis. Finally, EK-mediated changes in chromatin state and transcriptional output differed dramatically among cell lines further supporting a cell context-specific role of NSD2 EK. These results demonstrate a unique role of NSD2 EK in mediating clonal fitness through pleiotropic mechanisms dependent on the genetic and epigenetic landscape. IMPLICATIONS: NSD2 EK mutation leads to drug resistance and a clonal advantage in childhood B-ALL.


Asunto(s)
Resistencia a Antineoplásicos , Perfilación de la Expresión Génica/métodos , N-Metiltransferasa de Histona-Lisina/genética , Mutación , Recurrencia Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Represoras/genética , Animales , Ciclo Celular , Línea Celular Tumoral , Niño , Progresión de la Enfermedad , Epigénesis Genética , Células HEK293 , Humanos , Ratones , Análisis de Secuencia de ARN , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Proc Natl Acad Sci U S A ; 117(10): 5394-5401, 2020 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-32094176

RESUMEN

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 , Riesgo
16.
Mol Cancer Ther ; 18(10): 1887-1895, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31358663

RESUMEN

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5'-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.


Asunto(s)
5'-Nucleotidasa/metabolismo , Resistencia a Antineoplásicos , Mercaptopurina/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Línea Celular Tumoral , Células HEK293 , Humanos , Cinética , Mercaptopurina/química , Mercaptopurina/farmacología , Metabolómica , Modelos Biológicos , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología
19.
Blood ; 131(22): 2466-2474, 2018 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-29572377

RESUMEN

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.


Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Leucemia/tratamiento farmacológico , Mercaptopurina/uso terapéutico , Hidrolasas Diéster Fosfóricas/genética , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/toxicidad , Sistemas CRISPR-Cas , Niño , Cálculo de Dosificación de Drogas , Evaluación Preclínica de Medicamentos , Eliminación de Gen , Edición Génica , Genotipo , Humanos , Leucemia/genética , Leucemia/patología , Mercaptopurina/administración & dosificación , Mercaptopurina/toxicidad , Ratones , Ratones Noqueados , Pirofosfatasas/genética
20.
Leukemia ; 32(6): 1393-1403, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29535428

RESUMEN

Activating mutations in NT5C2, a gene encoding cytosolic purine 5'-nucleotidase (cN-II), confer chemoresistance in relapsed acute lymphoblastic leukemia. Here we show that all mutants became independent of allosteric effects of ATP and thus constitutively active. Structural mapping of mutations described in patients demonstrates that 90% of leukemia-specific allelles directly affect two regulatory hotspots within the cN-II molecule-the helix A region: residues 355-365, and the intersubunit interface: helix B (232-242) and flexible interhelical loop L (400-418). Furthermore, analysis of hetero-oligomeric complexes combining wild-type (WT) and mutant subunits showed that the activation is transmitted from the mutated to the WT subunit. This intersubunit interaction forms structural basis of hyperactive NT5C2 in drug-resistant leukemia in which heterozygous NT5C2 mutation gave rise to hetero-tetramer mutant and WT proteins. This enabled us to define criteria to aid the prediction of NT5C2 drug resistance mutations in leukemia.


Asunto(s)
5'-Nucleotidasa/genética , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , 5'-Nucleotidasa/química , Resistencia a Antineoplásicos , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Conformación Proteica , Subunidades de Proteína/química , Recurrencia
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