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Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.
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Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , Masculino , Femenino , VIH-1/genética , Viremia , Provirus/genética , Provirus/metabolismo , Infecciones por VIH/tratamiento farmacológico , Linfocitos T CD4-Positivos , ARN Viral , Carga ViralRESUMEN
Non-suppressible HIV-1 viremia (NSV) can occur in persons with HIV despite adherence to combination antiretroviral therapy (ART) and in the absence of significant drug resistance. Here, we show that plasma NSV sequences are comprised primarily of large clones without evidence of viral evolution over time. We defined proviruses that contribute to plasma viremia as "producer", and those that did not as "non-producer". Compared to ART-suppressed individuals, NSV participants had a significantly larger producer reservoir. Producer proviruses were enriched in chromosome 19 and in proximity to the activating H3K36me3 epigenetic mark. CD4+ cells from NSV participants demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, NSV participants showed no elevation in HIV-specific CD8+ cell responses and producer proviruses were enriched for HLA escape mutations. We identified critical host and viral mediators of NSV that represent potential targets to disrupt HIV persistence and promote viral silencing.
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Chronic hepatitis C can lead to cirrhosis and hepatocellular carcinoma. We studied the safety and immunogenicity of a novel therapeutic hepatitis C virus (HCV) genotype 1a/1b consensus DNA vaccine, INO-8000, encoding HCV NS3, NS4A, NS4B, and NS5A proteins alone or co-administered with DNA-encoding IL12 (INO-9012), a human cytokine that stimulates cellular immune function, in individuals with chronic hepatitis C. This was a phase I, multisite dose-escalation trial with an expansion cohort evaluating doses of 0, 0.3, 1.0, and 3.0 mg of INO-9012 (IL12 DNA) as an addition to 6.0 mg of (INO-8000; HCV DNA vaccine). Vaccines were administered by intramuscular injection followed by electroporation at study entry and at weeks 4, 12, and 24. HCV-specific CD4+ and CD8+ T-cell immune responses were measured by IFNγ ELISpot and flow cytometry-based assays. Transient, mild-to-moderate injection site reactions unrelated to IL12 DNA dose were common. Increases in HCV-specific IFNγ production occurred in 15/20 (75%) participants. Increases in the frequency of HCV-specific CD4+ and CD8+ T cells occurred at all dose levels, with the greatest increases seen at 1.0 mg of INO-9012. HCV-specific CD8+ and CD4+ T-cell activities increased in 16/18 (89%) and 14/17 (82%) participants with available data, respectively. The vaccine regimen was safe and induced HCV-specific CD4+ and CD8+ cellular immune responses of modest magnitude in most HCV-infected participants. The addition of 1.0 mg of IL12 DNA provided the best enhancement of immune responses. The vaccine regimen had little effect on controlling HCV viremia. PREVENTION RELEVANCE: The administration of IL12 DNA along with a hepatitis C viral antigen DNA vaccine enhanced the HCV-specific immune responses induced by the vaccine in individuals with chronic hepatitis C, an important cause of hepatocellular carcinoma. IL12 could be an effective adjuvant in vaccines targeting HCV and other oncogenic viruses.
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Carcinoma Hepatocelular , Hepatitis C Crónica , Hepatitis C , Neoplasias Hepáticas , Vacunas de ADN , Humanos , Vacunas de ADN/efectos adversos , Vacunas de ADN/genética , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Carcinoma Hepatocelular/prevención & control , Proteínas no Estructurales Virales/genética , Neoplasias Hepáticas/prevención & control , Hepatitis C/prevención & control , Hepacivirus/genética , ADN , Interleucina-12RESUMEN
Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. SIGNIFICANCE: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.
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Deriva y Cambio Antigénico , Leucemia-Linfoma Linfoblástico de Células Precursoras , Niño , Epítopos/uso terapéutico , Humanos , Inmunoterapia , Inotuzumab Ozogamicina , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
BACKGROUND: A vaccine against SARS-CoV-2 is of high urgency. Here the safety and immunogenicity induced by a DNA vaccine (INO-4800) targeting the full length spike antigen of SARS-CoV-2 are described. METHODS: INO-4800 was evaluated in two groups of 20 participants, receiving either 1.0 mg or 2.0 mg of vaccine intradermally followed by CELLECTRA® EP at 0 and 4 weeks. Thirty-nine subjects completed both doses; one subject in the 2.0 mg group discontinued trial participation prior to receiving the second dose. ClinicalTrials.gov identifier: NCT04336410. FINDINGS: The median age was 34.5, 55% (22/40) were men and 82.5% (33/40) white. Through week 8, only 6 related Grade 1 adverse events in 5 subjects were observed. None of these increased in frequency with the second administration. No serious adverse events were reported. All 38 subjects evaluable for immunogenicity had cellular and/or humoral immune responses following the second dose of INO-4800. By week 6, 95% (36/38) of the participants seroconverted based on their responses by generating binding (ELISA) and/or neutralizing antibodies (PRNT IC50), with responder geometric mean binding antibody titers of 655.5 [95% CI (255.6, 1681.0)] and 994.2 [95% CI (395.3, 2500.3)] in the 1.0 mg and 2.0 mg groups, respectively. For neutralizing antibody, 78% (14/18) and 84% (16/19) generated a response with corresponding geometric mean titers of 102.3 [95% CI (37.4, 280.3)] and 63.5 [95% CI (39.6, 101.8)], in the respective groups. By week 8, 74% (14/19) and 100% (19/19) of subjects generated T cell responses by IFN-É£ ELISpot assay with the median SFU per 106 PBMC of 46 [95% CI (21.1, 142.2)] and 71 [95% CI (32.2, 194.4)] in the 1.0 mg and 2.0 mg groups, respectively. Flow cytometry demonstrated a T cell response, dominated by CD8+ T cells co-producing IFN-É£ and TNF-α, without increase in IL-4. INTERPRETATION: INO-4800 demonstrated excellent safety and tolerability and was immunogenic in 100% (38/38) of the vaccinated subjects by eliciting either or both humoral or cellular immune responses. FUNDING: Coalition for Epidemic Preparedness Innovations (CEPI).
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: Background: Recurrent respiratory papillomatosis (RRP) is a rare disorder characterized by the generation of papillomas of the aerodigestive tract, usually associated with human papilloma virus (HPV) subtypes 6, 11. INO-3106 is a DNA plasmid-based immunotherapy targeting E6 and E7 proteins of HPV6, in order to create a robust immune T cell response. METHODS: Testing of INO-3016 in animal models confirmed immunogenicity of the DNA-based therapy. A single-site open-label Phase 1 study was initiated for patients with HPV6-positive RRP. Patients were dosed with INO-3106 with or without INO-9012, a DNA plasmid immunotherapy that encodes IL-12, delivered intramuscularly (IM) in combination with electroporation (EP) with the CELLECTRA® device. Patients received an escalating dose of INO-3106, 3 mg once and then 6 mg for three additional doses, each dose three weeks apart, with the third and fourth doses co-administered with INO-9012. The primary objective of the study was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. The secondary objective was to determine cellular immune responses to INO-3106 with and without INO-9012. Exploratory objectives included preliminary clinical efficacy to the therapy. RESULTS: Three patients were enrolled in this study, of which two had RRP. Study therapy was well-tolerated, with no related serious adverse events and all related adverse events (AEs) were low-grade. Injection site pain was the most common related AE reported. Immunogenicity was evidenced by multiple immune assays showing engagement and expansion of an HPV6-specific cellular response, including cytotoxic T cells. Preliminary efficacy was demonstrated in patients with RRP in the form of reduction in need for surgical intervention for papilloma growth. Prior to intervention, both patients required surgical intervention approximately every 180 days. One patient demonstrated a greater than three-fold increase in surgery avoidance (584 days) and the other patient remains completely surgery-free as of the last contact at 915 days, a greater than 5-fold increase in surgery interval. CONCLUSION: INO-3106 with and without INO-9012 was well tolerated, immunogenic and demonstrated preliminary efficacy in patients with HPV6-associated RRP aerodigestive lesions. Further clinical study is indicated.
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BACKGROUND: Nonlive vaccine approaches that are simple to deliver and stable at room temperature or 2-8°C could be advantageous in controlling future Ebola virus (EBOV) outbreaks. Using an immunopotent DNA vaccine that generates protection from lethal EBOV challenge in small animals and nonhuman primates, we performed a clinical study to evaluate both intramuscular (IM) and novel intradermal (ID) DNA delivery. METHODS: Two DNA vaccine candidates (INO-4201 and INO-4202) targeting the EBOV glycoprotein (GP) were evaluated for safety, tolerability, and immunogenicity in a phase 1 clinical trial. The candidates were evaluated alone, together, or in combination with plasmid-encoded human cytokine interleukin-12 followed by in vivo electroporation using either the CELLECTRA® IM or ID delivery devices. RESULTS: The safety profile of all 5 regimens was shown to be benign, with the ID route being better tolerated. Antibodies to EBOV GP were generated by all 5 regimens with the fastest and steepest rise observed in the ID group. Cellular immune responses were generated with every regimen. CONCLUSIONS: ID delivery of INO-4201 was well tolerated and resulted in 100% seroreactivity after 2 doses and elicited interferon-γ T-cell responses in over 70% of subjects, providing a new approach for EBOV prevention in diverse populations. Clinical Trials Registration. NCT02464670.
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Vacunas contra el Virus del Ébola/efectos adversos , Vacunas contra el Virus del Ébola/inmunología , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Vacunas de ADN/efectos adversos , Vacunas de ADN/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/inmunología , Ebolavirus/inmunología , Electroporación/métodos , Femenino , Glicoproteínas/inmunología , Voluntarios Sanos , Fiebre Hemorrágica Ebola/inmunología , Humanos , Inyecciones Intradérmicas/métodos , Interleucina-12/inmunología , Masculino , Persona de Mediana Edad , Temperatura , Vacunación/métodos , Adulto JovenRESUMEN
Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded â¼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.
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Empalme Alternativo , Linfocitos B/metabolismo , Regulación Leucémica de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea A1/genética , Degradación de ARNm Mediada por Codón sin Sentido , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones no Traducidas 3' , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adulto , Linfocitos B/patología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Línea Celular Tumoral , Niño , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Exones , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Intrones , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Cultivo Primario de Células , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Factores de Empalme Serina-Arginina/antagonistas & inhibidores , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
PARP inhibitors (PARPis) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success. We hypothesized that RAD52-mediated DNA repair remains active in PARPi-treated BRCA-deficient tumor cells and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show that RAD52 inhibitors (RAD52is) attenuated single-strand annealing (SSA) and residual homologous recombination (HR) in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with inhibitors or dominant-negative mutants caused synergistic accumulation of DSBs and eradication of BRCA-deficient but not BRCA-proficient tumor cells. Remarkably, Parp1-/-;Rad52-/- mice are normal and display prolonged latency of BRCA1-deficient leukemia compared with Parp1-/- and Rad52-/- counterparts. Finally, PARPi+RAD52i exerted synergistic activity against BRCA1-deficient tumors in immunodeficient mice with minimal toxicity to normal cells and tissues. In conclusion, our data indicate that addition of RAD52i will improve therapeutic outcome of BRCA-deficient malignancies treated with PARPi.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Animales , Proteína BRCA1/deficiencia , Proteína BRCA2/deficiencia , Reparación del ADN/efectos de los fármacos , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Recombinación Homóloga/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Proteína Recombinante y Reparadora de ADN Rad52/antagonistas & inhibidores , Proteína Recombinante y Reparadora de ADN Rad52/deficiencia , Mutaciones Letales Sintéticas , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
Prolonged exit from quiescence by hematopoietic stem cells (HSCs) progressively impairs their homeostasis in the bone marrow through an unidentified mechanism. We show that Rb proteins, which are major enforcers of quiescence, maintain HSC homeostasis by positively regulating thrombopoietin (Tpo)-mediated Jak2 signaling. Rb family protein inactivation triggers the progressive E2f-mediated transactivation of Socs3, a potent inhibitor of Jak2 signaling, in cycling HSCs. Aberrant activation of Socs3 impairs Tpo signaling and leads to impaired HSC homeostasis. Therefore, Rb proteins act as a central hub of quiescence and homeostasis by coordinating the regulation of both cell cycle and Jak2 signaling in HSCs.
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Células Madre Hematopoyéticas/metabolismo , Homeostasis/genética , Proteína de Retinoblastoma/genética , Proteína p107 Similar a la del Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma/genética , Proteína 3 Supresora de la Señalización de Citocinas/genética , Animales , Ciclo Celular/genética , División Celular/genética , Proliferación Celular/genética , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Perfilación de la Expresión Génica/métodos , Immunoblotting , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones Noqueados , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Interferencia de ARN , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Trombopoyetina/farmacología , Activación TranscripcionalRESUMEN
Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.
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Antineoplásicos/farmacología , Proliferación Celular , Leucemia/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Cricetinae , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Genes BRCA1 , Genes BRCA2 , Genes Letales , Genes abl , Humanos , Leucemia/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Embrionarias de Ratones/fisiología , Ftalazinas/farmacología , Piperazinas/farmacología , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.
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Crisis Blástica/genética , Genes Supresores de Tumor , Genes abl , Leucemia Experimental/genética , Leucemia Mieloide de Fase Crónica/genética , Proteínas Oncogénicas v-abl/fisiología , Proteínas de Fusión Oncogénica/fisiología , Proteínas Proto-Oncogénicas c-abl/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Crisis Blástica/tratamiento farmacológico , Crisis Blástica/enzimología , Crisis Blástica/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Citostáticos/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Inestabilidad Genómica , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Imidazoles/farmacología , Imidazoles/uso terapéutico , Leucemia Experimental/tratamiento farmacológico , Leucemia Experimental/enzimología , Leucemia Experimental/patología , Leucemia Mieloide de Fase Crónica/tratamiento farmacológico , Leucemia Mieloide de Fase Crónica/enzimología , Leucemia Mieloide de Fase Crónica/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Proteínas Oncogénicas v-abl/antagonistas & inhibidores , Proteínas Oncogénicas v-abl/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Estrés Oxidativo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-abl/genética , Piridazinas/farmacología , Piridazinas/uso terapéutico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.