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Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.
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Proteína BRCA1 , Daño del ADN , Leucemia , Animales , Ratones , Proteína BRCA2 , ADN/metabolismo , Leucemia/enzimología , Leucemia/genética , ADN Polimerasa thetaRESUMEN
While current treatment regimens for acute leukemia can dramatically improve patient survival, there remains room for improvement. Due to its roles in cell differentiation, cell survival, and apoptotic signaling, modulation of the cyclic AMP (cAMP) pathway has provided a meaningful target in hematological malignancies. Several studies have demonstrated that gene expression profiles associated with increased pro-survival cAMP activity or downregulation of various pro-apoptotic factors associated with the cAMP pathway are apparent in acute leukemia patients. Previous work to increase leukemia cell intracellular cAMP focused on the use of cAMP analogs, stimulating cAMP production via transmembrane-associated adenylyl cyclases, or decreasing cAMP degradation by inhibiting phosphodiesterase activity. However, targeting cyclic nucleotide efflux by ATP-binding cassette (ABC) transporters represents an unexplored approach for modulation of intracellular cyclic nucleotide levels. Preliminary studies have shown that inhibition of cAMP efflux can stimulate leukemia cell differentiation, cell growth arrest, and apoptosis, indicating that targeting cAMP efflux may show promise for future therapeutic development. Furthermore, inhibition of cyclic nucleotide transporter activity may also contribute multiple anticancer benefits by reducing extracellular pro-survival signaling in malignant cells. Hence, several opportunities for drug repurposing may exist for targeting cyclic nucleotide transporters.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Reposicionamiento de Medicamentos/métodos , Leucemia/tratamiento farmacológico , Animales , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , HumanosRESUMEN
Activating mutations in cytokine receptors and transcriptional regulators govern aberrant signal transduction in T-cell lineage acute lymphoblastic leukemia (T-ALL). However, the roles played by suppressors of cytokine signaling remain incompletely understood. We examined the regulatory roles of suppressor of cytokine signaling 5 (SOCS5) in T-ALL cellular signaling networks and leukemia progression. We found that SOCS5 was differentially expressed in primary T-ALL and its expression levels were lowered in HOXA-deregulated leukemia harboring KMT2A gene rearrangements. Here, we report that SOCS5 expression is epigenetically regulated by DNA methyltransferase-3A-mediated DNA methylation and methyl CpG binding protein-2-mediated histone deacetylation. We show that SOCS5 negatively regulates T-ALL cell growth and cell cycle progression but has no effect on apoptotic cell death. Mechanistically, SOCS5 silencing induces activation of JAK-STAT signaling, and negatively regulates interleukin-7 and interleukin-4 receptors. Using a human T-ALL murine xenograft model, we show that genetic inactivation of SOCS5 accelerates leukemia engraftment and progression, and leukemia burden. We postulate that SOCS5 is epigenetically deregulated in T-ALL and serves as an important regulator of T-ALL cell proliferation and leukemic progression. Our results link aberrant downregulation of SOCS5 expression to the enhanced activation of the JAK-STAT and cytokine receptor-signaling cascade in T-ALL.
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Epigénesis Genética , Quinasas Janus/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Factores de Transcripción STAT/genética , Proteínas Supresoras de la Señalización de Citocinas/genética , Animales , Línea Celular , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Quinasas Janus/metabolismo , Células Jurkat , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Tratamiento con ARN de Interferencia/métodos , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models.
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Apoptosis/efectos de los fármacos , Toxinas Bacterianas/farmacología , Exotoxinas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Adolescente , Adulto , Animales , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
A 17-year-old girl with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with persistent minimal residual disease (MRD) who underwent standard chemotherapy was found to have a BCR-ABL1-like gene expression pattern. Genome sequencing revealed a JAK2 mutation not previously described in BCP-ALL and a potential therapeutic target. Due to concern for an on-therapy relapse, the JAK2 inhibitor ruxolitinib was incorporated into a modified chemotherapy backbone to achieve complete remission prior to stem cell transplant. Treatment was well tolerated and she had undetectable MRD prior to a matched allogeneic stem cell transplant and remained in remission at day +100.
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Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Terapia Molecular Dirigida/métodos , Medicina de Precisión/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Pirazoles/uso terapéutico , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Mutación/genética , Neoplasia Residual/tratamiento farmacológico , Nitrilos , Pirimidinas , Trasplante de Células Madre , Resultado del TratamientoRESUMEN
Adhesion G protein-coupled receptor F5 (ADGRF5) is involved inthe neoplastic transformation of some cancer types. However, the significance of ADGRF5 expression signature and the impact of signaling pathways mediated by ADGRF5 during neoplastic transformation of the colon and colorectal cancer (CRC) progression has been poorly examined. Using Gene Expression Omnibus and The Cancer Genome Atlas datasets, we showed that ADGRF5 is overexpressed in the colons of patients with CRC. In line, combined analysis of ADGRF5 expression with clinical characterization revealed an increased expression of ADGRF5 in patients with more advanced stages of CRC compared to patients with early stages of CRC. The Spearman correlation analysis documented numerous genes positively and negatively correlated with the expression pattern of ADGRF5 in the colon of patients with CRC. In the colon of CRC patients, the expression signature of ADGRF5 was associated with genes participating in phosphatidylinositol 3-kinase/Akt, focal adhesion, cell adhesion molecules, and ribosome signaling pathways. Of note, ADGRF5 expression correlated with the levels of tumor-infiltrating immune cells in the colon of CRC patients. Moreover, we found that CRC patients with high expression of ADGRF5 had a significantly lower probability of overall survival and disease-free survival. In conclusion, our results support the prognostic value of ADGRF5 and its potent therapeutic implication in CRC.
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Neoplasias Colorrectales , Receptores Acoplados a Proteínas G , Humanos , Adhesión Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Metástasis de la NeoplasiaRESUMEN
Studies of cytotoxicity allow to elucidate the mechanisms by which chemical compounds influence cells and tissues. On the basis of the structural analogy between platinum(II) and palladium(II) complexes, a variety of studies on palladium(II) compounds as potential anticancer drugs have been carried out (1, 2). The cytotoxicity was evaluated by MTT assay. Abilities of trans-palladium(II) complex containing diethyl (pyridin-2-ylmethyl)phosphates as non-leaving ligands (trans-[PdCl2(2-pmOpe 2)]) to induce apoptosis and necrosis in normal lymphocytes, A549 cells and HT29 cell lines were performed by use of fluorochrome staining. The obtained results revealed, that the new trans-palladium(II) complex was more cytotoxic against A549 and HT29 tumor cells than on the normal lymphocytes in vitro. The novel complex induces apoptosis in all tested cells, but in lymphocytes to a lesser degree. The compound tested also induced significant amounts of necrotic cells, which exceeded the level of apoptotic cell fractions. The results demonstrate that the trans-Pd(II) complex showed substantial cytotoxic activity against A549 and HT29 tumor cells and indicate that the new trans-palladium(II) complex effectively inhibited cancer cells growth.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias/patología , Organofosfatos/farmacología , Paladio/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HT29 , Humanos , Linfocitos/efectos de los fármacos , Necrosis , Organofosfatos/toxicidad , Paladio/toxicidadRESUMEN
Cytokines are pleiotropic signaling molecules that execute an essential role in cell-to-cell communication through binding to cell surface receptors. Receptor binding activates intracellular signaling cascades in the target cell that bring about a wide range of cellular responses, including induction of cell proliferation, migration, differentiation, and apoptosis. The Janus kinase and transducers and activators of transcription (JAK/STAT) signaling pathways are activated upon cytokines and growth factors binding with their corresponding receptors. The SOCS family of proteins has emerged as a key regulator of cytokine signaling, and SOCS insufficiency leads to constitutive activation of JAK/STAT signaling and oncogenic transformation. Dysregulation of SOCS expression is linked to various solid tumors with invasive properties. However, the roles of SOCS in hematological malignancies, such as leukemia, are less clear. In this review, we discuss the recent advances pertaining to SOCS dysregulation in leukemia development and progression. We also highlight the roles of specific SOCS in immune cells within the tumor microenvironment and their possible involvement in anti-tumor immunity. Finally, we discuss the epigenetic, genetic, and post-transcriptional modifications of SOCS genes during tumorigenesis, with an emphasis on leukemia.
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Involvement of the Central Nervous System (CNS) in acute leukemia confers poor prognosis and lower overall survival. Existing CNS-directed therapies are associated with a significant risk of short- or long-term toxicities. Leukemic cells can metabolically adapt and survive in the microenvironment of the CNS. The supporting role of the CNS microenvironment in leukemia progression and dissemination has not received sufficient attention. Understanding the mechanism by which leukemic cells survive in the nutrient-poor and oxygen-deprived CNS microenvironment will lead to the development of more specific and less toxic therapies. Here, we review the current literature regarding the roles of metabolic reprogramming in leukemic cell adhesion and survival in the CNS.
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Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.
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ADN Metiltransferasa 3A/genética , Reparación del ADN , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Resistencia a Antineoplásicos/genética , Mutación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Técnicas de Silenciamiento del Gen , Genotipo , Humanos , Leucemia , Ratones , Ratones Transgénicos , Modelos Biológicos , Células Madre Neoplásicas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animales , Línea Celular Tumoral , Quimiotaxis de Leucocito , Niño , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Progresión de la Enfermedad , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Hematopoyesis , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Técnicas In Vitro , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Biogénesis de Organelos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined. RESULTS: V. parahaemolyticus deployed its TTSS1 to induce activation of the JNK, p38 and ERK MAPK in human epithelial cells. VP1680 was identified as the TTSS1 effector protein responsible for MAPK activation in Caco-2 cells and the activation of JNK and ERK by this protein was important in induction of host cell death. V. parahaemolyticus actively induced IL-8 secretion in a response mediated by TTSS1. A role for VP1680 and for the ERK signalling pathway in the stimulation of IL-8 production in epithelial cells by V. parahaemolyticus was established. Interestingly, TTSS2 inhibited IL-8 mRNA transcription at early stages of interaction between the bacterium and the cell. CONCLUSIONS: This study demonstrated that V. parahaemolyticus activates the three major MAPK signalling pathways in intestinal epithelial cells in a TTSS1-dependent manner that involves the TTSS1 effector VP1680. Furthermore VP1680 and JNK and ERK activation were needed for maximal cytotoxicity of the bacterium. It was shown that V. parahaemolyticus is a strong inducer of IL-8 secretion and that induction reflects a balance between the effects of TTSS1 and TTSS2. Increases in IL-8 secretion were mediated by TTSS1 and VP1680, and augmented by ERK activation. These results shed light on the mechanisms of bacterial pathogenesis mediated by TTSS and suggest significant roles for MAPK signalling during infection with V. parahaemolyticus.
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Interacciones Huésped-Patógeno , Proteínas de Transporte de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidad , Factores de Virulencia/metabolismo , Muerte Celular , Línea Celular , Células Epiteliales/microbiología , Humanos , Interleucina-8/metabolismo , Transporte de Proteínas , Regulación hacia ArribaRESUMEN
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.
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Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Indazoles/farmacología , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Sulfonamidas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
For children and young adults with T-lineage acute lymphoblastic leukemia (T-ALL), event free survival following relapse is < 10%. We recently showed that rearrangements of the mixed lineage leukemia gene (KMT2A-R) are associated with induction failure and an inferior survival in T-ALL. Because there are currently no molecular features that inform treatment strategies in T-ALL, we hypothesized that transcriptional alterations related to KMT2A-R and MLLT10-R T-ALL could identify biologically relevant genes and signaling pathways for the development of targeted therapies for these groups of patients. We analyzed microarray data from a retrospective cohort of 100 T-ALL patients to identify novel targets for KMT2A (n = 12) or MLLT10 (n = 9) chimeras. We identified 330 probe sets that could discriminate between these groups, including novel targets, like RUNX2, TCF4 or MYO6. The results were further validated in two independent data sets and the functional networks were analyzed to identify pathways that may be of pathogenic or therapeutic relevance.
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Cancer cells often accumulate spontaneous and treatment-induced DNA damage i.e. potentially lethal DNA double strand breaks (DSBs). Targeting DSB repair mechanisms with specific inhibitors could potentially sensitize cancer cells to the toxic effect of DSBs. Current treatment for glioblastoma includes tumor resection followed by radiotherapy and/or temozolomide (TMZ) - an alkylating agent inducing DNA damage. We hypothesize that combination of PARP inhibitor (PARPi) with TMZ in glioblastoma cells displaying downregulation of DSB repair genes could trigger synthetic lethality. In our study, we observed that PARP inhibitor (BMN673) was able to specifically sensitize DNA ligase 4 (LIG4)-deprived glioblastoma cells to TMZ while normal astrocytes were not affected. LIG4 downregulation resulting in low effectiveness of DNA-PK-mediated non-homologous end-joining (D-NHEJ), which in combination with BMN673 and TMZ resulted in accumulation of lethal DSBs and specific eradication of glioblastoma cells. Restoration of the LIG4 expression caused loss of sensitivity to BMN673+TMZ. In conclusion, PARP inhibitor combined with DNA damage inducing agents can be utilized in patients with glioblastoma displaying defects in D-NHEJ.
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Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.
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Antineoplásicos/farmacología , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos/métodos , Citometría de Flujo/métodos , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
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
The dynamic development of metal-containing anticancer drugs has started since the discovery of cis-diamminedichloroplatinum(II). For many years it was believed that trans platinum(II) compounds were non-active as antitumour agents because trans-diamminedichloroplatinum is biologically inactive although it binds to DNA and also forms monoadducts and cross-links. In the present work the ability of a novel platinum(II) compound trans-[PtCl(2)(4-pmOpe)(2)] to induce DNA damage in human non-small cell lung cancer cells A549 was examined using the alkaline comet assay. The obtained results revealed that the novel trans platinum(II) complex induced DNA strand breaks, which were effectively repaired during 2h of post-incubation, and cross-links which remained unrepaired under these test conditions. Apart from that, the modified comet assay with incubation with proteinase K was used to verify the ability of trans-[PtCl(2)(4-pmOpe)(2)] and cis-DDP to form DNA-protein cross-links. It has been proved that only trans-[PtCl(2)(4-pmOpe)(2)] complex exhibits the ability to induce DNA-protein cross-links. The results suggest a different mechanism of action of this compound in comparison to cis-DDP. It seems that trans geometry and the presence of two diethyl (pyridin-4-ylmethyl)phosphates as non-leaving ligands can determine dissimilar properties of the adducts formed on DNA and the different mechanism of action of trans-[PtCl(2)(4-pmOpe)(2)] and in consequence the efficacy in killing cancer cells.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , ADN de Neoplasias/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Organofosfatos/farmacología , Compuestos Organoplatinos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Reparación del ADN , ADN de Neoplasias/genética , Humanos , Neoplasias Pulmonares/genética , Azul de Tripano/químicaRESUMEN
The pre-B cell receptor (pre-BCR) is an immature form of the BCR critical for early B lymphocyte development. It is composed of the membrane-bound immunoglobulin (Ig) heavy chain, surrogate light chain components, and the signaling subunits Igα and Igß. We developed monovalent quantum dot (QD)-labeled probes specific for Igß to study the behavior of pre-BCRs engaged in autonomous, ligand-independent signaling in live B cells. Single-particle tracking revealed that QD-labeled pre-BCRs engaged in transient, but frequent, homotypic interactions. Receptor motion was correlated at short separation distances, consistent with the formation of dimers and higher-order oligomers. Repeated encounters between diffusing pre-BCRs appeared to reflect transient co-confinement in plasma membrane domains. In human B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, we showed that frequent, short-lived, homotypic pre-BCR interactions stimulated survival signals, including expression of BCL6, which encodes a transcriptional repressor. These survival signals were blocked by inhibitory monovalent antigen-binding antibody fragments (Fabs) specific for the surrogate light chain components of the pre-BCR or by inhibitors of the tyrosine kinases Lyn and Syk. For comparison, we evaluated pre-BCR aggregation mediated by dimeric galectin-1, which has binding sites for carbohydrate and for the surrogate light chain λ5 component. Galectin-1 binding resulted in the formation of large, highly immobile pre-BCR aggregates, which was partially relieved by the addition of lactose to prevent the cross-linking of galectin-BCR complexes to other glycosylated membrane components. Analysis of the pre-BCR and its signaling partners suggested that they could be potential targets for combination therapy in BCP-ALL.
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Receptores de Células Precursoras de Linfocitos B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Células Precursoras de Linfocitos B/inmunología , Multimerización de Proteína/inmunología , Transducción de Señal/inmunología , Línea Celular Tumoral , Supervivencia Celular/inmunología , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Precursoras de Linfocitos B/patología , Proteínas Proto-Oncogénicas c-bcl-6/inmunología , Quinasa Syk/inmunología , Familia-src Quinasas/inmunologíaRESUMEN
Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3'-5'-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing.