RESUMO
Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Neoplasias Pulmonares/genética , Proliferação de Células/genética , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. ⢠Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. ⢠ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. ⢠The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. ⢠The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Quinases , Humanos , Proteínas Quinases/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Doxorrubicina/farmacologia , Dano ao DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Chronic myelomonocytic leukemia (CMML) is a hematological neoplasm characterized by monocytosis, splenomegaly, thrombocytopenia, and anemia. Moreover, it is associated with SRSF2 mutations and, rarely, with CSF3R variants. We present the case of an 84-year-old patient with persistent anemia and monocytosis. Due to the presence of dysmorphic granulocytes, monocyte atypia, and myeloid precursors in the peripheral blood cells, the patient was subjected to a bone marrow examination. The diagnosis was consistent with CMML type 2. The Hemocoagulative test showed an increase in fibrinolysis markers. Next-generation targeted sequencing showed TET2 and SRSF2 mutations, along with an unexpected CSF3R germline missense variant, rarely encountered in CMML. The patient started Azacitidine treatment and achieved normal hemostatic process values. In conclusion, we identified a heterozygous germline mutation that, together with TET2 and SRSF2 variants, was responsible for the hemorrhagic manifestation.
Assuntos
Anemia , Leucemia Mielomonocítica Crônica , Humanos , Idoso de 80 Anos ou mais , Leucemia Mielomonocítica Crônica/complicações , Leucemia Mielomonocítica Crônica/genética , Mutação em Linhagem Germinativa , Predisposição Genética para Doença , Mutação , Células Germinativas , Receptores de Fator Estimulador de Colônias/genéticaRESUMO
The plasmacytoma variant translocation 1 (PVT1) is a long non-coding RNA gene involved in human disease, mainly in cancer onset/progression. Although widely analysed, its biological roles need to be further clarified. Notably, functional studies on PVT1 are complicated by the occurrence of multiple transcript variants, linear and circular, which generate technical issues in the experimental procedures used to evaluate its impact on human disease. Among the many PVT1 transcripts, the linear PVT1 (lncPVT1) and the circular hsa_circ_0001821 (circPVT1) are frequently reported to perform similar pathologic and pro-tumorigenic functions when overexpressed. The stimulation of cell proliferation, invasion and drug resistance, cell metabolism regulation, and apoptosis inhibition is controlled through multiple targets, including MYC, p21, STAT3, vimentin, cadherins, the PI3K/AKT, HK2, BCL2, and CASP3. However, some of this evidence may originate from an incorrect evaluation of these transcripts as two separate molecules, as they share the lncPVT1 exon-2 sequence. We here summarise lncPVT1/circPVT1 functions by mainly focusing on shared pathways, pointing out the potential bias that may exist when the biological role of each transcript is analysed. These considerations may improve the knowledge about lncPVT1/circPVT1 and their specific targets, which deserve further studies due to their diagnostic, prognostic, and therapeutic potential.
Assuntos
MicroRNAs , Neoplasias , RNA Longo não Codificante , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , MicroRNAs/genética , Neoplasias/genética , Neoplasias/patologia , Fosfatidilinositol 3-Quinases , Prognóstico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Non coding RNAs (ncRNAs) have emerged as regulators of human carcinogenesis by affecting the expression of key tumor suppressor genes and oncogenes. They are divided into short and long ncRNAs, according to their length. Circular RNAs (circRNAs) are included in the second group and were recently discovered as being originated by back-splicing, joining either single or multiple exons, or exons with retained introns. The human Plasmacytoma Variant Translocation 1 (PVT1) gene maps on the long arm of chromosome 8 (8q24) and encodes for 52 ncRNAs variants, including 26 linear and 26 circular isoforms, and 6 microRNAs. PVT1 genomic locus is 54 Kb downstream to MYC and several interactions have been described among these two genes, including a feedback regulatory mechanism. MYC-independent functions of PVT1/circPVT1 have been also reported, especially in the regulation of immune responses. We here review and discuss the role of both PVT1 and circPVT1 in the hematopoietic system. No information is currently available concerning their transforming ability in hematopoietic cells. However, present literature supports their cooperation with a more aggressive and/or undifferentiated cell phenotype, thus contributing to cancer progression. PVT1/circPVT1 upregulation through genomic amplification or rearrangements and/or increased transcription, provides a proliferative advantage to malignant cells in acute myeloid leukemia, acute promyelocytic leukemia, Burkitt lymphoma, multiple myeloma (linear PVT1) and acute lymphoblastic leukemia (circPVT1). In addition, PVT1 and circPVT1 regulate immune responses: the overexpression of the linear form in myeloid derived suppressor cells induced immune tolerance in preclinical tumor models and circPVT1 showed immunosuppressive properties in myeloid and lymphoid cell subsets. Overall, these recent data on PVT1 and circPVT1 functions in hematological malignancies and immune responses reflect two faces of the same coin: involvement in cancer progression by promoting a more aggressive phenotype of malignant cells and negative regulation of the immune system as a novel potential therapy-resistance mechanism.
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Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Imunidade/imunologia , RNA Circular/genética , RNA Longo não Codificante/genética , Neoplasias Hematológicas/patologia , HumanosRESUMO
This study was conducted to evaluate the expression of fibrinogen receptors on platelets of Philadelphia-negative chronic myeloproliferative neoplasm (MPN) patients. We collected blood samples from 40 consecutive MPN patients and healthy volunteers. We performed flow cytometry analysis of P-selectin expression and integrin beta-3, activation of glycoprotein (GP) IIb/IIIa and fibrinogen receptor exposure (PAC-1 binding). Surprisingly, we found a very low PAC-1 binding capacity in MPN patients; however, the expression of PAC-1 was almost completely recovered with aspirin intake. We hypothesize that the hypercoagulable states observed in MPN patients could depend on a primarily plasma-driven impairment of fibrin turnover and thrombin generation.
Assuntos
Aspirina/uso terapêutico , Fibrinogênio/uso terapêutico , Transtornos Mieloproliferativos/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspirina/farmacologia , Plaquetas , Doença Crônica , Fibrinogênio/farmacologia , Humanos , Pessoa de Meia-IdadeRESUMO
The incidence of hepatocellular carcinoma deriving from metabolic dysfunctions has increased in the last years. Sirtuin- (SIRT-3), phospho-mammalian target of rapamycin (p-mTOR) and hypoxia-inducible factor- (HIF-1α) are involved in metabolism and cancer. However, their role in hepatocellular carcinoma (HCC) metabolism, drug resistance and progression remains unclear. This study aimed to better clarify the biological and clinical function of these markers in HCC patients, in relation to the presence of metabolic alterations, metformin therapy and clinical outcome. A total of 70 HCC patients were enrolled: 48 and 22 of whom were in early stage and advanced stage, respectively. The expression levels of the three markers were assessed by immunohistochemistry and summarized using descriptive statistics. SIRT-3 expression was higher in diabetic than non-diabetic patients, and in metformin-treated than insulin-treated patients. Interestingly, p-mTOR was higher in patients with metabolic syndrome than those with different etiology, and, similar to SIRT-3, in metformin-treated than insulin-treated patients. Moreover, our results describe a slight, albeit not significant, benefit of high SIRT-3 and a significant benefit of high nuclear HIF-1α expression in early-stage patients, whereas high levels of p-mTOR correlated with worse prognosis in advanced-stage patients. Our study highlighted the involvement of SIRT-3 and p-mTOR in metabolic dysfunctions that occur in HCC patients, and suggested SIRT-3 and HIF-1α as predictors of prognosis in early-stage HCC patients, and p-mTOR as target for the treatment of advanced-stage HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/sangue , Neoplasias Hepáticas/metabolismo , Sirtuína 3/sangue , Serina-Treonina Quinases TOR/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/complicações , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/uso terapêutico , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/complicações , Masculino , Metformina/administração & dosagem , Metformina/uso terapêutico , Pessoa de Meia-IdadeRESUMO
BACKGROUND: In chronic lymphocytic leukemia (CLL) disease onset and progression are influenced by the behavior of specific CD4+ T cell subsets, such as T regulatory cells (Tregs). Here, we focused on the phenotypic and functional characterization of Tregs in CLL patients to improve our understanding of the putative mechanism by which these cells combine immunosuppressive and effector-like properties. METHODS: Peripheral blood mononuclear cells were isolated from newly diagnosed CLL patients (n = 25) and healthy volunteers (n = 25). The phenotypic and functional characterization of Tregs and their subsets was assessed by flow cytometry. In vitro analysis of TH1, TH2, TH17 and Tregs cytokines was evaluated by IFN-γ, IL-4, IL-17A and IL-10 secretion assays. The transcriptional profiling of 84 genes panel was evaluated by RT2 Profiler PCR Array. Statistical analysis was carried out using exact non parametric Mann-Whitney U test. RESULTS: In all CLL samples, we found a significant increase in the frequency of IL-10-secreting Tregs and Tregs subsets, a significant rise of TH2 IL-4+ and TH17 IL-17A+ cells, and a higher percentage of IFN-γ/IL-10 and IL-4/IL-10 double-releasing CD4+ T cells. In addition, we also observed the up-regulation of innate immunity genes and the down-regulation of adaptive immunity ones. CONCLUSIONS: Our data show that Tregs switch towards an effector-like phenotype in CLL patients. This multifaceted behavior is accompanied by an altered cytokine profiling and transcriptional program of immune genes, leading to a dysfunction in immune response in the peripheral blood environment of CLL patients.
Assuntos
Imunossupressores/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos T Reguladores/imunologia , Imunidade Adaptativa , Idoso , Idoso de 80 Anos ou mais , Candida albicans/fisiologia , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Imunidade Inata , Interferon gama/metabolismo , Interleucina-23/sangue , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/genética , Subpopulações de Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Recently, interest has been increasing for human decellularized matrices, due to their ability to reduce numerous side effects related to hernia repair. To date, only animal studies investigated the biological interaction post-implant of human decellularized matrices for soft tissue repair. Therefore, the aim of this study was to evaluate the morphological response one year post implant of human decellularized matrix, through morphological analysis of human biopsies. The histological and ultrastructural results revealed a perfect cellular repopulation and neoangiogenesis, with minimal inflammatory response and a well-organized collagen matrix. The results have indicated that this scaffold can be an effective treatment for hernia.
Assuntos
Derme Acelular , Herniorrafia/métodos , Matriz Extracelular/ultraestrutura , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-IdadeRESUMO
Although bone metastases represent a major challenge in the natural history of breast cancer (BC), the complex interactions involved have hindered the development of robust in vitro models. The aim of this work is the development of a preclinical model of cancer and bone stromal cells to mimic the bone microenvironment. We studied the effects on osteoclastogenesis of BC cells and Mesenchymal stem cells (MSC) cultured alone or in combination. We also analyzed: (a) whether the blockade of the Epithelial Growth Factor Receptor (EGFR) pathway modified their influence on monocytes towards differentiation, and (b) the efficacy of bone-targeted therapy on osteoclasts. We evaluated the osteoclastogenesis modulation of human peripheral blood monocytes (PBMC) indirectly induced by the conditioned medium (CM) of the human BC cell line SCP2, cultured singly or with MSC. Osteoclastogenesis was evaluated by TRAP analysis. The effect of the EGFR blockade was assessed by treating the cells with gefitinib, and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western Blot (WB). We observed that SCP2 co-cultured with MSC increased the differentiation of PBMC. This effect was underpinned upon pre-treatment of the co-culture with gefitinib. Co-culture of SCP2 with MSC increased the expression of both the bone-related marker Receptor Activator of Nuclear Factor κB (RANK) and EGFR in BC cells. These upregulations were not affected by the EGFR blockade. The effects of the CM obtained by the cells treated with gefitinib in combination with the treatment of the preosteoclasts with the bone-targeted agents and everolimus enhanced the inhibition of the osteoclastogenesis. Finally, we developed a fully human co-culture system of BC cells and bone progenitor cells. We observed that the interaction of MSC with cancer cells induced in the latter molecular changes and a higher power of inducing osteoclastogenesis. We found that blocking EGFR signaling could be an efficacious strategy for breaking the interactions between cancer and bone cells in order to inhibit bone metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Comunicação Celular/efeitos dos fármacos , Receptores ErbB , Células-Tronco Mesenquimais/metabolismo , Monócitos/metabolismo , Proteínas de Neoplasias , Osteoclastos/metabolismo , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Humanos , Células-Tronco Mesenquimais/patologia , Monócitos/patologia , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Osteoclastos/patologiaRESUMO
Shape memory alloys based on NiTi have found their main applications in manufacturing of new biomedical devices mainly in surgery tools, stents and orthopedics. Porous NiTi can exhibit an engineering elastic modulus comparable to that of cortical bone (12-17 GPa). This condition, combined with proper pore size, allows good osteointegration. Open cells porous NiTi was produced by self propagating high temperature synthesis (SHS), starting from Ni and Ti mixed powders. The main NiTi phase is formed during SHS together with other Ni-Ti compounds. The biocompatibility of such material was investigated by single culture experiment and ionic release on small specimen. In particular, NiTi and porous NiTi were evaluated together with elemental Ti and Ni reference metals and the two intermetallic TiNi3, Ti2Ni phases. This approach permitted to clearly identify the influence of secondary phases in porous NiTi materials and relation with Ni-ion release. The results indicated, apart the well-known high toxicity of Ni, also toxicity of TiNi3, whilst phases with higher Ti content showed high biocompatibility. A slightly reduced biocompatibility of porous NiTi was ascribed to combined effect of TiNi3 presence and topography that requires higher effort for the cells to adapt to the surface.
Assuntos
Ligas/síntese química , Materiais Biocompatíveis/química , Temperatura Alta , Níquel/química , Polimerização , Titânio/química , Ligas/química , Ligas/farmacologia , Células Cultivadas , Humanos , Teste de Materiais , Níquel/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Porosidade , Pós , Propriedades de Superfície , Titânio/farmacologiaRESUMO
Extracellular vesicles (EVs) act as molecular mediators in the tumor microenvironment, by shuttling information contained within malignant cells and functioning as regulators of the immune system. Circular (circ)RNAs are characterized by a closed loop-like structure that makes them more stable in the extracellular milieu and suitable to be packaged inside EVs. circPVT1 (hsa_circ_0001821) showed an oncogenic role in several cancer types and immunosuppressive properties in myeloid and lymphoid cell subsets. In this study, we characterized EVs from acute myeloid leukemia (AML) patients in terms of size, concentrations, surface markers and circPVT1 cargo. We showed that circPVT1 is overexpressed by primary blast cells from newly-diagnosed AML patients compared with hematopoietic stem-progenitor cells and is released as cell-free RNA in the plasma. We isolated EVs from the plasma of AML patients and healthy subjects by size exclusion chromatography and characterized them by nanoparticle tracking analysis. EVs from patients' plasma are larger compared with those from healthy subjects and their surface profile is characterized by higher levels of the leukemic cell markers CD133, CD105, CD49e and other immune-related epitopes, with differences according to AML molecular profile. Moreover, digital PCR analysis revealed that circPVT1 is more abundant inside EVs from the plasma of AML patients compared with healthy subjects. Our findings provide new insights on the features and content of AML EVs and suggest a role of circPVT1 in the crosstalk between AML cells and the tumor microenvironment.
Assuntos
Vesículas Extracelulares , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/metabolismo , Vesículas Extracelulares/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Comunicação Celular , Microambiente Tumoral/genéticaRESUMO
The members of the Poly(ADP-ribose) polymerase (PARP) superfamily are involved in several biological processes and, in particular, in the DNA damage response (DDR). The most studied members, PARP1, PARP2 and PARP3, act as sensors of DNA damages, in order to activate different intracellular repair pathways, including single-strand repair, homologous recombination, conventional and alternative non-homologous end joining. This review recapitulates the functional role of PARPs in the DDR pathways, also in relationship with the cell cycle phases, which drives our knowledge of the mechanisms of action of PARP inhibitors (PARPi), encompassing inhibition of single-strand breaks and base excision repair, PARP trapping and sensitization to antileukemia immune responses. Several studies have demonstrated a preclinical activity of the current available PARPi, olaparib, rucaparib, niraparib, veliparib and talazoparib, as single agent and/or in combination with cytotoxic, hypomethylating or targeted drugs in acute leukemia, thus encouraging the development of clinical trials. We here summarize the most recent preclinical and clinical findings and discuss the synthetic lethal interactions of PARPi in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite the low frequency of genomic alterations of PARP and other DDR-related genes in acute leukemia, selective vulnerabilities have been reported in several disease subgroups, along with a "BRCAness phenotype." AML carrying the RUNX1-RUNX1T1 or PML-RARA fusion genes or mutations in signaling genes (FLT3-ITD in combination with TET2 or TET2 and DNMT3A deficiency), cohesin complex members (STAG2), TP53 and BCOR as co-occurring lesions, IDH1/2 and ALL cases expressing the TCF3-HLF chimera or TET1 was highly sensitive to PARPi in preclinical studies. These data, along with the warning coming from the observation of cases of therapy-related myeloid malignancies among patients receiving PARPi for solid tumors treatment, indicate that PARPi represents a promising strategy in a personalized medicine setting. The characterization of the clonal and subclonal genetic background and of the DDR functionality is crucial to select acute leukemia patients that will likely benefit of PARPi-based therapeutic regimens.
Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Animais , Antineoplásicos/farmacologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Terapia de Alvo Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Hepatitis B- and C-virus (HBV and HCV) infections contribute to hepatocellular carcinoma (HCC) development through several different mechanisms. In addition to a diverse molecular background, HCC subtypes also show differences in their metabolic profiles, suggesting that prevention and treatment might require the integration of multiple different approaches. We here analyzed the response of two HCC cell lines representative of different virus-related etiology, namely Hep3B (HBV+) and HUH7 (permissive to HCV replication) to sorafenib treatment. Our findings suggest that virus-related specificities influence treatment response in HCC, along with molecular, metabolic and microenvironmental factors. These differences have to be taken into account in the design of future clinical trial aimed to improve HCC patients' outcome.
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In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.
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Adrenomedullin (ADM) is a hypotensive and vasodilator peptide belonging to the calcitonin gene-related peptide family. It is secreted in vitro by endothelial cells and vascular smooth muscle cells, and is significantly upregulated by a number of stimuli. Moreover, ADM participates in the regulation of hematopoietic compartment, solid tumors and leukemias, such as acute myeloid leukemia (AML). To better characterize ADM involvement in AML pathogenesis, we investigated its expression during human hematopoiesis and in leukemic subsets, based on a morphological, cytogenetic and molecular characterization and in T cells from AML patients. In hematopoietic stem/progenitor cells and T lymphocytes from healthy subjects, ADM transcript was barely detectable. It was expressed at low levels by megakaryocytes and erythroblasts, while higher levels were measured in neutrophils, monocytes and plasma cells. Moreover, cells populating the hematopoietic niche, including mesenchymal stem cells, showed to express ADM. ADM was overexpressed in AML cells versus normal CD34+ cells and in the subset of leukemia compared with hematopoietic stem cells. In parallel, we detected a significant variation of ADM expression among cytogenetic subgroups, measuring the highest levels in inv(16)/t(16;16) or complex karyotype AML. According to the mutational status of AML-related genes, the analysis showed a lower expression of ADM in FLT3-ITD, NPM1-mutated AML and FLT3-ITD/NPM1-mutated cases compared with wild-type ones. Moreover, ADM expression had a negative impact on overall survival within the favorable risk class, while showing a potential positive impact within the subgroup receiving a not-intensive treatment. The expression of 135 genes involved in leukemogenesis, regulation of cell proliferation, ferroptosis, protection from apoptosis, HIF-1α signaling, JAK-STAT pathway, immune and inflammatory responses was correlated with ADM levels in the bone marrow cells of at least two AML cohorts. Moreover, ADM was upregulated in CD4+ T and CD8+ T cells from AML patients compared with healthy controls and some ADM co-expressed genes participate in a signature of immune tolerance that characterizes CD4+ T cells from leukemic patients. Overall, our study shows that ADM expression in AML associates with a stem cell phenotype, inflammatory signatures and genes related to immunosuppression, all factors that contribute to therapy resistance and disease relapse.
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Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Dano ao DNA/genética , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatina/genética , Feminino , Genômica/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Prognóstico , Adulto Jovem , CoesinasRESUMO
Tumor protein p53 is a key regulator of several cellular pathways, including DNA repair, cell cycle and angiogenesis. Kevetrin exhibits p53dependent as well asindependent activity in solid tumors, while its effects on leukemic cells remain unknown. The aim of the present study was to analyze the response of acute myeloid leukemia (AML) cell lines (TP53 wildtype: OCIAML3 and MOLM13; and TP53mutant: KASUMI1 and NOMO1) to kevetrin at a concentration range of 85340 µM. The cellular and molecular effects of the treatment were analyzed in terms of cell growth, viability [Annexin Vpropidium iodide (PI) staining] and cell cycle alterations (PI staining). Gene expression profiling, western blotting and immunofluorescence were performed to elucidate the pathways underlying kevetrin activity. Pulsed exposure exerted no effect on the wildtype cells, but was effective on mutant cells. After continuous treatment, significant cell growth arrest and apoptosis were observed in all cell lines, with TP53mutant models displaying a higher sensitivity and p53 induction. Kevetrin also displayed efficacy against TP53 wildtype and mutant primary AML, with a preferential cytotoxic activity against blast cells. Gene expression profiling revealed a common core transcriptional program altered by drug exposure and the downregulation of glycolysis, DNA repair and unfolded protein response signatures. These findings suggest that kevetrin may be a promising therapeutic option for patients with both wildtype and TP53mutant AML.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Idoso , Idoso de 80 Anos ou mais , Anexina A5/genética , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Mutação/efeitos dos fármacos , Cultura Primária de CélulasRESUMO
INTRODUCTION: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. METHODS: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. RESULTS: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. CONCLUSION: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.
RESUMO
Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.