RESUMO
We report on the antileukemic activity of homoharringtonine (HHT) in T-ALL. We showed that HHT inhibited NOTCH/MYC pathway and induced a significantly longer survival in T-ALL mouse and patient-derived xenograft models, therefore supporting HHT as a promising agent for T-ALL.
RESUMO
ABSTRACT: The T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.
Assuntos
Proliferação de Células , Leucemia Linfocítica Crônica de Células B , Proteínas com Domínio T , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Humanos , Camundongos , Linfócitos B/patologia , Linfócitos B/metabolismo , Linfócitos B/imunologia , Camundongos Knockout , Regulação Leucêmica da Expressão Gênica , NF-kappa B/metabolismoRESUMO
Neddylation is a sequential enzyme-based process which regulates the function of E3 Cullin-RING ligase (CRL) and thus degradation of substrate proteins. Here we show that CD8+ T cells are a direct target for therapeutically relevant anti-lymphoma activity of pevonedistat, a Nedd8-activating enzyme (NAE) inhibitor. Pevonedistat-treated patient-derived CD8+ T cells upregulated TNFα and IFNγ and exhibited enhanced cytotoxicity. Pevonedistat induced CD8+ T-cell inflamed microenvironment and delayed tumor progression in A20 syngeneic lymphoma model. This anti-tumor effect lessened when CD8+ T cells lost the ability to engage tumors through MHC class I interactions, achieved either through CD8+ T-cell depletion or genetic knockout of B2M. Meanwhile, loss of UBE2M in tumor did not alter efficacy of pevonedistat. Concurrent blockade of NAE and PD-1 led to enhanced tumor immune infiltration, T-cell activation and chemokine expression and synergistically restricted tumor growth. shRNA-mediated knockdown of HIF-1α, a CRL substrate, abrogated the in vitro effects of pevonedistat, suggesting that NAE inhibition modulates T-cell function in HIF-1α-dependent manner. scRNA-Seq-based clinical analyses in lymphoma patients receiving pevonedistat therapy demonstrated upregulation of interferon response signatures in immune cells. Thus, targeting NAE enhances the inflammatory T-cell state, providing rationale for checkpoint blockade-based combination therapy.
Assuntos
Antineoplásicos , Linfoma , Humanos , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Linfoma/tratamento farmacológico , Ciclopentanos/farmacologia , Ciclopentanos/uso terapêutico , Proteína NEDD8 , Microambiente Tumoral , Enzimas de Conjugação de UbiquitinaRESUMO
Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS.
Assuntos
Rabdomiossarcoma , Humanos , Carcinogênese/genética , Linhagem Celular Tumoral , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Fatores de Transcrição , Transformação Celular Neoplásica , Diferenciação CelularRESUMO
Hematopoietic stem (HSC) and progenitor (HPC) cell fate is governed by intrinsic and extrinsic parameters. We examined the impact of hematopoietic niche elements on HSC and HPC function by analyzing the combined effect of osteoblasts (OBs) and stromal cells (SCs) on Lineage(-)Sca-1(+)CD117(+) (LSK) cells. CFU expansion and marrow repopulating potential of cultured Lineage(-)Sca-1(+)CD117(+) cells were significantly higher in OB compared with SC cultures, thus corroborating the importance of OBs in the competence of the hematopoietic niche. OB-mediated enhancement of HSC and HPC function was reduced in cocultures of OBs and SCs, suggesting that SCs suppressed the OB-mediated hematopoiesis-enhancing activity. Although the suppressive effect of SC was mediated by adipocytes, probably through up-regulation of neuropilin-1, the OB-mediated enhanced hematopoiesis function was elaborated through Notch signaling. Expression of Notch 2, Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, and Deltex was increased in OB cultures and suppressed in SC and OB/SC cultures. Phenotypic fractionation of OBs did not segregate the hematopoiesis-enhancing activity but demonstrated that this function is common to OBs from different anatomic sites. These data illustrate that OBs promote in vitro maintenance of hematopoietic functions, including repopulating potential by up-regulating Notch-mediated signaling between HSCs and OBs.
Assuntos
Medula Óssea/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Osteoblastos/citologia , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Comunicação Celular , Diferenciação Celular/fisiologia , Proliferação de Células , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
We report here on a novel pro-leukemogenic role of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) that interferes with microRNAs (miRNAs) biogenesis in acute myeloid leukemia (AML) blasts. We showed that FLT3-ITD interferes with the canonical biogenesis of intron-hosted miRNAs such as miR-126, by phosphorylating SPRED1 protein and inhibiting the "gatekeeper" Exportin 5 (XPO5)/RAN-GTP complex that regulates the nucleus-to-cytoplasm transport of pre-miRNAs for completion of maturation into mature miRNAs. Of note, despite the blockage of "canonical" miRNA biogenesis, miR-155 remains upregulated in FLT3-ITD+ AML blasts, suggesting activation of alternative mechanisms of miRNA biogenesis that circumvent the XPO5/RAN-GTP blockage. MiR-155, a BIC-155 long noncoding (lnc) RNA-hosted oncogenic miRNA, has previously been implicated in FLT3-ITD+ AML blast hyperproliferation. We showed that FLT3-ITD upregulates miR-155 by inhibiting DDX3X, a protein implicated in the splicing of lncRNAs, via p-AKT. Inhibition of DDX3X increases unspliced BIC-155 that is then shuttled by NXF1 from the nucleus to the cytoplasm, where it is processed into mature miR-155 by cytoplasmic DROSHA, thereby bypassing the XPO5/RAN-GTP blockage via "non-canonical" mechanisms of miRNA biogenesis.
Assuntos
Citoplasma/metabolismo , Leucemia Mieloide Aguda/patologia , MicroRNAs/biossíntese , Ribonuclease III/metabolismo , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Ribonuclease III/genética , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co-culture with Lin(-)Sca1(+)c-kit(+) (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin(-)Sca1(+) cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB.
Assuntos
Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Células-Tronco Hematopoéticas/fisiologia , Osteoblastos/fisiologia , Animais , Linhagem da Célula , Técnicas de Cocultura , Células-Tronco Hematopoéticas/citologia , Camundongos , Osteoblastos/químicaAssuntos
Tirosina Quinase da Agamaglobulinemia , Humanos , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Linfócitos B/metabolismo , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patologia , Linfoma de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , ProteóliseRESUMO
Osteoblasts (OBs) exert a prominent regulatory effect on hematopoietic stem cells (HSCs). We evaluated the difference in hematopoietic expansion and function in response to co-culture with OBs at various stages of development. Murine calvarial OBs were seeded directly (fresh) or cultured for 1, 2, or 3 weeks prior to seeding with 1000 Lin-Sca1 + cKit+ (LSK) cells for 1 week. Significant increases in the following hematopoietic parameters were detected when comparing co-cultures of fresh OBs to co-cultures containing OBs cultured for 1, 2, or 3 weeks: total hematopoietic cell number (up to a 3.4-fold increase), total colony forming unit (CFU) number in LSK progeny (up to an 18.1-fold increase), and percentage of Lin-Sca1+ cells (up to a 31.8-fold increase). Importantly, these studies were corroborated by in vivo reconstitution studies in which LSK cells maintained in fresh OB co-cultures supported a significantly higher level of chimerism than cells maintained in co-cultures containing 3-week OBs. Characterization of OBs cultured for 1, 2, or 3 weeks with real-time PCR and functional mineralization assays showed that OB maturation increased with culture duration but was not affected by the presence of LSK cells in culture. Linear regression analyses of multiple parameters measured in these studies show that fresh, most likely more immature OBs better promote hematopoietic expansion and function than cultured, presumably more mature OBs and suggest that the hematopoiesis-enhancing activity is mediated by cells present in fresh OB cultures de novo.