RESUMEN
Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Factor 4F Eucariótico de Iniciación/genética , Prohibitinas , Genes myc , ARN Mensajero/genéticaRESUMEN
Chronic lymphocytic leukemia (CLL) cells are highly dependent on interactions with the immunosuppressive tumor microenvironment (TME) for survival and proliferation. In the search for novel treatments, pro-inflammatory cytokines have emerged as candidates to reactivate the immune system. Among those, interleukin 27 (IL-27) has recently gained attention, but its effects differ among malignancies. Here, we utilized the Eµ-TCL1 and EBI3 knock-out mouse models as well as clinical samples from patients to investigate the role of IL-27 in CLL. Characterization of murine leukemic spleens revealed that the absence of IL-27 leads to enhanced CLL development and a more immunosuppressive TME in transgenic mice. Gene-profiling of T-cell subsets from EBI3 knock-out highlighted transcriptional changes in the CD8+ T-cell population associated with T-cell activation, proliferation, and cytotoxicity. We also observed an increased anti-tumor activity of CD8+ T cells in the presence of IL-27 ex vivo with murine and clinical samples. Notably, IL-27 treatment led to the reactivation of autologous T cells from CLL patients. Finally, we detected a decrease in IL-27 serum levels during CLL development in both pre-clinical and patient samples. Altogether, we demonstrated that IL-27 has a strong anti-tumorigenic role in CLL and postulate this cytokine as a promising treatment or adjuvant for this malignancy.
Asunto(s)
Interleucina-27 , Leucemia Linfocítica Crónica de Células B , Animales , Humanos , Ratones , Linfocitos T CD8-positivos , Citocinas , Inmunosupresores , Leucemia Linfocítica Crónica de Células B/patología , Ratones Transgénicos , Subgrupos de Linfocitos T/patología , Microambiente TumoralRESUMEN
While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.
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Autofagia/fisiología , Quimiocina CCL5/metabolismo , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Melanoma/metabolismo , Melanoma/patología , Animales , Beclina-1/metabolismo , Línea Celular Tumoral , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BL , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismoAsunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Sulfonamidas/farmacología , Serina-Treonina Quinasas TORRESUMEN
In this issue of Blood, Yeomans et al identify MYC as an important target for translational regulation in chronic lymphocytic leukemia (CLL) cells after B-cell receptor (BCR) stimulation and show that current therapies suppress this induction.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos B/inmunología , HumanosRESUMEN
Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.
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Exosomas/patología , Fibroblastos/patología , Leucemia Linfocítica Crónica de Células B/patología , Células del Estroma/patología , Anciano , Anciano de 80 o más Años , Supervivencia Celular , Células Cultivadas , Exosomas/inmunología , Exosomas/metabolismo , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , MicroARNs/metabolismo , Persona de Mediana Edad , Neovascularización Patológica/inmunología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Transporte de Proteínas , Transducción de Señal , Células del Estroma/inmunología , Células del Estroma/metabolismoAsunto(s)
Antígenos CD/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Antígenos CD/genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Ratones , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Receptor de Muerte Celular Programada 1/genética , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
The differentiation of human peripheral blood monocytes into macrophages can be reproduced ex vivo by culturing the cells in the presence of colony-stimulating factor 1 (CSF1). Using microarray profiling to explore the role of microRNAs (miRNAs), we identified a dramatic decrease in the expression of the hematopoietic specific miR-142-3p. Up- and down-regulation of this miRNA in primary human monocytes altered CSF1-induced differentiation of monocytes, as demonstrated by changes in the expression of the cell surface markers CD16 and CD163. One of the genes whose expression is repressed by miR-142-3p encodes the transcription factor Early Growth Response 2 (Egr2). In turn, Egr2 associated with its co-repressor NGFI-A (Nerve Growth Factor-Induced gene-A) binding protein 2 (NAB2) binds to the pre-miR-142-3p promoter to negatively regulate its expression. Interestingly, the expression of miR-142-3p is abnormally low in monocytes from patients with the most proliferative forms of chronic myelomonocytic leukemia (CMML), and miR-142-3p re-expression in CMML dysplastic monocytes can improve their differentiation potential. Altogether, miR-142-3p which functions in a molecular circuitry with Egr2 is an actor of CSF1-induced differentiation of human monocytes whose expression could be altered in CMML.
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Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , MicroARNs/genética , Monocitos/efectos de los fármacos , Monocitos/fisiología , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Células K562 , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/metabolismo , Leucemia Mielomonocítica Crónica/patología , Macrófagos/citología , Macrófagos/metabolismo , MicroARNs/biosíntesis , MicroARNs/metabolismo , Monocitos/citología , Monocitos/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin.
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Sistema Hematopoyético/citología , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Línea Celular , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/genética , Humanos , Poliadenilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Early cancer detection and disease stratification or classification are critical to successful treatment. Accessible, reliable, and informative cancer biomarkers can be medically valuable and can provide some relevant insights into cancer biology. Recent studies have suggested improvements in detecting malignancies by the use of specific extracellular microRNAs (miRNAs) in plasma. In chronic lymphocytic leukemia (CLL), an incurable hematologic disorder, sensitive, early, and noninvasive diagnosis and better disease classification would be very useful for more effective therapies. We show here that circulating miRNAs can be sensitive biomarkers for CLL, because certain extracellular miRNAs are present in CLL patient plasma at levels significantly different from healthy controls and from patients affected by other hematologic malignancies. The levels of several of these circulating miRNAs also displayed significant differences between zeta-associated protein 70 (ZAP-70)(+) and ZAP-70(-) CLL. We also determined that the level of circulating miR-20a correlates reliably with diagnosis-to-treatment time. Network analysis of our data, suggests a regulatory network associated with BCL2 and ZAP-70 expression in CLL. This hypothesis suggests the possibility of using the levels of specific miRNAs in plasma to detect CLL and to determine the ZAP-70 status.
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Biomarcadores de Tumor/sangre , Leucemia Linfocítica Crónica de Células B/sangre , MicroARNs/sangre , ARN Neoplásico/sangre , Anciano , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/terapia , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/sangre , Proteína Tirosina Quinasa ZAP-70/sangreRESUMEN
Despite being the most common adult leukemia in the western world, Chronic Lymphocytic Leukemia (CLL) remains a life-threatening and incurable disease. Efforts to develop new treatments are highly dependent on the availability of appropriate mouse models for pre-clinical testing. The Eµ-TCL1 mouse model is the most established pre-clinical approach to study CLL pathobiology and response to treatment, backed by numerous studies highlighting its resemblance to the most aggressive form of this malignancy. In contrast to the transgenic Eµ-TCL1 model, employing the adoptive transfer of Eµ-TCL1-derived splenocytes in immunocompetent C57BL/6 mice results in a comparably rapid (e.g., leukemic development within weeks compared to months in the transgenic model) and reliable model mimicking CLL. In this chapter, we would like to provide readers with a thoroughly optimized, detailed, and comprehensive protocol to use the adoptive transfer Eµ-TCL1 model in their research.
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Traslado Adoptivo , Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B , Ratones Endogámicos C57BL , Animales , Leucemia Linfocítica Crónica de Células B/terapia , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Traslado Adoptivo/métodos , Ratones Transgénicos , Bazo , Humanos , Proteínas Proto-OncogénicasRESUMEN
Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers. For complete details on the use and execution of this protocol, please refer to Gargiulo et al.1.
RESUMEN
Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.
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Glutamina , Piruvato Quinasa , Piruvato Quinasa/metabolismo , Glutamina/metabolismo , Glucólisis , Carbono , Serina/metabolismoAsunto(s)
Apoptosis/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/patología , Proteínas Represoras/metabolismo , Tiazoles/farmacología , Animales , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Ratones , Prohibitinas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Tiazoles/metabolismo , Activación Transcripcional/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world. It is characterized by a high dependency on interactions with the surrounding immune landscape, highlighting its suitability for immune-mediated therapeutic interventions. We recently revealed that the cytokine IL-27 exerts a strong anti-tumor role in CLL through a T-cell-mediated mechanism.
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Interleucina-27 , Leucemia Linfocítica Crónica de Células B , Adulto , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Citocinas , Linfocitos T , Terapia de InmunosupresiónRESUMEN
Hairy cell leukemia (HCL) is an incurable, rare lymphoproliferative hematological malignancy of mature B cAlthough first line therapy with purine analogues leads to positive results, almost half of HCL patients relapse after 5-10 years, and standard treatment may not be an option due to intolerance or refractoriness. Proliferation and survival of HCL cells is regulated by surrounding accessory cells and soluble signals present in the tumor microenvironment, which actively contributes to disease progression. In vitro studies show that different therapeutic approaches tested in HCL impact the tumor microenvironment, and that this milieu offers a protection affecting treatment efficacy. Herein we explore the effects of the tumor microenvironment to different approved and experimental therapeutic options for HCL. Dissecting the complex interactions between leukemia cells and their milieu will be essential to develop new targeted therapies for HCL patients.
RESUMEN
Small extracellular vesicle (sEV, or exosome) communication among cells in the tumor microenvironment has been modeled mainly in cell culture, whereas their relevance in cancer pathogenesis and progression in vivo is less characterized. Here we investigated cancer-microenvironment interactions in vivo using mouse models of chronic lymphocytic leukemia (CLL). sEVs isolated directly from CLL tissue were enriched in specific miRNA and immune-checkpoint ligands. Distinct molecular components of tumor-derived sEVs altered CD8+ T-cell transcriptome, proteome, and metabolome, leading to decreased functions and cell exhaustion ex vivo and in vivo. Using antagomiRs and blocking antibodies, we defined specific cargo-mediated alterations on CD8+ T cells. Abrogating sEV biogenesis by Rab27a/b knockout dramatically delayed CLL pathogenesis. This phenotype was rescued by exogenous leukemic sEV or CD8+ T-cell depletion. Finally, high expression of sEV-related genes correlated with poor outcomes in CLL patients, suggesting sEV profiling as a prognostic tool. In conclusion, sEVs shape the immune microenvironment during CLL progression. SIGNIFICANCE: sEVs produced in the leukemia microenvironment impair CD8+ T-cell mediated antitumor immune response and are indispensable for leukemia progression in vivo in murine preclinical models. In addition, high expression of sEV-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients. See related commentary by Zhong and Guo, p. 5. This article is highlighted in the In This Issue feature, p. 1.
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Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/genética , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Transcriptoma , Inmunidad , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Microambiente Tumoral/genéticaRESUMEN
Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
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Metilenotetrahidrofolato Deshidrogenasa (NADP) , Neoplasias , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , TetrahidrofolatosRESUMEN
Recently, small extracellular vesicles (sEVs) secreted in vivo from chronic lymphocytic leukemia (CLL) preclinical murine models were characterized. Leukemia microenvironment sEV (LME-sEVs) selectively target CD8+ T-cells, inducing exhaustion and hampering anti-tumor immune response. Additionally, a sEV-related gene expression correlated with patient treatment-free survival, overall survival and clinical parameters.
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Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Animales , Linfocitos T CD8-positivos , Vesículas Extracelulares/metabolismo , Humanos , Evasión Inmune , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/metabolismo , Ratones , Microambiente TumoralRESUMEN
Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.