ABSTRACT
Autologous T-cell-based therapies, such as CAR-T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) which correlates with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aims to investigate mechanisms by which CLL cells suppress (CAR) T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion and that specifically re-stimulation of (CAR-) T cells in presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, co-culture of T cells with CD40-activated CLL cells did not result in T-cell dysfunction and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40-signaling cascade revealed that the SRC-kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of SIGLEC-10-ligands CD24 and CD52, which was prevented by dasatinib; suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced (CAR) T-cell dysfunction upon co-culture with resting CLL cells. These results demonstrate that T cells derived from CLL patients can be reinvigorated by manipulating CLL-T cell interactions. Targeting CD24- and CD52-mediated CLL-T cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.
ABSTRACT
The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.
Subject(s)
Chromosomes, Human, Pair 3 , Histone Deacetylase Inhibitors , Leukemia, Myeloid, Acute , MDS1 and EVI1 Complex Locus Protein , Proteogenomics , Animals , Female , Humans , Mice , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromosomes, Human, Pair 3/genetics , Gene Expression Regulation, Leukemic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , MDS1 and EVI1 Complex Locus Protein/metabolism , MDS1 and EVI1 Complex Locus Protein/genetics , Proteogenomics/methods , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Xenograft Model Antitumor AssaysABSTRACT
NOTCH1 PEST domain mutations are often seen in hematopoietic malignancies, including T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). These mutations play a key role in the development and progression of lymphoproliferative tumors by increasing the Notch signaling and, consequently, promoting cell proliferation, survival, migration, and suppressing apoptosis. There is currently no specific treatment available for cancers caused by NOTCH1 PEST domain mutations. However, several NOTCH1 inhibitors are in development. Among these, inhibition of the Sarco-endoplasmic Ca2+-ATPase (SERCA) showed a greater effect in NOTCH1-mutated tumors compared to the wild-type ones. One example is CAD204520, a benzimidazole derivative active in T-ALL cells harboring NOTCH1 mutations. In this study, we preclinically assessed the effect of CAD204520 in CLL and MCL models and showed that NOTCH1 PEST domain mutations sensitize cells to the anti-leukemic activity mediated by CAD204520. Additionally, we tested the potential of CAD204520 in combination with the current first-line treatment of CLL, venetoclax, and ibrutinib. CAD204520 enhanced the synergistic effect of this treatment regimen only in samples harboring the NOTCH1 PEST domain mutations, thus supporting a role for Notch inhibition in these tumors. In summary, our work provides strong support for the development of CAD204520 as a novel therapeutic approach also in chronic lymphoproliferative disorders carrying NOTCH1 PEST domain mutations, emerging as a promising molecule for combination treatment in this aggressive subset of patients.
Subject(s)
Hematologic Neoplasms , Leukemia, Lymphocytic, Chronic, B-Cell , Lymphoproliferative Disorders , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Lymphoproliferative Disorders/drug therapy , Lymphoproliferative Disorders/genetics , Mutation , Receptor, Notch1/geneticsABSTRACT
The mechanisms underlying the success of propranolol in the treatment of infantile hemangioma (IH) remain elusive and do not fully explain the rapid regression of hemangiomatous lesions following drug administration. As autophagy is critically implicated in vascular homeostasis, we determined whether ß-blockers trigger the autophagic flux on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro. MATERIAL AND METHODS: Fresh tissue specimens, surgically removed for therapeutic purpose to seven children affected by proliferative IH, were subjected to enzymatic digestion. Cells were sorted with anti-human CD31 immunolabeled magnetic microbeads. Following phenotypic characterization, expanded Hem-ECs, at P2 to P6, were exposed to different concentrations (50 µM to 150 µM) of propranolol, atenolol or metoprolol alone and in combination with the autophagy inhibitor Bafilomycin A1. Rapamycin, a potent inducer of autophagy, was also used as control. Autophagy was assessed by Lysotracker Red staining, western blot analysis of LC3BII/LC3BI and p62, and morphologically by transmission electron microscopy. RESULTS: Hem-ECs treated with either propranolol, atenolol or metoprolol displayed positive LysoTracker Red staining. Increased LC3BII/LC3BI ratio, as well as p62 modulation, were documented in ß-blockers treated Hem-ECs. Abundant autophagic vacuoles and multilamellar bodies characterized the cytoplasmic ultrastructural features of autophagy in cultured Hem-ECs exposed in vitro to ß-blocking agents. Importantly, similar biochemical and morphologic evidence of autophagy were observed following rapamycin while Bafilomycin A1 significantly prevented the autophagic flux promoted by ß-blockers in Hem-ECs. CONCLUSION: Our data suggest that autophagy may be ascribed among the mechanisms of action of ß-blockers suggesting new mechanistic insights on the potential therapeutic application of this class of drugs in pathologic conditions involving uncontrolled angiogenesis.
Subject(s)
Hemangioma , Propranolol , Adrenergic beta-Antagonists/pharmacology , Amines , Atenolol/pharmacology , Atenolol/therapeutic use , Autophagy , Cell Proliferation , Child , Endothelial Cells , Hemangioma/pathology , Humans , Macrolides , Metoprolol/therapeutic use , Propranolol/pharmacology , Propranolol/therapeutic use , Sirolimus/pharmacologyABSTRACT
Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in acute lymphoblastic leukemia (ALL), suggesting new opportunities for therapeutic interventions. In this study, we identified G9a/EHMT2 as a potential target in T-ALL through the intersection of epigenome-centered shRNA and chemical screens. We subsequently validated G9a with low-throughput CRISPR-Cas9-based studies targeting the catalytic G9a SET-domain and the testing of G9a chemical inhibitors in vitro, 3D, and in vivo T-ALL models. Mechanistically we determined that G9a repression promotes lysosomal biogenesis and autophagic degradation associated with the suppression of sestrin2 (SESN2) and inhibition of glycogen synthase kinase-3 (GSK-3), suggesting that in T-ALL glycolytic dependent pathways are at least in part under epigenetic control. Thus, targeting G9a represents a strategy to exhaust the metabolic requirement of T-ALL cells.