Search Results (171)

To further investigate the role of PKCλ/ι in Schistosoma japonicum-induced hepatic fibrosis, we employed a CD4⁺ T-cell-specific PKCλ/ι conditional knockout (KOSJ) mouse model, with wild-type (WTSJ) mice used as controls. Transcriptomic profiling of hepatic mRNA was used to reveal the immune regulatory mechanisms underlying the role of PKCλ/ι in the hepatic fibrosis caused by S. japonicum infection. Flow cytometry, RT–qPCR and ELISA were used to analyze the effects of PKCλ/ι on Tfh and Tfr cells, and single-cell RNA sequencing was used to elucidate the interactions between Tfr and B cells. The results showed that PKCλ/ι deficiency led to altered BCR signaling gene expression, reduced germinal center activity, and decreased anti-SEA antibody levels. Tfh cells and key factors including IL-21, CXCR5, and ICOS were downregulated, while Tfr cells and IL-10⁺ B cells increased. Additionally, hepatic neutrophils decreased and Treg/Tfr ratios rose, with enhanced IL-10-mediated cellular crosstalk. These findings indicate that PKCλ/ι deficiency attenuates liver fibrosis by inhibiting Tfh differentiation, promoting Tfr function, and activating IL-10-producing Breg cells, suggesting its potential as a therapeutic target. Full article

Insights into the state of individual cells within a living organism are essential for identifying diseases and abnormalities. The internal state of a cell is reflected in its morphological features and changes in the localization of intracellular molecules. Using this information, it is possible to infer the state of the cells with high precision. In recent years, technological advancements and improvements in instrument specifications have made large-scale analyses, such as single-cell analysis, more widely accessible. Among these technologies, imaging flow cytometry (IFC) is a high-throughput imaging platform that can simultaneously acquire information from flow cytometry (FCM) and cellular images. While conventional FCM can only obtain fluorescence intensity information corresponding to each detector, IFC can acquire multidimensional information, including cellular morphology and the spatial arrangement of proteins, nucleic acids, and organelles for each imaging channel. This enables the discrimination of cell types and states based on the localization of proteins and organelles, which is difficult to assess accurately using conventional FCM. Because IFC can acquire a large number of single-cell morphological images in a short time, it is well suited for automated classification using machine learning. Furthermore, commercial instruments that combine integrated imaging and cell sorting capabilities have recently become available, enabling the sorting of cells based on their image information. In this review, we specifically highlight practical applications of IFC in four representative areas: cell cycle analysis, protein localization analysis, immunological synapse formation, and the detection of leukemic cells. In addition, particular emphasis is placed on applications that directly contribute to elucidating molecular mechanisms, thereby distinguishing this review from previous general overviews of IFC. IFC enables the estimation of cell cycle phases from large numbers of acquired cellular images using machine learning, thereby allowing more precise cell cycle analysis. Moreover, IFC has been applied to investigate intracellular survival and differentiation signals triggered by external stimuli, to monitor DNA damage responses such as γH2AX foci formation, and more recently, to detect immune synapse formation among interacting cells within large populations and to analyze these interactions at the molecular level. In hematological malignancies, IFC combined with fluorescence in situ hybridization (FISH) enables high-throughput detection of chromosomal abnormalities, such as BCR-ABL1 translocations. These advances demonstrate that IFC provides not only morphological and functional insights but also clinically relevant genomic information at the single-cell level. By summarizing these unique applications, this review aims to complement existing publications and provide researchers with practical insights into how IFC can be implemented in both basic and translational research. Full article

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder caused by the BCR::ABL1 fusion gene, resulting from a reciprocal translocation between chromosomes 22 and 9. Quantification of BCR::ABL1 transcript levels in peripheral blood by RT-qPCR represents the gold standard for molecular response (MR) monitoring, providing essential clinical information on treatment efficacy. Xpert^® BCR-ABL Ultra is a fully automated in vitro diagnostic test that quantitatively detects e13a2 and e14a2 BCR::ABL1 transcripts using a single-use cartridge that integrates RNA extraction, cDNA synthesis, nested real-time PCR, and signal detection within a rapid, closed, and user-friendly system. In this study, we evaluated Xpert^® BCR-ABL Ultra as an alternative to validated systems currently used by four highly specialized Italian laboratories affiliated with the Italian national laboratory network for CML. A total of 129 peripheral blood samples from CML patients at various disease stages, along with two external quality control materials, were analyzed. We assessed the test’s repeatability, specificity, and stability. Concordance of BCR::ABL1%IS values generated by the different methods was evaluated using EUTOS criteria and Bland–Altman analysis. Finally, MR value concordance was analyzed based on European LeukemiaNet recommendations or calculated using the formula 2 − log₁₀(BCR::ABL1%IS). Xpert^® BCR-ABL Ultra demonstrated high repeatability and stability. The BCR::ABL1%IS values obtained with this assay showed strong concordance with those generated by local reference methods, and MR classifications were consistent across platforms. These findings confirm the robustness, accuracy, and efficiency of the Xpert^® BCR-ABL Ultra assay, supporting its use as a reliable alternative to currently validated systems for the routine clinical monitoring of CML patients. Full article

Chronic myeloid leukemia (CML) is a blood disorder caused by a genetic alteration that creates the BCR-ABL fusion gene, leading to continuous activation of cell growth signals and uncontrolled proliferation of the blood cells. Imatinib (IMA) resistance remains a major obstacle in CML treatment. Integrins, particularly integrin α2 (ITGA2), have been associated with cancer progression and drug resistance. In the current study, we investigated the role of ITGA2 in IMA resistance using IMA-sensitive K562 (K562S) and IMA-resistant K562 (K562R) cells. Our findings showed that ITGA2 is overexpressed in K562R cells and ITGA2 inhibitor E7820 (2.5 µM) treatment significantly decreased cell viability and induced apoptosis in both sensitive and resistant cells. Combination treatment with E7820 and imatinib enhanced pro-apoptotic gene expression (BAX, BIM) and decreased anti-apoptotic BCL2 levels in imatinib-resistant K562R cells. Flow cytometry confirmed ITGA2 inhibition at the protein level, and rhodamine assays revealed reduced MDR1 activity in treated cells. These results demonstrate that targeting ITGA2 may overcome imatinib resistance and offer a novel therapeutic strategy for CML. Full article

Background/Objectives: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the BCR–ABL fusion gene, whose constitutive tyrosine kinase activity drives leukemogenesis. Although tyrosine kinase inhibitors (TKIs) have revolutionized treatment, drug resistance and leukemic stem cell persistence remain major challenges. Natural compounds such as polyphenols have shown potential in modulating key oncogenic pathways in CML. Results: Polyphenols such as resveratrol, quercetin, curcumin, and epigallocatechin gallate (EGCG) demonstrated significant antiproliferative and pro-apoptotic effects in CML cell lines, including imatinib-resistant models. These effects were mediated through the modulation of signaling pathways, including PI3K/Akt, STAT5, and MAPK; inhibition of BCR–ABL expression; induction of oxidative stress; and the enhancement of apoptosis via mitochondrial and caspase-dependent mechanisms. Some polyphenols also showed synergistic activity with TKIs, potentiating their efficacy and overcoming resistance. Conclusions: Preclinical evidence supports the role of polyphenols as potential adjuvants in CML therapy, particularly in drug-resistant contexts. Their pleiotropic molecular actions and low toxicity profile make them promising candidates for integrative oncology. Nonetheless, clinical translation requires further investigation through well-designed trials assessing efficacy, safety, and pharmacokinetics. Full article

(1) Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder driven by the BCR::ABL oncoprotein. During the chronic phase, Philadelphia chromosome-positive hematopoietic stem cells generate proliferative myeloid cells with various stages of maturation. Despite this expansion, leukemic stem cells (LSCs) retain self-renewal capacity via asymmetric cell divisions, sustaining the stem cell pool. Quiescent LSCs are known to be resistant to tyrosine kinase inhibitors (TKIs), potentially through BCR::ABL-independent signaling pathways. We hypothesize that dysregulation of genes governing asymmetric division in LSCs contributes to disease progression, and that their expression pattern may serve as a prognostic marker during the chronic phase of CML. (2) Methods: Genes related to asymmetric cell division in the context of hematopoietic stem cells were extracted from the PubMed database with the keyword “asymmetric hematopoietic stem cell”. The collected relative gene set was tested on two independent bulk transcriptome cohorts and the results were confirmed by single-cell RNA sequencing. (3) Results: The expression of genes involved in asymmetric hematopoietic stem cell division was found to discriminate disease phases during CML progression in the two independent transcriptome cohorts. Concordance between cohorts was observed on asymmetric molecules downregulated during blast crisis (BC) as compared to the chronic phase (CP). This downregulation during the BC phase was confirmed at single-cell level for SELL, CD63, NUMB, HK2, and LAMP2 genes. Single-cell analysis during the CP found that CD63 is associated with a poor prognosis phenotype, with the opposite prediction revealed by HK2 and NUMB expression. The single-cell trajectory reconstitution analysis in CP samples showed CD63 regulation highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, and PDIA3, known to be implicated in hematopoietic malignancies. (4) Conclusion: Regulation of CD63, a tetraspanin involved in the asymmetric division of hematopoietic stem cells, was found to be associated with poor prognosis during CML progression and could be a potential new therapeutic target. Full article

There have been multiple approved agents for relapsed/refractory (r/r) Diffuse Large B-cell Lymphoma (DLBCL) over the last 8 years. The majority of these therapies act on specific signaling pathways in malignant B-cells. These signaling pathways stem from the B-cell receptor (BCR), Toll-Like Receptor (TLR), PI3K/AKT/mTOR, BCL-2, and XPO-1. In addition, novel therapies that target extracellular proteins (CD19, CD20, CD30, ROR1, and PD-1) have been developed. The purpose of this review is to discuss the various therapies that target these pathways and highlight the success and shortcomings of these novel agents. Full article

The BCR-ABL1 chimeric oncoprotein plays a pivotal role in the pathogenesis of Chronic Myeloid Leukemia (CML) as its constitutive kinase activity transforms the hematopoietic stem cell, promoting pro-survival signaling. We and others have previously shown that the manipulation of BCR-ABL1 catalytic activity modulates its intracellular localization, thereby transforming the culprit of CML into a pro-apoptotic protein that selectively kills leukemic cells. Here, we investigated the role of the BCR coiled-coil and C2 domains on BCR-ABL1 intracellular localization and leukemogenic potential. We performed a bioinformatic analysis that identified two putative nuclear localization signals (NLSs) in BCR. Using recombinant DNA strategies, we generated multiple BCR and BCR-ABL1 mutants that were ectopically expressed in human cells. The intracellular localization of each construct was analyzed by immunofluorescence, while their biological activity was investigated employing proliferation and transforming assays. We show that BCR displays two nuclear localization signals functionally inactivated by the coiled-coil and C2 domains. The removal of these regions reactivated the nuclear migration of both BCR and BCR-ABL1 mutants. Moreover, BCR-ABL1 constructs devoid of the coiled-coil and C2 domains displayed reduced transforming potential in Ba/F3 cells and in primary human CD34+ progenitors. Finally, we demonstrate that the deletion of the C2 domain compromises TKI efficacy. Our findings identify two nuclear localization signals in the BCR sequence that are functionally suppressed by the coiled-coil and C2 domains. Targeting these regions may provide additional therapeutic strategies to manipulate both BCR-ABL1 intracellular localization and kinase activity. Full article

Currently, there are two major theories regarding the pathogenesis of sepsis: hyperimmune and hypoimmune. The hyperimmune theory suggests that a cytokine storm causes the symptoms of sepsis. On the contrary, the hypoimmune theory suggests that immunosuppression causes the manifestations of sepsis. By conducting a microarray analysis on peripheral leukocytes from patients with sepsis, this study found that hyperactivity of TH17 immunity was noted in sepsis patients. Innate immunity-related genes are significantly upregulated, including CD14, TLR1,2,4,5,8, HSP70, CEBP proteins, AP1 (JUNB and FOSL2), TGFB1, IL6, TGFA, CSF2 receptor, TNFRSF1A, S100A binding proteins, CCR2, FPR2, amyloid proteins, pentraxin, defensins, CLEC5A, whole complement machinery, CPD, NCF, MMP, neutrophil elastase, caspases, IgG and IgA Fc receptors (CD64, CD32), ALOX5, PTGS, LTB4R, LTA4H, and ICAM1. The majority of adaptive immunity genes were downregulated, including MHC-related genes, TCR genes, granzymes/perforin, CD40, CD8, CD3, TCR signaling, BCR signaling, T and B cell-specific transcription factors, NK killer receptors, and TH17 helper-specific transcription factors (STAT3, RORA, and REL), as well as Treg-related genes, including TGFB1, IL15, STAT5B, SMAD2/4, CD36, and thrombospondin. The findings of this study show that Th17 with Treg over-presentation play an important role in the pathophysiology of sepsis. Full article

The Janus kinase 2 (JAK2) protein fulfills an important role in hematopoiesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, as it provides the genetic driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs), which are clinically manifested as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common cause of MPNs is the mutation of JAK2 V617F in the JAK2 gene, which results in increased cell proliferation. However, both the pathogenesis and treatment regimen of BCR::ABL1-negative MPNs remain poorly understood. The aim of the present study was to establish K562 cell lines with a point mutation in exon 14 (JAK2p.V617F) using CRISPR/Cas9 technology. The modified JAK2 V617F cell lines were examined for the gene mutation using droplet digital PCR (DDPCR), and the presence of the mutation was confirmed by DNA sequencing. Modified cells were characterized by measuring JAK2 gene expression and the extent of cell proliferation. Interferon α2a (IFN-α2a) and arsenic trioxide were also administered to the cells to explore their potential effects. The JAK2 V617F-mutated cells were found to exhibit a higher level of JAK2 gene expression compared with the wild type. Interestingly, a significant increase in the proliferation rate was observed with the modified cells compared with the wild type cells (p < 0.001), as assessed from the JAK2 gene expression levels. Furthermore, the treatments with IFN-α2a and arsenic trioxide led to the preferential suppression of the cell proliferation rate of the K562 expressing mutant JAK2 cells compared with the wild type cells, and this suppression occurred in a dose-dependent manner(p < 0.01). Moreover, the modified cells were able to differentiate into megakaryocyte-like cells following stimulation with phorbol 12 myristate 13 acetate (PMA). Taken together, the results of the present study have shown that the CRISPR/Cas9-modified JAK2 V617F model may be used as a disease model in the search of novel therapies for MPNs. Full article

Background: Chronic-Phase Chronic Myeloid Leukemia (C-PCML) is defined by the presence of the BCR-ABL1 fusion gene, which encodes a tyrosine kinase protein that drives the uncontrolled proliferation and survival of leukemic stem cells (LSCs). Nilotinib, a tyrosine kinase inhibitor, targets the activity of BCR-ABL1 by reducing aberrant signaling pathways, which drive the regeneration of LSCs. Despite nilotinib’s action, a population of resilient LSCs persist in the bone marrow (BM) and can indeed drive relapse and progression in CML patients. Methods: Our study investigated the gene expression profiling (GEP) of BM CD34+/lin− cells from 79 CP-CML patients at diagnosis, compared to the BM CD34+/lin− cells from the same patients after 12 months of nilotinib treatment and to the normal counterpart cells from 10 donors (CTRLs). Results: GEP analyses identified 3012 significantly differentially expressed genes across these comparisons. Among these, we focused on certain key genes associated with eight crucial KEGG pathways: CML, cell cycle, JAK-STAT, PI3K-Akt, MAPK, Ras, NF-kB, and ABC transporters. Within these pathways, we observed the up-regulation of several genes at diagnosis compared to both 12 months of nilotinib treatment and the CTRLs. Conclusions: We observed that certain transcriptome features present at diagnosis persisted after 12 months of nilotinib treatment, compared to CTRLs. This suggests that nilotinib may exert selective pressure, potentially supporting the survival and self-renewal of LSCs. Future insights into these pathways could help identify therapeutic targets to improve outcomes in CML. Full article

Backgroung/objectives: Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype of malignant lymphoma and is a heterogeneous disease with various gene and chromosomal abnormalities. The development of novel therapeutic treatments has improved DLBCL prognosis, but patients with early relapse or refractory disease have a poor outcome (with a mortality of around 40%). Metabolic reprogramming is a hallmark of cancer cells. Fatty acid (FA) metabolism is frequently altered in cancer cells and recently emerged as a critical survival path for cancer cell survival. Methods: We first performed the metabolic characterization of an extended panel of DLBCL cell lines, including lipid droplet content. Then, we investigated the effect of drugs targeting FA metabolism on DLBCL cell survival. Further, we studied how the combination of drugs targeting FA and either mitochondrial metabolism or mTOR pathway impacts on DLBCL cell death. Results: Here, we reveal, using a large panel of DLBCL cell lines characterized by their metabolic status, that targeting of FA metabolism induces massive DLBCL cell death regardless of their OxPhos or BCR/glycolytic subtype. Further, FA drives resistance of DLBCL cell death induced by mitochondrial stress upon treatment with either metformin or L-asparaginase, two FDA-approved antimetabolic drugs. Interestingly, combining inhibition of FA metabolism with that of the mTOR oncogenic pathway strongly potentiates DLBCL cell death. Conclusion: Altogether, our data highlight the central role played by FA metabolism in DLBCL cell survival, independently of their metabolic subtype, and provide the framework for the use of drugs targeting this metabolic vulnerability to overcome resistance in DLBCL patients. Full article

Chronic lymphocytic leukemia (CLL) cells depend on microenvironment niches for proliferation and survival. The adhesion of tumor cells to stromal cells in such niches triggers the activation of signaling pathways crucial for their survival, including B-cell receptor (BCR) signaling. While inhibitors of Bruton’s tyrosine kinase (BTKi) have shown efficacy in patients with CLL by disrupting these interactions, acquired resistance and toxicity remain a challenge during long-term therapy. Thus, identifying additional therapeutic modalities is important. Previously, we demonstrated that 5-lipoxygenase (5-LOX) pathway inhibitors reduced mantle cell lymphoma (MCL) cell adhesion to stromal cells, motivating us to investigate their potential in the context of CLL. We employed an ex vivo co-culture model to study CLL cell adhesion to stromal cells in the absence and presence of 5-LOX pathway inhibitors (zileuton and MK886) as well as the BTKi ibrutinib that was included for comparative purposes. Our findings demonstrated that different CLL samples adhere to stromal cells differentially. We observed a variable decrease in CLL cell adhesion to stromal cells following the inhibition of the 5-LOX pathway across a spectrum of patient samples that was distinct to the spectrum for ibrutinib. Positive and negative correlations were shown between the clinical and genetic features of the CLL samples and their level of adherence to stromal cells in both the absence and presence of the tested inhibitors. These results suggest the 5-LOX pathway as a candidate for assessment as a new therapeutic target in CLL. Full article

BCR-ABL1 kinase is a critical driver of chronic myeloid leukemia (CML) pathophysiology. The approval of allosteric inhibitor asciminib brings new hope for overcoming drug resistance caused by mutations in the ATP-binding site. To expand the chemical diversity of BCR-ABL1 kinase inhibitors with positive anti-tumor effect with asciminib, structure-based virtual screening and molecular dynamics simulations were employed to discover novel scaffolds. This approach led to the identification of a series of N-(2-acetamidobenzo[d]thiazol-6-yl)-2-phenoxyacetamide derivatives as new BCR-ABL1 inhibitors. The most potent compound, 10m, demonstrated inhibition of BCR-ABL-dependent signaling and showed an anti-tumor effect against K562 cells, with an IC₅₀ value of 0.98 μM. Compound 10m displayed powerful synergistic anti-proliferation and pro-apoptotic effects when combined with asciminib, highlighting its potential as a promising lead for the development of potential BCR-ABL inhibitors. Full article

The proliferation and survival of chronic lymphocytic leukemia (CLL) cells are heavily dependent on B-cell receptor (BCR) signaling and resistance to apoptosis. Approvals of multiple covalent Bruton’s tyrosine kinas inhibitors (cBTKis) as well as the B-cell lymphoma-2 inhibitor (BCL2i) venetoclax targeting these pathways have revolutionized the treatment of CLL and small lymphocytic lymphoma (SLL). The superiority of these treatments over chemoimmunotherapy has been proven in phase III studies in both treatment-naïve and relapsed refractory settings, leading to the majority of patients with CLL being treated sequentially with cBTKis and the BCL2i venetoclax as their first- and second-line therapies. While most patients with CLL respond for many years to these sequenced treatments, they are unfortunately not curative. There remains an unmet need for effective treatment options for patients who progressed after treatment with both cBTKis and BCL2i, also referred to as double refractory patients. Treatment options for double refractory CLL has improved recently with the approval of the non-covalent BTK inhibitor (ncBTKi) pirtobrutinib as well as the CD19 targeted chimeric antigen receptor T-cell (CAR T-cell) therapy lisocabtagene maraleucel (liso-cel). These recently approved treatment options for patients with CLL with at least two prior lines of therapy have fortunately demonstrated efficacy for double refractory CLL. Additionally, there are several novel treatment options in clinical development, including bi-specific antibodies, second-generation BCL2is, new ncBTKis, and BTK degraders. Understanding resistance mechanisms to existing cBTKis and venetoclax can potentially inform us of the best utilization of available treatment options for double refractory CLL and provide a personalized approach for these patients. In this review, a challenging example of a double refractory patient with CLL will serve as the basis for a review of available literature on the treatment of double refractory CLL/SLL. Full article