Search Results (77)

(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

Casein Kinase II (CK2) is a ubiquitously present serine/threonine kinase essential for mammalian development. CK2 holoenzyme is a tetramer with two highly related catalytic subunits (α or α’) and two regulatory ß subunits. Global deletion of the α or β subunit in mice is embryonically lethal. We and others have shown that CK2 is overexpressed in leukemia cells and plays an important role in cell cycle, survival, and resistance to the apoptosis of leukemia stem cells (LSCs). To study the role of CK2α in adult mouse hematopoiesis, we generated hematopoietic cell-specific CK2α-conditional knockout mice (Vav-iCreCK2 ^f/f). Here we report the generation and validation of a novel mouse model that lacks CK2α in the hematopoietic compartment. Vav-iCreCK2α ^f/f mice were viable without dysmorphic features and showed a mild phenotype under baseline conditions. In Vav-iCreCK2α ^f/f mice, the blood count showed a significant decrease in total red blood cells and platelets. The spleen was enlarged in Vav-iCreCK2α ^f/f mice with evidence of extramedullary hematopoiesis. HSC and early progenitor cell compartments showed expansion in CK2α-null bone marrow, suggesting that the absence of CK2α impaired their proliferation and differentiation. Given the established roles of CK2 in cell cycle regulation and the findings reported here, further functional studies are warranted to investigate the role of CK2α in HSC self-renewal and differentiation. This mouse model serves as a valuable tool for understanding the role of CK2α in normal and malignant hematopoiesis. Full article

Acute Myeloid Leukemia (AML) is characterized by aggressive proliferation and metabolic reprogramming that support its survival and resistance to therapy. This review explores the metabolic distinctions between AML cells and normal hematopoietic stem cells (HSCs), emphasizing the role of altered mitochondrial function, oxidative phosphorylation (OXPHOS), and biosynthetic pathways in leukemic progression. AML cells exhibit distinct metabolic vulnerabilities, including increased mitochondrial biogenesis, reliance on glycolysis and amino acid metabolism, and unique signaling interactions that sustain leukemic stem cells (LSCs). These dependencies provide potential therapeutic targets, as metabolic inhibitors have demonstrated efficacy in disrupting AML cell survival while sparing normal hematopoietic cells. We examine current and emerging metabolic therapies, such as inhibitors targeting glycolysis, amino acid metabolism, and lipid biosynthesis, highlighting their potential in overcoming drug resistance. However, challenges remain in translating these strategies into clinical practice due to AML’s heterogeneity and adaptability. Further research into AML’s metabolic plasticity and precision medicine approaches is crucial for improving treatment outcomes. Understanding and exploiting AML’s metabolic vulnerabilities could pave the way for novel, more effective therapeutic strategies. Full article

Despite advances in outcome, one third of children with acute myeloid leukemia (AML) relapse, and less than half will achieve long-term survival. Relapse in AML has been shown to be driven in part by leukemic stem cells (LSCs), highlighting the unmet medical need to better characterize and target this therapy-resistant cell population. Micro-array profiling of pediatric AML subpopulations (LSCs and leukemic myeloblasts) and their healthy counterparts revealed nidogen-1 (NID1) as expressed in both leukemic subpopulations while absent in the hematopoietic stem cell. Using the TARGET dataset including pediatric AML patients (n = 1025), NID1 expression showed a correlation with worse event-free survival and KMT2A rearrangements. Drug response profiling of a NID1 knockdown model demonstrated differential sensitivity to HSP90 inhibition. RNA sequencing and gene set enrichment analysis between NID1^high and NID1^low phenotypes showed involvement of NID1 in mitochondrial metabolic pathways known to be enriched in LSCs. Altogether, this study highlights NID1 as a novel oncogene associated with worse EFS and metabolic LSC phenotype in AML. NID1 could serve as a biomarker and aid in further mapping LSCs to establish therapeutic strategies tackling the high relapse rates in pediatric AML. 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

Background: Acute myeloid leukemia (AML) is a heterogeneous disease highly resistant to chemotherapeutic agents. Leukemia stem cells (LSCs) can enter a dormant state and avoid apoptosis in the protective niche of the bone marrow (BM) microenvironment. Moreover, bone marrow stromal cells protect leukemia cells by promoting pro-survival signaling pathways and drug resistance. Therefore, attenuating interactions between leukemia cells and BM cells may have a positive therapeutic effect. Objectives: In this work, we hypothesized that sondages may inhibit the adhesion of leukemia cells to the bone marrow by inhibiting the Hedgehog (Hh) signaling pathway. The Hedgehog pathway is a key therapeutic target in AML due to its role in leukemic cell growth and survival. Methods: We investigated the effects of sonidegib on the adhesion of individual OCI-AML3 cells to a bone marrow stromal spheroid derived from the HS-5 cell line. For this purpose, we precisely determined the minimum cell-to-cell adhesion time using optical tweezers under normoxic (21% of O₂) and hypoxic (1% of O₂) conditions. Results: Our results demonstrated that sonidegib significantly increased the minimum cell-to-cell adhesion time necessary for leukemic cells to establish adhesive bonds with bone marrow stromal cells, thereby indicating a reduction in their adhesive properties. Additionally, we showed that sonidegib is particularly effective at hypoxic oxygen concentrations. Conclusions: The results obtained in this study suggest that sonidegib, through its modulation of the Hedgehog signaling pathway, holds promise as a potential therapeutic approach to target leukemic cell adhesion within the bone marrow microenvironment. Full article

Background/Objectives: Treatment with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) has revolutionized disease management and has transformed CML from a life-threatening disease to a chronic condition for many patients. However, overcoming resistance, particularly related to leukemic stem cells (LSC) that can persist even when the bulk of the leukemic cells are eliminated, remains a significant challenge. Methods: K562 and KU812 cell lines were treated in vitro with the TKI Imatinib (IM). Gene expression, protein analysis, and metabolomic screening were conducted to investigate the ability of the drug to enhance stem cell (SC) features. Moreover, a gene ontology analysis was performed on different available datasets, to further consolidate our data. Results: 48 h of IM treatment can significantly increase the expression of genes related to SC self-renewal, particularly SOX2 and OCT 3/4. Interestingly, these modulations occur in cells that remain alive after drug treatment and that displayed features consistent with leukemia stem-like CML cells, suggesting that SC genes levels are crucial even in cell population survived upon TKI treatment. Moreover, after in silico analysis of available data, we observed an enrichment of SOX2/NANOG and OCT 3/4 signatures after TKI treatment, thus strengthening our results. Conclusions: Our results confirmed the relevance of LSC features after TKI treatment, highlighting the need for more effective and potentially curative strategies targeting LSCs to overcome resistance in CML. Full article

Acute myeloid leukemia (AML) is an aggressive malignancy that poses significant challenges due to high rates of relapse and resistance to treatment, particularly in older populations. While therapeutic advances have been made, survival outcomes remain suboptimal. The evolution of DNA and RNA sequencing technologies, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-Seq), has significantly enhanced our understanding of AML at the molecular level. These technologies have led to the discovery of driver mutations and transcriptomic alterations critical for improving diagnosis, prognosis, and personalized therapy development. Furthermore, single-cell RNA sequencing (scRNA-Seq) has uncovered rare subpopulations of leukemia stem cells (LSCs) contributing to disease progression and relapse. However, widespread clinical integration of these tools remains limited by costs, data complexity, and ethical challenges. This review explores recent advancements in DNA/RNA sequencing in AML and highlights both the potential and limitations of these techniques in clinical practice. Full article

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however, CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs, leading to disease relapse. To prevent this, new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34⁺CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore, the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C₃₃H₆₁NO₅S) is a structurally simplified analog of JCI-20679, the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here, we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34⁺ CML cells isolated from the bone marrow of untreated CML patients. Taken together, these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs, and may improve the treatment of CML. Full article

Background: A major issue in Chronic Myeloid Leukemia (CML) is the persistence of quiescent leukemia stem cells (LSCs) in the hematopoietic niche under tyrosine kinase inhibitor (TKI) treatment. Results: Here, using CFSE sorting, we show that low-proliferating CD34+ cells from CML patients in 3D co-culture hide under HS27A stromal cells during TKI treatment—a behavior less observed in untreated cells. Under the same conditions, Ba/F3p210 cells lose their spontaneous motility. In CML CD34+ and Ba/F3p210 cells, while Rac1 is completely inhibited by TKI, RhoA remains activated but is unable to signal to ROCK. Co-incubation of Ba/F3p210 cells with TKI, SKF-96365 (a calcium channel inhibitor), and EGF restores myosin II activation and amoeboid motility to levels comparable to untreated cells, sustaining the activation of ROCK. In CFSE+ CD34+ cells containing quiescent leukemic stem cells, co-incubation of TKI with SKF-96365 induced the expulsion of these cells from the HS27A niche. Conclusions: This study underscores the role of RhoA in LSC behavior under TKI treatment and suggests that SKF-96365 could remobilize quiescent CML LSCs through reactivation of the RhoA/ROCK pathway. Full article

The association between leukemic stem cells (LSCs) and leukemia development has been widely established in the context of genetic alterations, epigenetic pathways, and signaling pathway regulation. Hematopoietic stem cells are at the top of the bone marrow hierarchy and can self-renew and progressively generate blood and immune cells. The microenvironment, niche cells, and complex signaling pathways that regulate them acquire genetic mutations and epigenetic alterations due to aging, a chronic inflammatory environment, stress, and cancer, resulting in hematopoietic stem cell dysregulation and the production of abnormal blood and immune cells, leading to hematological malignancies and blood cancer. Cells that acquire these mutations grow at a faster rate than other cells and induce clone expansion. Excessive growth leads to the development of blood cancers. Standard therapy targets blast cells, which proliferate rapidly; however, LSCs that can induce disease recurrence remain after treatment, leading to recurrence and poor prognosis. To overcome these limitations, researchers have focused on the characteristics and signaling systems of LSCs and therapies that target them to block LSCs. This review aims to provide a comprehensive understanding of the types of hematopoietic malignancies, the characteristics of leukemic stem cells that cause them, the mechanisms by which these cells acquire chemotherapy resistance, and the therapies targeting these mechanisms. Full article

Tyrosine kinase inhibitors (TKIs), such as imatinib (IM), increase the survival of chronic myeloid leukemia (CML) patients but do not eradicate the disease as leukemia stem cells (LSCs) with primitive and quiescent signatures persist after TKI monotherapy, driving disease relapse. Using single-cell publicly available transcriptomic data, we investigated potentially tractable vulnerabilities in this persistent CML LSC population. GAS2 is significantly upregulated when comparing LSCs from CML patients in remission to normal hematopoietic stem cells (HSCs). A topoisomerase IIβ inhibitor, XK469, was proposed to be repurposed as a candidate small-molecule inhibitor of GAS2, and its effect was investigated in cell line models in combination with IM in vitro. Alone, XK469 could induce cell cycle arrest/differentiation in CML cells and reduce cell viability. In combination with IM, XK469 significantly increased CML cell apoptosis and reduced CML cell clonogenic capacity. These results suggest that GAS2 is a targetable vulnerability in CML LSCs and that using XK469 in combination with TKI potentiates the sensitivity of CML cells to IM. Full article

Acute myeloid leukemia (AML) is a heterogenous blood cancer with a dismal prognosis. It emanates from leukemic stem cells (LSCs) arising from the genetic transformation of hematopoietic stem cells (HSCs). LSCs hold prognostic value, but their molecular and immunophenotypic heterogeneity poses challenges: there is no single marker for identifying all LSCs across AML samples. We hypothesized that imaging flow cytometry (IFC) paired with artificial intelligence-driven image analysis could visually distinguish LSCs from HSCs based solely on morphology. Initially, a seven-color IFC panel was employed to immunophenotypically identify LSCs and HSCs in bone marrow samples from five AML patients and ten healthy donors, respectively. Next, we developed convolutional neural network (CNN) models for HSC-LSC discrimination using brightfield (BF), side scatter (SSC), and DNA images. Classification using only BF images achieved 86.96% accuracy, indicating significant morphological differences. Accuracy increased to 93.42% when combining BF with DNA images, highlighting differences in nuclear morphology, although DNA images alone were inadequate for accurate HSC-LSC discrimination. Model development using SSC images revealed minor granularity differences. Performance metrics varied substantially between AML patients, indicating considerable morphologic variations among LSCs. Overall, we demonstrate proof-of-concept results for accurate CNN-based HSC-LSC differentiation, instigating the development of a novel technique within AML monitoring. Full article

Using multi-color flow cytometry analysis, we studied the immunophenotypical differences between leukemic cells from patients with AML/MDS and hematopoietic stem and progenitor cells (HSPCs) from patients in complete remission (CR) following their successful treatment. The panel of markers included CD34, CD38, CD45RA, CD123 as representatives for a hierarchical hematopoietic stem and progenitor cell (HSPC) classification as well as programmed death ligand 1 (PD-L1). Rather than restricting the evaluation on a 2- or 3-dimensional analysis, we applied a t-distributed stochastic neighbor embedding (t-SNE) approach to obtain deeper insight and segregation between leukemic cells and normal HPSCs. For that purpose, we created a t-SNE map, which resulted in the visualization of 27 cell clusters based on their similarity concerning the composition and intensity of antigen expression. Two of these clusters were “leukemia-related” containing a great proportion of CD34⁺/CD38⁻ hematopoietic stem cells (HSCs) or CD34⁺ cells with a strong co-expression of CD45RA/CD123, respectively. CD34⁺ cells within the latter cluster were also highly positive for PD-L1 reflecting their immunosuppressive capacity. Beyond this proof of principle study, the inclusion of additional markers will be helpful to refine the differentiation between normal HSPCs and leukemic cells, particularly in the context of minimal disease detection and antigen-targeted therapeutic interventions. Furthermore, we suggest a protocol for the assignment of new cell ensembles in quantitative terms, via a numerical value, the Pearson coefficient, based on a similarity comparison of the t-SNE pattern with a reference. Full article

Chronic myeloid leukemia is a multistep, multi-lineage myeloproliferative disease that originates from a translocation event between chromosome 9 and chromosome 22 within the hematopoietic stem cell compartment. The resultant fusion protein BCR::ABL1 is a constitutively active tyrosine kinase that can phosphorylate multiple downstream signaling molecules to promote cellular survival and inhibit apoptosis. Currently, tyrosine kinase inhibitors (TKIs), which impair ABL1 kinase activity by preventing ATP entry, are widely used as a successful therapeutic in CML treatment. However, disease relapses and the emergence of resistant clones have become a critical issue for CML therapeutics. Two main reasons behind the persisting obstacles to treatment are the acquired mutations in the ABL1 kinase domain and the presence of quiescent CML leukemia stem cells (LSCs) in the bone marrow, both of which can confer resistance to TKI therapy. In this article, we systemically review the structural and molecular properties of the critical domains of BCR::ABL1 and how understanding the essential role of BCR::ABL1 kinase activity has provided a solid foundation for the successful development of molecularly targeted therapy in CML. Comparison of responses and resistance to multiple BCR::ABL1 TKIs in clinical studies and current combination treatment strategies are also extensively discussed in this article. Full article