Primary Human Leukemia Stem Cell (LSC) Isolation and Characterization.

Leukemia stem cells (LSC) are thought to be the basis of leukemia progression since they are highly resistant to conventional chemotherapy. LSC isolation is critical in experimental studies, drug development, and application. Due to their likely hematopoietic stem cell (HSC) origin, LSCs have surface antigens that are similar to HSC. Surface markers such as CD34, CD123, CD133, and CD33 have been used extensively to assess LSCs. LSCs could be separated from other cells using magnetic selection (MS) or flow cytometry selection (FCS) methods using these markers. Understanding the role of LSCs in cancer progression and how to therapeutically target them in vitro and in vivo is critical for the development of LSC-targeting drug candidates. In this chapter, we set out to describe the primary human LSC purification and characterization processes used on patient samples with leukemia and lymphoma.

The relevance of the hematopoietic niche for therapy resistance in acute myeloid leukemia.

The expansion of acute myeloid leukemia (AML) blasts not only suppresses normal hematopoiesis, but also alters the microenvironment. The interplay of different components of the bone marrow gives rise to altered metabolic states and activates signaling pathways which lead to resistance and impede effective therapy. Therefore, the underlying processes and mechanisms represent attractive therapeutic leverage points for overcoming therapy resistance in AML. Here, we briefly discuss resistance mechanisms based on cell interactions and secreted soluble factors in the hematopoietic niche and provide an overview of niche-related therapeutic targets currently undergoing preclinical and clinical investigation which may help improve the outcome in AML therapy.

Racial and ethnic disparities in acuity of presentation among children with newly diagnosed acute leukemia.

We evaluated disparities in disease burden, organ dysfunction, vital signs, and timing of therapy in children newly presenting with acute leukemia. Among 899 patients with acute leukemia diagnosed at two large children's hospitals, a priori lab-based definitions of high disease burden, infection risk, renal dysfunction, and coagulopathy were applied to electronic health record data. Black patients with acute myeloid leukemia had increased prevalence of elevated white blood cell count and uric acid; Black patients with acute lymphoblastic leukemia demonstrated increased prevalence of coagulopathy. Black patients' presentation more frequently included multiple lab abnormalities consistent with advanced physiologic dysfunction. No differences were found in days to therapy initiation.

Regulatory role of RBM39 in acute myeloid leukemia: Mediation through the PI3K/AKT pathway.

BACKGROUND: Acute myeloid leukemia (AML) presents ongoing therapeutic challenges due to its intricate molecular pathogenesis. This study aimed to elucidate the role of RNA binding motif protein 39 (RBM39) in AML cell proliferation, apoptosis, and chemosensitivity, and its potential modulation of the PI3K/AKT pathway. METHODS: In vitro and in vivo experiments were conducted using AML cell lines (K562 and U937) and bone marrow mononuclear cells (BM-MNCs) from AML patients and healthy donors. RBM39 mRNA and protein levels were measured using qRT-PCR and Western blotting. Cells were transfected with sh-RBM39 or sh-control, and then treated with daunorubicin (DNR) or homoharringtonine (HHT) at varied concentrations. Cell proliferation, chemosensitivity, and apoptosis were assessed through CCK-8 assay and Annexin V-APC/PI staining. RNA sequencing identified differentially expressed genes (DEGs) post RBM39 knockdown. An in vivo xenograft AML model using E7070, a selective RBM39 inhibitor, was employed to evaluate RBM39 modulation effects. RESULTS: Elevated RBM39 levels were found in AML patients and cell lines compared to controls. RBM39 knockdown promoted apoptosis, curtailed cell proliferation, and enhanced chemosensitivity to DNR and HHT in vitro. Drug-resistant or relapsed AML patients displayed higher RBM39 levels. RNA sequencing after RBM39 knockdown revealed downregulated PI3K/AKT signaling. The xenograft model validated in vitro results, as E7070 treatment suppressed AML xenograft growth via RBM39-mediated PI3K/AKT pathway suppression. CONCLUSION: RBM39 plays a pivotal role in AML progression through the PI3K/AKT signaling pathway. Targeting RBM39, potentially with E7070, could inhibit proliferation and induce apoptosis in AML cells, offering a promising avenue for future AML research and treatment.

Discovery of a potent and selective CBP bromodomain inhibitor (Y08262) for treating acute myeloid leukemia.

The bromodomain of CREB (cyclic-AMP response element binding protein) binding protein (CBP) is an epigenetic "reader" and plays a key role in transcriptional regulation. CBP bromodomain is considered to be a promising therapeutic target for acute myeloid leukemia (AML). Herein, we report the discovery of a series of 1-(indolizin-3-yl)ethan-1-one derivatives as potent, and selective CBP bromodomain inhibitors focused on improving cellular potency. One of the most promising compounds, 7e (Y08262), inhibits the CBP bromodomain at the nanomolar level (IC50 = 73.1 nM) with remarkable selectivity. In addition, the new inhibitor also displays potent inhibitory activities in AML cell lines. Collectively, this study provides a new lead compound for further validation of CBP bromodomain as a molecular target for AML drug development.

Busulfan and subsequent malignancy: An evidence-based risk assessment.

BACKGROUND: The incidence of secondary malignancies associated with busulfan exposure is considered low, but has been poorly characterized. Because this alkylating agent is increasingly utilized as conditioning prior to gene therapy in nonmalignant hematologic and related disorders, more precise characterization of busulfan's potential contribution to subsequent malignant risk is warranted. PROCEDURE: We conducted a literature-based assessment of busulfan and subsequent late effects, with emphasis on secondary malignancies, identifying publications via PubMed searches, and selecting those reporting at least 3 years of follow-up. RESULTS: We identified eight pediatric and 13 adult publications describing long-term follow-up in 570 pediatric and 2076 adult hematopoietic cell transplant (HCT) recipients. Secondary malignancies were reported in 0.5% of pediatric HCT recipients, with no cases of myelodysplastic syndrome (MDS) or acute myelocytic leukemia (AML). Fatal secondary malignancies were reported in 0.8% of 1887 evaluable adult HCT recipients, and an overall incidence of secondary malignancies of 4.8% was reported in a subset of 389 evaluable adult patients. We also reviewed long-term results from eight publications evaluating lentiviral- and human promotor-based HSC-targeted gene therapy in 215 patients with nonmalignant conditions, in which busulfan/treosulfan monotherapy or busulfan/fludarabine was the only conditioning. Two malignancies were reported in patients with sickle cell disease (SCD), one of which was potentially busulfan-related. No additional malignancies were reported in 173 patients with follow-up of 5-12 years. CONCLUSION: The incidence of busulfan-related secondary malignancies is low, and likely to be substantially less than 1% in pediatric transplant recipients, especially those receiving busulfan monotherapy for nonmalignant conditions other than SCD.

Discovery of (R)-4-(8-methoxy-2-methyl-1-(1-phenylethy)-1H-imidazo[4,5-c]quinnolin-7-yl)-3,5-dimethylisoxazole as a potent and selective BET inhibitor for treatment of acute myeloid leukemia (AML) guided by FEP calculation.

The functions of the bromodomain and extra terminal (BET) family of proteins have been proved to be involved in various diseases, particularly the acute myeloid leukemia (AML). In this work, guided by free energy perturbation (FEP) calculation, a methyl group was selected to be attached to the 1H-imidazo[4,5-c]quinoline skeleton, and a series of congeneric compounds were synthesized. Among them, compound 10 demonstrated outstanding activity against BRD4 BD1 with an IC50 value of 1.9 nM and exhibited remarkable antiproliferative effects against MV4-11 cells. The X-ray cocrystal structure proved that 10 occupied the acetylated lysine (KAc) binding cavity and the WPF shelf of BRD4 BD1. Additionally, 10 displayed high selectivity towards BET family members, effectively inhibiting the growth of AML cells, promoting apoptosis, and arresting the cell cycle at the G0/G1 phase. Further mechanistic studies demonstrated that compound 10 could suppress the expression of c-Myc and CDK6 while enhancing the expression of P21, PARP, and cleaved PARP. Moreover, 10 exhibited remarkable pharmacokinetic properties and significant antitumor efficacy in vivo. Therefore, compound 10 may represent a new, potent and selective BET bromodomain inhibitor for the development of therapeutics to treat AML.

Liquid Chromatography-Tandem Mass Spectrometry-Based Therapeutic Monitoring of Plasma Atovaquone Concentrations in Pediatric Patients.

Atovaquone is an FDA-approved antiparasitic and antifungal therapeutic that is currently used as a prophylactic agent to prevent Pneumocystis carinii pneumonia (PCP) infections in acute myeloid leukemia (AML) patients after receiving hematopoietic stem cell transplantation (HSCT). Recent studies have shown that atovaquone has shown potential as an anticancer agent. The high variability in atovaquone bioavailability prompts the need for therapeutic drug monitoring, especially in pediatric patients. The goal of our study was to develop and validate the performance of an assay to quantify atovaquone plasma concentrations collected from pediatric cancer patients. Briefly, an organic-based solvent system is used to precipitate protein and extract the atovaquone content from each patient-derived plasma sample. After completing a second stage of sample dilution (5000-fold overall), a 2 µL volume of the plasma extract is analyzed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based bioanalytical method described.

Intravital Microscopy to Study the Effect of Matrix Metalloproteinase Inhibition on Acute Myeloid Leukemia Cell Migration in the Bone Marrow.

Hematopoiesis is the process through which all mature blood cells are formed and takes place in the bone marrow (BM). Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. AML progression causes drastic remodeling of the BM microenvironment, making it no longer supportive of healthy hematopoiesis and leading to clinical cytopenia in patients. Understanding the mechanisms by which AML cells shape the BM to their benefit would lead to the development of new therapeutic strategies. While the role of extracellular matrix (ECM) in solid cancer has been extensively studied during decades, its role in the BM and in leukemia progression has only begun to be acknowledged. In this context, intravital microscopy (IVM) gives the unique insight of direct in vivo observation of AML cell behavior in their environment during disease progression and/or upon drug treatments. Here we describe our protocol for visualizing and analyzing MLL-AF9 AML cell dynamics upon systemic inhibition of matrix metalloproteinases (MMP), combining confocal and two-photon microscopy and focusing on cell migration.

A real-world analysis of outcomes and healthcare costs of patients on perindopril/indapamide/amlodipine single-pill vs. multiple-pill combination in Italy.

OBJECTIVES: This analysis compared adherence, cardiovascular (CV) events and all-cause mortality incidence, and healthcare costs among hypertensive patients treated with perindopril (PER)/indapamide (IND)/amlodipine (AML) in single-pill combination (SPC) vs. multiple-pill combination, in a real-world setting in Italy. METHODS: In this observational retrospective analysis of Italian administrative databases, adult patients treated with PER/IND/AML between 2010 and 2020 were divided into two cohorts: single-pill vs. multiple-pill. Patient data were available for at least one year before and after index date. Propensity score matching (PSM) was applied to reduce selection bias. Adherence was defined as proportion of days covered: non-adherence, <40%; partial adherence, 40-79%, and adherence ≥80%. Mortality incidence and CV events as single, or composite, endpoints were evaluated after first year of follow-up. Healthcare cost analyses were performed from the perspective of the Italian National Health Service. RESULTS: Following PSM, the single-pill cohort included 12 150 patients, and the multiple-pill cohort, 6105. The SPC cohort had a significantly higher percentage of adherent patients vs. the multiple-pill cohort (59.9% vs. 26.9%, P  < 0.001). Following the first year of follow-up, incidence of all-cause mortality, and combined endpoint of all-cause mortality and CV events were lower in the SPC cohort compared with multiple-pill cohort. Average annual direct healthcare costs were lower in the single-pill cohort (€2970) vs. multiple-pill cohort (€3642); cost of all drugs and all-cause hospitalizations were major contributors. CONCLUSION: The SPC of PER/IND/AML, compared with multiple-pill combination, is associated with higher adherence to medication, lower incidence of CV events and mortality, and reduced healthcare costs.

Exploring the cytotoxicity mechanisms of copper ions and copper oxide nanoparticles in cells from the excretory system.

Copper oxide nanoparticles (CuO NPs) are widely applied in various products, including food, cosmetic, biomedical, and environmental goods. Despite their broad use, potential risks are still associated with these NPs, therefore, the aim of this study is to delve deeper into the cytotoxic effects of 85 nm CuO NPs on kidney MDCK and liver AML-12 cells, representing cell models from the excretory system. Our findings pointed out that the viability of both cell lines decreased in a concentration-dependent manner when exposed to CuO NPs. Additionally, CuO NPs induced the overproduction of reactive oxygen species (ROS) and caused depolarization of the mitochondrial membrane, thereby arresting the cell cycle at the G2/M phase in MDCK and AML-12 cells. Importantly, unlike others our study uncovered distinctive forms of cellular death induced by CuO NPs in these cell lines. MDCK cells exhibited a combination of apoptosis and autophagy while early apoptosis was predominant in AML-12 cells. Moreover, the role of Cu2+ ions and CuO NPs in exerting cytotoxic effects was investigated, revealing that MDCK cells were affected by both copper ions and NPs. In contrast, AML-12 cells experienced toxic effects solely from CuO NPs. These findings provide crucial insights into the different cell death mechanisms caused either by CuO NPs or Cu2+ ions in excretory system cells in vitro. Nevertheless, further research is needed to explore the underlying mechanisms at the in vivo level, ensuring the safe use of CuO NPs. The results suggest that specific concentrations of metal oxide NPs can impact the physiology of cells within the excretory system of various mammals, including humans, and pave the way for comparing the toxic effects between ions and nanoparticles for further nanotoxicological studies.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

[Clinical characteristics and prognosis of patients with therapy-related myelodysplastic syndrome and acute myeloid leukemia arising from malignant tumors].

Objective: To investigate the clinical characteristics, cytogenetics, molecular biology, treatment, and prognosis of patients with therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) secondary to malignancies. Methods: The clinical data of 86 patients with t-MDS/AML in West China Hospital of Sichuan University between January 2010 and April 2023 were retrospectively analyzed. The clinical characteristics, primary tumor types, and tumor-related therapies were analyzed. Results: The study enrolled a total of 86 patients with t-MDS/AML, including 67 patients with t-AML, including 1 patient with M(0), 6 with M(1), 27 with M(2), 9 with M(3), 12 with M(4), 10 with M(5), 1 with M(6), and 1 with M(7). Sixty-two patients could be genetically stratified, with a median overall survival (OS) of 36 (95% CI 22-52) months for 20 (29.9%) patients in the low-risk group and 6 (95% CI 3-9) months for 10 (14.9%) in the intermediate-risk group. The median OS time was 8 (95% CI 1-15) months in 32 (47.8%) patients in the high-risk group. For patients with non-acute promyelocytic leukemia (APL) and AML, the median OS of the low-risk group was 27 (95% CI 18-36) months, which was significantly longer than that of the non-low-risk group (χ(2)=5.534, P=0.019). All 9 APL cases were treated according to the initial treatment, and the median OS was not reached, and the 1-, 2-, and 3-year OS rates were 100.0%, (75.0±6.2) %, and (75.0±6.2) % respectively. Of the 58 patients with non-APL t-AML (89.7%), 52 received chemotherapy, and 16 achieved complete remission (30.8%) after the first induction chemotherapy. The 1-, 2-, and 3-year OS rates of the non-APL t-AML group were (42.0 ± 6.6) %, (22.9±5.7) %, and (13.4±4.7) %, respectively. The median OS of patients who achieved remission was 24 (95% CI 18-30) months, and the median OS of those who did not achieve remission was 6 (95% CI 3-9) months (χ(2)=10.170, P=0.001). Bone marrow CR was achieved in 7 (53.8%) of 13 patients treated with vineclar-containing chemotherapy, with a median OS of 12 (95% CI 9-15) months, which was not significantly different from that of vineclar-containing chemotherapy (χ(2)=0.600, P=0.437). In 19 patients with t-MDS, the 1-, 2-, and 3-year OS rates were (46.8±11.6) %, (17.5±9.1) %, and (11.7±9.1) % with a median OS of 12 (95% CI 7-17) months, which was not significantly different from that in t-AML (χ(2)=0.232, P=0.630) . Conclusions: Breast cancer, bowel cancer, and other primary tumors are common in patients with t-MDS/AML, which have a higher risk of adverse genetics. Patients with APL had a high induction remission rate and a good long-term prognosis, whereas patients without APL had a low remission rate and a poor long-term prognosis.