Proliferation Of Myeloid Progenitor Cells Research Articles

In Vivo Chemical Screening in Zebrafish Embryos Identified FDA-Approved Drugs That Induce Differentiation of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy.

A prediction model for reactivation of Langerhans cell histiocytosis based on machine-learning algorithms.

Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplasm characterized by the clonal proliferation of myeloid progenitor cells. The reactivation rate of LCH exceeds 30%. However, an effective prediction model to predict reactivation is lacking. To select potential prognostic factors of LCH and construct an easy-to-use predictive model based on machine-learning algorithms. Clinical records of LCH inpatients in the Second Xiangya Hospital of Central South University, from 2008 to 2022, were retrospectively studied. Seventy-six patients were classified into a reactivated/progressive group or a stable group. Clinical features and laboratory outcomes were compared, and machine-learning algorithms were used for building prognostic prediction models. Clinical classification (single-system LCH, multiple-system LCH, and central nervous system/lung LCH), level of anemia, bone involvement, skin involvement, and elevated monocyte count were the best performing factors and were finally chosen for the construction of the prediction models. Our results show that the above-mentioned five factors can be used together in a prediction model for the prognosis of LCH patients. The major limitations of this study include its retrospective nature and the relatively small sample size.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Improving Anti-Tumor Efficacy of CAR T-Cell Therapy for Acute Myeloid Leukemia (AML): Results from a Multi-Drug Interaction Screening Approach

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and impaired differentiation of myeloid progenitor cells. With a 5-year overall survival of approx. 30%, the prognosis of patients with acute myeloid leukemia remains poor, in particular for those with relapse or refractory disease. The adoptive transfer of T-cells genetically modified to express a chimeric antigen receptor (CAR) have shown impressive remission rates in patients with B-cell lymphoid malignancies. In contrast, CAR T-cell therapy for AML has induced durable remission only in a minority of patients so far, highlighting the need for strategies to improve anti-tumor efficacy and persistence of AML-specific CAR T-cell products. Adapting an established high throughput screening platform, we aimed to identify compounds with the potential to enhance CAR T-cell mediated cytotoxicity through interaction with tumor and/or CAR T-cells. Methods: We established a high throughput platform to determine the cytotoxicity of four different AML-specific CAR T-cell products targeting CD33 (CD33.CD28.41BB.zeta), CD70 (CD27.zeta), CLL1 (CLL1.CD28.zeta) and EMR2 (also known as ADGRE2; EMR2.41BB.zeta) in the presence of 32 compounds at five different concentrations in a 10,000-fold concentration range. CAR T-cells were generated by transduction of activated T-cells derived from peripheral blood mononuclear cells (PBMCs) of three healthy donors with a gamma retroviral vector containing the different CAR constructs. On day 11 after activation, CAR T-cells and non-CAR-transduced control T-cells were incubated with luciferase-expressing target cells at an effector-target ratio of 1:4 in 384-well plates. Viability of target cells cultured with CAR T-cells, control T-cells or in the absence of T-cells was calculated after 24 and 48 hours using a luciferase signal intensity assay. Combinatorial toxicities were calculated with the Bliss Independence model for synergistic effect and the highest single agent model for additive combination effect. Results: In quality analysis, the intensity of luminescence signals in control wells (tumor only, non-CAR-transduced control T-cells) of different plates was similar and principal component analysis (PCA) of samples derived from different days (batches) did not show significant differences ruling out relevant edge or batch effects that could negatively impact the screening results. Of all tested compounds birinapant, a peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of apoptosis protein (IAP) family proteins and the hypomethylating agent (HMA) decitabine showed the strongest improvement of CAR T cell cytotoxicity in an additive but also in a synergistic manner. These effects were evident in CAR T-cell products derived from all different T cell donors without significant differences. Depending on the structural composition of the CAR construct, the strongest drug-induced improvement of CAR T-cell cytotoxicity was seen either after 24 or 48 hours. The third generation CAR T- cell construct (CD33.CD28.41BB.zeta) showed enhanced cytotoxicity already after 24 hours, whereas significant anti-leukemic efficacy of CD27.zeta, CLL1.CD28.zeta and EMR2.41BB.zeta CAR T-cells became evident at the later 48 hours timepoint. Importantly, co-treatment with birinapant and decitabine particularly enhanced the cytotoxicity of EMR2-specific CAR T-cells which exhibited the lowest baseline anti-tumor efficacy compared to all other CAR constructs highlighting the potential of distinct chemical compounds to improve the functionality of AML-specific CAR T-cells. Conclusion: Our data suggest that high throughput drug interaction screens are a reliable approach to investigate the impact of chemical compounds on the functionality of AML-specific CAR T-cell products. HMA and SMAC mimetics are promising candidates that may potentially enhance the cytotoxicity of CAR T-cells. Ongoing studies are evaluating the effect of HMAs and SMAC mimetics on CAR T-cell proliferation and anti-tumor efficacy against AML cell lines and primary AML patient samples upon serial antigen stimulation. Furthermore, in-depth proteogenomic characterization to identify molecular signatures of enhanced functionality are currently being performed.

Real-World Treatment Patterns and Overall Survival Among Newly Diagnosed and Relapsed/Refractory Acute Myeloid Leukemia (AML) Patients: A Retrospective Cohort Study Using Claims Data

Introduction: Acute myeloid leukemia (AML) is a malignant disorder of the bone marrow, characterized by abnormal clonal proliferation and differentiation arrest of myeloid progenitor cells. Prognosis is generally poor and worsens with age. While curative therapies are available, they are often unavailable to older patients and those with significant comorbidities due to high morbidity and mortality associated with intensive therapy. Since 2017, seven new systemic therapies have been approved for various AML patient populations, including those unfit for intensive therapy. This study aims to characterize first-line (1L) and second-line (2L) treatment patterns and outcomes in newly diagnosed (ND) and relapsed/refractory (R/R) AML patients, in the context of these newly available therapies. Methods: A large US administrative claims database (Optum) was used to identify ND AML patients between January 1, 2016, and August 31, 2022. The “ND index date” was defined as the first date a non-R/R AML diagnosis code was observed. To confirm that the initial AML diagnosis represented a true case of AML, ≥2 subsequent AML diagnoses within 60 days post-index were required. A washout period of 12 months was used to confirm no previous AML diagnosis. ND patients were required to have continuous enrollment from 1 year prior to the index until ≥30 days after the index date or death, whichever was earlier. A subgroup of R/R patients was then identified: all ND cohort members who 1) had an R/R AML diagnosis code, 2) had continuous enrollment between ND index date and ≥30 days after the RR index date, and 3) did not receive 1L hematopoietic stem cell transplant (HSCT) after the ND index date were eligible for inclusion. In this subgroup, the “RR index date” was defined as the first date an R/R AML diagnosis code was observed. Demographic characteristics, clinical characteristics, and treatment patterns over time were reported descriptively. Treatment regimens over time were described using the following classification: HSCT, intensive chemotherapy without targeted agents, intensive chemotherapy with targeted agents, hypomethylating agent (HMA) therapy only, other targeted therapy, Venetoclax-based therapy, unspecified/other treatment, no treatment/supportive care only. Overall survival (OS) was described using the Kaplan-Meier estimator. All analyses were stratified by line of therapy and transplant status. Results: We identified 5,135 ND AML patients and 934 R/R AML patients (ND cohort: mean age at initial AML diagnosis 73.5 years, mean follow-up time 6.1 months). ND patients receiving transplant in 1L tended to be younger and had fewer comorbidities than those not receiving transplant ( Table 1). Over the entire study period, 61% of ND patients and 69% of R/R patients received some treatment for AML; older patients were more likely to receive supportive care only. In 1L, Venetoclax-based and intensive chemotherapy without targeted agents were the most common treatment regimens (21% and 17% of all ND patients). In 2L, Venetoclax-based and HMA-only therapies were most common (17% and 16% of all R/R patients). Increases in the use of novel therapies (Venetoclax, intensive chemotherapy with targeted agents, or other targeted therapies) were observed in both 1L and 2L over the study period ( Figure 1). 1-year OS among ND patients was 32% in non-transplant recipients, and 84% in transplant recipients; among R/R patients, 1-year OS from RR index date was 21% in non-transplant recipients and 81% in transplant recipients. Conclusions: The findings from the present study indicate substantial disease burden and ongoing unmet need among ND and R/R AML patients in the USA. While treatment patterns for AML have changed significantly following the advent of novel therapies (with 41% of 1L patients and 31% of 2L patients being treated with novel therapies in 2022), OS remained low. Notably, around 1/3 of patients with RR AML did not receive active treatment; these patients tended to be older and were more frail. Increasing off-label use of Venetoclax in 2L further underscores high unmet need for patients who require more effective treatment options.

Effect of the Serum Level of Interleukin-6 and Interleukin-10 on Chemotherapy in Acute Myeloid Leukemia Iraqi Patients

Background: Acute myeloid leukemia (AML), a cancer of the myeloid line of blood cells, It is characterized by malignant clonal proliferation and differentiation of immature myeloid progenitor cells that differentiate into malignant myeloblasts which cannot function like normal blood cells. Normally, cytokines are secreted by different types of cells to regulate the immune response, but in AML patients, cytokines can be produced by both leukemic blasts and immune system cells and their role in the pathogenesis is not clear .Blood cells and their marrow based progenitors are exquisitely responsive to their environment, and cytokines are an essential part of it. The overexpression of cytokines in leukemia patients declines in complete remission suggesting that these events are dependent on AML activity. Aim of the study is to determine the serum levels of IL-6, IL-10 in newly diagnosed, under treatment and relapsed acute myeloid leukemia Iraqi patients and show their relation to response to chemotherapy and used as a prognostic markers in the evaluation of the therapy making them highly applicable to routine clinical laboratories. Methods: Serum levels of IL-6, IL-10 were estimated by using ELISA kit, in 120 patients with AML, That divided into 40 patients for each group (newly diagnosed, under treatment and relapsed), depending on the stages of the patients with chemotherapy, from February 2022 to April 2023 from Baghdad Teaching Hospital, Baghdad, Iraq. Forty healthy controls were also enrolled in this study. Results: IL-6 and IL-10 levels were significantly higher in newly diagnosed and relapsed patients with AML than in control group and their levels decreased when patients responded to chemotherapy. Conclusion: The current study showed that the serum concentrations of IL-6 and IL-10 in AML patients increased before chemotherapy and began to decreased after therapy. These results may be used as a prognostic markers for the success of chemotherapy and could offer an interesting approach for treatment of AML.

CYLD stimulates macrophage phagocytosis of leukemic cells through STAT1 signalling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most aggressive hematopoietic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells within the bone marrow. Tumor suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme, which suppresses inflammatory response in macrophages. Macrophages have a central role in the defense against foreign substances and circulating cancer cells by their professional phagocytic capacity. Little is known about contributions of CYLD to changes in biological properties of human macrophages and its involvement in AML. The present study, therefore, explored whether macrophage functions in healthy individuals and AML patients are influenced by CYLD. To this end, ninety-two newly diagnosed AML patients and 80 healthy controls were recruited. The mRNA expression levels of inflammation-related genes were evaluated by real-time PCR, cell maturation, phagocytosis and apoptosis assays by flow cytometry and secretion of inflammatory cytokines by ELISA. As a result, AML patients with the low CYLD expression were significantly higher in M4/M5 than other subtypes according to the FAB type. The low CYLD expression was also closely associated with older patients and enhanced level of LDH in AML. Moreover, treatment of normal macrophages with CYLD siRNA enhanced activation of STAT-1, leading to increases in expressions of maturation markers and IL-6 production as well as suppression in cell apoptosis and phagocytosis, while macrophage phagocytosis from AML M4/M5b was higher than that from healthy controls upon CYLD siRNA transfection through STAT1 signalling. In conclusion, the inhibitory effects of CYLD on macrophage functions are expected to affect the immune response in AML.

Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells.

Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient's prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.

The Class IIA Histone Deacetylase (HDAC) Inhibitor TMP269 Downregulates Ribosomal Proteins and Has Anti-Proliferative and Pro-Apoptotic Effects on AML Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML.

Seasonal plasticity in immunocompetent cytokines (IL-2, IL-6, and TNF-α), myeloid progenitor cell (CFU-GM) proliferation, and LPS-induced oxido-inflammatory aberrations in a tropical rodent Funambulus pennanti: role of melatonin.

In seasonal breeders, photoperiods regulate the levels of circulatory melatonin, a well-known immunomodulator and an antioxidant. Melatonin is known to play a complex physiological role in maintaining the immune homeostasis by affecting cytokine production in immunocompetent cells. In this study, we have quantified seasonal and temporal variations in immunocompetent cytokines-IL-2, IL-6, and TNF-α-and circulatory corticosterone along with in- vitro proliferation of bone marrow-derived granulocyte macrophage-colony forming unit (CFU-GM) progenitor cells of a tropical seasonal breeder Funambulus pennanti (northern palm squirrel). Transient variations in antioxidant status of seasonal breeders might be due to the fluctuations associated with immunity and inflammation. Further,to establish a direct immunomodulatory effect of photoperiod, we recorded the LPS-induced oxidative and inflammatory responses of squirrels by housing them in artificial photoperiodic chambers mimicking summer and winter seasons respectively. We observeda marked variation in cytokines level, melatonin, and corticosterone , and CFU-GM cell proliferation during summer and winter seasons. High Peripheral melatonin levels directly correlated with cytokineIL-2 levels, and inversely correlated with TNF-α, and circulatory corticosterone level. LPS-challenged squirrels housed in short photoperiod (10L:14D; equivalent to winter days) showed a marked reduction in the components of the inflammatory cascade, CRP, TNF-α, IL-6, NOx, NF-κB, Cox-2, and PGES, with an overall improvement in antioxidant status when compared to squirrels maintained under a long photoperiod (16L:8D; equivalent to summer days). Our results underline the impact of seasonality, photoperiod, and melatonin in maintaining an intrinsic redox-immune homeostasis which helps the animal to withstand environmental stresses.

Multiplex Drug Screening Identifies a Novel Therapeutic Backbone for the Treatment of Acute Myeloid Leukaemia

Introduction: Acute Myeloid Leukemia (AML) is a blood cancer characterized by excessive proliferation, abnormal self-renewal, and/or differentiation defects of myeloid progenitor cells. Genomic sequencing has shown heterogeneous subpopulations characterized by distinct genetic and epigenetic alterations, suggesting a combination of therapies are required to effectively treat this disease. Combination therapies are a preferable and superior approach for the treatment of leukaemia: they can work in a synergistic manner allowing a lower therapeutic dose to be achieved, minimising potential long-term effects. Combination therapies can overcome adaptive resistance by targeting multiple disease drivers. To identify novel and effective drug combinations for the treatment of AML, we performed in vitro and ex vivo sensitivity profiling against a panel of 1128 pairwise combinations. Methods: The combinations were designed using a multiplex screening protocol established in our lab from a panel of 48 compounds representing standard of care chemotherapy and other near clinic agents, the doses used for each compound were an approximate IC30 based on published data for in vitro cell death. Using our all-pairs testing algorithm, compounds were grouped into pools of 5 compounds per well generating 1128 (48 x (48-1)/2) pairwise combinations to be accommodated in 172 individual wells. Concurrently, with the combination screen, a single-agent screen of the 48 compounds was performed. The biological readout for this experiment was CellTox™ Green Cytotoxicity Assay, fluorescence intensity was evaluated following 72-hour incubation. Four AML cell lines (OCI-AML3, THP-1, HL60 and SKM-1) representing a spectrum of cytogenetic abnormalities were used for this screening approach. Leukemic cells were isolated from the peripheral blood (PB) or bone marrow (BM) of fresh, diagnostic patient samples and ex vivo sensitivity profiling assessed. For each combination a robust Z-score was calculated using normalised, relative fluorescence values. In parallel, cell viability was calculated using a cytotoxic control as maximum cell death. Combinations which demonstrated a Z-score >2 and/or a cell viability of <30% were deconvoluted into 10 pairwise and 10 triple combinations to identify a clinically feasible combination. Results: Five, 5-compound combinations were taken forward for deconvolution in the OCI-AML3 cells. This identified the pairwise combination of dactinomycin, a chemotherapy medication and vorinostat, a histone deacetylase (HDAC) inhibitor, by demonstrating a significant reduction in cell viability (p=0.039). Combination Index (CI) values, calculated using the Chou and Talalay method, indicated a synergistic (CI=<1) relationship in OCI-AML3 cells. Previous studies indicate that dactinomycin is an effective therapeutic option for AML patients with a NPM1 gene mutation. Given the NPM1 mutant status of the OCI-AML3, we validated using 4 AML cell lines (OCI-AML2, HL60, Kasumi-1 and MV4-11) all with WT NPM1. Kasumi-1 and MV4-11 cells showed a significant reduction in cell viability with this combination (p=0.0003 and p=0.025, respectively). This was accompanied with a significant increase in Annexin V positivity in both of these cell lines (p=<0.005 and p=<0.0001, respectively). The highest scoring 5-compound combination across patient samples was combination 114 (Average Z-Score = 1.983), comprising enasidenib, idarubicin, pracinostat, ruxolitinib and dactinomycin. In 57% of the patient samples deconvolution identified the triple combination of a therapeutic backbone of idarubicin, an anthracycline chemotherapy and pracinostat, a HDAC inhibitor, with the addition of a "patient” specific third agent. Validation confirmed that the combination of idarubicin and practinostat can significantly reduce viability in comparison to the single agents (p=<0.05). Conclusion: In this study we demonstrate the utility of a novel multiplex screening approach with a custom panel of compounds to identify combination therapies for the treatment of AML. The combination of an anti-tumour antibiotic chemotherapy (dactinomycin and idraubicin) and a HDAC inhibitor (vorinostat and pracinostat) demonstrated the greatest efficacy following in vitro and ex vivo sensitivity profiling. Further investigations are required to elucidate the mechanism of action of these novel combinations.

Molecular Pathogenesis of Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are chronic hematological malignancies characterized by increased proliferation of MPN stem and myeloid progenitor cells with or without bone marrow fibrosis that typically lead to increased peripheral blood cell counts. The genetic and cytogenetic alterations that initiate and drive the development of MPNs have largely been defined, and we summarize these here. In recent years, advances in understanding the pathogenesis of MPNs have defined a long-preclinical phase in JAK2-mutant MPN, identified genetic loci associated with MPN predisposition and uncovered mechanistic insights in CALR-mutant MPN. The integration of molecular genetics into prognostic risk models is well-established in myelofibrosis and ongoing studies are interrogating the prognostic implications of concomitant mutations in ET and PV. Despite all these advances, the field is deficient in clonally selective therapies to effectively target the MPN clone at any stage of disease, from pre-clinical to advanced. Although the biological understanding of the pathogenesis of MPNs has progressed quickly, substantial knowledge gaps remain, including in the molecular mechanisms underlying MPN progression and myelofibrotic transformation. An ongoing goal for the MPN field is to translate advances in biological understanding to improved treatments for patients.

Over expression of mTOR gene predicts overall survival in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) is defined as heterogenous disease, it contains heterogenous leukemic stem cells with various degree of cell differentiation. The perturbation of genes involved in myeloid progenitor cell growth, differentiation and proliferation lead to morphologic dysplasia, maturation arrest, ineffective hematopoiesis hence the cytopenias and propensity to develop into acute myeloid leukemia (AML). Heterogeneous subsets of MDS patients have been defined by their clinical and biologic abnormalities. These different features lead to the development of different prognostic system; however, these approaches are limited in predicting clinical course, and management of patients remains challenging given the uncertainty of the time course of disease progression. It is of importance to identify transcriptomic marker causing maturational and differentiation arrest which could help in understanding the pathogenesis of disease. We have studied differential gene expression profiles (GEPs) in CD34 + marrow cells from myelodysplastic syndrome (MDS) patients (n = 14) and control CD34 + cells using Affymetrix Human Clariom S microarray with 20,000 well annotated genes. We found 4165 genes significantly (p < 0.05) differentially expressed in MDS. Using stringent bioinformatics analyses, we were able to identify few genes (MAPK8, JUNB, mTOR) which were differentially upregulated i.e. 5.39, 73.61 and 2.7 fold change observed in MDS than control and also validated (n = 60) these genes by RT - qPCR. Kaplan - Meier survival analysis indicated that MAPK8 and JUNB could be poor prognostic marker as patients with increased expression showed poor survival, whereas surprisingly mTOR increased expression proved to be good prognostic marker. The correlation analysis showed that the level of gene (MAPK8, JUNB, mTOR) expression was significantly (p ≤ 0.05) associated with frequency of genetic lesions. Interestingly the increased expression of MAPK8 was significantly accompanied with ASXL1 gene mutation. Our study showed an elevation of TNF and AMPK signalling pathways in MDS. TNF signalling might be mediating the proliferative advantage to myeloid clonal cells (mutation carrying cells) over normal cells, whereas, AMPK signalling could be acting as protector against it (favouring normal cells). Hence it would be interesting to explore the functions and pathways associated with mTOR, AMPK, MAPK8 and JUNB in myelopoiesis related diseases like MDS.

141 (PB021) - Novel iron-mediated cell death (Ferroptosis) inducer, HSB-1216, suppress acute myeloid leukemia growth

Acute myeloid leukemia (AML) is a heterogeneous and intrinsically resistant myeloid stem cell neoplasm, occurring in about 14 000 patients in the US annually. AML is characterized by the unbridled proliferation of myeloid progenitor cells and a block in normal differentiation.

P979: EXOSOMES IN POLYCYTHEMIA VERA: “MINI PLATELETS“ WITH THROMBOGENIC POTENTIAL

Background: Polycythemia vera (PV) is characterized by clonal proliferation of myeloid progenitor cells that is caused by activating mutation in the Janus Kinase (JAK)2 gene. Patients with PV tend to develop thrombotic complications across the vasculature tree. Yet, how PV-clonal cells induce pro-thrombotic environment is still under investigation. Secreted by all types of cells, exosomes are nano-scaled particles which carry a molecular cargo that reflects their cell of origin. Exosomes travel in blood, may reach distant sites and affect various physiological and pathological processes. These features led us to think that PV-derived exosomes (hereafter PV-exosomes) carry the mutated JAK2 oncogene and contribute to the thrombotic manifestations seen in PV. Aims: Determine whether PV-exosomes carry mutated JAK2 transcripts. Determine the prothrombotic potential of PV-exosomes and their effect on endothelium. Methods: Exosomes were isolated from eythroleukemia (HEL 92.1.7) cell line (positive for mutated JAK2) and from patients with PV by serial ultracentrifugation. To confirm the presence of exosomes, electron microscopy images were obtained and to quantify their concentration nanoscale tracking analysis was applied (Malvern). The exosomes were stained with the FM-4-11 lipophilic dye and their uptake by HUVECs and HaCaT cells was followed by flow cytometry. To determine the presence of JAK2 transcript, a 465 bp long DNA segment within exon14 of JAK2 was amplified by qRT-PCR and by Sanger sequencing JAK2 mutational status was determined. Thrombin generation assay (TGA) (Ceveron®, Alpha, Technoclone) was used to assess the thrombotic potential of PV-exosomes. Platelet activation status was assessed by the expression of PAC-1, CD-62 and CD-63 in platelets and Trans Epithelial Electric Resistance (TEER) (Electric cell substrate impedance sensing device, Applied Biophysics) was used to assay endothelial dysfunction in HUVECs. Results: We isolated exosomes from the sera of 24 healthy donors and 31 patients with PV. We found the presence of the mutated JAK2 mRNA in all samples confirming that the mutated JAK2 transcript is packaged into sera-exosomes. Yet, while healthy donors’ derived exosomes carry only the wild type JAK2 mRNA, we detected both the wild type and the mutated JAK2 isoforms in patients with PV. We showed that PV-exosomes induced thrombin generation. Then we compared PV-exosomes and healthy-individuals’ derived exosomes and found that thrombin generation potential of PV-exosomes is higher in all measured parameters (p< 0.05 in all parameters). Furthermore, using various platelet markers (CD62, CD63, PAC1) we confirmed that PV-exosomes induced platelet activation. Together, these findings suggest that PV-exosomes may promote thrombosis directly. Under physiological conditions the integrity of the vascular wall prevents uncontrolled thrombi. Therefore, we wondered whether PV-exosomes induce endothelial dysfunction, and in this way contribute indirectly to the pro-thrombotic phenotype. PV-exosomes were taken up by HUVEC endothelial cells in a dose- and time- dependent manner and TEER assay revealed that exosomal uptake by HUVECs induced endothelial dysfunction by breaking the tight junction between adjacent endothelial cells. Summary/Conclusion: Exosomes may act as “mini platelets” that promote thrombosis in patients with PV. PV-exosomes may promote thrombosis either directly by activating platelets and inducing thrombin generation or indirectly by inducing endothelial dysfunction.

Abstract 3849: Exosomes in polycythemia vera: "mini platelets" with oncogenic and thrombogenic potential

Abstract Polycythemia vera (PV) is a classical "myeloproliferative neoplasm" characterized by clonal proliferation of myeloid progenitor cells caused by their acquisition of a mutation in the JAK2 gene. Its common complications are thrombosis and second primary malignancies. Secreted from virtually all cell types, exosomes are extracellular vesicles that carry bioactive cargo derived from their cells of origin. When engulfed, their cargo may alter the phenotype of the recipient cells. For example, neoplastic exosomes modulate the cellular makeup of bystander cells. Therefore, since mutated JAK2 is present in virtually all PV-patients, we hypothesize that PV-derived exosomes (hereafter PV-exo) carry the mutated JAK2 oncogene and are taken up by non-clonal cells, thus contributing to the thrombotic manifestations and oncogenic potential of patients with PV. We isolated and characterized exosomes from PV-patients and from the Human erythroleukemia (HEL) cell line (hereafter HEL-exo). We exposed normal cells implicated in thrombosis and skin cells to these exosomes and examined the cells' phenotypic changes and activation after exposure. Using Sanger sequencing we discovered that both PV-exo and HEL-exo, like their parental cells, carry the mutated JAK2 transcripts, allowing clonal cells to export oncogenes to distant, non-clonal sites. Our preliminary findings suggest that these exosomes promote pro-coagulant and malignant phenotypes in multiple ways. Thrombin generation assay pointed to a significantly higher generation of thrombin in the presence of PV-exo than those derived from healthy donors. Similarly, flow cytometry analysis revealed that PV-exo increased the expression of platelet activation markers. Moreover, Trans Endothelial Electrical Resistance (TEER) assay results suggest that HEL-exo induces endothelial dysfunction. Furthermore, using the Sulforhodamine B assay, we found that exposure to HEL-exo increased the proliferation of normal keratinocytes. In summary, we developed the concept of exosomes as “mini metastasis” that might promote disease manifestations either directly or indirectly. Since unlike solid tumors, hematological malignancies do not metastasize, exosomes derived from neoplastic hematological cells may fulfill this function. Such exosomes may spread oncogenes (e.g., mutated JAK2), damage non-clonal tissues (e.g., endothelial dysfunction) or act as a miniature reflection of their cells of origin (e.g., exosomes as mini platelets) in a parallel way to "real" metastasis; this concept may affect treatment related decisions. For example, by reducing clonal burden, early treatment even in “low risk” patients may prevent exosomal spread and exosomal-dependent damage to distant tissues. The results of our study highlight PV-exo as promoters of PV-complications, thrombosis and malignant transformation. Citation Format: Orit Uziel, Adi Shacham-Abulafia, Galia Spectre, Ester Ziv, Karyn Revital Geiger, Zinab Sarsor, Neria Ron, Einat Beery, Pia Raanani, Shoshana Revel-Vilk, Mira Naamad, Uri Rozovski. Exosomes in polycythemia vera: "mini platelets" with oncogenic and thrombogenic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3849.

Therapeutic Management of Patients with FLT3 + Acute Myeloid Leukemia: Case Reports and Focus on Gilteritinib Monotherapy

Acute myeloid leukemia is a malignant disorder of the bone marrow, characterized by differentiation, clonal expansion, and uncontrolled proliferation of malignant myeloid progenitor cells and by several molecular and genetic abnormalities. A mutation of FMS-like tyrosine kinase 3 gene can be observed in about one-third of cases of acute myeloid leukemia. Two FLT3 inhibitors are actually approved for FLT3 mutated acute myeloid leukemia: midostaurin, a multikinase first generation inhibitor with lower affinity for FLT3 binding, and gilteritinib fumarate, a potent second-generation inhibitor of both FLT3-ITD and TKD. Gilteritinib is a new effective and well-tolerated drug for patients with relapsing or refractory FLT3-positive acute myeloid leukemia. Thanks to its efficacy, low toxicity, its good manageability (oral formulation), this drug is suitable for all the patients, including elderly frail patient with concomitant therapies or pre-existing or underlying diseases, and can be used also in the outpatient setting, reducing risks and costs related to the hospitalization. We report and discuss seven cases of different patients with FLT3 positive acute myeloid leukemia successfully managed with gilteritinib in the real clinical practice.

Dermatologic Manifestations of Systemic Diseases in Childhood

Dermatologic Manifestations of Systemic Diseases in Childhood

Essential Roles of Transcription Factor MEF2D in the Maintenance of MLL-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease with uncontrolled proliferation of myeloid progenitor cells and a failure of proper cell differentiation. Chromosomal translocations of the KMT2A (MLL) gene are found in about 10% AMLs affecting both children and adults. Patients harboring MLL rearrangement (MLL-r) have a high relapse rate and poor overall survival, emphasizing an unmet need to understand MLL-r AML pathogenesis and develop new therapeutic means. Through a comparative analysis of super-enhancers in AML cell lines and primary samples, we discovered that MLL-r leukemia cells preferentially gain super-enhancer signals at the MEF2D gene. MEF2D is aberrantly activated in MLL-r AML patient samples and predicts poor disease outcomes, implying a potential oncogenic role of MEF2D. Indeed, depletion of MEF2D inhibits human MLL-r leukemia growth and induces profound differentiation. Mechanistically, MEF2D directly represses CEBPE, a master regulator of myeloid differentiation. CEBPE depletion could rescue the growth defect and cell differentiation induced by MEF2D loss. We also demonstrated that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced anti-leukemia effects. Interestingly, MEF2C, a target of MLL fusion oncoproteins, is by far the most studied MEF2 family member with essential roles in MLL-r AML. Our genetic, biochemical, and integrative genomic data shows that MEF2D and MEF2C, both upregulated in MLL-r AML, interact with each other, colocalize genome-widely, and co-regulate target gene expression. We are currently investigating the molecular mechanisms underlying the interdependent function between MEF2 paralogs in MLL-r AML. Collectively, our findings suggest that MEF2D is a novel transcriptional dependency in MLL-r AML and uncover the MEF2-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block. DisclosuresNo relevant conflicts of interest to declare.