Purpose: Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm. Recent evidence has shown the bone marrow microenvironment in patients with AML to be intrinsically hypoxic. Adaptive cellular responses by leukemia cells to survive under low oxygenation also confer chemoresistance. We therefore asked whether therapeutic exploitation of marrow hypoxia via the hypoxia-activated nitrogen mustard prodrug, TH-302, could effectively inhibit AML growth.Experimental Design: We assessed the effects of hypoxia and TH-302 on human AML cells, primary samples, and systemic xenograft models.Results: We observed that human AML cells and primary AML colonies cultured under chronic hypoxia (1% O 2 , 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic controls. TH-302 treatment resulted in dose-and hypoxia-dependent apoptosis and cell death in diverse AML cells. TH-302 preferentially decreased proliferation, reduced HIF-1a expression, induced cell-cycle arrest, and enhanced double-stranded DNA breaks in hypoxic AML cells. Hypoxia-induced reactive oxygen species by AML cells were also diminished. In systemic human AML xenografts (HEL, HL60), TH-302 [50 mg/kg intraperitoneally (i.p.) 5 times per week] inhibited disease progression and prolonged overall survival. TH-302 treatment reduced the number of hypoxic cells within leukemic bone marrows and was not associated with hematologic toxicities in nonleukemic or leukemic mice. Later initiation of TH-302 treatment in advanced AML disease was as effective as earlier TH-302 treatment in xenograft models.Conclusions: Our results establish the preclinical activity of TH-302 in AML and provide the rationale for further clinical studies of this and other hypoxia-activated agents for leukemia therapy.
Bone loss is common in advanced cirrhosis, although the precise mechanisms underlying bone loss in cirrhosis are unknown. We studied the profile and functionality of bone‐forming cells and bone‐building proteins in bone marrow (BM) of individuals with cirrhosis (n = 61) and individuals without cirrhosis as normal controls (n = 50). We also performed dual energy X‐ray absorptiometry for clinical correlation. BM mesenchymal cells (MSCs) were analyzed for colony‐forming units‐fibroblasts and their osteogenic (fibronectin‐1 [FN1], insulin‐like growth factor binding protein 3 [IGFBP3], collagen type 1 alpha 1 chain [COL1A1], runt‐related transcription factor 2 [RUNX2], and alkaline phosphatase, liver [ALPL]) and adipogenic ( adiponectin, C1Q, and collagen domain containing [ADIPOQ], peroxisome proliferator‐activated receptor gamma [PPARγ], and fatty acid binding protein 4 [FABP4]) potentials. Colony‐forming units‐fibroblasts were lower in patients with cirrhosis (P = 0.002) than in controls. Cirrhotic BM‐MSCs showed >2‐fold decrease in osteogenic markers. Compared to controls, patients with cirrhosis showed fewer osteocytes (P = 0.05), osteoblasts, chondroblasts, osteocalcin‐positive (osteocalcin+) area, clusters of differentiation (CD)169+ macrophages (P < 0.001, each), and nestin+ MSCs (P = 0.001); this was more apparent in Child‐Turcotte‐Pugh (CTP) class C than A (P < 0.001). Multivariate logistic regression showed low nestin+ MSCs (P = 0.004) as a predictor of bone loss. Bone‐resolving osteoclasts were comparable among CTP groups, but >2‐fold decreased anti‐osteoclastic and increased pro‐osteoclastic factors were noted in patients with CTP C compared to CTP A. Bone‐building proteins (osteocalcin [P = 0.008], osteonectin [P < 0.001], and bone morphogenic protein 2 [P = 0.001]) were decreased while anti‐bone repair factors (fibroblast growth factor 23 [P = 0.015] and dipeptidyl peptidase 4 [P < 0.001]) were increased in BM and peripheral blood; this was more apparent in advanced cirrhosis. The dual energy X‐ray absorptiometry scan T score significantly correlated with the population of osteoblasts, osteocytes, MSCs, and CD169+ macrophages. Conclusion: Osteoprogenitor cells are substantially reduced in patients with cirrhosis and more so in advanced disease. Additionally, increased anti‐bone repair proteins enhance the ineffective bone repair and development of osteoporosis in cirrhosis. Hepatology Communications 2018;0:0‐0)
We examined whether potent vascular endothelial growth factor (VEGF) blockade mediated by aflibercept, a decoy VEGF receptor (VEGFR) 1/2 moiety with stronger affinity for VEGF than bevacizumab, resulted in antileukemia effects and enhanced the efficacy of systemic chemotherapy. The efficacy of aflibercept alone and in combination with doxorubicin was evaluated in human VEGF-expressing acute myeloid leukemia (AML) cell lines and primary cells xenotransplanted into immunodeficient mice. Aflibercept reduced primary VEGF/VEGFR-positive AML colony formation growth in vitro and inhibited AML xenograft growth up to 93% in association with antiangiogenic and antiproliferative effects, hypoxia, and VEGF sequestration in multiple models. High VEGF-A expression by AML cells promoted in vivo xenograft growth and aflibercept sensitivity. Aflibercept therapy slowed disease progression in two systemic human AML xenograft models and reduced peripheral leukemia disease in a primary relapsed AML model in NOD/SCID/IL2Rγnull mice. Combination aflibercept and doxorubicin enhanced antitumor effects in local xenograft models. Sequential aflibercept followed by doxorubicin resulted in progressive anthracycline accumulation in marrow and extramedullary AML sites and resulted in 2-fold higher drug levels 24 hours after administration. In contrast, tissues (tumor, plasma, marrow) treated with chemotherapy only showed progressive drug clearance over time. Combination aflibercept and doxorubicin also resulted in vascular narrowing, decreased vessel number, and perivascular apoptosis. These data suggest that inefficient drug delivery by leukemia-associated vasculature may mediate chemoresistance and support further clinical evaluation of combination aflibercept and anthracycline therapy in refractory/relapsed AML patients. Mol Cancer Ther; 9(10);
Timing of the anti-angiogenic agent with respect to the chemotherapeutic agent may be crucial in determining the success of combination therapy in cancer. We investigated the effects of sequential therapy with the potent VEGF inhibitor, aflibercept, and doxorubicin (DOX) in preclinical acute myeloid leukemia (AML) models. Mice were engrafted with human HL-60 and HEL-luciferase leukemia cells via S.C. and/or I.V. injection and treated with two to three doses of aflibercept (5-25 mg/kg) up to 3-7 days prior to doxorubicin (30 mg/kg) administration. Leukemia growth was determined by local tumor measurements (days 0-16) and systemic bioluminescent imaging (days 0-28) in animals receiving DOX (3 mg/kg) with or without aflibercept. A PK/PD model was developed to characterize how prior administration of aflibercept altered intratumoral DOX uptake. DOX concentration-time profiles were described using a four-compartment PK model with linear elimination. We determined that intratumoral DOX concentrations were 6-fold higher in the aflibercept plus DOX treatment group versus DOX alone in association with increased drug uptake rates (from 0.125 to 0.471 ml/h/kg) into tumor without affecting drug efflux. PD modeling demonstrated that the observed growth retardation was mainly due to the combination of DOX plus TRAP group; 0.00794 vs. 0.0043 h(-1). This PK/PD modeling approach in leukemia enabled us to predict the effects of dosing frequency and sequence for the combination of anti-VEGF and cytotoxic agents on AML growth in both xenograft and marrow, and may be useful in the design of future rational combinatorial dosing regimens in hematological malignancies.
Background Sepsis is common in cirrhosis and is often a result of immune dysregulation. Specific stimuli and pathways of inter‐cellular communications between immune cells in cirrhosis and sepsis are incompletely understood. Immune cell‐derived extracellular vesicles (EV) were studied to understand mechanisms of sepsis in cirrhosis. Methods Immune cell‐derived EV were measured in cirrhosis patients [Child‐Turcotte‐Pugh (Child) score A, n = 15; B n = 16; C n = 43 and Child‐C with sepsis (n = 38)], and healthy controls (HC, n = 11). In vitro and in vivo functional relevance of EV in cirrhosis and associated sepsis was investigated. Results Monocyte, neutrophil and hematopoietic stem cells associated EV progressively increased with higher Child score (P < .001)and correlated with liver disease severity indices (r2 > 0.3, P < .001), which further increased in Child C sepsis than without sepsis(P < .001); monocyte EV showing the highest association with disease stage [P = .013; Odds ratio‐4.14(1.34‐12.42)]. A threshold level of monocyte EV of 53/µl predicted mortality in patients of Child C with sepsis [Odds ratio‐6.2 (2.4‐15.9), AUROC = 0.76, P < .01]. In vitro EV from cirrhotic with sepsis compared without sepsis, induced mobilization arrest in healthy monocytes within 4 hours (P = .004), reduced basal oxygen consumption rate (P < .001) and induced pro‐inflammatory genes (P < .05). The septic‐EV on adoptive transfer to C57/BL6J mice, induced sepsis‐like condition within 24 h with leukocytopenia (P = .005), intrahepatic inflammation with increased CD11b + cells (P = .03) and bone marrow hyperplasia (P < .01). Conclusion Extracellular vesicles induce functional impairment in circulating monocytes and contribute to the development and perpetuation of sepsis. High levels of monocyte EV correlate with mortality and can help early stratification of sicker patients.
Selenium, a known antioxidant used in chemoprevention, has recently been shown to improve the efficacy of chemotherapy agents and mediate anti-angiogenic effects in preclinical solid tumor models. Prior data has also shown that sodium selenite can induce apoptosis of human acute myeloid leukemia (AML) cells in vitro. While the use of high non-toxic doses of selenium has been demonstrated to effectively enhance the therapeutic efficacy and selectivity of various anti-cancer drugs in multiple solid tumor xenograft models (Cao et al, Clin Canc Res10: 2561–2569, 2004), the in vivo effects of selenium compounds in hematological malignancies have not been extensively explored. Our laboratory has previously demonstrated the preclinical efficacy of VEGF inhibitors in human leukemia xenograft models. Here we investigated the in vitro and in vivo effects of seleno-L-methionine (SLM), alone and in combination with chemotherapy and anti-VEGF antibody, in human AML. The mechanism of action, dosage, and time course of SLM effects on AML cells (ML-2 and HL60) in vitro were determined using colorimetric (CCK-8) viability, tritiated thymidine proliferation, and Annexin-V flow cytometric assays. SLM therapy resulted in dose dependent inhibition of AML proliferation with marked induction of apoptosis and a 67–92% reduction in viable cells at 400–500 μM. Ten million AML cells (ML-2 and HL60-luciferase) were then subcutaneously engrafted into immunodeficient (SCID/SCID) mice. ML-2 tumor bearing mice were treated with PBS (vehicle), SLM (200 mcg p.o. daily), cytarabine (Ara-C 2 mg i.p. for 3 days), or combination SLM + Ara-C therapy for over two weeks. SLM showed no effective modulation of therapeutic efficacy and toxicity in AML tumor models, when combined with Ara-C. Neither SLM nor Ara-C alone or in combination resulted in significant reduction of AML growth. ML-2 and HL60-luciferase tumor bearing mice were then treated with PBS, SLM (200 mcg p.o. daily), anti-VEGF antibody bevacuzimab (BV 100mcg i.p. twice weekly), or SLM + BV for three weeks. Disease progression was monitored by tumor volume, end tumor weight, and bioluminescent imaging. Although SLM alone was ineffective in reducing HL60 leukemia growth, SLM + BV combination therapy effectively induced a 75% reduction in mean tumor volume after 21 days of treatment which was statistically greater than BV therapy alone (62% reduction). Synergistic effects of SLM+BV therapy was also confirmed by differences in mean tumor weights and bioluminescent imaging as compared with single agent BV and SLM treated mice. Similar results were seen in the ML-2 AML xenograft model. Conclusions: SLM improved the anti-angiogenic and anti-tumor effects of anti-VEGF therapy with bevacizumab in human AML xenograft models. Additional studies investigating the mechanisms of action of these synergistic effects will be presented.
Background and Aims Cirrhosis patients exhibit cytopenia, and, at times refractory neutropenia to granulocyte colony-stimulating factor (G-CSF), which acts through the CSF3-receptor (CSF3R), and changes in CSF3R can affect the response. We conducted this study to assess the CSF3R status and its relevance in cirrhotic patients. Methods Cirrhotic patients ( n =127) and controls ( n =26) with clinically indicated bone marrow (BM) examination were studied. BM assessment was done by qRT-PCR and immunohistochemistry (IHC) for CSF3R . Circulating G-CSF, CSF3R, and carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) were measured. BM hematopoietic precursor cells and their alterations were examined by flow cytometry. The findings were validated in liver cirrhosis patients who received G-CSF for severe neutropenia. Results The mean age was 48.6±13.4 years, and 80.3% were men. Circulatory CSF3R reduction was noted with the advancement of cirrhosis, and confirmed by qRT-PCR and IHC in BM. CSF3R decline was related to decreased hematopoietic stem cells (HSCs) and downregulation of CSF3R in the remaining HSCs. Cocultures confirmed that CEACAM1 led to CSF3R downregulation in BM cells by possible lysosomal degradation. Baseline low peripheral blood-(PB)-CSF3R also predisposed development of infections on follow-up. Decreased CSF3R was also associated with nonresponse to exogenous G-CSF treatment of neutropenia. Conclusions Advanced liver cirrhosis was associated with low CSF3R and high CEACAM1 levels in the BM and circulation, making patients prone to infection and inadequate response to exogenous G-CSF.
Background and aimPeripheral cytopaenias and dyspoiesis are common in cirrhosis; however, the prevalence of dyspoiesis and its contribution in cirrhosis-related cytopaenias has not been studied. We aimed to study the bone marrow (BM) dyspoiesis and its impact on peripheral blood cell counts and refractory anaemia in patients with cirrhosis.Patients and methodsWe reviewed all the BM aspirates and biopsies of cirrhotic cases, done from 2011 to 2018 for clinical indications. Dyspoiesis was considered if >5% of the precursor cells of any of the three lineages showed dyspoietic changes. Primary haematological or non-haematological malignancies, chronic kidney disease, drug intake, acute and chronic hepatitis and granulomatous disease were excluded.ResultsOf 608 these, 82 cases (13.5%) showed dyspoiesis in the BM precursors. There was no difference in age (p=0.16), gender (p=0.58) and spleen size (p=0.35) in cases with or without dyspoiesis. Majority of the cases had dyspoiesis in erythroid series (62, 75.6%) and megakaryocytes (15, 18.2%). Dyspoiesis was more prominent in alcoholics 44 cases (53.6%) and autoimmune diseases 13 cases (15.8%). Erythroid hyperplasia (47.7±14.4 vs 40±11.1; p<0.001) was more in cases with dyserythropoiesis, indicating ineffective erythropoiesis. Patients with dyspoiesis had lower haemoglobin (7.5±1.9 vs 9.3±2.2 g/dL, p<0.001). 54 (8.07%) had refractory anaemia with dyspoiesis present in 48 (88.8%) (p<0.01). Dyspoiesis was independently associated with refractory anaemia when adjusted for age, gender, aetiology and liver disease severity.ConclusionsBM dyspoiesis, especially dyserythropoiesis, is associated with severe refractory anaemia in patients with cirrhosis and requires new therapeutic approaches.
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