No abstract

Abstract(–)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

No abstract

Green tea catechins are bioactive polyphenol compounds which have attracted significant attention for their diverse biological activities and potential health benefits. Notably, epigallocatechin-3-gallate (EGCG) has emerged as a potent apoptosis inducer through mechanisms involving caspase activation, modulation of Bcl-2 family proteins, disruption of survival signaling pathways and by regulating the redox balance, inducing oxidative stress. Furthermore, emerging evidence suggests that green tea catechins can modulate epigenetic alterations, including DNA methylation and histone modifications. In addition to their apoptotic actions, ROS signaling effects and reversal of epigenetic alterations, green tea catechins have shown promising results in promoting the differentiation of leukemia cells. This review highlights the comprehensive actions of green tea catechins and provides valuable insights from clinical trials investigating the therapeutic potential of green tea catechins in leukemia treatment. Understanding these multifaceted mechanisms and the outcomes of clinical trials may pave the way for the development of innovative strategies and the integration of green tea catechins into clinical practice for improving leukemia patient outcomes.

Background: Primary Myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) characterized by increased myeloid proliferation and associated with mutations that induce tyrosine-kinase activation mainly via JAK-STAT pathway, culminating in extensive bone marrow (BM) fibrosis in the course of disease progression. In contrast to the monoclonal origin of hematopoietic cells, fibroblasts proliferation is polyclonal, and mediators involved in fibrosis, neoangiogenesis and osteosclerosis seem to be involved in disease progression. Metformin (MTF) is a biguanide that exerts selective antineoplastic activity in a variety of malignancies, through its action on nutrients privation and hypoxia, leading to apoptosis. In JAK2-mutated cell lines, MTF reduced cell viability, proliferation and clonogenicity, while in Jak2V617F knock-in-induced mice, MTF reduced Ba/F3 JAK2V617F tumor burden and splenomegaly. These data suggest that MTF could have a therapeutic effect in PMF patients. Aims: To conduct an open label phase II study to evaluate MTF effects on BM fibrosis, inflammation mediators, JAK-STAT pathway activation and disease progression in PMF patients. Methods: PMF non-diabetic adults were eligible. Subjects with severe renal function impairment were not included. Patients received MTF (Glifage XR®) in rising doses until a maximum of 2500mg PO daily, according to tolerance. Primary endpoint was BM fibrosis reversion. Secondary endpoints included reduction of inflammation and downregulation of the JAK-STAT pathway. Clinical data was systematically compiled. Blood and BM samples were collected at the time points: pretreatment (0), 3 mo and 6 mo. Collagen was evaluated in BM biopsy specimens by Masson's trichrome stain: three representative areas from each slide were analyzed and the collagen/sample area was quantified using Image J software; the mean percentage of each slide was used for statistics. IL-6, IL-8 and TNF-α levels were analyzed in BM samples using multiplex assay. Phosphorylation status of intracellular proteins STAT3 and STAT5 was analyzed by flow cytometry and the percentage of cells was recorded using FlowJo software. In order to evaluate gene modulation following MTF exposure, samples at time points 0 and 6 mo were analyzed by PCR array for insulin signaling genes (PAHS-030Z, Qiagen). Genes with ±1.5 fold-change in both directions were selected for validation. For each experiment, statistical analysis was performed and a p value <0.05 was considered statistically significant. Results were expressed as medians (min-max). This trial was approved by the Institutional and National Review Board; written informed consent was obtained from all subjects. Results: 11 patients (aged 40-84y) were included. Two subjects had early treatment discontinuation due to non-related causes. The median exposure to MTF was 10 mo (5-11) and the median dose was 2000mg/day (1500-2500mg). The most frequent adverse event was diarrhea (n=3). No life threatening event occurred. A reduction in BM collagen area percentage was observed comparing pretreatment biopsies (26.9% (14.8-53.1%)) versus 3 months (3.8% (2.3-4.0%), p=0.062) and versus 6 months of MTF use (0.84% (0.12-17.1%), p=0.125), however, this result was not statistically significant probably due to the low number of patients analyzed (n=5). IL-6, IL-8 and TNF-α levels did not differ between time points. Flow cytometry analysis demonstrated a trend in STAT3 phosphorylation decrease when comparing pretreatment samples versus 6 months of MTF use, though this result was not statistically significant (p=0.06). Mean fluorescence intensity for pSTAT3 was: pretreatment 10.53 ± 5.75, 3 mo 7.34 ± 2.4, 6 mo 5.41 ± 1.14; and for pSTAT5: pretreatment 14.03 ± 7.41; 3 mo 10.71 ± 7.74; 6 mo 6.03 ± 1.41. PCR array for insulin signaling genes showed 21 genes downregulated after 6 months of MTF treatment, including genes previously associated with MPN phenotype: INS (0 and 6 mo treatment fold-decrease: 0.18), NOS2 (0.24), VEGFA (0.34), LEP (0.34), IGFBP1 (0.38) and IRS2 (0.62). Conclusions: In this study, metformin showed to be a safe and well-tolerated drug. Our preliminary results demonstrated a trend in BM collagen reduction in PMF patients following metformin treatment. Downregulation of important genes associated with MPN phenotype was also noted. The trial is ongoing and these results will be validated at other time points for all subjects. Disclosures Pagnano: Abbvie: Consultancy; Pint Pharma: Consultancy; Sandoz: Consultancy.

Background: Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by myeloid expansion associated with elevation of cytokines involved in fibrosis, angiogenesis, and osteosclerosis, leading to progressive fibrous connective tissue deposition in the bone marrow (BM) and BM failure. Although JAK2, CALR and MPL mutations are frequently seen in PMF, patients' molecular heterogeneity and the lack of an expressive clinical and laboratory response following JAK1/2 inhibitors suggest that other factors, such as unknown protein interactions, additional mutations or epigenetic mechanisms may be involved in the progression of PMF. Metformin (MTF) is an anti-diabetic drug, which has been described to possess anti-cancerous properties through the modulation of the AMPK/TORC1 pathway, thereby causing apoptosis in neoplastic cells. Previous reports demonstrated that MTF significantly reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice. In this context, our goal was to evaluate the effects of MTF treatment in PMF patients. Aims: To report final results of an open label phase II trial (FIBROMET), which evaluated outcomes of PMF patients after 24mo on MTF treatment. Methods: PMF non-diabetic adults were eligible. Patients received MTF in increasing doses until a maximum of 2500mg PO daily, according to tolerance. Primary endpoint was BM fibrosis reversion. Secondary endpoints included reduction of inflammation and downregulation of the JAK-STAT pathway. Samples were collected at the time points: screening (0), 3mo, 6mo, 12mo, 18mo and 24mo. The extent of collagen deposits in BM biopsies was semi-quantitatively assessed with Masson's trichrome stainings, following the recommendations of the European Consensus on grading of BM fibrosis (grades 0, 1, 2 or 3). The levels of CXCL4, sIL-2Ra, IP-10, VEFG-A, MIG, MCP-1, MIP-1b, FGF-2, IL-1RA, IL-5, IL-6, IL-8, IL-15, IL-18, TNFa and TGFb1 were analyzed in BM samples using multiplex assay. Phosphorylation status of intracellular proteins STAT3 and STAT5 was analyzed by flow cytometry and the percentage of cells was recorded using FlowJo software. This trial was approved by the Institutional and National Review Board; written informed consent was obtained from all subjects. REBEC registry number: RBR-52ty66. Results: 11 patients (aged 40-84y) were included between Aug/2018- Feb/2019. Two subjects had early treatment discontinuation due to non-related causes. One patient had disease progression after 12mo of treatment and was submitted to BM transplantation. The median exposure to MTF was 21 mo (3-24) and the median dose was 2500mg/day (1500-2500mg). The most frequent adverse event was diarrhea. No life threatening event occurred. BM collagen deposits were independently evaluated by two hematopathologists with an overall agreement of 80.64% (grade 0: 100%, grade 1: 62.5%, grade 2: 60.0%, grade 3: 90.0%); discordant cases were submitted to joint review. BM collagen deposits did not change when different time points were compared. After 24mo of MTF treatment, a significant 35.4% reduction in IL-5 levels was observed (p=0.03); a 36% reduction in MCP-1 levels was also observed, however this finding was not statistically significant (p=0.06). Flow cytometry analysis demonstrated a STAT3 phosphorylation decrease when comparing screening samples versus 6, 12, 18 and 24 mo of MTF use (all p<0.05), as well as a STAT5 phosphorylation decrease when comparing screening samples versus 6 and 12 mo (all p<0.05). Mean fluorescence intensity for pSTAT3 was: screening 12.01±2.58, 3mo 7.24±1.19, 6mo 4.40±0.24, 12mo 6.31±0.47, 18 mo 6.89±1.01, 24mo 5.71±1.18; and for pSTAT5: screening 15.32±3.59, 3mo 11.50±3.74, 6mo 4.67±0.45, 12mo 6.25±0.58, 18mo 6.24±0.81, 24mo 4.98±0.72. Conclusions: Final results of the FIBROMET trial demonstrated that, in our study population, metformin was not capable of reversing established bone marrow fibrosis in PMF patients. However, a significant downregulation of the JAK-STAT pathway and reduction of cytokine secretion was observed. Finally, metformin showed to be a safe and well-tolerated drug. Phase III studies are required to confirm our results and to evaluate whether MTF treatment in early PMF phases could delay the progression of BM fibrosis through the downregulation of the JAK-STAT pathway. Disclosures Pagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture. OffLabel Disclosure: Metformin for Primary Myelofibrosis treatment.

Background: (-)-Epigallocatechin-3-gallate (EGCG) is a gallate ester obtained by the condensation of gallic acid with the (3R)-hydroxy group of (-)-epigallocatechin. This component, extracted from green tea, has multiple effects on signal transduction pathways and enzyme activities which could enhance apoptosis and suppress of cell proliferation, invasion, angiogenesis and metastasis in cancers. This study aims to evaluate the effect of EGCG in an experimental model of leukemia (PML-RARα mice). Methods: NOD.CB17-Prkdcscid/J mice (12-16 weeks old) received 2Gy irradiation followed by transplantation of leukemia cells obtained from hCG-PML-RARα transgenic mice by i.v. injection in the caudal vein. Establishment of disease was confirmed at day 12 through presence of leukocytosis (>30x103/µL), and/or anemia (<10g/dL), and/or thrombocytopenia (<500x103/µL), associated to the presence of blasts in blood. At 12th day, mice (n=10/group) were randomly selected to receive EGCG (25mg/kg/day) (Cayman Chemical Co., Michigan, USA) or vehicle i.p. for five consecutive days. Mice were then sacrificed and peripheral blood, bone marrow and spleens were collected for flow cytometry and western blot analysis. All experiments were approved by the Ethical Committee for Animal Experimentation of Institution (nº3995-1/A). Results: Hematological analysis revealed that EGCG treatment reversed leukocytosis (54.09±57.71 vs 11.45±16.08; p=0.0371), anemia (9.60±1.50 vs 11.32±1.36; p=0.0155) and thrombocytopenia (238.5±146.43 vs 475.8±247.91; p=0.0179) and prolonged survival of PML/RARα mice (13 vs 15 days; p=0.0017). Notably, EGCG reduced leukemia immature cells (CD45+CD34+) (8.04±2.49 vs 5.13±1.58; p=0.0060) and promyelocytes (CD45+CD117+) (73.54±12.85 vs 56.26±15.93; p=0.0157) in bone marrow of mice whereas increased mature myeloid cells (CD11b+Gr-1+) (6.15±3.00 vs 14.60±7.83; p=0.0051), possibly by inducing cellular differentiation. These results were corroborated by the reduction in promyelocytes (45.97±11.72 vs 30.29±11.01; p=0.0154), and the increase in neutrophils (CD45+Gr-1+) (38.20±14.34 vs 54.88±14.25; p=0.0178) and monocytes (CD45+CD11b+) (60.22±18.87 vs 76.79±15.59; p=0.0463) detected in peripheral blood. We then evaluated the effect of EGCG on cellular differentiation by studying degradation of PML/RARα oncoprotein. EGCG increased the percentage of cells with aggregated PML bodies stain in the bone marrow of PML-RARα mice, suggestive of higher degradation of oncoprotein, parallel to a reduction in PIN1 expression in bone marrow cells. Higher intracellular levels of reactive oxygen species (ROS) were also detected in leukemia immature cells (2101±1025 vs 3544±614; p=0.0051), promyelocytes (1765±1176 vs 3090±1282; p=0.0271) and neutrophils (1830±1093 vs 3532±1157; p=0.0033) of bone marrow. These results are consistent with literature data demonstrating that the ablation of PIN1 and/or induction of ROS could trigger PML/RARα degradation. EGCG has been reported to inhibit PIN1, a peptidyl isomerase overexpressed and/or over activated in human cancers, which is described as a key target in PML/RARα. Furthermore, apoptosis was detected in spleen cells of PML-RARα mice (5.97±4.19 vs 10.42±3.54; p=0.0197) in parallel to increased expression of BAX, reduced expression of BCL-2, and reduction of spleen weight (0.5587±0.05 vs 0.3949±0.10; p=0.0085). Conclusion: Collectively, our results support further evaluation of EGCG in clinical trials for acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Beneficial effects of green tea (GT) consumption have been described, including the ability to reduce cancer development. Polyphenols are the main chemical constituents of GT extract and have been identified as the most effective substances that can inhibit tumorigenesis. Acute myeloid leukemia is an aggressive hematologic malignancy and there is no sufficient evidence that supports a protective role of tea intake on its development. In this concern, the aim of this study was to investigate GT effects in acute promyelocytic leukemia (APL) mice. A total of 1 × 106 leukemic cells obtained from hCG-PML-RARa transgenic mice were injected in the tail vein of 12- to 16-week-old NOD.CB17-Prkdcscid/J mice, after 4-6 h of sublethal cobalt irradiation with 2 Gy. The hematologic counts were monitored weekly, and the following criteria were used for the diagnosis of leukemia: presence of at least 1% of blast in peripheral blood associated with leukocytosis above 30 000 cells/L, hemoglobin levels below 10 g/dL, and thrombocytopenia below 500 × 103 cells/L (He et al, 1997). Twelve days after transplantation, mice were then submitted to daily oral treatment (gavage) with 250 mg/kg/day GT or vehicle only (water) for 5 consecutive days and were sacrificed; bone marrow (BM) and spleens were collected to the assays. Treatment with GT significantly increased the mean number of apoptotic cells in the BM (29.4 ± 5.2 vs untreated 21.0 ± 2.1 %, P < 0.05) and spleen (13.9 ± 3.1 vs untreated 9.2 ± 1.9 % P < 0.05) of mice, evaluated by Annexin V-FITC/PI. GT induced an increase in the median fluorescence intensity (MFI) of cleaved caspase-3 in the BM (83.9 ± 3.6 vs untreated 72.6 ± 4.7, P < 0.05) and in the spleen (75.5 ± 28.2 vs untreated 55.8 ± 7.3, P < 0.01); cleaved caspase-8 in the BM (117.3 ± 9.9 vs untreated 89.1 ± 12.3, P < 0.005) and in the spleen (118.0 ± 31.5 vs untreated 81.5 ± 14.8, P < 0.001); and cleaved caspase-9 in the BM (138.2 ± 52.4 vs untreated 85.8 ± 12.9, P < 0.001) and in the spleen (121.7 ± 49.2 vs untreated 76.5 ± 21.9, P < 0.001) of leukemic mice. Moreover, GT treatment reduced the percentage of CD34+ hematopoietic progenitor cells (32.4 ± 2.3 vs untreated 41.0 ± 0.5 %) as well as of CD117+ cells (33.4 ± 3.7 vs untreated 44.2 ± 1.8 %). We then evaluated the phenotype of cells infiltrated in the spleen. Interestingly, we found that GT induces a decrease in the percentage of CD117+ (40.7 ± 0.3 vs leukemic 44.6 ± 0.9 %) and Gr-1 cells (60.8 ± 0.2 vs untreated 65.6 ± 0.5 %) present in the spleen. We then analyzed the effects of GT in the production of intracellular ROS in the BM subpopulations of CD34+, CD117+ and Gr-1+ cells from leukemic mice. Significant increases in the median fluorescence intensity (MFI) of intracellular ROS production by Gr-1 cells of GT-treated mice were observed (670 ± 103 vs untreated 428.5 ± 5.2). Interestingly, GT induced a reduction of MFI of intracellular ROS production in the CD34+ (167.5 ± 27.1 vs untreated 405.5 ± 73.3) and CD117+ (360 ± 142 vs untreated 1635 ± 40.4) cells. We then studied the expression and localization of CXCR4 and HIF-1α proteins. Studies have shown that ROS increases expression of CXCR4 in cancer and immune cells (Li et al, 2009; Lin et al, 2011; Chetram et al, 2011; 2013) through nuclear translocation of HIF-1α (Lee et al, 2002; Salmeen et al, 2003). Our results showed that GT decreased the MFI of CXCR4 in the leukemic mice (9028 ± 1367 vs untreated 4196 ± 970). Reduction of the nuclear translocation of HIF-1α in GT-treated mice was also observed, using the ImageStream imaging flow. In conclusion, GT treatment in APL mice induces apoptosis of cells in the BM and spleen, confirmed by activation of caspase-3, -8 and -9, probably by modulating the production of intracellular ROS in the leukemic cells. Although GT and its polyphenols are well known as antioxidants, there is evidence that some of the effects of these compounds may be related to induction of oxidative stress in immune cells, which might be responsible for the induction of apoptosis of tumor cells. These pro-oxidant properties may also induce endogenous antioxidant systems in normal tissues that offer protection against cancer. On the other hand, it is possible that, in leukemic cells, which has excessive ROS, the antioxidant effect of GT become more evident. Several potential mechanisms have been proposed including both antioxidant and pro-oxidant effects to polyphenol compounds, but questions remain concerning the relevance of these mechanisms to cancer prevention. Disclosures Torello: Fundação de Amparo à Pesquisa do Estado de São Paulo - Fapesp: Research Funding; Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq: Research Funding. Shiraishi:University of Campinas: Employment.