scholarly journals HO-1 Promotes Resistance to EZH2 Inhibitor through the Prb-E2F Pathway: Corelated with the Conversion of Myelodysplastic Syndrome to Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5401-5401
Author(s):  
Zhengchang He ◽  
Jishi Wang
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Tumor Cells ◽  
Myeloid Leukemia ◽  
Control Group ◽  
Apoptosis Rate ◽  
Experimental Group ◽  
Very High ◽  
Acute Myeloid

Myelodysplastic syndrome (MDS) patients would have a chance to become acute myeloid leukemia (AML) when undergoing chemotherapy or waiting for the hematopoietic stem cell transplantation. These patients were not sensitive to demethylation therapy and we should explore deeper mechanisms. According to the WPSS scoring system, we divided 58 MDS patients into four different groups. Real-time PCR results showed the expression of EZH2 or HO-1 in MDS patients were higher than that in normal donors (P<0.05). Even HO-1 and EZH2 were simultaneously increased in some patients, especially in high-risk and extremely very high-risk groups. The linear correlation analysis result of them was 0.42 (P<0.05). In addition, Laser scanning confocal microscopy results also indicated that they were both present in the nucleus of tumor cells. Therefore, we speculated that there was a correlation between EZH2 and HO-1 in MDS patients. Using the High-throughput sequencing to analyze MDS cells, we found that the conversion of MDS to AML may be related to EZH2. The EZH2 in converted MDS patients were significantly higher than that of other MDS patients (P<0.05). Among these patients, we also found that HO-1 and EZH2 were also positively correlated. We found the new EZH2 inhibitor JQEZ5 could significantly promote tumor cells apoptosis in this part of patients. When the concentration of JQEZ5 was 10 umol/mL, the apoptosis rate of tumor cells reached 46.7% after 24 hours (P<0.05). Apoptosis rate was positively correlated with the concentration of JQEZ5 (P<0.05). And tumor cells were significantly inhibited in the G0/G1 cell phase. Cell cycle regulatory genes (CDK4 and CDK6) and apoptosis regulatory genes (Caspase-3 and Caspase-9) would changed. The expression of P15 and P53 would also be affected. In order to verify the malignant degree of MDS cells whether be related to the expression of EZH2. We injected MDS cells into 10 mice. Compared to the control group, MDS cells that highly express EZH2 infiltrated the spleen of experimental mice. Interestingly, the spleens of the experimental group were significantly reduced (0.2CM-0.4CM) and the spleens weight of the experimental group was reduced by 0.028g-0.12g compared to the control group spleens weight. These cells also significantly shortened the survival days of mice and reduced their body weight. Although control mice could survived for 30 days without disease, the time of the experimental mice was significantly shortened (18-25 days). Even One of them survived only 15 days. The results of immunohistochemistry indicated that EZH2 was related to the pRB-E2F pathway. Using the E2F inhibitor HLM006474, we proved HO-1 could regulated EZH2 through the pRB-E2F pathway. Our previous experiments indicated that HO-1 could help leukemia cells resistance and proliferation. The effect of JQEZ5 would be affected when we used hemin and zinc protoporphyrin to regulate HO-1 in MDS cells. The EZH2 was significantly inhibited by JQEZ5 after HO-1 was silenced by siRNAs. Also, the apoptosis rate of MDS cells increased and the cell cycle was arrested in the G0/G1 phase. However, when HO-1 expression was up-regulated by lentivirus, the effects of JQEZ5 were attenuated. MDS patients are frequently in a state of HO-1 enrichment during chemotherapy. HO-1 stimulates MDS patients to transcribe and activate excess EZH2 through pRB-E2F pathway, which increases the chances of becoming AML. Therefore, the conversion of MDS may be attributed to EZH2. In addition, considering HO-1 could promote the expression of EZH2, HO-1 may be a target for enhancing the effects of EZH2 inhibitors on MDS and the influence of HO-1 should be considered in the treatment of patients with high-risk and extremely very high-risk MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals HO-1 promotes resistance to an EZH2 inhibitor through the pRB-E2F pathway: correlation with the progression of myelodysplastic syndrome into acute myeloid leukemia

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Zhengchang He ◽  
Siyu Zhang ◽  
Dan Ma ◽  
Qin Fang ◽  
Liping Yang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Tumor Cells ◽  
High Throughput ◽  
Myeloid Leukemia ◽  
High Throughput Sequencing ◽  
Ezh2 Expression ◽  
Very High ◽  
Acute Myeloid

Abstract Background Myelodysplastic syndrome (MDS) can progress to acute myeloid leukemia (AML), and conventional chemotherapy (decitabine) does not effectively inhibit tumor cells. Enhancer of zeste homologue 2 (EZH2) and Heme oxygenase-1 (HO-1) are two key factors in patients resistance and deterioration. Methods In total, 58 MDS patients were divided into four groups. We analyzed the difference in HO-1 and EZH2 expression among the groups by real-time PCR. After treatment with Hemin or Znpp IX, flow cytometry was used to detect apoptosis and assess the cell cycle distribution of tumor cells. Following injection of mice with very high-risk MDS cells, spleen and bone marrow samples were studied by immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining. MDS cells overexpressing EZH2 and HO-1 were analyzed by high-throughput sequencing. The effect of HO-1 on the pRB-E2F pathway was analyzed by Western blotting. The effects of decitabine on P15INK4B and TP53 in MDS cells after inhibiting HO-1 were detected by Western blotting. Results Real-time PCR results showed that EZH2 and HO-1 expression levels were higher in MDS patients than in normal donors. The levels of HO-1 and EZH2 were simultaneously increased in the high-risk and very high-risk groups. Linear correlation analysis and laser scanning confocal microscopy results indicated that EZH2 was related to HO-1. MDS cells that highly expressed EZH2 and HO-1 infiltrated the tissues of experimental mice. IHC results indicated that these phenomena were related to the pRB-E2F pathway. High-throughput sequencing indicated that the progression of MDS to AML was related to EZH2. Using the E2F inhibitor HLM006474 and the EZH2 inhibitor JQEZ5, we showed that HO-1 could regulate EZH2 expression. HO-1 could stimulate the transcription and activation of EZH2 through the pRB-E2F pathway in MDS patients during chemotherapy, which reduced TP53 and P15INK4B expression. Conclusions EZH2 was associated with HO-1 in high-risk and very high-risk MDS patients. HO-1 could influence MDS resistance and progression to AML.

Study on mechanism of Tim-3 on immune escape in benzene-induced acute myeloid leukemia

2020 ◽  
Author(s):  
qiong Ning ◽  
xiangxin li ◽  
Xiangdong Jian ◽  
Xiaopeng He
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Immune Escape ◽  
Serum Levels ◽  
Control Group ◽  
M2 Macrophages ◽  
Experimental Group ◽  
Group Serum ◽  
Acute Myeloid

Abstract To study the mechanism of Tim-3 on immune escape in benzene-induced acute myeloid leukemia (AML), to provide potential targets of clinical monitoring and intervention of hematological toxicity in benzene-induced AML . C3H/He mice were randomly divided into control group and experimental group. Serum levels of IL-12 in the experimental group were significantly lower than that in the control group. Serum levels of TGF-β1 in the experimental group were significantly higher than that in the control group( p <0.05). The proportion of Tim-3 positive CD14 + monocytes of bone marrow and spleen in the experimental group were both significantly higher than that in the control group ( p <0.05) by Flow cytometry (FCM). Compared with the control group, the expression of Tim-3 on (M1+M2) macrophages of bone marrow in the experimental group significantly increased by immunofluorescence assay. The expression of type M2 macrophages in (M1+M2) macrophages of bone marrow and spleen tissues in the experimental group were both higher than that in the control group. The expression levels of p-PI3K, p-AKT and p-mTOR in the experimental group were all significantly higher than that in the control group. Tim-3 was highly expressed in macrophages in benzene-induced AML. It promoted the activation of PI3K/AKT/mTOR signaling pathway, stimulated the secretion of anti-inflammatory cytokines, and inhibited the secretion of pro-inflammatory cytokines. High expression of Tim-3 changed the phenotype and function of macrophages by promoting the macrophages polarization, thus inducing negative immune response in the tumor microenvironment and tumor immune escape.

Karyotypic Complexity In Acute Myeloid Leukemia In The Context Of Adverse Prognosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 489-489 ◽  
Author(s):  
Friedrich Stölzel ◽  
Brigitte Mohr ◽  
Michael Kramer ◽  
Christoph Röllig ◽  
Tilmann Bochtler ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Normal Karyotype ◽  
Control Group ◽  
Cytogenetic Aberrations ◽  
First Remission ◽  
The Uk ◽  
Acute Myeloid

Abstract Introduction Cytogenetic analysis is a mandatory component in the diagnostic evaluation of acute myeloid leukemia (AML) providing information regarding the biology of the disease including response or resistance to therapy. One of the cytogenetic markers which reflect an adverse outcome in conventional chemotherapy regimens is the complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities. In AML two definitions have been established which differ in the perception of unbalanced aberrations as well as the number of single aberrations. The ELN classification scheme adopts three unrelated abnormalities while the UK MRC recently recommended four abnormalities as the most informative cut-off of complexity in the context of an adverse prognosis. The aim of this work was to study the best cut-off defining complexity (3 vs. 4) in AML with other cytogenetic high-risk markers. Methods The databases of three clinical multicentric, randomized, and prospective SAL trials (NCT 00180115, 00180102, and 00180167) were analyzed for AML patients with multiple cytogenetic aberrations as well as normal karyotypes (control group). Unbalanced abnormalities were counted as two aberrations according to the recommendations of the MRC (i.e. a single unbalanced translocation leading to gain and loss of chromosomal material as two unique abnormalities). The following single aberrations associated with an adverse prognosis according to ELN as well as UK MRC recommendations were included: inv(3), t(3;3), abn(3q), -5, del(5q), t(5q), t(6;9), -7, add(7q)/del(7q), t(11;v)(q23;v) (except t(9;11)), and abnl(17p). Results Complete data were analyzed from 2056 patients: normal karyotype (NK) n=1590, three aberrations (K3) n=65, ≥ four aberrations (K4) n=355, t(8;21)/inv(16)/t(16;16) and at least two additional aberrations n=46. All four groups differed significantly in 5–year overall survival (OS): 35% [95% CI 32–37], 19% [95% CI 9–29], 7% [95% CI 4–10], 67% [95% CI 53–81], respectively, p≤0.001. The K4 group had a significant inferior 5–year OS as compared to the K3 group, 19% [95% CI 9–29] and 7% [95% CI 4–10], p≤0.001. HSCT was performed in first remission in 25% of patients with K3 (n=16) and 17% of patients with K4 (n=59) (p=n.s.). As demonstrated earlier, multiple aberrations additional to the good risk anomalies (t(8;21), inv(16), or t(16;16)) did not impact on the favourable prognosis of the respective group. In the K3 and K4 groups single adverse risk abnormalities were found in 55% (abnl(17p) 12%) and 83% (abnl(17p) 37%) in these patients, respectively. A hyperdiploid karyotype (HDK) with gains of whole chromosomes without any structural aberration or monosomy was present in 14% of K3 and 3% of K4-patients. Interestingly, HDK with three trisomies as well as ≥ four trisomies led to a survival similar to K4 patients without HDK. Therefore, the K3 group lost its inferior survival as compared to NK when patients with adverse risk, which induce a worse prognosis per se, as well as HDK were excluded (5y–OS: 29% [9–44] vs. 35%, [95% CI 32–37], p=n.s.). HDK patients or patients with additional single adverse risk abnormalities had a worse survival compared to NK (5y–OS: 11%, [95% CI 0–32], p=0.012; and 15%, [95% CI 3–28], p=0.004 vs. 35%, [95% CI 32–37], respectively). In contrast, when comparing the K4 group after exclusion of adverse risk and HDK patients to NK, the K4 group remained its inferior OS as compared to NK, p<0.001. Conclusions Hence, our investigation confirms and therefore favors the ≥4 cut-off of complexity in the context of an adverse prognosis as proposed by the MRC with the exception of HDK patients. HDK patients should be considered as high-risk independent of the level of complexity. Whether K3 patients without single adverse risk abnormalities and HDK should be treated as intermediate risk, as suggested by our results, needs to be investigated prospectively in clinical trials. Disclosures: Platzbecker: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Allogeneic Transplantation Versus Chemotherapy as Postremission Therapy for Acute Myeloid Leukemia: A Prospective Matched Pairs Analysis

2014 ◽  
Vol 32 (4) ◽  
pp. 288-296 ◽  
Author(s):  
Matthias Stelljes ◽  
Utz Krug ◽  
Dietrich W. Beelen ◽  
Jan Braess ◽  
Maria C. Sauerland ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Myeloid Leukemia ◽  
Allogeneic Transplantation ◽  
Rank Test ◽  
Control Group ◽  
Matched Pairs ◽  
Postremission Therapy ◽  
Matching Criteria ◽  
Acute Myeloid

Purpose The majority of patients with acute myeloid leukemia (AML) who achieve complete remission (CR) relapse with conventional postremission chemotherapy. Allogeneic stem-cell transplantation (alloSCT) might improve survival at the expense of increased toxicity. It remains unknown for which patients alloSCT is preferable. Patients and Methods We compared the outcome of 185 matched pairs of a large multicenter clinical trial (AMLCG99). Patients younger than 60 years who underwent alloSCT in first remission (CR1) were matched to patients who received conventional postremission therapy. The main matching criteria were AML type, cytogenetic risk group, patient age, and time in first CR. Results In the overall pairwise compared AML population, the projected 7-year overall survival (OS) rate was 58% for the alloSCT and 46% for the conventional postremission treatment group (P = .037; log-rank test). Relapse-free survival (RFS) was 52% in the alloSCT group compared with 33% in the control group (P < .001). OS was significantly better for alloSCT in patient subgroups with nonfavorable chromosomal aberrations, patients older than 45 years, and patients with secondary AML or high-risk myelodysplastic syndrome. For the entire patient cohort, postremission therapy was an independent factor for OS (hazard ratio, 0.66; 95% CI, 0.49 to 0.89 for alloSCT v conventional chemotherapy), among age, cytogenetics, and bone marrow blasts after the first induction cycle. Conclusion AlloSCT is the most potent postremission therapy for AML and is particularly active for patients 45 to 59 years of age and/or those with high-risk cytogenetics.

scholarly journals High Dose Flamsa, CLAG or FLAG-Based Sequential Conditioning Regimen Followed By Allogeneic Hematopoietic Transplantation Results in Favorable Outcome for High Risk Acute Myeloid Leukemia, Myelodysplastic Syndrome, Myeloproliferative Neoplasia Patients : A Multicenter Study in Singapore

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Lip Leong Chong ◽  
Yang Liang Boo ◽  
Yin Jie Koh ◽  
Michelle Poon ◽  
Yeh Ching Linn ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Cumulative Incidence ◽  
Myeloid Leukemia ◽  
Conditioning Regimen ◽  
Unrelated Donors ◽  
Sequential Regimen ◽  
Very High ◽  
Acute Myeloid

Introduction: Allogeneic hematopoietic cell transplant (allo-HCT) is an effective consolidative treatment for patients with certain hematological malignancies and gives the best outcome when done in remission. However, patients with refractory acute myeloid leukemia (AML), certain forms of myeloproliferative neoplasia (MPN), and myelodysplastic syndrome (MDS) deemed unable to achieve remission with standard induction are often excluded from allo-HCT with conventional conditioning regimen as pre-transplant remission could not be achieved. Recently, a sequential transplant approach, as developed by the Munich group, comprising of intensive cytoreductive chemotherapy FLAMSA (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, had been successfully used for high-risk (HR) AML/MDS with promising results. Methods: We studied 56 patients (median age, 52 years; range 17-68) with HR AML (n=45), as defined by refractory, relapsed disease, secondary leukemia, complete remission with adverse-risk cytogenetics according to ELN criteria, or high/very high risk refined Disease Risk Index (DRI), MPN (n=2), and HR MDS (n=9) according to IPSS-R, undergoing allo-HCT using the sequential transplant approach in 2 transplant centers in Singapore between January 2009 and June 2020. The sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either FLAMSA (n=17), FLAG +/- Ida [fludarabine/cytarabine/granulocyte colony stimulating factor (GCSF) +/- idarubicin] (n=23), or CLAG (clofarabine +/- cytarabine +/- GCSF) (n=15), followed by reduced intensity (RIC) (N=48) or myeloablative (MAC) (N=8) conditioning regimen. All patients received peripheral blood stem cell from matched related donors (N=30), mismatched related donors (N=3), matched unrelated donors (N=16), or mismatched unrelated donors (N=7). Post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. Thymoglobuline or post-transplant cyclophosphamide were added for GVHD prophylaxis in unrelated donor transplant and mismatched related donors, respectively. Results: The median time to neutrophil &gt; 1000/μL was 11 days (range, 8-19). With a median follow-up of 44 months (range, 1-123), the Kaplan-Meier estimate of overall (OS) and leukemia-free (LFS) survivals at 5 years were 49% (95% CI, 35-62) and 37% (95% CI, 23-52), respectively. The 2-years cumulative incidence of relapse and non-relapse mortality (NRM) were 47% (95% CI, 32-60) and 13% (95% CI, 6-24), respectively. Patients receiving FLAG or CLAG-based sequential regimen showed lower cumulative incidence of NRM (2-year cumulative incidence for NRM: 5% vs 29%; p=0.018), and similar relapse (2-year cumulative incidence for relapse: 49% vs 53%; p=0.64), as compared to patients given FLAMSA-based sequential regimen, resulting in a trend towards more favourable OS (5-year OS: 53% vs 41%; p=0.29) and LFS (5-year LFS: 46% vs 20%; p=0.08). In multivariable analysis, only refined DRI showed significant impact on OS (p=0.04), but has no significant impact on LFS, NRM, and relapse. The 5-year OS for patients with intermediate/high risk and very high risk DRI were 59% and 27%, respectively (p=0.017), and the corresponding 5-year LFS were 46%, and 20% (p=0.06), respectively (Figure 1 & Figure 2). The intensity of conditioning regimen did not significantly impact on OS, LFS, relapse, and NRM. Conclusions: Sequential transplant conditioning with FLAMSA, FLAG or CLAG followed by allo-HCT is an effective strategy in overcoming the dismal prognosis of HR AML, MDS and MPN, and enabling favourable long-term disease-free survival. More studies on effective strategies such as post-transplant maintenance therapy with prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. Disclosures No relevant conflicts of interest to declare.

Activity and Mechanism of Action of AS1411 in Acute Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1604-1604 ◽  
Author(s):  
Weiwei Chen ◽  
Vijayalakshmi Sridharan ◽  
Sridharan Soundararajan ◽  
Yoko Otake ◽  
Robert Stuart ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Clinical Trial ◽  
Acute Myeloid Leukemia ◽  
Tumor Cells ◽  
Phase Ii ◽  
Myeloid Leukemia ◽  
S Phase ◽  
Mrna Levels ◽  
Cytotoxic Effects ◽  
Acute Myeloid

Abstract AS1411 is a 26 base unmodified DNA aptamer which is currently in a Phase II clinical trial for the treatment of acute myeloid leukemia (AML) and will shortly enter a Phase II trial in renal cell carcinoma (RCC). AS1411 has previously been shown to have cytostatic (a result of arrest in S phase of the cell cycle) and cytotoxic effects on tumor cells, but not normal cells. AS1411 binds to nucleolin, which is overexpressed in the cytoplasm and on the surface of tumor cells. Treatment of MV4-11 cells (an AML-derived cell line) with low micromolar concentrations of AS1411 results in cell growth inhibition and a reduction in viability; this effect becomes apparent after 48 hours incubation. Co-immunoprecipitation of nucleolin and AS1411 from MV4-11 cells treated with radiolabeled AS1411 confirmed previous work that AS1411 binds to nucleolin. In addition, confocal microscopy showed that nucleolin is present in the cytoplasm and the plasma membrane of MV4-11 cells. Incubation of these cells with AS1411 decreased nucleolin mRNA levels, and nucleolin protein was decreased in both cytoplasm and nucleus to approximately 10% of control levels after 96 hours. The reduction in nucleolin levels started to appear at around 48 hours and may reflect cells arresting in S phase of the cell cycle. Nucleolin binds to an ARE sequence in the 3′-untranslated region of Bcl-2 mRNA resulting in stabilization of the mRNA. Using gel shift assays with a radiolabeled fragment of Bcl-2 mRNA we show here that AS1411 interferes with this binding. In addition, levels of Bcl-2 mRNA in MV4-11 cells decrease to 10% of control levels after 48 hours incubation with AS1411 and the level of Bcl-2 protein also decreases in comparison to control cells. Finally, mononuclear cells isolated from blood samples of AML patients were incubated with AS1411 for 96 hours and viability determined. In all samples, AS1411 caused a reduction in cell viability at low micromolar concentrations, similar to those required to kill cell lines. This work therefore demonstrates that AS1411 has both cytostatic and cytotoxic effects on AML cells and underpins the dosing regime of AS1411 in the ongoing AML clinical trial.

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