MEK1 inhibition sensitizes primary acute myelogenous leukemia to arsenic trioxide-induced apoptosis

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73 gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative DeltaNp73 isoforms, thereby failing to elevate the TA/DeltaNp73 ratio. Conversely, treatment with PD184352 reduces the level of DeltaNp73 and blunts the arsenic-mediated up-regulation of DeltaNp73, thus causing an increase in the TA/DeltaNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1 (p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.     

热休克蛋白90抑制乙型肝炎病毒复制的初步研究

目的 了解热休克蛋白90 (HSP90)对HBV复制的影响并探讨其可能的分子机制. 方法 构建人HSP90真核表达质粒pXF3H-HSP90 (HA-HSP90),与HBV复制型质粒HBV1.3共转HepG2细胞,ELISA检测细胞上清液HBsAg表达水平,Southern blot检测HBV复制中间体的表达.将HA-HSP90表达质粒或HSP90 siRNA转染HepG2细胞,实时定量逆转录聚合酶链反应检测白细胞介素(IL)-1β、IL-6以及干扰素应答基因1(IFIT1)的表达.将TANK结合激酶1(TBK1)siRNA、HBV1.3、HA-HSP90共染HepG2细胞,Southern blot检测HBV复制中间体的表达.多组间数据比较采用单因素方差分析,两两比较采用LSD检验. 结果 成功构建了表达HSP90的真核表达质粒HA-HSP90.转染HA-HSP90的HepG2细胞内高水平表达外源蛋白,而未转染质粒的细胞内没有检测到外源蛋白的表达.转染HA-HSP90质粒组细胞上清液中的HBsAg表达量吸光度值为0.124±0.033,明显低于转染空载体pXF3H组的0.340±0.011及未转染组的0.615±0.069(F=61.013,P＜0.05).过表达HSP90也能抑制HBV复制,转染HA-HSP90组、转染空载体pXF3H组与未转染组HBV复制中间体表达量吸光度值分别为108.92±8.59、872.45±13.00和7856.37±60.23,差异有统计学意义(F=28260.00,P＜0.05).在HepG2细胞内高表达HSP90能诱导高水平的IFIT1产生,其中HA-HSP90组与空载体对照pXF3H组IFIT1表达的2-△△CT值分别为82.139±0.919和5.579±0.644,差异有统计学意义(F=13.108,P＜0.05),但未检测到IL-1 p与IL-6的诱导表达.下调HepG2细胞中干扰素信号通路分子TBK1不影响HSP90对HBV的抑制,其中HSP90组与siTBK1+ HSP90组HBV复制中间体条带灰度值分别为1952.64±67.88和2366.64±71.16 (F=31.30,P＞0.05). 结论 HSP90能够抑制HBV的复制与蛋白表达,这种抑制作用不依赖于TBK1通路,也与IL-1β信号通路无关.     

Repression of oxidative phosphorylation sensitizes leukemia cell lines to cytarabine

Objectives: Leukemia is a group of bone marrow cancers and drug resistance is one of the challenges in treatment. Caffeic acid phenethyl ester’s (CAPE’s) anti-proliferative and apoptotic properties have been reported in leukemia cell lines. However, CAPE’s effect on drug resistance and cellular metabolism is still unknown. Thus, in this study, we aimed to explore CAPE’s effect on drug resistance and oxidative phosphorylation (oxphos).

Methods: Leukemia cell lines NB-4, HL-60, and K562 were treated with CAPE. ATP-based cell viability assay was used. For gene expression studies, RNAs were isolated and reverse transcribed. To investigate CAPE’s effect on mitochondrial dysfunction in AML cell lines, we examined oxygen consumption rates (OCRs) in our cell lines.

Results: We found 5?μM CAPE sensitized all cell lines to cytarabine. This similar effect was also observed in the Decitabine-resistant K562 cell line. However, no difference was seen in MDR1 expression upon CAPE treatment in all cell lines. OCR significantly decreased upon CAPE treatment in all cell lines, while the expression of key regulatory glycolytic enzymes increased in K562 and NB-4 cell lines. Expression of STAT3 also changed upon CAPE treatment.

Discussion: Our results suggested that CAPE alters cellular metabolism by decreasing oxphos and increasing glycolysis in K562 and NB-4 cells. Furthermore, CAPE treatment altered STAT3 expression regarding alterations in oxphos and aerobic glycolysis.

Conclusion: Our results suggest a new property of CAPE, which is oxphos repression, and a presumptive link between altered metabolism and drug resistance.     

Detection of cytarabine resistance in patients with acute myelogenous leukemia using flow cytometry

Cytarabine (Ara-C) is currently used in the treatment of adult acute myeloid leukemia (AML). To predict the results of induction chemotherapy, it could be useful to detect leukemic cells that are resistant to Ara-C in patients with AML. Using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM), we have developed a cell resistance index to Ara-C (RI). The technique has been applied to 121 bone marrow (BM) samples from patients with de novo AML treated by a regimen containing Ara-C and daunorubicin (DNR). Ninety-seven patients achieved a complete remission (CR), and 24 patients did not and were considered drug-resistant (DR). The BM cells collected at diagnosis were cultured for 48 hours and underwent BrdUrd/DNA analysis. Among 25 patients with no or very low proliferative activity (<3% of cells in S-phase), the proportion of DR patients (nine of 25) was significantly higher than in a second group of 96 patients with detectable proliferative activity (15 of 96) (P < .025). Within this second group, there was a first group of nine patients with high RI values, which included only DR patients; a second group of 63 patients with low RI values, which included 62 CR patients; and a third group of 24 patients with intermediate RI values, which included 19 CR and five DR patients. In view of this series, our results show that it is possible to detect a majority of DR patients treated by Ara-C.     

Concomitant inhibition of DNA methyltransferase and BCL-2 protein function synergistically induce mitochondrial apoptosis in acute myelogenous leukemia cells

DNA methylation and BLC-2 are potential therapeutic targets in acute myeloid leukemia (AML). We investigated pharmacologic interaction between the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) and the BCL-2 inhibitor ABT-737. Increased BCL-2 expression determined by reverse phase protein analysis was associated with poor survival in AML patients with unfavorable cytogenetics (n?=?195). We found that 5-AZA, which itself has modest apoptotic activity, acts synergistically with ABT-737 to induce apoptosis. The 5-AZA/ABT-737 combination enhanced mitochondrial outer membrane permeabilization, as evidenced by effective conformational activation of BAX and ?ψ_m loss. Although absence of p53 limited apoptotic activities of 5-AZA and ABT-737 as single agents, the combination synergistically induced apoptosis independent of p53 expression. 5-AZA down-regulated MCL-1, known to mediate resistance to ABT-737, in a p53-independent manner. The 5-AZA/ABT-737 combination synergistically induced apoptosis in AML cells in seven of eight patients. 5-AZA significantly reduced MCL-1 levels in two of three samples examined. Our data provide a molecular rationale for this combination strategy in AML therapy.     

Abolition of stress-induced protein synthesis sensitizes leukemia cells to anthracycline-induced death

Anthracycline action has been thought to involve the neosynthesis of proapoptotic gene products and to therefore depend on protein synthesis for optimal effect. We found that inhibition of general, but not rapamycin-sensitive (cap-dependent), protein synthesis in the preapoptotic period enhanced anthracycline-induced acute myelogenous leukemia (AML) cell death, both in vitro and in several animal AML models. Pre-apoptotic anthracycline-exposed AML cells had altered translational specificity, with enhanced synthesis of a subset of proteins, including endoplasmatic reticulum chaperones. The altered translational specificity could be explained by perturbation (protein degradation, truncation, or dephosphorylation) of the cap-dependent translation initiation machinery and of proteins control-ing translation of specific mRNAs. We propose that judiciously timed inhibition of cap-independent translation is considered for combination therapy with anthracyclines in AML.     

Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells

In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90α-null DCs and mice. CD8α(+) DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90α-null mice. Ag translocation into the cytosol was diminished in HSP90α-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol.     

High-dose cytarabine for the treatment of blastic phase chronic myelogenous leukemia

High-dose cytarabine chemotherapy regimens were given to 22 patients for the treatment of blastic phase chronic myelogenous leukemia. Bone marrow aplasia occurred in 21 of these patients; in one patient the marrow was not cleared of blasts. In five patients (26%), blastic phase promptly recurred. Eight patients (37%) died of infection or hemorrhage during the period of marrow aplasia before bone marrow recovery. Seven patients achieved complete remission and one achieved partial remission, but the duration of the remission was brief [median, 98 days (range, 52-345)]. One patient received consolidation therapy with an additional course of high-dose cytarabine and maintained remission for 345 days. These results suggest that alternative approaches to the treatment need to be explored.     

Hypocholesterolemia in acute myelogenous leukemia

Plasma-cholesterol concentrations were determined in 85 acute myelogenous leukemia patients. Measurements were repeated in 28 cases during remission. Mean plasma-cholesterol concentration (+/- SD) at diagnosis was 3.95 mmol/l (+/- 1.29). 47 patients (55.3%) had hypocholesterolemia (less than 3.87 mmol/l). Among the main clinical, hematologic and biochemical parameters, only high leukocyte counts were correlated with hypocholesterolemia. As far the FAB subtypes are concerned, the lowest cholesterol levels were observed in leukemias with monocytic component. However, although the same FAB subtypes showed significantly higher leucocytes counts than the other subtypes, both parameters were independently related to low cholesterol levels. Remission was associated with a significant increase in cholesterol levels in those patients with low cholesterol concentrations or high leukocyte counts at diagnosis. These results support the idea that initial hypocholesterolemia in acute myelogenous leukemia is related to the tumoral mass present at diagnosis.     

Therapy of acute myelogenous leukemia

Over the past 10 years, there have been substantial advances in the treatment of AML. Intensive induction chemotherapy using 7-day courses of cytarabine and daunorubicin or amsacrine produce remission in 60% to 85% of patients. Median remission duration is 9 to 16 months. In some series, 20% to 40% of patients are in continuous remission for 2 years or more; many of these patients remain in remission for 5 years or longer and some may be cured. Bone marrow transplantation has evolved as a useful therapeutic modality capable of achieving long-term survival in some circumstances in which chemotherapy is relatively ineffective. Its precise role in the initial therapy of AML remains to be defined, but it is likely to be beneficial in selected patients. These data indicate substantial recent progress in the treatment of this disease, which was almost uniformly fatal 30 years ago. The fact that most patients relapse within 1 to 2 years reflects a lack of progress in developing effective postremission therapy. Maintenance chemotherapy, immunotherapy, and CNS prophylaxis have little role in AML. It is unclear whether consolidation or intensification extend remissions or increase the proportion of long-term survivors; controlled randomized trials should answer this question within the next few years. Future progress in the treatment of AML awaits the development of more sensitive methods for detecting residual leukemia, more effective use of current therapeutic modalities and the introduction of new effective drugs. Most data suggest that early intensive treatment is of key importance for achieving cures. However, we cannot presently distinguish between patients cured by initial treatment and those who required further chemotherapy.     

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34⁺ cells, especially the LSC-enriched CD34⁺CD38⁻ subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34⁺ cells but not those from normal CD34⁺ cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide–down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.Acute myelogenous leukemia (AML) represents a group of clonal hematopoietic stem cell disorders, in which a small subpopulation of leukemia stem cells (LSCs) are responsible for the accumulation of large numbers of immature myeloblasts in the bone marrow of AML patients. In addition to their crucial roles in leukemia initiation and progression, LSCs are also responsible for the high frequency of relapse that is characteristic of current AML therapies. Of patients receiving treatment with curative intent, less than one-half will achieve long-term survival (1). Similar to normal hematopoietic stem cells (HSCs), LSCs exhibit stem cell-like characteristics such as the capacity for self-renewal, differentiation potential, and relative quiescence (2, 3). The quiescent feature renders LSCs resistant to conventional chemotherapeutic agents that predominantly target proliferating rather than quiescent cells (1). For this reason, it is not surprising that relapse occurs in the majority of cases; this is further supported by recent studies showing that AML patients with LSCs enrichment have worse clinical outcomes (4–7). It is therefore crucial that therapies be developed targeting the quiescent and drug-resistant LSCs.Despite the similarities shared by LSCs and HSCs, LSCs often possess several unique features as well, which may provide important hints for designing LSC-targeted therapy. For instance, LSCs are usually associated with the abnormal expression of CD markers (e.g., CD44, CD47, CD96, and CD123), constitutive activation of nuclear factor κB (NF-κB), active Wnt/β-catenin signaling, and elevated levels of interferon regulatory factor-1 (IRF-1) and death-associated protein kinase (DAPK) (8–12). Most recently, emerging evidence points to oxidative signaling as being a two-edged sword in AML: moderate levels of reactive oxygen species (ROS) are important for driving disease, whereas higher levels result in cell death (13–15). The dual roles of oxidative signaling suggest that LSCs, in comparison with normal HSCs, are more vulnerable to ROS-generating agents. Accordingly, pharmacological agents favoring the generation of ROS are worth exploring in LSC-targeted therapy. Indeed, ROS induction has been shown as a critical mechanism for the selective eradication of LSCs by several compounds, such as parthenolide (PTL), dimethyl-aminoparthenolide (DMAPT), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), and 4-hydroxynonenal (HNE) (16–19).Another promising agent that could be used in this regard is fenretinide, a synthetic retinoid that lacks a carboxyl functional group likely necessary for retinoid receptor activity (20). We and others have previously demonstrated that fenretinide, unlike classical retinoids that often induce differentiation, triggers apoptotic effects; it is largely achieved through the generation of ROS (21–24), enhanced cellular ceramide, and/or ganglioside D3 (25). Moreover, several key stem cell survival-associated signaling pathways, such as NF-κB, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), have been reported to be inactivated in the fenretinide-induced apoptosis in different cancer cell types (25, 26); this further suggests the therapeutic value of fenretinide in targeting cancer stem cells.Fenretinide has been used clinically for some time as an effective chemopreventive agent for various cancers (27). It can significantly reduce the risk of breast cancer and small cell lung cancer (28, 29), suggesting an ability to prevent the development of cancer and/or eliminate early-stage malignant cells (likely cancer-initiating cells). Furthermore, long-term clinical trials have demonstrated only minimal side effects in patients receiving fenretinide (28, 30–32). In particular, no significant hematopoietic toxicity has been observed in patients treated with fenretinide (28). To illustrate the potential value of fenretinide in AML therapies, in this study, we defined the fenretinide effects on primary AML CD34⁺ cells and LSC-enriched AML CD34⁺CD38⁻ cells.     

High-dose cytarabine and daunorubicin induction and postremission chemotherapy for the treatment of acute myelogenous leukemia in adults

Seventy consecutive adult patients with acute myelogenous leukemia (AML), median age 44 years, received high-dose cytarabine (3 g/m2 every 12 hours for 12 doses) followed by daunorubicin (45 mg/m2 daily for three doses) for remission induction. A single, identical course was planned for postremission therapy. Complete remission (CR) was achieved in 63 patients (90%, 95% confidence interval [CI] 83% to 97%), 60 after a single course. Eight patients were selected to undergo elective bone marrow transplantation (BMT) during first CR. Of the remaining 55 patients, 40 (73%) underwent planned post-CR therapy; 15 patients did not, owing to early relapse, excessive toxicity from the induction chemotherapy, or refusal. Nineteen patients, including 13 who received planned post-CR therapy, remain in continuous CR at a median follow-up of 5.2 years (range 3.0 to 7.1 years). The 5-year actuarial leukemia-free survival was 30% (95% Cl, 19% to 42%) for all patients achieving CR and 32% (95% Cl, 19% to 47%) for the 40 patients who received the planned post-CR chemotherapy. Analysis of various putative prognostic factors for CR and overall and leukemia-free survival showed significance for a previous history of myelodysplasia, higher initial leukocyte counts, certain French-American-British (FAB) types, and certain abnormal karyotypes. None of these factors was consistently significant regarding the above parameters, although small patient numbers in certain analyses may have obscured significant associations. Myelosuppression was occasionally prolonged after remission induction and especially post-CR therapy. Severe cerebellar toxicity was observed in 13 patients; in 11 cases, this toxicity was fully reversible. Other serious complications were infrequent. Intensive chemotherapy with high-dose cytarabine and daunorubicin has substantial antileukemic activity in adult AML, and may represent an improvement over conventional therapy. Relapses were common, however, even in patients who received planned therapy, and substantial toxicity was observed. The optimum use of this regimen in AML remains to be determined.     

Intensive sequential chemotherapy with mitoxantrone and continuous infusion etoposide and cytarabine for previously treated acute myelogenous leukemia

Intensive sequential chemotherapy with mitoxantrone, 12 mg/m2/d on days 1 through 3, etoposide, 200 mg/m2/d as a continuous infusion on days 8 through 10, and cytarabine, 500 mg/m2/d as a continuous infusion on days 1 through 3 and 8 through 10 was administered to 72 patients aged less than 60 years with previously treated acute myelogenous leukemia (AML). Forty patients had refractory AML (nonresponse to prior therapy, early first relapse, or multiple relapse) and 32 had late first relapse. Sixty-one percent of patients, with a 95% confidence interval (CI) ranging from 49% to 72%, achieved complete remission (CR), including 45% (CI: 30% to 62%) of refractory patients and 81% (CI: 64% to 93%) of late first relapse patients. Twenty-nine percent of patients (CI: 19% to 41%) did not respond to therapy and 10% (CI: 4% to 19%) died from therapy-related toxicity. Median duration of aplasia was 30 days. Nonhematologic WHO grade 3 or more toxicity included sepsis (57% of patients), vomiting (10%), mucositis (35%), diarrhea (7%), skin rash (6%), and hyperbilirubinemia (11%). Postinduction therapy was attempted in 36 of 44 CR patients: 16 of them received a second course of the same regimen, 7 received maintenance chemotherapy, 4 underwent autologous bone marrow transplantation (BMT), and 9 allogeneic BMT. At a median follow-up of 20 months, 23 of the 44 complete remitters have relapsed, 1 to 14 months after achievement of CR, including 19 of 31 patients not undergoing BMT. Median survival is 7 months with 16% (CI: 4% to 28%) projected survival at 47 months. Median disease-free survival is 6 months with 21% (CI: 3% to 39%) of CR patients projected to remain disease-free at 46 months. Twenty-six percent (CI: 13% to 43%) of the evaluable patients who did not receive transplantation had inversion of CR duration. Among patients younger than 50 years, there was no significant difference in disease-free survival between patients receiving postinduction chemotherapy and those receiving BMT. We conclude that this chemotherapy regimen is highly efficient and could be used as first-line therapy in young patients with AML.