Therapeutic Strategy For Acute Leukemia Research Articles

Effects of Methionine Restriction on Proliferation, Cell Cycle, and Apoptosis of Human Acute Leukemia Cells

To investigate the effects of methionine restriction on proliferation, cell cycle and apoptosis of human acute leukemia cells. Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of methionine restriction on HL-60 and Jurkat cells proliferation. The effect of methionine restriction on cell cycle of HL-60 and Jurkat cells was examined by PI staining. Annexin V-FITC / PI double staining was applied to detect apoptosis of HL-60 and Jurkat cells following methionine restriction. The expression of cell cycle-related proteins cyclin B1, CDC2 and apoptosis-related protein Bcl-2 was evaluated by Western blot assay. Methionine restriction significantly inhibited the proliferation of HL-60 and Jurkat cells in a time-dependent manner (HL-60: r =0.7773, Jurkat: r =0.8725), arrested the cells at G2/M phase (P < 0.001), and significantly induced apoptosis of HL-60 and Jurkat cells (HL-60: P < 0.001; Jurkat: P < 0.05). Furthermore, Western blot analysis demonstrated that methionine restriction significantly reduced the proteins expression of Cyclin B1 (P < 0.05), CDC2 (P < 0.01) and Bcl-2 (P < 0.001) in HL-60 and Jurkat cells. Acute leukemia cells HL-60 and Jurkat exhibit methionine dependence. Methionine restriction can significantly inhibit the proliferation, promote cell cycle arrest and induce apoptosis of HL-60 and Jurkat cells, which suggests that methionine restriction may be a potential therapeutic strategy for acute leukemia.

Patient-derived intrafemoral orthotopic xenografts of peripheral blood or bone marrow from acute myeloid and acute lymphoblastic leukemia patients: clinical characterization, methodology, and validation

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are malignant clonal diseases of the hematopoietic system with an unsatisfactory overall prognosis. The main obstacle is the increased resistance of AML and ALL cells to chemotherapy. The development and validation of new therapeutic strategies for acute leukemia require preclinical models that accurately recapitulate the genetic, pathological, and clinical features of acute leukemia. A patient-derived orthotopic xenograft (PDOX) model is established using surgical orthotopic implantation. They closely resemble human tumor progression and microenvironment and are more reliable translational research tools than subcutaneous-transplant models. In this study, we established PDOX models by direct intrafemoral injection of bone marrow and peripheral blood cells from AML and ALL patients, characterized their pathology, cytology, and genetics, and compared the model's characteristics and drug responsiveness with those of the corresponding patients.

Inhibition of DNA damage response pathway using combination of DDR pathway inhibitors and radiation in treatment of acute lymphoblastic leukemia cells.

An alarming increase in acute lymphoblastic leukemia cases among children and adults has attracted the attention of researchers to discover new therapeutic strategies with a better prognosis. In cancer cells, theDNA damage response (DDR) pathway elements have been recognized to protect tumor cells from various stresses and cause tumor progression;targeting these DDR members is an attractive strategy for treatment of cancers. The inhibition of theDDR pathway in cancer cells for the treatment of cancers has recently been introduced. Hence, effective treatment strategies are needed for this purpose. Chemotherapy in combination with radiotherapy is considered a potential therapeutic strategy for acute leukemia. This review aims to assess the synergistic effects of these inhibitors with irradiation for the treatment of leukemia.

Small molecule inhibition of Dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells

Intensive chemotherapy for acute leukemia can usually induce complete remission, but fails in many patients to eradicate the leukemia stem cells responsible for relapse. There is accumulating evidence that these relapse-inducing cells are maintained and protected by signals provided by the microenvironment. Thus, inhibition of niche signals is a proposed strategy to target leukemia stem cells but this requires knowledge of the critical signals and may be subject to compensatory mechanisms. Signals from the niche require receptor-mediated endocytosis, a generic process dependent on the Dynamin family of large GTPases. Here, we show that Dynole 34-2, a potent inhibitor of Dynamin GTPase activity, can block transduction of key signalling pathways and overcome chemoresistance of leukemia stem cells. Our results provide a significant conceptual advance in therapeutic strategies for acute leukemia that may be applicable to other malignancies in which signals from the niche are involved in disease progression and chemoresistance.

Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of Kv1.3 potassium channels, AKT and ERK1/2 signaling

BackgroundTreatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine.MethodsWe analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients’ acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA.ResultsOur study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis.ConclusionsOur study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia.

The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

BackgroundThere are some limitations of standard chemotherapy for acute leukemia. Vincristine and doxorubicin are commonly used for acute leukemia, but they may induce serious side effects such as cardiomyopathy and neurotoxicity. Furthermore, chemotherapy resistance occurs more and more frequently. Therefore, effective treatment strategies are needed. Histone deacetylase 6 inhibition is considered as a potential therapeutic strategy for acute leukemia, since it is observed that HDAC6 is overexpressed in acute leukemia and regulates tumor survival. Combination therapy for cancer is used to minimize adverse drug effects, reduce drug dosage, enhance efficacy, and prevent drug resistance. In order to improve efficacy of chemotherapy agents of acute leukemia, this study will investigate the effects of combination MPT0G211, a novel histone deacetylase 6 inhibitor, with doxorubicin or vincristine on human acute leukemia cells.ResultsMPT0G211 combined with doxorubicin induces DNA damage response on human acute myeloid leukemia cells. MPT0G211 can additionally increase Ku70 acetylation and release BAX to mitochondria. Ectopic expression of HDAC6 successively reversed the apoptosis triggered by the combined treatment. Moreover, co-treatment of MPT0G211 and vincristine may alter microtubule dynamics, triggering acute lymphoblastic leukemia cells arrest in mitotic phase followed by induction of the apoptotic pathway. Finally, MPT0G211 plus doxorubicin or vincristine can significantly improve the tumor growth delay in a tumor xenograft model.ConclusionsCollectively, our data highlighted that MPT0G211 in combination with chemotherapy drugs has significant anticancer activity, suggesting a novel strategy for the treatment of acute leukemia.

HOXA9 Reprograms the Enhancer Landscape to Promote Leukemogenesis

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis invivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

High-affinity small molecular blockers of mixed lineage leukemia 1 (MLL1)-WDR5 interaction inhibit MLL1 complex H3K4 methyltransferase activity

MLL1-WDR5 protein-protein interaction is essential for MLL1 H3K4 methyltransferase activity. Targeting MLL1 enzymatic activity to regulate expression level of MLL-dependent genes represents a therapeutic strategy for acute leukemia harboring MLL fusion proteins. Herein we reported a series of biphenyl compounds disturbed MLL1-WDR5 interaction. These compounds effectively inhibited MLL1 histone methyltransferase (HMT) activity in vitro and in MV4-11 cell line. The representative compound 30 (DDO-2084) inhibited proliferation and induced apoptosis of MV4-11 cells through deregulating expression level of Hoxa9 and Meis-1 genes, which emphasized our compounds were on-target. Optimization of compound 30 led to high-affinity inhibitors. Especially, compound 42 (DDO-2117, IC50 = 7.6 nM) bearing an amino and a 4-aminobutanamido group was the most potent inhibitor reported to-date, and showed the most potent inhibitory activity (IC50 = 0.19 μM) in HMT assay.

Study of prognostic significance of marrow angiogenesis assessment in patients with de novo acute leukemia

BackgroundAngiogenesis is the highly ordered formation of new blood vessels from pre-existing vessels. It is seen throughout growth, in wound healing, menses, and is important in cancer, where pro- and antiangiogenic signals can be released by cancer cells, endothelial cells, stromal cells, blood, and the extracellular matrix.Aim of the study is to use standardized method for counting blood vessels to verify the significance and prognostic value of assessing marrow angiogenesis at diagnosis of de novo acute leukemia.Subjects and methodsThe study included 70 newly diagnosed acute leukemia cases and a control group composed of 35 bone marrow biopsy sections obtained from breast cancer patients. Examination of CD34 immunohistochemically stained sections for the assessment of marrow angiogenesis by quantification of its microvessel density (MVD).ResultsMVD was significantly increased in acute leukemia patients in comparison to control group (P-value <0.001). Increased MVD was associated with unfavorable outcome.ConclusionThe study demonstrated an evidence of increased angiogenesis in acute leukemia detected by high bone marrow MVD which may play a significant role in leukemic process. Understanding its role may help in designing new therapeutic strategies for acute leukemia.

Inhibitory effect of combination of rapamycin and 5-Aza-dC on Nalm-6 cells and the underlying mechanisms

Objective To investigate the effects and underlying mechanisms of combination of mTOR inhibitor rapamycin(RAPA)and DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine(5-Aza-dC)on the cell proliferation and apoptosis of human acute leukemia cell lines Nalm-6 for providing a new promising targeted therapeutic strategy for acute leukemia. Methods The recovered Nalm-6 cells were cultured to exponential phase, and divided into groups in which RAPA and 5-Aza-dC with different concentrations or their combinations. The effects on cell proliferation were analyzed by CCK-8 assay. Apoptosis was detected by flow cytometry with Annexin V-PE/7-AAD staining. Western blot analysis was performed to examine the expression of TSC2 protein and phosphorylation of mTOR. Results RAPA and 5-Aza-dC could inhibit the proliferative activity of Nalm-6 cells in a dose dependent manner(P < 0.05). Combination of 5-Aza-dC with RAPA exerted a synergistic anti-poliferative effect on Nalm-6 cells(CI < 1). apoptosis was induced by RAPA and 5-Aza-dC in Nalm-6 cells(P < 0.05). Compared with RAPA alone groups, combined groups could observably increase the apoptosis rate of Nalm-6 cells(P < 0.05). In Nalm-6 cells, 5-Aza-dC could increase the expression of TSC2 protein. Though RAPA and 5-Aza-dC individually inhibited the phosphorylation of mTOR, combination of them were more significantly effective in inhibiting mTOR phosphorylation. Conclusions Combination of RAPA and 5-Aza-dC exerted a synergistic effect on inhibition of cell proliferation and induction of cell apoptosis in Nalm-6 cells. The mechanisms may be associated with the regulation of the proteins expression of mTOR pathway. Key words: Leukemia; Rapamycin; 5-aza-2'-deoxycytidine; Mammalian target of rapamycin

Prognostic significance of B-cell lymphoma 2 expression in acute leukemia: A systematic review and meta-analysis.

A number of studies have provided estimates of the correlation between B-cell lymphoma 2 (Bcl-2) expression and its clinical significance in acute leukemia (AL); however, the results have been heterogeneous. In order to clarify the prognostic significance of Bcl-2 status in patients with AL, a systematic review and meta-analysis of 5 published studies including a total of 665 subjects was performed. The reported frequency of Bcl-2 expression was 0-99.00%. Bcl-2-positive patients had a higher median white blood cell count compared to Bcl-2-negative patients. Additionally, Bcl-2-negative patients had >2-fold higher odds of achieving complete remission (CR) compared to Bcl-2-positive patients. The summary hazard ratio of Bcl-2 negativity/positivity for CR was 0.62 [95% confidence interval: 0.53-0.81, P<0.001]. Although this meta-analysis was based on data abstracted from observational studies, our results may justify the use of risk-adapted therapeutic strategies for AL according to the Bcl-2 expression status.

Abstract C250: Concurrent targeting Bcl-2/Bcl-xL and MDM2 as a new therapeutic strategy for acute leukemia.

Abstract Acquired resistance of tumor cells to anticancer drugs is a major cause of cancer therapy failure in the clinic. In this study, we have investigated the acquired resistance mechanisms in vitro and in vivo for two novel anticancer drugs currently in clinical trials, a Bcl-2/Bcl-xL inhibitor ABT-263 and a MDM2-p53 interaction inhibitor MI-77301/SAR405838. In adult relapsed ALL (RS4;11) xenograft model, both ABT-263 and SAR405838 induced rapid and complete tumor regression but tumors regrew shortly after treatment cessation for both drugs. In pediatric AML (MV4;11) xenograft model, ABT-263 induced tumor growth inhibition and SAR405838 induced rapid and complete tumor regression but tumors also regrew after treatment stopped. Analysis of RS4;11 and MV4;11 resistant tumors to both drugs show that tumor cells evade apoptosis induction by the MDM2 inhibitor by mutation of p53 gene or compromised p53 function, whereas in RS4;11 tumor cells lose their sensitivity to the Bcl-2/Bcl-xL inhibitor through down-regulation of pro-apoptotic Bcl-2 family member BAX. Interestingly, RS4;11 tumors which became resistant to SAR405838, retained sensitivity to ABT-263 and rapid tumor regression was observed upon treatment with ABT-263. Similarly, in both RS4;11 and MV4;11 models, tumor cells which became highly resistant to ABT-263 were still very sensitive to SAR405838 and tumors resistant to the treatment of ABT-263 underwent rapid and complete tumor regression upon treatment with SAR405838. Furthermore, in both leukemia xenograft mouse models, sequential treatment with ABT-263 followed by SAR405838 achieved rapid and complete tumor regression with extended tumor free survival compared to the single agents. Additionally, the combination of SAR405838 and ABT-263 achieved a higher degree of long-term tumor regression than either agent alone. Our study provides new insights into the resistant mechanisms for both Bcl-2/Bcl-xL and MDM2 inhibitors in acute leukemia. Importantly, our preclinical data suggest that the combination and sequential treatment of these two distinct classes of novel apoptosis-inducing agents should be explored as an innovative treatment strategy for acute leukemia in the clinic with the goal to overcome acquired drug resistance and to improve the long-term clinical outcome for acute leukemia patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C250. Citation Format: C. Gianna Hoffman-Luca, Donna McEachern, Daniel Ziazadeh, Yu-Jun Zhao, Wei Sun, Laurent Debussche, Shaomeng Wang. Concurrent targeting Bcl-2/Bcl-xL and MDM2 as a new therapeutic strategy for acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C250.

Heatshockprotein-60/90 Are Overexpressed in Leukemia Stem Cells and Stabilize Oncogene Signaling.

AbstractAbstract 2472 Introduction:Using a global phosphoproteome screen, we have recently identified heat shock proteins (HSPs) to be crucial effectors to stabilize KIT oncoprotein function in the presence of tyrosine kinase inhibitors in mutant-KIT leukemia models (Kampa-Schittenhelm et al., ASH annual meeting 2010). We now show that HSP60 and (p)HSP90 are frequently expressed in acute leukemia, arguing for an integrative function of HSPs to stabilize leukemia-driving oncoproteins. Importantly, HSPs are highly expressed in leukemia stem cells (LSCs), which provides a rationale for therapy insensitivity. Targeting HSPs may therefore be an attractive strategy to target the LSC clone. Methods:Intracellular protein levels of (p)HSPs in leukemic blasts were studied by flow cytometry. Three dimensional detection of the putative CD34+/CD38-/HSP+ leukemia stem/progenitor cell fraction was performed using -FITC, -PE and APC-coupled secondary antibodies using standard techniques. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Immunoblot experiments to assess IPI-504 interaction on the chaperone level were set up using standard protocols including a LICOR imaging system. Results:Of 16 evaluated patients with AML, 50% and 56% demonstrated significant HSP90 and HSP60 expression levels (with a threshold level set as > 3× of the relative mean fluorescence compared to IgG controls), respectively. Interestingly, HSP expression was predominantly seen in patients with core–binding factor leukemia (associated with KIT mutations). Moreover, five evaluable freshly harvested patient samples were used to assess the putative CD34+/CD38-/HSP90 and HSP60 population by flow cytometry. Tantalizingly, all tested CD34+/CD38- samples revealed high co-expression of HSPs. Consequently, an HSP90 inhibitor (IPI-504) potently inhibited cellular proliferation in in vitro leukemia models as well as freshly harvested leukemia cells at highly variable doses starting in low nanomolar ranges up to lower micromolar concentrations with regard to IC50s. Several cell lines with defined leukemia-driving oncogenes (MOLM14 – FLT3 ITD, HMC1.1 – KIT V560G, Kasumi1 – KIT N822H) were treated with IPI-504, and degradation of FLT3 and KIT was confirmed by immunoblotting. Consequently, oncogene-degradation led to induction of apoptosis in the nanomolar range which was confirmed in ex vivo blasts. Noteworthy, treating isolated freshly harvested CD34+/CD38- progenitor cells lead to potent reduction of the viable cell fraction. Conclusion:HSPs are frequently expressed in acute leukemias, including LSCs, arguing for a function as mechanism to protect and uphold LSC function in the presence of cell stress such as antileukemic treatment. Targeting the HSP-expressing stem cell pool may be an attractive novel therapeutic strategy in acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Successful Treatment of Acute Leukemia with t(4; ll) in an Infant with Congenital Hypothyroidism

We describe a case of acute leukemia with t(4;11) (q21;q23) in a 3-month-old girl suffering from congenital hypothyroidism. The blast cells were cytochemically and immunologically classifiable as acute lymphoblastic leukemia of an early B-cell lineage (HLA-DR +, B4 +, CALLA-). but we treated this patient with a protocol designed mainly for acute nonlymphocytic leukemia, employing Adriamycin, vincristine, and cytosine arabinoside. Although the prognosis of this type of leukemia is known to be extremely poor, our patient is currently alive and in continuous complete remission lasting 35 months to date. This case may demonstrate a potential alternative therapeutic strategy for acute leukemia with t(4;11) as well as clinical evidence for the mixed-lineage characteristics of this condition. However, the pathogenetic role of congenital hypothyroidism in the development of acute leukemia is still uncertain.