Pathway In Acute Lymphoblastic Leukemia Research Articles

Association between high-dose methotrexate-induced toxicity and polymorphisms within methotrexate pathway genes in acute lymphoblastic leukemia.

Methotrexate (MTX) is a folic acid antagonist, the mechanism of action is to inhibit DNA synthesis, repair and cell proliferation by decreasing the activities of several folate-dependent enzymes. It is widely used as a chemotherapy drug for children and adults with malignant tumors. High-dose methotrexate (HD-MTX) is an effective treatment for extramedullary infiltration and systemic consolidation in children with acute lymphoblastic leukemia (ALL). However, significant toxicity results in most patients treated with HD-MTX, which limits its use. HD-MTX-induced toxicity is heterogeneous, and this heterogeneity may be related to gene polymorphisms in related enzymes of the MTX intracellular metabolic pathway. To gain a deeper understanding of the differences in toxicity induced by HD-MTX in individuals, the present review examines the correlation between HD-MTX-induced toxicity and the gene polymorphisms of related enzymes in the MTX metabolic pathway in ALL. In this review, we conclude that only the association of SLCO1B1 and ARID5B gene polymorphisms with plasma levels of MTX and MTX-related toxicity is clearly described. These results suggest that SLCO1B1 and ARID5B gene polymorphisms should be evaluated before HD-MTX treatment. In addition, considering factors such as age and race, the other exact predictor of MTX induced toxicity in ALL needs to be further determined.

The role of E3 ubiquitin ligase WWP2 and the regulation of PARP1 by ubiquitinated degradation in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) has been a huge threat for people's health and finding effective target therapy is urgent and important. WWP2, as one of E3 ubiquitin ligase, is involved in many biological processes by specifically binding to substrates. PARP1 plays a role in cell apoptosis and is considered as a therapeutic target of certain cancers. In this study, we firstly found that WWP2 expressed higher in newly diagnosed ALL patients comparing with complete remission (CR) ALL patients and normal control people, and WWP2 in relapse ALL patients expressed higher than normal control people. WWP2 expression was related with the FAB subtype of ALL and the proportion of blast cells in bone marrow blood tested by flow cytometry. We demonstrated knockout WWP2 inhibited the ALL growth and enhanced apoptosis induced by Dox in vitro and vivo for the first time. WWP2 negatively regulated and interacted with PARP1 and WWP2 mechanically degraded PARP1 through polyubiquitin-proteasome pathway in ALL. These findings suggested WWP2 played a role in ALL development as well as growth and apoptosis, and also displayed a regulatory pathway of PARP1, which provided a new potential therapeutic target for the treatment of ALL.

Calcicoptosis Induced by Purple Sweet Potato Anthocyanins through the Nonosmotic Regulation of the NFAT5/S100A4-S100A9 Pathway in Acute Lymphoblastic Leukemia.

Purple sweet potato is considered an abundant, inexpensive, and ideal source of anthocyanins. Purple sweet potato anthocyanins (PSPAs) have been shown to possess high antimutagenicity and antitumor effects due to the abundance of acylated anthocyanins. However, the effect and underlying mechanism of PSPA effects in acute lymphoblastic leukemia (ALL), especially T-cell acute lymphoblastic leukemia (T-ALL), remain unclear. In this study, the antileukemic effects of PSPAs and the underlying molecular mechanisms were evaluated by in vitro and in silico assays. PSPAs extracted from ten cultivars were analyzed and quantified. Anthocyanins from Nanzi 018, which showed the best antileukemic effect, were selected to analyze the underlying mechanism. First, the PSPAs potently reduced cell viability and induced apoptosis. Additionally, the PSPAs sharply increased intracellular Ca2+ levels, which resulted in calcium overload in T-ALL cells. Furthermore, on the basis of bioinformatics analyses, we focused on an osmotically regulated transcription factor, NFAT5. Molecular docking preliminarily indicated that PSPA molecules bound and interacted with the NFAT5 protein. Western blot analyses confirmed that PSPAs elicited calcium overload by nonosmotic regulation of NFAT5/S100A4-S100A9 pathway activation. Moreover, pretreatment with a NFAT5 inducer confirmed that PSPAs targeted NFAT5 and affected p38/NF-κB/Bcl-2/Caspase-3 axis activation. This study demonstrates that PSPAs exert their antileukemic effects through calcicoptosis induction by targeting NFAT5.

Molecular effects of genistein, as a potential anticancer agent, on CXCR-4 and VEGF pathway in acute lymphoblastic leukemia.

Vascular endothelial growth factor (VEGF) is one of the angiogenic mediators that can be secreted by leukemic cells and plays an important role in tumor invasion and metastasis. Another important agent contributing to the relapse of ALL is C-X-C chemokine receptor type-4 (CXCR-4), expression of this receptor in cancer cells has been related to metastasis. It has been identified that genistein-a soy-derived isoflavonoid-has anti-angiogenesis functions. We aimed to show the effects of this compound on VEGF and CXCR-4 in Acute lymphoblastic leukemia (ALL) cell models. The cytotoxicity of Genistein was measured using the MTS colorimetric assay. After being treated with Genistein, the expression of VEGF in mRNA and protein levels was measured in MOLT-4 and Jurkat cells. We also used flow cytometry assay to determine the expression of CXCR-4 in cell surfaces. We found that Genistein decreased cell viability in two cell models while was more effective on MOLT-4 cells. After Genistein-treatment, surface expression levels of CXCR-4 were decreased, while VEGF secretion and mRNA expression levels were increased in MOLT-4 and Jurkat cells. The results suggest that Genistein may not be a reliable choice for the treatment of ALL; however, this different identified pattern can be useful for the recognition of VEGF and CXCR-4 modulators and thus for planning new treatments for leukemia and other VEGF related disorders.

The Role Played by Wnt/β-Catenin Signaling Pathway in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic neoplastic disorder that arises from the clonal expansion of transformed T-cell or B-cell precursors. Thanks to progress in chemotherapy protocols, ALL outcome has significantly improved. However, drug-resistance remains an unresolved issue in the treatment of ALL and toxic effects limit dose escalation of current chemotherapeutics. Therefore, the identification of novel targeted therapies to support conventional chemotherapy is required. The Wnt/β-catenin pathway is a conserved signaling axis involved in several physiological processes such as development, differentiation, and adult tissue homeostasis. As a result, deregulation of this cascade is closely related to initiation and progression of various types of cancers, including hematological malignancies. In particular, deregulation of this signaling network is involved in the transformation of healthy HSCs in leukemic stem cells (LSCs), as well as cancer cell multi-drug-resistance. This review highlights the recent findings on the role of Wnt/β-catenin in hematopoietic malignancies and provides information on the current status of Wnt/β-catenin inhibitors with respect to their therapeutic potential in the treatment of ALL.

EXT1, Regulated by MiR-665, Promotes Cell Apoptosis via ERK1/2 Signaling Pathway in Acute Lymphoblastic Leukemia

BackgroundEXT1 is an endoplasmic reticulum-resident glycosyl transferase whose intracellular expression alters the biosynthesis and distribution of heparan sulfate. EXT1 is regarded as a classic tumor suppressor. MiR-665 can act as either an oncogene or tumor-suppressing gene in different tumors. The aim of the current study was to determine the function and molecular mechanisms of EXT1 and miR-665 in acute lymphoblastic leukemia (ALL).Material/MethodsEXT1 expression in ALL was evaluated by real-time polymerase chain reaction (RT-PCR) and western blotting. The effects of EXT1 in ALL were explored by Cell Counting Kit-8 (CCK-8)/EdU assays, western blotting, flow cytometry, and in vivo tumorigenesis assays. Label-free quantification was used to detect differentially expressed proteins in EXT1-overexpressing Reh cells.ResultsEXT1 expression is downregulated in ALL and negatively correlated with miR-665 expression. Moreover, low EXT1 and high miR-665 expression levels in adult ALL bone marrow tissues are correlated with poor patient survival. Our study showed that EXT1 modulates the proliferation and apoptosis of ALL cells in vitro and in vivo and that miR-665 promotes cell growth and inhibits apoptosis by suppressing EXT1. EXT1 promotes cell apoptosis via deactivating the ERK1/2 pathway.ConclusionsIn conclusion, this study is the first to confirm the association between low EXT1 levels and several clinical features of ALL. Low bone marrow EXT1 levels independently predict poor prognoses in adult ALL patients. Thus, our study suggests that EXT1- or miR-665-targeted strategies can confer the therapeutic effect of promoting apoptosis by deactivating the ERK1/2 pathway.

Abstract 1289: AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. We previously uncovered that ALL are vulnerable to metabolic/energy stress and ER-stress via AMP-activated protein kinase (AMPK) activation. In order to identify genome wide metabolic stress and AMPK transcriptionally regulated genes, we used RNA-Seq and compared mRNA profiles in ALL cells treated with the adenosine analog AICAR, an activator of AMPK. RNA-Seq data indicated that high dose AICAR (15 mM/45 min) induced a robust downregulation of a cluster of genes known as the immediate early genes (IEGs), which are critical for cell survival, proliferation and adaptation. AICAR-induced downregulation on IEG expression was dose- and time-dependent, and observed in other cell types (HEK293T, Hela, MEF), indicating this mechanism is conserved in mammalian cells. Using MEF AMPKα2 and AMPKα1/α2 knockout cell lines, we found that these alterations were AMPK-independent. Characterization of AICAR’s mechanism of action identified the protein kinase D1 (PKD1) as responsible for these effects. PKD1 is a Ser/Thr protein kinase involved in many cellular processes important for cancer development and progression, including proliferation, survival, apoptosis, invasion, cell adhesion and angiogenesis. We uncovered that high dose AICAR significantly inhibited PKD1 activation (Ser-910) at the plasma membrane which prevented its nuclear translocation. When PKD1 activity was pharmacologically inhibited by CRT0066101 or downregulated by shRNA, we observed similar IEGs’ downregulation in ALL cells. Conversely, the effect of AICAR on IEGs’ expression was abrogated by PMA, a direct activator of PKD1. In addition, when PKD1 was overexpressed in HEK293T cells, AICAR-induced IEG’s downregulation was partially restored. Using a kinase assay, we found that AICAR, but not ZMP, directly inhibited PKD1 kinase activity. Further, we determined that AICAR suppressed phosphorylation and nuclear export of PKD1-targeted histone deacetylases HDAC4/5, which led to decreased histone H3 acetylation at the IEGs’ promoter region. Finally, ChIP-qPCR indicated that AICAR-induced PKD1 inhibition prevented NF-κB recruitment to IEGs’ promoters. Inhibition of PKD1 activity led to decreased cell proliferation and promoted apoptosis in ALL cells. To confirm the in vivo relevance of our data, single agent and combination experiments using our NSG ALL mouse model are underway. Taken together, we have identified a novel AMPK-independent mechanism leading to AICAR’s inhibition of PKD1-mediated ALL survival. Consequently, co-targeting PDK1 and other pro-survival stress response pathways in ALL cells offers novel strategies to overcome therapeutic resistance. Citation Format: Anna Shvab, Guangyan Sun, Bin Li, Felipe Beckedorff, Guy J. Leclerc, Ramin Shiekhattar, Julio C. Barredo. AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1289.

Effects of Rituximab on JAK-STAT and NF-κB signaling pathways in acute lymphoblastic leukemia and chronic lymphocytic leukemia

Abstract Objectives Rituximab is a monoclonal antibody that targets the B-lymphocyte surface antigen CD20. It is used in the treatment of some diseases including B-cell chronic lymphocytic leukemia (B-CLL). There are a lot of data regarding effect of Rituximab on lymphoma cells. But, there is no satisfactory information about the effect of Rituximab on the signaling pathways in leukemia cells. In this study, it was aimed to understand the effect of Rituximab on JAK-STAT and NF-κB signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) and B-CLL. Material and methods Apoptotic effect of Rituximab in the TANOUE (B-ALL) and EHEB (B-CLL) cell lines were evaluated by using the Annexin V method. mRNA expression levels of STAT3 and RelA were analysed by quantitative RT-PCR (Q-PCR). Alterations in STAT3 and RelA protein expressions were detected by using a chromogenic alkaline phosphatase assay after Western Blotting. Results Rituximab had no apoptotic effect on both cell lines. Complement-mediated cytotoxicity was only detected in EHEB cells. mRNA and protein expressions of STAT3 and RelA genes were decreased following Rituximab treatment. Conclusion Our preliminary results suggest that the use of Rituximab might be effective in B-ALL though both signaling pathways.

Akt2 Mediates Glucocorticoid Resistance in Acute Lymphoblastic Leukemia through FoxO3a/Bim Axis and Serves As a Direct Target for Resistance Reversal

Background: Glucocorticoids (GCs) are common components in chemotherapeutic protocols for acute lymphoblastic leukemia (ALL). A major obstacle in GC therapy, however, is the gradual acquisition of apoptotic resistance in ALL cells repeatedly treated with these hormones. Previous reports indicate that 15-30% of pediatric ALL samples are resistant to GCs, while in refractory childhood ALL, the prevalence of GC resistance is as high as 70%. Identification of specific molecular mechanisms driving resistance to GC and targeting downstream molecules may lead to the development of new therapeutic strategies.Results: FoxO transcription factor FoxO3a has been shown to regulate apoptosis in lymphocytes. Unphosphorylated FoxO3a can be upregulated by dexamethasone (DEX), which subsequently translocates into the nucleus, upregulates Bim expression and induces apoptosis. In our current study, we cultured a GC-sensitive T-ALL cell line CCRF-CEM with a specific concentration of DEX over multiple passages and obtained a highly resistant cell line, designated CEM-DR. We observed this T-ALL cell line acquires resistance to DEX-mediated killing through abnormal activation of Akt, resulting in inhibition of the FoxO3a/Bim pathway (A). Pharmacologic inhibition of Akt with Akt inhibitor IV effectively restores sensitivity to DEX of CEM-DR by enhancing the FoxO3a/Bim pathway, but shows significant hepatotoxicity with increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo (B). Common hematologic toxicities and hepatic toxicities with increased AST and ALT of Akt inhibitor have also been reported in the treatment of solid tumors in humans, partially limiting their clinical applicability (Becher OJ et al, Pediatr Blood Cancer 2017). There are two closely related, highly conserved homologues of Akt: Akt-1 and -2, which differ in enzyme function. Our study aimed to investigate the potential role of Akt isoforms Akt1 and Akt2 in the mechanism of GC resistance and explore a more direct and specific target for resistance reversal.By western blot analysis we observed the expression of Akt2 in intrinsically resistant T-ALL cells Jurkat was significantly higher than that in sensitive CCRF-CEM cells, while Akt1 expression in aforementioned cell lines was similar. The expression of Akt2 also increased synchronously with the increase of half maximal inhibitory concentration (IC50) of DEX in CEM-DR cells, further indicating that Akt2 expression increases in secondarily resistant lymphocytes. A significantly elevated expression of Akt2 not Akt1 were shown in relapsed/refractory ALL patients when compared with the newly diagnosed patients. To detect if Akt2 was able to directly interact with FoxO3a, co-immunoprecipitation assay was employed. MYC-FoxO3a was co-transfected with Flag-Akt1 or Flag-Akt2 into HEK293T cells. The result demonstrated the more presence of Flag-AKT2 than Flag-Akt1 in MYC-FoxO3a immunoprecipitates, suggesting that AKT2 as the major regulator directly interacting with FoxO3a. Reciprocal immunoprecipitation experiments confirmed the closer association between Flag-Akt2 and MYC-FoxO3a (C). Then we used siRNA to down-regulate Akt1 or Akt2 expression in resistant T-ALL cells; we observed GC-induced apoptosis increased significantly (D) after down-regulation of Akt2 expression, along with the expression of p-FoxO3a decreased (E) .To examine the therapeutic role of Akt isoform specific inhibitors, we treated resistant T-ALL cell with A-674563 (Akt1 inhibitor), CCT128930 (Akt2 inhibitor) or Akti1/2 (Akt1/2 inhibitor). Selective inhibition of Akt2 with CCT128930 more significantly enhances the FoxO3a/Bim signaling pathway, increases DEX-mediated killing of resistant T-ALL cells (F) and effectively reverses GC resistance than Akt1 inhibitor in vitro and in vivo. When exploring the potential influence on viscera by Akt isoform inhibitors in vivo, we found Akt2 inhibitor did not significantly influence hematopoiesis, cardiac and renal functions. Notably, Akt2 inhibitor did not show hepatic toxicities of increased serum ALT, AST and total bilirubin which appear in Akt1 or Akt1/2 inhibition.Conclusions: Akt2 might serve as a more direct and specific kinase mediating GC resistance through FoxO3a/Bim-signaling pathway in ALL, and targeting Akt2 with CCT128930 may be explored as a promising therapeutic strategy for resistance reversal. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Challenges in Targeting Signaling Pathways in Acute Lymphoblastic Leukemia by NGS Approaches: An Update.

The identification and study of genetic alterations involved in various signaling pathways associated with the pathogenesis of acute lymphoblastic leukemia (ALL) and the application of recent next-generation sequencing (NGS) in the identification of these lesions not only broaden our understanding of the involvement of various genetic alterations in the pathogenesis of the disease but also identify new therapeutic targets for future clinical trials. The present review describes the main deletions, amplifications, sequence mutations, epigenetic lesions, and new structural DNA rearrangements detected by NGS in B-ALL and T-ALL and their clinical importance for therapeutic procedures. We reviewed the molecular basis of pathways including transcriptional regulation, lymphoid differentiation and development, TP53 and the cell cycle, RAS signaling, JAK/STAT, NOTCH, PI3K/AKT/mTOR, Wnt/β-catenin signaling, chromatin structure modifiers, and epigenetic regulators. The implementation of NGS strategies has enabled important mutated genes in each pathway, their associations with the genetic subtypes of ALL, and their outcomes, which will be described further. We also discuss classic and new cryptic DNA rearrangements in ALL identified by mRNA-seq strategies. Novel cooperative abnormalities in ALL could be key prognostic and/or predictive biomarkers for selecting the best frontline treatment and for developing therapies after the first relapse or refractory disease.

Glycogen Synthase Kinase-3β (GSK-3β) is a Critical Factor in Intracellular Signaling Pathways in Acute Lymphoblastic Leukemia

Glycogen Synthase Kinase-3β (GSK-3β) is a Critical Factor in Intracellular Signaling Pathways in Acute Lymphoblastic Leukemia

Glycogen Synthase Kinase-3β (GSK-3β ) is a Critical Factor in Intracellular Signaling Pathways in Acute Lymphoblastic Leukemia

Research in Medical & Engineering Sciences Glycogen Synthase Kinase-3β(GSK-3β ) is a Critical Factor in Intracellular Signaling Pathways in Acute Lymphoblastic Leukemia Cristian Fabian Layton Tovar* Autonomous University of Mexico State, Mexico *Corresponding author: Cristian Fabian Layton Tovar, General Agustín Olachea 1401, Ocho Cedros, Toluca, México, CP 50170 Submission: November 11, 2017; Published: December 19, 2017 DOI: 10.31031/RMES.2017.03.000552 ISSN : 2576-8816Volume3 Issue1

Co-existence of IL7R high and SH2B3 low expression distinguishes a novel high-risk acute lymphoblastic leukemia with Ikaros dysfunction.

Acute lymphoblastic leukemia (ALL) remains the leading cause of cancer-related death in children and young adults. Compared to ALL in children, adult ALL has a much lower cure rate. Therefore, it is important to understand the molecular mechanisms underlying high-risk ALL and to develop therapeutic strategies that specifically target genes or pathways in ALL. Here, we explored the IL7R and SH2B3 expression in adult ALL and found that IL7R is significantly higher and Sh2B3 lower expressed in B-ALL compared to normal bone marrow control, and the IL7RhighSH2B3low is associated with high-risk factors, and with high relapse rate and low disease-free survival rate in the patients. We also found that Ikaros deletion was associated with the IL7RhighSH2B3low expression pattern and Ikaros directly binds the IL7R and SH2B3 promoter, and suppresses IL7R and promotes SH2B3 expression. On the other hand, casein kinase inhibitor, which increases Ikaros function, inhibits IL7R and stimulates SH2B3 expression in an Ikaros dependent manner. Our data indicate that IL7RhighSH2B3low expression distinguishes a novel subset of high-risk B-ALL associated with Ikaros dysfunction, and also suggest the therapeutic potential for treatment that combines casein kinase inhibitor, as an Ikaros activator, with drugs that target the IL7R signaling pathway.

RRNA synthesis inhibitor, CX-5461, activates ATM/ATR pathway in acute lymphoblastic leukemia, arrests cells in G2 phase and induces apoptosis.

Ribosome biogenesis is a fundamental cellular process and is elevated in cancer cells. As one of the most energy consuming cellular processes, it is highly regulated by signaling pathways in response to changing cellular conditions. Many of the regulators of this process are aberrantly activated in various cancers. Recently two novel rRNA synthesis inhibitors, CX-5461 and BMH-21, have been shown to selectively kill cancer cells while sparing normal cells. Here, we tested the effectiveness of pre-rRNA synthesis inhibitor CX-5461 on acute lymphoblastic leukemia cells with different cytogenetic abnormalities. Acute lymphoblastic leukemia cells are more sensitive to rRNA synthesis inhibition compared to normal bone marrow cells. CX-5461 treated cells undergo caspase-dependent apoptosis independent of their p53 status. More-over, CX5461, activates checkpoint kinases and arrests cells in G2 phase of cell cycle. Finally, overcoming this G2 arrest by inhibiting ATR kinase leads to robust cell killing. These results show that CX-5461 can be even more potent in combination with ATR inhibitors.

Towards precision medicine in childhood leukemia – Insights from mutationally activated cytokine receptor pathways in acute lymphoblastic leukemia

The successful therapy of childhood leukemia has been characterized by careful personalized adaptation of therapy by risk stratification. Yet almost all drugs are relatively non-specific. To achieve greater precision in therapy, druggable targets and specific targeting drugs are necessary. Here we review the recent discoveries of cytokine receptors and their signaling components in high risk leukemias and the potential approaches to target them.

Targeting signaling pathways in acute lymphoblastic leukemia: new insights

The genetics of acute lymphoblastic leukemia are becoming well understood and the incidence of individual chromosomal abnormalities varies considerably with age. Cytogenetics provide reliable risk stratification for treatment: high hyperdiploidy and ETV6-RUNX1 are good risk, whereas BCR-ABL1, MLL rearrangements, and hypodiploidy are poor risk. Nevertheless, some patients within the good- and intermediate-risk groups will unpredictably relapse. With advancing technologies in array-based approaches (single nucleotide polymorphism arrays) and next-generation sequencing to study the genome, increasing numbers of new genetic changes are being discovered. These include deletions of B-cell differentiation and cell cycle control genes, as well as mutations of genes in key signaling pathways. Their associations and interactions with established cytogenetic subgroups and with each other are becoming elucidated. Whether they have a link to outcome is the most important factor for refinement of risk factors in relation to clinical trials. For several newly identified abnormalities, including intrachromosomal amplification of chromosome 21 (iAMP21), that are associated with a poor prognosis with standard therapy, appropriately modified treatment has significantly improved outcome. After the successful use of tyrosine kinase inhibitors in the treatment of BCR-ABL1-positive acute lymphoblastic leukemia, patients with alternative ABL1 translocations and rearrangements involving PDGFRB may benefit from treatment with tyrosine kinase inhibitors. Other aberrations, for example, CRLF2 overexpression and JAK2 mutations, are also providing potential novel therapeutic targets with the prospect of reduced toxicity.

The NEDD8 Conjugation Pathway Inhibitor MLN4924 Activates AMPK and Induces The ER Stress/UPR-Mediated Cell Death Pathway In Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and cure rates for patients with resistant/relapsed ALL remain poor. In addition, many childhood cancer survivors experience significant long-term therapy related toxicity, highlighting the need for novel targeted anti-leukemic agents. We recently uncovered that ALL cells are vulnerable to undergo apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms (Mol Cancer Res 2012;10:969-978). We reported that the AMP activated protein kinase (AMPK) is activated in response to ER stressors (metformin, 2-DG, tunicamycin), and its activation suppressed the UPR preventing ALL cells from processing misfolded/unfolded proteins in the ER lumen, leading to apoptosis. In search for novel agents targeting AMPK, we investigated the anti-leukemic activity of a new AMP analogue, MLN4924, in Bp- and T-ALL cell line models. This agent targets the ubiquitin-like protein NEDD8 conjugation pathway leading to inhibition of cullin-based E3 ligase dependent proteasomal degradation of proteins essential for cancer cell growth and survival. We demonstrated that MLN4924 induced dose-dependent cell growth inhibition and apoptosis in both Bp-ALL (EC50 = 372 nM, p<0.0001 for MLN4924-treated NALM6 cells vs. controls) and T-ALL (EC50 = 421 nM, p<0.0001 for MLN4924-treated CCRF-CEM cells vs. controls) cell line models. To investigate the mechanisms of cell death induced by MLN4924 in ALL cells, we determined its effect on AMPK, UPR, and NF-κB signaling pathways using Western immunoblots. Our results show that MLN4924 induced phosphorylation of AMPK (Thr172) and down-regulated its downstream target p-mTOR (Ser2448), suggesting that MLN4924 as an AMP-analogue can allosterically induce activation of AMPK and affect downstream AMPK-associated signaling pathways. As expected, MLN4924 decreased significantly the expression of NEDDylated cullins confirming the inhibitory effect of MLN4924 on the NEDD8 conjugation pathway. We also demonstrated that MLN4924 induced ER stress as evidenced by increased expression of the main UPR regulator GRP78 and the UPR cell death mediator CHOP, suggesting that MLN4924 induces cell death in ALL cell models via an UPR-mediated mechanism. Additional Western blots revealed that MLN4924 inhibited the degradation of the inhibitor of NF-κB, p-IκBα (Ser32), with concomitant down-regulation of phosphorylated RelA (p65) at Ser 536, suggesting that stabilization of p-IκBα (Ser32) following inhibition of NEDD8 conjugation pathway by MLN4924 prevents NF-κB activation in ALL. To further characterize the cytotocixity of MLN4924 in ALL cells, we examined the effects of MLN4924 on ROS generation. Using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorogenic dye, we did not detect ROS induction in MLN4924-treated ALL cells and co-treatment of ALL cells with the antioxidant N-Acetyl-L-Cysteine (NAC) did not rescue MLN4924's cytotoxicity in ALL cells. In contrast to data reported in AML (Blood 2010; 115:3796-3800), our findings suggest that ROS does not mediate MLN4924-induced cell death in ALL. Taken together, our data indicate that MLN4924 induces cell death in ALL cells by inhibiting the NEDD8 conjugation pathway and activating AMPK, which respectively trigger down-regulation of the NF-κB pathway and ER stress/UPR-mediated cell death. These findings provide a rationale to further investigate the mechanism of action of MLN4924 either alone or in combination with agents that target the NF-κB pathway and ER stress/UPR pathways in ALL. Disclosures:No relevant conflicts of interest to declare.

Differential Effects of Selective Inhibitors Targeting the PI3K/AKT/mTOR Pathway in Acute Lymphoblastic Leukemia

PurposeAberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.Experimental DesignWe compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.ResultsDual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.ConclusionsInhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.

Epigenetic Regulation of Cell Signaling Pathways in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a heterogeneous cancer that is characterized by rapid and uncontrolled proliferation of immature B- or T-lymphoid precursors. Although ALL has been regarded as a genetic disease for many years, the crucial importance of epigenetic alterations in leukemogenesis has become increasingly evident. Epigenetic mechanisms, which include DNA methylation and histone modifications, are critical for gene regulation during many key biological processes. Here, we review the cell signaling pathways that are regulated by DNA methylation or histone modifications in ALL. Recent studies have highlighted the fundamental role of these modifications in ALL development, and suggested that future investigation into the specific genes and pathways that are altered by epigenetic mechanisms can contribute to the development of novel drug-based therapies for ALL.

Curcumin potentiates antitumor activity of l-asparaginase via inhibition of the AKT signaling pathway in acute lymphoblastic leukemia

l-asparaginase (L-ASP) is a universal component of therapy for acute lymphoblastic leukemia (ALL). Curcumin is a naturally occurring yellow pigment that is derived from the rhizome of Curcuma longa. In this study, we evaluated the cytotoxicity of the combined treatment of L-ASP and curcumin in three ALL cell lines. Synergistic cytotoxicity was observed in all three cell lines following the combined treatment of curcumin and L-ASP. Our results revealed that curcumin significantly enhanced the antitumor effect of L-ASP in the three ALL cell lines compared to that for L-ASP alone ( p < 0.05). Curcumin and L-ASP co-treatments induced apoptosis, via activation and cleavage of caspase-8 and BID cleavage accompanied by release of cytochrome c and activation of caspase-9/3, compared to the group treated with only L-ASP and the control group. Furthermore, the combination of curcumin and L-ASP led to significant reductions in phosphorylated AKT and expression of AKT-regulated gene products (FoxO1, GSK3β, IKKα, NF-κB, XIAP) compared with the group treated with only L-ASP and the control group. Overall, our findings suggest that curcumin potentiates the antitumor effects of L-ASP in acute lymphoblastic leukemia by constitutively inhibiting AKT and AKT-regulated gene products.