Apoptosis In Acute Lymphoblastic Leukemia Research Articles

Molecular Mechanism of circVRK1 Regulating the Proliferation and Apoptosis of Acute Lymphoblastic Leukemia KOCL44 Cells by Targeting miR-4428

To elucidate the role of circVRK1 and its interaction with miR-4428 in regulating proliferation and apoptosis in acute lymphoblastic leukemia (ALL) cells. KOCL44 ALL cells were cultured in vitro, and experimental groups included pcDNA, pcDNA-circVRK1, anti-miR-NC, anti-miR-4428, si-NC, si-circVRK1, pcDNA-circVRK1+miR-NC, and pcDNA-circVRK1+miR-4428. The expression levels of circVRK1 and miR-4428 were detected using qRT-PCR. CCK-8 assays and flow cytometry were used to assess cell proliferation and apoptosis, respectively. The dual luciferase reporter assays were employed to investigate the interaction between circVRK1 and miR-4428, with groups categorized as WT-circVRK1+miR-NC, WT-circVRK1+miR-4428, MUT-circVRK1+miR-NC, and MUT-circVRK1+ miR-4428. Western blotting was utilized to detect the expression levels of Ki-67, cleaved caspase-3, and cleaved caspase-9 proteins. Compared to the pcDNA group, circVRK1 expression was up-regulated in the pcDNA-circVRK1 group (P<0.05). Compared to transfection with pcDNA or anti-miR-NC, transfection with pcDNA-circVRK1 or anti-miR-4428 led to decreased cell viability and Ki-67 protein levels in KOCL44 cells (P<0.05), and increased apoptosis rates and levels of cleaved caspase-3 and cleaved caspase-9 (P<0.05). circVRK1 was found to negatively regulate miR-4428 expression, with this effect observed only in the WT-circVRK1 group. miR-4428 levels were lower in the pcDNA-circVRK1 group compared to the pcDNA group (P<0.05) and higher in the si-circVRK1 group compared to the si-NC group (P<0.05). Co-transfection with pcDNA-circVRK1+miR-4428 resulted in increased cell viability (P<0.05) and Ki-67 expression (P<0.05), and decreased apoptosis rates and levels of cleaved caspase-3 and cleaved caspase-9 (P<0.05) compared to co-transfection with pcDNA-circVRK1+miR-NC. Overexpression of circVRK1 reduces the proliferation ability of acute ALL cells and induces cell apoptosis by downregulating miR-4428 expression.

PKA inhibition is a central step in D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a hematologic cancer that mostly affects children. It accounts for over a quarter of ALL pediatric cancers, causing most of the cancer death among children. Previously, we demonstrated that D,L-methadone causes ALL cell apoptosis via μ-opioid receptor 1 (OPRM1)-triggered ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i. However, the precise mechanism by which D,L-methadone induces ER Ca2+ release remains to be defined. Here, we show that in ALL cells, D,L-methadone-induced ER Ca2+ release is blocked by inhibition of Gαi, but not Gβϒ, indicating that the process is dependent on Gαi. Activation of adenylyl cyclase (AC) with forskolin or treatment with 8-CPT-cAMP blocks D,L-methadone-induced ER Ca2+ release, indicating that the latter results from Gαi-dependent downregulation of AC and cAMP. The 14-22 amide (myr) PKA inhibitor alone elicits ER Ca2+ release, and subsequent treatment with D,L-methadone does not cause additional ER Ca2+ release, indicating that PKA inhibition is a key step in D,L-methadone-induced ER Ca2+ release and can bypass the D,L-methadone-OPRM1-AC-cAMP step. This is consistent with the decrease in PKA-dependent (i) inhibitory PLCβ3 Ser1105 phosphorylation that leads to PLCβ3 activation and ER Ca2+ release, and (ii) BAD Ser118 phosphorylation, which together ultimately result in caspase activation and apoptosis. Thus, our findings indicate that D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in ALL cells is mediated by Gαi-dependent downregulation of the AC-cAMP-PKA-PLCβ3/BAD pathway. The fact that 14-22 amide (myr) alone effectively kills ALL cells suggests that PKA may be targeted for ALL therapy.

Royal jelly induces ROS-mediated apoptosis in acute lymphoblastic leukemia (ALL)-derived Nalm-6 cells: Shedding light on novel therapeutic approaches for ALL.

Until recently, a conventional chemotherapy regimen for Acute lymphoblastic leukemia (ALL) is considered an efficient therapeutic method in children. However, suboptimal long-term survival rates in adults, disease relapse, and drug-induced toxicities require novel therapeutic agents for ALL treatments. Today, natural products with pharmacological benefits play a significant role in treating different cancers. Among the most valued natural products, honey bees' royal jelly (RJ) is one of the most appreciated which has revealed anti-tumor activity against different human cancers. This study aimed to evaluate anti-leukemic properties and the molecular mechanisms of RJ cytotoxicity on ALL-derived Nalm-6 cells. The metabolic activity was measured by MTT assay. Apoptosis, cell distribution in the cell cycle, and intracellular reactive oxygen species (ROS) level were investigated using flow cytometry analysis. Moreover, quantitative real-time PCR (qRT-PCR) was performed to scrutinize the expression of various regulatory genes. RJ significantly decreased the viability of Nalm-6 cells but had no cytotoxic effect on normal cells. In addition, RJ induced ROS-mediated apoptosis by up-regulating pro-apoptotic genes while decreasing anti-apoptotic gene expression. The results outlined that ROS-dependent up-regulation of FOXO4 and Sirt1 inhibits the cells' transition to the S phase of the cell cycle through p21 up-regulation. The qRT-PCR analysis of autophagy-related gene expression also demonstrated that RJ induced BECN1 mediated autophagy in Naml-6 cells. Taken together, this study showed that RJ can be utilized as a potent natural substance to induce ALL cells' programmed cell death. However, further studies are required to examine this compound's pharmaceutical application.

MG132 inhibits proliferation and induces apoptosis of acute lymphoblastic leukemia via Akt/FOXO3a/Bim pathway.

Acute lymphoblastic leukemia (ALL) is one of the most common pediatric cancers, characterized by the malignant proliferation of leukemic cells. Despite advancements in treatment, the prognosis for refractory and relapsed ALL remains poor, underscoring the need for novel therapeutic targets and approaches. To investigate the anti-leukemic properties of MG132, MTS assays were employed to assess cell viability, and flow cytometry was used to evaluate apoptosis. Mechanistic studies, including qRT-PCR, Western blotting, and lentivirus-mediated FOXO3a knockdown, were conducted to explore MG132's effects on the Akt/FOXO3a/Bim signaling pathway. A xenograft mouse model was utilized to validate the in vivo efficacy of MG132 in suppressing tumor growth. MG132 inhibited cell proliferation and induced apoptosis in both ALL cell lines and primary cells in a concentration-dependent manner. Mechanistic studies revealed that MG132 promoted FOXO3a nuclear localization by suppressing Akt phosphorylation and preventing FOXO3a degradation, leading to increased Bim expression. Furthermore, FOXO3a knockdown significantly reduced MG132's anti-proliferative effects. In vivo, MG132 markedly inhibited tumor growth in the xenograft model. These findings suggest that MG132 exerts potent anti-leukemic effects through modulation of the Akt/FOXO3a/Bim axis, offering a promising therapeutic avenue for treating ALL.

MiR-29a-3p Targets Hepatoma-Derived Growth Factor to Inhibit the Proliferation and Promote the Apoptosis of E6-1 Cells

To investigate the regulatory effect of miRNA-29a-3p (miR-29a-3p ) on hepatoma-derived growth factor (HDGF), and its influences on the proliferation and apoptosis of acute lymphoblastic leukemia (ALL) cell line E6-1. Thirty-five patients with ALL treated in our hospital from January 2017 to January 2019 were selected as research objects, and 35 adults who underwent physical examination in the same period were selected as healthy control group. The miR-29a-3p overexpression vector, miR-29a-3p inhibitory expression vector, and miR-29a-3p and HDGF co-overexpression vector were transfected into E6-1 cells. The expression levels of miR-29a-3p and HDGF mRNA were detected by RT-qPCR. The expression of protein was detected by Western blot. The targeting relationship between miR-29a-3p and HDGF was detected by dual luciferase reporter assay. The cell proliferation was detected by CCK-8 method, while apoptosis detected by flow cytometry. Compared with healthy control group, HDGF was highly expressed in serum of patients with ALL and leukemia cells HuT 78, E6-1, CCRF-CEM, while miR-29a-3p was low expressed (P<0.05). After overexpression of miR-29a-3p , the expression levels of CyclinD1 and Bcl-2 in leukemia cells E6-1 were significantly reduced, while the expression levels of p21 and Bax were significantly increased (P<0.05). The activity of E6-1 cells was also significantly reduced, while the apoptosis rate of E6-1 cells was significantly increased (P<0.05). miR-29a-3p could target and regulate the expression of HDGF, while overexpression of HDGF reversed the inhibitory effect of miR-29a-3p overexpression on the proliferation and promotion effect on the apoptosis of leukemia cells E6-1. Overexpression of miR-29a-3p can inhibit the proliferation and promote the apoptosis of ALL cells E6-1, and its mechanism may be related to the regulation of HDGF expression.

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: B-cell acute lymphoblastic leukemia (ALL) harboring KMT2A-rearrangements (KMT2A-R) is a high-risk ALL subtype with significant relapse risk and poor overall survival across the age spectrum. While new antibody-based and cellular immunotherapies have revolutionized the therapeutic paradigm for many patients with relapsed ALL, application of these therapies has proven particularly challenging to date in infants given their immune system immaturity and in older adults prone to high-grade immunotherapy toxicities. Unlike other ALL subtypes, KMT2A-R ALL has an extremely low frequency of co-mutations, which makes it particularly difficult to find new treatment options. To identify targetable kinases in KMT2A-R ALL, we performed a domain-specific kinome-wide CRISPR screen and identified the serine/threonine kinase DYRK1A as required for the survival of KMT2A-R ALL. Importantly, DYRK1A appears to be non-essential for normal tissues per the Cancer Dependency MAP, suggesting potential for a therapeutic window via DYRK1A targeting. Results:In vitro treatment studies using three different menin inhibitors (MI-2-2 / MI-503 / VTP50469) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A at the RNA and the protein level. We further analyzed ChIP-Seq data and identified that oncogenic KMT2A-R directly binds to the DYRK1A promoter, indicating that DYRK1A is directly regulated by the oncogenic KMT2A fusion. Interestingly, the DYRK1A RNA and protein levels were elevated in KMT2A-R ALL cell lines and patient-derived xenograft (PDX) samples compared to non-KMT2A-R ALL cases. To test the consequence of direct pharmacologic DYRK1A inhibition on cell proliferation and apoptosis, we treated KMT2A-R ALL cells with the DYRK1A inhibitors EHT1610 and harmine. Interestingly, we observed that pharmacologic inhibition of DYRK1A with both drugs did not induce apoptosis but induced potent leukemic cell growth inhibition in vitro and in vivo. In preliminary studies, we further observed that pharmacologic inhibition of DYRK1A or genetic deletion resulted in MYC upregulation and hyperphosphorylation of ERK. It has been demonstrated that premature signaling activation in developing B cells induces cell cycle arrest as a protective mechanism to prevent the development of autoimmune diseases. We hypothesized that DYRK1A inhibitor-induced ERK hyperactivation and consequent cell cycle arrest remains a conserved mechanism in KMT2A-R ALL. Supporting our hypothesis, combining DYRK1A inhibitors with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibition does not induce apoptosis and cells restart to proliferate after DYRK1A inhibitor withdrawal, we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. Interestingly, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. BIM is a pro-apoptotic molecule that is inactive when bound by the anti-apoptotic molecule BCL2. Given that DYRK1A inhibition results in MYC upregulation, we performed a new Western blot analysis and validated that EHT1610 and harmine both induce BIM overexpression in KMT2A-R ALL cell lines. To test if combined DYRK1A and BCL2 inhibition will lead to enhanced KMT2A-R ALL apoptosis, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, combining both drugs synergistically killed KMT2A-R ALL cells in vitro. More importantly, we validated the anti-leukemic effect of harmine in combination with venetoclax in mice injected with a KMT2A-R PDX case. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Potential Synergistic Effects of the Bioflavonoid Quercetin with the Apoptosis‐Inducer, TRAIL

Acute lymphoblastic leukemia (ALL) mutations in lymphocyte precursor cells cause continuous production of lymphocytes. TRAIL, TNF‐related apoptosis‐inducing ligand, stimulates apoptosis in a range of cancer cells. TRAIL is being used more frequently in cancer research because of its directed anti‐cancer abilities, however cancer cells often become resistant to TRAIL treatment. Plant flavonoids such as Kaempferol have been shown to boost the effects of TRAIL and reduce resistance. This study examines the use of both Kaempferol and Quercetin, a plant flavonoid found in several fruits, as co‐treatments with TRAIL in inducing apoptosis in ALL. While the flavonoids have been used in combination with other traditional cancer drugs, little has been done to examine how combinations of flavonoids and molecules like TRAIL affect apoptosis in ALL. We observed an effective concentration for quercetin of 2.5 uM which produced 80% apoptosis in ALL cells. Kaempferol’s effective concentration was 100 uM, resulting in 37% cell death. TRAIL, at concentrations of 50 and 100 nM, showed no effect on Molt‐4 cells, primarily due to Molt‐4’s known resistance to TRAIL. Co‐treatment with quercetin and TRAIL showed suppression of ALL viability as did a triple combination treatment. Co‐treatments also resulted in modification of anti‐ and pro‐apoptotic protein expression. These results indicate that quercetin and other flavonoids may improve the effectiveness of TRAIL, even in TRAIL‐resistant cancer cell lines.

Targeting Apoptosis in ALL.

While the treatment of acute lymphoblastic leukemia (ALL) has improved significantly over the last 30years, the majority of adult patients will have their disease relapse. The BCL-2 gene was initially discovered from follicular lymphoma research; however, the BH3 family of proteins has is emerging to be crucial in patients with ALL due to their reliance on the balance of these pro-apoptotic and anti-apoptotic proteins in the BH3 family. We discuss apoptosis in ALL, the reliance mechanisms, drug development in this space, and areas for future research. The first drugs that were developed to inhibit the BCL-2 pathway include both venetoclax (BCL-2 specific inhibitor) and navitoclax (BCL-2, BCL-XL, and BCL-W). These drugs show promise and have obtained complete remissions, minimal residual disease negative status, and have been used as a bridge to allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia and chronic lymphocytic leukemia. There are multiple ongoing clinical trials looking to assess the use of BCL-2 inhibition with chemotherapy, targeted therapies, and bi-specific T-cell engager therapies not only in both frontline and relapsed refractory ALL but also in consolidation and maintenance phases. There is still a large need for improvement of ALL outcomes in adult patients. Research has shown that ALL depends on the BCL-2 family of proteins for cell survival and proliferation. Targeting this pathway with BCL-2 inhibition has led to encouraging results, and future research is aimed at incorporating this targeted therapy into current treatment paradigms.

MAGI2-AS3 restrains proliferation, glycolysis, and triggers apoptosis in acute lymphoblastic leukemia via regulating miR-452-5p/FOXN3 pathway

Objective(s):MAGI2-AS3 is a cancer suppressor gene of multiple malignancies. Acute lymphoblastic leukemia (ALL) is an important type of leukemia that especially occurs in children. Our work evaluated the modulation of MAGI2-AS3 in ALL.Materials and Methods:qPCR and Western blotting were adopted for detection of target molecular expression. Growth and apoptosis were determined by CCK8 assay and Annexin V/PI staining. Glycolysis was detected by commercial kits. The direct binding between miR-452-5p and MAGI2-AS3 or FOXN3 was assessed by luciferase reporter assay. Tumor growth was measured in nude mice in vivo.Results:MAGI2-AS3 was down-regulated in ALL. Enforced expression of MAGI2-AS3 inhibited growth and glycolysis while promoting apoptosis of ALL cells. Moreover, MAGI2-AS3 up-regulated FOXN3 via sponging miR-452-5p. FOXN3 depletion abrogated MAGI2-AS3-mediated anti-cancer action. More importantly, MAGI2-AS3 repressed ALL cell growth in nude mice through regulation of miR-452-5p/FOXN3. Conclusion:MAGI2-AS3 inhibits ALL development via modulating miR-452-5p/FOXN3.

Extracellular vesicles carrying miRNA-181b-5p affects the malignant progression of acute lymphoblastic leukemia

ObjectiveTo investigate how serum extracellular vesicles (EVs)-carried miRNA-181b-5p affected the proliferation, cell cycle and apoptosis of acute lymphoblastic leukemia (ALL) cells.MethodsDifferentially expressed miRNAs related to ALL were screened by bioinformatics analysis, and the localization of target miRNA was searched by its expression. qRT-PCR was adopted to confirm the expression of miRNA-181b-5p. Flow cytometry and fluorescence microscopy were applied to evaluate EVs internalization. MTT assay was employed to verify the proliferation of ALL cells. Cell cycle and apoptosis were analyzed by flow cytometry. Transwell assay was applied to evaluate migration and invasion abilities.ResultsHigh expression of miRNA-181b-5p was proved in ALL cell lines, and miRNA-181b-5p enriched in the exosomes and vesicles of blood cells. In the meantime, it was found that EVs carrying miRNA-181b-5p could be internalized by ALL cells and thus the expression of miRNA-181b-5p was up-regulated. Cell function assays showed that the proliferation, migration, invasion abilities of ALL cell lines were promoted in miRNA-181b-5p mimic group or the group co-culturing ALL-derived EVs and BALL-1 cell lines. The percentage of cells in G0/G1 phase was reduced and cell apoptosis was also inhibited.ConclusionmiRNA-181b-5p carried by EVs in peripheral blood of ALL patients can enter ALL cells and thus promote the malignancy of ALL cells.

MicroRNA-221 regulates cell activity and apoptosis in acute lymphoblastic leukemia via regulating PTEN.

T cell acute lymphoblastic leukemia (T-ALL), an aggressive and heterogeneous malignancy originating from T cell precursors (thymocytes), accounts for ~15% of all ALL cases in children and for ~25% in adults. The present study aimed to investigate the role of microRNA-221 (miR-221) in the regulation of cell viability and apoptosis of human T-ALL cells and its related regulatory mechanisms. To perform this investigation, miR-221 was upregulated or knocked down in human T-ALL cells (Jurkat cells) using miR-221 mimic or inhibitor, respectively. Then, cell viability was determined using a 3-(4,5-dimethylthiahiazol-2-y1)-2,5-diphenytetrazolium bromide assay, cell invasion and migration were analyzed via Transwell assays, and cell apoptosis was detected using flow cytometry. It was found that transfection with a miR-221 inhibitor significantly inhibited Jurkat cell viability, migration and invasion, and induced Jurkat cell apoptosis. Whereas, transfection with the miR-221 mimic resulted in the opposite effects. Besides, the results showed that phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was a target of miR-221. Moreover, it was observed that the effects of the miR-221 inhibitor on Jurkat cell viability, migration and invasion, and cell apoptosis were significantly eliminated by PTEN-small interfering RNA. In addition, it was shown that the phosphatidylinositol 3-kinase/AKT pathway was involved in the effect of miR-221 on Jurkat cells. In conclusion, the data indicated that miR-221 existed as an oncogene in T-ALL, and its downregulation could inhibit the development of ALL by targeting PTEN. Therefore, miR-221 may be a novel potential therapeutic target for ALL.

Role of Autophagy and Apoptosis in Acute Lymphoblastic Leukemia.

Background:Acute lymphoblastic leukemia (ALL) is a malignant disease characterized by an excessive number of immature lymphocytes, including immature precursors of both B- and T cells. ALL affects children more often than adults. Immature lymphocytes lead to arrested differentiation and proliferation of cells. Its conventional treatments involve medication with dexamethasone, vincristine, and other anticancer drugs. Although the current first-line drugs can achieve effective treatment, they still cannot prevent the recurrence of some patients with ALL. Treatments have high risk of recurrence especially after the first remission. Currently, novel therapies to treat ALL are in need. Autophagy and apoptosis play important roles in regulating cancer development. Autophagy involves degradation of proteins and organelles, and apoptosis leads to cell death. These phenomena are crucial in cancer progression. Past studies reported that many potential anticancer agents regulate intracellular signaling pathways.Methods:The authors discuss the recent research findings on the role of autophagy and apoptosis in ALL.Results:The autophagy and apoptosis are widely used in the treatment of ALL. Most studies showed that many agents regulate autophagy and apoptosis in ALL cell models, clinical trials, and ALL animal models.Conclusions:In summary, activating autophagy and apoptosis pathways are the main strategies for ALL treatments. For ALL, combining new drugs with traditional chemotherapy and glucocorticoids treatments can achieve the greatest therapeutic effect by activating autophagy and apoptosis.

LncRNA-MALAT1 regulates proliferation and apoptosis of acute lymphoblastic leukemia cells via miR-205-PTK7 pathway.

Long non-coding RNA (lncRNA) MALAT1 has been confirmed to function as an oncogene in various solid tumors. MALAT1 level has been shown to be upregulated in relapsed acute lymphoblastic leukemia (ALL) patients, but the mechanism is unclear. This study aims to investigate the functional roles and underlying mechanisms of MALAT1 in ALL. MALAT1 and miR-205 expression were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). MTT assay and flow cytometry were performed to evaluate cell proliferation and apoptosis, respectively. Protein level of protein tyrosine kinase-7 (PTK7) was detected by Western blot assay. Dual luciferase reporter assay was conducted to confirm the binding of MALAT1 and miR-205, as well as miR-205 and PTK7. The levels of MALAT1 and PTK7 were upregulated in ALL samples. In contrast, miR-205 level was downregulated in ALL in ALL samples. Moreover, MALAT1 silencing or miR-205 overexpression restrained proliferation and promoted apoptosis of ALL cells. Mechanistically, MALAT1 sponged miR-205 to regulate PTK7 expression. In summary, MALAT1 affected ALL cell proliferation and apoptosis via regulating miR-205-PTK7 axis. Our results suggest that MALAT1-miR-205-PTK7 axis participates in the proliferation and apoptosis of ALL, which may provide a potential treatment target for ALL.

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion.

Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL. Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson's correlation. Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas. These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis.

Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells.

Acute lymphoblastic leukaemia(ALL) is the most frequent childhood cancer and, although it is highly treatable, resistance to therapy, toxicity and side effects remain challenging. The synthetic glucocorticoid(GC) dexamethasone(Dex) is commonly used to treat ALL, the main drawback of which is the development of resistance to this treatment. The aim of the present study was to investigate potential molecular circuits mediating resistance and sensitivity to GC‑induced apoptosis in ALL. The leukaemia cell lines CEM‑C7‑14, CEM‑C1‑15 and MOLT4 treated with chloroquine(CLQ), thapsigargin(TG) and rotenone(ROT) were used to explore the roles of autophagy, endoplasmic reticulum(ER) stress/unfolded protein response(UPR) and reactive oxygen species(ROS) generation in the response to GC treatment. ROS levels were associated with increased cell death and mitochondrial membrane potential in rotenone‑treated CEM cells. Autophagy inhibition by CLQ exhibited the strongest cytotoxic effect in GC‑resistant leukaemia. Autophagy may act as a pro‑survival mechanism in GC‑resistant leukaemia since increasing trends in beclin‑1 and microtubule‑associated protein1 light chain3α levels were detected in CEM‑C1‑15 and MOLT4 cells treated with Dex, whereas decreasing trends in these autophagy markers were observed in CEM‑C7‑14 cells. The intracellular protein levels of the ER stress markers glucose‑regulated protein(GRP)78 and GRP94 were stimulated by Dex only in the GC‑sensitive cells, suggesting a role of these chaperones in the GC‑mediated ALL cell death. Increased cell surface levels of GRP94 were recorded in CEM‑C7‑14 cells treated with combination of Dex with TG compared with those in cells treated with TG alone, whereas decreasing trends were observed in CEM‑C1‑15 cells under these conditions. Taken together, the results of the present study demonstrated that autophagy may be a pro‑survival mechanism in GC‑resistant leukaemia, and by modulating intracellular and surface GRP94 protein levels, Dex is involved in the regulation of ER stress/UPR‑dependent cell death and immune surveillance. These observations may be of clinical importance if confirmed in patients.

Gene Expression and Resistance to Glucocorticoid-Induced Apoptosis in Acute Lymphoblastic Leukemia: A Brief Review and Update.

Resistance to glucocorticoid (GC)-induced apoptosis in Acute Lymphoblastic Leukemia (ALL), is considered one of the major prognostic factors for the disease. Prednisolone is a corticosteroid and one of the most important agents in the treatment of acute lymphoblastic leukemia. The mechanics of GC resistance are largely unknown and intense ongoing research focuses on this topic. The aim of the present study is to review some aspects of GC resistance in ALL, and in particular of Prednisolone, with emphasis on previous and present knowledge on gene expression and signaling pathways playing a role in the phenomenon. An electronic literature search was conducted by the authors from 1994 to June 2019. Original articles and systematic reviews selected, and the titles and abstracts of papers screened to determine whether they met the eligibility criteria, and full texts of the selected articles were retrieved. Identification of gene targets responsible for glucocorticoid resistance may allow discovery of drugs, which in combination with glucocorticoids may increase the effectiveness of anti-leukemia therapies. The inherent plasticity of clinically evolving cancer justifies approaches to characterize and prevent undesirable activation of early oncogenic pathways. Study of the pattern of intracellular signal pathway activation by anticancer drugs can lead to development of efficient treatment strategies by reducing detrimental secondary effects.

Berberine promotes XIAP-mediated cells apoptosis by upregulation of miR-24-3p in acute lymphoblastic leukemia.

Background: Berberine (BBR) has gained considerable attention because of its anti-tumor activity. BBR can induce apoptosis of acute lymphoblastic leukemia (ALL) cells through the MDM2/p53 pathway. However, the effects of BBR on those ALL patients with p53 deficiency remain unclear.Results: We found that BBR reduced ALL cell viability and induced apoptosis in p53-null EU-4 and p53-mutant EU-6 cells by downregulating X-linked inhibitor of apoptosis protein (XIAP), which is increased in ALL tissues and cells. BBR-induced cell apoptosis was attenuated by inhibition of XIAP that was controlled by PIM-2. Mechanistic studies showed that BBR treatment induced an enhancement of miR-24-3p. PIM-2 is a direct target of miR-24-3p. Blockade of PIM-2 or miR-24-3p reversed BBR-induced cell apoptosis. In vivo studies, BBR remarkably alleviated leukemia conditions in a EU4 xenograft mouse model, whereas inhibition of miR-24-3p significantly reversed the effects of BBR in the leukemia condition.Conclusions: miR-24-3p/PIM-2/XIAP signaling contributes to BBR-mediated leukemia mitigation in p53-defect ALL, which should be further developed as a treatment strategy in ALL patients with p53 deficiency.Methods: Cell viability and apoptosis were determined using CCK-8 and TUNEL assays, respectively. The dual-luciferase reporter gene system was used to determine the interaction between miR-24-3p and 3′-untranslated regions (UTRs) of PIM-2.

Synergistic action of dual IGF1/R and MEK inhibition sensitizes childhood acute lymphoblastic leukemia (ALL) cells to cytotoxic agents and involves downregulation of STAT6 and PDAP1

Heterogeneous upregulation of multiple prosurvival pathways underlies resistance to damage-induced apoptosis in acute lymphoblastic leukemia (ALL) cells despite normal p53 responses. Here, we show that the dual combination of insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1/R) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition using AG1024 + U0126 can sensitize apoptosis-resistant ALL cells to ionizing radiation-induced DNA damage irrespective of effect of single pathway inhibition in vitro. This AG1024 + U0126 combination also significantly potentiates the ability of the core chemotherapy compounds vincristine, dexamethasone, and daunorubicin to kill ALL cells in vitro. Evidence of the synergistic action of AG1024 + U0126 in samples with variable basal levels of phosphorylated IGF1/Rβ and ERK1/2 suggested additional targets of this drug combination. Consistent with this, gene expression profiling identified 32 "synergy genes" differentially targeted by IGF1/R + MEK inhibition and, among these, Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) were the most differentially downregulated cluster. Pearson correlation analysesrevealed that STAT6 and PDAP1 display significant expression codependency and a common expression pattern linked with other key "synergy" genes, supporting their predicted role in an STAT6-ERK-nuclear factor kappa beta (NF-κB) network. Knockdown studies revealed that loss of STAT6, but not PDAP1, impinges on the cell cycle, causing reduced numbers of viable cells. In combination with daunorubicin, STAT6 loss has an additive effect on cell killing, whereas PDAP1 loss is synergistic, indicating an important role of PDAP1 in the cellular response to this anthracycline. Inhibition of STAT6 or PDAP1 may therefore represent a potential novel therapeutic strategy for resistant ALL by enhancing sensitivity to chemotherapy.

Inhibition of MDM2 by a Rhein-Derived Compound AQ-101 Suppresses Cancer Development in SCID Mice.

A novel small-molecule anthraquinone (AQ) analogue, AQ-101, which was synthesized through chemical modification of the core structures of rhein, exhibited potent anticancer activity. In the present study, we evaluated the cancer-inhibiting mechanism of AQ-101 and tested the therapeutic potential of this compound for treating cancer in mice. We found that AQ-101 was able to induce MDM2 protein degradation through a self-ubiquitination and proteasome-mediated mechanism. This AQ-101-induced MDM2 downregulation led to activation of p53, which contributed to apoptosis of acute lymphoblastic leukemia (ALL), especially those with a wild-type p53 phenotype and MDM2 expression in vitro and in vivo When given for a period of 2 weeks (20 mg/kg/day, 3×/week), AQ-101 inhibited development of ALL in nude or SCID mice with a human ALL xenograft and achieved cure by the end of the 5-month experiment. Importantly, AQ-101 showed minimal or no inhibitory effect on normal human hematopoiesis in vitro and was well tolerated in vivo in animal models. Given that MDM2-overexpressing cancers are commonly refractory to current treatment options, our study results suggest that further development of AQ-101 is warranted, as it represents a potentially new, safe anticancer drug with a novel strategy for targeting MDM2. Mol Cancer Ther; 17(2); 497-507. ©2017 AACR.