NB4 Cells Research Articles

Circadian clock protein Bmal1 accelerates acute myeloid leukemia by inhibiting ferroptosis through the EBF3/ALOX15 axis.

Acute myeloid leukemia (AML) is a major leukemia with high mortality. Ferroptosis is an important regulator of cancers. However, the role of ferroptosis and its regulatory mechanisms in AML remain largely unknown. In this study, we reported elevated brain and muscle ARNT-Like protein-1 (Bmal1) expression in AML patients and cell lines, and its upregulation indicated the poor survival of patients. The correlation analysis showed that Bmal1 expression was closely correlated with cytogenetics and the French-American-British subtypes, but was not correlated with age, gender and white blood cells. RSL3 reduced Bmal1 expression in HL-60 and NB4 cells. Malondialdehyde, total iron, Fe2+ , glutathione and lipid peroxidation were examined to evaluate ferroptosis. Overexpression of Bmal1 repressed RSL3-induced ferroptosis in AML cells. Bmal1 recruited Enhancer of zeste homolog 2 (EZH2) to the Early B cell factor 3 (EBF3) promoter and enhanced its methylation, thus suppressing EBF3 expression. Moreover, the knockdown of Bmal1 sensitized AML cells to RSL3-induced ferroptosis, and it was counteracted by EBF3 knockdown. Furthermore, EBF3 bound to the Arachidonate 15-pipoxygenase (ALOX15) promoter to enhance its expression, and overexpression of EBF3 enhanced RSL3-induced ferroptosis dependent on ALOX5. We established a subcutaneous AML xenograft tumor model and reported that knockdown of Bmal1 and overexpression of EBF3 restrained AML growth by promoting ALOX15-mediated ferroptosis in vivo. Collectively, Bmal1 inhibits RSL3-induced ferroptosis by promoting EZH2-mediated EBF3 methylation and suppressing the expression of EBF3 and ALOX15, thus accelerating AML.

Sirtuin Inhibitor Cambinol Induces Cell Differentiation and Differently Interferes with SIRT1 and 2 at the Substrate Binding Site.

Epigenetic mechanisms finely regulate gene expression and represent potential therapeutic targets. Cambinol is a synthetic heterocyclic compound that inhibits class III histone deacetylases known as sirtuins (SIRTs). The acetylating action that results could be crucial in modulating cellular functions via epigenetic regulations. The main aim of this research was to investigate the effects of cambinol, and its underlying mechanisms, on cell differentiation by combining wet experiments with bioinformatics analyses and molecular docking simulations. Our in vitro study evidenced the ability of cambinol to induce the differentiation in MCF-7, NB4, and 3T3-L1 cell lines. Interestingly, focusing on the latter that accumulated cytoplasmic lipid droplets, the first promising results related to the action mechanisms of cambinol have shown the induction of cell cycle-related proteins (such as p16 and p27) and modulation of the expression of Rb protein and nuclear receptors related to cell differentiation. Moreover, we explored the inhibitory mechanism of cambinol on human SIRT1 and 2 performing in silico molecular simulations by protein-ligand docking. Cambinol, unlike from other sirtuin inhibitors, is able to better interact with the substrate binding site of SIRT1 than with the inhibition site. Additionally, for SIRT2, cambinol partially interacts with the substrate binding site, although the inhibition site is preferred. Overall, our findings suggest that cambinol might contribute to the development of an alternative to the existing epigenetic therapies that modulate SIRTs.

Silymarin in combination with ATRA enhances apoptosis induction in human acute promyelocytic NB4 cells

Although all-trans retinoic acid (ATRA) is an efficient pattern in acute promyelocytic leukemia (APL) therapy, further studies are required due to the extant clinical limitations of ATRA. It has been reported that Silymarin, an anti-cancer herbal substance extracted from milk thistle (Silybum marianum), is able to regulate apoptosis in various types of cancer cells through different mechanisms of action. This study investigated the apoptosis-inducing effect of Silymarin (SM) alone and in combination with ATRA on human acute promyelocytic NB4 cells. Examination using MTT assay indicated that SM treatment leads to growth inhibition in NB4 cells in a dose-dependent manner. The IC50 values of SM and ATRA were calculated 90 μM and 2 μM, respectively. Cell cycle analysis by flow cytometry revealed that a more increase in the sub-G1 phase (a sign of apoptosis) when cells were exposed to SM in combination with ATRA. The incidence of apoptosis was confirmed through Hoechst 33258 staining and Annexin V-FITC analysis. The results showed that Silymarin enhances ATRA-induced apoptosis. The flow cytometric analysis also indicated an enhancement in levels of ROS in the treated cells with both compounds. The real-time PCR illustrated that SM targets apoptosis by down-regulation in Survivin and Bcl-2 while up-regulation in Bax. The findings showed that the combination of the two compounds is more effective in the induction of apoptosis in NB4 cells. Molecular docking studies indicated that Sylibin, as a primary compound of the SM, binds to the BH3 domain of Bcl-2 and the BIR domain of Survivin with various affinities. Based on the findings, it seems that SM used alone and in combination with ATRA may be beneficial for inducing apoptosis in APL cells.

Expression pattern and prognostic implication of zinc homeostasis-related genes in acute myeloid leukemia.

Zinc homeostasis is regulated by the SLC39A/ZIP, SLC30A/ZnT, and metallothionein (MT) protein families. The association of zinc homeostasis with acute myeloid leukemia (AML) is unclear. We previously demonstrated that zinc depletion by TPEN triggers apoptosis in NB4 AML cells with the degradation of PML-RARα oncoprotein, suggesting that zinc homeostasis may be associated with AML. The primary aim of this study was to explore the expression pattern and prognostic roles of zinc homeostasis-related genes in AML. Bioinformatics analyses were performed using integrated datasets from the TCGA and GTEx projects. The GEPIA tool was used to analyze the differential expression of zinc homeostasis-related genes. Correlations between zinc homeostasis-related genes were assessed with Spearman's correlation coefficient. OncoLnc was used to evaluate the prognostic roles of zinc homeostasis-related genes with Kaplan-Meier and Cox regression models. In both NB4 and U937 cells, the transcriptional regulation of zinc homeostasis-related genes by zinc depletion was detected through qPCR. We found that multiple ZIPs, ZnTs, and MTs were differentially expressed and correlated in AML tumors. In AML patients, higher expression of ZIP4 and lower expression of ZnT5 and ZnT7 predicted poorer survival. We further found that zinc depletion by TPEN upregulated ZIP7, ZIP9, ZIP10, ZIP13, and ZnT7 and downregulated ZIP14, ZnT1, ZnT6, and most of the positively expressed MTs in both NB4 and U937 AML cells. Our findings suggest high expression of ZIP4 and low expression of ZnT5 and ZnT7 as potential risk factors for the prognosis of AML. Zinc homeostasis may be a potential therapeutic target for AML, deserving further exploration.

The interaction between Hsp90-mediated unfolded protein response and autophagy contributes to As3+/ Se4+ combination-induced apoptosis of acute promyelocytic leukemia cells

The interaction between the unfolded protein response (UPR) and autophagy plays either pro-survival or pro-apoptotic roles in the treatment of acute promyelocytic leukemia (APL). Our previous study has shown that the combination therapy of arsenite (As3+) and selenite (Se4+) induces apoptosis in APL NB4 cells, although the mechanisms are not clear. Here, we demonstrate that the interaction between heat shock protein 90 (Hsp90)-mediated UPR and autophagy is the core module for As3+/Se4+ combination-induced apoptosis. Hsp90 overexpression and knockdown assays indicate that Hsp90 inhibition by PERK modulates two branches of the UPR, leading to the activation of ATF4 and CHOP, causing the degradation of IRE1α and the dephosphorylation of eIF2α, thereby contributing to switching the cytoprotective UPR into an apoptotic pathway. Assays using pretreatment with inducers and inhibitors of endoplasmic reticulum stress (ERS) and autophagy reveal that autophagy is stimulated by ERS but suppressed by As3+/Se4+ combination via the mTOR signaling pathway. However, inhibition of autophagy decreases GRP78 expression and eIF2α phosphorylation, thereby further promoting ERS-induced apoptosis. Moreover, As3+/Se4+ combination blocks hepatic infiltration in an APL-NCG mouse model of extramedullary infiltration. Taken together, these findings provide novel agents and therapeutic approaches for APL.

Abstract 385: Synergistic effects of MIA-602 and its potential for use in ATRA/ATO Resistant APL

Abstract Introduction: Acute Myeloid Leukemia (AML) is a malignant neoplastic disease arising from myeloid cell lines. AML is characterized by the proliferation of immature, nonfunctional cells in the bone marrow. Acute Promyelocytic Leukemia (APL) is a subtype of AML characterized by proliferation of immature promyelocytes. Growth Hormone Releasing Hormone (GHRH) is a hypothalamic peptide that stimulates the release of Growth Hormone from the anterior pituitary. This study aimed to investigate the role of MIA-602, a growth hormone-releasing hormone receptor (GHRH-R) antagonist as front line-combination therapy in the K-562 in-vitro model of AML as well as its potential for treating ATRA/ATO resistant APL. Design: Western blot analysis was used to detect the GHRH-R in both K-562 and NB4 cell lines. K562 cells were maintained as a cell suspension in the RPMI medium, with 10% heat-inactivated fetal bovine serum and gentamicin. Doxorubicin treatment concentrations were 0.005, 0.01, and 0.05 μg/μl. Combination therapy concentrations were 0.005, 0.01, and 0.05 μg/μl of doxorubicin and 5 μmol/L of MIA-602. Proliferation was observed and recorded at 24h and 48h. Wild type NB4 cells and NB4 cells resistant to both ATRA and ATO (NB4-RAA) were cultured in suspension. Cell lysates were prepared after incubation with MIA-602. Cell viability was measured at 24, 48, and 72h. Flow cytometry was also performed to assess expression of CD-56. Results: Incubation of cells with doxorubicin showed a decrease in proliferation in a dose-dependent manner. Co-incubation of K562 cells with MIA-602 and doxorubicin showed a significantly greater reduction in proliferation (p<0.05). Co-administration of doxorubicin and MIA-602 was shown to have a synergistic effect, when compared to doxorubicin alone at all concentrations at 24 and 48h. Viability of both NB4 cell lines decreased at similar levels at 24 h and 48 h when exposed to concentrations of MIA-602 higher than 0.05 μmol/L (p < 0.05). No difference in cell viability was found between naïve NB4 and resistant NB4-RAA when exposed to the same concentrations of MIA-602. Results of flow cytometry revealed significantly increased expression of CD-56 (>5.8-fold; p,0.05) in NB4-RAA cells as compared to the parent cell line. Discussion: Co-administration of doxorubicin and the GHRH-R antagonist MIA-602 was shown to have a synergistic effect in reducing proliferation of the K-562 AML cell line. Use of MIA-602 in front-line combination therapy with anthracycline-based regimens may help prevent the acquisition of resistance in AML. CD-56 plays an essential role in the regulation of cell survival and stress resistance. Aberrant expression of CD-56 is also associated with reduced complete remission rates, higher relapse rates, and poor overall survival. The equal effectiveness of MIA-602 in cells with increased expression of CD-56 may provide an important therapeutic option in the setting of resistance. Citation Format: Ravinder S. Chale, Mario Rodriguez, Stephanie M. Almeida, Shelly Sclatter, Anastasia Singaready, Andrew V. Schally, Joaquin J. Jimenez. Synergistic effects of MIA-602 and its potential for use in ATRA/ATO Resistant APL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 385.

Abstract 333: Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability

Abstract Classically, the interleukin-23 receptor (IL-23R) is expressed on the surface of T cells where activation by its ligand, interleukin-23 (IL-23) leads to T cell activation through the JAK/STAT pathway. Here, we identify a novel localization and function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. IL-23R was identified as a potential target through InterLinked Therapeutics’ ITX machine learning platform. IL-23R was identified through this unbiased in silico screen as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed that IL-23R protein was overexpressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression in normal hematopoietic cells. Consistent with prior studies, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were detected on the surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. We confirmed intracellular localization of IL-23R in AML cells using 4 different antibodies targeted against 4 different epitopes of the receptor. We used BioID mass spectrometry to identify novel protein interactors with intracellular IL-23R and identified 61 interacting proteins. 36 of those 61 were nuclear or cytoplasmic in localization and the largest subset was associated with mitotic spindle regulation. By Proximity Ligation Assay, we confirmed the interactions between endogenous IL-23R with known mitotic spindle proteins NUMA, TMEM201, BAG6, and TACC1 in OCI-AML2 cells and primary AML samples. By confocal microscopy, we discovered that IL-23R co-localized with the mitotic spindle and centrosomes in AML cells. Genetic knockout of IL-23R increased spindle defects with multipolarity, lagging chromosomes, and spindle orientation errors. Interestingly, heterodimeric partner of IL-23R, IL12Rβ1 was also detected to be co-localized with mitotic spindles but the cytokine, IL-23, could not be detected. Knockdown of IL-23R also reduced cell growth in OCI-AML2, TEX, K562, NB4, and U937 cells. IL-23R knockdown also reduced the clonogenic growth and engraftment efficiency of TEX leukemia cells into murine bone marrow. In summary, intracellular IL-23R is a critical regulator of mitosis in AML and is necessary for AML viability. This study highlights a novel function and localization of IL-23R in AML and identifies a potential therapeutic target. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil MacLean, Marcela Gronda, Brandon D. Brown, Brian Raught, Andrea Arruda, Hong Chang, Mark D. Minden, Li Zhang, Steven M. Kornblau, Vito Spadavecchio, Aaron D. Schimmer. Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 333.

Regulation of matrix metalloproteinase-9 during monopoiesis and zinc deficiency by chromatin remodeling

IntroductionMatrix metalloproteinase-9 (MMP-9) cleaves various extracellular matrix proteins, hence significantly contributes to numerous physiological but also pathological processes. Monocytic differentiation is associated with increased MMP-9 gene expression. Interestingly, MMP-9 upregulation during monocytic differentiation is paralleled by a decline in intracellular zinc levels. Hence, an influence of zinc on the regulation of MMP-9 expression may exist. Although, previous studies suggest a vital role of zinc regarding MMP-9 activity, the possible relevance of zinc homeostasis during transcriptional regulation of MMP-9 for example via epigenetic mechanisms is rather unclear. AimThis study aims to find a correlation between zinc deficiency and MMP-9 transcriptional regulation, focusing on epigenetics as the possible mechanism behind zinc deficiency-induced changes. MethodsThe effect of differentiation and zinc deficiency on MMP-9 expression and MMP9 promoter accessibility was investigated using the acute promyelocytic cell line NB4. Intracellular free zinc levels were detected by flow cytometry. MMP-9 gene expression was measured by real-time PCR and ELISA. Analysis of chromatin structures was done using chromatin accessibility by real-time PCR (CHART) assay. ResultsDuring monocytic differentiation of NB4 cells, the decrease in intracellular zinc levels was paralleled by an increased production of MMP-9. Assessment of chromatin structure revealed increased accessibility of certain regions within the MMP-9 promoter in differentiated cells. Interestingly, upregulated activation-induced MMP-9 gene expression as well as a more accessible MMP-9 promoter were in zinc-deficient NB4 cells whereas zinc resupplementation reversed the effects. ConclusionThese data demonstrate an important role of epigenetic mechanisms in regulating MMP-9 expression under zinc deficiency. This could provide an encouraging step to expand the research on using zinc for the treatment of various pathological conditions such as inflammatory, vascular and autoimmune diseases resulting from MMP-9 deregulation.

The Effect of NSAIDS and Oral Hypoglycemic Agent on Leukemia and Lymphoma Cell Lines

Cancer is regarded as global burden and a serious health challenge. Its occurrences increased due to population aging and the prevalence of risk factors. This study investigated the eff ect of metformin and aspirin on two cell lines SR (diff use large B cell lymphoma) and NB4 (promyelocytic leukemia). The eff ect of metformin as monotherapy on SR was 28.43% viable, while on NB4, only 44.4% were viable. However, the eff ect of aspirin on SR and NB4 cell lines were decreased; the percentage of cell viability were 27.2 and 41.2%, respectively. In conclusion, there is signifi cant eff ect of metformin and aspirin on SR cell line and only mild eff ect on NB4. These may be useful drugs in old diabetic patients with diff use large B cell lymphoma that may reduce the chemotherapy dose

AURKB Geni miR-34a-5p ve let-7b-5p Tarafından Hedeflendiğinden AML Hücre Proliferasyonunda Rol Oynar mı?

Objective: The production of normal blood cells in the bone marrow is interrupted in AML, which is characterized by the proliferation and accumulation of leukemic blasts. Therefore, patients experience anemia and thrombocytopenia. When gene expression of Aurora kinases, which is reported to be highly expressed in AML, decreases, it may be possible to alleviate the clinical findings in AML. In this study, it was aimed to examine the relationship of AURKB with important miRNAs that have the potential to regulate gene expression. 
 Method: HL60 and NB4 cells were transfected with important tumor suppressor miRNAs miR-34a-5p and let-7b-5p mimics. Then, its effects on proliferation were examined with WST-8 technique and its effects on AURKB gene expression were examined with qRT-PCR.
 Results: It was determined that these miRNAs negatively regulated proliferation in both AML cell lines and downregulated the expression level of the Aurora kinase B (AURKB) gene in the miRNA transfected group compared to the control group.
 Conclusion: In conclusion, it was determined that miR-34a-5p and let-7b-5p could regulate AURKB expression in AML cells. Therefore, it was thought that these miRNAs may have an important potential as a therapeutic biomarker in preventing excessive cell division and poor prognosis in AML.

Tocotrienols-enriched Self-nanoemulsifying Drug Delivery System Enhances the Antileukemic Activity of All-trans Retinoic Acid but not Electrocardiogram Alterations Evoked by Its Combination with Arsenic Trioxide

All-trans retinoic acid and arsenic trioxide are the leading choices for the treatment of acute promyelocytic leukemia. Notwithstanding the impressive differentiative properties of all-trans retinoic acid and the apoptotic properties of arsenic trioxide, some problems still occur in acute promyelocytic leukemia treatment. These problems are due to patients' relapses, mainly related to changes in the ligand-binding domain of RARα (retinoic acid receptor α) and the cardiotoxic effects caused by arsenic trioxide. We previously developed a self-nanoemulsifying drug delivery system enriched with tocotrienols to deliver all-trans retinoic acid (SNEDDS-TRF-ATRA). Herein, we have evaluated if tocotrienols can help revert ATRA resistance in an APL cell line (NB4-R2 compared to sensitive NB4 cells) and mitigate the cardiotoxic effects of arsenic trioxide in a murine model. SNEDDS-TRF-ATRA enhanced all-trans retinoic acid cytotoxicity in NB4-R2 (resistant) cells but not in NB4 (sensitive) cells. Moreover, SNEDDS-TRF-ATRA did not significantly change the differentiative properties of all-trans retinoic acid in both NB4 and NB4-R2 cells. Combined administration of SNEDDS-TRF-ATRA and arsenic trioxide could revert QTc interval prolongation caused by ATO but evoked other electrocardiogram alterations in mice, such as T wave flattening. Therefore, SNEDDS-TRF-ATRA may enhance the antileukemic properties of all-trans retinoic acid but may influence ECG changes caused by arsenic trioxide administration. SNEDDS-TRF-ATRA presents cytotoxicity in resistant APL cells (NB4-R2). Combined administration of ATO and SNEDDS-TRF-ATRA in mice prevented the prolongation of the QTc interval caused by ATO but evoked ECG abnormalities such as T wave flattening.

Anti-leukemic effect of menthol, a peppermint compound, on induction of apoptosis and autophagy.

Menthol, a natural compound in peppermint leaves, has several biological activities, including antioxidant, anti-inflammatory, antiviral, antibacterial and anticancer properties. This study revealed the anti-leukemic effects and its underlying mechanisms of the menthol related apoptosis signaling pathway and autophagy in both NB4 and Molt-4 leukemic cell lines. Both leukemic cells were treated with menthol in various concentration. Cell viability was assessed using MTT assay, whereas apoptosis and autophagy were analyzed by flow cytometry using Annexin V-FITC/PI and anti-LC3/FITC antibodies staining, respectively. Apoptotic and autophagic related gene and protein expression were detected using RT-qPCR and western blot analysis, respectively. Moreover, STITCH database was used to predicts the interaction between menthol and proposed proteins. Menthol significantly decreased cell viability in NB4 and Molt-4 cell lines in dose dependent manner. In combination of menthol and daunorubicin, synergistic cytotoxic effects were observed in leukemic cells. However, there was a minimal effect found on normal, peripheral blood mononuclear cells (PBMCs). Moreover, menthol significantly induced apoptosis induction via upregulation of caspase-3, BAX, p53 and downregulation of MDM2 mRNA expression. Autophagy was also induced by menthol through upregulating ATG3 and downregulating mTOR mRNA expression. For protein expression, menthol significantly increased caspase-3 whereas decreased mTOR in both leukemic cells. Conclusions. These results suggest that menthol exhibits cytotoxic activities by inhibition of cell proliferation, induction of apoptosis and autophagy through activating the caspase cascade, altering BAX and p53/MDM2, and regulating autophagy via the ATG3/mTOR signaling pathway.

Caspase-9-mediated cleavage of vimentin attenuates the aggressiveness of leukemic NB4 cells.

Vimentin is a main type 3 intermediate filament protein. It seems that abnormal expression of vimentin is contributed to the appearance of the aggressive feature of cancer cells. So that it has been reported that malignancy and epithelial-mesenchymal transition in solid tumors, and poor clinical outcomes in patients with lymphocytic leukemia and acute myelocytic leukemia have been associated with the high expression of vimentin. Vimentin is a non-caspase substrate of caspase-9 although its cleavage by caspase-9 in biological processes has not been reported. In the present study, we sought to understand whether vimentin cleavage mediated by caspase-9 could reverse the malignancy in leukemic cells. Herein, to address the issue, we investigated vimentin changes in differentiation and took advantage of the inducible caspase-9 (iC9)/AP1903 system in human leukemic NB4 cells. Following the transfection and treatment of the cells using the iC9/AP1903 system, vimentin expression, cleavage, and subsequently, the cell invasion and the relevant markers such as CD44 and MMP-9 were evaluated. Our results revealed the downregulation and cleavage of vimentin which attenuates the malignant phenotype of the NB4 cells. Considering the favorable effect of this strategy in keeping down the malignant features of the leukemic cells, the effect of the iC9/AP1903 system in combination with all-trans-retinoic acid (ATRA) treatment was evaluated. The obtained data prove that iC9/AP1903 significantly makes the leukemic cells more sensitive to ATRA.

Roles of PADI4 in the expression of cytokines involved in inflammation and adhesion in differentiated NB4 cells treated with ATRA.

Differentiation syndrome (DS) is a common complication in patients with acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA). However, the target of ATRA during DS in patients with APL remains to be elucidated. Therefore, the current study aimed to investigate the role of peptidylarginine deiminase 4 (PADI4) in the differentiation of ATRA-induced NB4 APL cells. The results showed that PADI4 was significantly upregulated in peripheral blood samples derived from patients with APL DS compared with patients with APL only. In addition, whether ATRA could enhance the expression levels of PADI4 in NB4 cells in vitro was subsequently investigated. The results also showed that PADI4 overexpression promoted the differentiation of NB4 cells treated with ATRA, which was reversed after PADI4 silencing. To uncover the potential mechanisms underlying the above process, PADI4 overexpression induced the secretion of inflammation-related cytokines, such as TNF-α, IL-1β, IL-8, C-C motif chemokine (CCL)2, CCL4, CCR1 and intercellular adhesion molecule-1 in ATRA-treated NB4 cells. However, PADI4 knockdown in the same cells had the opposite effect. The above findings indicated that PADI4 could be involved in the differentiation of ATRA-induced NB4 cells and upregulation of cytokines.

Cytotoxic Effects of Darinaparsin, a Novel Organic Arsenical, against Human Leukemia Cells.

To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T-cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G2/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G2/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G2/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer.

System analysis of surface CD markers during the process of granulocytic differentiation.

Plasma membrane proteins with extracellular-exposed domains are responsible for transduction of extracellular signals into intracellular responses, and their accessibility to therapeutic molecules makes them attractive targets for drug development. In this work, using omics technologies and immunochemical methods, we have studied changes in the content of markers of clusters of differentiation (CD markers) of neutrophils (CD33, CD97, CD54, CD38, CD18, CD11b, CD44, and CD71) at the level of transcripts and proteins in NB4, HL-60 and K562 cell lines, induced by the treatment with all-trans-retinoic acid (ATRA). Transcriptomic analysis revealed the induction of CD38, CD54, CD11b, and CD18 markers as early as 3 h after the addition of the inducer in the ATRA-responsive cell lines HL-60 and NB4. After 24 h, a line-specific expression pattern of CD markers could be observed in all cell lines. Studies of changes in the content of CD antigens by means of flow cytometry and targeted mass spectrometry (MS) gave similar results. The proteomic profile of the surface markers (CD38, CD54, CD11b, and CD18), characteristic of the NB4 and HL-60 lines, reflects different molecular pathways for the implementation of ATRA-induced differentiation of leukemic cells into mature neutrophils.

Kermanian propolis induces apoptosis through upregulation of Bax/Bcl-2 ratio in acute myeloblastic leukemia cell line (NB4).

Propolis is a viscous resinous honeybee-produced substance with numerous medicinal functions; its composition and texture varies according to the geographic location. It is considered to be a promising natural source for the management and prevention of various pathological conditions. Although several studies have exhibited the anti-cancer activity of different types of propolis, the tumor-suppressing potential of Kermanian propolis against leukemia cell lines has remained poorly understood. Therefore, the current experiment was aimed to reveal the anti-tumor activity of this bioactive compound both as monotherapy and combined therapy with cytarabine against an acute myeloid leukemia (AML) cell line, NB4. Following the treatment of NB4 cells with either Kermanian propolis (5, 10, 20, 40, 80, 160, and 320 μg/mL), cytarabine (0.1, 0.25, 0.5, 0.75, 1, and 2 mM), or their combination (40 and 80 μg/mL of Kermanian propolis along with 0.1, 0.25, and 0.5 mM of cytarabine), colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to measure the viability (%) of the cells. Next, to examine the apoptotic rate and the pattern of corresponding gene expression (Bcl-2, Bax, p53, and p21), Annexin-V/PI staining by flow cytometry and quantitative Real-Time polymerase chain reaction assays were performed, respectively. We perceived significant apoptosis induction in a dose-dependent manner following the treatment with Kermanian propolis, cytarabine, and also their combination in the NB4 cell line. In addition, the combined treatment was associated with lower expression of the anti-apoptotic gene (Bcl-2) and higher expression of the pro-apoptotic genes (p53, Bax, and p21) in comparison to mono treatments. The synergistic anti-tumor activity induced by the combination of Kermanian propolis and cytarabine presents a novel and encouraging option for AML treatment.

Synergistic effects of PI3K inhibition and pioglitazone against acute promyelocytic leukemia cells.

Although pioglitazone, a well-known anti-diabetic agent, has recently established itself as a pillar of cancer treatment, its therapeutic value could be attenuated by the aberrant activation of the PI3K/Akt pathway. To evaluate whether the PI3K/Akt suppression in leukemic cells could potentiate the anti-leukemic effects of pioglitazone. To assess the anti-leukemic effects of PI3K/Akt inhibitors on anti-leukemic effects of pioglitazone, we used MTT and trypan blue assays. Flow cytometric analysis and qRT-PCR were also applied to evaluate cell cycle and apoptosis. The resulting data revealed that upon PPARγ stimulation in different leukemic cell lines using pioglitazone, the survival and the proliferative capacity of the cells were significantly halted. Then, we evaluated the impact of the PI3K/Akt axis on the effectiveness of the drug in the most sensitive leukemic cell line; NB4 cells. Our results showed that treatment of NB4 cells with the PI3K inhibitors increased the sensitivity of leukemic cells to pioglitazone to the degree that even lower concentrations of the agent succeeded to induce apoptotic as well as the anti-proliferative effects. Moreover, it seems that PI3K inhibition could potentiate the anti-leukemic effect of pioglitazone through induction of p21-mediated sub-G1 cell cycle arrest and altering the balance between the pro-and anti-apoptotic genes. This study sheds light on the significance of the PI3K/Akt pathway in APL cell sensitivity to pioglitazone and proposed that the presence of the PI3K inhibitor in the therapeutic regimen containing pioglitazone could be promising in the treatment of this malignancy.

The Exportin, XPO2, Is a Novel Therapeutic Target in Pediatric, Adolescent and Young Adult AML

The active transport of proteins across the nuclear membrane is regulated by nuclear transport receptors termed importins and exportins, depending on their direction of transport. Nuclear transport receptors contribute to cancer development, through abnormal localization of protein cargos. Increased expression of the exportin, exportin-1/chromosome region maintenance 1 (XPO1/CRM1), has been associated with high risk AML and shorter survival times and XPO1 inhibitors show signs of efficacy in AML. While previous data demonstrate a functional role for XPO1 in AML, less is known about the exportin, XPO2, (alias CSE1L). Despite XPO2 RNAi depletion screens demonstrating a higher genetic dependence in leukemia versus solid tumors/non-hematologic malignancies (p = 0.0394), the functional impact of inhibiting XPO2 has never been studied in leukemia. We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes in AML, along with KPNB1 and XPO1 (DepMap 22Q2 Public+Score, Chronos, www.depmap.org). An RNAi depletion screen confirmed these findings, with XPO2 identified within the top 0.2% of essential genes in AML (Achilles+DRIVE+Marcotte, DEMETER2, www.depmap.org). Gene dependency scores indicated a greater reliance on XPO2, compared to XPO1 (p <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (p = 0.0014) and overall survival (p = 0.00091) in pediatric and adolescent/young adult (AYA) AML patients from the TARGET dataset. Analyzing the BeatAML dataset, increased XPO2 mRNA expression was also associated with decreased overall survival in AML patients <40 years, but not in patients >40 years (p <0.0001). We analyzed protein expression databases and identified XPO2 as the highest expressed member of the exportin family in AML cells lines and AML primary patient samples. By immunoblot analysis, increased expression of XPO2 was detected in 7/7 primary AML samples and 8 AML cell lines (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX and U937), compared to normal hematopoietic cells. XPO2 mRNA was also increased in 5/5 primary AML samples and 8/8 AML cell lines, compared to normal hematopoietic cells, as measured by qRT-PCR. To confirm the functional importance of XPO2 in AML, we knocked down XPO2 with shRNA in AML cell lines. Genetic silencing of XPO2 decreased growth and viability of OCI-AML2, TEX and NB4 cells. Gambogic acid (GA), a natural product from Garcinia hanburyi, has been identified as a covalent inhibitor of XPO2-mediated nuclear transportation. In OCI-AML2, TEX and NB4 cell lines, gambogic acid decreased growth and viability of AML cell lines at nanomolar concentrations (TEX cells IC50 = 369nM, OCI-AML2 cells IC50 = 433nM, NB4 cells IC50 = 161nM). To uncover the mechanism of how XPO2 mediates cell death in AML we interrogated a publicly available BioID database (PXD007976) and performed gene set enrichment analysis on XPO2 targets using gprofiler. Processes related to ribosome biogenesis were significantly enriched in XPO2 cargo, whereas XPO1 cargo were enriched for processes related to snRNA transcription and transport (p <0.05). In summary, we identified XPO2 as an essential gene for AML. XPO2 is preferentially increased in AML cell lines and primary patient samples compared to normal hematopoietic cells. Increased XPO2 expression is associated with worse clinical outcomes in pediatric and AYA patients with AML. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemia initiation and progression. Thus, this work demonstrates a new dependency of AML on XPO2, as well as highlights a new therapeutic target for this disease.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.