Apoptosis In Acute Myeloid Leukemia Research Articles

Homoharringtonine enhances cytarabine-induced apoptosis in acute myeloid leukaemia by regulating the p38 MAPK/H2AX/Mcl-1 axis

Acute myeloid leukaemia (AML) is a fatal haematopoietic malignancy and is treated with the conventional combination of cytarabine (Ara-C) and daunorubicin (Dau). The survival rate of AML patients is lower due to the cardiotoxicity of daunorubicin. Clinically, homoharringtonine (HHT) plus Ara-C has been reported to be equally effective as Dau plus Ara-C in some types of AML patients with less toxic effects. We utilized the clinical use of homoharringtonine in combination with Ara-C to test its combination mechanism. We found that the insensitivity of AML cells to cytarabine-induced apoptosis is associated with increased Mcl-1 stability and p38 inactivation. HHT downregulates Mcl-1, phosphorylates H2AX and induces apoptosis by activating p38 MAPK. Inactivation of p38 through inhibitors and siRNA blocks apoptosis, H2AX phosphorylation and Mcl-1 reduction. HHT enhances Ara-C activation of the p38 MAPK signalling pathway, overcoming Ara-C tolerance to cell apoptosis by regulating the p38/H2AX/Mcl-1 axis. The optimal ratio of HHT to Ara-C for synergistic lethality in AML cells is 1:4 (M/M). HHT synergistically induces apoptosis in combination with Ara-C in vitro and prolongs the survival of xenografts. We provide a new mechanism for AML treatment by regulating the p38 MAPK/H2AX/Mcl-1 axis to improve cytarabine therapy.

The Combination of Oolonghomobisflavan B and Diallyl Disulfide Induces Apoptotic Cell Death via 67-kDa Laminin Receptor/Cyclic Guanosine Monophosphate in Acute Myeloid Leukemia Cells.

Diallyl disulfide (DADS) is a well-known principal functional component derived from garlic (Allium sativum) that has various health benefits. Previously, we identified a 67-kDa laminin receptor, a receptor for oolong tea polyphenol oolonghomobisflavan B (OHBFB). However, its molecular mechanisms still remain to be elucidated. Here, we show that DADS synergistically enhanced the effect of the oolong tea polyphenol oolonghomobisflavan B (OHBFB), which induces apoptosis in acute myeloid leukemia (AML) cancer cells without affecting normal human peripheral blood mononuclear cells (PBMCs). The underlying mechanism of OHBFB-induced anti-AML effects involves the upregulation of the 67-kDa laminin receptor/endothelial nitric oxide synthase/cyclic guanosine monophosphate (cGMP)/protein kinase c delta (PKCδ)/acid sphingomyelinase (ASM)/cleaved caspase-3 signaling pathway. In conclusion, we show that the combination of OHBFB and DADS synergistically induced apoptotic cell death in AML cells through activation of 67LR/cGMP/PKCδ/ASM signaling pathway. Moreover, in this mechanism, we demonstrate DADS may reduce the enzyme activity of phosphodiesterase, which is a negative regulator of cGMP that potentiates OHBFB-induced AML apoptotic cell death without affecting normal PBMCs.

Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1β, Transforming Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Mitochondrial regulation of GPX4 inhibition-mediated ferroptosis in acute myeloid leukemia.

Resistance to apoptosis in acute myeloid leukemia (AML) cells causes refractory or relapsed disease, associated with dismal clinical outcomes. Ferroptosis, a mode of non-apoptotic cell death triggered by iron-dependent lipid peroxidation, has been investigated as potential therapeutic modality against therapy-resistant cancers, but our knowledge of its role in AML is limited. We investigated ferroptosis in AML cells and identified its mitochondrial regulation as a therapeutic vulnerability. GPX4 knockdown induced ferroptosis in AML cells, accompanied with characteristic mitochondrial lipid peroxidation, exerting anti-AML effects in vitro and in vivo. Electron transport chains (ETC) are primary sources of coenzyme Q10 (CoQ) recycling for its function of anti-lipid peroxidation in mitochondria. We found that the mitochondria-specific CoQ potently inhibited GPX4 inhibition-mediated ferroptosis, suggesting that mitochondrial lipid redox regulates ferroptosis in AML cells. Consistently, Rho0 cells, which lack functional ETC, were more sensitive to GPX4 inhibition-mediated mitochondrial lipid peroxidation and ferroptosis than control cells. Furthermore, degradation of ETC through hyperactivation of a mitochondrial protease, caseinolytic protease P (ClpP), synergistically enhanced the anti-AML effects of GPX4 inhibition. Collectively, our findings indicate that in AML cells, GPX4 inhibition induces ferroptosis, which is regulated by mitochondrial lipid redox and ETC.

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance.

Microvesicles Derived from Human Placental Mesenchymal Stem Cells Induce Autophagy and Apoptosis in Acute Myeloid Leukemia

Microvesicles Derived from Human Placental Mesenchymal Stem Cells Induce Autophagy and Apoptosis in Acute Myeloid Leukemia

Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells

To investigate the effect of long non-coding RNA LINC01268 on apoptosis of acute myeloid leukemia (AML) cells and related mechanisms. The expression levels of LINC01268 and miR-217 in peripheral blood samples from AML patients and AML cell lines HL-60 and KG-1 were detected by qRT-PCR. HL-60 cells were divided into pcDNA3.1-NC, pcDNA3.1-LINC01268, si-NC, si-LINC01268, miR-NC, miR-217 mimics, si-LINC01268 + inhibitor-NC and si-LINC01268+ miR-217 inhibitor groups. The mRNA expressions of LINC01268 and miR-217 were detected by qRT-PCR. The targeting relationship between LINC01268 and miR-217 was detected by dual-luciferase reporter assay. Cell viability was detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The expression of cell cycle and apoptosis-related proteins p21, Bcl-2, Bax, caspase-3 and PI3K/AKT signaling pathway-related proteins were detected by Western blot. The expression of LINC01268 in peripheral blood samples of AML patients and AML cell lines HL-60 and KG-1 was increased (P < 0.05), and the expression of miR-217 was decreased (P < 0.05). Compared with si-NC group and miR-NC group, the viability of HL-60 cells was decreased in si-LINC01268 group and miR-217 mimics group (P < 0.05), the proportion of cells in G1 phase and apoptosis rate were increased (P < 0.05), the protein expression levels of p21, Bax and caspase-3 were increased (P < 0.05), while the protein expression level of Bcl-2 was decreased (P < 0.05). LINC01268 targeted and negatively regulated the expression of miR-217, and inhibiting the expression of miR-217 partially reversed the effects of LINC01268 interference on the viability, cell cycle and apoptosis of HL-60 cells. Interference with LINC01268 could inhibit the activity of PI3K/AKT signaling pathway. Inhibiting the expression of miR-217 could partially reverse the inhibition of LINC01268 interference on PI3K/AKT signaling pathway. LINC01268 is highly expressed and miR-217 is lowly expressed in AML cells. LINC01268 can promote the activity of PI3K/AKT signaling pathway, increase the survival rate and inhibit the apoptosis of AML cells by targeting miR-217 expression.

The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia

Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.

Casein Kinase II Inhibitor Sensitizes the Anti-Tumor Effect of EZH2 Inhibitor By Suppressing FOSL1 Expression in Acute Myeloid Leukemia

Background More effective combination therapy is emerging needed owing to the high mortality and poor outcome in acute myeloid leukemia (AML). The genomic amplification and overexpression of Enhancer of zeste homolog 2 (EZH2) occur in AML patients, and high expression of the Casein kinase 2 (CK2) is associated with poor survival in AML patients. Both EZH2 inhibitor and CK2 inhibitors are reported to inhibit cell proliferation and induce apoptosis in AML, however, the anti-leukemia effect of EZH2 inhibitor in combination with CK2 inhibitor has not been reported. The present study aims to investigate the effect of the combination of the EZH2 inhibitor DZNeP with CK2 inhibitor CX4945 in AML and possible underlying mechanisms. Methods A total of 40 newly diagnosed AML patients and 40 bone marrow control from healthy donors were obtained from our institute with the approval of the Ethics Committee. Cell Counting Kit-8 assay (CCK8), Cell cycle assay, and apoptosis assays were applied in THP-1 AML cells treated with control, DZNeP, CX4945, and CX4945+DZNeP. Global transcriptome analysis by RNA-seq was performed in U937 cells treated with 2μM DZNeP; 12.5μM CX4945 or vehicle for 48 hours. FOSL1 mRNA level was examined by qPCR; R2 genomics analysis and visualization application were used for comparison of the expression difference and the Kaplan-Meier overall survival analysis was conducted by the Gene Expression Profiling Interactive Analysis (GEPIA) platform. Results DZNeP only has little effect on cell proliferation arrest in THP-1 cells, but it significantly sensitized the effect of CX4945, and CalcuSyn analysis showed the synergistic effect of the CX4945+DZNeP (Fig.1A). The treatment of CX4945+DZNeP combination induced the G0/G1 phase arrest in THP-1 cells versus single-drug control (Fig. 1B). The combination also significantly increased the % of the apoptotic cells in THP-1 cells as well as the AML-5b primary cells from the patient compared to single-drug controls (p&amp;lt;0.0001) (Fig.1C). These data indicate the synergistic anti-tumor effect of CX4945+DZNep combination in AML. To understand the molecular mechanism underlying the synergy, the global transcriptome was identified in U937 cells. The significantly different expression genes (DEGs) were identified upon CX4945 treatment (∣log2FC∣&amp;gt;1.5, FDR&amp;lt;0.05), or DZNeP treatment (∣log2FC∣&amp;gt;1.0, FDR&amp;lt;0.05) versus vehicle-control; and 1908 overlapped DEGs are identified upon either drug treatment, in which 596 were intersected (429 genes changed in the same direction but 167 changed in the opposite (Fig. 1D). KEGG enrichment analyses showed the overlapped DEGs upon the drug treatment mainly involved in cell cycle, DNA replication, and MAPK signaling pathway. (Fig.1E). It is worth noting that FOSL1 is dramatically downregulated in U937 cells upon treatment of either DZNeP or CX4945 (Fig1.D); and qPCR data showed that the mRNA level of FOSL1 is significantly downregulated by CX4945+DZNeP, DZNeP or CX4945 compared to the vehicle control (p&amp;lt;0.0001) in THP-1 cells (Fig. 1F). These data indicated that the combination exerts its anti-tumor effect by down-regulation of FOSL1 expression in AML. FOSL1 is a key component of the AP-1 transcriptional complex, and overexpression of FOSL1 is associated with invasion, migration, proliferation, and stemness in human solid cancers. We also examined the FOSL1 mRNA level in 40 newly diagnosed patients with AML versus 40 normal bone marrow controls. Results showed the mRNA level of FOSL1 is significantly higher in AML patients than that of normal controls (p&amp;lt;0.001) (Fig1.G). Metadata analysis showed that FOSL1 high expression is associated with short overall survival (p=0.033) (Fig.1H). These results not only suggested the oncogenic role of FOSL1 in AML but also further supported the CX4945+DZNeP combination-induced anti-tumor effect mediated by suppressing FOSL1 expression in AML. The mechanism model is summarized in Fig. 1E. Conclusion The combination of EZH2 inhibitor DZNeP and CK2 inhibitor CX4945 exerts a potent synergistic effect on proliferation arrest, cell cycle arrest, and apoptosis in AML by suppressing FOSL1 expression. Our results reveal the oncogenic role of FOSL1 in AML. Our data also provide experimental hints for the potential clinical trial of the novel combination in AML patients.

Synergistic Effect of CK2 Inhibitor CX-4945 with HDACi Chidamide on Cell Proliferation Arrest and Apoptosis in Acute Myeloid Leukemia

Background CX-4945 is an ATP-competitive inhibitor of CK2, which is currently being used in clinical trials to treat various cancer types. Chidamide (CHI), a novel histone deacetylase inhibitor (HDACi), is now involved in the salvage treatment of relapsed/refractory acute myeloid leukemia (AML) in a clinical trial. The anti-leukemia effects of either single drug have been reported by inducing cell apoptosis through suppressing multi-oncogenic pathways in human tumor cells. However, whether and how the combination of the two drugs exerts synergistic anti-tumor effects remains undetermined. This study is to investigate the synergistic therapeutic effects of CX-4945 in combination with CHI in AML. Methods Cell Counting Kit-8 (CCK-8) cell proliferation assay, Annexin V apoptosis assay, and Propidium Iodide cell cycle assay were performed in AML cells in the presence of CX-4945 only, CHI only, CX-4945 + CHI, and vehicle control for 48h. RNA-seq was performed in U937 cells treated with CX-4945 and CHI, respectively; the differentially expressed genes (DEGs) were identified by R studio, and pathway enrichment of the DEGs was analyzed with Metascape online tool. Data were expressed as the mean ± standard deviation. Ordinary one-way ANOVA statistical analysis and Log-rank test was employed to determine the significant difference between groups. A synergistic effect was determined by the combination indices (CIs) with CalcuSyn software. Results The 50% inhibiting concentration (IC50) of CX-4945 on the U937 and THP-1 cell proliferation is 7.435±0.066μmol/L and 6.787±0.153μmol/L. The IC50 of CHI on the U937 and THP-1 cell proliferation is 3.009±0.32μmol/L and 3.782±0.239μmol/L, respectively. The combination of various doses of CHI with either IC50 or 1/2 IC50 of CX-4945 significantly decreases the cell viability of the cells versus single-drug control and CalcuSyn analysis showed the high synergistic therapeutic effects of CHI with CX-4945 (Fig.1A). The combination of (4µM) CHI with (8µM) CX-4945 significantly induced the G0/G1 cell cycle arrest (p &amp;lt;0.001, Fig.1B) and elevated the % of apoptotic cells (p &amp;lt;0.0001, Fig.1C) in the AML cells compared to either single drug alone. Moreover, the combination of (1.6µM) CHI with (8µM) CX-4945 significantly increased the % of apoptotic cells in the primary blasts from the AML patient versus either single drug alone (p&amp;lt;0.0001, Fig.1D). To further understand the underlying mechanism of the synergy, RNA-seq analysis was performed in U937 cells treated with either CHI or CX-4945, respectively (Fig.2A). The DEGs are achieved upon either drug treatment, and the 76 up-regulated and 411 down-regulated overlapped DEGs were identified (Fig.2B). Pathways analysis showed that the overlapped DEGs are enriched in cell cycle progress, apoptosis, and multiple oncogenic signaling including cMyc and PI3K/AKT signaling pathways (Fig.2C). cMyc and PI3K/AKT-dependent signaling pathways are involved in myeloid cell proliferation. Also, our ChIP-seq data indicated obvious Ikaros binding peaks in the promoter region of cMyc and PIK3CD and they are the direct targets of Ikaros (data not shown). cMyc and PIK3CD are at the top of the overlapped DEGs, indicating the critical roles of the cMyc and PIK3CD underlying the mechanism of the combination in AML. Indeed, the mRNA level of cMyc and PIK3CD is significantly down-regulated upon the combination treatment versus single drug control (Fig.2D or data not shown). Consistently, the mRNA level of PIK3CD is significantly higher expressed in AML patients versus that in bone marrow samples of the healthy donors (p&amp;lt;0.05, Fig.2E) and the AML patients with high PIK3CD high expression have shorter OS than that with low expression (p &amp;lt;0.05, Fig.2F). These data support the hypothesis that the combination exerts the anti-tumor effect by suppression of cMyc and PIK3CD expression in AML. Conclusions The combination of CHI and CX-4945 has a synergistic effect on cell proliferation arrest, apoptosis, and cell cycle arrest by suppressing cMyc and PI3K signaling in AML cells. Our data provide experimental evidence for the combination as a potential option for AML therapy. More pre-clinical studies will be explored for future clinical trials.

Novel Mechanisms of Venetoclax Resistance in Acute Myeloid Leukemia Based on Genomic Rearrangements

Introduction: Venetoclax (VEN), a BCL-2 inhibitor, effectively induces apoptosis in acute myeloid leukemia (AML) cells. Resistance to VEN in the treatment of AML can occur through various mechanisms. Some of the identified resistance mechanisms include: expansion or gain of clones with activation of the receptor tyrosine kinase (RTK) pathway; disruption of TP53 function, a tumor suppressor gene known to play a critical role in cell cycle regulation and apoptosis; selection of AML populations with monocytic differentiation, possibly altering cellular characteristics and response to treatment; overexpression of the MCL1 gene, which encodes for a protein that can counteract the pro-apoptotic effects of venetoclax; switch to lipid metabolism, an adaptive response by leukemia cells that may confer resistance to venetoclax; mutations in BCL-2 and BAX genes, which could affect the activity of these key regulators of apoptosis. While these mechanisms have been extensively studied, the role of rearrangements, often combined with genomic events, has not been fully explored. The INTHEMA protocol (NCT04298892) aimed to identify new VEN resistance mechanisms in patients with relapsed/refractory AML treated with VEN in combination with hypomethylating agents and to provide addressable molecular pathways useful for restoring drug sensitivity or offer novel therapeutic opportunities. Methods: Informed consent was obtained from 21 consecutive patients. Samples were collected from these patients with relapse or refractory disease after VEN + hypomethylating agent treatment. TruSight RNA Pan-Cancer analysis was performed to investigate fusion genes (using capture technology covering 1385 genes), RNA expression, and expressed mutations for each sample. Results: Among the 21 patients, 17 (81%) showed at least one putative mechanism of VEN resistance (as illustrated in the figure). These mechanisms included co-occurring mutations and genomic rearrangements. RTK mutations were found in 14 patients (67%), with specific mutations observed in NRAS, PTPN11, CBL, FLT3, JAK2, cKIT, EPHB6, FGFR and PDGFRb. One patient had a BAX mutation (p.G67R; BH3 domain), three patients exhibited a switch to a monocytic phenotype, and two patients had marker chromosomes. Gene fusions were identified in eight patients (38%): in particular, we discovered a cryptic rearrangement of MECOM (TBL1XR1-MECOM and ETV6_MECOM) in 2 patients, a novel BCL11B fusion ( BCL11B-GSDMC), a CCND2-ETV6 BIRC6-LTBP1, the reciprocal NUP98-NSD1, the canonical CBFB-MYH11 and additional transcripts with less defined roles ( PSPC1-ZMYM2, PER1-DDX5, ELL-SUGP2). Interestingly, most of the genes involved in traslocations, with a potential driver role in therapy resistance ( MECOM, ETV6, CCND2, BCL11B, NUP98) also showed high expression levels in patients harboring the traslocation. Unsupervised clustering of RNA expression data revealed two main patient groups. The first group (24%) demonstrated an “activating-like” signature, characterized by overexpression of CSF3R and IKZF, suggesting a potential target for tyrosine kinase inhibitors. The second group exhibited a “self-renewal like” signature, suggesting a possible benefit from histone deacetylase inhibitors, as previously published. Patients with gene fusions were predominantly clustered within the “self-renewal like” group, particularly in a subgroup (subgroup 4) showing overexpression of HOXA genes. Conclusions: Through deep transcriptomic characterization combined with conventional diagnostics, this analysis uncovered novel mechanisms of VEN resistance while confirming established ones. The distinct gene expression patterns may help tailor targeted therapies, with patients showing the “activating-like” signature potentially benefiting from tyrosine kinase inhibitors and those with the “self-renewal like” signature possibly responding well to histone deacetylase inhibitors. Furthermore, HOXA gene overexpression presents an exciting therapeutic opportunity for selected patients. Further research and clinical validation are needed to translate these findings into actionable clinical strategies.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

PD-1/PD-L1 Interaction Regulates BCL2, KI67, BAX, and CASP3, Altering Proliferation, Survival, and Apoptosis in Acute Myeloid Leukemia

Programmed death ligand‑1 (PD‑L1) is a pivotal inhibitory checkpoint ligand known to induce T-cell exhaustion via interaction with the programmed death‑1 (PD‑1) receptor. Beyond this, PD-L1’s intrinsic signaling pathways within cancer cells warrant further exploration. This study aims to elucidate the effect of PD-L1 stimulation on the proliferation, survival, and apoptosis of acute myeloid leukemia (AML) cell lines.&#x0D; Two human AML cell lines, HL-60 and THP-1 were cultured and treated with phorbol 12-myristate 13-acetate (PMA) to induce PD-L1overexpression. Post-treatment PD-L1 expression was confirmed via flow cytometry. Subsequently, cell surface PD-L1 was stimulated using a recombinant PD-1, 24 hours post-PMA treatment. The expression alterations in pivotal genes including BCL2, MKI67, BAX, and CASP3 were monitored using quantitative real-time polymerase chain reaction 24 and 48 hours post-treatment. Additionally, annexin-V through flow cytometry.&#x0D; Findings reveal that PD-L1 stimulation augments AML cell proliferation and survival by enhancing MKI67 and BCL2 expressions while concurrently inhibiting cell apoptosis due to decreased BAX and CASP3 expression following PD-L1 stimulation. Notably, stimulated cells expressed exhibited reduced annexin-V compared to control cells.&#x0D; This study underscores that PD-L1 stimulation fosters AML cell proliferation and survival while impeding cell apoptosis. The results hold potential implications for targeting PD-L1 in AML treatment strategies.

Mesenchymal Stem Cell-Derived Exosomal miRNA-222-3p Increases Th1/Th2 Ratio and Promotes Apoptosis of Acute Myeloid Leukemia Cells.

Interferon regulatory factor 2 (IRF2) participates in the differentiation of immune T cells. Bone marrow mesenchymal stem cell (BM-MSC)-derived exosomes can secret mRNA, miRNAs, and proteins to regulate tumor microenvironment. The present study focused on the miRNA/IRF2 axis in regulating Th1/Th2 ratio and cell apoptosis in acute myeloid leukemia (AML). The flow cytometry analysis was performed to examine the Th1/Th2 ratio and AML apoptosis in vivo and in vitro. The contents of Interferon γ (IFN-γ) and Interleukin-4 (IL-4) were measured using enzyme-linked immunosorbent assay. StarBase was used to predict the potential binding site between miR-222-3p and the 3' untranslated region of IRF2. Luciferase reporter assay was applied for validating the combination of miR-222-3p and IRF2. BM-MSC exosomes were successfully isolated. BM-MSC exosomes increased Th1/Th2 ratio and promoted apoptosis of AML cells. Further analysis showed that IRF2 was targeted by miR-222-3p. Overexpression of miR-222-3p promoted Th1/Th2 ratio and AML cell apoptosis. IRF2 partially reversed the effect that is exerted by miR-222-3p on Th1/Th2 ratio and AML cell apoptosis. Overexpression of miR-222-3p promoted Th1/Th2 ratio and caspase 3 expression in vivo. To sum up, miR-222-3p promotes Th1/Th2 ratio and AML cell apoptosis by regulating IRF2 expression, which provided crucial targets for the treatment of AML.

Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax

IntroductionThe emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332).Materials and methodsTo determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems.ResultsXL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis.ConclusionTaken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

Retracted: lncRNA GAS5 Induces Cell Apoptosis in Acute Myeloid Leukemia by Targeting Nrf2.

[This retracts the article DOI: 10.1155/2022/5178122.].

BCL2L13 protein prevents apoptosis in acute myeloid leukemia cells

Abstract Objectives The B cell lymphoma-2 (BCL-2) protein group is essential for regulating apoptosis. BCL2L13 harbors all BH domains present in the BCL-2 protein family in addition to a BHNo domain comprising 250 amino acids at its C-terminal. BCL2L13 is highly expressed in AML and is involved in apoptosis. We investigated the possibility of discovering BCL2L13 as a treatment target for acute leukemia. Methods We reduced the expression of BCL2L13 in Mono Mac 6 (MM6) cells using shRNA and overexpressed BCL2L13 in THP-1 cells. MM6 and THP-1 cells were treated with staurosporine (STS) to confirm the role of BCL2L13 in apoptosis. Results In acute myeloid leukemia (AML) cells, BCL2L13 is involved in inhibiting apoptosis as evidenced by its strikingly augmented expression in these cells. BCL-2 was downregulated and cleaved Caspase 3, and sphingosine-1-phosphate phosphatase 1 (SGPP1) was upregulated when MM6 cells with knockdown BCL2L13 were treated with STS, which significantly increased their apoptosis. Furthermore, mitochondrial membrane potential decreased in MM6 cells in response to the downregulation of BCL2L13. In BCL2L13-depleted MM6 cells, the amount of cytochrome c increased in the cytoplasm. The THP-1 cells overexpressing BCL2L13 and treated with STS showed a significant decrease in the expression of genes related to apoptosis induction, resulting in decreased apoptosis. Conclusion BCL2L13 inhibits apoptosis in AML, and BCL2L13-specific inhibition might serve as a new strategy for treating this condition.

Research Progress on Gene Synthesis and Anticancer and Lipid-lowering Mechanism of Monacolin K.

Monacolin K (MK), also known as lovastatin (LOV), is a secondary metabolite synthesized by Monascus in the later stage of fermentation and is the main component of functional red yeast rice (RYR). The structure of MK is similar to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), and it can competitively bind to 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGCR), thus reducing the level of blood lipids. MK can affect the expression of MAPK, PI3K/AKT, and NF-κB pathway, prepare conjugates with other compounds, and enhance the sensitivity of cancer cells to chemotherapeutic drugs so as to induce apoptosis of acute myeloid leukemia, prostate cancer, breast cancer, lung cancer, gastric cancer, and liver cancer. Combined with the synthetic route of MK, this paper summarizes the latest lipid-lowering and anticancer mechanism of MK, and provides a reference for the application of MK in medicine.