Chronic Myelogenous Leukemia Cell Line Research Articles

Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

Herpesvirus entry mediator (HVEM) is a molecular switch that can modulate immune responses against cancer. The significance of HVEM as an immune checkpoint target and a potential prognostic biomarker in malignancies is still controversial. This study aims to determine whether HVEM is an immune checkpoint target with inhibitory effects on anti-tumor CD4+ T cell responses in vitro and whether HVEM gene expression is dysregulated in patients with acute lymphocytic leukemia (ALL). HVEM gene expression in tumor cell lines and peripheral blood mononuclear cells (PBMCs) from ALL patients and healthy controls was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Tumor cells were left untreated (control) or were treated with an HVEM blocker before co-culturing with CD4+ T cells in vitro in a carboxyfluorescein succinimidyl ester (CFSE)-dependent proliferation assay. HVEM expression was upregulated in the chronic myelogenous leukemia cell line (K562) (FC = 376.3, p = 0.086) compared with normal embryonic kidney cells (Hek293). CD4+ T cell proliferation was significantly increased in the HVEM blocker-treated K562 cells (p = 0.0033). Significant HVEM differences were detected in ALL PBMCs compared with the controls, and these were associated with newly diagnosed ALL (p = 0.0011) and relapsed/refractory (p = 0.0051) B cell ALL (p = 0.0039) patients. A significant differentiation between malignant ALL and the controls was observed in a receiver operating characteristic (ROC) curve analysis with AUC = 0.78 ± 0.092 (p = 0.014). These results indicate that HVEM is an inhibitory molecule that may serve as a target for immunotherapy and a potential ALL biomarker.

Novel Mn(II) and Cu(II) metal complexes with sulfa drug-derived ligands as potent antimicrobial and anticancer agents: In vitro studies, ADMET profile and molecular docking

The present work deals with the synthesis, characterization, and pharmaceutical potential evaluation of new biologically active Mn(II) and Cu(II) complexes with Schiff bases derived from sulfamethoxazole. The coordination mode of the complexes, geometry, and general formula were determined using elemental analysis, emission atomic spectroscopy, molar conductance, magnetic measurements, TGA, FTIR, electronic absorption spectroscopy, SEM-EDX, and EPR spectroscopy. The results indicate that the Schiff base act as bidentate ligands binding to the metal ions via the nitrogen atoms of the sulfonamide and oxazole groups which leads to a distorted octahedral geometry around the central metals.The in vitro antimicrobial properties of the Schiff base ligands and their respective Mn(II) and Cu(II) complexes were evaluated against Gram-negative and Gram-positive (Escherichia coli klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis)) bacteria, and two fungal strains (Candida albicans, Aspergillus niger). The results assert that the complexes display excellent antimicrobial properties with low MIC values. Additionally, DPPH-scavenging assay results suggest that the coordination compounds could act as efficient antioxidant species.The in vitro cytotoxicity assay conducted on chronic myelogenous leukaemia cell lines (K562) showed that the coordination compounds exhibit potent anticancer activities. Furthermore, the pharmaceutical properties, including pharmacokinetics and toxicity, were examined by ADMET simulations and molecular docking studies were employed to assess interactions between the complexes and drug targets. The in vitro experimental data and in silico computational analysis revealed that the ligands and their respective coordination compounds as noteworthy candidates for drug development.

Evaluation the Anti-leukemia effect of Methanol extracts of Camellia cuongiana on the K562 cell line

This study aims to examine the anti-proliferation effects of Camellia cuongiana methanolic extracts on the K562 chronic myelogenous leukaemia cell line. The crude extract was obtained by soaking the sample with methanol, following concentration using a rotary evaporator. Trypan blue exclusion test was performed in five concentrations of extract to detect the anti-cancer characteristic of Camellia cuongiana. Morphological changes in the treatment cell were observed using a phase-contrast microscope. After 72 hours of treatment, the extracts inhibited cell proliferation, with IC50 values of 72.26±5.75µg/mL. Increasing the extract concentration decreased the number of living cells. The most frequently observed abnormal morphology was cell shrinkage. Evidently, Camellia cuongiana could inhibit cancer cell proliferation and induce programmed cell death.

Effect of Extracellular Vesicles Derived From Peripheral Blood Mononuclear Cells on K562 Leukemia Cell Line

Recently, extracellular vesicles (EVs) are generating considerable interest in terms of their ability. EVs defined themselves as a route for intercellular communication between the origin cells and the recipient ones by transferring information. This paper investigates the influence of peripheral blood mononuclear cells derived EVs on proliferation and apoptosis of the chronic myelogenous leukemia cell line k562. Trypan blue staining was used to detect cell viability subsequently, metabolic activity was assessed by the MTT assay. Cell apoptosis and cell cycle progression were evaluated using flowcytometric assay after treatment of k562 cell line with MNC derived EVs. Our results showed that MNC-EVs have no inhibitory effect on k562 cell growth and proliferation. Our data did not reveal any significant variations in the case of enhancing k562 cell line growth following treatment with MNC derived EVs. It seems tumour-derived extracellular vesicles support tumour cells growth by communicating with each other through their extracellular vesicles.

Inhibition of LATS kinases reduces tumorigenicity and increases the sensitivity of human chronic myelogenous leukemia cells to imatinib

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study, we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype, IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI), a specific LATS inhibitor, or shRNA targeting LATS1/2 significantly reduced clonogenicity, increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore, depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together, our results suggest that LATS could be one of the key factors contributing to the rapid proliferation, reduced apoptosis, and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM.

L‐lysine Janus Dendrimer Conjugate with Naproxen and Mefenamic Acid and its Anticancer Activity

AbstractDendritic architectures have potential use as excellent carriers for drug delivery due to their multifunctionality, biopermeability, and biodegradability properties. Furthermore, their conjugation with non‐steroidal anti‐inflammatory drugs (NSAIDs) results in a synergetic effect improving their anticancer activity. Here we report the synthesis and cytotoxic activity of novel Janus dendrimer conjugates with naproxen and mefenamic acid moieties at the periphery. Diethanolamine dendrons were convergently synthesized through a protection‐deprotection sequence with naproxen as a terminal group and finally coupled to triethylene glycol as a linker. On the other hand, mefenamic acid was covalently joined to the L‐lysine. The dendron and dendrimer‐conjugates with naproxen and mefenamic acid showed high inhibition and selectivity against PC‐3 (human prostatic adenocarcinoma), and K‐562 (human chronic myelogenous leukemia) cell lines, but low cytotoxicity against normal cell lines. To explain the molecular mechanisms involved in the anticancer activity of dendrimer conjugates, our research will be continued.

Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line.

Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Tyrosine Kinase Inhibitor Profiling Using Multiple Forskolin-Responsive Reporter Cells.

We have developed a highly sensitive promoter trap vector system using transposons to generate reporter cells with high efficiency. Using an EGFP/luciferase reporter cell clone responsive to forskolin, which is thought to activate adenylate cyclase, isolated from human chronic myelogenous leukemia cell line K562, we found several compounds unexpectedly caused reporter responses. These included tyrosine kinase inhibitors such as dasatinib and cerdulatinib, which were seemingly unrelated to the forskolin-reactive pathway. To investigate whether any other clones of forskolin-responsive cells would show the same response, nine additional forskolin-responsive clones, each with a unique integration site, were generated and quantitatively evaluated by luciferase assay. The results showed that each clone represented different response patterns to the reactive compounds. Also, it became clear that each of the reactive compounds could be profiled as a unique pattern by the 10 reporter clones. When other TKIs, mainly bcr-abl inhibitors, were evaluated using a more focused set of five reporter clones, they also showed unique profiling. Among them, dasatinib and bosutinib, and imatinib and bafetinib showed homologous profiling. The tyrosine kinase inhibitors mentioned above are approved as anticancer agents, and the system could be used for similarity evaluation, efficacy prediction, etc., in the development of new anticancer agents.

Inhibition of casein kinase 2 induces cell death in tyrosine kinase inhibitor resistant chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL oncogene. Despite the high performance of treatment with tyrosine kinase inhibitors (TKI), about 30% of patients develop resistance to the therapy. To improve the outcomes, identification of new targets of treatment is needed. Here, we explored the Casein Kinase 2 (CK2) as a potential target for CML therapy. Previously, we detected increased phosphorylation of HSP90β Serine 226 in patients non-responding to TKIs imatinib and dasatinib. This site is known to be phosphorylated by CK2, which was also linked to CML resistance to imatinib. In the present work, we established six novel imatinib- and dasatinib-resistant CML cell lines, all of which had increased CK2 activation. A CK2 inhibitor, CX-4945, induced cell death of CML cells in both parental and resistant cell lines. In some cases, CK2 inhibition also potentiated the effects of TKI on the cell metabolic activity. No effects of CK2 inhibition were observed in normal mononuclear blood cells from healthy donors and BCR-ABL negative HL60 cell line. Our data indicate that CK2 kinase supports CML cell viability even in cells with different mechanisms of resistance to TKI, and thus represents a potential target for treatment.

Visinin-like 1, a novel target gene of the Wnt/β-catenin signaling pathway, is involved in apoptosis resistance in colorectal cancer.

Abnormal activation of Wnt/β-catenin signaling is associated with various aspects of cancer development. This study explored the roles of novel target genes of the Wnt/β-catenin signaling pathway in cancer cells. Using the haploid chronic myelogenous leukemia cell line HAP1, RNA sequencing (RNA-seq) was performed to identify genes whose expression was increased by APC disruption and reversed by β-catenin knockdown (KD). The regulatory mechanism and function of one of the candidate genes was investigated in colorectal cancer (CRC) cells. In total, 64 candidate genes whose expression was regulated by Wnt/β-catenin signaling were identified. Of these candidate genes, the expression levels of six were reduced by β-catenin KD in HCT116 CRC cells in our previous microarray. One of these genes was Visinin-like 1 (VSNL1), which belongs to the neuronal calcium-sensor gene family. The expression of VSNL1 was regulated by the β-catenin/TCF7L2 complex via two TCF7L2-binding elements in intron 1. VSNL1 KD-induced apoptosis in VSNL1-positive CRC cells. Additionally, forced expression of wild-type VSNL1, but not a myristoylation, Ca2+ -binding, or dimerization-defective mutant, suppressed the apoptosis induced by camptothecin and doxorubicin in VSNL1-negative CRC cells. Our findings suggest that VSNL1, a novel target gene of the Wnt/β-catenin signaling pathway, is associated with apoptosis resistance in CRC cells.

Abstract 3832: Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line

Abstract Introduction: Medicinal plants have been used in traditional medicine practices since ancient times. Mayan traditional medicine uses herbs as treatment to illness that can be related to cancer disease such as Phoradendron wattii. As part of the search for novel cancer treatment two triterpene compounds were isolated from P. wattii: 3-epi-betulinic acid and lupenone with preliminary activity on leukemias. We investigated the cancer treatment abilities of 3-epi-betulinic acid and lupenone on human chronic myelogenous leukemia (K562) cell line and monocytes isolated from peripheral blood using flow cytometry methods. Results: Lupenone showed low effect on both cells with an IC50 of 177.60 ± 5.07 µg/mL and a selectivity index (SI) of 0.10 times more on K-562 cell line than on monocytes. Nevertheless, 3-epi-betulinic acid showed a moderate anti-CML activity with an IC50 of 38.74 ± 5.07 µg/mL, having a SI of 4.10 on K-562 cells when compared to monocytes. These results indicate that 3-epi-betulinic acid was 41 times more selective for K-562 cell line than lupenone and it also was 7.45 more selective than etoposide as positive control. Additionally, 3-epi-betulinic acid increased cell population in the G0/G1 phase of the cell cycle and apoptotic cells, and it inhibited cell proliferation in a dose-dependent manner. Furthermore, the relative expression of genes associated with apoptosis Bax and caspase 3 increased compared to control. Conclusion: Combined, these results suggest that 3-epi-betulinic acid plays an important regulatory role of cell cycle and apoptosis. Finally, the 3-epi-betulinic acid investigated in this work is potentially a cheap, safe, and effective alternative to chemotherapy in cancer treatment. Citation Format: Rosa E. Moo-Puc, Shirley A. Calderón Sauri, Jairo R. Villanueva-Toledo, Sergio R. Peraza-Sánchez, Antonieta Chávez-González. Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line. [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 3832.

Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice.

Every bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia. Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at rest and at the end of an acute bout of cycling exercise. Flow cytometry and single-cell RNA sequencing was performed to identify phenotypic and transcriptomic differences between resting and exercise-mobilized cells using a targeted gene expression panel curated for human immunology. PBMCs were injected into the tail vein of xenogeneic NSG-IL-15 mice and subsequently challenged with a luciferase tagged chronic myelogenous leukemia cell line (K562). Tumor growth (bioluminescence) and xenogeneic graft-versus-host disease (GvHD) were monitored bi-weekly for 40-days. Exercise preferentially mobilized NK-cell, CD8+ T-cell and monocyte subtypes with a differentiated and effector phenotype, without significantly mobilizing CD4+ regulatory T-cells. Mobilized effector lymphocytes, particularly effector-memory CD8+ T-cells and NK-cells, displayed differentially expressed genes and enriched gene sets associated with anti-tumor activity, including cytotoxicity, migration/chemotaxis, antigen binding, cytokine responsiveness and alloreactivity (e.g. graft-versus-host/leukemia). Mice receiving exercise-mobilized PBMCs had lower tumor burden and higher overall survival (4.14E+08 photons/s and 47%, respectively) at day 40 compared to mice receiving resting PBMCs (12.1E+08 photons/s and 22%, respectively) from the same donors (p<0.05). Human immune cell engraftment was similar for resting and exercise-mobilized DLI. However, when compared to non-tumor bearing mice, K562 increased the expansion of NK-cell and CD3+/CD4-/CD8- T-cells in mice receiving exercise-mobilized but not resting lymphocytes, 1-2 weeks after DLI. No differences in GvHD or GvHD-free survival was observed between groups either with or without K562 challenge. Exercise in humans mobilizes effector lymphocytes with an anti-tumor transcriptomic profile and their use as DLI extends survival and enhances the graft-versus-leukemia (GvL) effect without exacerbating GvHD in human leukemia bearing xenogeneic mice. Exercise may serve as an effective and economical adjuvant to increase the GvL effects of allogeneic cell therapies without intensifying GvHD.

A phytotherapic blend immunity-6™ inhibits myeloid leukemic cells 2 activation involving purinergic signaling

Leukemia is among the most common types of hematological cancers and the use of herbal medicines to prevent and treat leukemia are under quick development. Among several molecular pathways involved in leukemia pathogenesis and exacerbations, purinergic signaling is revealed as a key component. In the present study, the effects of two doses (5 ug/mL and 10 ug/mL) of Immunity-6™, a phytocomplex composed by beta-glucan, green tea (Camelia sinensis), chamomile (Matricaria chamomilla), and ascorbic acid (vitamin C) was tested in vitro, using chronic myelogenous leukemia cell line (K-562; 5 ×104/mL/well), which were challenged with lipopolysaccharide (LPS; 1 ug/mL) for 24 h. The results demonstrated that both doses of Immunity-6™ inhibited ATP release (p < 0.001) and P2×7 receptor at mRNA levels expression (p < 0.001). Purinergic inhibition by Immunity-6™ was followed by reduced release of proinflammatory cytokines IL-1beta (p < 0.001) and IL-6 (p < 0.001), while only 5 ug/mL of Immunity-6™ reduced the release of TNF-alpha (p < 0.001). Beyond to inhibit the release of pro-inflammatory cytokines, both doses of Immunity-6™ induced the release of anti-inflammatory cytokine IL-10 (p < 0.001), while only the higher dose (10 ug/mL) of Immunity-6™ induced the release of anti-inflammatory IL-1ra (p < 0.05) and klotho (p < 0.001). Thus, Immunity-6™ may be a promising adjuvant in the treatment of leukemia and further clinical trials are guaranteed.

SLC25A40 Facilitates Anticancer Drug Resistance in Human Leukemia K562 Cells.

The chronic myelogenous leukemia cell line, K562/ADM is derived from the K562 cell line, which is resistant to doxorubicin (alias, adriamycin: ADM). P-glycoprotein levels are significantly higher in K562/ADM cells than in K562 cells. The overexpression of p-glycoprotein has been shown to cause drug resistance. Therefore, the present study investigated a novel mechanism underlying the drug resistance of K562/ADM cells. A gene ontology analysis demonstrated that the expression of solute carrier (SLC)-mediated transmembrane transport genes was significantly higher in K562/ADM cells than in K562 cells. The expression level of a member of the SLC family, SLC25A40 was higher in K562/ADM cells than in K562 cells. SLC25A40 is located near the ABCB1 gene. A real-time PCR analysis showed that the expression of SLC25A40, ABCB4, and ADAM22 was up-regulated. These genes are located close to SLC25A40. The down-regulation of SLC25A40 significantly decreased the mitochondrial concentration of glutathione and cell proliferation. Collectively, the present results demonstrated that the expression of SLC25A40 was up-regulated in K562/ADM cells, which enhanced to cell proliferation, and that the expression of SLC25A40 affected drug resistance to ADM.

A novel tumor-targeting strain of Xenorhabdus stockiae exhibits potent biological activities.

Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema presenting two distinct forms in their life cycle, and can produce a broad range of bioactive compounds. In this study, a novel Xenorhabdus stockiae strain HN_xs01 was isolated from a soil sample via an entrapment method using Galleria melonella nematodes. The supernatants of strain HN_xs01 exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, and insecticidal properties against Helicoverpa armigera larvae, and antitumor properties as well. Moreover, three linear rhabdopeptides (1, 2 and 3) were identified from strain HN_xs01 using nuclear magnetic resonance analysis, which exhibited significant cytotoxic activity against the human epithelial carcinoma cell line A431 and the human chronic myelogenous leukemia cell line K562. Some bacteria have been reported to colonize the tumor region, and we determined that HN_xs01 could grow in tumor xenografts in this study. HN_xs01 invaded and replicated in B16 melanoma cells grafted into C57BL/6 mice, resulting in tumor inhibition. Moreover, strain HN_xs01 not only merely aggregated in the tumor environment, but also prevented pulmonary metastasis. It caused fragmentation of vessels and cell apoptosis, which contributed to its antitumor effect. In conclusion, X. stockiae HN_xs01 is a novel tumor-targeting strain with potential applications in medicinal and agricultural fields.

Synthesis and Biological Evaluation of Imadazo[1,2-a]pyrazines as Anticancer and Antiviral Agents through Inhibition of CDK9 and Human Coronavirus.

In this work, novel imadazo[1,2-a]pyrazine derivatives were synthesized and evaluated as CDK9 inhibitors. The results of CDK9 assay showed that the derivatives with pyridin-4-yl in position 2 and benzyl in position 3 of imadazo[1,2-a]pyrazine 3c displayed the most potent CDK9 inhibitory activity with IC50 of 0.16 µM. The anti-proliferative effect of the new compounds was examined against breast cancer (MCF7), colorectal cancer (HCT116), and chronic myelogenous leukaemia (K652) cell lines. The data of MTT assay showed that the cytotoxic effect of the inhibitors is correlated to their inhibitory activity against CDK9. Compound 3c exhibited the most potent cytotoxicity effect with average IC50s of three cell lines of 6.66 µM. The drug likeness properties of 3c were predicated in silico and demonstrated that 3c have reasonable physiochemical and pharmacokinetic properties. Selected derivatives were assessed in antiviral assay against human coronavirus 229E. The results of this assay showed that the derivative with pyridin-4-yl in position 2 and cyclohexyl in position 3 of imadazo[1,2-a]pyrazine 3b exhibited the most potent anti-coronaviral activity with IC50 of 56.96 µM and selectivity index of 7.14. The target predication result revealed that 3b showed high affinity to protease enzyme. Docking studies of 3b with COVID-19 main protease was conducted and showed good binding affinity, which confirmed the in vitro assay data.

Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway.

Metabolic regulation of cancer cell growth via AMP-activated protein kinase (AMPK) activation is a widely studied strategy for cancer treatment, including leukemias. Recent notions that naturally occurring compounds might have AMPK activity led to the search for nutraceuticals with potential AMPK-stimulating activity. We found that hydroxycitric acid (HCA), a natural, safe bioactive from the plant Garcinia gummi-gutta (cambogia), has potent AMPK activity in chronic myelogenous leukemia (CML) cell line K562. HCA is a known competitive inhibitor of ATP citrate lyase (ACLY) and is widely used as a weight loss inducer. We found that HCA was able to inhibit the growth of K562 cells in in vitro and in vivo xenograft models. At the mechanistic level, we identified a direct interaction between AMPK and ACLY that seems to be sensitive to HCA treatment. Additionally, HCA treatment resulted in the co-activation of AMPK and the mammalian target of rapamycin (mTOR) pathways. Moreover, we found an enhanced unfolded protein response as observed by activation of the eIF2α/ATF4 pathway that could explain the induction of cell cycle arrest at the G2/M phase and DNA fragmentation upon HCA treatment in K562 cells. Overall, these findings suggest HCA as a nutraceutical approach for the treatment of CMLs.

Abstract 3775: Inhibition of mitochondrial translation by the marine natural product elatol shows potent antileukemia activity

Abstract Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Recent studies have shown that some forms of hematologic malignancies (Ashton et al., 2018) and solid tumors (Molina et al., 2018) have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation. Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins leading to selective lethality in hematologic malignancies reliant on oxidative phosphorylation (Norberg et al., 2017). Combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant chronic myelogenous leukemia (CML), both in vitro and in vivo (Kuntz et al., 2017). We have previously reported that elatol, the major compound from algae Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary CML cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells treated with single-digit micromolar concentrations of elatol for short periods revealed strong downregulation of mitochondrion-encoded proteins (no transcriptional effect). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 2-10x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines. Through sedimentation property analysis using sucrose gradients, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. Moreover, elatol treatment leads to activation of the integrated stress response (ISR) evidenced by induction of ATF4 and CHOP. We found that elatol activates the ISR through mitochondrial stress in which cleaved DELE1 binds to HRI to phosphorylate eIF2α, a mechanism described in Fessler et al., 2020. In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis and mechanistically different from chloramphenicol. Tigecycline’s compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol’s greatly improved potency provides a potential starting point for further optimization of this paradigm. Citation Format: Tyler A. Cunningham, Priyanka Maiti, Antonio Barrientos, Jonathan H. Schatz. Inhibition of mitochondrial translation by the marine natural product elatol shows potent antileukemia activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3775.

MiR-494 in Extracellular Vesicles as a Potent Biomarker of Chronic Myeloid Leukemia Treatment with Tyrosine Kinase Inhibitors

Extracellular vesicles (EVs) are nano-sized particles released from cells and transferring molecules (proteins, lipids and nucleic acids such as mRNA, tRNA and miRNA) to recipient cells. Surface antigens and components are important for the functions as cell-to-cell communication of EVs. Thus, EVs are useful biomarkers for various diseases including leukemias and other types of malignancies. We evaluated whether miRNAs in EVs released from chronic myelogenous leukemia (CML) cells could be used for diagnosis. Microarray analysis of miRNAs in EVs obtained from the culture supernatants of two CML cell lines showed that miR-494 and miR-373-5p were significantly decreased by tyrosine kinase inhibitor for BCR-ABL1. Validation analysis with Taqman-based qRT-PCR of whole serum obtained patients with CML in the chronic phase (n = 5) did not show a significant difference in miR-494 levels compared to the CML accelerated phase and blast crisis patients (n = 5). However, the levels of miR-494 were 2.9-fold higher in the accelerated phase or blast crisis than in the chronic phase (p &lt; 0.05). These results indicate that it is important to measure miR-494 using only EVs rather than whole serum. Our data suggest that EV-miR-494 is a useful biomarker of CML progression and evaluation of response to tyrosine kinase inhibitors.

Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells.

In this study, extracts from non‐psychoactive Cannabis sativa L. varieties were characterized by means of ultra high‐performance liquid chromatography coupled with high‐resolution mass spectrometry (UHPLC‐HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)‐rich extract inhibited cell proliferation of K562 cell line in a dose‐dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.