Chronic Myeloid Leukemia Cell Lines Research Articles

Tern-701 (HS-10382) Is a Potent Inhibitor of BCR::ABL1 and Is Synergistic with Active Site Tyrosine Kinase Inhibitors

CONCLUSIONS Although tyrosine kinase inhibitors (TKIs) targeting the ATP-binding site of the BCR::ABL1 oncoprotein are effective therapeutics for chronic myeloid leukemia (CML), patients often develop drug resistance due to ATP-site mutations that inhibit drug binding. TERN-701 is a potent and highly selective inhibitor of BCR::ABL1 that is designed to bind the allosteric myristoyl site on the kinase, circumventing resistance to active site mutations, with potential for synergistic combination with active site TKIs. Competition binding screens were used to assess the selectivity of TERN-701 on more than 450 targets spanning the human kinome. The potency of TERN-701 against the proliferation of wild type and mutant CML cell lines, as well as a panel of 102 cancer cell lines, was assessed using CellTiter-Glo®. Synergy between TERN-701 and active site TKIs was assessed in vitro with interactions quantified using Bliss, combination, and curve-shift analyses. Prospective mutagenesis experiments were conducted by treating BaF/3 cells engineered to overexpress the BCR::ABL1 kinase with various concentrations of both TERN-701 and the TKI imatinib, followed by observation of clonal outgrowth of treated cells over time. We have previously shown that TERN-701 is a potent inhibitor of native CML cell lines such as K562. Here, we show that TERN-701 inhibited cell proliferation in various clinically relevant mutant CML cell lines (including T315I) with IC 50s ranging from 5 to 26 nM and 70 to 1200 nM against active site and myristoyl site mutations, respectively. TERN-701 was highly selective for BCR::ABL1, with no appreciable activity (>50%) against >450 purified kinase targets. Against a panel of cancer cell lines, TERN-701 was more selective than the comparator allosteric BCR::ABL1 inhibitor asciminib while maintaining similar potency. In vitro combination studies previously revealed that TERN-701 works synergistically with multiple TKIs in the non-mutant K562 cell line. Here, expanded studies using KCL22-s and BCR::ABL1-T315I mutant cell lines identified strong synergistic interactions between TERN-701 and active site TKIs, including ponatinib and dasatinib. Furthermore, prospective mutagenesis studies showed that the combination of TERN-701 and imatinib resulted in greater inhibition of the outgrowth of resistant clones relative to either compound alone. TERN-701 is a potent and highly selective allosteric inhibitor of BCR::ABL1 that can act synergistically with active site TKIs in vitro, even against the T315I gatekeeper mutation that confers resistance to all approved active site TKIs except ponatinib, which has known safety liabilities. These data support the continued development of TERN-701 for the treatment of CML using both monotherapy and combination approaches.

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

Novel Role of Mitochondrial Folate Metabolism in Cell Fate Decisions and Leukaemogenesis

Folate-mediated one carbon (1C) metabolism sustains proliferation of rapidly dividing cells through its direct contribution to nucleotide biosynthesis. In brief, new 1C units are donated from the amino acid serine (the primary source of 1C units), while folate molecules function as 1C unit carriers. Accordingly, antifolates have been used in the treatment of several malignancies, particularly in the field of haemato-oncology. Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder that arises in a single haematopoietic stem cell with the introduction of the BCR::ABL1 oncoprotein. Although it is generally accepted that CML is driven by leukaemic stem cells (LSCs) that are insensitive to standard therapy due to their quiescent/slow-cycling status, the role of folate metabolism in LSCs remains undescribed. Transcriptomic analysis of patient-derived CML LSCs (CD34 +38 -) revealed a significant upregulation of folate metabolism genes, including the mitochondrial hydroxymethyltransferase (SHMT2; p≤0.05), when compared with normal counterparts. Furthermore, CML stem/progenitor (CD34 +) cells displayed a significant increase in the exchange rate of formate (folate intermediate necessary for nucleotide synthesis) compared to the secretome of normal CD34 + cells (p<0.05), suggesting an upregulation of 1C metabolism in therapy resistant CML cells. Following SHMT2 knockout (KO), we observed a strong antiproliferative effect and significantly impaired tumour formation in CML cell line xenograft models (p<0.01), verifying that mitochondrial 1C metabolism is not dispensable for CML cells. Metabolic characterisation using liquid chromatography-mass spectrometry revealed that both genetic and pharmacological inhibition (using the SHMT1/2 inhibitor, SHIN1) of 1C metabolism results in a significant decrease in de novo purine synthesis. Furthermore, we uncovered activation of AMPK signalling and suppression of mTORC1 activity following mitochondrial 1C metabolism inhibition. Notably, formate supplementation or reconstitution of purine levels was sufficient to reverse the effect on AMPK and mTORC1. Phenotypically, SHIN1 treatment induced the expression of erythropoiesis markers CD71 and Glycophorin A in CML cells, including patient derived CD34 + cells. AMPKα1/α2 KO revealed that the increased expression of these markers was independent of AMPK activity. Mechanistically, we discovered that reconstitution of purine levels (adenine) and mTORC1 activity prevented erythroid differentiation following SHMT1/2 inhibition, highlighting that depletion of 1C units drives differentiation of leukaemic cells through mTORC1-mediated purine sensing. Of clinical relevance, combination of SHIN1 with imatinib, a frontline treatment for CML patients, decreased the number of therapy-resistant CML LSCs in a patient-derived xenograft model (p<0.05). Lastly, stable isotope-assisted metabolomics using 13C 315N 1-serine revealed that imatinib reduced labelling into purine nucleotides, but it maintained labelling into glutathione (GSH), the most abundant antioxidant in the cell, and its oxidised derivate (GSSG). Through generation of glycine, folate metabolism directly contributes to the GSH pool. These data suggest that standard therapy does not affect the incorporation of serine into GSH, implying that the synergistic effect with folate metabolism inhibition might be related to impeded antioxidant defence. Overall, our findings highlight a novel role for mitochondrial folate metabolism in stem cell fate decisions and leukaemogenesis. This work is available as a preprint: https://www.biorxiv.org/content/10.1101/2022.12.21.521404v1

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Hydroquinone-selected chronic myelogenous leukemia cells are sensitive to chloroquine-induced cytotoxicity via MCL1 suppression and glycolysis inhibition

Previous studies have provided evidence that repeated exposure to the benzene metabolite hydroquinone (HQ) induces malignant transformation and increases basal autophagy in the chronic myeloid leukemia (CML) cell line K562. This study explored the cytotoxicity of the autophagy inhibitor chloroquine (CQ) on parental and HQ-selected K562 (K562/HQ) cells. CQ triggered apoptosis in these cells independently of inhibiting autophagic flux; however, in K562/HQ cells, CQ-induced cytotoxicity was higher than in K562 cells. Mechanistically, CQ-induced NOXA upregulation led to MCL1 downregulation and mitochondrial depolarization in K562/HQ cells. MCL1 overexpression or NOXA silencing attenuated CQ-mediated cytotoxicity in K562/HQ cells. CQ triggered ERK inactivation to increase Sp1, NFκB, and p300 expression, and co-assembly of Sp1, NFκB, and p300 in the miR-29a promoter region coordinately upregulated miR-29a transcription. CQ-induced miR-29a expression destabilized tristetraprolin (TTP) mRNA, which in turn reduced TTP-mediated NOXA mRNA decay, thereby increasing NOXA protein expression. A similar mechanism explained the CQ-induced downregulation of MCL1 in K562 cells. K562/HQ cells relied more on glycolysis for ATP production than K562 cells, whereas inhibition of glycolysis by CQ was greater in K562/HQ cells than in K562 cells. Likewise, CQ-induced MCL1 suppression and glycolysis inhibition resulted in higher cytotoxicity in CML KU812/HQ cells than in KU812 cells. Taken together, our data confirm that CQ inhibits MCL1 expression through the ERK/miR-29a/TTP/NOXA pathway, and that inhibition of glycolysis is positively correlated to higher cytotoxicity of CQ on HQ-selected CML cells.

Asciminib Enhances Its Treatment Efficacy Synergistically in the Treatment of Chronic Myeloid Leukemia Harboring ABL1 Kinase Domain Mutation When Combined with a Reduced Dose of Ponatinib, Dasatinib, or Bosutinib, but Not with Nilotinib or Imatinib

Background: Asciminib (ASC) monotherapy has been approved for chronic myeloid leukemia (CML) treatment in the patients who failed at least 2 lines of tyrosine kinase inhibitor (TKI) therapy. Theoretically, dual inhibition of the ABL1 kinase protein is plausible by combining ASC with ATP-binding pocket inhibitors (ABPIs) including Imatinib (IMA), Dasatinib (DAS), Nilotinib (NIL), Bosutinib (BOS) or Ponatinib (PON). A phase 1 study determined the appropriate dose of ASC with fixed doses of ABPIs (IMA, DAS, NIL). However, this approach of using full dose ABPIs with variable dose of ASC would raise a concern of tolerability given that all the toxicity from the doses of ABPI will be augmented by adding even half dose of ASC, evidenced by the FASCINATION trial (Ernst et al. EHA 2023). Questions still remained such as what is the best dose for combination? Is the combination of full dose ASC with reduced dose ABPIs effective? Is this combination effective to other ABL1 kinase domain mutant CML? For the ABL1 kinase domain (KD) mutant CML, which ABPI and what dose is more effective and more synergistic than other ABPIs? Methods and Materials: To investigate the combined effect of ASC with ABPIs, we determined the IC 50 values for ASC and ABPIs in the wild-type (WT) BaF3 BCR-ABL+ cell line, and utilized the IC 50 as baselines throughout the study. From the baseline IC 50s of ASC (3nM), IMA (150nM), NIL (10nM), BOS (10nM), DAS (0.8nM) and PON (0.6nM), serially diluted ASC alone, ABPIs alone or in combination were administered to BaF3 cell lines. We conducted IC 50 determining experiment in different combination of ASCs/ABPIs/combination in different concentration (conc) on eleven different KD mutant (KDM) BaF3 cell lines, including WT, G250E, E255K, T315A, M351T, F317L, F317V, H396P, Y253F, M244V, and T315I mutant cell lines. ZIP synergy scores for these cell lines were analyzed using SynergyFinder. Results We observed that the addition of 3 nM ASC to ABPI at baseline IC 50 conc led to a higher response (% cell growth inhibition) compared to treatment with ABPI alone at baseline conc, irrespective of the mutation. Moreover, the combined treatment of ABPI at half baseline conc with 3nM ASC demonstrated similar or even higher effects than ABPI alone at IC 50 baseline conc. These results suggest that double blockade allows for the reduction of ABPI dose by half while maintaining cell inhibition activity. According to its sensitivity to ASC monotherapy and combination, we have stratified the cell lines into 3 categories. High sensitive cell lines to ASC monotherapy (i.e. WT, G250E, and E255K) achieving 100% of response rate when combined with ABPI. Also, T315A mutation showed a significant synergistic effect (ZIP = 12.91~21.75) with the combination of ASC 3nM with a half conc of ABPI, resulting in 1.90-2.43 times higher inhibitory effect than ABPI monotherapy.Intermediate sensitive cell lines (i.e. M351T and F317L) displaying 69-85% inhibition efficiency with ASC 6nM combined with ABPI, without significant increases in all ABPI conc.Low sensitive cell lines (i.e. F317V, H396P, Y253F, M244V, and T315I) exhibiting unique resistance profiles that varied with different combinations. Notably, F317V and F317L mutations showed resistance to DAS. When combined with ASC 3nM, F317V showed restored sensitivity with IMA or NIL than with DAS, while F317L better with DAS. Moreover, the H396P, Y253F, and M244V mutations demonstrated higher sensitivities with BOS or DAS. Interestingly, in the combination treatment with ASC at 3 or 6 nM, with exception of the F317V and T315I mutations, both DAS and PON exhibited a potent efficacy. High resistance of T315I to ABPIs can be overcome with combination of ASC at baseline conc with reduced dose PON. In the case of T315I, the combination of 3nM ASC with a half conc PON resulted in a higher inhibitory effect than baseline conc PON alone. Conclusion: The present study demonstrated that a half of baseline conc ABPIs in combination with ASC is as effective as other ABPIs not just in WT but also in other ABL1 KDM CML cell lines. Different CML cell lines harboring different ABL KDM has different treatment spectrum on optimal ABPI drug as well as optimal drug conc. This result will be useful to design future clinical trial of dual blockade of ASC with other ABPIs considering the ABL KDM profiles. It also emphasizes the importance of ABL KDM testing prior to ASC monotherapy or combination with other ABPIs.

Dynamic Single-Cell RNA-Seq Reveals Mechanism of Selinexor-Resistance in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm caused by BCR-ABL chimeric oncogene. The administration of tyrosine kinase inhibitors (TKI) has been shown to significantly improve the prognosis of the CML cases. TKI-resistance may lead to progression of the disease into accelerated and blast phases. To tackle this problem, there are currently several strategies that can be employed to manage or overcome the difficulties associated with TKI-resistance. Selinexor is a small molecule inhibitor that targets the nuclear transporter Exportin 1 (XPO1). This inhibition leads to disruptions in various signaling pathways involved in tumor cell cycle regulation, DNA repair, and apoptosis. In CML, Selinexor can increase the nuclear-cytoplasmic distribution ratio of BCR/ABL, cause cell cycle arrest, inhibit the proliferation of CML cell line K562, and exhibit anti-tumor properties in mouse model. When combined with imatinib, Selinexor can effectively disrupt the nuclear-cytoplasmic transport signal of leukemia stem cells, resulting in reduced formation of clones and increased apoptosis. In this work, we were aiming at investigating the mechanism of drug-resistance of Selinexor in CML cell lines K562. We performed single cell dynamic transcriptome sequence to analysis intratumor heterogeneity of subpopulation of CML and identified key gene expression changes that occur as Selinexor-resistance. We generated Selinexor-resistant (SR) lines by culturing K562 cell lines with increasing concentrations of Selinexor. At first, we conducted an analysis of differential gene expression between the parental group and various levels of SR groups. Our findings revealed differential expression patterns in MT2A, TFPI, MTND3, and HMGCS1 in total RNA, as well as MT-TW, DNAJB1, and HSPB1 in newly synthesized RNA, within both the parental and drug-resistant groups. These results suggest that mitochondrial metabolism and heat shock proteins play a significant role in the regulation of cellular drug resistance. Furthermore, we found hallmark signaling pathways of epithelial mesenchymal transition and protein secretion upregulated in SR groups. Through the application of pseudo-time analysis, it was observed that cluster 6 persisted in both the parental and drug-resistant groups, and displayed characteristics indicative of tumor stem cells. Notably, a set of distinct genes were identified that distinguish cluster 6 from the other clusters, these genes are associated with lysosomes and ferroptosis pathways by KEGG enrichment analysis. Next, it was determined that the gene set in the newly synthesized RNA exhibits a consistent upregulation by the expression pattern clustering analysis. Within this gene set, we revealed an enrichment of the endoplasmic reticulum protein processing and ferroptosis signaling pathway. Finally, to provide additional evidence for the ferroptosis-related pathway in the SR group, we observed that the expression of ferroptosis-inhibitory molecules FTH1 and SLC7A11 increased gradually with the development of drug resistance. Conversely, the expression of ferroptosis-driving molecules HMGB1 and MTDH decreased gradually. Subsequently, in vitro experiments were conducted to validate that the combination of the ferroptosis inducer RSL3 can effectively overcome Selinexor resistance. In summary, this study identified a subgroup exhibiting tumor stem cell properties. Additionally, it demonstrated that the combination of ferroptosis inducer can improve resistance to Selinexor. Through single-cell dynamic transcriptome RNA-seq technology, this investigation elucidated the mechanism of resistance to Selinexor in the K562 cell line, highlighting the potential involvement ferroptosis in drug resistance. These findings offer novel insights for the treatment of CML.

Apoptotic and antiproliferative effects of Urtica dioica L. extract on K562 chronic myeloid leukemia cell line

Apoptotic and antiproliferative effects of Urtica dioica L. extract on K562 chronic myeloid leukemia cell line

In-house glutamine based compound loaded poly-(D-L-lactide-coglycolide) nanoparticles as a possible candidate for the management of chronic myeloid leukemia

The development of targeted medicines such tyrosine kinase inhibitors (TKIs) have turned chronic myeloid leukemia (CML) from a potentially fatal illness into a manageable chronic condition for many individuals. But in case of TKIs, some serious drug induced adverse events as well as drug resistance were reported. In continuation of our earlier work, we have synthesized two p-nitrobenzenesulfonyl-D-glutamine derivatives which were screened against human CML cell line (K562). In comparison with earlier reported p-nitrobenzenesulfonyl-L-glutamine derivatives (Compound 1 and Compound 2), newly synthesized compounds showed more than 3 times better activity against K562 cell line. The best active compound (Compound 3) was encapsulated into Poly-(D-L-lactide-coglycolide) nanoparticles (NPs). The NPs were characterized and screened against K562 and human PBMC cell line. The IC50 value of NP of Compound 3 was found in the nanomolar range (522 nM) against K562 cell line which is more than 10 times better active than Compound 3. DNA deformation assay also indicates cell shrinkage and nuclear fragmentation which are associated with apoptosis-mediated cell death. The prepared NPs of Compound 3 (NP3) are very much less toxic against normal human PBMC (IC50 value > 500 µM). The current study implies that NP3 holds promise for managing chronic myeloid leukemia.

Leukemia cells accumulate zinc for oncofusion protein stabilization

Acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) are both hematological malignancies characterized by genetic alterations leading to the formation of oncofusion proteins. The classical chromosomal aberrations in APL and CML result in the PML-RARα and BCR-ABL1 oncofusion proteins, respectively. Interestingly, our flow cytometric analyses revealed elevated free intracellular zinc levels in various leukemia cells, which may play a role in stabilizing oncofusion proteins in leukemia and thus support cell proliferation and malignancy. Long-term zinc deficiency resulted in the degradation of PML-RARα in NB4 cells (APL cell line) and of BCR-ABL1 in K562 cells (CML cell line). This degradation may be explained by increased caspase 3 activity observed in zinc deficient cells, whereas zinc reconstitution normalized the caspase 3 activity and abolished zinc deficiency-induced oncofusion protein degradation. In NB4 cells, fluorescence microscopic images further indicated enlarged and enriched lysosomes during zinc deficiency, suggesting increased rates of autophagy. Moreover, NB4 cells exhibited increased expression of the zinc transporters ZIP2, ZIP10 and ZnT3 during zinc deficiency and revealed excessive accumulation of zinc in contrast to healthy peripheral blood mononuclear cells (PBMCs), when zinc was abundantly available extracellularly. Our results highlight the importance of altered zinc homeostasis for some characteristics in leukemia cells, uncover potential pathways underlying the effects of zinc deficiency in leukemia cells, and provide potential alternative strategies by which oncofusion proteins can be degraded.

Effect of asciminib and vitamin K2 on Abelson tyrosine-kinase-inhibitor-resistant chronic myelogenous leukemia cells

BackgroundAbelson (ABL) tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML); however, many patients develop resistance during ABL TKI therapy. Vitamin K2 (VK2) is a crucial fat-soluble vitamin used to activate hepatic coagulation factors and treat osteoporosis. Although VK2 has demonstrated impressive anticancer activity in various cancer cell lines, it is not known whether VK2 enhances the effects of asciminib, which specifically targets the ABL myristoyl pocket (STAMP) inhibitor.MethodIn this work, we investigated whether VK2 contributed to the development of CML cell lines. We also investigated the efficacy of asciminib and VK2 by using K562, ponatinib-resistant K562 (K562 PR), Ba/F3 BCR–ABL, and T315I point mutant Ba/F3 (Ba/F3 T315I) cells.ResultsBased on data from the Gene Expression Omnibus (GEO) database, gamma-glutamyl carboxylase (GGCX) and vitamin K epoxide reductase complex subunit 1 (VKORC1) were elevated in imatinib-resistant patients (GSE130404). UBIA Prenyltransferase Domain Containing 1 (UBIAD1) was decreased, and K562 PR cells were resistant to ponatinib. In contrast, asciminib inhibited CML cells and ponatinib resistance in a dose-dependent manner. CML cells were suppressed by VK2. Caspase 3/7 activity was also elevated, as was cellular cytotoxicity. Asciminib plus VK2 therapy induced a significantly higher level of cytotoxicity than use of each drug alone. Asciminib and VK2 therapy altered the mitochondrial membrane potential.ConclusionsAsciminib and VK2 are suggested as a novel treatment for ABL-TKI-resistant cells since they increase treatment efficacy. Additionally, this treatment option has intriguing clinical relevance for patients who are resistant to ABL TKIs.

CD4 T cells in chronic myeloid leukemia present MHC class II-dependent and IFN-γ-dependent cytotoxic capacity.

At present, many therapeutic schemes have been used to improve the prognosis of patients with chronic myeloid leukemia (CML), but response remains poor in a small group of patients. CD4 T cell-mediated cytotoxicity has been found in various autoimmune diseases. This study analyzed the characteristics of CD4 T cell mediated cytotoxicity in CML patients and healthy people. The cytotoxicity of CD4 T cells was tested in using two CML cell lines, including the MHC class II-deficient K562 cells and the MHC class II-expressing KU812 cells. CD4 T cell-mediated lysis was minimal in K562 cells but was much higher in KU812 cells. In CML patients, the level of CD4 T cell-mediated lysis was limited to a certain level. Interestingly, pre-treating KU812 cells with IFN-γ could significantly elevate the expression of MHC class II and elevate the level of CD4 T cell-mediated lysis. Overall, these data indicated CD4 T cells could become a potential candidate for cytotoxic elimination of CML cells.

Phenotypic and functional characterization of subpopulation of Imatinib resistant chronic myeloid leukemia cell line

PurposeChronic myeloid leukemia (CML) is a hematological malignancy characterized by the presence of BCR-ABL protein. Imatinib (IMA) is considered as the first line therapy in management of CML which particularly targets the BCR-ABL tyrosine kinase protein. However, emergence of resistance to IMA hinders its clinical efficiency. Hence, identifying novel targets for therapeutic approaches in CML treatment is of great importance. Here, we characterize a new subpopulation of highly adherent IMA-resistant CML cells that express stemness and adhesion markers compared to naive counterparts. Materials and methodsWe performed several experimental assays including FISH, flow cytometry, and gene expression assays. Additionally, bioinformatics analysis was performed by normalized web-available microarray data (GSE120932) to revalidate and introduce probable biomarkers. Protein-protein interactions (PPI) network was analyzed by the STRING database employing Cytoscape v3.8.2. ResultsOur findings demonstrated that constant exposure to 5 ​μM IMA led to development of the adherent phenotype (K562R-adh). FISH and BCR-ABL expression analysis indicated that K562R-adh cells were derived from the original cells (K562R). In order to determine the role of various genes involved in epithelial–mesenchymal transition (EMT) and stem cell characterization, up/down-regulation of various genes including cancer stem cell (CSC), adhesion and cell surface markers and integrins were observed which was similar to the findings of the GSE120932 dataset. ConclusionTreating CML patients with tyrosine kinase inhibitors (TKIs) as well as targeting adhesion molecules deemed to be effective approaches in prevention of IMA resistance emergence which in turn may provide promising effects in the clinical management of CML patients.

Occurrence of L1M Elements in Chromosomal Rearrangements Associated to Chronic Myeloid Leukemia (CML): Insights from Patient-Specific Breakpoints Characterization.

Chronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML.

Dual inhibition of STAT3 and STAT5 may overcome imatinib resistance in chronic myeloid leukemia

ABSTRACTBackground:Clinical outcome of patients with chronic myeloid leukemia (CML) has improved dramatically since the introduction of tyrosine kinase inhibitors such as imatinib mesylate (IM). However, approximately 20-30% of patients experience IM resistance. SH-4-54, which targets the SH2 domains of both proteins STAT3 and STAT5, has been reported to exhibit anticancer activity in solid tumors. However, the roles of SH-4-54 in CML remain unclear. The aim was to explore whether SH-4-54 could overcome IM resistance and identify novel targets for CML.Methods:Cell viability was measured by CCK-8 assays after treatment of K562 and K562R cells with different concentrations of SH-4-54. Annexin V-FITC and PI were applied to assess the effects of SH-4-54 on cell apoptosis. Effects of SH-4-54 on the expression of proteins downstream of BCR::ABL1 were assessed by western blotting (WB). Effects of SH-4-54 on gene expression profile of CML cells were analyzed by Next generation sequence (NGS).Results:SH-4-54 inhibited the growth of CML cell lines with increasing concentration. SH-4-54 cytotoxic effects correlated with a significant induction of apoptosis. The results of WB analysis showed the downstream proteins of BCR::ABL1, such as STAT3 and STAT5, decreased after SH-4-54 treatment; moreover, the phosphorylation of both proteins were inhibited in dose-dependent manner. Using NGS, we obtained Mrna expression profiles in SH-4-54 treated K562 and K562R cells and identified differentially expressed mRNAs. Among these, STAT3 and STAT5 were markedly downregulated.Conclusion:SH-4-54 may overcome IM resistance and represent a promising novel approach to improve the outcome of CML.

KOENZİM Q0 İNSAN KRONİK MYELOİD LÖSEMİ K562 HÜCRELERİNİN PROLİFERASYONUNU ENGELLER VE MAPK VE AKT SİNYAL YOLAKLARINI MODÜLE EDER

Objective: This study evaluated the antiproliferative and pro-apoptotic effects of coenzyme Q0 (CoQ0) in human chronic myeloid leukemia K562 cell line. Material and Method: The cytotoxic effect of CoQ0 on human chronic myeloid leukemia cell line, K562 was determined by MTT test. The activity of caspase-3, expression of proteins involved in apoptosis, MAPK and AKT signaling pathways were determined with enzymatic assay and western blot analysis, respectively. Result and Discussion: Results showed that CoQ0 inhibited cell viability of K562 cells at 5 μM and higher concentrations and Bax protein expression was significantly decreased at 12.5 μM concentration of CoQ0. However, CoQ0 did not significantly affect caspase 3 activity and Bcl-2 protein expression. p-c-Raf (Ser259) protein expression was significantly decreased at 12.5 μM of CoQ0. Treatment with 10 μM of CoQ0 induced significantly phosphorylation of p38 MAPK and 12.5 μM CoQ0 caused a nonsignificant decrease in p-ERK1/2 protein expression in K562 cell line. Interestingly, in K562 cells, phosphorylation of Akt (Ser473) was diminished at 12.5 μM of CoQ0, with no change observed in p-Akt (Thr308) protein expression among groups. In conclusion, CoQ0 inhibited cell proliferation and suppressed phosphorylation of c-Raf (Ser259), Akt (Ser473), but not ERK1/2 in K562 cells. There is still a need for new insights into the anticancer mechanisms of CoQ0 and develop treatment strategies for chronic myeloid leukemia.

Modulating retinoid-X-receptor alpha (RXRA) expression sensitizes chronic myeloid leukemia cells to imatinib in vitro and reduces disease burden in vivo.

Introduction: The ligand-activated transcription factors, nuclear hormone receptors (NHRs), remain unexplored in hematological malignancies except for retinoic acid receptor alpha (RARA). Methods: Here we profiled the expression of various NHRs and their coregulators in Chronic myeloid leukemia (CML) cell lines and identified a significant differential expression pattern between inherently imatinib mesylate (IM)-sensitive and resistant cell lines. Results: Retinoid-X-receptor alpha (RXRA) was downregulated in CML cell lines inherently resistant to IM and in primary CML CD34+ cells. Pre-treatment with clinically relevant RXRA ligands improved sensitivity to IM in-vitro in both CML cell lines and primary CML cells. This combination effectively reduced the viability and colony-forming capacity of CML CD34+ cells in-vitro. In-vivo, this combination reduced leukemic burden and prolonged survival. Overexpression (OE) of RXRA inhibited proliferation and improved sensitivity to IM in-vitro. In-vivo, RXRA OE cells showed reduced engraftment of cells in the bone marrow, improved sensitivity to IM, and prolonged survival. Both RXRA OE and ligand treatment markedly reduced BCR::ABL1 downstream kinase activation, activating apoptotic cascades and improving sensitivity to IM. Importantly, RXRA OE also led to the disruption of the oxidative capacity of these cells. Conclusion: Combining IM with clinically available RXRA ligands could form an alternative treatment strategy in CML patients with suboptimal response to IM.

HDAC inhibitor chidamide overcomes drug resistance in chronic myeloid leukemia with the T315i mutation through the Akt-autophagy pathway.

Currently, therapy for Chronic Myeloid Leukemia (CML) patients with the T315I mutation is a major challenge in clinical practice due to its high degree of resistance to first- and second-generation Tyrosine Kinase Inhibitors (TKIs). Chidamide, a Histone Deacetylase Inhibitor (HDACi) drug, is currently used to treat peripheral T-cell lymphoma. In this study, we investigated the anti-leukemia effects of chidamide on the CML cell lines Ba/F3 P210 and Ba/F3 T315I and primary tumor cells from CML patients with the T315I mutation. The underlying mechanism was investigated, and we found that chidamide could inhibit Ba/F3 T315I cells at G0/G1 phase. Signaling pathway analysis showed that chidamide induced H3 acetylation, downregulated pAKT expression and upregulated pSTAT5 expression in Ba/F3 T315I cells. Additionally, we found that the antitumor effect of chidamide could be exerted by regulating the crosstalk between apoptosis and autophagy. When chidamide was used in combination with imatinib or nilotinib, the antitumor effects were enhanced compared with chidamide alone in Ba/F3 T315I and Ba/F3 P210 cells. Therefore, we conclude that chidamide may overcome T315I mutation-related drug resistance in CML patients and works efficiently if used in combination with TKIs.

Utilization of Dairy By-Products as a Source of Functional and Health Compounds-The Role of Ovine Colostrum and Milk Whey on Chronic Myeloid Leukemia Cells.

Nowadays, the search for food products that promote consumers' health has gained interest, and dairy by-products, due to their biological quality, could have a prominent position among products with health benefits. However, little is known about their activity on cancer cells. This study aimed to provide evidence about the effect of ovine colostrum and milk whey on K562 cells, a model of the human chronic myeloid leukemia cell line. The exposure of K562 cells to a single administration of sheep by-products at different concentrations for three days and three treatments for three days was carried out. Using a flow cytometric approach, we found that CD235a expression remained stable in the cells exposed to ovine whey (milk and colostrum) at concentrations ranging from 1 ng/mL to 100 μg/mL, after three days from one or three administrations, respectively. A significant reduction in fluorescent cells was observed in the populations exposed to 1 mg/mL of both milk and colostrum at the same time points. In these conditions, the size and granularity of the leukemic cells also changed, with a substantial reduction in the number of actively dividing cells in the S phase of the cell cycle. This phenomenon was highlighted by the Annexin V/PI cytofluorimetric test, which is able to provide quantitative results regarding the population of cells in early or late apoptosis or necrotic cells after exposure to a single dose or three doses of colostrum or sheep whey for three days, respectively. This report showed that both colostrum and milk whey were able to modify the phenotypic profile and cell cycle of the K562 cell line, inducing apoptosis at the highest concentration.

Cytotoxic and immunomodulatory potential of a novel [2-(4-(2,5-dimethyl-1H-pyrrol-1-yl)−1H-pyrazol-3-yl)pyridine] in myeloid leukemia

Cancer ranks among the leading causes of mortality worldwide. However, the efficacy of commercially available anticancer drugs is compromised by the emerging challenge of drug resistance. This study aimed to investigate the anticancer and immunomodulatory potential of a recently developed a novel [2-(4-(2,5-dimethyl-1 H-pyrrol-1-yl)− 1 H-pyrazol-3-yl) pyridine]. The cytotoxic potential of the compound was assessed using the MTT assay on both cancerous HL60 (acute myeloid leukemia) and K562 (chronic myeloid leukemia) cell lines, as well as non-cancerous Vero cells and human peripheral blood mononuclear cells (PBMCs). A clonogenic assay was employed to evaluate the anticancer efficacy of the compound, while flow cytometry was utilized to investigate its effect on cell cycle arrest. Furthermore, the immunomodulatory potential of the compound was assessed by quantifying inflammatory and anti-inflammatory biomarkers in the supernatant of PBMCs previously treated with the compound. Our study revealed that the novel pyridine ensemble exhibits selective cytotoxicity against HL60 (IC50 = 25.93 µg/mL) and K562 (IC50 = 10.42 µg/mL) cell lines, while displaying no significant cytotoxic effect on non-cancerous cells. In addition, the compound induced a decrease of 18% and 19% in the overall activity of COX-1 and COX-2, respectively. Concurrently, it upregulated the expression of cytokines including IL4, IL6, IL10, and IL12/23p40, while downregulating INFγ expression. These findings suggest that the compound has the potential to serve as a promising candidate for the treatment of acute and chronic myeloid leukemias due to its effective antiproliferative and immunomodulatory activities, without causing cytotoxicity in non-cancerous cells.