BCR-ABL Tyrosine Kinase Activity Research Articles

A novel fluorescent probe based imprinted polymer-coated magnetite for the detection of imatinib leukemia anti-cancer drug traces in human plasma samples

Myeloid leukemia is a chronic cancer, which associated with abnormal BCR-ABL tyrosine kinase activity. Imatinib (IMB) acts as a tyrosine kinase inhibitor and averts tumor growth in cancer cells by controlling cell division, so it is urgent to develop an effective assay to detect and monitor its IMB concentration. Therefore, an innovative fluorescent biomimetic sensor is a promising sensing material that constructed for the efficient recognition of IMB and displays excellent selectivity and sensitivity stemming from molecularly imprinted polymer@Fe3O4 (MIP@Fe3O4). The detection strategy depends on the recognition of IMB molecules at the imprinted sites in the presence of coexisting molecules, which are then transferred to the fluorescence signal. The synthesized MIP@Fe3O4 was characterized using Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Furthermore, computational studies of the band gap (EHOMO-ELUMO) of the monomers, IMB, and their complexes were performed. These results confirmed that the copolymer is the most appropriate and has high stability (Binding energy; 0.004 x 10-19 KJ) and low reactivity. A comprehensive linear response over IMB concentrations from 5 × 10-6 mol/L to 8 × 10-4 mol/L with a low detection limit of 9.3 × 10-7 mol/L was achieved. Furthermore, the proposed technique displayed long-term stability (over 2 months), high intermediate precision (RSD<2.1 %), good reproducibility (RSD <1.9 %), and outstanding selectivity toward IMB over analogous molecules with similar chemical and spatial structure (no interference by 100 to 150-fold of the competitors). Owing to these merits, the proposed fluorescence sensor was utilized to detect IMB in drug tablets and human plasma, and satisfactory results (99.3–100.4 %) were obtained. Thus, the synthesized fluorescence sensor is a promising platform for IMB sensing in various applications.

Synthesis of Novel Nilotinib Analogues and Biological Evaluation of Their Antiplatelet Activity and Functionality towards Cancer Cell Proliferation In Vitro.

Nilotinib, a second-generation tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia (CML), inhibits Bcr-Abl tyrosine kinase activity and proliferation of Bcr-Abl-expressing cells, as well as other malignancies. In the present study, new nilotinib analogues were synthesized and fully characterized. A platelet aggregation assay was performed, and the expression of P-selectin and PAC-1, as well as the effect on the proliferation of healthy endothelial cells, were evaluated. The expression and antimetastatic effects of E-cadherin and N-cadherin were assessed. The analogues inhibited platelet aggregation in a statistically significant manner compared to nilotinib, while they exhibited a strong inhibitory effect on P-selectin and PAC-1 expression when activated by AA. All three analogues caused arrest in the mitosis phase of the HepG2 cell cycle, while analogue-1 exhibited the most potent apoptotic effect compared to nilotinib. Interestingly, none of them promoted apoptosis in HUVECs. All the analogues reduced the expression of E- and N-cadherin in different amounts, while the analogues-1 and -3 exhibited similar antimigratory effects on HepG2 cells. The results of this study reveal considerable potential to develop new tyrosine kinase inhibitors with improved antiplatelet and antitumor properties.

Clinical efficacy and safety of flumatinib versus dasatinib combined with multi-drug chemotherapy in adults with Philadelphia-positive acute lymphoblastic leukemia

Introduction: Flumatinib, a highly selective ABL kinase inhibitor, exhibits stronger inhibition of intracellular BCR-ABL tyrosine kinase activity, compared to Imatinib. However, there is limited research comparing the real-world efficacy and safety of flumatinib and dasatinib in patients with Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Objective: Investigating the differences in therapeutic efficacy and safety between flumatinib and dasatinib in combination with multi-drug chemotherapy for the treatment of newly diagnosed Ph+ ALL. Method: In this study, we assessed 43 patients with newly diagnosed Ph+ ALL (20 in the flumatinib group, 23 in the dasatinib group). Results: There were no significant differences in gender, age, fusion gene type, initial blood routine, bone marrow blast cell ratio or chromosome karyotype between the two groups. Within 1 month, there were no significant differences in the complete response (CR), major molecular response (MMR) or minimal residual disease (MRD) negativity rate between the flumatinib and dasatinib groups. Similarly, within 3 months, there were no significant differences in CR or MMR rates between the two groups. However, the rates of complete molecular response (CMR) and MRD negativity within 3 months were significantly higher in the flumatinib group, compared to the dasatinib group (P < 0.05). Additionally, the flumatinib group exhibited fewer adverse reactions compared to the dasatinib group. Conclusion: These findings suggest that flumatinib is a safe and effective tyrosine kinase inhibitor (TKI) for achieving CMR and MRD negativity in patients with Ph+ ALL, as supported by this small series of patients.

AS1041, a novel derivative of marine natural compound Aspergiolide A, induces senescence of leukemia cells via oxidative stress-induced DNA damage and BCR-ABL degradation

Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of BCR-ABL tyrosine kinase. Imatinib was approved for CML therapy, however, BCR-ABL-dependent drug resistance, especially BCR-ABL-T315I mutation, restricts its clinical application. In this study, we reported anthraquinone lactone AS1041, a synthesized derivative of marine natural compound Aspergiolide A, showed anti-leukemia effect in vitro and in vivo by promoting cell senescence. Mechanistic study revealed the pro-senescence effect of AS1041 was dependent on oxidative stress-induced DNA damage, and the resultant activation of P53/P21 and P16INK4a/Rb. Also, AS1041 promoted ubiquitin proteasome system (UPS)-mediated BCR-ABL degradation, which also contributed to AS1041-induced senescence. In vivo, AS1041-induced senescence promoted tumor growth inhibition. In summary, the in vitro and in vivo antitumor effect of AS1041 suggests it can serve as a pro-senescence agent for alternative antileukemia therapy and imatinib-resistant cancer therapy by enhancing cellular oxidative stress and BCR-ABL degradation.

METHYLATION PATTERN OF THE RUNX3 GENE AND ITS IMPACT ON RESPONSE TO IMATINIB THERAPY IN CHRONIC MYELOID LEUKEMIA

BackgroundChronic Myelogenous Leukemia (CML) is a clonal stem cell disease with deregulated tyrosine kinase activity of BCR-ABL. DNA methylation in gene promoters influences disease development, therapeutic response, and clinical outcome in various diseases.&#x0D; ObjectivesThis study aimed to investigate the methylation pattern in promoter regions of the runt-related transcription factor 3 (RUNX3) gene and its effect on the response to imatinib therapy, among CML patients.&#x0D; Patients and MethodsBlood samples were collected from 100 participants from November 2020 to February 2022, in two groups 75 samples of CML patients and 25 samples of healthy controls, genomic DNA was extracted from the samples by using the salting-out method, then Isolated DNA was treated with sodium bisulfite. By Methylation-Sensitive High-Resolution Melting (MS-HRM), the samples were analyzed.&#x0D; ResultsThe promoter sequence methylation level is significantly higher (P&lt;0.05) in DNA samples from CML patients than in the control group, the methylation level in most of the healthy control samples 96% was detected between 0-24% while in CML patients was 16%, in 25-49%, RUNX3 methylation levels in Health individuals were 4%, moreover in CML patients were 24% also, in 50-100% of methylation level, health control was 0%, in CML patient showed 60%. According to methylation detection of RUNX3 among Imatinib resistance and good response, the results of IM resistance were detected between 0-40% was 15.21%, Furthermore, in good response was 79.31%. In other hand at 41-100% RUNX3 methylation levels in IM resistance was 84.78%, while a good response was recorded at 20.68%.&#x0D; ConclusionThe study concluded that RUNX3 gene hypermethylation often occurs in CML patients. Additionally, there is a strong correlation between RUNX3 hypermethylation and poor imatinib respondents. We found that both IM resistance and CML patients have higher levels of RUNX3 gene hypermethylation. Moreover, research on a larger sample size is required to validate these findings, which may serve as possible indicators of disease progression and imatinib resistance.

EICOSANOIDS AS HALLMARKS OF CHRONIC MYELOID LEUKEMIA PROGRESSION AND RESISTANCE TO TKI

Chronic myeloid leukemia (CML) is a Ph-positive myeloproliferative neoplasm giving rise to BCR-ABL1 oncogene. Usually, the disease is diagnosed in the chronic phase (CML-CP) with uncontrolled clonal expansion and high white blood cell count. Without therapy intervention, CML progress to advanced phases (accelerated and blastic phases; CML-AP). The Tyrosine Kinase Inhibitors (TKI) are the first-line therapeutic approach for CML. TKI targets the BCR-ABL tyrosine kinase activity and restrain the disease. The knowledge about the mechanisms involved in disease progression and resistance to TKI, beyond BCR-ABL1 , is limited. The relation among TKI treatment, disease progression and eicosanoids pattern in CML is unknown. Lipid mediators were quantified in healthy individuals (Controls; n = 9) as well as in CML patients in CP (n = 10), CML-AP (n = 13) and CML patients in molecular remission post-TKI therapy (RM; n = 6) using Liquid Chromatography/Mass Spectrometry (LC/MS). Mass spectral data were acquired with negative electrospray ionization. ProteinWizard was used to convert files to be used in bioinformatic subsequent analysis. Mummichog software was used for metabolic pathway enrichment analysis. Heatmaps were generated with the R package gplots and hierarchical clustering was performed with the R package amap with Spearman distance method and ward linkage algorithm. Volcano and bubble plots were obtained with the package ggplot2. We described the metabolomic profiles of CML in different phases and post-TKI therapy. The metabolic patterns were able to distinguish the CML patients in different phases and resistant to TKI. The metabolomic results suggested an association between reprogramming in lipid metabolism and CML progression and resistance to TKI therapy. CML patients showed to be a lower producer of some eicosanoids compared to controls. Interestingly, AP-CML produced less 12-HETE and EPA than CP-CML patients. CML patients in molecular remission produced more EPA in comparison with each other group. Taken together, the results established the eicosanoid profile of CML patients who progress to advanced phases and fail to respond to TKI.

Research Status, Synthesis and Clinical Application of Recently Marketed and Clinical BCR-ABL Inhibitors.

Tyrosine kinases expressed by BCR-ABL fusion genes can cause changes in cell proliferation, adhesion, and survival properties, which are the main causes of chronic myelogenous leukemia (CML). Inhibiting the activity of BCR-ABL tyrosine kinase has become one of the effective methods for the treatment of chronic myelogenous leukemia. Initially, imatinib was the first small molecule of BCR-ABL tyrosine kinases inhibitors (TKIs) for the effective treatment of chronic myelogenous leukemia. Later, due to the emergence of various BCR-ABL mutations, especially T315I mutation, imatinib developed strong resistance. The second-generation kinase inhibitors dasatinib and nilotinib were able to overcome most of the mutation resistance but not T315I mutations. Therefore, in order to further overcome the problem of drug resistance, new types of KTIs such as flumatinib and radotinib have been developed, providing more options for clinical treatment. Some new drugs have entered clinical trials. In this review, two new BCRABL inhibitors (flumatinib and radotinib) and five new BCR-ABL inhibitors have been introduced into the clinical market in recent years. We reviewed their research status, synthesis methods, and clinical applications.

Bioactive Lipids as Chronic Myeloid Leukemia's Potential Biomarkers for Disease Progression and Response to Tyrosine Kinase Inhibitors.

Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that expresses the Philadelphia chromosome and constitutively activated Bcr-Abl tyrosine kinase in hematopoietic progenitor cells. Bcr-Abl tyrosine-kinase inhibitors (TKI) do not definitively cure all CML patients. The efficacy of TKI is reduced in CML patients in the blastic phase—the most severe phase of the disease—and resistance to this drug has emerged. There is limited knowledge on the underlying mechanisms of disease progression and resistance to TKI beyond BCR-ABL1, as well as on the impact of TKI treatment and disease progression on the metabolome of CML patients. The present study reports the metabolomic profiles of CML patients at different phases of the disease treated with TKI. The plasma metabolites from CML patients were analyzed using liquid chromatography, mass spectrometry, and bioinformatics. Distinct metabolic patterns were identified for CML patients at different phases of the disease and for those who were resistant to TKI. The lipid metabolism in CML patients at advanced phases and TKI-resistant patients is reprogrammed, as detected by analysis of metabolomic data. CML patients who were responsive and resistant to TKI therapy exhibited distinct enriched pathways. In addition, ceramide levels were higher and sphingomyelin levels were lower in resistant patients compared with control and CML groups. Taken together, the results here reported established metabolic profiles of CML patients who progressed to advanced phases of the disease and failed to respond to TKI therapy as well as patients in remission. In the future, an expanded study on CML metabolomics may provide new potential prognostic markers for disease progression and response to therapy.

Effect of HSP90AB1 and CC domain interaction on Bcr-Abl protein cytoplasm localization and function in chronic myeloid leukemia cells

BackgroundThe fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). After moving into the nucleus, the fusion protein can induce apoptosis of CML cells. The coiled-coil domain (CC domain) of Bcr-Abl protein plays a central role in the subcellular localization. However, how CC domain affects subcellular localization of Bcr-Abl remains unclear.MethodsHerein, the key proteins interacting with the Bcr-Abl CC domain were screened by immunoprecipitation binding mass spectrometry. The specific site of Bcr-Abl CC domain binding to target protein was predicted by Deep Viewer. Immunoprecipitation assay was used to confirmed the specific sites of protein binding. IF and western blot were used to observe the subcellular localization of target protein. Western blot was used to examine the protein changes. CCK-8, clonal formation test and FCM cycle detection were used to observe the effect of inhibitor on the proliferation ability of CML cells. FCM apoptosis detection was used to observe the level of cells apoptosis.ResultsHSP90AB1 interacts with Bcr-Abl CC domain via N-terminal domain (NTD), preventing the transport of Bcr-Abl protein to the nucleus and maintaining the activation of Bcr-Abl tyrosine kinase. The nucleus-entrapped Bcr-Abl markedly inhibits the proliferation and induces apoptosis of CML cells by activating p73 and repressing the expression of cytoplasmic oncogenic signaling pathways mediated by Bcr-Abl. Moreover, the combination of 17AAG (Tanespimycin) with Leptomycin B (LMB) considerably decreased the proliferation of CML cells.ConclusionOur study provides evidence that it is feasible to transport Bcr-Abl into the nucleus as an alternative strategy for the treatment of CML, and targeting the NTD of HSP90AB1 to inhibit the interaction with Bcr-Abl is more accurate for the development and application of HSP90 inhibitor in the treatment of CML and other Bcr-Abl-addicted malignancies.F7yTRqYkfkXw_k-2eiVFmgVideo abstract

Combinational Treatment of Imatinib and Allicin Synergistically Inhibits the Growth of BCR-ABL-Positive Leukemia and the Emergence of Drug-Resistant Cells

Imatinib mesylate (Gleevec, Glivec, STI571) is used as a first-line therapy for chronic myeloid leukemia (CML) to inhibit the tyrosine kinase activity of BCR-ABL. Although imatinib has greatly improved CML patient prognosis, imatinib-resistant cells often develop in about 30-40% of patients after treatment, causing major therapeutic failure. Several second and third generation tyrosine kinase inhibitors, such as dasatinib, nilotinib and ponatinib, have been developed, but none of them are able to completely block the development of drug-resistance. Therefore, new therapeutic approaches are in need to prevent or reduce the emergence of drug-resistant cells in the early stages of imatinib therapy.In this study, we show that the combinational treatment of allicin, an active component in garlic, and imatinib effectively blocks imatinib-resistance in K562 BCR-ABL-positive leukemia cells. Both imatinib and allicin had dose-dependent effects on cell viability during the first 3 days of treatment. However, the growth inhibitory effect did not last and resulted in re-growth of cells when treated with allicin alone (2 microgram/ml) or 200 nM of imatinib, which is close to the IC50 value in our K562 cells determined on day 3. In contrast, the combination of allicin with imatinib still inhibited cell growth by day 6. Wright-Giemsa stainings showed that the combinational treatment of imatinib and allicin also caused erythroid differentiation.To identify the molecular mechanisms of imatinib and allicin's actions, we analyzed RNA expression after single and combinational treatments with imatinib and allicin. The results demonstrated that both imatinib and allicin increased the expression of alpha -globin and gamma -globin. The expression of glycophorin A (GYPA), a marker of erythroid differentiation, was synergistically increased by the combinational treatment of imatinib and allicin. One of the notable changes we observed was a decrease of p21 (cyclin-dependent kinase inhibitor 1A; CDKN1A) expression when cells were treated with the IC50 dosage of imatinib. Surprisingly, when allicin was treated together with imatinib, the p21 decrease by imatinib was completely inhibited. Allicin alone did not increase p21, suggesting that allicin does not control p21 expression directly, but instead antagonizes the action of imatinib downregulating p21.More importantly, a long-term treatment of K562 cells with imatinib and allicin showed that allicin was able to completely block the emergence of imatinib-resistant cells in vitro. Viable cells started to grow again within 12 days when treated with imatinib or allicin alone. However, no resistant cells were observed after up to 40 days when cells were cultured with the combinational treatment.Taken together, the combinational treatment of imatinib and allicin has a synergistic effect on blocking leukemia cell expansion as they enhance erythroid differentiation and prevent cell cycle progression of residual resistant cells by attenuating the negative effects of imatinib on p21 expression. Our findings suggest that combining imatinib and allicin could be a potential therapeutic to prevent drug-resistance in BCR-ABL-positive leukemia. DisclosuresNo relevant conflicts of interest to declare.

Blast MRD CML 1 Trial: Blockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease (MRD) in Chronic Myeloid Leukemia (CML)- a Phase II Study of Adding the Anti-PD-1 Pembrolizumab to Tyrosine Kinase Inhibitors in Patients with Chronic Myeloid Leukemia and Persistently Detectable Minimal Residual Disease: A Trial of the ECOG-ACRIN Cancer Research Group (EA9171)

Blast MRD CML 1 Trial: Blockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease (MRD) in Chronic Myeloid Leukemia (CML)- a Phase II Study of Adding the Anti-PD-1 Pembrolizumab to Tyrosine Kinase Inhibitors in Patients with Chronic Myeloid Leukemia and Persistently Detectable Minimal Residual Disease: A Trial of the ECOG-ACRIN Cancer Research Group (EA9171)

P19INK4d inhibits proliferation and enhances imatinib efficacy through BCR-ABL signaling pathway in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a kind of myeloproliferative disorder caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs), imatinib and its derivatives, have achieved great progress in the treatment of CML. However, many CML patients do not respond to TKIs alone. p19INK4d, a cyclin-dependent kinase inhibitor, plays important roles in proliferation, DNA damage repair, apoptosis and cell differentiation, but its role in CML is unknown. Herein, we found that the expression of p19INK4d in CML patients was significantly lower than that in healthy controls. p19INK4d overexpression inhibits cell proliferation through cell cycle arrest, and cooperates with imatinib to inhibit CML more effectively in vitro and in vivo. Mechanistically, p19INK4d decreased the expression of BCR-ABL and its downstream molecules p-Mek1/2, moreover, the expression of Gli-1, c-myc, MUC1, Shh and TC48 also reduced significantly. Collectively, p19INK4d inhibits proliferation and enhances imatinib efficacy in the treatment of CML. These findings maybe have implications for developing potential targets to increase imatinib sensitivity for CML.

Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence, proliferation and TKI resistance.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR‐ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR‐ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1‐mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell‐mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.

Review on Pathophysiological and Pharmacotherapeutic approach on Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell malignant clonal disorder resulting in elevation of erythroid cells and platelets in peripheral blood and clear myeloid hyperplasia in the bone marrow. Philadelphia (Ph) chromosome detection forms the major diagnosis in CML. It displays the translocation t(9; 22) that generates the oncogene bcr/abl. Constitutive tyrosine kinase activity is been exhibited by the oncoprotein (BCR/ABL) which supports growth and continued existence in cells of CML. Incidence remains almost constant worldwide and it is the most commonest in India majorly in adults. Clinically, Chronic Phase (CP), Accelerated Phase (AP), and Blast Phase (BP) are the three phases of CML. The drugs used in the treatment of CML are known as Tyrosine Kinase Inhibitors (TKI'S). The primary drug which directly target BCR-ABL tyrosine kinase activity was Imatinib. Imatinib resistance is the major drawback of this drug due to mutations. Imatinib binding is prevented by BCR-ABL kinase domain mutations, BCR-ABL gene amplification or over-expression, clonal progression, and reduced bioavailability of imatinib or cell exposure. Stem cell transplantation is the only curative approach till date. In patients who cannot undergo transplantation, the BCR-ABL tyrosine kinase inhibitor STI571 (Gleevec, Imatinib), immune therapy or interferon-alpha and other cytoprotective drugs are used. Currently available data shows that Imatinib is a better compound compared to second line drugs in producing complete molecular and cytogenetic responses. The resistance against STI571 has been reported which has to be tested with mutational analysis for the treatment for using an accurate regimen. Current researchers are consequently challenging to inhibit or work against STI571 resistance by the development of new molecules or combination of molecules contributing for further improvement in the treatment of CML and for better understanding of resistant mechanisms. These strategies, whether will guide to curative treatment in CML in the future remains unclear.

MiR-96 acts as a tumor suppressor via targeting the BCR-ABL1 oncogene in chronic myeloid leukemia blastic transformation

MicroRNA-mediated posttranscriptional regulation is an important epigenetic regulatory mechanism of gene expression, and its dysregulation is involved in the development and progression of a variety of malignancies, including chronic myeloid leukemia (CML). The BCR-ABL1 fusion gene is not only the initiating factor of CML, but it is also an important driving factor for blastic transformation. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 tyrosine kinase activity, represented by imatinib, are currently the first-line treatment for CML. However, due to primary resistance or secondary resistance caused by mutations in the BCR-ABL1 kinase domain, TKIs cannot completely prevent the progression of CML; thus, the study of BCR-ABL1 gene expression regulation is of great significance. In this study, bioinformatics analysis and our results showed that miR-96 could directly bind to the 3'UTR region of BCR-ABL1 to regulate fusion protein expression, thereby regulating its downstream signaling pathway activity. We also found that miR-96 was downregulated during the progression from the chronic phase (CML-CP) to the blast crisis (CML-BC). Downregulation of miR-96 could promote the proliferation and participate in the cell differentiation of CML-BC cells. Additionally, we found that the novel histone deacetylase drug chidamide and the DNA methyltransferase inhibitor decitabine could restore the low expression of miR-96 in CML cells, and there were two abnormal hypermethylated sites in the promoter region of miR-96 in CML, suggesting that its low expression might be at least partially regulated by epigenetic mechanisms. In addition, re-expression of miR-96 could increase the sensitivity of CML-BC cells to imatinib. Thus, miR-96 functions as a tumor suppressor, and re-expression of this microRNA might have therapeutic benefits in CML blastic transformation.

PF398 KNOCKDOWN OF LASP1 IN CXCR4 EXPRESSING CML CELLS PROMOTES CELL PERSISTENCE, PROLIFERATION AND TKI RESISTANCE

Background:Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by a fusion protein, the constitutively active BCR–ABL tyrosine kinase, leading to expansion of differentiated granulocytes. Although tyrosine kinase inhibitors (TKI) have significantly improved patient care, minimal residual disease (MRD) remains a clinical challenge. Recently, the LIM and SH3 domain protein 1 (LASP1) has been identified as a novel BCR–ABL substrate, which is hyperphosphorylated at Y171 in CML cells. LASP1 is associated with proliferation, migration, tumorigenesis and chemoresistance. Furthermore, the protein can bind to the chemokine receptor 4 (CXCR4), known to be involved in homing of leukemic stem cells and relapse of CML patients.Aims:These two LASP1‐influenced disease promoting effects prompted us to investigate the effect of LASP1 and CXCR4 on processes involved in initiation, progression and maintenance of CML in order to identify and exploit BCR‐ABL‐independent pathways and other extrinsic factors, which may help to eradicate MRD.Methods:Using a CRISPR/Cas9‐based LASP1 knockout system and a lentivirus‐mediated CXCR4 overexpression in the CML cell line K562, the biological influence of CXCR4 and LASP1 was characterized in apoptosis, viability, cell cycle, migration and adhesion assays in the presence of nilotinib. Furthermore, comprehensive transcriptomic microarray data from a public database, immunofluorescence imaging and biochemical analysis were employed to elucidate the roles of LASP1 in CML.Results:Microarray analysis revealed that LASP1 is significantly lower expressed in samples of CML patients not responding to imatinib therapy. The in vitro data indicate that not only the overexpression of CXCR4, but also knockdown of LASP1 contributed to proliferation, reduced apoptosis and impaired cell migration. Additionally, we show that LASP1 depletion leads to decreased cytokine release and reduced natural killer (NK)‐cell mediated cytotoxicity. Finally, we describe a phosphorylation‐dependent loss of direct binding between pLASP1‐Y171 and CXCR4 in CML, which most likely results in receptor destabilization.Summary/Conclusion:Taken together, our results indicate that in CML, reduced levels of LASP1 alone and especially in combination with high CXCR4 expression contribute to TKI resistance and stem cell persistence.

A Search for the Protonation Model with Thermodynamic Dissociation Constants and (Extra)-Thermodynamics of Nilotinib Hydrochloride (TASIGNA)

Nilotinib hydrochloride (AMN107, TASIGNA, Novartis) is used to treat adults with chronic myeloid leukemia (CML), a type of leukemia. It is a novel, orally active BCR-ABL tyrosine kinase inhibitor derived from aminopyrimidine that is 30 times more effective against CML cells than is Imatinib. The nonlinear regression of the A versus pH spectra with REACTLAB and SQUAD84 and of the pH-titration curve with ESAB determined the four close and consecutive dissociation constants in the 11 steps of the newly proposed procedure. Prediction of pKa performed by MARVIN, PALLAS and ACD/Percepta determined the protonation sites. The sparingly soluble nilotinib hydrochloride denoted as L forms four water-soluble LH+, $${\text{LH}}_{2}^{2 + }$$ , $${\text{LH}}_{3}^{3 + }$$ , and $${\text{LH}}_{4}^{4 + }$$ cations. Although the adjusted pH has less effect on the absorbance changes in the chromophore, four thermodynamic dissociation constants were reliably determined: $${\text{p}}K_{\text{a1}}^{\text{T}}$$ = 3.60 ± 0.04, $${\text{p}}K_{\text{a2}}^{\text{T}}$$ = 4.42 ± 0.07, $${\text{p}}K_{\text{a3}}^{\text{T}}$$ = 4.71 ± 0.04, and $${\text{p}}K_{\text{a4}}^{\text{T}}$$ = 4.84 ± 0.03 at 25 °C and $${\text{p}}K_{\text{a1}}^{\text{T}}$$ = 3.61 ± 0.11, $${\text{p}}K_{\text{a2}}^{\text{T}}$$ = 4.29 ± 0.18, $${\text{p}}K_{\text{a3}}^{\text{T}}$$ = 4.49 ± 0.02, and $${\text{p}}K_{\text{a4}}^{\text{T}}$$ = 5.05 ± 0.03 at 37 °C, and by regression analysis of potentiometric titration curves with ESAB, $${\text{p}}K_{\text{a1}}^{\text{T}}$$ = 3.74 ± 0.01, $${\text{p}}K_{\text{a2}}^{\text{T}}$$ = 4.05 ± 0.01, $${\text{p}}K_{\text{a3}}^{\text{T}}$$ = 4.25 ± 0.01, and $${\text{p}}K_{\text{a4}}^{\text{T}}$$ = 4.91 ± 0.20 at 25 °C and $${\text{p}}K_{\text{a1}}^{\text{T}}$$ = 3.63 ± 0.03, $${\text{p}}K_{\text{a2}}^{\text{T}}$$ = 3.96 ± 0.03, $${\text{p}}K_{\text{a3}}^{\text{T}}$$ = 4.18 ± 0.03, and $${\text{p}}K_{4 1}^{\text{T}}$$ = 4.81 ± 0.05 at 37 °C. Positive enthalpy values ΔH° at 25 °C showed that the dissociation process is endothermic and is accompanied by heat absorption. Inasmuch as the entropy values of the dissociation process ΔS° at 25 °C and 37 °C were negative, the dissociation process is reversible.

Monitoring the chronic myeloid leukemia patients between 2008 and 2018; the experience of the Hematology and Bone Marrow Transplantation Unit Târgu-Mureș

Chronic myeloid leukemia is a clonal myeloproliferative disorder characterized by the BCR-ABL gene rearrangement with translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase inhibitor became the standard frontline therapy. The molecular monitoring is essential. We studied the chronic myeloid leukemia patients at the Clinical Hematology and Bone Marrow Transplant Unit Tg-Mures between 2008 and 2018. We followed 59 patients, median age of 45 years, female:male ratio 1.5:1. 80% of the patients were in chronic phase. Sokal score was low in 61%, intermediate 27% and high in 12% of the patients. The median follow-up time was 5 years and 9 months. 59% of the patients reached molecular remission (average time 11 months). The cumulative overall survival was 80% at 5 years and 76% at 10 years. The overall survival according to disease phase was 98%, 85%, 20%; according to Sokal score it was 91%, 66%, 51%. The cumulative progression-free survival was 75% at 5 years and 50% at 10 years. Only 8% of the low risk patients are progressing opposite to 77% of the high risk patients. The cumulative probability to maintain the molecular remission for 5 years is 100%, for 10 years 91% and for 15 years 52%. A rising level of BCR-ABL is an early indication of the loss of response identifying the patients who need close monitoring and therapeutic change. Orv Hetil. 2019; 160(2): 67-72.

Expression analysis of Akirin-2, NFκB-p65 and β-catenin proteins in imatinib resistance of chronic myeloid leukemia

ABSTRACTObjective: Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and β-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and β-catenin in imatinib resistance of CML and to test if any direct physical protein–protein interaction exists between NFkB and both β-catenin and Akirin-2.Methods: RT–PCR and western blot were performed to determine Akirin-2, NFκB-p65 and β-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and β-catenin proteins to MDR1 promoter region, respectively.Results: β-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein–protein interaction could not be found between NFκB-p65 and Akirin-2 or β-catenin proteins. Nuclear β-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells.Discussion: We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and β-catenin genes may provide an opportunity to overcome imatinib resistance in CML.

Taxodione induces apoptosis in BCR-ABL-positive cells through ROS generation

Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are hematopoietic malignancies caused by the constitutive activation of BCR-ABL tyrosine kinase. Although direct BCR-ABL inhibitors, such as imatinib, were initially successful in the treatment of leukemia, many patients developed drug resistance over time due to the gatekeeper mutation of BCR-ABL T315I. In the present study, we found that taxodione, a quinone methide diterpene isolated from Taxodium distichum, significantly induced apoptosis in human myelogenous leukemia-derived K562 cells, which were transformed by BCR-ABL. Taxodione reduced the activities of mitochondrial respiratory chain (MRC) complexes III and V, which appeared to induce the production of reactive oxygen species (ROS). N-acetylcysteine (NAC), an antioxidant agent, canceled taxodione-induced ROS production, reductions in MRC activities, particularly complex V, and apoptotic cell death. Furthermore, in K562 cells treated with taxodione, BCR-ABL and its major signaling molecules, such as STAT5 and Akt were sequestered in mitochondrial fraction, and their localization changes decrease their abilities to stimulate cell proliferation, suggesting that these actions seem to be a mechanism how taxodione functions as an anti-tumor drug. Strikingly, NAC canceled these taxodione-caused anti-cancer effects. Taxodione induced apoptosis in transformed Ba/F3 cells induced not only by BCR-ABL, but also T315I-mutated BCR-ABL through the generation of ROS. Collectively, the present results suggest that in the treatment of leukemia, taxodione has potential as a compound with high efficacy to overcome BCR-ABL T315I mutation-mediated resistance in leukemia cells.