BCR-ABL Expression Research Articles

Hematological profile and its correlation with reverse transcription–polymerase chain reaction-based serial quantitative detection of BCR-ABL in chronic myeloid leukemia patients at a referral cancer center

Abstract BACKGROUND: There are variations in the hematological profile among the Indian population affected with chronic myeloid leukemia (CML). The range of white blood cell count (WBC) was 0.46 × 109/mm3 to 1.86 × 109/mm3. Similarly, range for hemoglobin was 9–11 g/dl. WBC count can be as low as 0.18 × 109/mm3 even though CML is a myeloproliferative disorder. OBJECTIVE: The objective of this study was to correlate BCR-ABL expression with the hematological and myeloid proliferative parameters. METHODS: The present study was a single-center, hospital-based follow-up study among 66 cases of CML. The sample of patients was analyzed for BCR-ABL gene expression; hematological parameters and myeloid proliferative parameters such as band forms, myelocytes, metamyelocytes, and blast were also measured. Some patients reported twice, some thrice, and the total count after all follow-up for all 66 cases was 179. RESULTS: The mean age was 43.15 years. Men were more than women with male-to-female ratio of 1.6:1. The mean hemoglobin was lesser at 10.8 g/dl. Median total count was normal at 5610 (interquartile range = 2750) per μl. Mean neutrophils, lymphocytes, monocytes, basophils, and platelets were normal. The eosinophil count was raised. The BCR-ABL gene expression was positively and significantly correlated with total count. It was negatively and significantly correlated with lymphocytes and monocytes. It was not correlated with other hematological parameters. All the myeloid proliferative parameters, namely, band forms, myelocytes, metamyelocytes, and blast, were positively and significantly correlated with BCR-ABL gene expression. CONCLUSION: Total count and all myeloid proliferative parameters were positively and significantly correlated with BCR-ABL gene expression. Lymphocytes and monocytes were negatively correlated. These parameters can be used to track the disease progression in patients with CML.

Combined targeting of GPX4 and BCR-ABL tyrosine kinase selectively compromises BCR-ABL+ leukemia stem cells

BackgroundIn the ongoing battle against BCR-ABL+ leukemia, despite significant advances with tyrosine kinase inhibitors (TKIs), the persistent challenges of drug resistance and the enduring presence of leukemic stem cells (LSCs) remain formidable barriers to achieving a cure.MethodsIn this study, we demonstrated that Disulfiram (DSF) induces ferroptosis to synergize with TKIs in inhibiting BCR-ABL+ cells, particularly targeting resistant cells and LSCs, using cell models, mouse models, and primary cells from patients. We elucidated the mechanism by which DSF promotes GPX4 degradation to induce ferroptosis through immunofluorescence, co-immunoprecipitation (CO-IP), RNA sequencing, lipid peroxidation assays, and rescue experiments.ResultsHere, we present compelling evidence elucidating the sensitivity of DSF, an USA FDA-approved drug for alcohol dependence, towards BCR-ABL+ cells. Our findings underscore DSF’s ability to selectively induce a potent cytotoxic effect on BCR-ABL+ cell lines and effectively inhibit primary BCR-ABL+ leukemia cells. Crucially, the combined treatment of DSF with TKIs selectively eradicates TKI-insensitive stem cells and resistant cells. Of particular note is DSF’s capacity to disrupt GPX4 stability, elevate the labile iron pool, and intensify lipid peroxidation, ultimately leading to ferroptotic cell death. Our investigation shows that BCR-ABL expression induces alterations in cellular iron metabolism and increases GPX4 expression. Additionally, we demonstrate the indispensability of GPX4 for LSC development and the initiation/maintenance of BCR-ABL+ leukemia. Mechanical analysis further elucidates DSF’s capacity to overcome resistance by reducing GPX4 levels through the disruption of its binding with HSPA8, thereby promoting STUB1-mediated GPX4 ubiquitination and subsequent proteasomal degradation. Furthermore, the combined treatment of DSF with TKIs effectively targets both BCR-ABL+ blast cells and drug-insensitive LSCs, conferring a significant survival advantage in mouse models.ConclusionIn summary, the dual inhibition of GPX4 and BCR-ABL presents a promising therapeutic strategy to synergistically target blast cells and drug-insensitive LSCs in patients, offering potential avenues for advancing leukemia treatment.

Study of TRAIL and SAHA Co-Treatment on Leukemia K562 Cell Line.

TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) is an attractive agent being considered a potential cancer treatment. It attaches to its death receptors, leading many cancer cells to apoptosis. However, some malignancies indicate substantial resistance to TRAIL, challenging anticancer scientists. Herein, combination therapy with TRAIL plus SAHA (Suberoyl Anilide Hydroxamic Acid) was conducted to evaluate the capability of SAHA to overcome TRAIL resistance in the leukemia K562 cell line. First, the IC50 for SAHA was calculated (2 µM) at 12, 24, 48, and 72 h of treatment using MTT assay. Second, the K562 cells were treated with concentrations of 50 and 100 nM of TRAIL and 2 μM of SAHA separately and together for 24, 48, and 72 h and the survival of these cells was evaluated by Flowcytometry following the annexin-V and PI staining. To demonstrate the non-toxicity of the combined treatment for normal cells, the HEK-293 cell line was treated with the TRAIL 100 nM and SAHA 2 μM combined and separated at the same periods. In the end, by performing real-time PCR, the amount of candidate genes' expression implicated in TRAIL resistance, and the levels of BCR-ABL expression was measured. The drug dosages were not toxic to normal cells. SAHA plus TRAIL strongly triggered apoptosis in K562 cells after 24, 48, and 72 h of exposure. Furthermore, it was shown that DR4, DR5, and CHOP expressions were enhanced, and PI3K, Akt, ERK, STAT3, c-FLIPL, NF-κB, and BCR-ABL expressions were decreased by SAHA in K562 cells. Our study indicated that SAHA combined with TRAIL can increase the sensitivity of K562 leukemic cells to TRAIL by suppressing intracellular anti-apoptotic molecules and augmenting the expressions of DR4/DR5 and CHOP.

Bcr-Abl Inhibitory Activities of Imatinib Derivatives

Background: Imatinib, a frontline targeted-therapeutic agent for patients with chronic myelogenous leukemia (CML), was a synthetic tyrosine kinase inhibitor approved by the US Food and Drug Administration. Objective: To expand the structural diversity of Bcr-Abl inhibitors, we synthesized nine novel imatinib analogues (1a-1i) and evaluated their cytostatic effects against human cancer cell lines in vitro. Method: Imatinib and its analogues were successfully synthesized by an improved method in six main steps. Inhibitory activities of all compounds were evaluated on K562, HL-60, MCF-7, A549 and PBMC in vitro. Then, the effect of the most active compound was studied using flow cytometer, real-time qPCR and western blot experiments to determine its mechanism action. Finally, the molecular docking of the most active compound were determined. Results: The IC50 of one imatinib analogue (compound 1h) for K562 and HL-60 was lower than imatinib itself. Further studies indicate that the pro-apoptotic effect of compound 1h on K562 cells was stronger than imatinib over a range of concentrations. Importantly, the real-time qPCR and western blot experiments showed that compound 1h was superior to imatinib in inhibiting Bcr-Abl expression. The structure-activity relationship was analyzed by determining the inhibitory rate of each imatinib analogue. Introducing benzene (A ring) and piperidine (E ring) rings instead of pyridine (A ring) and piperazine (E ring) in imatinib significantly enhanced the potency of imatinib against K562 and HL-60 cell lines, which is consistent with the docking results. Conclusion: Imatinib analogue 1h showed a better inhibitory effect on leukemia cell lines than other cell lines, which was consistent with the imatinib-like structure, moreover, had little effect on PBMC. Overall, we conclude that compound 1h has the potential to treat chronic myeloid leukemia.

The interplay of knowledge, motivation, and treatment response in medication adherence among patients with chronic myeloid leukemia treated with Imatinib

Chronic Myeloid Leukemia (CML) requires consistent medication adherence to Imatinib (IM) for optimal outcomes, however, adherence to oral chemotherapy is challenging. This observational study explores the relationship between patient knowledge, motivation, and adherence to IM therapy, and their collective impact on clinical outcomes. A prospective, observational study was conducted with 101 CML patients. The 6-Item Morisky Medication Adherence Scale (MMAS-6) was used to assess adherence, motivation, and knowledge levels. The study found that high motivation was significantly associated with lower BCR-ABL expression (p = 0.025). Patients with high knowledge and motivation had a 71% favorable response rate, compared to 0% in those with low knowledge and motivation (p = 0.01). As conclusion both patient motivation and knowledge are crucial for favorable treatment outcomes in CML. High levels of both significantly correlate with better clinical responses. Tailored interventions to enhance patient knowledge and motivation are essential.

Abstract 670: Preclinical candidate LAE120, a novel selective USP1 inhibitor shows effective anticancer and combination activity with PARP inhibitor

Abstract The Fanconi anemia (FA) and translesion synthesis (TLS) are DNA damage tolerance and repair pathways that have been found to be regulated by reversible ubiquitination in which ubiquitin-specific protease 1 (USP1) plays a key role. Numerous studies have indicated that USP1 inhibitors have therapeutic effects on tumors with homologous recombination deficiency (HRD), including mutations in BRCA1/2. Due to the emergence of resistance to PARP inhibitors in clinical applications, new strategies are needed to address this issue. Here we present LAE120, a novel, allosteric and highly potent USP1 inhibitor, displayed monotherapy potency and combination activity with PARP inhibitor in HRD cancers. LAE120 has a unique tail group distinct from all the published USP1 inhibitors and is predicted to induce a different conformational change at the USP1 allosteric site. In the biochemical assay, LAE120 strongly inhibited USP1 enzymatic activity with IC50 of 5.6 nM. LAE120 potently inhibited proliferation of BRCA-mutated MDA-MB-436 breast cancer cell (IC50:19 nM) and exhibited anti-proliferative activity in NCI-H1792 (IC50: 69 nM) and K562 (IC50: 38 nM) cell lines. In the xenograft model of MDA-MB-436 cells with BRCA mutations, LAE120 demonstrated robust single-agent tumor inhibitory activity (TGI: 88%, 10 mg/kg BID) and a favorable dose-dependent response (TGI: 106%, 25 mg/kg BID). We also observed strong tumor-inhibition efficacy of LAE120 as a single agent in the K562 xenograft model with Bcr-Abl expression (TGI: 86%, 10 mg/kg BID). Current research indicates that USP1 inhibitors exhibit synergistic activity in combination with PARP inhibitors and re-sensitizes PARP refractory tumors. Similar results have been confirmed in the xenograft model of BRCA-mutated MDA-MB-436 cells. The combination of LAE120 and Olaparib (PARP inhibitor) yielded a more robust and durable anti-tumor efficacy at low dose (TGI: 110%, LAE120, 5 mg/kg BID + Olaparib, 50 mg/kg QD). LAE120 also displayed favorable ADME properties and PK profiles. Preclinical-tox studies indicated the molecule was well tolerated without significant toxicities and hematological observations. Both in vitro and in vivo data strongly support further investigation of LAE120, whether as a single agent or in combination therapy. Citation Format: Jintao Wang, Yan Chen, Junyan Chen, Ling Jiang, Xiaofen Lin, Chaojun Cai, Minhua Zhang, Ming Li, Justin Gu. Preclinical candidate LAE120, a novel selective USP1 inhibitor shows effective anticancer and combination activity with PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 670.

Zinc as a potential regulator of the BCR-ABL oncogene in chronic myelocytic leukemia cells

BackgroundGenerally, decreased zinc in the serum of tumor patients but increased zinc in tumor cells can be observed. However, the role of zinc homeostasis in myeloid leukemia remains elusive. BCR-ABL is essential for the initiation, maintenance, and progression of chronic myelocytic leukemia (CML). We are currently investigating the association between zinc homeostasis and CML. MethodsGenes involved in zinc homeostasis were examined using three GEO datasets. Western blotting and qPCR were used to investigate the effects of zinc depletion on BCR-ABL expression. Furthermore, the effect of TPEN on BCR-ABL promoter activity was determined using the dual-luciferase reporter assay. MRNA stability and protein stability of BCR-ABL were assessed using actinomycin D and cycloheximide. ResultsTranscriptome data mining revealed that zinc homeostasis-related genes were associated with CML progression and drug resistance. Several zinc homeostasis genes were affected by TPEN. Additionally, we found that zinc depletion by TPEN decreased BCR-ABL mRNA stability and transcriptional activity in K562 CML cells. Zinc supplementation and sodium nitroprusside treatment reversed BCR-ABL downregulation by TPEN, suggesting zinc- and nitric oxide-dependent mechanisms. ConclusionOur in vitro findings may help to understand the role of zinc homeostasis in BCR-ABL regulation and thus highlight the importance of zinc homeostasis in CML.

Sorafenib combination with imatinib enhances apoptotic and cell cycle arrest effects with no effect on viability and BCR-ABL expression of K-562 cells

Chronic myeloid leukemia (CML) is one of the myeloproliferative disorders that affect hematopoiesis. Historically, many therapies have been used for the management of this disease with moderate response. The discovery of tyrosine kinase inhibitors (TKIs), marked by the introduction of imatinib, revolutionized the therapy of CML. Resistance to imatinib, however, marks CML cells in refractory disease. Additionally, advanced stages of CML may still be refractory to available therapies. Sorafenib is an inhibitor of multiple intracellular and receptor protein kinases that is approved by the FDA for the treatment of advanced hepatocellular carcinoma patients and renal cell cancer. Other TKIs, such as erlotinib and sunitinib, are under study for the treatment of CML patients. In this study, the synergistic effect of the combination of imatinib and sorafenib on the K-562 CML cell line was evaluated. We used viability assay, cell cycle analysis, apoptosis assay, reverse transcription quantitative real-time PCR detect the expression of Breakpoint Cluster Region – abelson fusion oncogene and to evaluate the interaction of the combination. The study showed that the addition of sorafenib to imatinib did not produce a significant synergistic effect on the viability of cells. The combination, however, had a significant effect on cell cycle arrest and induction of apoptosis with no effect on the reduction of BCR-ABL mRNA expression on K-562 cells.

Olverembatinib (HQP1351) Enhances Antitumor Effects of Chemotherapy in Philadelphia Chromosome-Positive (Ph⁺) Acute Lymphoblastic Leukemia (ALL)

Introduction Ph⁺ ALL is the most common subtype of B-cell- (B-) ALL in adults. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML) cells. However, treatment with TKIs fails to maintain disease remission in Ph⁺ B-ALL. Although the complete hematologic remission rate is very high, cytogenetic and molecular remission rates are low, so that TKIs are still considered as complements to chemotherapy and bridges to allogeneic stem cell transplantation. Olverembatinib is a novel, third-generation TKI with more potent activity against BCR-ABL1 and overcomes various resistant mutations, including the “gatekeeper” T315I, which confers resistance against all first- and second-generation TKIs. The aim of this study was to assess whether olverembatinib combined with chemotherapeutic agents vincristine or doxorubicin can promote anti-cancer effects in Ph⁺ ALL cells and explore the potential mechanism of action of the combinations. Methods Cell-based antiproliferation assays to evaluate the activities of olverembatinib alone or in combination with vincristine or doxorubicin were conducted using the CellTiter-Glo® Luminescent Cell Viability assay kits. Drug induced cell apoptosis was measured by flow cytometry. Potential mechanism of action analysis was conducted using western blot to evaluate the protein expression involved in BCR-ABL1 and apoptosis signaling pathways. Results Cell-based antiproliferation studies showed synergistic activity of olverembatinib in combination with vincristine or doxorubicin in Ph⁺ ALL SUP-B15 cells compared with single agents after 72 hours of treatment. The percentage of apoptotic cells induced by 24-hour treatment of olverembatinib (100 nM), vincristine (300 nM), and the combination were 27.7%, 27.2%, and 40.7%, respectively, while the percentage of apoptotic cells induced by 24-hour treatment of doxorubicin (100 nM) alone and in combination with olverembatinib (100 nM) were 35.3 and 74.1, respectively (apoptotic cells % in DMSO control group is 6.74). The combination significantly increased cell apoptosis compared with single agents ( p < 0.0001). Mechanistically, the combination of olverembatinib with vincristine showed synergistic effects in inhibiting protein expression and phosphorylation of BCR-ABL1, and downstream signal transducer and activator of transcription 5 (STAT5). The synergistic effects were also seen in decreased phosphorylation of AKT and ERK1/2. In addition, the combination significantly decreased protein levels of antiapoptotic MCL-1, BCL-2, and BCL-xL and increased protein levels of proapoptotic BAX and p53 upregulated modulator of apoptosis (PUMA) in a dose-dependent manner, which led to marked augmentation of cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). A similar pattern was also observed when olverembatinib was combined with doxorubicin. Conclusions Olverembatinib, a novel, third-generation TKI, in combination with chemotherapeutic agents vincristine or doxorubicin decreased MCL-1, BCL-2, and BCL-xL and increased BAX and PUMA in a dose-dependent manner, the combinations have demonstrated synergistic antitumor effects by enhancing apoptosis and antiproliferation in Ph⁺ ALL cells, which may provide an alternative approach for treatment of patients with Ph⁺ ALL.

Biological Role of Extracellular Vesicles in Myeloid Neoplasms: A Systematic Review of the Current Literature

Background and aim: Extracellular vesicles (EVs) are lipid-bilayer membrane vesicles secreted by a variety of cells that carry bioactive molecules like proteins, lipids and nucleic acids. They are of increasing interest as they play an important biological role in cancer by a multitude of cell-extrinsic and cell-intrinsic mechanisms. However, the investigations of their biology in myeloid malignancies has not been systematically reviewed so far. Therefore, the aim of this review was to summarize the currently published literature in the field and provide an overview of their potential clinical applications. Method: We performed a systematic literature research with defined search strings in the Embase, Medline and PubMed Central databases and used a standardized data-extraction form to collect all relevant information. Results: 63 of 1529 studies met the inclusion/exclusion criteria and could be assigned to seven main investigational categories ( A). They comprised studies on their effect on i) prothrombotic activity (n=13), ii) tumor-microenvironment (n=14), iii) malignant transformation (n=5), iv) tumor progression (n=4), v) chemotherapy-resistance (n=5) as well as their use as vi) biomarkers (n=14) and vii) pharmacotherapeutic carriers (n=3) ( B). The most relevant biological mechanism is the transfer of microRNA (miRNA) by EVs from different clonal and non-clonal cells, which target the microenvironment, vascular system, as well as immune cells. All mechanisms identified in our review were assigned to one of the seven main categories and the most relevant are summarized in C: i) Prothrombotic activity: Leukaemic cells shed prothrombotic EVs containing tissue-factor (TF) and anionic phosphatidylserine (PS), which facilitate the recruitment of coagulation factors and a pro-coagulant state ( Zannoni, 2019). Reduction of EVs from platelets and leucocytes after chemotherapy attenuates this pro-coagulant state ( Van Aalderen, 2011). ii) Tumor microenvironment: EVs derived from MDS cells can disrupt the osteolineage differentiation of bone marrow mesenchymal stromal cells (BM-MSCs) and impair hematopoiesis ( Hayashi, 2022). iii) Malignant transformation: EVs containing BCR-ABL1 mRNA are integrated into BM-MSCs, which induce BCR-ABL1 expression and secretion of TGF-β1 leading to enhanced proliferation of BM-MSCs ( Fu, 2017). iv) Tumor progression: EVs derived from leukemic cells promote tumor progression by reducing apoptosis and tumor immune-surveillance. v) Chemotherapy resistance: AML-cells expel PEGylated liposomal doxorubicin (PLD) through EVs thus increasing their resistance ( Hekmatirad, 2021). vi) Disease biomarkers: AML patients at diagnosis have a higher number of EVs carrying the myeloblast markers CD34, CD117, CD33 and HLA-DR than AML patients in remission or healthy controls ( Tzoran, 2015). Based on differential abundancy and the content of proteins, lipids and nucleid acids EVs may be suitable as biomarkers for diagnosis, prognosis, and disease monitoring. vii) Pharmacotherapeutic role: Cytotoxic T-lymphocytes are more effective in killing the parental leukaemic cells after being exposed to their cell-derived EVs. The mechanism is related to leukemia antigen presentation on EVs to dendritic cells (DCs) using ICAM1 and Hsp70 ( Shen C, 2011). Finally, EVs can be used as carriers for bioactive compounds and may expand their potential use for cell-type specific delivery of drugs. Conclusions: EVs open the window for the future investigation of novel pathophysiological mechanisms that will improve our understanding of their role in the biology of myeloid malignancies, Moreover, they hold promise as potential biomarkers and drug carriers for cell-type specific treatment

ABL001 (asciminib) Efficiently Targets Transplantable BCR::ABL1 Lymphoid Blast Crisis in the Scl-Tta-p210-BCR::ABL1 Mouse Model

Objectives: This study aimed at investigating the therapeutic efficacy of ABL001 in the ScltTA-p210-BCR::ABL1 transgenic mouse model. ABL001 (asciminib) is a novel allosteric inhibitor of ABL kinase demonstrating its particular efficacy against BCR::ABL1 mutations that emerge during treatment with traditional TKIs. However, the activity and tolerability of ABL001 in a primary mouse model of BCR::ABL1 disease remain unknown. In addition, resistance mutations against ABL001 were observed in the myristoyl pocket of BCR::ABL1, but their emergence has not been analyzed in BCR::ABL1+ murine hematopoietic cells. Importantly, leukemic stem cells (LSCs) expressing BCR::ABL1 have developed mechanisms to evade eradication by tyrosine kinase inhibitors, underscoring the necessity for alternative therapeutic approaches. Methods: 5-fluorouracil- (5-FU-) treated bone marrow (BM) cells harvested from FVB/N-tg(ScltTA)(tetO- p210-BCR::ABL1) double-transgenic (dtg) donor mice (CD45.1+) were transplanted into lethally irradiated recipient mice (CD45.2+). One week after transplantation, tetracycline (tet) was removed from the drinking water to induce Bcr-Abl1 expression. Following an additional week, mice were treated daily with ABL001 or vehicle control by oral gavage (30 mg/kg body weight). Therapeutic effects were assessed using flow cytometry (peripheral blood, spleen, and BM), histological examination, RNA analysis and DNA sequencing of the myristoyl pocket of BCR::ABL1. Secondary transplants using splenocytes were performed to analyze whether ABL001 treatment was able to suppress BCR::ABL1-positive repopulating malignant stem cells. Additionally, we assessed potential ABL001-resistant subclones after secondary transplantation by subjecting the mice to further ABL001 treatment, modeling clinically relevant mutations in BCR::ABL1-positive disease. Results: Within 3 weeks after tet withdrawal, the transplanted mice developed an aggressive B-lymphoblastic p210-BCR::ABL1-positive disease, mimicking lymphoid blast crisis, as described previously (Koschmieder et al., Blood, 2005). Interestingly, ABL001 treatment significantly reduced this BCR::ABL1-driven phenotype as indicated by suppression of malignant B cells and restoration of normal hematopoiesis. ABL001 also significantly improved survival compared to the vehicle control group (p=0.026). Histological analysis confirmed the alleviation of the BCR::ABL1-driven phenotype in spleen and BM. Furthermore, ABL001 treatment significantly reduced BCR::ABL1 transcripts to background levels, demonstrating its ability to suppress BCR::ABL1-induced disease in the BM. The efficacy of ABL001 in reducing the malignant clone correlated with normalization of spleen weight. Essentially, ABL001 treatment normalized the phenotypic long-term repopulating hematopoietic stem cell (LT-HSC) population in the BM, suggesting restoration of the stem cell pool and targeting malignant LT-HSCs. This was supported by secondary transplantation experiments, showing that several mice receiving ABL001-treated splenocytes failed to develop leukemia, while all mice who received vehicle-treated cells did. Moreover, none of the secondary transplanted mice who were treated with ABL001 developed leukemia. Interestingly, Sanger sequencing of the BCR::ABL1 myristoyl pocket region demonstrated wildtype sequences and the absence of resistance mutations. Conclusion: Our study demonstrates the efficacy of ABL001 in ameliorating p210-BCR::ABL1-induced lymphoid blast crisis by specifically targeting BCR::ABL1-positive malignant stem cells and restoring normal hematopoiesis in a transgenic mouse model. These promising findings highlight the potential of ABL001 as an alternative therapeutic strategy for first-line treatment of BCR::ABL1-driven leukemia, including B-lymphoblastic blast crisis. Furthermore, since TKI treatment alone has not proven successful in patients with lymphoid blast crisis, this now opens new avenues for the in vivo-testing of novel combination treatments of ABL001 with chemotherapy or other targeting agents.

A Deeply Quiescent Subset of CML LSC depend on FAO yet Avoid Deleterious ROS by Suppressing Mitochondrial Complex I.

Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures. Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset. Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib. We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.

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.

BCR-ABL promotes hematopoietic stem and progenitor cell formation in embryonic stem cells

Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.

The mRNA Expression of PTEN, LEF1, JAK3, LC3 and p62/SQSTM1 Genes in Patients with Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a progressive myeloproliferative disorder resulting from forming a chimeric BCR-ABL gene. The proteins derived from this gene can affect some genes from various signaling pathways such as PI3K/AKT/Wnt/catenin/JAK/Stat involved in proliferation, differentiation, cell death, and genes related to autophagy. Imatinib is the first-line treatment for CML patients, with durable and proper responses in Iranian children and adult CML patients. Hence, we aimed to evaluate the mRNA expression of some selected key genes from those pathways in patients with CML before and under treatment. In the case-control study, the mRNA expression of PTEN, LEF1, JAK3, LC3 and p62 genes were measured in 51 CML patients (6 patients before treatment and 45 patients under treatment with imatinib mesylate) and 40 healthy controls using the Real-time PCR method. The mRNA expression of PTEN and P62 were significantly higher in newly diagnosed patients than in controls (P<0.0001 and P = 0.0183, respectively), while the expression of the LC3 gene was significantly lower in the untreated newly diagnosed group than in control subjects (P = 0.0191). The expression level of PTEN, LEF1, JAK3 and P62 genes were significantly decreased in patients under treatment than in the group before treatment (P = 0.0172, P = 0.0002, P = 0.0047 and P = 0.0038, respectively). A positive correlation was seen between the gene expression of P62 and BCR-ABL in the patients under treatment (r 0529, P = 0.016). Our findings showed that the changes in expression of these genes were related to the patient's treatment. Due to the key role of these genes in proliferation, differentiation and tumor suppression, it is proposed that these genes may be helpful for follow-up of treatment in CML patients.

TSPAN32 suppresses chronic myeloid leukemia pathogenesis and progression by stabilizing PTEN

We report herein that TSPAN32 is a key node factor for Philadelphia (Ph+) leukemia pathogenesis. We found that TSPAN32 expression was repressed by BCR-ABL and ectopic TSPAN32 expression upon Imatinib treatment inhibited the proliferation of Ph+ cell lines. Tspan32 overexpression significantly prevented BCR-ABL induced leukemia progression in a murine model and impaired leukemia stem cell (LSC) proliferation. LSCs represent an obstacle for chronic myeloid leukemia (CML) elimination, which continually replenish leukemia cells and are associated with disease relapse. Therefore, the identification of essential targets that contribute to the survival and self-renewal of LSCs is important for novel curative CML. Mechanistically, TSPAN32 was shown to interact with PTEN, increased its protein level and caused a reduction in PI3K-AKT signaling activity. We also found that TSPAN32 was repressed by BCR-ABL via the suppression of an important transcription factor, TAL1. Ectopic expression of TAL1 significantly increased TSPAN32 mRNA and protein level, which indicated that BCR-ABL repressed TSPAN32 transcription by decreasing TAL1 expression. Overall, we identified a new signaling axis composed of “BCR-ABL-TAL1-TSPAN32-PTEN-PI3K-AKT”. Our findings further complement the known mechanisms underlying the transformation potential of BCR-ABL in CML pathogenesis. This new signaling axis also provides a potential means to target PI3K-AKT for CML treatment.

Hypoxia-inducible factor 1α inhibitor induces cell death via suppression of BCR-ABL1 and Met expression in BCR-ABL1 tyrosine kinase inhibitor sensitive and resistant chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) has a markedly improved prognosis with the use of breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitors (BCR-ABL1 TKIs). However, approximately 40% of patients are resistant or intolerant to BCR-ABL1 TKIs. Hypoxia-inducible factor 1α (HIF-1α) is a hypoxia response factor that has been reported to be highly expressed in CML patients, making it a therapeutic target for BCR-ABL1 TKI-sensitive CML and BCR-ABL1 TKI-resistant CML. In this study, we examined whether HIF-1α inhibitors induce cell death in CML cells and BCR-ABL1 TKI-resistant CML cells. We found that echinomycin and PX-478 induced cell death in BCR-ABL1 TKIs sensitive and resistant CML cells at similar concentrations while the cell sensitivity was not affected with imatinib or dasatinib in BCR-ABL1 TKIs resistant CML cells. In addition, echinomycin and PX-478 inhibited the c-Jun N-terminal kinase (JNK), Akt, and extracellular-regulated protein kinase 1/2 (ERK1/2) activation via suppression of BCR-ABL1 and Met expression in BCR-ABL1 sensitive and resistant CML cells. Moreover, treatment with HIF-1α siRNA induced cell death by inhibiting BCR-ABL1 and Met expression and activation of JNK, Akt, and ERK1/2 in BCR-ABL1 TKIs sensitive and resistant CML cells. These results indicated that HIF-1α regulates BCR-ABL and Met expression and is involved in cell survival in CML cells, suggesting that HIF-1α inhibitors induce cell death in BCR-ABL1 TKIs sensitive and resistant CML cells and therefore HIF-1α inhibitors are potential candidates for CML treatment. [BMB Reports 2023; 56(2): 78-83].

Comparative Expression Analysis of Breakpoint Cluster Region-Abelson Oncogene in Leukemia Patients.

Leukemia is a proliferative disorder of myeloid and lymphoid cells that may lead to death. Different types of leukemia have been reported, and several genetic and environmental factors are involved in their development. The Philadelphia chromosome causes the most common mutation known as breakpoint cluster region-Abelson oncogene (BCR-ABL1), which shows abnormal protein tyrosine kinase (PTK) activity. Basically, this activity is accountable for activating multiple pathways, including the inhibition of cell differentiation, controlled proliferation, and cell death. As a result of the absence of kinase activity, this mutation leads to the uncontrolled proliferation of leukocytes, causing chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), and chronic lymphocytic leukemia (CLL). This study aimed to evaluate the level of BCR-ABL1 expression in patients with these types of leukemias through qPCR. In brief, PBMCs were isolated from blood samples of patients, RNA was extracted from PBMCs, cDNA was synthesized, and the transcript levels of BCR-ABL1 in patients with each type of leukemia were determined by qPCR. The clinical, demographical, and experimental data were analyzed among CML, AML, and ALL patients. Results: The BCR-ABL1 expression levels are variable in all studied groups and are 90, 30-35, and 1-2.5% in CML, ALL, and AML, respectively. Demographic characteristics such as gender, BMI, age, family history, and clinical parameters along with CBC are also associated with the prevalence and diagnosis of leukemia. In a comparative expression analysis, the expression of BCR-ABL1 is onefold high in AML, but four- and sevenfold high in ALL and CML, respectively, as compared with normal levels. Conclusions: In this study, a significant difference was observed in the expression levels of BCR-ABL1 between CML (p = 0.0043) and ALL (p = 0.0006) and between CML and AML groups, and a high expression of BCR-ABL1 was noted in CML as compared with ALL and AML.

Curcumin Decreases Viability and Inhibits Proliferation of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia Cell Lines.

Chronic myeloid leukemia (CML) is a myeloproliferative haematological malignancy characterized by constitutive activation of BCR-ABL1 tyrosine kinase in the majority of patients. BCR-ABL1 expression activates signaling pathways involved in cell proliferation and survival. Current treatment options for CML include tyrosine kinase inhibitors (TKI) with resistance as a major issue. Various treatment options for overcoming resistance are being investigated. Among them, phytochemical curcumin could play an important role. Curcumin has been found to exhibit anti-cancerous effects in various models, including CML, through regulation of multiple molecular signaling pathways contributing to tumorigenesis. We have evaluated curcumin's effects on imatinib-sensitive LAMA84S and K562, as well as imatinib-resistant LAMA84R cell lines. Our results indicate a significant dose-dependent decrease in cell viability and proliferation of imatinib-sensitive and imatinib-resistant cell lines after curcumin treatment. Suppression of key signaling molecules regulating metabolic and proliferative events, such as Akt, P70S6K and NF-kB, was observed. Increased expression of caspase-3 suggests the potential pro-apoptotic effect of curcumin in the imatinib-resistant CML model. Additional in silico molecular docking studies revealed binding modes and affinities of curcumin with different targets and the results are in accordance with in vitro findings. Altogether, these results indicate the potential role of curcumin in the treatment of CML.

Targeting a Highly Deregulated eIF4F Translation Initiation Complex Sensitises IM-Resistant Cells to Tyrosine Kinase Inhibitors and Effectively Suppresses BCR-ABL1 Protein Expression

Background We previously reported that Abelson helper integration site-1 (AHI-1) is highly deregulated in chronic myeloid leukemia (CML) leukemic stem cells (LSCs) and mediates tyrosine kinase inhibitor (TKI) resistance. AHI-1 physically interacts with BCR-ABL1 and its SH3 domain interacts with BCR-ABL1 substrates, like dynamin-2, which regulate leukemic properties of CML LSCs. However, the molecular and biological roles of AHI-1 and its interacting partners in mediating TKI resistance remain largely unknown. Objective To investigate the molecular functions of AHI-1 and its SH3 domain in regulation of TKI resistance, using advanced antibody microarray analysis. Methods A high content antibody microarray was performed in BCR-ABL1+ cells co-transduced with wild-type (WT) AHI-1 or the deletion of AHI-1 SH3 domain (SH3Δ) with or without imatinib (IM). Changes in antibody signals for protein expression or phosphorylation were determined using limma and pathway enrichment analysis by g:Profiler. eIF4G1 genetic inhibition by lentiviral-mediated shRNA or pharmacological inhibition by SBI-756, was performed in TKI-resistant cells to assay translation initiation activity by proximity ligation assay (PLA), o-propargyl-puromycin (OPP), and polysome profiling. Results The antibody microarray analysis revealed that WT AHI-1 cells have the greatest number of changes in the phospho-proteome and proteome compared to BCR-ABL1+ cells and AHI-1 SH3Δ cells with and without IM. Pathway enrichment analysis identified that the targets with significantly increased differential antibody signal after IM treatment in WT AHI-1 cells were related to the regulation of translation initiation complex (p < 0.0001). Interestingly, our RNA-seq dataset analysis further identified several eukaryotic initiation factor 4F (eIF4F) complex members to be significantly increased in CD34+ CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex (2-fold, p = 0.001). WT AHI-1 cells also showed increased expression and phosphorylation of eIF4G1 (>2-fold) and eIF4B (>2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, and cyclin D3 (a downstream protein of eIF4F translational activity) as compared to BCR-ABL1+ cells, by immunoblotting. These results were similarly demonstrated in IM-resistant cells as compared to IM-sensitive cells (2-5-fold, p < 0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p < 0.0001) and increased TKI sensitivity in IM-resistant cells (p < 0.0021). These cells showed reduced levels of eIF4F complex formation by PLA assay (p = 0.013) and reduced protein expression of cyclin D3 (40%, p = 0.0036) and BCR-ABL1 (70%). Similarly, IM-resistant cells were more sensitive to SBI-756 treatment, an eIF4G1 inhibitor (50% reduction, p = 0.0001) than IM-sensitive cells (30% reduction); these effects were enhanced by a combination of SBI-756 and IM (80%, p < 0.02). Furthermore, SBI-756 treatment reduced PLA foci formation (p = 0.014) and global protein synthesis rates (p < 0.0001). Polysome profiling demonstrated that IM-resistant cells had higher levels of translation activity, which was inhibited by SBI-756 treatment. Most interestingly, SBI-756 treatment reduced protein expression of BCR-ABL1 (70%) and cyclin D3 (40%) in these cells. Conclusion We have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells. eIF4G1 inhibitor treatment sensitises IM-resistant cells to TKI and reduces BCR-ABL1 protein expression, providing potential treatment strategies to overcome TKI resistance.