Resistant Chronic Myeloid Leukemia Research Articles

BCAT1 contributes to the development of TKI-resistant CML.

Although most of chronic myeloid leukemia (CML) patients can be effectively treated by the tyrosine kinase inhibitors (TKIs), such as Imatinib, TKI-resistance still occurs in approximately 15-17% of cases. Although many studies indicate that branched chain amino acid (BCAA) metabolism may contribute to the TKI resistance in CML, the detailed mechanisms remains largely unknown. The cell proliferation, colony formation and in vivo transplantation were used to determined the functions of BCAT1 in leukemogenesis. Quantitative real-time PCR (RT-PCR), western blotting, RNA sequencing, BCAA stimulation in vitro were applied to characterize the underlying molecular mechanism that control the leukemogenic activity of BCAT1-knockdown cells. In this report, we revealed that branched chain amino acid transaminase 1 (BCAT1) is highly enriched in both mouse and human TKI-resistant CML cells. Leukemia was almost completely abrogated upon BCAT1 knockdown during transplantation in a BCR-ABLT315I-induced murine TKI-resistant CML model. Moreover, knockdown of BCAT1 led to a dramatic decrease in the proliferation of TKI-resistant human leukemia cell lines. BCAA/BCAT1 signaling enhanced the phosphorylation of CREB, which is required for maintenance of TKI-resistant CML cells. Importantly, blockade of BCAA/BCAT1 signaling efficiently inhibited leukemogenesis both in vivo and in vitro. These findings demonstrate the role of BCAA/BCAT1 signaling in cancer development and suggest that targeting BCAA/BCAT1 signaling is a potential strategy for interfering with TKI-resistant CML.

Effects of imidazole derivatives on cellular proliferation and apoptosis in myeloid leukemia.

Acute promyelocytic leukemia (APL) is the sub-type of Acute myeloid leukemia (AML) which is described by differentiation block at promyelocytic stage and t(15; 17) translocation with All trans retinoic acid (ATRA) and arsenic trioxide (ATO) as standard treatments. Chronic myeloid leukemia (CML) translocation t (19; 22) causes a rise in granulocytes and their immature precursors in the blood. Different mutations cause resistance to first-line tyrosine kinase therapies in CML. Beside drug resistance, leukemia stem cells (LSC) are critical resources for relapse and resistance in APL and CML. The drug toxicity and resistant profile associated with LSC and current therapeutics of APL and CML necessitate the development of new therapies. Imidazoles are heterocyclic nitrogen compounds with diverse cellular actions. The purpose of this research was to assess the anti-leukemic properties of four novel imidazole derivatives including L-4, L-7, R-35, and R-NIM04. Pharmacological and biochemical approaches were used which showed that all four imidazole derivatives interfere with the NB4 cells proliferation, an APL cell line, while only L-7 exhibit anti-proliferative activity against K562 cells, a CML cell line. The anti-proliferative effect of imidazole derivatives was linked to apoptosis induction. Further real-time polymerase chain reaction (RT-PCR) analysis revealed downregulation of AXL-Receptor Tyrosine Kinase (AXL-RTK) and target genes of Wnt/beta-catenin pathway like c-Myc, Axin2 and EYA3. An additive effect was observed after combinatorial treatment of L-7 with standard drugs ATRA or Imatinib on the proliferation of NB4 and K562 cells respectively which was related to further downregulation of target genes of Wnt/beta catenin pathway. Imidazole derivatives significantly reduce proliferation of NB4 and K562 cells by inducing apoptosis, down regulating of AXL-RTK and Wnt/β-catenin target genes.

Inhibition of Tumoral VISTA to Overcome TKI Resistance via Downregulation of the AKT/mTOR and JAK2/STAT5 Pathways in Chronic Myeloid Leukemia.

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment landscape for chronic myeloid leukemia (CML). However, TKI resistance poses a significant challenge, leading to treatment failure and disease progression. Resistance mechanisms include both BCR::ABL1-dependent and BCR::ABL1-independent pathways. The mechanisms underlying BCR::ABL1 independence remain incompletely understood, with CML cells potentially activating alternative signaling pathways, including the AKT/mTOR and JAK2/STAT5 pathways, to compensate for the loss of BCR::ABL1 kinase activity. This study explored tumoral VISTA (encoded by VSIR) as a contributing factor to TKI resistance in CML patients and identified elevated tumoral VISTA levels as a marker of resistance and poor survival. Through in vitro and in vivo analyses, we demonstrated that VSIR knockdown and the application of NSC-622608, a novel VISTA inhibitor, significantly impeded CML cell proliferation and induced apoptosis by attenuating the AKT/ mTOR and JAK2/STAT5 pathways, which are crucial for CML cell survival independent of BCR::ABL1 kinase activity. Moreover, VSIR overexpression promoted TKI resistance in CML cells. Importantly, the synergistic effect of NSC-622608 with TKIs offers a potent therapeutic avenue against both imatinib-sensitive and imatinib-resistant CML cells, including those harboring the challenging T315I mutation. Our findings highlight the role of tumoral VISTA in mediating TKI resistance in CML, suggesting that inhibition of VISTA, particularly in combination with TKIs, is an innovative approach to enhancing treatment outcomes in CML patients, irrespective of BCR::ABL1 mutation status. This study not only identified a new pathway contributing to TKI resistance but also revealed the possibility of targeting tumoral VISTA as a means of overcoming this significant clinical challenge.

Morphology, Morphometry, and Immunohistochemical Profile of Megakaryocytes and Bone Marrow Microenvironment in Disease Progression and Therapy Resistance in Chronic Myeloid Leukemia.

Background Tyrosine kinase inhibitors have revolutionized the treatment of chronic myeloid leukemia (CML) since the beginning of the century. However, resistance to therapy and the progression of disease tend to occur in certain patients. The bone marrow microenvironment may play a role in the disease outcome. Megakaryocytes have multiple roles in the regulation and maintenance of the hematopoietic stem cell microenvironment. In the current study, we evaluated the association of megakaryocyte morphology, morphometry, and microenvironment with disease progression and therapy resistance in CML. Methodology Megakaryocyte morphology and morphometry were analyzed and compared between the different phases (chronic and advanced) at diagnosis in 150 cases of BCR-ABL-positive CML. All CML-CP patients (n = 119) were followed up on tyrosine kinase inhibitor therapy for a minimum of 15 months and classified based on their treatment outcome as a response, resistance to therapy, or progression of disease based on standard criteria. Immunohistochemistry on a bone marrow trephine biopsy was done for vascular endothelial growth factor (VEGF), FOXP3, CD150, CD48, CD44, osteopontin, CXCL12, N-cadherin, PDL-1, and IL-7, and their expression on megakaryocytes and their association with treatment outcome was evaluated. Results The morphology and morphometry of megakaryocytes showed a heterogeneous population in CML. Morphology and morphometric parameters, when compared between the chronic and advanced phases of disease at diagnosis, did not show any statistical difference. Megakaryocytes were variably positive for VEGF, FOXP3, CD150, CD48, osteopontin, N-cadherin, CXCL12, CD44, PDL-1, and IL-7. However, only CD44-positive megakaryocytes were statistically associated with the treatment outcome. The patients with a higher expression of CD44 megakaryocytes progressed to the advanced phase of the disease during therapy compared to those who responded. Conclusion Megakaryocyte morphology and morphometry were heterogeneous in CML; however, they did not show any significant difference with either the phase of the disease or with treatment outcomes. Among the various immunohistochemical markers of the microenvironment, only CD44-positivity on megakaryocytes was associated with poor treatment outcomes.

Molecular biomarkers of leukemia: convergence-based drug resistance mechanisms in chronic myeloid leukemia and myeloproliferative neoplasms.

Leukemia represents a diverse group of hematopoietic neoplasms that can be classified into different subtypes based on the molecular aberration in the affected cell population. Identification of these molecular classification is required to identify specific targeted therapeutic approaches for each leukemic subtype. In general, targeted therapy approaches achieve good responses in some leukemia subgroups, however, resistance against these targeted therapies is common. In this review, we summarize molecular drug resistance biomarkers in targeted therapies in BCR::ABL1-driven chronic myeloid leukemia (CML) and JAK2-driven myeloproliferative neoplasms (MPNs). While acquisition of secondary mutations in the BCR::ABL1 kinase domain is the a common mechanism associated with TKI resistance in CML, in JAK2-driven MPNs secondary mutations in JAK2 are rare. Due to high prevalence and lack of specific therapy approaches in MPNs compared to CML, identification of crucial pathways leading to inhibitor persistence in MPN model is utterly important. In this review, we focus on different alternative signaling pathways activated in both, BCR::ABL1-mediated CML and JAK2-mediated MPNs, by combining data from in vitro and in vivo-studies that could be used as potential biomarkers of drug resistance. In a nutshell, some common similarities, especially activation of PDGFR, Ras, PI3K/Akt signaling pathways, have been demonstrated in both leukemias. In addition, induction of the nucleoprotein YBX1 was shown to be involved in TKI-resistant JAK2-mediated MPN, as well as TKI-resistant CML highlighting deubiquitinating enzymes as potential biomarkers of TKI resistance. Taken together, whole exome sequencing of cell-based or patients-derived samples are highly beneficial to define specific resistance markers. Additionally, this might be helpful for the development of novel diagnostic tools, e.g., liquid biopsy, and novel therapeutic agents, which could be used to overcome TKI resistance in molecularly distinct leukemia subtypes.

BTK acts as a modulator of the response to imatinib in chronic myeloid leukemia.

The use of tyrosine kinase inhibitors, such as imatinib, against the chronic myeloid leukemia (CML)-causing kinase BCR::ABL1 has become the model for successful targeted therapy. Nevertheless, drug resistance remains a clinical problem. Analysis of genome-wide expression and genetic aberrations of an in vitro imatinib-resistant CML cell line revealed downregulation of Bruton's tyrosine kinase (BTK), predominantly associated with B cell malignancies, and a novel BTK kinase domain variant in imatinib resistance. This raised the question of the role of BTK in imatinib-resistant CML. In the present study, BTK downregulation and the presence of the BTK variant c.1699_1700delinsAG p.(Glu567Arg) were confirmed in imatinib resistance in vitro. Similarly, BTK inhibition or small interfering RNA-mediated BTK knockdown reduced imatinib susceptibility by 84 and 71%, respectively. BTK overexpression was detrimental to CML cells, as proliferation was significantly reduced by 20.5% under imatinib treatment. In addition, BTK rescue in imatinib-resistant cells restored imatinib sensitivity. The presence of the BTK p.(Glu567Arg) variant increased cell numbers (57%) and proliferation (37%) under imatinib exposure. These data demonstrate that BTK is important for the development of imatinib resistance in CML: Its presence increased drug response, while its absence promotes imatinib resistance. Moreover, the BTK p.(Glu567Arg) variant abrogates imatinib sensitivity. These findings demonstrate a context-dependent role for BTK as an oncogene in B cell malignancies, but as a tumor suppressor in other neoplasms.

S1P Signaling Genes as Prominent Drivers of BCR-ABL1-Independent Imatinib Resistance and Six Herbal Compounds as Potential Drugs for Chronic Myeloid Leukemia.

Imatinib (IM), a breakthrough in chronic myeloid leukemia (CML) treatment, is accompanied by discontinuation challenges owing to drug intolerance. Although BCR-ABL1 mutation is a key cause of CML resistance, understanding mechanisms independent of BCR-ABL1 is also important. This study investigated the sphingosine-1-phosphate (S1P) signaling-associated genes (SphK1 and S1PRs) and their role in BCR-ABL1-independent resistant CML, an area currently lacking investigation. Through comprehensive transcriptomic analysis of IM-sensitive and IM-resistant CML groups, we identified the differentially expressed genes and found a notable upregulation of SphK1, S1PR2, and S1PR5 in IM-resistant CML. Functional annotation revealed their roles in critical cellular processes such as proliferation and GPCR activity. Their network analysis uncovered significant clusters, emphasizing the interconnectedness of the S1P signaling genes. Further, we identified interactors such as BIRC3, TRAF6, and SRC genes, with potential implications for IM resistance. Additionally, receiver operator characteristic curve analysis suggested these genes' potential as biomarkers for predicting IM resistance. Network pharmacology analysis identified six herbal compounds-ampelopsin, ellagic acid, colchicine, epigallocatechin-3-gallate, cucurbitacin B, and evodin-as potential drug candidates targeting the S1P signaling genes. In summary, this study contributes to efforts to better understand the molecular mechanisms underlying BCR-ABL1-independent CML resistance. Moreover, the S1P signaling genes are promising therapeutic targets and plausible new innovation avenues to combat IM resistance in cancer clinical care in the future.

Identification of exosomal microRNAs and related hub genes associated with imatinib resistance in chronic myeloid leukemia.

Chemotherapy resistance is a major obstacle in cancer therapy, and identifying novel druggable targets to reverse this phenomenon is essential. The exosome-mediated transmittance of drug resistance has been shown in various cancer models including ovarian and prostate cancer models. In this study, we aimed to investigate the role of exosomal miRNA transfer inchronic myeloid leukemia drug resistance. For this purpose, firstly exosomes were isolated from imatinib sensitive (K562S) and resistant (K562R) chronic myeloid leukemia (CML)cells and named as Sexo and Rexo, respectively. Then, miRNA microarray was used to compare miRNA profiles of K562S, K562R, Sexo, Rexo, and Rexo-treated K562S cells. According to our results, miR-125b-5p and miR-99a-5p exhibited increased expression in resistant cells, their exosomes, and Rexo-treated sensitive cells compared to their sensitive counterparts. On the other hand, miR-210-3p and miR-193b-3p were determined to be the two miRNAs which exhibited decreased expression profile in resistant cells and their exosomes compared to their sensitive counterparts. Gene targets, signaling pathways, and enrichment analysis were performed for these miRNAs by TargetScan, KEGG, and DAVID. Potential interactions between gene candidates at the protein level were analyzed via STRING and Cytoscape software. Our findings revealed CCR5, GRK2, EDN1, ARRB1, P2RY2, LAMC2, PAK3, PAK4, and GIT2 as novel gene targets that may play roles in exosomal imatinib resistance transfer as well as mTOR, STAT3, MCL1, LAMC1, and KRAS which are already linked to imatinib resistance. MDR1 mRNA exhibited higher expression in Rexo compared to Sexo as well as in K562S cells treated with Rexo compared to K562S cells which may suggest exosomal transfer of MDR1 mRNA.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Integrated functional genomic screening to bypass TKI resistance in chronic myeloid leukemia

CML is readily treatable with tyrosine kinase inhibitors (TKIs); however, resistance occurs, with the disease curable in only ∼15%-20% of patients. Using integrated functional genomics, Adnan Awad etal.1 identify agents effective against CML stem cells and describe mechanisms underlying TKI resistance.

High-dose nicotinamide, a histone deacetylase inhibitor (Sirtuin-1), can prevent emergence of treatment resistance in chronic myeloid leukemia – A perspective

Chronic myeloid leukemia (CML) is now widely treated using tyrosine kinase inhibitors (TKI). These TKIs can annihilate dividing cells, but they have no effect on quiescent stem cells. These quiescent stem cells slowly give rise to treatment resistance in the form of mutations. T315I is one such mutation that is resistant to most of the TKI’s and treating this acquired kinase domain mutation i.e T315I, is often costly. Nicotinamide is histone deacetylase inhibitor. It inhibits SIRT-1(Sirtuin-1). High dose nicotinamide, when used with TKI, will not only potentiate TKI action, but also annihilate quiescent stem cells thereby preventing the emergence of treatment resistance in CML. We propose a perspective article on using high dose nicotinmaide along with TKI to prevent emergence of treatment resistance. Thus going by the famous idiom “prevention is better than cure”,we suggest trial on high dose nicotinamide with TKI in CML.

Mechanisms underlying therapeutic resistance of tyrosine kinase inhibitors in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a malignant clonal disease involving hematopoietic stem cells that is characterized by myeloid cell proliferation in bone marrow and peripheral blood, and the presence of the Philadelphia (Ph) chromosome with BCR-ABL fusion gene. Treatment of CML has dramatically improved since the advent of tyrosine kinase inhibitors (TKI). However, there are a small subset of CML patients who develop resistance to TKI. Mutations in the ABL kinase domain (KD) are currently recognized as the leading cause of TKI resistance in CML. In this review, we discuss the concept of resistance and summarize recent advances exploring the mechanisms underlying CML resistance. Overcoming TKI resistance appears to be the most successful approach to reduce the burden of leukemia and enhance cures for CML. Advances in new strategies to combat drug resistance may rapidly change the management of TKI-resistant CML and expand the prospects for available therapies.

Literature Review of Hematology Division The Mechanism of Imanitib Resistance in Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) refers to a kind of malignancycharacterized by the clonal proliferation of myeloid leukocytes in the bone marrow. The World Health Organization (WHO) classifies CML as a Myeloproliferative Neoplasm (MPN) identified by the proliferation of granulocyte cells without differentiation disorders. As a result, peripheral blood smears display varying levels of differentiation within the granulocyte series. Furthermore, the translocation between chromosomes 9 and 22 gives rise to the Philadelphia chromosome (Ph) (BCR-ABL1). Imatinib mesylate (GleevecTM), a chemotherapeutic belonging to the protein kinase inhibitor group, is the first-generation Tyrosine Kinase Inhibitor (TKI) used for treating chronic phase CML. Imatinib mesylate suppresses cancer cell signals and inhibits a sequence of chemical events that contribute to cell growth and development. It achieves this by binding to the ATP binding region, trapping it in a self-inhibited or closed conformation, and exerting non-competitive suppression on protein enzyme activities. Consequently, this procedure leads to the inhibition of leukemogenesis-promoting signaling pathways.Imatinib resistance poses a significant challenge, and it can be classified as primary or secondary resistance based on the onset time. Depending on the mechanism, resistance can be characterized as BCR-ABL1-independent or BCR-ABL1-dependent. The most prevalent mechanism of imatinib resistance is the mutation of the ABL kinase domain, followed by BCR-ABL1 amplification and overexpression. In cases of inadequate response or treatment failure, the European Leukemia Network (ELN) recommends mutation screening before transitioning to second-generation Tyrosine Kinase Inhibitors (TKIs). Mutations of the BCR-ABL1 kinase domain can be analyzed using alternative examination methods such as Sanger sequencing, Next-Generation Sequencing (NGS), and digital Polymerase Chain Reaction (dPCR).Various methods have been employed to enhance therapy response or treat TKI-resistant patients, including increasing the dose of Imatinib, utilizing next-generation Tyrosine Kinase Inhibitors, and resorting to bone marrow transplantation.

Mechanism of Late Recurrences of Chronic Myeloid Leukemia after Discontinuation of TKI Therapy: BCR::ABL1 Ins35bp Loss-of-Function Splicing Mutation and Regulatory T Cells

Introduction: Treatment-free remission (TFR) as one of the goals of treatment of BCR::ABL-positive chronic phase chronic myeloid leukemia (CML) has been proposed. Actually, approximately 40-60% of CML patients maintain TFR without recurrence after discontinuation of tyrosine kinase inhibitors (TKIs). In some of such patients, there are fluctuated cases in which BCR::ABL positive clones continue to be detected for long periods at levels of international scale (IS) BCR::ABL < 0.1%. Such fluctuated cases show no apparent molecular recurrence and maintain TFR for a long time, but they have also been shown to be more likely to eventually develop late recurrence (Haematologica 2022, Blood adv. 2020). We have reported that a higher percentage of PD-1 positive CD8 + T cells before and after TKI discontinuation is associated with molecular recurrence, and together with the possibility of assessing host immune response capacity after TKI discontinuation by using regulatory T (Treg) cells (Fujioka, et al. Cancers 2021). On the other hands, mutations in BCR::ABL caused by the alternative splicing, such as BCR::ABL Ins35bp, are known to be one of the TKI resistance in CML. Loss of BCR::ABL kinase activity is thought to result in loss of TKI sensitivity and thus failure to achieve deep molecular remission (Yuda, et al. Cancer Science 2017). In this study, we analyzed the mutations that cause splicing variants such as BCR::ABL Ins35bp and host immune response in CML patients to elucidate molecular mechanisms in the long-term maintenance of TFR and late recurrence. Methods: Twenty-four CML patients who discontinued TKI treatment for any reason at Akita University Hospital were included in the study. The trends of IS level after TKI discontinuation were monitored, and immune profile analysis by multi-color flow cytometry (FCM) was performed in all patients. Mutation analysis of the BCR::ABL region including splicing variants was performed in selected patients. Mutation analysis was performed on samples with sufficient cell count and IS > 0.0032% (MR 4.5), and the BCR::ABL region of these samples was amplified by long nested PCR and then analyzed by the next-generation sequencer (NGS). Results: First, BCR::ABL Ins35bp was evaluated by mutation analysis. Among CML patients who underwent mutation analysis, BCR::ABL Ins35bp was detected in all cases in which the samples could perform analysis at newly diagnosis (ND). Even after TKI discontinuation, a certain percentage of BCR::ABL Ins35bp was detected in all patients, and the percentage of BCR::ABL Ins35bp increased at the time of recurrence regardless of whether early or late. These results suggest that function-dead BCR::ABL Ins35bp clones may remain in CML patients with detectable BCR::ABL under and after TKI treatment, and eventually leading to recurrence with transcriptional promotion of native BCR::ABL. On the other hand, there were cases in which the BCR::ABL Ins35bp was lost at the time of recurrence. There is a possibility of immunological involvement in such cases. CML patients with detectable BCR::ABL in the TFR for a long time had a decreased immunosuppressive Treg cells, while analysis using late recurrence increased Treg cells prior to recurrence (figure). These suggest the presence of cases in which early recurrence is suppressed by host immune responses. Conclusion: Although the loss of function mutations such as BCR::ABL Ins35bp was previously thought to occur after TKI administration as a cause of TKI resistance, this analysis reveals that the clone is included from the ND before TKI treatment. Examining the BCR::ABL Ins35bp clone at ND may be possible to predict the possibility of recurrence after TFR. Host immune capacity, as assessed by the kinetics of Treg after TKI discontinuation, may contribute to prolonged TFR. Thus Treg monitoring may be important in predicting molecular recurrence, especially in BCR::ABL Ins35bp cases.

In Vitro Evidence of Synergistically Enhanced Efficacy of Axitinib When Combined with Asciminib in T315I Mutated Chronic Myeloid Leukemia

Background: Development of ABL1 kinase domain (KD) mutation is one of resistance mechanism to ATP-binding pocket inhibitor (ABPI) therapy. T315I mutation in ABL KD confers broad-spectrum resistance to all 1st- and 2nd-generation ABPIs including Imatinib (IMA), Dasatinib (DAS), Nilotinib (NIL) and Bosutinib (BOS). Ponatinib (PON), a 3 rd generation inhibitor, can overcome T315I-mediated resistance in chronic myeloid leukemia (CML) treatment. However, its increased risk of cardiovascular toxicity limits the broader use of PON in the clinic. Asciminib (ASC) is a Specifically Targeting ABL1 Myristoyl Pocket (STAMP) inhibitor and has been approved for CML treatment in the patients who failed at least two previous lines of TKI therapy. ASC, which blocks the myristoylate pocket in ABL1 KD protein, can potentially overcome ABL1 KD mutation (KDM)-mediated tyrosine kinase inhibitor (TKI) resistance by combining with ABPIs which blocks ATP binding pockets in ABL1 KD protein. However, in vitro evidence to support double blockade approach is still to be further investigated including combination with ABPIs as well with other drug compounds. Axitinib (AXI), which is approved for the treatment of advanced renal cell carcinoma (RCC), is an ABPI that can specifically bind to the ATP-binding pocket in ABL1 KD protein structurally harboring the T315I mutation. In the Cancer Therapeutics Response Portal (CTRP) database, which integrates drug compound and cell line experiment databases, AXI showed very low IC 50 in CML cell lines compared to other cancer cell lines, including RCC. The present study attempted to provide in vitro evidence of the synergistic enhancement of therapeutic efficacy of AXI combined with ASC to overcome T315I mutated CML. Methods and Materials: We analyzed IC 50 values for 649 cell lines tested with AXI using the CTRPv2 database. The drug response of AXI in K562/Wild-Type (WT), K562/T315I mut, BaF3/WT, and BaF3/T315I mut cell lines was evaluated using the WST-8 assay. From the measured IC 50 value in those cell lines as the baseline concentration (conc), AXI and ASC conc were serially diluted. The dose-response matrix and ZIP synergy score were analyzed using SynergyFinder. Results We compared the sensitivity of 649 different cell lines to AXI in the CTRPv2 database and confirmed that the IC 50 of AXI in CML (median IC 50 = 0.31 uM) is 96 times lower than that of various cell lines derived from RCC (median IC 50 = 29.85 uM) (Fig. 1A). This result implies that AXI is more effectively inhibit CML cells at a lower conc than RCC cell lines or other cell lines. When measuring the inhibitory activityof AXI (Fig. 1B), AXI treatment showed 2.22 times lower IC 50 in the K562/T315I mut (IC 50 = 111.6nM) than in the K562/WT (IC 50 = 248nM). Also AXI treatment showed 4.73 times lower IC 50 in the BaF3/T315I mut (IC 50 = 84nM) than in the BaF3/WT (IC 50 = 397.4nM). Overall, the T315I carrying CML cell lines were much more sensitive to lower dose AXI than wild type CML cell lines. In the dose-response matrix analysis (Fig. 1C), AXI treatment alone at 100 nM inhibited the growth of the K562/T315 mut cell line by 38.42%, and ASC treatment alone at 100 nM, by 36.52%. Of note, the combination of AXI at 25nM and ASC at 12.5nM inhibited cell growth by 38.1%, and the combination of AXI at 12.5nM and ASC at 25nM showed 38.42% inhibition, which is similar to the inhibition achieved by 100nM of each drug alone. The result suggests that the combination of reduced dose of each drug, by 25% or 12.5% from the baseline IC 50 dose, showed equivalent inhibitor activity to the baseline IC 50 concentration of each drug monotherapy in the K562/T315 mut cell line. Similarly, when AXI and ASC were combined at a reduced concentration by a half or a quarter and tested in the BaF3/T315I mut cell line, a higher inhibitory activity was observed compared to the baseline IC 50 concentration of each drug monotherapy. The ZIP score was calculated as 31.7 and 20.64 in K562/T315 mut and BaF3/T315I mut cell line. Given that ZIP score was above 10 each, synergistic enhancement of inhibitory activity was demonstrated against T315I mutant CML cells between AXI and ASC. Conclusion: This result suggests synergistically enhanced inhibitory activity of dual blockade using AXI combined with ASC specifically for T315I mutant CML. This approach will be promising against CML cells carrying compound mutation with T315I mutation, which is highly resistant even to Ponatinib or Asciminib.

Tgrx-678, a Novel Allosteric Inhibitor of BCR-ABL1, Demonstrates Preclinical Anti-Leukemia Activity, High Oral Bioavailability and Synergism with Ponatinib to Suppress the Highly Resistant Compound Mutations

Introduction Clinical resistance to the treatment of BCR-ABL1-positive chronic myeloid leukemia (CML) patients is often conferred by mutation(s) in the ATP-binding site of ABL1 kinase domain. ABL1 is subject to auto-inhibition mediated by myristoylation-triggered conformational change, which can be exploited to overcome resistance, as validated by allosteric inhibitors such as GNF2, GNF5 and asciminib. TGRX-678, a novel allosteric inhibitor designed to target the myristoyl pocket of ABL1, is currently being investigated in a phase I clinical trial (NCT05434312). Herein we report the selectivity, potency, anti-leukemia activity and in vivo pharmacokinetic (PK)/pharmacodynamic(PD) characteristics of TGRX-678. Methods For in vitro study, ABL1b 65-534 fragments were used following the protocol for Z'-LYTE kinase assay kit (Thermo Fisher). For cellular assays, CML cell lines or engineered Ba/F3 cells were incubated with titrating TGRX-678 for 72 hrs, then CellTiter Glo reagent (Promega) was added and the chemiluminescence was measured. For in vivo studies, SD rats received TGRX-678 or asciminib and plasma samples were collected for analysis. NOD-SCID mice were inoculated with CML or Ba/F3-BCR-ABL1 T315I cells and received oral TGRX-678. Tumor volumes and body weights were recorded for 3 to 5 weeks. Results TGRX-678 exhibited minimum off-target inhibition at 1 μM in a panel of 298 kinases, but inhibited the enzymatic activity of both ABL1 and the T315I mutant with IC50 values of 2.96 nM and 1.15 nM, respectively. The anti-proliferative activity of TGRX-678 was determined in CML cell lines including K562, KU812, KCL22-S, KCL22-R, LAMA-84 and MEG-01 with IC50 values ranging from 1.1 nM to 6.56 nM. TGRX-678 had no cytotoxicity in 29 cell lines representing other hematological malignancies and solid tumors. Using engineered Ba/F3 cell lines, TGRX-678 was active against the native BCR-ABL1 (IC50 = 4.11 nM) and the clinically prominent mutants including G250E (IC50 = 2.49 nM), Q252H (IC50 = 2.38 nM), Y253H (IC50 = 5.6 nM), E255K (IC50 = 1.01 nM), E255V (IC50 = 2.73 nM) and T315I (IC50 = 66.1 nM). The cellular activity of TGRX-678 was attenuated by mutations in the myristoyl pocket such as A337V, P465S, V468F and I502L or mutations in the SH3-kinase domain interface including P223S and K294E, underpinning its allosteric mode of action. In SD rats, oral administration of TGRX-678 at 20 mg/kg achieved a maximum plasma concentration (C max) of 2369 ng/mL, which was 1.3-fold higher than that of asciminib. The overall exposure (AUC last) of TGRX-678 (23,147 h·ng/mL) was also 1.7-fold higher than that of asciminib. The oral bioavailability of TGRX-678 was 62%. In xenograft-bearing mice, oral dosing of TGRX-678 at 1 mg/kg QD (for KU812) or 3 mg/kg QD (for K562) was sufficient to completely inhibit tumor growth. In the Ba/F3-BCR-ABL1 T315I xenograft mice, TGRX-678 induced dose-dependent tumor regression and reduced tumor volume by 67% at 45 mg/kg QD dose. We further examined the synergistic effect of TGRX-678 and ponatinib combination. While each agent was inactive to the Y253H/T315I mutant, the combination of TGRX-678 and ponatinib inhibited cell proliferation at suboptimal concentrations. The addition of TGRX-678 dramatically lowered the IC90 for ponatinib from 1489 nM to 60.9 nM, whereas asciminib only reduced the IC90 to 165 nM (Figure A). Similarly, while ponatinib alone had limited effect on cell viability of the E255V/T315I, Q252H/T315I and T315M mutants, the addition of TGRX-678 at a clinically achievable concentration of 720 nM significantly reduced cell viability to 0-10 % (Figures B-D). In comparison, the addition of asciminib at a much higher concentration of 6000 nM only reduced cell viability to 5-29% (Figures B-D). Conclusion Our findings support that TGRX-678, a novel allosteric inhibitor of BCR-ABL1, is potent against the CML cells and cell lines harboring clinical ATP-site mutations with minimum non-specific cytotoxicity. TGRX-678 exhibits a favorable PK profile and anti-tumor activity in vivo. Furthermore, TGRX-678 and ponatinib synergize to overcome the clinically challenging resistance conferred by T315M or T315I-inclusive compound mutations. This data warrant further clinical investigation of TGRX-678 for the treatment of resistant or refractory CML 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

Loss of the vitamin D receptor triggers senescence in chronic myeloid leukemia via DDIT4-mediated DNA damage.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy driven by the fusion gene BCR::ABL1. Drug resistance to tyrosine kinase inhibitors (TKIs), due to BCR::ABL1 mutations and residual leukemia stem cells (LSCs), remains a major challenge in CML treatment. Here, we revealed the requirement of the vitamin D receptor (VDR) in the progression of CML. VDR was upregulated by BCR::ABL1 and highly expressed in CML cells. Interestingly, VDR knockdown inhibited the proliferation of CML cells driven by both BCR::ABL1 and TKI-resistant BCR::ABL1 mutations. Mechanistically, VDR transcriptionally regulated DDIT4 expression; reduced DDIT4 levels upon VDR knockdown triggered DNA damage and senescence via p53 signaling activation in CML cells. Furthermore, VDR deficiency not only suppressed tumor burden and progression in primary CML mice but also reduced the self-renewal capacity of CML-LSCs. Together, our study demonstrated that targeting VDR is a promising strategy to overcome TKI resistance and eradicate LSCs in CML.

Circ_SIRT1 upregulates ATG12 to facilitate Imatinib resistance in CML through interacting with EIF4A3

Imatinib is the current gold standard for patients with chronic myeloid leukemia (CML). However, the primary and acquired drug resistance seriously limits the efficacy. To identify novel therapeutic target in Imatinib-resistant CML is of crucial clinical significance. CircRNAs have been demonstrated the essential regulatory roles in the progression and drug resistance of cancers. In this study, we identified a novel circRNA (circ_SIRT1), derived from the SIRT1, which is up-regulated in CML. The high expression of circ_SIRT1 is correlated with drug resistance in CML. Knockdown of circ_SIRT1 regulated K562/R cells viability, invasion and apoptosis. Besides, the inhibition of circ_SIRT1 attenuated autophagy level and reduced IC50 to Imatinib of K562/R cells. Mechanistically, circ_SIRT1 directly binds to the transcription factor Eukaryotic Translation Initiation Factor 4A3(EIF4A3) and regulated EIF4A3-mediated transcription of Autophagy Related 12 (ATG12), thereby affecting Imatinib resistance and autophagy level. Overexpression of ATG12 reversed the regulative effects induced by knockdown of circ_SIRT1. Taken together, our findings revealed circ_SIRT1 acted as a potential tumor regulator in CML and unveiled the underlying mechanism on regulating Imatinib resistance. circ_SIRT1 may serve as a novel therapeutic target and provide crucial clinical implications for Imatinib-resistant CML treatment.

Retracted: lncRNA HOTTIP Recruits EZH2 to Inhibit PTEN Expression and Participates in IM Resistance in Chronic Myeloid Leukemia.

[This retracts the article DOI: 10.1155/2022/9993393.].