Imatinib-resistant Cell Lines Research Articles

Synthesis and anticancer activity of cinnoline sulphonamides and 4‐heteroyclic derivatives: Cross‐coupling approach

AbstractA series of novel cinnoline sulphonamide derivatives and 4‐substuted cinnoline derivatives (13a‐h, 16a‐h and 17–31 total 33 analogues) were designed based on scaffold hopping techniques and evaluated for their antileukemic activity on wild type K562 as well as imatinib resistant cell lines (K562‐IR1 and K562‐IR2). Out of 33 analogues, five compounds (19, 22, 23, 28, and 31) exhibited potent antileukemic activity. An easy and efficient approach to synthesize numerous cinnoline derivatives by the various cross‐coupling reactions of 4‐chlorocinnoline has been developed and also demonstrated that metal‐free cross couplings give high product yield compared with metal catalyzed cross couplings.

Abstract 3142: NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models

Abstract c-Kit (CD117) is a type III receptor tyrosine kinase (RTK), which is known to be activated upon binding to its cognate ligand stem cell factor (SCF) to induce critical signaling pathways involved in cell proliferation, survival, and differentiation. Indicative of poor prognosis, overexpression and activating mutations of c-Kit have been implicated in a variety of human cancers including small cell lung cancer (SCLC), gastrointestinal stromal tumors (GIST), and acute myelogenous leukemia (AML). While imatinib, a small molecule c-Kit inhibitor, has been a beneficial treatment for GIST, resistance eventually develops to the drug, moreover, shows limited efficacy in c-Kit overexpressing tumors such as SCLC. Herein, we developed NN3201, a novel c-Kit targeting antibody-drug conjugate (ADC), comprised of fully human 2G4 antibody with a picomolar binding affinity to c-Kit and cytotoxic monomethyl auristatin E (MMAE) as the payload. NN3201 displayed potent in vitro cytotoxicity in various wild-type and mutant c-Kit positive SCLC and GIST cell lines with IC50 in the nanomolar range. In addition, NN3201 was rapidly internalized and inhibited c-Kit-mediated signaling., NN3201 treatment resulted in the increased proportions of the sub G1 and G2/M in the c-Kit positive cancer cell lines, indicating that release of MMAE induced cell cycle arrest. Moreover, bystander effect was confirmed by co-culturing c-Kit high and negative cell lines. Most importantly, in mouse xenograft models, NN3201 showed a remarkable tumor growth inhibition of several wild type, mutant c-Kit positive, and imatinib resistant SCLC and GIST cell lines. Enhanced anti-tumor activity and delayed tumor growth were observed in an SOC-treated (etoposide and carboplatin) SCLC xenograft model, by NN3201 in comparison to second line SOC (topotecan or irinotecan). In the GLP study, repeated intravenous administration of NN3201 was well tolerated at all doses (0.5, 1, 2 mg/kg Q3Wx3) without unscheduled death. Dose-dependent, yet transient, cytotoxicity of NN3201 was observed in the bone marrow. We confirmed HNSTD (Highest Non-Severely Toxic Dose) of NN3201 to be 2 mg/kg through exploratory and GLP repeat dose toxicity studies as no serious irreversible toxicity was observed. Accumulating data suggest that NN3201 is a promising therapeutic alternative for the treatment of SCLC and GIST regardless of wild-type or activating mutations in c-Kit. Citation Format: Chansik Kim, Jin Gu Cho, TaeMin Wi, Jiwoo Moon, Han-Jik Ko, Jina Lee, Jin Woo Park, Yeonjy Lee, Sanghee Kim, Jin-Ock Kim, Jaeyoung Song, Sun-Hwa Lee, Sang Gyu Park. NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models [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 3142.

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.

Metformin exerts antileukemic effects by modulating lactate metabolism and overcomes imatinib resistance in chronic myelogenous leukemia.

Imatinib is the frontline treatment option in treating chronic myelogenous leukemia (CML). Hitherto, some patients relapse following treatment. Biochemical analysis of a panel of clonally derived imatinib-resistant cells revealed enhanced glucose uptake and ATP production, suggesting increased rates of glycolysis. Interestingly, increased lactate export was also observed in imatinib-resistant cell lines. Here, we show that metformin inhibits the growth of imatinib-resistant cell lines as well as peripheral blood mononuclear cells isolated from patients who relapsed following imatinib treatment. Metformin exerted these antiproliferative effects by inhibiting MCT1 and MCT4, leading to the inhibition of lactate export. Furthermore, glucose uptake and ATP production were also inhibited following metformin treatment due to the inhibition of GLUT1 and HK-II in an AMPK-dependent manner. Our results also confirmed that metformin-mediated inhibition of lactate export and glucose uptake occurs through the regulation of mTORC1 and HIF-1α. These results delineate the molecular mechanisms underlying metabolic reprogramming leading to secondary imatinib resistance and the potential of metformin as a therapeutic option in CML.

Abstract 4872: DCC-3116, a first-in-class selective ULK1/2 inhibitor of autophagy, in combination with the KIT inhibitor ripretinib induces complete regressions in GIST preclinical models

Abstract Background: Cancer cells activate autophagy as an adaptive stress response (ASR) mechanism to therapies targeting the RTK/RAS/MAPK/PI3K pathways, limiting antitumor response. Autophagy is initiated through ULK1/2 kinases and is triggered by inhibitors of the MAPK and PI3K pathways. Most gastrointestinal stromal tumors (GIST) are driven by mutations in KIT kinase. KIT signals through MAPK/PI3K pathways, suppressing ULK1/2 kinases and autophagy1,2,3. Inhibition of mutant KIT reverses this suppression, activating autophagy and cancer cell survival. Approved therapies for GIST include imatinib, sunitinib, regorafenib, ripretinib, and avapritinib. Treatment with these inhibitors is initially successful, but drug resistance can develop either through KIT secondary mutations or ASR pathways including autophagy. DCC-3116 is a selective, potent, first-in-class investigational inhibitor of the ULK1/2 in clinical development in combination with targeted therapies that activate the autophagic ASR pathway. Herein we demonstrate that ULK1/2 and autophagy are activated upon treatment with ripretinib in KIT mutant GIST models A combination of ripretinib with DCC-3116 inhibits autophagy in vitro and leads to complete tumor regressions in preclinical models of GIST. Methods: Inhibition of ULK1/2 in cell assays was measured using an ELISA for the ULK substrate phospho-ATG13 (pATG13). Autophagic flux was measured by monitoring mCherry/GFP tagged LC3 protein in GIST cells. Xenograft studies were performed at CROs. Results: Ripretinib treatment led to the activation of ULK1/2 by 2-3-fold in mutant KIT GIST cell lines. DCC-3116 inhibited both ripretinib-induced and basal pATG13 with IC50 values of 12-32 nM. Treatment of GIST-T1 cells with ripretinib increased autophagic flux 3-fold. DCC-3116 potently inhibited flux with an IC50 value of 38 nM. Ripretinib also induced pATG13 and autophagic flux (1.5-2.5-fold) in multiple imatinib-resistant cell lines, which was inhibited by DCC-3116 with IC50 values between 8-189 nM. In a GIST T1 PK/PD model, DCC-3116 inhibited ULK1/2-mediated pATG13. The combination of DCC-3116 with ripretinib resulted in complete tumor regressions in comparison to single agent treatment in GIST preclinical models. Conclusions: These data demonstrate preclinically that, like other receptor tyrosine kinase inhibitors1, ripretinib activates ULK1/2-mediated autophagy as an ASR resistance mechanism which is inhibited by DCC-3116, providing the rationale to study the combination of DCC-3116 with ripretinib in GIST patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04892017).

Co-targeting of ACK1 and KIT triggers additive anti-proliferative and -migration effects in imatinib-resistant gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GIST) harbor mutated receptor tyrosine kinase (RTK) KIT/PDGFRA, which provides an attractive therapeutic target. However, a majority of GISTs ultimately develop resistance to KIT/PDGFRA inhibitor imatinib, multiple therapeutic targets will be identified as a reasonable strategy in imatinib-resistant GISTs. Biological mechanisms of non-RTK activated CDC42 associated kinase 1 (ACK1) are still unclear, which has been found to be activated in GISTs. In the current report, ACK1 overexpression is demonstrated in GIST cell lines and biopsies. RNA-seq analysis and immunoblotting show that ACK1 expression is dependent on imatinib treatment time in GIST-T1 cell line. The colocalization/complex of KIT and ACK1 in GIST cells are observed, and ACK1 activation is in a partially KIT and CDC42 dependent manner. Treatment with a specific ACK1 inhibitor AIM-100 or ACK1 siRNA, mildly suppresses cell viability, but markedly inhibits cell migration in imatinib sensitive and in imatinib resistant GIST cell lines, which is associated with inactivation of PI3K/AKT/mTOR and RAF/MAPK signaling pathways, and inhibition of epithelial-mesenchymal transition, evidencing upregulation of E-cadherin and downregulation of ZEB1, N-cadherin, vimentin, snail, and/or β-catenin after treatment with AIM-100 or ACK1/CDC42 shRNAs. Combination inhibition of ACK1 and KIT results in additive effects of anti-proliferation and pro-apoptosis as well as cell cycle arrest, and inhibition of invasiveness and migration in vitro and in vivo, compared to either intervention alone through dephosphorylation of KIT downstream intermediates (AKT, S6, and MAPK). Our data suggest that co-targeting of ACK1 and KIT might be a novel therapeutic strategy in imatinib-resistant GIST.

Combination of pimitespib (TAS-116) with sunitinib is an effective therapy for imatinib-resistant gastrointestinal stromal tumors.

Despite the effectiveness of imatinib, most gastrointestinal stromal tumors (GISTs) develop resistance to the treatment, mainly due to the reactivation of KIT tyrosine kinase activity. Sunitinib, which inhibits the phosphorylation of KIT and vascular endothelial growth factor (VEGF) receptor, has been established as second-line therapy for GISTs. The recently-developed heat shock protein 90 (HSP90) inhibitor pimitespib (PIM; TAS-116) demonstrated clinical benefits in some clinical trials; however, the effects were limited. The aim of our study was therefore to clarify the effectiveness and mechanism of the combination of PIM with sunitinib for imatinib-resistant GISTs. We evaluated the efficacy and mechanism of the combination of PIM with sunitinib against imatinib-resistant GIST using imatinib-resistant GIST cell lines and murine xenograft models. In vitro analysis demonstrated that PIM and sunitinib combination therapy strongly inhibited growth and induced apoptosis in imatinib-resistant GIST cell lines by inhibiting KIT signaling and decreasing auto-phosphorylated KIT in the Golgi apparatus. In addition, PIM and sunitinib combination therapy enhanced antitumor responses in the murine xenograft models compared to individual therapies. Further analysis of the xenograft models showed that the combination therapy not only downregulated the KIT signaling pathway but also decreased the tumor microvessel density. Furthermore, we found that PIM suppressed VEGF expression in GIST cells by suppressing protein kinase D2 and hypoxia-inducible factor-1 alpha, which are both HSP90 client proteins. In conclusion, the combination of PIM and sunitinib is effective against imatinib-resistant GIST via the downregulation of KIT signaling and angiogenic signaling pathways.

Alvespimycin Inhibits Heat Shock Protein 90 and Overcomes Imatinib Resistance in Chronic Myeloid Leukemia Cell Lines.

Heat shock protein 90 (HSP90) facilitates folding and stability and prevents the degradation of multiple client proteins. One of these HSP90 clients is BCR-ABL, the oncoprotein characteristic of chronic myeloid leukemia (CML) and the target of tyrosine kinase inhibitors, such as imatinib. Alvespimycin is an HSP90 inhibitor with better pharmacokinetic properties and fewer side effects than other similar drugs, but its role in overcoming imatinib resistance is not yet clarified. This work studied the therapeutic potential of alvespimycin in imatinib-sensitive (K562) and imatinib-resistant (K562-RC and K562-RD) CML cell lines. Metabolic activity was determined by the resazurin assay. Cell death, caspase activity, mitochondrial membrane potential, and cell cycle were evaluated by means of flow cytometry. Cell death was also analyzed by optical microscopy. HSPs expression levels were assessed by western blotting. Alvespimycin reduced metabolic activity in a time-, dose-, and cell line-dependent manner. Resistant cells were more sensitive to alvespimycin with an IC50 of 31 nM for K562-RC and 44 nM for K562-RD, compared to 50 nM for K562. This drug induced apoptosis via the mitochondrial pathway. In K562 cells, alvespimycin induced cell cycle arrest in G0/G1. As a marker of HSP90 inhibition, a significant increase in HSP70 expression was observed. Our results suggest that alvespimycin might be a new therapeutic approach to CML treatment, even in cases of resistance to imatinib.

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.

The Activity of Novel BCR-ABL Small-Molecule Degraders Containing Pyrimidine Rings and Their Role in Overcoming Drug Resistance.

Inducing protein degradation by proteolysis-targeting chimeras (PROTACs) has gained tremendous momentum in the field for its promise in the discovery and development of new therapies. Based on our previously reported PROTAC BCR-ABL degraders, we designed and synthesized additional 4 PROTAC compounds with a novel linker that contains pyrimidine rings. Molecular and cellular studies have shown that different linkers affect the degradation activity of small-molecule degraders on the target protein of BCR-ABL. We screened out a lead compound, DMP11, with stable physicochemical properties and high activity. Preliminary evaluation of its pharmacodynamics in vitro model showed that it has a good inhibitory effect on imatinib-resistant chronic myeloid leukemia cell lines, as has been shown in animal models. Our preliminary research into the mechanism of DMP11 found that DMP11 can overcome drug resistance by simultaneously inhibiting the targets of BCR-ABL and SRC-family kinase (SFK).

Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells.

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20–30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinib-resistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 μM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.

[The Effect of hnRNPK/Beclin1 Signaling on the Drug Resistance of Imatinib in Ph+ Leukemia].

To explore the effect of hnRNPK/Beclin1 signaling on the drug resistance of imatinib in Ph+ leukemia. Expression level of hnRNPK was verified in the imatinib resistant and sensitive Ph+ leukemia cell lines by using Western blot. hnRNPK expression was down-regulated by using RNAi. Expression level of LC3I/II and Beclin1 were detected by Western blot and the sensitivity of imatinib was analyzed by CCK-8 assay before and after modulation of hnRNPK expression. hnRNPK showed overexpressed in imatinib resistant leukemia cell line. After the expression level of hnRNPK was down-regulated by RNAi, the sensitivity of drug resistance lines to imatinib restored, while the expression level of LC3I/II and Beclin1 were consistant with the modulation of hnRNPK expression. hnRNP K/Beclin1 signaling may be involved in the development of imatinib resistance in Ph+ leukemia through the regulation of autophagy.

Combination of Elacridar with Imatinib Modulates Resistance Associated with Drug Efflux Transporters in Chronic Myeloid Leukemia.

Multidrug resistance (MDR) development has emerged as a complication that compromises the success of several chemotherapeutic agents. In chronic myeloid leukemia (CML), imatinib resistance has been associated with changes in BCR-ABL1 and intracellular drug concentration, controlled by SLC and ABC transporters. We evaluate the therapeutic potential of a P-glycoprotein and BCRP inhibitor, elacridar, in sensitive (K562 and LAMA-84) and imatinib-resistant (K562-RC and K562-RD) CML cell lines as monotherapy and combined with imatinib. Cell viability was analyzed by resazurin assay. Drug transporter activity, cell death, cell proliferation rate, and cell cycle distribution were analyzed by flow cytometry. Both resistant models presented an increased activity of BCRP and P-gP compared to K562 cells. Elacridar as monotherapy did not reach IC50 in any CML models but activated apoptosis without cytostatic effect. Nevertheless, the association of elacridar (250 nM) with imatinib overcomes resistance, re-sensitizing K562-RC and K562-RD cells with five and ten times lower imatinib concentrations, respectively. Drug combination induced apoptosis with increased cleaved-caspases-3, cleaved-PARP and DNA damage, reduced cell proliferation rate, and arrested CML cells in the S phase. These data suggest that elacridar combined with imatinib might represent a new therapeutic option for overcoming TKI resistance involving efflux transporters.

Identification of Novel Imatinib-Resistant Genes in Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are common ICC precursor sarcomas, which are considered to be a potential malignant mesenchymal tumor driven by specific KIT or PDGFRA signals in the gastrointestinal tract. The standard treatment for GIST without metastasis is surgical resection. GIST with metastasis is usually treated with tyrosine kinase inhibitors (TKIs) only but cannot be cured. The TKI imatinib is the main drug of GIST drug therapy. In adjuvant therapy, the duration of imatinib adjuvant therapy is 3 years. It has been proved that imatinib can improve the overall survival time (OS). However, many GIST patients develop drug resistance due to the long-term use of imatinib. We were forced to look for new strategies to treat GIST. The purpose of the current academic work is to study the drug-resistant genes of imatinib and their potential mechanisms. A total of 897 differentially expressed genes (DEGs) were found between imatinib-sensitive cell line GIST882 and imatinib-resistant cell line GIST430 by RNA sequencing (RNA-seq). After analyzing the DEGs, 10 top genes were selected (NDN, FABP4, COL4A1, COLEC11, MEG3, EPHA3, EDN3, LMO3, RGS4, and CRISP2). These genes were analyzed by RT-PCR, and it was confirmed that the expression trend of FABP4, COL4A1, and RGS4 in different imatinib-resistant cell lines was in accord with the GEO database. It is suggested that these genes may play a potential role in the clinical diagnosis and treatment of imatinib resistance in GIST.

Metformin and Thymoquinone Synergistically Inhibit Proliferation of Imatinib-Resistant Human Leukemic Cells.

Chemotherapy resistance is one of the major challenges in cancer treatment, including leukemia. A massive array of research is evaluating combinations of drugs directed against different intracellular signaling molecules to overcome cancer resistance, increase therapy effectiveness, and decrease its adverse effects. Combining chemicals with proven safety profiles, such as drugs already used in therapy and active substances isolated from natural sources, could potentially have superior effects compared to monotherapies. In this study, we evaluated the effects of metformin and thymoquinone (TQ) as monotherapy and combinatorial treatments in chronic myeloid leukemia (CML) cell lines sensitive and resistant to imatinib therapy. The effects were also evaluated in primary monocytic acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) cells. Both compounds induced a dose- and time-dependent decrease of viability and proliferation in tested cells. Metformin had similar IC50 values in imatinib-sensitive and imatinib-resistant cell lines. IC50 values of TQ were significantly higher in imatinib-resistant cells, but with a limited resistance index (2.4). Synergistic effects of combinatorial treatments were observed in all tested cell lines, as well as in primary cells. The strongest synergistic effects were observed in the inhibition of imatinib-resistant cell line proliferation. Metformin and TQ inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and induced apoptosis in tested cell lines and primary cells. The enhanced effects of combinatorial treatments on the induction of apoptosis were more dominant in imatinib-resistant compared to imatinib-sensitive CML cells. Primary cells were more sensitive to combinatorial treatments compared to cell lines. A combination of 1.25 mM metformin and 0.625 µM TQ increased the levels of cleaved poly (ADP-ribose) polymerase (PARP), decreased the levels of proliferation regulatory proteins, and inhibited protein kinase B (Akt) and NF-κB signaling in primary CLL cells. This study demonstrates that combinatorial treatments of imatinib-resistant malignant clones with metformin and TQ by complementary intracellular multi-targeting represents a promising approach in future studies.

Discovery of Anticancer Hybrid Molecules by Supervised Machine Learning Models and in Vitro Validation in Drug Resistant Chronic Myeloid Leukemia Cells.

The concept of hybrid drugs for targeting multiple aberrant pathways of cancer, by combining the key pharmacophores of clinically approved single-targeted drugs, has emerged as a promising approach for overcoming drug-resistance. Here, we report the design of unique hybrid molecules by combining the two pharmacophores of clinically approved BCR-ABL inhibitor (ponatinib) and HDAC inhibitor (vorinostat) and results of in vitro studies in drug-resistant CML cells. Robust 2D-QSAR and 3D-pharmacophore machine learning supervised models were developed for virtual screening of the hybrid molecules based on their predicted BCR-ABL and HDAC inhibitory activity. The developed 2D-QSAR model showed five information rich molecular descriptors while the 3D-pharmacophore model of BCR-ABL showed five different chemical features (hydrogen bond acceptor, donor, hydrophobic group, positive ion group, and aromatic rings) and the HDAC model showed four different chemical features (hydrogen bond acceptor, donor, positive ion group, and aromatic rings) for potent BCR-ABL and HDAC inhibition. Virtual screening of the 16 designed hybrid molecules identified FP7 and FP10 with better potential of inhibitory activity. FP7 was the most effective molecule with predicted IC50 using the BCR-ABL based 2D-QSAR model of 0.005 μM and that of the HDAC model of 0.153 μM, and that using the BCR-ABL based 3D-pharmacophore model was 0.02 μM and that with HDAC model was 0.014 μM. In vitro study (dose-response relationship) of FP7 in wild type and imatinib-resistant CML cell lines harboring Thr315Ile or Tyr253His mutations showed growth inhibitory IC50 values of 0.000 16, 0.0039, and 0.01 μM, respectively. This molecule also showed better biocompatibility when tested in whole blood and in PBMCs as compared to ponatinib or vorinostat.

Impact of metformin on tyrosine kinase inhibitor response in chronic myeloid leukemia.

ObjectiveOral tyrosine kinase inhibitors (TKIs) are first line therapy for chronic myeloid leukemia (CML). A complete cytogenetic response (CCyR) correlates with increased overall survival, however only 66%–88% of patients achieve CCyR after one year of TKI treatment. Because TKI therapy alone cannot eliminate CML stem cells, strategies aimed at achieving faster and deeper responses are needed to improve long-term survival. Metformin is a widely prescribed glucose-lowering agent for patients with diabetes and in preclinical studies, has been shown to suppress cell viability, induce apoptosis, and downregulate the mTORC1 signaling pathway in imatinib resistant CML cell lines (K562R). This study aims to investigate the utility of metformin added to TKI therapy in patients with CML.Data SourcesAn observational study at an academic medical center (Salt Lake City, UT) was performed for adults with newly diagnosed, chronic-phase CML to evaluate attainment of CCyR from TKI therapy with or without concomitant metformin use. Descriptive analyses were used to describe baseline characteristics and attainment of response to TKI therapy.Data SummaryFifty-nine patients were evaluated. One hundred percent (5 of 5) in the metformin group and 73.6% (39 of 54) in the non-metformin group achieved CCyR. Approximately 20% of patients in both groups relapsed (defined by a loss of CCyR during study) after a median 34.5 months of follow-up.ConclusionsFuture research is warranted to validate these findings and determine the utility of metformin added to TKI therapy.

[The Relationship between MicroRNA Expression Profiling in Imatinib-Resistant Cell Line K562/G and Potential Mechanism through FOXO3/Bcl-6 Signaling Pathway].

To investigate the drug resistant related FOXO3/Bcl-6 signaling pathway in K562/G cell line and its related microRNA(miRNA) mechanisms. The drug resistance potency of imatinib on K562/G was detected by MTT assay. The expression of FOXO3 and Bcl-6 proteins in K562 and K562/G cells was detected by Western blot. Real-time PCR (RT-PCR) was used to detect the expression of FOXO3 and Bcl-6 mRNA. The miRNA expression profiling in K562 and K562/G cells was analyzed by microarray technique, and the miRNA targeted to FOXO/Bcl-6 signaling pathway was identified. The expression of FOXO3 and Bcl-6 protein was significantly increased in K562/G cells as compared with that in K562 cells (P<0.01), the expression level of Bcl-6 mRNA showed no increase in K562/G cells. However, FOXO3 mRNA was up-regulated in K562/G cells (P<0.05). MiRNA microarray results showed that 109 miRNAs were expressed differentially in K562 and K562/G cells. The expression of 81 miRNAs were up-regulated while 28 miRNAs were down-regulated. Through reverse prediction by bioinformatics, miR-6718-5p, miR-5195-5p, miR-4711-3p, miR-4763-5p, miR-4664-5p and miR-3176 were related to FOXO/Bcl-6 signaling pathway. The FOXO3/Bcl-6 signaling pathway contributes to imatinib resistance in K562/G cell line, and the miRNA expression profiles showed significant differences between K562/G and K562 cells.

Research on the Mechanism of SIRT1/AMPK Inducing Resistance to Imatinib in K562 Cells By Inhibiting Srebp to Promote Lipid Metabolism

Background Leukemia stem cells (LSC) have the characteristics of independent of BCR-ABL kinase activity and metabolic reprogramming to support self-renewal. They are the root cause of the occurrence of chronic myeloid leukemia (CML), mediating TKI resistance and leading to clonal evolution. LSC self-renewal depends on the activity of SIRT1, the enhanced of SIRT1 induces LSC mitochondrial metabolic reprogramming. Studies have shown that SIRT1 and abnormally activated lipid metabolism are important reasons for inducing CML resistance to imatinib.Objective Study the mechanism of SIRT1 regulating lipid metabolism and intervention strategies under imatinib therapy.Method Liquid chromatography coupled with mass spectrometry (LC-MS) were used to analysis the lipid metabolism in imatinib-resistant CML cell line K562-R under low-glucose conditions. RT-qPCR and Western blot were used to detect the key enzymes related to lipid metabolism, and the SIRT1 inhibitor TV-6 was given to study the regulation mechanism of SIRT1 on lipid metabolism.Result Firstly, we found that the glycolysis/gluconeogenesis pathway and fatty acid metabolism were abnormal in K562-R cells by metabolomics, compared with normal culture conditions, the expression of SIRT1 and AMPK were significantly increased, the expression of fatty acid synthesis-related genes (SREBP, FASN, ACC, SCD1) was significantly reduced, and the fatty acid β oxidation rate-limiting enzyme CPT-1 is significantly increased inK562-R cells under low-glucose conditions. Therefore, in the low-glucose environment, the expression of the above genes was reversed after the application of the SIRT1 inhibitor TV-6.Conclusion K562-R imatinib-resistant cells can down-regulate the expression of SREBP through up-regulated SIRT1/AMPK pathway under low-glucose conditions, thereby inhibiting the expression of transcription factors involved in lipogenesis, reducing fatty acid synthesis, and providing the imatinib-resistant cells with the energy which required for proliferation.Keyword Chronic myeloid leukemia; Metabolomics; Lipid metabolism; SIRT1/AMPK DisclosuresNo relevant conflicts of interest to declare.

Altered apoptotic protein expressions characterize the survival of Bcr-Abl-independent drug-resistant chronic myeloid leukemia cell line

Apoptosis is a programmed cellular process that occurs in pathological and physiological pathways and it is one of the most studied topics in cell biology. To understand the underlying mechanism of apoptosis plays an important role in the molecular pathogenesis of many diseases including cancers. Chronic myeloid leukemia (CML) is a clonal myeloproliferative malignancy arising from the neoplastic transformation of the hematopoietic stem cell. Here, we used a Bcr-Abl-independent, imatinib-resistant K562 subpopulation (K562-IR) generated and characterized earlier in our laboratory. We showed that the proteins Bcl-2, Bim, RIP, p-MAPK(Erk1/Erk2) and NF-кB which play critical roles in cell death pathways are downregulated, Lamin A/C protein expression is upregulated in K562-IR derivative cells compared to K562 ancestral cells. Our data provide new information on the expression of apoptotic molecules in a Bcr-Abl-independent imatinib-resistant CML cell line.