Imatinib Uptake Research Articles

Renal Pharmacokinetic Adaptation to Cholestasis Causes Increased Nephrotoxic Drug Accumulation by Mrp6 Downregulation in Mice

In hepatic dysfunction, renal pharmacokinetic adaptation can be observed, although information on the changes in drug exposure and the interorgan regulation of membrane transporters in kidney in liver diseases is limited. This study aimed to clarify the effects of renal exposure to nephrotoxic drugs during cholestasis induced by bile duct ligation (BDL). Among the 11 nephrotoxic drugs examined, the tissue accumulation of imatinib and cisplatin in kidney slices obtained from mice 2 weeks after BDL operation was higher than that in sham-operated mice. The uptake of imatinib in the kidney slices of BDL mice was slightly higher, whereas its efflux from the slices was largely decreased compared to that in sham-operated mice. Proteomic analysis revealed a reduction in renal expression of the efflux transporter multidrug resistance-associated protein 6 (Mrp6/Abcc6) in BDL mice, and both imatinib and cisplatin were identified as Mrp6 substrates. Survival probability after cisplatin administration was reduced in BDL mice. In conclusion, the present study demonstrated that BDL-induced cholestasis leads to the downregulation of the renal basolateral efflux transporter Mrp6, resulting in drug accumulation in renal cells and promoting drug-induced renal injury.

OCT-1 Expression in Patients with Chronic Myeloid Leukemia: A Comparative Analysis with Respect to Response to Imatinib Treatment.

The introduction of tyrosine kinase inhibitors (TKI) has resulted in a significant improvement in the treatment of CML patients. However, some CML patients are resistant to imatinib therapy, the initial TKI therapy in the CML. Therefore, it is important to find prognostic markers for resistance. The OCT-1 gene involved in imatinib uptake is also suspected to cause imatinib resistance. The aim of this study was to investigate the role of OCT-1 in imatinib resistance by comparing OCT-1 expression levels in imatinib resistant and imatinib sensitive patients with chronic myeloid leukemia (CML). This study was conducted on 101 patients with CML [imatinib sensitive (n = 51) and imatinib resistant (n = 50)] who were treated with imatinib. Gene expression analysis was done using QRT-PCR. The relative expression levels of OCT-1 were calculated using 2(-ΔΔCT) method. OCT1 mRNA expression levels were 0.149 (0.011-2.532) and 0.119 (0.008-2.868) in imatinib-sensitive group and imatinib-resistant group, respectively. OCT-1 expression levels were not significantly different in the imatinib-sensitive group when compared to imatinib resistant group (p > 0.05). OCT-1 expression was also similar in BCR-ABL1 kinase domain mutation positive and negative cases (p > 0.05). The imatinib-resistant group had a higher rate of hydroxyurea or interferon-alpha treatment prior to imatinib therapy and a lower rate for first-line imatinib as the only treatment than the imatinib-sensitive group (p = 0.002 and p = 0.002, respectively). According to the results of our study, OCT-1 does not have a biomarker feature in the evaluation of imatinib response. In addition, the study should be performed in larger patient groups.

Targeting SOS1 overcomes imatinib resistance with BCR-ABL independence through uptake transporter SLC22A4 in CML

Resistance to the BCR-ABL inhibitor imatinib mesylate poses a major problem for the treatment of chronic myeloid leukemia. Imatinib resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, sometimes there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent imatinib mesylate resistance remains to be elucidated. SOS1, a guanine nucleotide exchange factor for Ras protein, affects drug sensitivity and resistance to imatinib. The depletion of SOS1 markedly inhibits cell growth either in vitro or in vivo and significantly increases the sensitivity of chronic myeloid leukemia cells to imatinib. Furthermore, LC-MS/MS and RNA-seq assays reveal that SOS1 negatively regulates the expression of SLC22A4, a member of the carnitine/organic cation transporter family, which mediates the active uptake of imatinib into chronic myeloid leukemia cells. HPLC assay confirms that intracellular accumulation of imatinib is accompanied by upregulation of SLC22A4 through SOS1 inhibition in both sensitive and resistant chronic myeloid leukemia cells. BAY-293, an inhibitor of SOS1/Ras, was found to depress proliferation and colony formation in chronic myeloid leukemia cells with resistance and BCR-ABL independence. Altogether these findings indicate that targeting SOS1 inhibition promotes imatinib sensitivity and overcomes resistance with BCR-ABL independence by SLC22A4-mediated uptake transport.

Thymoquinone chemosensitizes human colorectal cancer cells to imatinib via uptake/efflux genes modulation.

Imatinib (IM) is a pharmaceutical drug that inhibits tyrosine kinase enzymes that are responsible for the activation of many proteins by signal transduction cascades as c-Abl, c-Kit and the platelet-derived growth factor (PDGF) receptor. Thymoquinone (TQ) is an active constituent of Nigella sativa seeds. Thymoquinone benefits are attributed to its medicinal uses as antioxidant, anticancer and antimicrobial agent. This study aimed to investigate the impact of using TQ with IM in the HCT116 human colorectal cancer cell line model. The HCT116 cells were treated with IM or/and TQ in non-constant ratios, in which the fixed concentrations of TQ (5, 10 or 20µmol/L) were co-treated with various concentrations of IM (7.5-120µmol/L) for 24, 48 and 72hours. Imatinib-TQ interaction was analysed using CompuSyn software. The IC50 values for IM were 105, 72μmol/L after 48 and 72hours, respectively, and were significantly reduced to 7.3, 7 and 5.5μmol/L after combination with TQ (10μmol/L) and to 5.8, 5.6 and 4.6μmol/L after combination with TQ (20μmol/L) to 24, 48 and 72hours, respectively. The combination index (CI) and dose reduction index (DRI) values indicate a significant synergism in HCT-116 cells at different treatment time points. Thymoquinone significantly enhances the cellular uptake of IM in HCT116 cells in a time and concentration-dependent manner. A significant downregulation in ATP-binding cassette (ABC) subfamily B member 1 (ABCB1), ABC subfamily G member 2 (ABCG2) and human organic cation transporter 1 (hOCT1) genes was observed in the cells exposed to IM+TQ combination as compared to IM alone, which resulted in a substantial elevation in uptake/efflux ratio in combination group. In conclusion, TQ potentiates IM efficacy on HCT116 cells via uptake/efflux genes modulation.

High-resolution AP-SMALDI MSI as a tool for drug imaging in Schistosoma mansoni

Schistosoma mansoni is a parasitic flatworm causing schistosomiasis, an infectious disease affecting several hundred million people worldwide. Schistosomes live dioeciously, and upon pairing with the male, the female starts massive egg production, which causes pathology. Praziquantel (PZQ) is the only drug used, but it has an inherent risk of resistance development. Therefore, alternatives are needed. In the context of drug repurposing, the cancer drug imatinib was tested, showing high efficacy against S. mansoni in vitro. Besides the gonads, imatinib mainly affected the integrity of the intestine in males and females. In this study, we investigated the potential uptake and distribution of imatinib in adult schistosomes including its distribution kinetics. To this end, we applied for the first time atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) for drug imaging in paired S. mansoni. Our results indicate that imatinib was present in the esophagus and intestine of the male as early as 20 min after in vitro exposure, suggesting an oral uptake route. After one hour, the drug was also found inside the paired female. The detection of the main metabolite, N-desmethyl imatinib, indicated metabolization of the drug. Additionally, a marker signal for the female ovary was successfully applied to facilitate further conclusions regarding organ tropism of imatinib. Our results demonstrate that AP-SMALDI MSI is a useful method to study the uptake, tissue distribution, and metabolization of imatinib in S. mansoni. The results suggest using AP-SMALDI MSI also for investigating other antiparasitic compounds and their metabolites in schistosomes and other parasites.Graphical abstract

Comprehensive in-silico analysis of damage associated SNPs in hOCT1 affecting Imatinib response in chronic myeloid leukemia

Non-synonymous single nucleotide polymorphisms (nsSNPs) in hOCT1 (encoded by SLC22A1 gene) are expected to affect Imatinib uptake in chronic myeloid leukemia (CML). In this study, sequence homology-based genetic analysis of a set of 270 coding SNPs identified 18 nsSNPs to be putatively damaging/deleterious using eight different algorithms. Subsequently, based on conservation of amino acid residues, stability analysis, posttranscriptional modifications, and solvent accessibility analysis, the possible structural-functional relationship was established for high-confidence nsSNPs. Furthermore, based on the modeling results, some dissimilarities of mutant type amino acids from wild-type amino acids such as size, charge, interaction and hydrophobicity were revealed. Three highly deleterious mutations consisting of P283L, G401S and R402G in SLC22A1 gene that may alter the protein structure, function and stability were identified. These results provide a filtered data to explore the effect of uncharacterized nsSNP and find their association with Imatinib resistance in CML.

AB0626 IMATINIB FOR THE TREATMENT OF SYSTEMIC SCLEROSIS: RATIONALE, CLINICAL EVIDENCE AND FUTURE DEVELOPMENT

Background:Systemic sclerosis (SSc) is a chronic disorder of connective tissue affecting the skin and internal organs. The molecular mechanisms behind SSc are not entirely understood, but recent advances highlight key signalling pathways1(see Figure 1). Fibrosis disrupts tissue architecture resulting in organ dysfunction. This causes significant morbidity and mortality1, therefore there is a clear need for identifying efficacious antifibrotic treatment.Imatinib is a tyrosine kinase inhibitor with established use in some malignancies, and existing evidence that it can treat SSc2.Objectives:The aim of this literature review is to summarise the current evidence and future developments of imatinib as antifibrotic treatment in SSc.Methods:PubMed headings “systemic sclerosis”, “scleroderma”, “imatinib” and synonyms were used. See Figure 2.Results:Lead author and yearStudy designPopulationIntervention (imatinib dose given daily)ResultsHinchcliff ME. 2016Case study1 female (F) with an 8-month history of diffuse cutaneous systemic sclerosis (dcSSc)400mgSevere adverse events (AEs)Pope J. 2014Early phase proof of concept trial10 with dcSSc.9 given 400mg, 1 placeboPoor drug tolerabilityPrey S. 2012Double blind RCT28 with SSc15 given 400mg, 13 placeboNo improvementKhanna D. 2011Phase I/IIa pilot trial20 with SScUp to 600mgAEs presentPope J. 2011Double blind RCT10 with SSc.9 given 400mg, 1 placeboNo improvement, AEs presentGordon J. 2014Open label, single arm, extension phase clinical trial17 with average disease duration of 3.5 years100-400mg↓ modified Rodnan skin score (mRSS)Fraticelli P. 2014Phase II pilot trial30 with SSc200mg↑ lung functionGuo L. 2012Case series6 F, Chinese pts with SSc200mg↓ mRSS and ↑ lung functionDivekar AA. 2011Single centre, open-label study15 with SSc100mg up to 600mg/day↑ lung functionSpiera RF. 20111-year, phase IIa, single-arm, open-label clinical trial30 with dcSSc400mg↓ mRSS and ↑ lung functionFreyhaus H. 2009Case report58yo F400mg↑ lung functionChung L. 2009Case report2 with early dcSSc200mg↓ mRSS and ↑ lung functionVan Daele PL. 2008Case report69yo F400mg↓ mRSS and ↑ lung functionP. P. Sfikakis. 2008Case report24yo F400mg↓ mRSS and ↑ lung function9 studies showed imatinib had positive efficacy in the treatment of SSc. 5 showed no improvement or adverse effects.Conclusion:Overall, current evidence suggests that imatinib can be a useful drug to improve manifestations of SSc, for some. Despite inconclusive evidence, a dose-dependent relationship seems to exist for imatinib toxicities, with more research needed to ascertain a safe dose.Gene expression profiles may distinguish patients that can benefit from imatinib3. Also, Notch signalling could be exploited to increase imatinib uptake into fibroblasts, thereby increasing efficacy4.

Imatinib Uptake into Cells is Not Mediated by Organic Cation Transporters OCT1, OCT2, or OCT3, But is Influenced by Extracellular pH.

Cancer cells undergo genetic and environmental changes that can alter cellular disposition of drugs, notably by alterations of transmembrane drug transporters expression. Whether the influx organic cation transporter 1 (OCT1) encoded by the gene SLC221A1 is implicated in the cellular uptake of imatinib is still controversial. Besides, imatinib ionization state may be modulated by the hypoxic acidic surrounding extracellular microenvironment. To determine the functional contribution of OCTs and extracellular pH on imatinib cellular disposition. We measured imatinib uptake in two different models of selective OCTs drug transporter expression (transfected Xenopus laevis oocytes and OCT-expressing HEK293 human cells), incubated at pH 7.4 and 6, using specific mass spectrometry analysis. Imatinib cellular uptake occurred independently of OCT1- OCT2- or OCT3-mediated drug transport at pH 7.4. Uptake of the OCTs substrate tetraethylammonium in oocytes remained intact at pH 6, while the accumulation of imatinib in oocytes was 10-fold lower than at pH 7.4, irrespectively of OCTs expressions. In OCT1- and OCT2-HEK cells at pH 6, imatinib accumulation was reduced by 2- 3-fold regardless of OCTs expressions. Since 99.5% of imatinib at pH6 is under the cationic form, the reduced cellular accumulation of imatinib at such pH may be explained by the lower amount of uncharged imatinib remaining for passive diffusion across cellular membrane. Imatinib is not a substrate of OCTs 1-3 while the environmental pH modulates cellular disposition of imatinib. The observation that a slightly acidic extracellular pH influences imatinib cellular accumulation is important, considering the low extracellular pH reported in the hematopoietic leukemia/ cancer cell microenvironment.

Long-Term Patterns of Oral Anticancer Agent Adoption, Duration, and Switching in Patients With CML.

Oral tyrosine kinase inhibitors (TKIs) have been the standard of care for chronic myeloid leukemia (CML) since 2001. However, few studies have evaluated changes in the treatment landscape of CML over time. This study assessed the long-term treatment patterns of oral anticancer therapies among patients with CML. This retrospective cohort study included patients newly diagnosed with CML between January 1, 2000, and December 31, 2016, from 10 integrated healthcare systems. The proportion of patients treated with 5 FDA-approved oral TKI agents-bosutinib, dasatinib, imatinib, nilotinib, and ponatinib-in the 12 months after diagnosis were measured, overall and by year, between 2000 and 2017. We assessed the use of each oral agent through the fourth-line setting. Multivariable logistic regression estimated the odds of receiving any oral agent, adjusting for sociodemographic and clinical characteristics. Among 853 patients with CML, 81% received an oral agent between 2000 and 2017. Use of non-oral therapies decreased from 100% in 2000 to 5% in 2005, coinciding with imatinib uptake from 65% in 2001 to 98% in 2005. Approximately 28% of patients switched to a second-line agent, 9% switched to a third-line agent, and 2% switched to a fourth-line agent. Adjusted analysis showed that age at diagnosis, year of diagnosis, and comorbidity burden were statistically significantly associated with odds of receiving an oral agent. A dramatic shift was seen in CML treatments away from traditional, nonoral chemotherapy toward use of novel oral TKIs between 2000 and 2017. As the costs of oral anticancer agents reach new highs, studies assessing the long-term health and financial outcomes among patients with CML are warranted.

Abstract 2106: Role of organic cation transporter-1 (OCT-1) in drug resistance condition of ph+ chronic myeloid leukemia (CML)

Abstract A solute carrier (SLC) superfamily member, a major influx protein Organic Cation Transporter-1 (OCT-1), reported to be involved in influx of the Imatinib and other drugs in target cells. Despite the excellent responses to Imatinib and other tyrosine kinase inhibitors (TKIs) in CML, around a quarter of patients demonstrate primary resistance or suboptimal response within one year of therapy. Hence, present study mainly emphases the possible translocation mechanism of Imatinib, major TKIs and other chemotherapeutic agents that is mediated by OCT-1 transporter and OCT-1 gene expression level in progressive phases of CML. Homology based comparative modeling of OCT-1 protein, and its in-depth systemic interaction binding pattern analysis with Imatinib and other TKIs were conducted using computational modeling and molecular dynamics studies, respectively. Quantitative expression analysis of OCT-1 has also been evaluated in patients receiving Imatinib alone, hydroxyurea alone or both drug in different CML phases such as chronic, accelerated and blastic crisis phases for both in bone marrow and PBMNC samples (n=120). The interaction profile of Imatinib with OCT-1 revealed major conformational between ligand and protein interaction sites. However, generated protein-ligand complexes proceeded for molecular dynamics studies (100ns) that have confirmed the selectivity of Imatinib and other drugs towards the OCT-1 and possible translocation path of ligands in the OCT-1 cavities. In quantitative gene expression analysis, downregulation of OCT-1 expression as evident by decreased fold change values in patients with blastic crisis compared to chronic phase responsible for lower uptake of Imatinib into the cells. Hence, cells can not achieve optimum Imatinib inhibitory concentration which may be responsible for disease progression and ultimately leads to drug resistance. Thus, proposed combined computational and experimental approaches suggests OCT-1 mediated activation of bcr-abl independent mechanism, which may be potential confounder of developing Imatinib resistance in CML patients. Citation Format: Saumya Patel, Krupa Shah, Rakesh Rawal. Role of organic cation transporter-1 (OCT-1) in drug resistance condition of ph+ chronic myeloid leukemia (CML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2106.

Notch Signaling Activity Determines Uptake and Biological Effect of Imatinib in Systemic Sclerosis Dermal Fibroblasts

Tyrosine kinase inhibitors have emerged as a therapeutic option for rheumatic diseases such as systemic sclerosis (SSc). Because tyrosine kinases like c-Abl kinase are important for fibroblast activation and fibrosis development in SSc, the c-Abl inhibitor imatinib was proposed for SSc treatment. Transporters for organic cations have become increasingly recognized as an important determinant for uptake and efficacy of tyrosine kinase inhibitors. Therefore, we investigated the role of organic cation transporters in the uptake of imatinib. Moreover, the influence of important SSc pathogenetic factors, like PDGF and Notch pathway activation on these uptake processes, has been studied. We showed that organic cation transporters OCT1-3, novel organic cation transporters OCTN1/2, and the multidrug and toxin extrusion protein MATE1 are expressed in healthy dermal and SSc fibroblasts. Decreased expression levels of MATE1 and decreased imatinib uptake were measured in SSc fibroblasts. In small interfering RNA experiments, MATE1 was identified as key transporter for imatinib uptake and biological effect in dermal fibroblasts. Furthermore, PDGF reduced imatinib uptake by decreasing MATE1 expression in SSc fibroblasts, but not in healthy fibroblasts. Blocking the Notch pathway in SSc fibroblasts increased MATE1 transporter expression and imatinib uptake. In conclusion, MATE1-mediated transport governs therapeutic efficacy of imatinib in SSc.

PO-470 hOCT1 gene polymorphism M420del is associated with decreased response to imatinib in CML patients and amp; its effect is counteracted by M408V polymorphism

IntroductionHuman organic cation transporter1 (hOCT1,SLC22A1),an influx transporter,is responsible for the uptake of Imatinib into chronic myelod leukaemia (CML)cells. Some patients fail to achieve optimal molecular response to Imatinib, defined as major molecular response (MMR) i.e. BCR-ABL 1≤0.1% within 12 months of therapy. Variation in clinical response to Imatinib has been observed with two nonsynonymous SNPs in hOCT1 gene, namely M420del and M408V in some populations.Material and methods30 newly diagnosed BCR-ABL positive CML patients in chronic phase,and 30 healthy control subjects, all ethnic Indians, were recruited in the study. M420del and M408V SNPs were examined by allele specific PCR(AS-PCR) in DNA from PBMCs.After initiation of imatinib therapy, haematological response was monitored at regular intervals, and molecular response (BCR-ABL1/ABL1 ratio) assessed after 6 or 12 months.Results and discussionsMinor allele frequencies for M420del were 0.18 and 0.1 in CML patients and controls; for M408V 0.4 and 0.27 respectively, closely paralleling those reported in western population.No significant association between different genotypes of M420del and M408V was observed with either time to achieve complete haematological response (CHR) (p=0.341 for both SNPs),or presence of optimal/sub-optimal molecular responses(p=0.125, 0.629 for M420del and M408V respectively).To analyse the combined effect of these two SNPs, CML cases were divided into 4 groups.Patients with mutant (homo/heterozygous) M420del and wild type homozygous M408V, failed to achieve an optimal molecular response to imatinib, unlike those with mutant genotypes (homo/heterozygous) for both SNPs (p=0.02).ConclusionMutant M420del allele may be linked to poor outcome of imatinib treatment in CML,however simultaneous presence of mutant M408V allele appears to circumvent this effect.These SNPs in hOCT1 gene occur at reasonable frequencies in Indian population, to be of clinical interest as predictors of response to imatinib in CML.

Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity.

Molecular Response to Imatinib and Its Correlation with mRNA Expression Levels of Imatinib Influx Transporter (OCT1) in Indian Chronic Myeloid Leukemia Patients

Background and objectives:Imatinib mesylate is approved for the treatment of Chronic Myeloid Leukemia (CML). About 20% of patients with CML do not respond to treatment with Imatinib either initially or because of acquired resistance. In addition to mutated BCR-ABL1 kinase, the organic cation transporter1 (OCT1, encoded by SLC22A1) has been considered to contribute to Imatinib resistance in patients with chronic myeloid leukemia (CML). OCT1 has been reported to be the main influx transporter involved in Imatinib uptake into CML cells. To date, only a few studies have been reported on involvement of influx transporters in development of Imatinib resistance. Therefore this study was aimed to determine the expression level of Imatinib uptake transporter (OCT1) in CML patients and to correlate this level with molecular response.Methods:One hundred fifty eight patients on Imatinib were considered for gene expression analysis study for OCT1 gene. Total RNA was extracted from peripheral blood mononuclear cells. Complementary DNAs (cDNAs) were synthesized and Real Time Polymerase Chain Reaction (RQ-PCR) was performed.Results:High OCT1 expression was present in 81 (51.8%) patients and low OCT1 expression was in 77 (48.7%) patients. Low Sokal risk score group have a significantly high OCT1 expression (p=0.048). The rate of molecular response was higher in those with high OCT1 expression than in those with low OCT1 expression (p=0.05). Both event-free survival and median overall survival were significantly shorter in patients with low OCT1 expressions when compared to the patients with high OCT1 expression (p=0.03 and p=0.05).Conclusions:Our findings demonstrated that the mRNA expression level of OCT1 was significantly correlated with molecular response in CML patients. Based on these findings, present study believes that the pre-therapeutic higher expression of OCT1 may help to predict response to imatinib therapy in CML patients.

Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen

Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system.Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells.Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS).Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance.Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. DisclosuresMahon:ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

MATE1 regulates cellular uptake and sensitivity to imatinib in CML patients.

Although imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), 25–30% patients do not respond or relapse after initial response. Imatinib uptake into targeted cells is crucial for its molecular response and clinical effectiveness. The organic cation transporter 1 (OCT1) has been proposed to be responsible for this process, but its relevance has been discussed controversially in recent times. Here we found that the multidrug and toxin extrusion protein 1 (MATE1) transports imatinib with a manifold higher affinity. MATE1 mainly mediates the cellular uptake of imatinib into targeted cells and thereby controls the intracellular effectiveness of imatinib. Importantly, MATE1 but not OCT1 expression is reduced in total bone marrow cells of imatinib-non-responding CML patients compared with imatinib-responding patients, indicating that MATE1 but not OCT1 determines the therapeutic success of imatinib. We thus propose that imatinib non-responders could be identified early before starting therapy by measuring MATE1 expression levels.

Controlled uptake and release of imatinib from ultrasound nanoparticles Cu3(BTC)2 metal–organic framework in comparison with bulk structure

The porosity of metal–organic frameworks (MOFs) is an important point concerning the possible use of such functional materials for different purposes. In this work, we study uptake and release properties of imatinib (IM) from nano Cu(II)-MOF in comparison with bulk Cu(II)-MOF. To explore the absorption ability of the Cu(II)-MOF to IM, fresh sample of Cu3(BTC)2 was immersed in an aqueous solution of IM and were monitored in real time with UV/vis spectroscopy. Results show that the adsorbed quantity of IM over nano Cu3(BTC)2 (I) is much higher than those over a bulk Cu3(BTC)2 (II).

Peroxisome Proliferator‐Activated Receptor Ligands and Their Role in Chronic Myeloid Leukemia: Therapeutic Strategies

Imatinib therapy remains the gold standard for treatment of chronic myeloid leukemia; however, the acquired resistance to this therapeutic agent in patients has urged the scientists to devise modalities for overcoming this chemoresistance. For this purpose, initially therapeutic agents with higher tyrosine kinase activity were introduced, which had the potential for inhibiting even mutant forms of Bcr-Abl. Furthermore, coupling imatinib with peroxisome proliferator-activated receptor ligands also showed beneficial effects in chronic myeloid leukemia cell proliferation. These combination protocols inhibited cell growth and induced apoptosis as well as differentiation in chronic myeloid leukemia cell lines. In addition, peroxisome proliferator-activated receptors ligands increased imatinib uptake by upregulating the expression of human organic cation transporter 1. Taken together, peroxisome proliferator-activated receptors ligands are currently being considered as novel promising therapeutic candidates for chronic myeloid leukemia treatment, because they can synergistically enhance the efficacy of imatinib. In this article, we reviewed the potential of peroxisome proliferator-activated receptors ligands for use in chronic myeloid leukemia treatment. The mechanism of action of these therapeutics modalities are also presented in detail.

Cell penetrating peptides from agglutinin protein of Abrus precatorius facilitate the uptake of Imatinib mesylate

Targeted drug delivery is of paramount importance for cancer patients. Cell penetrating peptides (CPPs) have emerged as potent vehicles for this purpose. Herein, we demonstrate CPP- like properties of two peptides: NH2-SGASDDEEIAR-COOH (SR11) and NH2-ICSSHYEPTVRIGGR-COOH (IR15), derived from the tryptic digest of Abrus precatorius agglutinin. Both IR15 and SR11 were found to be non-toxic at lower doses (up to 50μg/ml). These two peptides entered into HeLa cells through lipid raft-mediated endocytosis within 15min and penetrated the nuclear membrane in 60min of incubation. Co-treatment of peptides (20μg/ml) and Imatinib (5μM) in HeLa cells increased uptake of the drug by ∼55% and lowered the IC50 value to one-third in comparison to the drug added exclusively. However, co-treatment of TAT peptide (standard CPP) did not alter the Imatinib uptake significantly. In summary, we have identified two novel CPPs from tryptic digest of Abrus agglutinin which increased the cellular uptake of Imatinib upon co-administration. Further studies may result in deciphering a novel mode of drug delivery.

OCT1 and imatinib transport in CML: is it clinically relevant?

Imatinib is a highly effective therapy for chronic phase-chronic myeloid leukaemia (CP-CML) patients; however, responses to frontline imatinib are variable. The human organic cation transporter 1 (OCT1; SLC22A1) has been reported to be the main influx transporter involved in imatinib uptake into CML cells. Furthermore, variation in the efficiency of imatinib influx via OCT1 has been demonstrated to result in the inter-patient variation observed in primary response to imatinib. Although studies have questioned the role of OCT1 in imatinib influx, these have been largely performed in non-clinical settings. Measuring both OCT1 mRNA levels and the functional activity of OCT1 in primary leukaemic cells has been demonstrated to predict molecular response and outcome in imatinib-treated CP-CML patients in several independent studies. Here, the role of OCT1 and OCT1 genetic variants in imatinib uptake and response prediction is summarised and data generated from model systems assessing the role of OCT1 in imatinib transport is discussed.