Imatinib Treatments Research Articles

Real-world efficacy and safety outcomes of imatinib treatment in patients with chronic myeloid leukemia: An Australian experience.

Tyrosine kinase inhibitors (TKI) have revolutionized the treatment of chronic myeloid leukemia (CML), but patients still experience treatment‐limiting toxicities or therapeutic failure. To investigate the real‐world use and outcomes of imatinib in patients with CML in Australia, a retrospective cohort study of patients with CML commencing imatinib (2001–2018) was conducted across two sites. Prescribing patterns, tolerability outcomes, and survival and molecular response were evaluated. 86 patients received 89 imatinib treatments. Dose modifications were frequently observed (12‐month rate of 58%). At last follow‐up, 62 patients (5‐year rate of 55%) had permanently discontinued imatinib treatment, of which 44 switched to another TKI (5‐year rate of 46%). Within 3 months of starting imatinib, 43% (95% CI, 32%–53%) of patients experienced imatinib‐related grade ≥3 adverse drug reactions (ADRs). Higher comorbidity score, lower body weight, higher imatinib starting dose, and Middle Eastern or North African ancestry were associated with a higher risk of grade ≥3 ADR occurrence on multivariable analysis (MVA). Estimated overall survival and event‐free survival rates at 3 years were 97% (95% CI, 92%–100%) and 81% (95% CI, 72%–92%), respectively. Cumulative incidence of major molecular response (MMR) at 3 years was 63% (95% CI, 50%–73%). On MVA, imatinib starting dose, ELTS score, BCR‐ABL1 transcript type, pre‐existing pulmonary disease, and potential drug–drug interactions were predictive of MMR. In conclusion, imatinib induced deep molecular responses that translated to good survival outcomes in a real‐world setting, but was associated with a higher incidence of ADRs, dose modifications and treatment discontinuations than in clinical trials.

LncRNA-HOTAIR activates autophagy and promotes the imatinib resistance of gastrointestinal stromal tumor cells through a mechanism involving the miR-130a/ATG2B pathway

Gastrointestinal stromal tumors (GISTs) are common neoplasms of the gastrointestinal tract that can be treated successfully using C-kit target therapy and surgery; however, imatinib chemoresistance is a major barrier to success in therapy. The present study aimed to discover alternative pathways in imatinib-resistant GISTs. Long noncoding RNAs (lncRNAs) are newly discovered regulators of chemoresistance. Previously, we showed that the lncRNA HOTAIR was upregulated in recurrent GISTs. In this study, we analyzed differentially expressed lncRNAs after imatinib treatment and found that HOTAIR displayed the largest increase. The distribution of HOTAIR in GISTs was shifted from nucleus to cytoplasm after imatinib treatments. The expression of HOTAIR was validated as related to drug sensitivity through Cell Counting Kit-8 assays. Moreover, HOTAIR was associated strongly with cell autophagy and regulated drug sensitivity via autophagy. Mechanistically, HOTAIR correlated negatively with miRNA-130a in GISTs. The downregulation of miRNA-130a reversed HOTAIR-small interfering RNA-induced suppression of autophagy and imatinib sensitivity. We identified autophagy-related protein 2 homolog B (ATG2B) as a downstream target of miR-130a and HOTAIR. ATG2B downregulation reversed the effect of pEX-3-HOTAIR/miR-130a inhibitor on imatinib sensitivity. Finally, HOTAIR was shown to influence the autophagy and imatinib sensitivity of GIST cells in mouse tumor models. Our results suggested that HOTAIR targets the ATG2B inhibitor miR-130a to upregulate the level of cell autophagy so that promotes the imatinib resistance in GISTs.

Long-term imatinib diminishes ovarian reserve and impacts embryo quality.

Tyrosine kinase inhibitors (TKIs) such as imatinib are commonly used chemotherapeutics, but the effects of long-term treatments on reproductive outlook for cancer survivors are unknown. The purpose of this study was to examine the effects of long-term imatinib treatments on follicle development and embryo quality. Since prospective studies are not possible in healthy humans, we have incorporated a commonly used mouse model. Adult female mice were treated with daily IP injections of imatinib for 4-6weeks. Liquid chromatography-mass spectrometry was used to measure imatinib in serum and ovarian tissues. At the end of treatments, females were superovulated and mated to yield fertilized embryos. Oocytes and embryos were collected from oviducts, assessed for development by microscopy, and fertilized embryos were cultured in vitro. Blastocysts were fixed and stained for differential cell counts. Long-term imatinib treatments caused a shift in follicle development, with imatinib-treated females having fewer primordial follicles, but an increase in primary and secondary follicles (P < 0.05). There was no effect on ovulation or fertilization rates. However, blastocysts from imatinib-treated females had fewer total cells (P < 0.05) and a significant shift from inner cell mass to increased trophectoderm cells. This pilot study indicates that long-term TKI treatments may have significant impact on ovarian reserve and embryo developmental capacity. More studies are needed in other model systems to determine the long-term impact of TKIs in patients. Knowing the potential effects of chemotherapeutics on reproductive outlook is critical for quality of life and more research is needed.

Inhibition of the Nrf2-TrxR Axis Sensitizes the Drug-Resistant Chronic Myelogenous Leukemia Cell Line K562/G01 to Imatinib Treatments.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

ALT-GIST: Randomized phase II trial of imatinib alternating with regorafenib versus imatinib alone for the first-line treatment of metastatic gastrointestinal stromal tumor (GIST).

11023 Background: Imatinib (IM) is the standard first-line treatment for advanced GIST and regorafenib (REG) is approved for third line therapy. We studied if an alternating regimen of two tyrosine kinase inhibitors, IM and REG, delays resistance to IM and improves outcomes. Methods: ALT-GIST (NCT02365441) is a randomised non-comparative phase II trial to investigate the efficacy of an alternating regimen (ALT) of 21-25 days of IM 400mg orally daily followed by a 3-7-day gap for washout followed by 21 days of REG 160 mg orally daily and a 7-day gap for washout. The control arm was continuous IM 400mg daily. Delayed recruitment led to revised endpoints of activity and safety. To assess clinical activity, the best objective tumor response (OTR) at 9 months was deemed to be an appropriate endpoint in the revised protocol. Results: Seventy-six eligible patients (ALT 40, IM 36) enrolled from June 2015 to September 2018 were evaluable for the OTR. The patients (pts) were predominately male (n = 51, 67%). Median age was 58 (range, 24-81) in the ALT arm and 65 (range, 35-82) in the IM arm. KIT was mutated in 63, PDGFR in 2, and wildtype in 5 tumors. Relative dose intensity in the ALT arm 102% for IM and 82% for REG and was 93% in the IM arm. Median follow-up time was 19.3 months (range 6.0-40.0).The best responses to the ALT and IM treatments were similar at 9 months, 1 vs 0 pts had complete response, 23 vs 23 partial response, 15 vs 13 stable disease, and the OTR was 60% (95% CI, 45-74%) and 64% (95% CI, 48-78%), respectively. Seven (18%) pts in ALT arm and 10 (28%) in IM arm discontinued treatment due to progressive disease. Seven pts (18%) in the ALT arm stopped protocol therapy due to unacceptable toxicity, and none in the IM arm. Fifteen (38%) pts in the ALT arm and 14 (38%) in the IM arm had serious adverse events, mostly grade 3. Progression free survival (PFS) at 1 year was ALT 0.86 (95%CI:0.69,0.94) and IMI 0.83 (95% CI 0.65-0.92), p logrank = 0.57. Conclusions: There was no meaningful difference in the primary endpoint of OTR and in PFS between the groups in this first analysis of ALT-GIST, and no unexpected safety signals. The study is ongoing and other endpoints will be reported in due course. Clinical trial information: ACTRN12614000950662.

Imatinib treatments have long-term impact on placentation and embryo survival

Imatinib is an oral chemotherapeutic used primarily to treat chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). The potential effects of cancer treatments on a patient’s future fertility are a major concern affecting the quality of life for cancer survivors. The effects of imatinib on future fertility are unknown. It is teratogenic. Therefore, patients are advised to stop treatment before pregnancy. Unfortunately, CML and GIST have high rates of recurrence in the absence of the drug, therefore halting imatinib during pregnancy endangers the mother. Possible long-term (post-treatment) effects of imatinib on reproduction have not been studied. We have used a mouse model to examine the effects of imatinib on the placenta and implantation after long-term imatinib exposure. We found significant changes in epigenetic markers of key imprinted genes in the placenta. There was a significant decrease in the labyrinth zone and vasculature of the placenta, which could impact fetal growth later in pregnancy. These effects on placental growth occurred even when imatinib was stopped prior to pregnancy. These results indicate potential long-term effects of imatinib on pregnancy and implantation. A prolonged wash-out period prior to pregnancy or extra monitoring for possible placental insufficiency may be advisable.

NKG2A Down-Regulation by Dasatinib Enhances Natural Killer Cytotoxicity and Accelerates Effective Treatment Responses in Patients With Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the presence of t(9;22) chromosomal translocation that results in BCR-ABL fusion gene. ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, are currently the front-line treatment options for CML. Recently, natural killer (NK) cell activation and expansion have been shown to be associated with optimal treatment responses for CML. To investigate the effects and mechanisms of these TKIs on NK cells, here we characterized activating and inhibitory NK receptors in CD3−CD16+CD56dim NK cells isolated from CML patients in chronic phase (CP). The expressions of activating NK receptors, such as NKG2D, natural cytotoxicity receptor (NCR) and DNAM-1, rebounded after successful TKI treatments for CML. In contrast, among the three surveyed inhibitory receptors (NKG2A, KIR2DL1, and KIR3DL1), only the expression of NKG2A was reverted and suppressed to a very low level by dasatinib, and not by imatinib or nilotinib. CML patients treated with dasatinib indeed expressed fewer NKG2A+ NK cells, which send negative signals for induction of NK cytotoxicity. For these dasatinib-treated patients, the duration to reach major molecular response (MMR) was shorter, and significantly correlated with individual's NKG2A+ NK cell number. This clinical relevance to NKG2A was not observed in treatments with imatinib or nilotinib. In line with dasatinib-specific down-regulation of NKG2A, NK cytotoxicity evaluated by the killing assay was also significantly higher in patients treated with dasatinib than in those treated with imatinib or nilotinib. The lower NK cytotoxicity from imatinib or nilotinib treatments could be reverted by NKG2A blockade using anti-NKG2A antibody. Further in vitro experiments revealed mechanistically that dasatinib could inactivate p38 mitogen-activated protein kinase (MAPK), and consequently affect nuclear import of GATA-3 and GATA-3 transcriptional activities for NKG2A. Our results highlight the dual effects of dasatinib in direct inhibition of ABL kinase and in immunomodulation through NKG2A down-regulation, contributing to accelerated molecular responses (MR) in CML.

Aryl hydrocarbon receptor (AHR) is a novel druggable pathway controlling malignant progenitor proliferation in chronic myeloid leukemia (CML).

Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)–derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the “AHR low” fraction and an upregulation of genes involved in stem cell maintenance in the “AHR high” fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.

Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2.

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs.

Imatinib Ameliorated Retinal Neovascularization by Suppressing PDGFR-α and PDGFR-β

Background/Aims: Platelet-derived growth factors (PDGFs) have emerged as pivotal in pathological angiogenesis, which is a hallmark of various tumors and retinal diseases. Here we evaluated the anti-angiogenic effect of imatinib, an inhibitor of PDGF receptors α and β (PDGFR-α and -β), in retinal neovascularization using an oxygen-induced retinopathy (OIR) model. Methods: The OIR model was established and given imatinib or vehicle treatments daily from P12 to P16. At the peak of angiogenesis at P17, the neovascularization area was quantified on retinal whole-mounts with isolectin B4 staining. Immunofluorescence staining and western blots were used to determine the effect of imatinib on different vascular cells and the pathway molecules involved. Results: Imatinib effectively suppressed pathological angiogenesis in OIR mice and reduced the number of all three types of vascular cells, including endothelial cells, pericytes, and smooth muscle cells. Moreover, the expression and activation of PDGFR-α and -β were inhibited by imatinib. The imatinib-treated OIR mice presented with reduced expression of other potent pro-angiogenic factors such as VEGF and FGF2. No obvious retinal or systemic side effects were observed in the imatinib treatment group. Conclusions: Imatinib appears to be safe and effective in suppressing retinal neovascularization. Targeting PDGFs/PDGFRs may also be important for anti-angiogenic treatment and offer a viable alternative treatment for retinal angiogenic diseases.

Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumor after failure with imatinib and sunitinib treatment: A meta-analysis.

Aims:This meta-analysis aimed to evaluate the safety and efficacy of regorafenib as a treatment for patients with advanced (metastatic and/or unresectable) gastrointestinal stromal tumor (AGIST) after developing resistance to imatinib and sunitinib.Methods:A literature search of databases such as PubMed, Embase, and Cochrane library was conducted up to February 2017. The pooled percentages and the corresponding 95% confidence intervals (CIs) were calculated using the Stata 11.0 software.Results:Four studies involving 243 patients with AGIST were included. Results revealed that approximately 49% (95% CI 30–67), 14% (95% CI 5–23), and 41% (95% CI 21–61) of patients with AGIST showed clinical benefit (including complete response), partial response, and stable disease, respectively, after regorafenib treatment, which was given after failure with imatinib and sunitinib treatments. No complete response was found in the included studies. Pooled progression-free survival was 6.58 months (95% CI 4.62–8.54). Hypertension (20%; 95% CI 7–33), hand–foot skin reaction (22%; 95% CI 17–27), and hypophosphatemia (18%; 95% CI 5–41) were common grade ≥3 regorafenib-related adverse events in patients treated with regorafenib after failure with imatinib and sunitinib treatments.Conclusions:Forty-nine per cent of patients with AGIST benefited after regorafenib treatment after the development of resistance to imatinib and sunitinib. More studies should be performed to improve the clinical survival of patients with AGIST. Close monitoring and appropriate management of grade ≥3 regorafenib-related adverse events should be considered during treatment.

Therapeutic drug monitoring and tyrosine kinase inhibitors

The therapeutic activity of drugs can be optimized by establishing an individualized dosage, based on the measurement of the drug concentration in the serum, particularly if the drugs are characterized by an inter-individual variation in pharmacokinetics that results in an under- or overexposure to treatment. In recent years, several tyrosine kinase inhibitors (TKIs) have been developed to block intracellular signaling pathways in tumor cells. These oral drugs are candidates for therapeutic drug monitoring (TDM) due to their high inter-individual variability for therapeutic and toxic effects. Following a literature search on PubMed, studies on TKIs and their pharmacokinetic characteristics, plasma quantification and inter-individual variability was studied. TDM is commonly used in various medical fields, including cardiology and psychiatry, but is not often applied in oncology. Plasma concentration monitoring has been thoroughly studied for imatinib, in order to evaluate the usefulness of TDM. The measurement of plasma concentration can be performed by various analytical techniques, with liquid chromatography-mass spectrometry being the reference method. This method is currently used to monitor the efficacy and tolerability of imatinib treatments. Although TDM is already being used for imatinib, additional studies are required in order to improve this practice with the inclusion of other TKIs.

Disruption of the Peripheral Lymphoid Niche Contributes to Lymphopenia in CML Patients Undergoing Imatinib Treatments

PURPOSEChronic myelogenous leukemia (CML) is a disorder affecting early hematopoietic stem cells (HSC) and is characterized by excessive proliferation and accumulation of myeloid progenitors and progeny in the periphery. During the chronic phase of the disease, CML patients are normally at low risk of developing infections but such complications tend to rise during the progression of the disease. Gleevec (imatinib mesylate) is currently administered as first line therapy for patients with Philadelphia chromosome-positive CML. Despite the relative high specificity of tyrosine kinase inhibitor (TKI) treatment towards the BCR-ABL fusion protein, off-target multikinase inhibitory effects occur and can interfere with normal hematopoiesis. This study was conducted to evaluate how myeloid and lymphoid immune homeostasis are affected by Imatinib mesylate. METHODSHealthy volunteer donors (n=25) and CML patients were recruited during their first visit at our CML clinic. Seven CML patients were treated with Imatinib (400mg). The median time of Gleevec treatment was 2.9 years (range: 0.5-10.9). The median time of remission post TKI was 1.1 years (range: 0.3-3). Phenotypic analysis of dendritic cell (DCs) subsets: plasmacytoid (pDCs) and myeloid type 1, 2 and 3 (mDC1, mDC2, mDC3) were evaluated by flow cytometry. Percentage and absolute numbers of naive and memory CD4+ and CD8+ T cells, NK cells and B cells were also evaluated. DCs were differentiated from purified CD34+ cells culturedwith GM-CSF (800 U/ml) or Flt3-L (50ng/ml), IL-4 (10 U/ml) and TNFa (50 U/ml), in the presence of varying concentrations of Imatinib mesylate (0 to 5µM/mL). TCR and IL-7 signaling were evaluated based on ERK-phosphorylation (-p) and STAT5-p after incubation with 3µM of Imatinib. RESULTS AND CONCLUSIONAt diagnosis, several CML patients have a deficit in DCs resulting from a severe skewing affecting BM progenitor cells. After initiating Gleevec therapy, normalization of stem cell progenitors occurs but DC counts remain well below normal levels in all CML patients. We demonstrated a direct and dose dependent interference of Imatinib on GM-CSF and Flt3-L pathways for DC differentiation from CD34+ stem cells. For T lymphocytes, Imatinib interfered with TCR and IL-7 signaling through the inhibition of ERK and STAT5 phosphorylation respectively. The failure to maintain adequate numbers of DCs combined to diminished homeostatic response to cytokines and TCR stimuli explains T cell lymphopenia in these patients. Such immune dysfunction is at least in part responsible for infectious complications that are often increased in patients treated with Imatinib. DisclosuresNo relevant conflicts of interest to declare.

Effects of acute and chronic sunitinib treatment on cardiac function and calcium/calmodulin-dependent protein kinase II.

Calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac contractile function and dysfunction and may be an unwanted secondary target for anti-cancer drugs such as sunitinib and imatinib that have been reported to alter cardiac performance. This study aimed to determine whether anti-cancer kinase inhibitors may affect CaMKII activity and expression when administered in vivo. Cardiovascular haemodynamics in response to acute and chronic sunitinib treatment, and chronic imatinib treatment, were assessed in guinea pigs and the effects compared with those of the known positive and negative inotropes, isoprenaline and verapamil. Parallel studies from the same animals assessed CaMKIIδ expression and CaMKII activity following drug treatments. Acute administration of sunitinib decreased left ventricular (LV) dP/dtmax. Acute administration of isoprenaline increased LVdP/dtmax dose-dependently, while LVdP/dtmax was decreased by verapamil. CaMKII activity was decreased by acute administration of sunitinib and was increased by acute administration of isoprenaline, and decreased by acute administration of verapamil. CaMKIIδ expression following all acute treatments remained unchanged. Chronic imatinib and sunitinib treatments did not alter fractional shortening; however, both CaMKIIδ expression and CaMKII activity were significantly increased. Chronic administration of isoprenaline and verapamil decreased LV fractional shortening with parallel increases in CaMKIIδ expression and CaMKII activity. Chronic sunitinib and imatinib treatment increased CaMKIIδ expression and CaMKII activity. As these compounds are associated with cardiac dysfunction, increased CaMKII expression could be an early indication of cellular cardiotoxicity marking potential progression of cardiac contractile dysfunction.

Overcoming Therapy Failure in Elderly Patients with Chronic Myeloid Leukemia

SUMMARY Chronic myeloid leukemia (CML) affects mainly older adults, as median age at diagnosis is 60–65 years. For a long time, survival of elderly CML patients has been shorter compared with younger patients. With the advent of the first tyrosine kinase inhibitor (TKI), imatinib, long-term outcome has significantly improved, including in the elderly, with rates of cytogenetic and molecular responses roughly equal to those attained in the young, as well as manageable toxicity. More recently, second-generation (dasatinib, nilotinib and bosutinib) and third-generation (ponatinib) TKIs were employed in CML patients failing imatinib or other front-line treatments. Despite a shorter follow-up, these TKIs showed remarkable activity in elderly patients. This review focuses on the therapeutic strategies to prevent and overcome treatment failure in elderly CML patients.

40: Inhibition of euchromatic histone methyltransferase 1 and 2 sensitizes chronic myeloid leukemia cells to interferon treatment

H3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response. We report the application of EHTM1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNa2a in all four CML cell lines tested but not three non-CML cell lines. Additionally, BIX01294 augments IFNa2a- and imatinib-mediated apoptosis in CML cells. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon response in CML cell lines. Our study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML.

Inhibition of euchromatic histone methyltransferase 1 and 2 sensitizes chronic myeloid leukemia cells to interferon treatment.

BackgroundH3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response.ResultsWe report the application of EHMT1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNα2a in four CML cell lines, K562, KCL22, BV173 and KT1 cells. Chromatin immunoprecipitation assay shows that BIX01294 treatment enhances type I interferon response by reducing H3K9me2 at the promoters of interferon-stimulated genes. Additionally, BIX01294 treatment augments IFNα2a- and imatinib-mediated apoptosis in CML cell lines. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon responsiveness in CML cell lines.ConclusionsOur study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML.

An Epigenetic Mechanism Of Imatinib Via Demethylation Of MiR-203

BackgroundMicroRNAs (miRNAs) are short noncoding RNAs regulating a variety of biological processes by post-transcriptionally silencing via targeting mRNA. Recently there are many reports demonstrating that epigenetic alterations correlate to the characteristics of tumor cells, and that miRNAs were also reported to be regulated by methylation of CpG islands within the promoter region. MiR-203 is epigenetically silenced in human BCR-ABL1-positive leukemic cell lines and primary chronic myelogenous leukemia (CML) cells by the methylation of promoter region. In this study, we analyzed the effect of imatinib, a tyrosine-kinase inhibitor specific to BCR-ABL1 protein, on the expression of miRNA in BCR-ABL1-positive cells. Materials & MethodsTwo CML cell lines (K562 and KU812) and one AML cell line (HL60) were treated with imatinib for 72 hours. Microarray analysis of miRNAs was conducted by 3D-Gene (TORAY) in K562 cells with/without imatinib. Methylation specific PCR and bisulfite direct sequencing was performed to evaluate methylation status of promoter region of miR-203. Validation of expressions of miRNAs, including miR-203, and mRNAs was analyzed by RT-qPCR. The expression of BCR-ABL protein was confirmed by Western blotting. The function of miR-203 for cell survival was evaluated by the transfection of anti-miRNA. ResultsThe microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment, in K562 cells. The demethylated state of the promoter region of miR-203, one of 48 miRNAs, was confirmed by bisulfite direct sequencing. The expression of BCR-ABL mRNA, which is one of the target of miR-203, was inhibited with imatinib to 52% and 26% of the level in control cultures in K562 cells and KU812 cells, respectively. The expression of BCR-ABL protein was also inhibited. The addition of anti-miR-203 increased the expression level of BCR-ABL protein to 68.1% in the K562 cell culture with imatinib treatment. The expression of DNA methyltransferase (DNMT) mRNA was analyzed, and the expressions of DNMT1 and DNMT3B were significantly decreased after imatinib treatments in CML cell lines, whereas the expression of DNMT3A was not changed. Discussion & ConclusionWe report, for the first time, that imatinib up-regulated miR-203 by inducing demethylation of the promoter region of miR-203 in CML cells. MiR-203 is the important miRNA to inhibit ABL1 and BCR-ABL1 mRNA, and imatinib-induced demethylation of miR-203 is the possible mechanism to suppress growth of BCR-ABL1-positive leukemic cells. It was suggested that the demethylation was partially caused by down regulation of DNMT1 and DNMT3B after imatinib treatments in CML cell lines. In conclusion, imatinib not only inhibits the activity of tyrosine kinase but also induces DNA demethylation of miR-203 in CML cells. Disclosures:No relevant conflicts of interest to declare.

PTMScan Direct: Identification and Quantification of Peptides from Critical Signaling Proteins by Immunoaffinity Enrichment Coupled with LC-MS/MS

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.

Rapid and Sustained Increase of LGL and Rare CMV-Reactivation During Dasatinib Treatments in CML

Abstract 2752Dasatinib has extensive inhibition profile on multiple kinases. Clonal expansion of large granular lymphocyte (LGL) is a noteworthy off-target phenomenon. In this study, we investigated the increase of LGL in peripheral blood during dasatinib treatments which were changed from imatinib treatments, in 25 chronic myelogenous leukemia (CML) patients in CP or AP. METHODS: The criteria of the LGL-increase was set by “increase of lymphocytes over 3,000/μL, and increase of LGL count over 1,500/μL” was found during the course of the treatment. Flow cytometry analyses were performed during the treatments in all cases, and also before the dasatinib treatments in 17 cases. T-cell receptor (TCR) clonal rearrangements for TCR-β, -γ, and -δ, were examined by PCR-based method during the treatments in all cases. Furthermore, comparison of LGL counts before and 2 hours after oral intake were investigated in all 25 cases. CMV activations were assessed in all cases by serum CMV-IgG, serum CMV-IgM, and by “CMV antigen test Teijin (HRP-C7)” which can detect CMV pp65 antigen in leukocytes. RESULTS: Median follow-up time was 11 months (range 3–28). (1) Fifteen out of the 25 patients (60%) showed increase of LGL, which had not been observed during preceding imatinib treatments. Median time until LGL count reached 1,500/μL was 11 weeks (range 6–44), and increased-LGL sustained as far as dasatinib was continued. In these 15 patients, all showed increase of CD56+, CD16+, CD3- NK-cells, and 11 patients also showed increase of CD3+, CD8+, CD4- T-cells. Evident increase of γδ-T cells was not observed. Clonal rearrangements of TCR-β gene were observed in 13 of 15 (87%), and TCR-γ in 12 (80%), and TCR-δ in 9 (60%) patients. Pleural effusions were observed in 9 out of 15 (60%) patients. Regarding clinical response, 4 cases newly achieved CMR, 7 maintained CMR (totally 73% achieved or maintained CMR), and 3 achieved or maintained MMR. (2) On the other hand, 10 out of 25 patients (40%) did not show increase of LGL. In these 10 patients, clonal rearrangements of TCR-β gene were observed in 4 of 10 (40%), and TCR-γ in 3 (30%), and TCR-Δ in 4 (40%) patients. Three patients showed no clonality for all the three TCR genes. Pleural effusions were observed in 2 out of 10 (20%) patients. Regarding clinical response, no case newly achieved CMR, 5 maintained CMR (totally 50% achieved or maintained CMR), and 4 achieved or maintained MMR. (3) LGL counts increased 2 hours after intake of dasatinib in all patients. Regarding the 15 patients who showed LGL increase during the treatment courses, LGL counts were 1,349±918/μL (mean±SD)(before), and 2,895±2,240/μL (2 hours after), indicating 2.1 times increase (P=0.006); total lymphocyte counts were 2,153±1,129/μL (before), and 4,456±3,219/μL (after), indicating 2.1 times increase (P=0.01). Regarding the 10 patients who did not show LGL increase during the treatment courses, LGL counts were 422±384/μL (before), and 805±419/μL (after), indicating 1.9 times increase (P=0.02); total lymphocyte counts were 1,094±556/μL (before), and 1,859±899/μL (after), indicating 1.7 times increase (P=0.03). (4) In the total 25 patients, serum CMV-IgM were negative in most of the cases (23 of 25; 92%). CMV HRP-C7 were completely negative (meaning undetectable) in most of the cases (23 of 25; 92%), and it was barely positive only in 2 cases (1 cell and 2 cells were positive out of ∼60,000 leukocytes examined, respectively). CONCLUSION: LGL lymphocytosis occurred in 60% of patients, which seems to be related with favorable molecular responses, and with relatively high incidence of pleural effusions. The TCR gene rearrangements were observed in both groups, but higher in the increase-LGL group than the non-increase-LGL group. Approximately 2-times rapid increase of LGL and total lymphocytes were observed 2 hours after intake of dasatinib, in all the patients, irrespective of the LGL-increase during the treatment courses. In the clinical setting, evident immunodeficiency was not observed in terms of CMV-reactivation. Disclosures:No relevant conflicts of interest to declare.