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

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Yingjun Deng ◽  
Xin Li ◽  
Jinxin Feng ◽  
Xiangliang Zhang
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Imatinib Resistance ◽  
Hexokinase 2 ◽  
Imatinib Resistant ◽  
First Choice ◽  
Long Term Treatment

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.

Proliferation Inhibition and Apoptosis Induction Of Imatinib Resistance Chronic Myeloid Leukemia Cells By Down-Regulated PPP2R5C

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5158-5158
Author(s):  
Qi Shen ◽  
Sichu Liu ◽  
Yu Chen ◽  
Lijian Yang ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Primary Cells ◽  
Healthy Individuals ◽  
Hoechst 33258 ◽  
Imatinib Resistance ◽  
Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. The prognosis in CML improved markedly after introduction of abl tyrosine kinase inhibitors (TKI), still a lot of CML patients die due to abl mutation related drug resistance and the blast crisis, moreover, de novo or secondary TKI-resistance is a significant problem in CML. The aim of the study is to down-regulate the PPP2R5C gene expression in imatinib-sensitive or imatinib-resistant chronic myeloid leukemia (CML) cell lines: K562, K562R (imatinib resistance without abl gene mutation), 32D-Bcr-Abl WT (imatinib sensitive, murine CML cell lines with wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistance, with abl gene T315I mutation) and primary cells from CML patients by RNA interference, thereby inhibit the CML cells proliferation and induce apoptosis. PPP2R5C-siRNAs numbered 799 or 991 were obtained by chemosynthesis. Non-silencing siRNA control (SC)-treated, mock-transfected, untreated cells were used as controls. PPP2R5C expression in mRNA levels from CML cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR. The PPP2R5C protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst 33258 stain and flow cytometry (FCM). Bone marrow mononuclear cells (BM-MNCs) from healthy individuals were transferred by PPP2R5C-siRNA-991. BFU-E, CFU-Meg and CFU-GM were performed from PPP2R5C-siRNA-991 treated BM-MNCs by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in BM-MNCs after PPP2R5C-siRNA transfection. The results showed that both PPP2R5C-siRNA-799 and PPP2R5C-siRNA-991 took best silencing results after nucleofection in all of four cells and primary cells from CML patients. The reduction about 2 to 7 folds in PPP2R5C mRNA level was observed in PPP2R5C-siRNA799 or PPP2R5C-siRNA991 treated cells. And PPP2R5C protein expression inhibition rate reached 38.08%-55.26% at 48 or 72 h after treatment. The proliferation rates of PPP2R5C-siRNA-799 or 991 treated CML cells were significantly decreased at 72 h (P < 0.05). PPP2R5C-siRNA-799 or 991 treated CML cells lines showed a significantly increase in AnnexinV/PI-positive cells (apoptosis) (P < 0.05), similar results in the morphological changes of apoptosis were found by Hoechst 33258 staining test. PPP2R5C gene mRNA expression levels in BM-MNCs from healthy individuals were significantly lower than that in K562 cells (P < 0.05), and the expression level was not significant changed after PPP2R5C-siRNA-991 transfection. The formation of BFU-E, CFU-Meg and CFU-GM from BM-MNCs showed no significant difference between PPP2R5C-siRNA-991 treatment and MOCK control group (P > 0.05). In conclusions, suppression of PPP2R5C by RNA interference could inhibit the proliferation and induce the apoptosis effectively in CML cells either in imatinib sensitive or imatinib resistance cell lines, while no significant effect of PPP2R5C-siRNA on the proliferation and differentiation of BM-MNCs in vitro, suggesting that PPP2R5C-siRNA might specially target on the CML cells. Down-regulating the PPP2R5C gene expression might be considered as a new target therapeutic strategy in CML, especially in imatinib-resistant CML. Disclosures: Li: This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.

scholarly journals A Review of the Management of Chronic Myeloid Leukemia with Dasatinib

2009 ◽  
Vol 1 ◽  
pp. CMT.S1167
Author(s):  
Joanna Gora-Tybor ◽  
Tadeusz Robak
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Therapeutic Option ◽  
Chronic Phase ◽  
Imatinib Resistant ◽  
First Choice ◽  
Multitargeted Tyrosine Kinase Inhibitor ◽  
Mutant Forms

Dasatinib is a multitargeted tyrosine kinase inhibitor (TKI), recommended for chronic myeloid leukemia (CML) in chronic, accelerated and blastic phases that are resistant or intolerant to imatinib. The recognition of BCR-ABL gene and corresponding protein with deregulated tyrosine kinase (TK) activity as crucial for malignant transformation in CML, led to synthesis of the small-molecule drugs designed to interfere with Bcr-Abl TK activation. The first TKI introduced into clinical practice in 1998, was imatinib mesylate, which became the first choice drug in chronic phase CML. However, approximately 20%-25% of patients initially successfully treated with imatinib, develop resistance to this drug, mainly because of selection of clones expressing mutant forms of BCR-ABL with impairing imatinib binding. Dasatinib is 325-fold more potent than imatinib against cell expressing wild-type BCR-ABL and showing activity against most imatinib-resistant Bcr-Abl mutants. Several clinical trials demonstrated that dasatinib is effective and generally well tolerated in imatinib resistant or intolerant CML and represents a promising therapeutic option for these patients.

scholarly journals Tyrosine kinase inhibitors and interferon‐α increase tunneling nanotube (TNT) formation and cell adhesion in chronic myeloid leukemia (CML) cell lines

2020 ◽  
Vol 34 (3) ◽  
pp. 3773-3791 ◽  
Author(s):  
Maria Omsland ◽  
Vibeke Andresen ◽  
Stein‐Erik Gullaksen ◽  
Pilar Ayuda‐Durán ◽  
Mihaela Popa ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Interferon Α ◽  
Tunneling Nanotube

Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways

2011 ◽  
Vol 52 (11) ◽  
pp. 2139-2147 ◽  
Author(s):  
Carine Tang ◽  
Lisa Schafranek ◽  
Dale B. Watkins ◽  
Wendy T. Parker ◽  
Sarah Moore ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Inhibitor Resistance ◽  
Myeloid Leukemia Cell

Combination Therapy with Tyrosine Kinase Inhibitors and Agents with Different Mechnisms of Actions Is Effective in a Patient with Chronic Myeloid Leukemia Harboring the T315l BCR - ABL1 Mutation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4282-4282
Author(s):  
Fabio P S Santos ◽  
Jorge Cortes ◽  
Charles Koller ◽  
Elias Jabbour
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Combination Therapy ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Specific Inhibitor ◽  
Cytogenetic Response ◽  
Molecular Response ◽  
T315i Mutation

Abstract Abstract 4282 Mutations of BCR-ABL1 have been observed in 50% of patients with chronic myeloid leukemia (CML) who develop resistance to imatinib. The gate-keeper mutation T315I is one of the mutations with universal resistance to imatinib and to the second-generation tyrosine kinase inhibitors (TKI) that are approved for the treatment of patients with imatinib failure. The use of new kinase inhibitors with in vitro activity against T315I mutation as well as other agents with different mechanisms of actions is being evaluated in clinical trials. We report the case of a 57-year old man that was diagnosed with CML in 2003. Patient received initial therapy with standard-dose imatinib that was subsequently increased to 800 mg daily. He did achieve a complete cytogenetic response (CCyR) 9 months post dose escalation. He was followed by RT-PCR for BCR-ABL1.. In May, 2007, the patient BCR-ABL1/ABL1 ratio increased to 16.38 but the patient remained in CCyR. BCR-ABL1 sequencing revealed the T315I mutation in 100% of cells (Figure 1). One month later the patient lost CCyR (5% Philadelphia-positive [Ph+] cells) and the BCR-ABL1/ABL1 ratio was 5.08. The patient was started on the T315I specific inhibitor KW-2449 (100 mg orally twice daily for 14 days, every 3 weeks). Patient had a progressive decline in percentage of cells with the T315I mutation (Figure 1). However, at the same time he had an increase in percentage of Ph+ cells. In September, 2007, three months after starting therapy with KW-2449, patient had no cytogenetic response (80% Ph+ cells, PCR for BCR-ABL1 ratio > 100) and the T315I mutation was undetectable. At that time, a new ABL1 sequencing revealed the F359I mutation (no quantification was done). Patient was maintained on KW-2449 for the next 6 months, without significant improvement in cytogenetic response nor BCR-ABL1 ratio, but the clone with the T315I mutation did not reappear. In February, 2008, the patient lost hematologic response and presented with an elevated white blood cell count of 22×109/L. The F359I mutation was still present. Therapy with KW-2449 was stopped and the patient started dasatinib 100 mg/day and Interferon-a 3,000,000 units. Three months later, the patient acheived CCyR with a BCR-ABL1/ABL1 ratio of 0.05. At the last evaluation, 16 months after the start of dasatinib and interferon combination, the patient was maintaining CCyR and major molecular response. In conclusion, this case illustrates the benefit of the use of combination therapy, mainly TKI and agent with different mechanism of action either sequentially (TKI followed by KW-2449) or concomitantly (TKI + interferon) in eradicating resistant disease with T315I clone. Figure 1 Serial Monitoring of Ph+ Cells, T315I Cells and BCR-ABL1/ABL1 Ratio Figure 1. Serial Monitoring of Ph+ Cells, T315I Cells and BCR-ABL1/ABL1 Ratio Disclosures: Cortes: Novartis: Research Funding. Jabbour:Novartis: Speakers Bureau; Bristol Myers Squibb : Speakers Bureau.

Targeting Isoprenylcysteine Carboxyl Methyltransferase to Overcome Resistance and Improve Responses in Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3273-3273
Author(s):  
Wen Tian Sun ◽  
Bee Ling Ng ◽  
Li Yi Yang ◽  
Mei Wang ◽  
William Hwang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Rho Gtpases ◽  
Myeloid Leukemia ◽  
Small Gtpases ◽  
Synergistic Activity ◽  
Carboxyl Methyltransferase ◽  
Imatinib Resistant ◽  
Cd34 Cells ◽  
Isoprenylcysteine Carboxyl Methyltransferase

Abstract Abstract 3273 Poster Board III-1 A strategy to overcome resistance of chronic myeloid leukemia (CML) to Bcr-Abl tyrosine kinase inhibitors (TKI) is by targeting alternative pathways. Increasing evidence suggests that activation of the Ras family of small GTPases is important in leukaemogenic transformation by Bcr-Abl. Ras and Rho GTPases play important roles in the initiation and progression of cancer by disrupting the normal regulation of specific intracellular signalling pathways, promoting cell cycle progression and resistance to apoptosis. The biological function of Ras and Rho GTPases is, to a large extent, dependent on prenylation, a three-step post-translational modification process. The first step is catalysed by farnesyl transferase or geranylgeranyl transferase and the final step is catalysed by isoprenylcysteine carboxyl methyltransferase (Icmt). We have found inhibition of Icmt by a novel selective inhibitor, cysmethynil, enhanced the anti-proliferative effect of imatinib in CML cell lines. The combination of cysmethynil and imatinib was synergistic in inducing apoptosis in the imatinib-sensitive K562 and Bafp210 CML cell lines and apoptosis was further increased when the drugs were given sequentially, with cysmethynil added after imatinib. This combination was also effective in the imatinib-resistant Bcr-Abl- and P-glycoprotein-overexpressing LAMA84 cell line. The synergy was also observed when cysmethynil was combined with dasatinib in both the imatinib-sensitive Bafp210 and imatinib-resistant Bafp210 Y253F mutant but not in the Bafp210 T315I mutant cell lines. The combination therapy was more effective than single agent in inducing apoptosis in treatment-naive primary CML CD34+ cells and was not toxic to cord blood CD34+ cells (Figure 1). Figure 1 Effect of imatinib and cysmethynil on apoptosis in CD34+ cells. Abbreviations: IM + Cys, imatinib and cysmethynil added concurrently; IM −> Cys, cysmethynil added 9 hours after imatinib; Cys −> IM, imatinib added 9 hours after cysmethynil Figure 1. Effect of imatinib and cysmethynil on apoptosis in CD34+ cells. Abbreviations: IM + Cys, imatinib and cysmethynil added concurrently; IM −> Cys, cysmethynil added 9 hours after imatinib; Cys −> IM, imatinib added 9 hours after cysmethynil Imatinib decreases p-CrkL through its inhibition of Bcr-Abl kinase. Interestingly, in both K562 and Bafp210, cysmethynil also decreases p-CrkL and when combined with imatinib, p-CrkL levels are reduced further (Figure 2). CrkL activation is dependent on a multimeric protein complex containing PI-3 kinase, docking protein 2, CrkL, Vav and the Rho GTPase, Rac. We hypothesise that inhibition of CrkL phosphorylation in the CML cell lines by cysmethynil is through the inhibition of Rac prenylation. This may be responsible for the synergistic activity observed when cysmethynil is combined with imatinib. In conclusion, our preliminary results suggest that the combination of a Bcr-Abl TKI and an Icmt inhibitor exerts a selective apoptotic effect in CML cells and this combination should be explored further as a useful tool to overcome resistance and improve responses in CML. Figure 2 Effect of imatinib and cysmethynil on p-CrkL in Bafp210. Figure 2. Effect of imatinib and cysmethynil on p-CrkL in Bafp210. Disclosures: Druker: MolecularMD: Equity Ownership; Novartis Pharmaceuticals: ; Bristol-Myers Squibb:.

Study of different mutations in chronic myeloid leukemia in India and their co-relation with drug resistance.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 7083-7083
Author(s):  
Priti Mitra ◽  
Swati Dasgupta ◽  
Chinmay Kumar Basu ◽  
Firoj Hossain Gharami ◽  
Subrata Mandal ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chromosomal Abnormalities ◽  
Mutation Screening ◽  
Resistance Mutation ◽  
Tyrosine Kinase Domain ◽  
Molecular Response ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Resistant Mutation

7083 Background: Emergence of ABL point mutations is the most frequent cause for imatinib resistance in CML. Aim of our study is to investigate two potential resistance mechanisms i.e.,mutations of BCR-ABL tyrosine kinase domain (TKD) and Additional Chromosomal Abnormalities during TKI treatment in CML. Methods: Karyotyping and BCR-ABL TKD mutation screening are performed in 100 imatinib resistant CML patients who were on imatinib at the time of loss of hematologic response, cytogenetic or molecular response. Imatinib–Resistance Mutation Analysis (Qualitative) were detected by Nested RTPCR and Sanger’s Sequencing. In 100 cases, 34 received escalated imatinib, 34 nilotinib and another 32 dasatinib. Results: In 100 BCR-ABL positive imatinib, nilotinib and dasatinib resistant cases, 11 different BCR-ABL TKD mutations were detected. Analysis revealed no mutations-43 cases, M351T-12 cases, G250E-10 cases, F317L-8 cases, M244V-5 cases, E255K-4 cases, V379I-4 cases, F359V-3 cases, H396R-3 cases, Y253F-3 cases, E355G-3 cases, T315I-2 cases. 11 novel mutations (F317L, G250E, M244V, Y253F, E255K, M351T, F359V, H396R, V379I, E355G, T315I) conferring imatinib resistance, 10 nilotinib–resistant mutations (M244V, F359V, T315I, E355G, G250E) and 8 dasatinib-resistant mutations (H396R, F317L, H396R, T315I, M351T) were seen in our patient population. T315I was found more frequently in cases on dasatinib than on imatinib therapy. Conclusions: T315I which confers resistance to all TKIs was detected only in 2/100 patients who demonstrated loss of response in our population. As compared with other western studies, incidence of T315I mutation was very low in our study. In addition analysis of mutation patterns at baseline may help in stratifying patients for treatment. For cases with TKI resistance, mutation and ACA screening may play role in identifying patients with poorer prognosis. In our practice if nilotinib–resistant mutation was detected, dasatinib was preferred and for dasatinib-resistant mutation, nilotinib was preferred. We are planning for using bosutinib, panotinib and omacetaxine (SC route) in third line therapy in imatinib resistant different mutation positive chronic myeloid leukemia.

Treatment of Pediatric Chronic Myeloid Leukemia in the Year 2010: Use of Tyrosine Kinase Inhibitors and Stem-Cell Transplantation

Hematology ◽  
2010 ◽  
Vol 2010 (1) ◽  
pp. 368-376 ◽  
Author(s):  
Meinolf Suttorp ◽  
Frédéric Millot
Keyword(s):  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Stem Cell Transplantation ◽  
Tyrosine Kinase ◽  
Cell Transplantation ◽  
Myeloid Leukemia ◽  
Second Generation ◽  
Young Patients ◽  
Hematopoietic Stem ◽  
First Choice

AbstractAllogeneic hematopoietic stem cell transplantation (allo-SCT) remains the only proven cure for chronic myeloid leukemia (CML), a rare malignancy in childhood. With the excellent results induced by the tyrosine kinase inhibitor (TKI) imatinib in adults in the last decade, the appropriate management of children with CML has also changed radically, and only a minority are now transplanted as a front-line treatment. Data on pediatric experiences with imatinib in CML from controlled trials remain very limited, but this review of available data describes the role of imatinib in children with CML, addressing: 1) the starting dose; 2) pharmacokinetics in childhood; 3) possible adverse effects, with a focus on the still-growing skeleton; 4) early monitoring of treatment efficacy in an attempt to avoid failure; 5) the timing of allo-SCT in children; and 6) treatment of CML relapse after allo-SCT. Because the characteristics of CML in children seem to overlap extensively with what is described in adult internal medicine, most answers and pediatric algorithms are adapted from the treatment of CML in adults. Today in 2010, allo-SCT in children should be postponed until CML becomes refractory to imatinib. The approach for young patients with suboptimal responses is unclear because data on the efficacy and safety of second-generation TKIs in childhood are almost entirely missing. Other than being included in a formal trial on second-generation TKIs, allo-SCT for patients failing imatinib remains the first choice.

Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy

Blood ◽  
2006 ◽  
Vol 109 (6) ◽  
pp. 2303-2309 ◽  
Author(s):  
Andreas Hochhaus ◽  
Hagop M. Kantarjian ◽  
Michele Baccarani ◽  
Jeffrey H. Lipton ◽  
Jane F. Apperley ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Therapeutic Option ◽  
Phase 1 ◽  
Treatment Options ◽  
Chronic Phase ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Dose Modifications

AbstractAlthough imatinib induces marked responses in patients with chronic myeloid leukemia (CML), resistance is increasingly problematic, and treatment options for imatinib-resistant or -intolerant CML are limited. Dasatinib, a novel, highly potent, oral, multitargeted kinase inhibitor of BCR-ABL and SRC family kinases, induced cytogenetic responses in a phase 1 study in imatinib-resistant or -intolerant CML and was well tolerated. Initial results are presented from a phase 2 study of 186 patients with imatinib-resistant or -intolerant chronic-phase CML (CML-CP) designed to further establish the efficacy and safety of dasatinib (70 mg twice daily). At 8-months' follow-up, dasatinib induced notable responses, with 90% and 52% of patients achieving complete hematologic and major cytogenetic responses (MCyR), respectively. Responses were long lasting: only 2% of patients achieving MCyR progressed or died. Importantly, comparable responses were achieved by patients carrying BCR-ABL mutations conferring imatinib resistance. Dasatinib also induced molecular responses, reducing BCR-ABL/ABL transcript ratios from 66% at baseline to 2.6% at 9 months. Nonhematologic adverse events were generally mild to moderate, and most cytopenias were effectively managed with dose modifications. Cross-intolerance with imatinib was not evident. To conclude, dasatinib induces notable responses in imatinib-resistant or -intolerant CML-CP, is well tolerated, and represents a promising therapeutic option for these patients. This trial was registered at www.clinicaltrials.gov as CA180013.

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