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Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

ABSTRACT: Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ?FRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ?FRET value and molecular response, a threshold of the ?FRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ?FRET value ?0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ?FRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).

SUBMITTER: Kondo T

PROVIDER: S-EPMC6029835 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

Kondo Takeshi T Fujioka Mari M Tsuda Masumi M Murai Kazunori K Yamaguchi Kohei K Miyagishima Takuto T Shindo Motohiro M Nagashima Takahiro T Wakasa Kentaro K Fujimoto Nozomu N Yamamoto Satoshi S Yonezumi Masakatsu M Saito Souichi S Sato Shinji S Ogawa Kazuei K Chou Takaaki T Watanabe Reiko R Kato Yuichi Y Takahashi Shuichiro S Okano Yoshiaki Y Yamamoto Joji J Ohta Masatsugu M Iijima Hiroaki H Oba Koji K Kishino Satoshi S Sakamoto Junichi J Ishida Yoji Y Ohba Yusuke Y Teshima Takanori T

Cancer science 20180529 7

Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated ...[more]

PMID: 29719934

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Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Project description:Drug resistance is an obstacle in the treatment of chronic myelogenous leukemia (CML), and is a common reason for treatment failure or disease progression. However, the underlying mechanisms of cyclophosphamide resistance remain poorly defined. In the present study, microarray data concerning cyclophosphamide‑sensitive and ‑resistant chronic myelogenous leukemia cell lines were analyzed. A total of 258 differentially‑expressed genes (DEGs) were identified between these two groups, from which 139 DEGs were upregulated and 119 were downregulated. Several candidate genes that were associated with cyclophosphamide resistance were also identified. These DEGs were subsequently classified using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis. A total of 487 biological processes and 17 KEGG pathways were revealed to be enriched. Furthermore, an interaction network was established to identify the core genes that regulated cyclophosphamide resistance. Signal transducer and activator of transcription 5A (STAT5A), FYN proto‑oncogene, Src family tyrosine kinase and spleen associated tyrosine kinase were revealed to be the hub genes in multiple enriched biological processes and signaling pathways, indicating that these were involved in mediating cyclophosphamide sensitivity in CML cells. The expression levels of 5 DEGs were also confirmed in two human CML cell lines (K‑562 and KU812) by reverse transcription‑quantitative polymerase chain reaction. Furthermore, selective knockdown of STAT5A and S100 calcium binding protein A4 (S100A4) recovered cyclophosphamide sensitivity in K‑562 cells, suggesting their involvement in drug resistance. The present study identified several potential genes and pathways contributing to cyclophosphamide resistance, and confirmed the involvement of STAT5A and S100A4 in drug resistance. These results enable improved understanding of the mechanisms underlying drug resistance in CML cells.

Dataset Information

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

Publications

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.

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