In vivo, <em>ex vivo</em> and <em>in vitro</em> dasatinib activity in chronic lymphocytic leukemia

Giannopoulos,Krzysztof; Karczmarczyk,Agnieszka; Karp,Marta; Bojarska‑Junak,Agnieszka; Kosior,Kamila; Kowal,Małgorzata; Tomczak,Waldemar; Hus,Marek; Machnicki,Marcin; Stokłosa,Tomasz

doi:10.3892/ol.2021.12546

In vivo, ex vivo and in vitro dasatinib activity in chronic lymphocytic leukemia

Authors:
- Krzysztof Giannopoulos
- Agnieszka Karczmarczyk
- Marta Karp
- Agnieszka Bojarska‑Junak
- Kamila Kosior
- Małgorzata Kowal
- Waldemar Tomczak
- Marek Hus
- Marcin Machnicki
- Tomasz Stokłosa
View Affiliations

Affiliations: Department of Experimental Hematooncology, Medical University of Lublin, Lublin 20‑093, Poland, Department of Clinical Immunology, Medical University of Lublin, Lublin 20‑093, Poland, Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin, Lublin 20‑093, Poland, Department of Immunology, Center for Biostructure Research, Medical University of Warsaw, Warsaw 02‑097, Poland
Published online on: February 12, 2021 https://doi.org/10.3892/ol.2021.12546
Article Number: 285
Copyright: © Giannopoulos et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Dasatinib inhibits the breakpoint cluster region‑Abelson murine leukemia 1 (BCR‑ABL1) gene along with other kinases known to be overexpressed and abnormally active in patients with chronic lymphocytic leukemia (CLL). The current study used primary leukemic cells obtained from 53 patients with CLL that were treated with dasatinib. A 2,3‑bis‑(2‑methoxy‑4‑nitro‑5‑sulfophenyl)‑2H‑tetrazolium‑5‑carboxanilide (XTT) assay and Annexin V staining was performed to assess the cytotoxic effects of dasatinib treatment. The XTT assay revealed that the median cytotoxicity of dasatinib was 8.30% (range, 0.00‑77.89%). Due to high dispersion of dasatinib activity, patients were divided into sensitive (n=27; 50.94%; median cytotoxicity, 22.81%) and resistant groups (n=26; 49.06%; median cytotoxicity, 0.00%). A median cytotoxicity of 8.30% was selected as a cut off value. Using Annexin V staining and flow cytometry on exemplary sensitive and resistant CLL samples, it was revealed that 17.71 and 1.84% of cells were apoptotic, respectively. The current study presented a case of a patient with concomitant occurrence of CLL and chronic myeloid leukemia (CML) with a major molecular response after dasatinib treatment. A simultaneous reduction of circulating CLL cells indicated in vivo anti‑CLL activity induced by dasatinib. After an in vitro culture of the patient's mononuclear cells with subsequent dasatinib treatment, a higher percentage of CLL cells undergoing apoptosis was obsevered when compared with untreated samples (38.19 vs. 21.99%, respectively). Similarly, the percentage of CLL apoptotic cells (ΔΨm^low) measured by chloromethyl‑X‑rosamine was higher after incubation with dasatinib (7.28%) than in the negative control (2.86%). In conclusion, dasatinib induced antileukemic effects against CML and CLL cells. The results of the current study indicated that dasatinib may induce apoptosis ex vivo, in vitro and in vivo in CLL.

View Figures

View References

1	Ghia P, Ferreri AM and Caligaris-Cappio F: Chronic lymphocytic leukemia. Crit Rev Oncol Hematol. 64:234–246. 2007. View Article : Google Scholar : PubMed/NCBI
2	Giannopoulos K: Biologia i rokowanie w przewlekłej białaczce limfocytowej. Acta Haematol Pol. 41:433–440. 2010.
3	Zenz T, Mertens D, Döhner H and Stilgenbauer S: Importance of genetics in chronic lymphocytic leukemia. Blood Rev. 25:131–137. 2011. View Article : Google Scholar : PubMed/NCBI
4	Kuckertz M, Patz M, Veldurthy A, Gehrke I, Claasen J, Frenzel LP, Wendtner CM, Hallek M and Krause G: Comparison of the effects of two kinase inhibitors, sorafenib and dasatinib, on chronic lymphocytic leukemia cells. Onkologie. 35:420–426. 2012.PubMed/NCBI
5	Stevenson FK and Caligaris-Cappio F: Chronic lymphocytic leukemia: Revelations from the B-cell receptor. Blood. 103:4389–4395. 2004. View Article : Google Scholar : PubMed/NCBI
6	Amrein PC: The potential for dasatinib in treating chronic lymphocytic leukemia, acute myeloid leukemia, and myeloproliferative neoplasms. Leuk Lymphoma. 52:754–763. 2011. View Article : Google Scholar : PubMed/NCBI
7	Winiarska M, Bojarczuk K, Pyrzynska B, Bil J, Siernicka M, Dwojak M, Bobrowicz M, Miazek N, Zapala P, Zagozdzon A, et al: Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies. MAbs. 6:1300–1313. 2014. View Article : Google Scholar : PubMed/NCBI
8	McCaig AM, Cosimo E, Leach MT and Michie AM: Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals. Br J Haematol. 153:199–211. 2011. View Article : Google Scholar : PubMed/NCBI
9	Amrein L, Hernandez TA, Ferrario C, Johnston J, Gibson SB, Panasci L and Aloyz R: Dasatinib sensitizes primary chronic lymphocytic leukaemia lymphocytes to chlorambucil and fludarabine in vitro. Br J Haematol. 143:698–706. 2008. View Article : Google Scholar : PubMed/NCBI
10	Contri A, Brunati AM, Trentin L, Cabrelle A, Miorin M, Cesaro L, Pinna LA, Zambello R, Semenzato G and Donella-Deana A: Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis. J Clin Invest. 115:369–378. 2005. View Article : Google Scholar : PubMed/NCBI
11	Gobessi S, Laurenti L, Longo PG, Sica S, Leone G and Efremov DG: ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells. Blood. 109:2032–2039. 2007. View Article : Google Scholar : PubMed/NCBI
12	Lin K, Glenn MA, Harris RJ, Duckworth AD, Dennett S, Cawley JC, Zuzel M and Slupsky JR: c-Abl expression in chronic lymphocytic leukemia cells: Clinical and therapeutic implications. Cancer Res. 66:7801–7809. 2006. View Article : Google Scholar : PubMed/NCBI
13	Hochhaus A, Kantarjian HM, Baccarani M, Lipton JH, Apperley JF, Druker BJ, Facon T, Goldberg SL, Cervantes F, Niederwieser D, et al: Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood. 109:2303–2309. 2007. View Article : Google Scholar : PubMed/NCBI
14	McCaig AM, Cosimo E, Leach MT and Michie AM: Dasatinib inhibits CXCR4 signaling in chronic lymphocytic leukaemia cells and impairs migration towards CXCL12. PLoS One. 7:e489292012. View Article : Google Scholar : PubMed/NCBI
15	Amrein L, Soulières D, Johnston JB and Aloyz R: p53 and autophagy contribute to dasatinib resistance in primary CLL lymphocytes. Leuk Res. 35:99–102. 2011. View Article : Google Scholar : PubMed/NCBI
16	Hallaert DYH, Jaspers A, van Noesel CJ, van Oers MH, Kater AP and Eldering E: c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: Implications for therapeutic targeting of chemoresistant niches. Blood. 112:5141–5149. 2008. View Article : Google Scholar : PubMed/NCBI
17	Kater AP, Spiering M, Liu RD, Doreen Te Raa G, Slinger E, Tonino SH, Beckers MM, Daenen S, Doorduijn JK, Lankheet NA, et al: Dasatinib in combination with fludarabine in patients with refractory chronic lymphocytic leukemia: A multicenter phase 2 study. Leuk Res. 38:34–41. 2014. View Article : Google Scholar : PubMed/NCBI
18	ten Hacken E, Scielzo C, Bertilaccio MTS, Scarfò L, Apollonio B, Barbaglio F, Stamatopoulos K, Ponzoni M, Ghia P and Caligaris-Cappio F: Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia. Blood. 121:2264–2273. 2013. View Article : Google Scholar : PubMed/NCBI
19	Giannopoulos K, Dmoszynska A, Kowal M, Wasik-Szczepanek E, Bojarska-Junak A, Rolinski J, Döhner H, Stilgenbauer S and Bullinger L: Thalidomide exerts distinct molecular antileukemic effects and combined thalidomide/fludarabine therapy is clinically effective in high-risk chronic lymphocytic leukemia. Leukemia. 23:1771–1778. 2009. View Article : Google Scholar : PubMed/NCBI
20	Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K, Castaneda S, Cornelius LAM, Das J, Doweyko AM, et al: Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem. 47:6658–6661. 2004. View Article : Google Scholar : PubMed/NCBI
21	Martinez Marignac VL, Smith S, Toban N, Bazile M and Aloyz R: Resistance to Dasatinib in primary chronic lymphocytic leukemia lymphocytes involves AMPK-mediated energetic re-programming. Oncotarget. 4:2550–2566. 2013. View Article : Google Scholar : PubMed/NCBI
22	Amrein PC, Attar EC, Takvorian T, Hochberg EP, Ballen KK, Leahy KM, Fisher DC, Lacasce AS, Jacobsen ED, Armand P, et al: Phase II study of dasatinib in relapsed or refractory chronic lymphocytic leukemia. Clin Cancer Res. 17:2977–2986. 2011. View Article : Google Scholar : PubMed/NCBI
23	Hochhaus A, Baccarani M, Silver RT, Schiffer C, Apperley JF, Cervantes F, Clark RE, Cortes JE, Deininger MW, Guilhot F, et al: European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia. 34:966–984. 2020. View Article : Google Scholar : PubMed/NCBI
24	Putowski M, Podgórniak M, Piróg M, Knap J, Zaleska J, Purkot J, Zawiślak J, Zakrzewska E, Karczmarczyk A, Własiuk P, et al: Prognostic impact of NOTCH1, MYD88, and SF3B1 mutations in Polish patients with chronic lymphocytic leukemia. Pol Arch Intern Med. 127:238–244. 2017.PubMed/NCBI
25	Bojarska-Junak A, Giannopoulos K, Kowal M, Dmoszyńska A and Roliński J: Comparison of methods for determining zeta-chain associated protein-70 (ZAP-70) expression in patients with B-cell chronic lymphocytic leukemia (B-CLL). Cytometry B Clin Cytom. 70:293–301. 2006. View Article : Google Scholar : PubMed/NCBI
26	Burger JA and O'Brien S: Evolution of CLL treatment-from chemoimmunotherapy to targeted and individualized therapy. Nat Rev Clin Oncol. 15:510–527. 2018. View Article : Google Scholar : PubMed/NCBI
27	Dühren-von Minden M, Übelhart R, Schneider D, Wossning T, Bach MP, Buchner M, Hofmann D, Surova E, Follo M, Köhler F, et al: Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature. 489:309–312. 2012. View Article : Google Scholar : PubMed/NCBI
28	Veldurthy A, Patz M, Hagist S, Pallasch CP, Wendtner CM, Hallek M and Krause G: The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes. Blood. 112:1443–1452. 2008. View Article : Google Scholar : PubMed/NCBI
29	Pitini V, Arrigo C and Altavilla G: Dasatinib induces a response in chronic lymphocytic leukemia. Blood. 113:4982009. View Article : Google Scholar : PubMed/NCBI
30	Garg RJ, Wierda W, Fayad L, Estrov Z, Bickel SM and O'Brien S: Phase II study of dasatinib in patients with relapsed CLL. Blood. 112:4197a2008. View Article : Google Scholar
31	Song Z, Lu P, Furman RR, Leonard JP, Martin P, Tyrell L, Lee FY, Knowles DM, Coleman M and Wang YL: Activities of SYK and PLCgamma2 predict apoptotic response of CLL cells to SRC tyrosine kinase inhibitor dasatinib. Clin Cancer Res. 16:587–599. 2010. View Article : Google Scholar : PubMed/NCBI
32	Hantschel O, Rix U, Schmidt U, Bürckstümmer T, Kneidinger M, Schütze G, Colinge J, Bennett KL, Ellmeier W, Valent P, et al: The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc Natl Acad Sci USA. 104:13283–13288. 2007. View Article : Google Scholar : PubMed/NCBI
33	Serpa M, Bendit I, Seguro F, Xavier F, Cavalcante M, Steinbaum D, Nardinelli L, Aldred VL, de Paula HM and Dorlhiac-Llacer PE: Response to dasatinib in a patient with concomitant chronic myeloid leukemia and chronic lymphocytic leukemia. Acta Haematol. 124:105–109. 2010. View Article : Google Scholar : PubMed/NCBI
34	Kim DH, Kamel-Reid S, Chang H, Sutherland R, Jung CW, Kim HJ, Lee JJ and Lipton JH: Natural killer or natural killer/T cell lineage large granular lymphocytosis associated with dasatinib therapy for Philadelphia chromosome positive leukemia. Haematologica. 94:135–139. 2009. View Article : Google Scholar : PubMed/NCBI
35	Mustjoki S, Ekblom M, Arstila TP, Dybedal I, Epling-Burnette PK, Guilhot F, Hjorth-Hansen H, Höglund M, Kovanen P, Laurinolli T, et al: Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy. Leukemia. 23:1398–1405. 2009. View Article : Google Scholar : PubMed/NCBI
36	Tsimberidou AM, Wen S, McLaughlin P, O'Brien S, Wierda WG, Lerner S, Strom S, Freireich EJ, Medeiros LJ, Kantarjian HM, et al: Other malignancies in chronic lymphocytic leukemia/small lymphocytic lymphoma. J Clin Oncol. 27:904–910. 2009. View Article : Google Scholar : PubMed/NCBI
37	Benjamini O, Jain P, Trinh L, Qiao W, Strom SS, Lerner S, Wang X, Burger J, Ferrajoli A, Kantarjian H, et al: Second cancers in patients with chronic lymphocytic leukemia who received frontline fludarabine, cyclophosphamide and rituximab therapy: Distribution and clinical outcomes. Leuk Lymphoma. 56:1643–1650. 2015. View Article : Google Scholar : PubMed/NCBI
38	Boddu P, Gibbons J, Burger J, Sivina M, Thakral B, Kanagal-Shamanna R and Ferrajoli A: Co-occurrence of chronic myeloid leukemia with chronic lymphocytic leukemia: A report of two cases. Leuk Lymphoma. 60:1568–1571. 2019. View Article : Google Scholar : PubMed/NCBI
39	Fischer K, Bahlo J, Fink AM, Goede V, Herling CD, Cramer P, Langerbeins P, von Tresckow J, Engelke A, Maurer C, et al: Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL: Updated results of the CLL8 trial. Blood. 127:208–215. 2016. View Article : Google Scholar : PubMed/NCBI
40	Tecchio C, Nichele I, Todeschini G, Pizzolo G and Ambrosetti A: Dasatinib-induced response in a rare case of chronic lymphocytic leukaemia associated with chronic myeloid leukaemia. Br J Haematol. 146:222–223. 2009. View Article : Google Scholar : PubMed/NCBI
41	Nagao T, Takahashi N, Kameoka Y, Noguchi S, Shinohara Y, Ohyagi H, Kume M and Sawada K: Dasatinib-responsive chronic lymphocytic leukemia in a patient treated for coexisting chronic myeloid leukemia. Intern Med. 52:2567–2571. 2013. View Article : Google Scholar : PubMed/NCBI