BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 690-698 ◽  
Author(s):  
Nicholas J. Donato ◽  
Ji Yuan Wu ◽  
Jonathan Stapley ◽  
Gary Gallick ◽  
Hui Lin ◽  
...  

Clinical studies have shown that the tyrosine kinase inhibitor STI571 effectively controls BCR-ABL–positive chronic myelogenous leukemia (CML). However, disease progression while on STI571 therapy has been reported, suggesting de novo or intrinsic resistance to BCR-ABL–targeted therapy. To investigate possible mediators of acquired STI571 resistance, K562 cells resistant to 5 μM STI571 (K562-R) were cloned and compared to the parental cell population. K562-R cells had reduced BCR-ABL expression and limited activation of BCR-ABL signaling cascades (Stat 5, CrkL, MAPK). STI571 failed to activate caspase cascades or to suppress expression of survival genes (bcl-xL) in resistant cells. Gene sequencing and tyrosine kinase activity measurements demonstrated that K562-R cells retained wild-type and active BCR-ABL tyrosine kinase that was inhibitable by in vitro incubation with STI571, suggesting that BCR-ABL was not coupled to proliferation or survival of K562-R cells. The src-related kinase LYN was highly overexpressed and activated in K562-R cells, and its inhibition reduced proliferation and survival of K562-R cells while having limited effects of K562 cells. Specimens taken from patients with advanced CML that progressed on STI571 therapy also were analyzed for LYN kinase expression, and they were found to be elevated to a level similar to that of K562-R cells. Comparison of samples from patients taken prior to and following STI571 failure suggested that expression and/or activation of LYN/HCK occurs during disease progression. Together, these results suggest that acquired STI571 resistance may be associated with BCR-ABL independence and mediated in part through overexpression of other tyrosine kinases.

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2202-2202
Author(s):  
Katharina M. Brauer ◽  
Daniela Werth ◽  
Karin von Schwarzenberg ◽  
Anita Bringmann ◽  
Lothar Kanz ◽  
...  

Abstract Imatinib mesylate (Gleevec®) is a specific tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. In the treatment of chronic myelogenous leukemia (CML) it has become indispensable and shows few side effects. Recently, it was shown that patients treated with imatinib showed impaired CTL responses in comparison to patients treated with IFN-α, which might be due to a reduced immunogenicity of CML cells or result from an inhibitory effect of imatinib on the function of antigen presenting cells and T lymphocytes. In the present study, we show that imatinib treatment leads to a downregulation of immunogenic antigens on the CML cells, which in turn inhibits the development of CML-specific cytotoxic T lymphocytes (CTLs). To achieve this, we treated the CML cell line K562 and an imatinib-resistant K562 variant, K562R, with imatinib or DMSO, isolated the total RNA and used it to electroporate monocyte-derived dendritic cells (DCs). These cells were then used as antigen presenting cells (APCs) for the induction of polyclonal CTL responses. The cytolytic activity of the CTLs was assayed in standard 51Cr-release assays and their fine specificity in IFNγ-Elispot assays. CTLs generated using RNA from imatinib-treated K562 cells were completely incapable of specific killing and did not react in Elispot assays, whereas those CTLs induced using RNA from K562 cells subjected to DMSO treatment as well as RNA from imatinib-treated K562R cells showed specific cytolytic activity against targets electroporated with RNA from CML cells and were able to recognize several CML-associated antigens, like survivin, PRAME, WT-1 and PR3 in Elispot assays. To confirm that this effect is mediated by BCR-ABL inhibition, we used specific siRNA against the bcr-abl fusion site b3a2 to downregulate the protein expression and found essentially the same results. Even in K562R cells, that constitutively overexpress BCR-ABL, targeting the expression of the protein directly by specific siRNA leads to an impairment of CTL induction. In order to confirm and expand these studies, we additionally analyzed the expression of antigens connected to immune responses to CML in Western Blot and Real-time PCR experiments. We found, that imatinib-mediated inhibition of BCR-ABL in K562 cells leads to a decreased expression of tumor antigens and cellular proteins including survivin, adipophilin, hTERT, WT-1, Bcl-xL and Bcl-2 in correlation to the decreased development of specific CTLs. Matching the results of the 51Cr-release assays, these effects were not observed in K562R cells. In primary CML cells subjected to imatinib a downregulation of hTERT and survivin could be detected, which corresponded to a decreased lysis of DCs electroporated with RNA from these cells in standard 51Cr-release assays. Our results demonstrate, that BCR-ABL directly influences the expression of immunogenic tumor associated antigens by its uncontrolled tyrosine kinase activity and therefore substantially contributes to the immunogenicity of CML cells.


2019 ◽  
Vol 3 (5) ◽  
pp. 857-864 ◽  
Author(s):  
Declan C T Lavoie ◽  
Marie-Eve Robinson ◽  
Donna Johnston ◽  
Marika Pagé ◽  
Victor N Konji ◽  
...  

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4818-4818
Author(s):  
Eugene McPherson ◽  
R. Shuklar ◽  
S.Y. Huang ◽  
M. Grimbel ◽  
E. Hazel

Abstract Imatinib mesylate (IM) is potent BCR/abl tyrosine kinase inhibitor in patients with chronic myelogenous leukemia (CML). It has remarkable frontline clinical effects in this disease, however, the leukemic cells become resistant to IM in both chronic and blast phases. BCR/abl kinase can induce reactive oxygen species (ROS) and promote self-mutation which subsequently render IM to resistance and failure to eliminate all leukemia cells. This mechanism of resistance in IM (mutation) is caused by oxidant damage to DNA with kinase domain mutations, reduced IM binding and kinase inhibition. Antioxidants (ascorbic acid, etc.) may help overcome IM resistance and restore sensitivity to IM via suppression of transcription factor Nrf2 that regulates the gene expression gammaglutamylcysteine ((g-GCS), the rate-limiting enzyme in glutathione (GSH) biosynthesis and detoxification. P-glycoprotein (P-gp) drug efflux can also exist and complete molecular response relapse occurs. Leukemic cells that are P-gp positive, and P-gp dependent decline of intracellular IM levels are associated with retained phosphorylation pattern of BCR/abl and loss of IM effect on apoptosis and cellular proliferation. Modulation of P-gp with HMG-CoA reductase inhibitor simvastatin may help restore IM cytotoxicity. We present a case of an 80+ year old female with CML-chronic phase-II (CML-CP-II) with concommitant cormorbidities of CAD, unstable angina, hypertension, and dyslipidemia treated with aspirin, simvastatin and started on IM 400mg daily. After two months of therapy she developed grade 3 neutropenia, lower extremity edemia, nausea/vomiting and fluid retention requiring IM interruption and supportive care with growth factors. IM dose reduction to 300mg daily and simvastatin 10 mg every other day. Soluble interleukin-2 receptor (sIL-2R) levels were elevated and trending down once proinflammatory cytokines were modulated. Real-time BCR-ABL/abl-PCR ratio increased insignificantly from 0.001% to 0.003%. Betacarotene level significantly decreased to 4, ascorbic level within normal limits, VEGF remained < 31 pg/ml (normal 31–86 pg/ml), fibrinogen level 309.90 mg/dl (normal 162–431), ESR 15 mm/hr, C-reactive protein 5.55 mg/dl and sIL-2R increased to > 3,000 U/mL (normal 200– 1100 U/mL). Betacarotene and ascorbic antioxidants dosage were increased and immunomodulation of preinflammatory cytokines ROS, sIL-2R and betacarotene normalized, 512 and 44 respectively. Serial measurement of BCR-ABL/abl ratio did not exceed 0.02% on three occasions or 0.05% on two occasions, therefore no molecular relapse and persistent low levels of BCR-ABL/abl ratio with no hematologic or cytogenetic relapse. We felt that an acute coronary syndrome with perturbation of CML with some IM resistance was developed. CONCLUSION: IM, a tyrosine kinase inhibitor may develop resistance when leukemic cells are positive with P-gp and oxidative stress increase ROS along with decreases in IM intracellular levels. Modulation by HMG -CoA reductase inhibitor (simvastatin) via a mechanism of inhibition of P-gp transport and antioxidants reduction of BCR/abl mutagenesis may allow IM to efficently restore normal hematopoiesis in CML patients.


RSC Advances ◽  
2016 ◽  
Vol 6 (115) ◽  
pp. 114096-114108 ◽  
Author(s):  
Xiaowei Huo ◽  
Yonghong Liao ◽  
Yu Tian ◽  
Li Gao ◽  
Li Cao

Chronic myelogenous leukemia (CML) is a hematopoietic malignancy caused by the constitutive activation of BCR–ABL tyrosine kinase.


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