CircBA1 derived from BCR-ABL fusion gene inhibits cell proliferation in chronic myeloid leukemia.
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ABSTRACT:
SUBMITTER: Wang J
PROVIDER: S-EPMC7819548 | biostudies-literature | 2021 Jan
REPOSITORIES: biostudies-literature
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CircBA1 derived from BCR-ABL fusion gene inhibits cell proliferation in chronic myeloid leukemia.
Wang Jin J Ma Hu-Lin HL Liu Wen-Rong WR Peng Yong Y Zhou Jian-Kang JK Yang Jin-Lin JL
Cancer communications (London, England) 20210114 1
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Project description:The BCR-ABL fusion gene plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). The BCR-ABL oncoprotein encoded by this fusion gene has been extensively studied. However, research on whether BCR-ABL also affects circular RNAs (circRNAs) is limited. This study aimed to explore the new fusion circRNAs produced by BCR-ABL and their role in CML cells. In this study, we identified a novel fusion circRNA, named F-circBA1, originating from BCR-ABL in K562 and K562/G01 cells using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Sanger sequencing. qRT-PCR of the nuclear RNA and cytoplasmic RNA were separated, indicating that F-circBA1 was mainly localized in the cytoplasm. Cell counting kit-8 assay and flow cytometry showed that F-circBA1 knockdown by shRNA prevented the proliferation of K562 and K562/G01 cells, and the cell cycle was arrested at G2/M. Mechanically, dual-luciferase reporter assay and western blotting assay showed that F-circBA1 sponged miR-148-3p and F-circBA1 silencing decreased CDC25B expression in vitro. Furthermore, the results of the murine leukemogenesis model showed that F-circBA1 knockdown suppressed leukemogenesis in vivo. Besides, we found the existence of F-circBA1 in some patients with BCR-ABL-positive CML. In conclusion, these results demonstrate the presence of F-circBA1 and its oncogenic role in CML cells.
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Project description:Chronic myeloid leukemia (CML) accounts for approximately 15% of new adult leukemia cases. The fusion gene BCR‑ABL is an important biological basis and target for CML. In the present study, a novel compound, ND‑09, was developed and its inhibitory effect and mechanism of action on CML growth were evaluated using RT‑PCR and western blot analysis. The results showed that ND‑09 demonstrated a high level of inhibitory action toward CML cells overexpressing BCR‑ABL and induced K562 cell apoptosis through the mitochondrial pathway. Notably, combined ND‑09 and BCR‑ABL siRNA treatment could better inhibit cell proliferation and induce apoptosis in K562 cells. Furthermore, this growth effect of BCR‑ABL siRNA could be fully rescued by transfection with BCR‑ABL. ND‑09 exhibited a good fit within BCR‑ABL and occupied its ATP‑binding pocket, thus altering BCR‑ABL kinase activity. Therefore, ND‑09 downregulated the phosphorylation of BCR‑ABL and ABL, ultimately inhibiting the downstream signaling pathways in K562 cells. These findings suggest that ND‑09 induces growth arrest in CML cells by targeting BCR‑ABL.
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Project description:Chronic myeloid leukemia was the first haematological neoplasia that benefited from a targeted therapy with imatinib nearly 15 years ago. Since then, several studies have investigated the role of genes, their variants (i.e., polymorphisms) and their encoded proteins in the pharmacokinetics and pharmacodynamics of BCR-ABL1 tyrosine kinase activity inhibitors (TKIs). Transmembrane transporters seem to influence in a significant manner the disposition of TKIs, especially that of imatinib at both cellular and systemic levels. In particular, members of the ATP-binding cassette (ABC) family (namely ABCB1 and ABCG2) together with solute carrier (SLC) transporters (i.e., SLC22A1) are responsible for the differences in drug pharmacokinetics. In the case of the newer TKIs, such as nilotinib and dasatinib, the substrate affinity of these drugs for transporters is variable but lower than that measured for imatinib. In this scenario, the investigation of genetic variants as possible predictive markers has led to some discordant results. With the partial exception of imatinib, these discrepancies seem to limit the application of discovered biomarkers in the clinical settings. In order to overcome these issues, larger prospective confirmative trials are needed.
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Project description:To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro.32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells.C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC₅₀ at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC₅₀ at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells.C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.
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Project description:At diagnosis, about 5% of Chronic Myeloid Leukemia (CML) patients lacks Philadelphia chromosome (Ph), despite the presence of the BCR/ABL rearrangement. Two mechanisms have been proposed about the occurrence of this rearrangement: the first one is a cryptic insertion between chromosomes 9 and 22; the second one involves two sequential translocations: a classic t(9;22) followed by a reverse translocation, which reconstitutes the normal morphology of the partner chromosomes. Out of 398 newly diagnosed CML patients, we selected 12 Ph-negative cases. Six Ph-negative patients treated with tyrosine kinase inhibitors (TKIs) were characterized, in order to study the mechanisms leading to the rearrangement and the eventual correlation with prognosis in treatment with TKIs. FISH analysis revealed cryptic insertion in 5 patients and classic translocation in the last one. In more detail, we observed 4 different patterns of rearrangement, suggesting high genetic heterogeneity of these patients. In our cases, the BCR/ABL rearrangement mapped more frequently on 9q34 region than on 22q11 region, in contrast to previous reports. Four patients, with low Sokal risk, achieved Complete Cytogenetic Response and/or Major Molecular Response after TKIs therapy. Therapy resistance was observed in one patient with duplication of BCR/ABL rearrangement and in another one with high risk. Even if the number patient is inevitably low, we can confirm that the rare Ph-negative CML patients do not constitute a "warning" category, meanwhile the presence of further cytogenetic abnormalities remains an adverse prognostic factor even in TKI era.
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Project description:BackgroundThe BCR-ABL fusion protein is the key factor that results in the occurrence of chronic myeloid leukemia (CML). Imatinib (IM) is a targeted inhibitor of BCR-ABL to achieve complete remission. However, remission failure occurs due to acquired resistance caused by secondary BCR-ABL mutations, underlining the need for novel BCR-ABL-targeting strategies. Circular RNAs (circRNAs) derived from tumor-related genes have been revealed as possible therapeutic targets for relevant cancers in recent investigations. In CML, the roles of this kind of circRNA are yet obscure.MethodsFirstly, RT-qPCR was used for determining circCRKL expression level in cell lines and clinical samples, RNase R and Actinomycin D were employed to verify the stability of circCRKL. Then shRNAs were designed to specifically knockdown circCRKL. The function of circCRKL in vitro was investigated using CCK-8, colony formation assay, and flow cytometry, while a CML mouse model was constructed to explore the function in vivo. Finally, a dual-luciferase reporter assay, RNA pull-down, RNA immunoprecipitation, and rescue experiments were conducted to investigate the mechanism of circCRKL functioning.ResultsHere, we determined circCRKL, which derives from CML-relevant gene CRKL, is over-expressed in BCR-ABL+ cells. Then we noticed knocking down circCRKL using shRNA lentivirus dampens the proliferation of BCR-ABL+ cells both in vitro and in vivo, and augments susceptibility of resistant cells to IM. Intriguingly, we observed that circCRKL has a considerable impact on the expression level of BCR-ABL. Mechanistically, circCRKL could behave like a decoy for miR-877-5p to enhance the BCR-ABL level, allowing BCR-ABL+ cells to maintain viability.ConclusionsOverall, the current study uncovers that circCRKL is specifically expressed and regulates BCR-ABL expression level via decoying miR-877-5p in BCR-ABL+ cells, highlighting that targeting circCRKL along with imatinib treatment could be utilized as a potential therapeutic strategy for CML patients.
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Project description:Chronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the bcr-abl chimeric oncogene, encoding a 210 kDa protein with constitutive tyrosine kinase activity. In spite of the efficiency of tyrosine kinase inhibitors (TKI; Imatinib), other strategies are explored to eliminate CML leukemia stem cells, such as calcium pathways.In this work, we showed that Store-Operated Calcium Entry (SOCE) and thrombin induced calcium influx were decreased in Bcr-Abl expressing 32d cells (32d-p210). The 32d-p210 cells showed modified Orai1/STIM1 ratio and reduced TRPC1 expression that could explain SOCE reduction. Decrease in SOCE and thrombin induced calcium entry was associated to reduced Nuclear Factor of Activated T cells (NFAT) nucleus translocation in 32d-p210 cells. We demonstrated that SOCE blockers enhanced cell mobility of 32d-p210 cells and reduced the proliferation rate in both 32d cell lines. TKI treatment slightly reduced the thrombin-induced response, but imatinib restored SOCE to the wild type level. Bcr-Abl is also known to deregulate Protein Kinase C (PKC), which was described to modulate calcium entries. We showed that PKC enhances SOCE and thrombin induced calcium entries in control cells while this effect is lost in Bcr-Abl-expressing cells.The tyrosine kinase activity seems to regulate calcium entries probably not directly but through a global cellular reorganization involving a PKC pathway. Altogether, calcium entries are deregulated in Bcr-Abl-expressing cells and could represent an interesting therapeutic target in combination with TKI.
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Project description:Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr-Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr-Abl+ cells reduced levels of the Bcr-Abl protein and also the phosphorylation of Tyr177 of Bcr-Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr-Abl+ cells with Jak2 inhibitors for 4-6?h but not with IM also reduced Bcr-Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr-Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr-Abl in immune complexes but did not reduce levels of Bcr-Abl, suggesting that the reduction of Bcr-Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr-Abl stability and oncogenic signaling in CML cells are under the control of Jak2.
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