Dynamic Interplay of miR-155-5p Transfer in the Mechanisms Underlying Ibrutinib Resistance in DLBCL through Extracellular Vesicles

doi:10.21203/rs.3.rs-3951865/v1

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Dynamic Interplay of miR-155-5p Transfer in the Mechanisms Underlying Ibrutinib Resistance in DLBCL through Extracellular Vesicles

https://doi.org/10.21203/rs.3.rs-3951865/v1

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Ibrutinib, a Bruton Tyrosine Kinase (BTK) inhibitor, has exhibited efficacy in various B-cell lymphoid malignancies. However, prolonged usage may lead to resistance, impacting treatment outcomes. The oncogenic microRNA, miR-155-5p is related with poor prognosis in B-cell lymphomas, prompting our investigation into the mechanism of acquired ibrutinib resistance in B-cell lymphoma cells. To generate ibrutinib-resistant OCI-Ly1 cells (OCI-Ly1-IbtR), continuous exposure to 1µM and 2µM of ibrutinib was employed. microRNA profiling of OCI-Ly1-IbtR was conducted, and exosomes were isolated using ultracentrifugation. Comparative studies of microRNA levels in cells and exosomes, and targets of up-regulated microRNAs in OCI-Ly1-IbtR were explored. Target validation involved transfection of candidate microRNA, and co-culture experiments utilized OCI-Ly1 with exosomes from OCI-Ly1-IbtR. Elevated miR-155-5p levels were observed in OCI-Ly1-IbtR and exosomes from OCI-Ly1-IbtR, correlating with AKT and NF-κB activation. MiR-155-5p transfection induced AKT/NF-κB pathway activation in OCI-Ly1, resulting in ibrutinib resistance, enhanced colony formation and sustained BTK activity. Primary cell lines from ibrutinib-refractory B-cell lymphoma patients exhibited similar signaling protein activation. Target evaluation identified KDM5B and DEPTOR as miR-155-5p targets, confirmed by downregulation after transfection. KDM5B and DEPTOR enrichment in Ago2 during ibrutinib resistance and miR-155-5p transfection was observed. Co-culture experiments demonstrated exosome-mediated miR-155-5p transfer, inducing ibrutinib resistance and KDM5B/DEPTOR downregulation in OCI-Ly1. Our findings suggest that miR-155-5p overexpression is associated with AKT and NF-κB pathway activation in ibrutinib-resistant cells, proposing a potential role for acquired miR-155-5p upregulation in B-cell lymphoma ibrutinib resistance.

B-Cell Lymphoma

Ibrutinib

Exosome

MicroRNA

KDM5B

DEPTOR

In B-cell lymphomas, aberrant B-cell receptor (BCR) signaling stands out as a pivotal mechanism fueling tumor proliferation and progression [1, 2]. Extensive research has explored BCR signaling as a therapeutic target in various B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and chronic lymphocytic leukemia (CLL) [3]. Given that Bruton's tyrosine kinase (BTK) plays a crucial role in the BCR signaling pathway, inhibiting BTK has emerged as a therapeutic approach for B-cell lymphomas marked by activated BCR signaling [4].

Ibrutinib, a pioneering oral covalent BTK inhibitor, gained approval from the US Food and Drug Administration for treating adult patients with diverse B-cell malignancies [5]. The clinical success of ibrutinib in several B-cell lymphomas stems from its effective blockade of BCR signaling. However, the continuous administration of ibrutinib may lead to the development of resistance [6–8]. While the mechanisms of ibrutinib resistance are not fully elucidated, potential factors include the C481S mutation at the ibrutinib binding site of BTK and activating mutations in phospholipase C gamma 2, a direct BTK substrate [9–11]. Beyond genomic complexities, microRNAs (miRNAs) have emerged as mediators of resistance to BTK inhibition. Notably, the overexpression of miR-494, miR-495, and miR-543 has been linked to acquired resistance to BTK inhibitors, regulating the PTEN/AKT/mTOR signaling axis in B-cell lymphomas [12].

MiRNAs, small single-stranded non-coding RNAs, play a crucial role as negative post-transcriptional regulators of gene expression through mRNA degradation or translational repression [13, 14]. Their binding to the 3’ untranslated region of target mRNA leads to repression or degradation, influencing the expression of approximately 60% of all human protein-coding genes [15]. About half of miRNA genes reside in cancer-associated genomic regions, underscoring their potential role in human cancer pathogenesis [16]. Moreover, miRNAs can be transferred through cell-to-cell communication in the tumor microenvironment, with exosomes—small extracellular vesicles with a diameter of 30–150 nm—acting as mediators [17]. Tumor cells release exosomes into the bloodstream, containing mainly small RNA, including microRNA [18, 19]. Cancer-associated miRNAs may act as oncogenic agents, modulating the expression of tumor suppressor genes or oncogenes [20].

The oncogenic miRNA, miR-155-5p, has been associated with poor prognosis in B-cell lymphomas like DLBCL, MCL, and CLL [21–23]. Considering its linkage to the AKT/mTOR pathway and chemo-resistance, the upregulation of miR-155-5p might activate alternative survival pathways in B cells. This pro-tumor effect of miR-155-5p could potentially be transferred through exosome-mediated interactions [24–26]. However, it remains unexplored whether ibrutinib resistance is associated with miR-155-5p in B-cell lymphomas. Therefore, our investigation delves into the mechanism of resistance to BTK inhibition using ibrutinib-resistant B-cell lymphoma cells, hypothesizing exosome-mediated miR-155-5p upregulation in tumor cells undergoing acquired resistance to ibrutinib.

Culture of ibrutinib-resistant B-cell lymphoma cells

For the generation of ibrutinib-resistant B-cell lymphoma cells, we employed the OCI-Ly1 B-cell lymphoma cell line, which was generously provided by Professor WS Kim. OCI-Ly1 cells were cultivated in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% fetal bovine serum (FBS) and 1% antibiotics. The culture of OCI-Ly1 cells involved continuous exposure to ibrutinib at concentrations of 1 µM and 2 µM.

To simulate the development of acquired ibrutinib resistance observed in clinical practice, the established ibrutinib-resistant OCI-Ly1 cells were perpetually cultured under the influence of ibrutinib. This approach aimed to replicate the conditions encountered during ibrutinib treatment and the subsequent emergence of resistance in a clinical setting.

Cell-derived exosome isolation

Following the cultivation of cells at a density of 4 x 10⁵ cells/mL in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% exosome-depleted fetal bovine serum (FBS) for a duration of 72 hours, the cell culture medium was harvested. The harvested medium underwent differential centrifugation steps, initially at 300 x g for 10 minutes at 4°C, followed by a subsequent centrifugation at 2000 x g for 10 minutes at 4°C. Subsequently, the concentrated cell culture medium was obtained through centrifugation at 3000 x g (4°C) utilizing an Allegra® X-15R centrifuge and an Amicon® Ultra-15 100 kDa device. Further purification involved a centrifugation step at 10,000 x g for 30 minutes at 4°C, followed by filtration using a 0.22µm filter (Milipore, Carrigtwohill, Co Cork, Ireland) to eliminate cell debris. The clarified cell culture medium underwent pelleting via ultracentrifugation at 110,000 x g for 2 hours at 4°C. The resulting pellet was resuspended in phosphate-buffered saline (PBS) and subjected to a final ultracentrifugation step at 110,000 x g for 70 minutes at 4°C. After removal of the supernatant, the ultimate pellet was resuspended in PBS.

Cell viability assay

Cell proliferation was evaluated employing the Cell Counting Kit-8 (DOJINDO Laboratories, Kumamoto, Japan). To assess the impact of the drug on cell viability, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and exposed to a gradient of ibrutinib concentrations (ranging from 0 to 10 µM) for a duration of 72 hours. Subsequently, 10 µL of CCK-8 solution was added to each well, and the plate was incubated for 2 hours at 37 ℃. Absorbance measurements were obtained at 450 nm using a microplate reader (ABS Plus, San Jose, CA, USA).

BTK assay

The impact of ibrutinib on BTK (Bruton's tyrosine kinase) activity was assessed to ascertain whether the drug influenced BTK function. For the BTK assay, OCI-Ly1 ibrutinib-resistant cells and OCI-Ly1 parental cells transfected with miRNAs at final concentrations of 100 nM were prepared. These cells were subsequently seeded into 6-well plates at a density of 1.2 × 10⁷ cells per well and exposed to 0.3 µM concentrations of ibrutinib for a duration of 72 hours. BTK activity was determined using the human BTK ELISA Kit (Abcam, Boston, MA, USA) following the manufacturer's instructions. The BTK assay results were quantified using a microplate reader (ABS Plus, San Jose, CA, USA) at 450 nm. This process aimed to elucidate any alterations in BTK activity resulting from exposure to ibrutinib and the influence of miR-N.C and miR-155-5p on BTK function.

Ago2 Immunoprecipitation

To conduct Ago2 immunoprecipitation (IP), OCI-Ly1 ibrutinib-resistant cells and cells transfected with miR-N.C and miR-155-5p at final concentrations of 50 nM were prepared. These cells were seeded into 6-well plates at a density of 5 × 10⁵ cells per well and incubated for 24 hours. Subsequently, the cells were lysed in PEB buffer (20 mM Tris-HCl [pH 7.4], 0.5% Nonident P-40, 150 mM KCl, and 5 mM MgCl₂). The lysed cells underwent immunoprecipitation using Dynabeads Protein G for Immunoprecipitation (Invitrogen, Carlsbad, CA), along with monoclonal Anti-AGO2 antibody produced in rat (Sigma-Aldrich, St. Louis, MO, USA #087M4841V), and Normal rat IgG utilized as a control IgG (Santa cruz biotechnology, Dallas, TX, USA #A2121), following the manufacturer's instructions. After elution from the beads, RNA was isolated using Phenol Acid. This procedure was designed to selectively isolate RNA associated with Ago2 protein, providing insights into miRNA-mediated regulatory mechanisms.

Luciferase

The 3′-UTR sequences of KDM5B and DEPTOR, harboring the miR-155-5p binding site, were retrieved from gene sequence databases such as TargetScan. The pmiRGLO plasmid vector (Promega) was employed to design both the wild-type and mutant-type constructs for KDM5B and DEPTOR. In brief, PCR amplification was conducted on KDM5B and DEPTOR using specific forward and reverse primers (Supplementary Table 1), followed by their cloning into the multiple cloning region of the pmiRGLO plasmid vector. The mutant-type constructs for KDM5B and DEPTOR were generated through site-directed mutagenesis. For reporter assays, HEK293T cells seeded at a density of 1 × 10⁵ cells/ml in a 24-well plate were transfected with either the wild-type or mutant-type KDM5B and DEPTOR plasmid vectors, along with miR-NC or miR-155-5p (50 nM), using Lipofectamine and Lipofectamine 3000. Firefly and Renilla luciferase activities were quantified through a luminometer (Turner BioSystems, Sunnyvale, CA, USA). The relative expression was calculated by averaging the ratio of firefly to Renilla luciferase activities. In parallel experiments, OCI-Ly1 parental cells were transfected with miRNA (final concentrations of 100 nM) using Lipofectamine™ RNAiMAX (Life Technologies Co., Carlsbad, CA, USA). miRCURY LNA miRNAs, negative control miRNA mimic, and miR-155-5p mimic (Life technologies, Carlsbad, CA, USA) were utilized. Furthermore, OCI-Ly1 parental cells were transfected with siRNA (final concentrations of 100 nM or 150 nM) using Lipofectamine™ RNAiMAX. Negative control siRNA, KDM5B siRNA, and DEPTOR siRNA (Dharmacon, Chicago, IL, USA) were employed in these experiments.

Up-regulation of miR-155-5p in ibrutinib-resistant B-cell lymphoma cells

Following continuous exposure of OCI-Ly1 cells to ibrutinib at concentrations of 1µM (Ly1R1) and 2µM (Ly1R2) for four weeks, the resulting ibrutinib-resistant B-cell lymphoma cells displayed over a 10-fold increase in IC50 values compared to the parental OCI-Ly1 cells (Fig. 1A). Exposure to 0.3µM ibrutinib for 72 hours revealed a decrease in BTK activity in parental OCI-Ly1, while no significant difference was observed in the ibrutinib-resistant Ly1R1 and Ly1R2 cells (Fig. 1B). Comparative miRNA profiling between parental OCI-Ly1 and Ly1R1 revealed more than a two-fold up-regulation of 23 different miRNAs, including miR-155-5p, in the ibrutinib-resistant cells (Fig. 1C). KEGG pathway analysis based on the up-regulated miRNAs in ibrutinib-resistant cells implicated their involvement in four major pathways (p53 signaling, MAPK signaling, AKT signaling, and B cell receptor signaling) (Fig. 1D). Validation by qRT-PCR confirmed the elevated expression of miR-155-5p in Ly1R1 and Ly1R2 compared to parental OCI-Ly1, aligning with increased phosphorylation of AKT, ERK, and NF-κB (Fig. 1E, F). Notably, PTEN levels remained insignificantly different between parental and ibrutinib-resistant OCI-Ly1 cells (Fig. 1G).

Enrichment of miR-155-5p in exosomes isolated from ibrutinib-resistant OCI-Ly1 cells

Exosomes isolated from OCI-Ly1-IbtR and parental OCI-Ly1 cells displayed typical morphology consistent with exosomes, along with the presence of exosome markers CD63 and CD81 (Fig. 2A, B). No significant differences in size and number of exosomes were observed based on the originating cell type (Fig. 2C). Exosomes derived from Ly1R1 and Ly1R2 exhibited an elevated expression of miR-155-5p and increased phosphorylation of AKT, ERK, and NF-κB (Fig. 2D, E). Exposure of parental cells to exosomes from ibrutinib-resistant cells confirmed the presence of exosomes in parental cells, suggesting a potential role for exosome-mediated transfer of miR-155-5p from ibrutinib-resistant tumor cells to parental tumor cells (Fig. 2F).

Pro-survival effect and resistance to ibrutinib after miR-155-5p upregulation

Transfection of miR-155-5p into parental OCI-Ly1 cells conferred resistance to ibrutinib treatment, mimicking the behavior of ibrutinib-resistant OCI-Ly1 cells. MiR-155-5p-transduced OCI-Ly1 cells displayed increased cell viability and persistent BTK activity following treatment with 0.3µM ibrutinib, unlike parental OCI-Ly1 cells that exhibited decreased cell viability and loss of BTK activity under the same conditions (Fig. 3A, B). Consistent with this pro-survival effect, miR-155-5p-transduced OCI-Ly1 cells demonstrated increased phosphorylation of AKT, p70S6K, ERK, and NF-κB signaling pathways, along with enhanced colony-forming activity (Fig. 3C, D). Up-regulation of miR-155-5p was also evident in primary tumor cells isolated from refractory DLBCL patients (Patient B and C) resistant to salvage therapy with ibrutinib, and the exosomes from their culture medium exhibited miR-155-5p enrichment compared to those with a favorable outcome (Patient A) (Fig. 3E). In line with the effects of miR-155-5p on B-cell lymphoma cells, increased phosphorylation of AKT, p70S6K, ERK, and NF-κB was observed in primary tumor cells of refractory patients (Patient B and C) compared to those with a favorable outcome (Patient A) (Fig. 3F), implying an association between up-regulated miR-155-5p and ibrutinib resistance. The scheme (Fig. 3G) illustrates the dynamic process of miR-155-5p transfer via EVs between ibrutinib-resistant and parental OCI-Ly1 cells, resulting in the acquisition of ibrutinib resistance in the parental cells. The feedback loop created by sustained miR-155-5p transfer and signaling pathway activation contributes to the persistence of the resistant phenotype in the parental cell population.

Exploration of miR-155-5p target genes related with ibrutinib resistance

To identify miR-155-5p target genes associated with ibrutinib resistance, down-regulated genes in ibrutinib-resistant OCI-Ly1 cells were explored. Comparative RNA sequencing analysis revealed fifty-two genes down-regulated more than two-fold in ibrutinib-resistant cells compared to parental cells (Fig. 4A). In silico analysis using miRDB predicted 701 genes with conserved miR-155-5p binding sites. Among these, KDM5B, DEPTOR, and KCNN3 overlapped with the down-regulated gene list in RNA sequencing of ibrutinib-resistant OCI-Ly1 (Fig. 4A). qRT-PCR and western blot analysis confirmed the downregulation of KDM5B and DEPTOR in ibrutinib-resistant OCI-Ly1 cells compared to parental OCI-Ly1 (Fig. 4B, C). An inverse association of cellular miR-155-5p with target genes KDM5B and DEPTOR was observed in miR-155-5p-transfected OCI-Ly1 cells (Fig. 4D). However, no significant difference in KCNN3 levels was observed in the qRT-PCR analysis of ibrutinib-resistant cells and miR-155-5p-transfected OCI-Ly1 cells (Supplementary 1A, B).

Validation of KDM5B and DEPTOR as a target of miR-155-5p

Ago2 immunoprecipitation (Ago2 IP) was performed to determine whether KDM5B and DEPTOR could be regulated by miR-155-5p. The levels of KDM5B and DEPTOR decreased in ibrutinib-resistant OCI-Ly1 cells (Fig. 5A). Similar results were observed in miR-155-5p-transfected OCI-Ly1 cells, where levels of KDM5B and DEPTOR decreased and Ago2 was enriched compared to parental control cells (Fig. 5B). Luciferase reporter vectors were created with wild-type and mutant-type constructs for KDM5B and DEPTOR, confirming direct targeting by miR-155-5p. The wild-type constructs, where miR-155-5p bound to the 3’-UTR sequences of KDM5B and DEPTOR, showed decreased levels of KDM5B and DEPTOR (Fig. 5C). However, no change was observed in the mutant-type constructs where miR-155-5p did not bind to the 3’-UTR sequences of KDM5B and DEPTOR (Fig. 5C). These results indicated that miR-155-5p directly targets KDM5B and DEPTOR. Conversely, no Ago2 enrichment was observed in the case of KCNN3, aligning with the lack of association between KCNN3 and miR-155-5p (Supplementary Fig. 1C).

Association of miR-155-5p target genes with cell growth

To assess the effect of knockdown of KDM5B and DEPTOR, OCI-Ly1 cells were transfected with a combination of KDM5B and DEPTOR siRNAs, each with four different binding sites (labeled as #1, #2, #3 and #4). qRT-PCR and Western blot analysis confirmed down-regulation of KDM5B and DEPTOR in the transfected cells (Fig. 6A, B). While the expression of KDM5B decreased in KDM5B siRNA-transfected cells, DEPTOR levels showed no significant down-regulation, consistent with the marginal effect of DEPTOR siRNA on RNA levels of DEPTOR (Fig. 6B). Cell growth increased after 24h, 48h, and 72h in KDM5B siRNA-transfected cells, with a slight increase in colony formation (Fig. 6C). In DEPTOR siRNA-transfected cells, a significant increase in cell growth and colony formation was observed (Fig. 6D).

Our results suggest a complex interplay between miR-155-5p, exosomes, and ibrutinib resistance in B-cell lymphomas. The current study focused on miR-155-5p, an oncogenic miRNA whose elevated expression has been associated with poor prognosis in B-cell lymphomas such as DLBCL, MCL, and CLL [21–23]. MiR-155-5p has been implicated in modulating the AKT/mTOR pathway and contributing to chemo-resistance [24–26]. Its role in ibrutinib resistance, however, has not been extensively studied until now. The study provides compelling evidence for the upregulation of miR-155-5p in ibrutinib-resistant B-cell lymphoma cells (OCI-Ly1-IbtR) compared to parental cells (OCI-Ly1). Moreover, the investigation extends beyond intracellular mechanisms and explores the role of exosomes in mediating miR-155-5p transfer. Exosomes derived from ibrutinib-resistant cells exhibited elevated levels of miR-155-5p, suggesting a potential mechanism for the transfer of miR-155-5p between tumor cells. This finding aligns with the emerging understanding of miRNA-enriched exosomes contributing to intercellular communication in the tumor microenvironment [17, 18]. Functional assays, including miR-155-5p transfection into parental OCI-Ly1 cells, demonstrated a pro-survival effect and resistance to ibrutinib treatment. This was accompanied by the activation of signaling pathways involving AKT, p70S6K, ERK, and NF-κB. Importantly, the study extended its observations to primary tumor cells derived from refractory DLBCL patients, providing clinical relevance to the findings. The study further delved into the potential targets of miR-155-5p in the context of ibrutinib resistance. Through RNA sequencing and in silico analysis, KDM5B and DEPTOR emerged as candidate genes downregulated in ibrutinib-resistant cells. Both KDM5B, a histone demethylase, and DEPTOR, an mTOR phosphatase, have been implicated in tumor suppression and negative regulation of cell proliferation [27–32]. Validation experiments, including Ago2 immunoprecipitation and luciferase reporter assays, provided evidence for the direct regulation of KDM5B and DEPTOR by miR-155-5p. The inverse correlation between miR-155-5p levels and KDM5B/DEPTOR expression supported the hypothesis that these genes are downstream targets of miR-155-5p. Functional consequences of KDM5B and DEPTOR downregulation were explored through siRNA transfection experiments. Knockdown of KDM5B and DEPTOR resulted in increased cell growth and colony formation, implicating these genes in the regulation of cell proliferation and survival.

Our study concludes that miR-155-5p upregulation, potentially mediated by exosomes, could activate the AKT and NF-κB pathway in ibrutinib-resistant cells. This suggests that acquired upregulation of miR-155-5p might be a crucial mechanism contributing to ibrutinib resistance in B-cell lymphomas. The findings underscore the clinical relevance of miR-155-5p in the context of ibrutinib resistance, especially in refractory DLBCL patients. The association of miR-155-5p with AKT/mTOR pathway activation and chemo-resistance aligns with previous reports implicating miRNAs in the modulation of therapeutic responses in cancer [12, 20]. While the study provides valuable insights, it is essential to acknowledge its limitations. The specific mechanisms governing exosome-mediated transfer of miR-155-5p and the comprehensive landscape of miRNA involvement in ibrutinib resistance warrant further exploration. MiR-155-5p might not be the only miRNA associated with ibrutinib-resistance in DLBCL. In our miRNA analysis results, miR-146-5p and miR-21-5p were also up-regulated in ibrutinib resistant cells and EVs. There are several reports that miR-146a-5p and miR-21-5p promotes macrophage and stromal cells to have pro-tumor phenotype [33, 34]. Therefore, the identification of other potential miRNAs involved in this process could unveil a more intricate network of regulatory mechanisms. In clinical situation, DLBCL cells may not only interact with each other but also with tumor microenvironment.

In conclusion, this study sheds light on the role of miR-155-5p and exosomes in mediating ibrutinib resistance in B-cell lymphomas. The findings provide a foundation for further investigations into the intricate molecular mechanisms governing miRNA-mediated resistance and highlight potential therapeutic targets, such as KDM5B and DEPTOR, in the context of BTK inhibition. Understanding these mechanisms is crucial for developing effective strategies to overcome ibrutinib resistance and improve outcomes for patients with B-cell lymphomas.

Funding declaration

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2019R1C1C1005945, and 2021R1A2C1007531).

Author Contribution

B.P. and M.E.C. wrote the main manuscript text. B.P., M.E.C. and K.J.R. perfomred experiments and C.P. prepared figures 1-6. S.E.Y., W.S.K. J.Y.H., and S.J.K. prepared clinical data. J.Y.H. and S.J.K. designed the study and experiments. All authors reviewed the manuscript.

Acknowledgments

We acknowledge all participants in this study and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT).

Data availability declaration

The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request.

Tanaka S, Baba Y (2020) B Cell Receptor Signaling. Adv Exp Med Biol 1254:23-36. https://doi.org/10.1007/978-981-15-3532-1_2
Burger JA, Wiestner A (2018) Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nat Rev Cancer 18:148-167. https://doi.org/10.1038/nrc.2017.121
Profitos-Peleja N, Santos JC, Marin-Niebla A, Roue G, Ribeiro ML (2022) Regulation of B-Cell Receptor Signaling and Its Therapeutic Relevance in Aggressive B-Cell Lymphomas. Cancers (Basel) 14. https://doi.org/10.3390/cancers14040860
Alu A, Lei H, Han X, Wei Y, Wei X (2022) BTK inhibitors in the treatment of hematological malignancies and inflammatory diseases: mechanisms and clinical studies. J Hematol Oncol 15:138. https://doi.org/10.1186/s13045-022-01353-w
Davids MS, Brown JR (2014) Ibrutinib: a first in class covalent inhibitor of Bruton's tyrosine kinase. Future Oncol 10:957-967. https://doi.org/10.2217/fon.14.51
George B, Chowdhury SM, Hart A, Sircar A, Singh SK, Nath UK, Mamgain M, Singhal NK, Sehgal L, Jain N (2020) Ibrutinib Resistance Mechanisms and Treatment Strategies for B-Cell lymphomas. Cancers (Basel) 12. https://doi.org/10.3390/cancers12051328
Lee YP, Jung YJ, Cho J, Ko YH, Kim WS, Kim SJ, Yoon SE (2023) A retrospective analysis of ibrutinib outcomes in relapsed or refractory mantle cell lymphoma. Blood Res 58:208-220. https://doi.org/10.5045/br.2023.2023208
Nakhoda S, Vistarop A, Wang YL (2023) Resistance to Bruton tyrosine kinase inhibition in chronic lymphocytic leukaemia and non-Hodgkin lymphoma. Br J Haematol 200:137-149. https://doi.org/10.1111/bjh.18418
Chiron D, Di Liberto M, Martin P, Huang X, Sharman J, Blecua P, Mathew S, Vijay P, Eng K, Ali S, Johnson A, Chang B, Ely S, Elemento O, Mason CE, Leonard JP, Chen-Kiang S (2014) Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma. Cancer Discov 4:1022-1035. https://doi.org/10.1158/2159-8290.CD-14-0098
Woyach JA, Furman RR, Liu TM, Ozer HG, Zapatka M, Ruppert AS, Xue L, Li DH, Steggerda SM, Versele M, Dave SS, Zhang J, Yilmaz AS, Jaglowski SM, Blum KA, Lozanski A, Lozanski G, James DF, Barrientos JC, Lichter P, Stilgenbauer S, Buggy JJ, Chang BY, Johnson AJ, Byrd JC (2014) Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib. N Engl J Med 370:2286-2294. https://doi.org/10.1056/NEJMoa1400029
Kanagal-Shamanna R, Jain P, Patel KP, Routbort M, Bueso-Ramos C, Alhalouli T, Khoury JD, Luthra R, Ferrajoli A, Keating M, Jain N, Burger J, Estrov Z, Wierda W, Kantarjian HM, Medeiros LJ (2019) Targeted multigene deep sequencing of Bruton tyrosine kinase inhibitor-resistant chronic lymphocytic leukemia with disease progression and Richter transformation. Cancer 125:559-574. https://doi.org/10.1002/cncr.31831
Kapoor I, Bodo J, Hill BT, Almasan A (2021) Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies. Cell Death Dis 12:1061. https://doi.org/10.1038/s41419-021-04353-9
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215-233. https://doi.org/10.1016/j.cell.2009.01.002
Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466:835-840. https://doi.org/10.1038/nature09267
Friedman RC, Farh KK, Burge CB, Bartel DP (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19:92-105. https://doi.org/10.1101/gr.082701.108
Zhang B, Pan X, Cobb GP, Anderson TA (2007) microRNAs as oncogenes and tumor suppressors. Dev Biol 302:1-12. https://doi.org/10.1016/j.ydbio.2006.08.028
Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, Antoniou A, Arab T, Archer F, Atkin-Smith GK, Ayre DC, Bach JM, Bachurski D, Baharvand H, Balaj L, Baldacchino S, Bauer NN, Baxter AA, Bebawy M, Beckham C, Bedina Zavec A, Benmoussa A, Berardi AC, Bergese P, Bielska E, Blenkiron C, Bobis-Wozowicz S, Boilard E, Boireau W, Bongiovanni A, Borras FE, Bosch S, Boulanger CM, Breakefield X, Breglio AM, Brennan MA, Brigstock DR, Brisson A, Broekman ML, Bromberg JF, Bryl-Gorecka P, Buch S, Buck AH, Burger D, Busatto S, Buschmann D, Bussolati B, Buzas EI, Byrd JB, Camussi G, Carter DR, Caruso S, Chamley LW, Chang YT, Chen C, Chen S, Cheng L, Chin AR, Clayton A, Clerici SP, Cocks A, Cocucci E, Coffey RJ, Cordeiro-da-Silva A, Couch Y, Coumans FA, Coyle B, Crescitelli R, Criado MF, D'Souza-Schorey C, Das S, Datta Chaudhuri A, de Candia P, De Santana EF, De Wever O, Del Portillo HA, Demaret T, Deville S, Devitt A, Dhondt B, Di Vizio D, Dieterich LC, Dolo V, Dominguez Rubio AP, Dominici M, Dourado MR, Driedonks TA, Duarte FV, Duncan HM, Eichenberger RM, Ekstrom K, El Andaloussi S, Elie-Caille C, Erdbrugger U, Falcon-Perez JM, Fatima F, Fish JE, Flores-Bellver M, Forsonits A, Frelet-Barrand A, Fricke F, Fuhrmann G, Gabrielsson S, Gamez-Valero A, Gardiner C, Gartner K, Gaudin R, Gho YS, Giebel B, Gilbert C, Gimona M, Giusti I, Goberdhan DC, Gorgens A, Gorski SM, Greening DW, Gross JC, Gualerzi A, Gupta GN, Gustafson D, Handberg A, Haraszti RA, Harrison P, Hegyesi H, Hendrix A, Hill AF, Hochberg FH, Hoffmann KF, Holder B, Holthofer H, Hosseinkhani B, Hu G, Huang Y, Huber V, Hunt S, Ibrahim AG, Ikezu T, Inal JM, Isin M, Ivanova A, Jackson HK, Jacobsen S, Jay SM, Jayachandran M, Jenster G, Jiang L, Johnson SM, Jones JC, Jong A, Jovanovic-Talisman T, Jung S, Kalluri R, Kano SI, Kaur S, Kawamura Y, Keller ET, Khamari D, Khomyakova E, Khvorova A, Kierulf P, Kim KP, Kislinger T, Klingeborn M, Klinke DJ, 2nd, Kornek M, Kosanovic MM, Kovacs AF, Kramer-Albers EM, Krasemann S, Krause M, Kurochkin IV, Kusuma GD, Kuypers S, Laitinen S, Langevin SM, Languino LR, Lannigan J, Lasser C, Laurent LC, Lavieu G, Lazaro-Ibanez E, Le Lay S, Lee MS, Lee YXF, Lemos DS, Lenassi M, Leszczynska A, Li IT, Liao K, Libregts SF, Ligeti E, Lim R, Lim SK, Line A, Linnemannstons K, Llorente A, Lombard CA, Lorenowicz MJ, Lorincz AM, Lotvall J, Lovett J, Lowry MC, Loyer X, Lu Q, Lukomska B, Lunavat TR, Maas SL, Malhi H, Marcilla A, Mariani J, Mariscal J, Martens-Uzunova ES, Martin-Jaular L, Martinez MC, Martins VR, Mathieu M, Mathivanan S, Maugeri M, McGinnis LK, McVey MJ, Meckes DG, Jr., Meehan KL, Mertens I, Minciacchi VR, Moller A, Moller Jorgensen M, Morales-Kastresana A, Morhayim J, Mullier F, Muraca M, Musante L, Mussack V, Muth DC, Myburgh KH, Najrana T, Nawaz M, Nazarenko I, Nejsum P, Neri C, Neri T, Nieuwland R, Nimrichter L, Nolan JP, Nolte-'t Hoen EN, Noren Hooten N, O'Driscoll L, O'Grady T, O'Loghlen A, Ochiya T, Olivier M, Ortiz A, Ortiz LA, Osteikoetxea X, Ostergaard O, Ostrowski M, Park J, Pegtel DM, Peinado H, Perut F, Pfaffl MW, Phinney DG, Pieters BC, Pink RC, Pisetsky DS, Pogge von Strandmann E, Polakovicova I, Poon IK, Powell BH, Prada I, Pulliam L, Quesenberry P, Radeghieri A, Raffai RL, Raimondo S, Rak J, Ramirez MI, Raposo G, Rayyan MS, Regev-Rudzki N, Ricklefs FL, Robbins PD, Roberts DD, Rodrigues SC, Rohde E, Rome S, Rouschop KM, Rughetti A, Russell AE, Saa P, Sahoo S, Salas-Huenuleo E, Sanchez C, Saugstad JA, Saul MJ, Schiffelers RM, Schneider R, Schoyen TH, Scott A, Shahaj E, Sharma S, Shatnyeva O, Shekari F, Shelke GV, Shetty AK, Shiba K, Siljander PR, Silva AM, Skowronek A, Snyder OL, 2nd, Soares RP, Sodar BW, Soekmadji C, Sotillo J, Stahl PD, Stoorvogel W, Stott SL, Strasser EF, Swift S, Tahara H, Tewari M, Timms K, Tiwari S, Tixeira R, Tkach M, Toh WS, Tomasini R, Torrecilhas AC, Tosar JP, Toxavidis V, Urbanelli L, Vader P, van Balkom BW, van der Grein SG, Van Deun J, van Herwijnen MJ, Van Keuren-Jensen K, van Niel G, van Royen ME, van Wijnen AJ, Vasconcelos MH, Vechetti IJ, Jr., Veit TD, Vella LJ, Velot E, Verweij FJ, Vestad B, Vinas JL, Visnovitz T, Vukman KV, Wahlgren J, Watson DC, Wauben MH, Weaver A, Webber JP, Weber V, Wehman AM, Weiss DJ, Welsh JA, Wendt S, Wheelock AM, Wiener Z, Witte L, Wolfram J, Xagorari A, Xander P, Xu J, Yan X, Yanez-Mo M, Yin H, Yuana Y, Zappulli V, Zarubova J, Zekas V, Zhang JY, Zhao Z, Zheng L, Zheutlin AR, Zickler AM, Zimmermann P, Zivkovic AM, Zocco D, Zuba-Surma EK (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7:1535750. https://doi.org/10.1080/20013078.2018.1535750
Berardocco M, Radeghieri A, Busatto S, Gallorini M, Raggi C, Gissi C, D'Agnano I, Bergese P, Felsani A, Berardi AC (2017) RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines. Oncotarget 8:82920-82939. https://doi.org/10.18632/oncotarget.20503
Lunavat TR, Cheng L, Kim DK, Bhadury J, Jang SC, Lasser C, Sharples RA, Lopez MD, Nilsson J, Gho YS, Hill AF, Lotvall J (2015) Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells--Evidence of unique microRNA cargos. RNA Biol 12:810-823. https://doi.org/10.1080/15476286.2015.1056975
Loh HY, Norman BP, Lai KS, Rahman N, Alitheen NBM, Osman MA (2019) The Regulatory Role of MicroRNAs in Breast Cancer. Int J Mol Sci 20. https://doi.org/10.3390/ijms20194940
Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, Jacobs S, Kroesen BJ, van den Berg A (2005) BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol 207:243-249. https://doi.org/10.1002/path.1825
He J, Xi Y, Gao N, Xu E, Chang J, Liu J (2021) Identification of miRNA-34a and miRNA-155 as prognostic markers for mantle cell lymphoma. J Int Med Res 49:3000605211016390. https://doi.org/10.1177/03000605211016390
Papageorgiou SG, Kontos CK, Diamantopoulos MA, Bouchla A, Glezou E, Bazani E, Pappa V, Scorilas A (2017) MicroRNA-155-5p Overexpression in Peripheral Blood Mononuclear Cells of Chronic Lymphocytic Leukemia Patients Is a Novel, Independent Molecular Biomarker of Poor Prognosis. Dis Markers 2017:2046545. https://doi.org/10.1155/2017/2046545
Mahesh G, Biswas R (2019) MicroRNA-155: A Master Regulator of Inflammation. J Interferon Cytokine Res 39:321-330. https://doi.org/10.1089/jir.2018.0155
Milane L, Singh A, Mattheolabakis G, Suresh M, Amiji MM (2015) Exosome mediated communication within the tumor microenvironment. J Control Release 219:278-294. https://doi.org/10.1016/j.jconrel.2015.06.029
Yang E, Wang X, Gong Z, Yu M, Wu H, Zhang D (2020) Exosome-mediated metabolic reprogramming: the emerging role in tumor microenvironment remodeling and its influence on cancer progression. Signal Transduct Target Ther 5:242. https://doi.org/10.1038/s41392-020-00359-5
Daniunaite K, Dubikaityte M, Gibas P, Bakavicius A, Rimantas Lazutka J, Ulys A, Jankevicius F, Jarmalaite S (2017) Clinical significance of miRNA host gene promoter methylation in prostate cancer. Hum Mol Genet 26:2451-2461. https://doi.org/10.1093/hmg/ddx138
Diener C, Hart M, Kehl T, Rheinheimer S, Ludwig N, Krammes L, Pawusch S, Lenhof K, Tänzer T, Schub D, Sester M, Walch-Rückheim B, Keller A, Lenhof HP, Meese E (2020) Quantitative and time-resolved miRNA pattern of early human T cell activation. Nucleic Acids Res 48:10164-10183. https://doi.org/10.1093/nar/gkaa788
Chan LH, Yan Q (2022) Awakening KDM5B to defeat leukemia. Proc Natl Acad Sci U S A 119:e2202245119. https://doi.org/10.1073/pnas.2202245119
Cui D, Dai X, Gong L, Chen X, Wang L, Xiong X, Zhao Y (2020) DEPTOR is a direct p53 target that suppresses cell growth and chemosensitivity. Cell Death Dis 11:976. https://doi.org/10.1038/s41419-020-03185-3
Jabłońska E, Białopiotrowicz E, Szydłowski M, Prochorec-Sobieszek M, Juszczyński P, Szumera-Ciećkiewicz A (2020) DEPTOR is a microRNA-155 target regulating migration and cytokine production in diffuse large B-cell lymphoma cells. Exp Hematol 88:56-67.e52. https://doi.org/10.1016/j.exphem.2020.07.005
Souza OF, Popi AF (2022) Role of microRNAs in B-Cell Compartment: Development, Proliferation and Hematological Diseases. Biomedicines 10. https://doi.org/10.3390/biomedicines10082004
Xi J, Huang Q, Wang L, Ma X, Deng Q, Kumar M, Zhou Z, Li L, Zeng Z, Young KH, Zhang M, Li Y (2018) miR-21 depletion in macrophages promotes tumoricidal polarization and enhances PD-1 immunotherapy. Oncogene 37:3151-3165. https://doi.org/10.1038/s41388-018-0178-3
De Veirman K, Wang J, Xu S, Leleu X, Himpe E, Maes K, De Bruyne E, Van Valckenborgh E, Vanderkerken K, Menu E, Van Riet I (2016) Induction of miR-146a by multiple myeloma cells in mesenchymal stromal cells stimulates their pro-tumoral activity. Cancer Lett 377:17-24. https://doi.org/10.1016/j.canlet.2016.04.024

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Dynamic Interplay of miR-155-5p Transfer in the Mechanisms Underlying Ibrutinib Resistance in DLBCL through Extracellular Vesicles

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Abstract

Figures

Introduction

Method

Culture of ibrutinib-resistant B-cell lymphoma cells

Cell-derived exosome isolation

Cell viability assay

BTK assay

Ago2 Immunoprecipitation

Luciferase

Results

Up-regulation of miR-155-5p in ibrutinib-resistant B-cell lymphoma cells

Enrichment of miR-155-5p in exosomes isolated from ibrutinib-resistant OCI-Ly1 cells

Pro-survival effect and resistance to ibrutinib after miR-155-5p upregulation

Exploration of miR-155-5p target genes related with ibrutinib resistance

Association of miR-155-5p target genes with cell growth

Discussion

Declarations

Author Contribution

Acknowledgments

References

Additional Declarations

Supplementary Files

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