Abstract 2857: PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies
Authors: Li Yue , Kapoor Isha , Zhu Huayuan , Hill Brian T. , Xu Wei , Almasan Alex 
Published: August 2018

Abstract
B-cell lymphoid malignancies, including CLL and DLBCL, are characterized by abnormal activation of Bruton's tyrosine kinase (BTK)-mediated B-cell receptor signaling that contributes to malignant transformation. Ibrutinib (IB) is an oral covalent BTK inhibitor that has shown impressive clinical activity. However, many IB-treated patients relapse over time with fulminant progression. We sought to define the molecular pathways that mediate Ib resistance by studying changes that occurred in vitro in cell lines and primary patient samples with prolonged exposure to IB. We found IB-increased apoptosis in a dose- and time-dependent manner, with upregulated levels of PTEN, BIM, and FOXO3a in Mec-1 and RIVA cells; followed by decreased phosphorylation (p) of Ser473-AKT and Ser253-FOXO3a. mRNA expression levels of Foxo3a and its targets Bim and Pten were significantly upregulated in a dose- and time-dependent manner. Interestingly, a subset of miRNAs, including miRNA-494 were significantly downregulated with upregulation of target transcripts Pten and Bim. Similar results were observed after IB treatment of primary CLL cells of naïve patients and those on new drug targets at high risk for relapse. To examine if miRNA-494-dependent PTEN regulation and activation of PI3K/AKT/FOXO3a signaling has a role in de novo and acquired resistance to IB in B-cell malignancies, we investigated the resistance mechanism that develops to IB therapy by generating IB-resistant (Ib-R) cells. Chronic exposure of RIVA cells to IB demonstrated AKT activation and increased pSer253-FOXO3a, in turn, leading to decreased PTEN and BIM levels; attributed to its decreased transcription as assessed by qRT-PCR in Ib-R RIVA cells. Interestingly, Ib-R RIVA cells had strikingly higher miRNA-494 expression and decreased Pten mRNA levels, indicating further regulation of PTEN/AKT/FOXO3a signaling. Moreover, unlike IB-sensitive CLL samples, patients with partial remission and Ib-R, before and after in vivo IB treatment, demonstrated significant upregulation of miRNA-494 and decreased Pten mRNA expression. The Ib-R cells were sensitized to treatment with IB by PI3K (idelalisib) and AKT (MK-2206) inhibition. MK-2206 downregulated pAKT and sensitized Ib-R cells through AKT-mediated PTEN activation. siRNA-mediated downregulation of AKT and FOXO3a significantly modulated cell survival, demonstrating the importance of these cell survival factors for Ib-R. Importantly, overexpression of miRNA-494 inhibitor increased Pten mRNA levels, further indicating regulation of apoptosis by AKT/FOXO3a signaling. Collectively, these findings reveal a novel mechanism of ibrutinib resistance in B-cell malignancies that modulates the expression and activity of tumor suppressor proteins and pro-apoptotic factors to confer drug resistance that could be effectively inhibited by a rational combination of therapeutic regimens.
Citation Format: Yue Li, Isha Kapoor, Huayuan Zhu, Brian T. Hill, Wei Xu, Alex Almasan. PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2857.