Abstract 260: Bromodomain and extra-terminal motif proteins regulate linear and circular PVT1 in acute myeloid leukemia cells under normoxia and hypoxia
Authors: Ghetti Martina , Vannini Ivan , Bruno Samantha , Liverani Chiara , Traversa Debora , Montanaro Lorenzo , Napolitano Roberta , Luserna di Rorà Andrea Ghelli , Ibrahim Toni , Storlazzi Clelia Tiziana , Martinelli Giovanni , Simonetti Giorgia 
Published: September 2020

Abstract
Bromodomain and extra-terminal motif-protein inhibitors (BETi) have shown an antileukemic effect in several subtypes of acute myeloid leukemia (AML), both in vitro and in vivo (Daniel Gerlach et al. 2018 Oncogene). The effect of BETi is mainly mediated by suppression of bromodomain-containing protein 4 (BRD4) activity, an epigenetic regulator that sustains MYC and c-KIT expression, with consequences on a number of cellular processes, including proliferation and apoptosis (Kazuki Homma et al. 2017 Blood). Moreover recent findings suggested that MYC regulates through a feedback loop, a long-non coding RNA, named PVT1, which is located in the same genomic region (8q24) and is often associated with its pro-tumorigenic role (Alberto L'Abbate et al. 2018 Leukemia). PVT1 also encodes for a circular isoform (circPVT1), with MYC-dependent and independent functions.
The aim of the present study is to investigate the role of BETi in the regulation of the circular and linear forms of PVT1 in AML and the functional consequences of their downregulation.
OCI-AML3 and KASUMI-1 AML cell lines were treated for 16 hours with BETi under normoxic and hypoxic conditions mimicking the bone marrow microenvironment. circPVT1 and linear PVT1 (PVT1) were quantified by qRT-PCR, using total RNA and di-polisomal fraction. circPVT1 and PVT1 total RNA significantly decreased in both cell lines after treatment with BETi, under normoxia and hypoxia. Notably, KASUMI-1 expressed higher PVT1 levels under hypoxia compared to normoxia. Interestingly, circPVT1 and PVT1 were also detected in the polysomal fraction. This result was confirmed by RNA in situ hybridization (RNA-ISH) of PVT1 isoforms in combination with the S6 ribosomal marker with PVT1 showing mostly a nuclear localization, while circPVT1 colocalizing with ribosomes. In addition, circPVT1 was silenced by small interfering (siRNA) in order to investigate the effects on apoptosis, proliferation, and its downstream targets in OCI-AML3 cells. circPVT1 knockdown slightly but significantly affected cell number, with a modest impact on apoptosis and no changes of MYC transcript. We then evaluated the consequences of circPVT1 silencing on the expression of PKR, which has been previously reported to have a role in the innate immunity and to be inhibited by circRNA (Chu-Xiao Liu et al. 2019 Cell). Of note, an increased PKR expression was detected after siRNA treatment.
Our findings show that circPVT1 and PVT1 are regulated by BETi, along with MYC, in AML cell lines under normoxia and hypoxia and that silencing of circPVT1 dampens cell proliferation. Therefore PVT1 and circPVT1 may contribute to AML pathogenesis and progression, and our results suggest that targeting circPVT1 may have therapeutic potentials in AML.
Citation Format: Martina Ghetti, Ivan Vannini, Samantha Bruno, Chiara Liverani, Debora Traversa, Lorenzo Montanaro, Roberta Napolitano, Andrea Ghelli Luserna di Rorà, Toni Ibrahim, Clelia Tiziana Storlazzi, Giovanni Martinelli, Giorgia Simonetti. Bromodomain and extra-terminal motif proteins regulate linear and circular PVT1 in acute myeloid leukemia cells under normoxia and hypoxia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 260.