Abstract 2450: Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation
Authors: Aysegul Balyimez, Yihong Guan, Emily Esakov, Shinjini Ganguly, Ofer Reizes, Daniel J. Lindner, Jaroslaw Maciejewski, Babal Jha, Omar Y. Mian
Journal: Cancer Research (2020)

Abstract Background: Epigenetic reader proteins maintain an imbalance between differentiation and self-renewal in cancer. Genetic alterations in the DNA methylation machinery (e.g., TET, DNMTs) and chromatin remodelers (e.g., KDMs, EZH1/2) are a hallmark of cancer. Methyl CpG binding domain protein 2 (MBD2) is an epigenetic reader protein which modulates regional gene transcription by recruiting co-repressor complexes to sites of CpG methylation. Our goal is to develop a novel class of anti-neoplastic epigenetic therapies targeting MBD2 to selectively reprogram tumor cells towards a terminally differentiated state and sensitize them to chemotherapy, radiation, and immunotherapy. Methods: To explore conserved mediators of MBD2 function in cancer, we used MBD2 targeting shRNA lentivirus to stably knockdown MBD2 in prostate cancer (PC3, DU145, LnCap), leukemia (SigM5, TET2WTK562 or TET2KOK562) and triple negative breast cancer (MDA-MB231-nanog-GFP) cell lines. Inhibition of MBD2 expression was confirmed by qRT-PCR and Western Blot. Proliferative potential was determined by cell counting and clonogenic potential was determined by methylcellulose-based colony forming assays. RNAseq analysis was performed on prostate cancer and leukemia cell lines. miRNA expression was analyzed using miScript miRNA PCR (Qiagen). In vivo tumor initiation capacity was analyzed using orthotropic and heterotopic xenografts in athymic NSG mice. Results: Our results show that MBD2 is required for the proliferation of triple negative breast cancer (TNBC), prostate cancer (PCa), and TET mutant leukemias (TML), in vitro. The functional mediators of MBD2's growth promoting effects were tissue/tumor context dependent. In leukemias, MBD2's growth promoting and tumor initiating effects were most pronounced in TET2 null cells (which accumulate 5mC). In TNBC, nanog-GFP reporter positive cells were more sensitive to MBD2 knockdown than reporter negative cells. In PCa and TNBC, RNAseq analysis revealed that knockdown of MBD2 led to downregulation of Myc pathway genes and increased the expression of Myc targeting microRNAs, miR33-5, miR34a, miR148a and miR363. Western blot analysis confirmed that MBD2 knockdown coordinately downregulated cMyc expression and activated p27 expression. We further demonstrated that inhibition of MBD2 diminished the tumor initiating capacity of TNBC and PCa in xenograft models and the in vivo engraftment rate of patient derived TET-/- AML. Conclusions: MBD2 knockdown diminished the proliferative capacity of PCa, TNBC and primary TET2 mutant leukemia cells in a genetic (TET2) and phenotypic (nanog+ stem/progenitor) context dependent manner. Delayed peak effect and altered differentiation markers after MBD2 inhibition suggest epigenetic reprogramming as the mechanism of growth suppression. MBD2 targets miRNA's upstream of cMyc in PCa and TNBC. MBD2 murine knockout models are developmentally normal, suggesting an acquired function in cancer with a favorable therapeutic window for targeting. Citation Format: Aysegul Balyimez, Yihong Guan, Emily Esakov, Shinjini Ganguly, Ofer Reizes, Daniel J. Lindner, Jaroslaw Maciejewski, Babal Jha, Omar Y. Mian. Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation [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 2450.