Abstract 2917: Differentiation therapy and the mechanisms that terminate malignant proliferation
Authors: Enane Francis O. , Gu Xiaorong , Korc Murray , Saunthararajah Yogen 
Published: August 2018

Abstract
Historically, chemotherapy often attempts to activate apoptosis in tumor cells even when master regulators of apoptosis (e.g., TP53) are inactivated by genetic mutations, leading to multi-drug resistance. A potential form of non-toxic therapy is to engage p53-independent terminal differentiation. Exponential proliferation in lineage committed progenitors, coordinated by the transcription factor MYC, is succeeded by terminal differentiation, whereas MYC-coordinated aberrant proliferation in human malignancies is not succeeded by terminal differentiation. The mechanisms that suppress terminal differentiation are mostly unclear, impeding development of differentiation therapies.
We performed a systematic review of published literature between January 2007 – January 2017 to identify genetic alterations that produce candidate molecular targets for differentiation induction in three treatment-recalcitrant cancers: hepatocellular carcinoma (HCC), ovarian cancer (OVC) and pancreatic ductal adenocarcinoma (PDAC). We then validated candidate molecular targets in-vitro with FANA molecules and CRISPR-Cas9, and by in-vivo validation of small molecule drugs in HCC.
By systematic review, lineage tracing studies suggested master transcription factors of cell fate and their cofactors as links to suppression of cell cycle exit by differentiation. Indeed, sequence analysis of HCC, OVC and PDAC identified frequent genetic haploinsufficiency in lineage transcription factors (e.g., GATA4 67% hetloss HCC, n=442, FOXO1 63% hetloss, OVC n=302, PDX1 58% hetloss, PDAC n=109), while their coactivators (e.g. ARID1A/B, and ARID3A/C) were frequently inactivated by genetic mutation/deletion. Interestingly, corepressors (e.g., DNMT1, EED, BAZ1A/B, and SUZ12) that cooperate with these transcription factors to turn off genes were frequently amplified but not inactivated. By immunoprecipitation and mass spectroscopy, we identified in HCC that corepressors (e.g., DNMT1) were aberrantly recruited to wild-type hepatocyte transcription factors (FOXA1/2) and this interaction epigenetically suppressed hepatocyte differentiation genes (e.g., HNF4A). Pre-clinical trials evaluating DNMT1 therapeutic efficacy - by DNMT1-FANA or DNMT1-CRISPR-Cas9 - suppressed HCC proliferation but increased hepatocyte differentiation. Non-cytotoxic treatment (0.2µM) of decitabine (DEC) - DNMT1 inhibitor - terminated HCC proliferation without inducing apoptosis. This inhibition enhanced FOXA1/2 interaction with coactivators, decreased corepressors, increased hepatocyte differentiation by decreasing MYC protein, increasing p27 protein and activating HNF4A. Using 0.2mg/Kg of DEC in vivo - in obesity induced mouse model of HCC - DEC reduced HCC tumor burden, without apoptosis induction. Thus, aberrant corepressors are rational molecular targets that engage terminal differentiation in cancer cells.
Citation Format: Francis O. Enane, Xiaorong Gu, Murray Korc, Yogen Saunthararajah. Differentiation therapy and the mechanisms that terminate malignant proliferation [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 2917.