Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here, we show an innovative RNA-based targeted approach to enhance endogenous albumin production whilst reducing liver tumour burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumours increased circulating serum albumin by over 30% showing evidence of improved liver function. Tumour burden decreased by 80% (p = 0.003) with a 40% reduction in a marker of pre-neoplastic transformation. Since C/EBPα has known anti-proliferative activities via retinoblastoma, p21 and cyclins; we used mRNA expression liver cancer specific microarray in C/EBPα-saRNA transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis and metastasis. Up-regulated genes were enriched for tumour suppressors and positive regulators of cell differentiation. A quantitative PCR and Western-blot analysis of C/EBPα-saRNA transfected cells suggested that in addition to the known anti-proliferative targets of C/EBPα, we also observed suppression of IL6R, c-Myc and reduced STAT3 phosphorylation. Conclusion We demonstrate for the first time that a novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumour burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

Prohibitin (PHB) is a cell cycle regulatory protein, known to repress E2F1-mediated gene activation via recruitment of transcriptional regulatory factors such as retinoblastoma and histone deacetylase 1 (HDAC1). We previously identified PHB as a target protein of androgen signaling in prostate cancer cells and showed that downregulation of PHB is required for androgen-induced cell cycle entry in these cells. We now present evidence that PHB, which has 54% homology at the protein level to the oestrogen receptor corepressor REA (repressor of oestrogen receptor activity), can repress androgen receptor (AR)-mediated transcription and androgen-dependent cell growth. Depletion of endogenous PHB resulted in an increase in expression of the androgen-regulated prostatespecific antigen gene. The repression appears to be specific to androgen and closely related receptors, as it is also evident for the glucocorticoid and progesterone, but not oestrogen, receptors. In spite of interaction of PHB with HDAC1, HDAC activity is not required for this repression. Although AR and PHB could be co-immunoprecipitated, no direct interaction was detectable, suggesting that PHB forms part of a repressive complex with the AR. Competition with the co-activator SRC1 further suggests that formation of a complex with AR, PHB and other cofactors is the mechanism by which repression is achieved. It appears then that repression of AR activity is one mechanism by which PHB inhibits androgendependent growth of prostate cells. Further, this study implies that the AR itself could, by mediating downregulation of a corepressor, be involved in the progression of prostate tumours to the hormone refractory stage.

Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells. A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity. This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic.

Purpose: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a firstin-class small activating RNA oligonucleotide drug which up-regulates C/EBP-α. Experimental Design:We conducted a phase I, open label, dose escalation trial of MTL-CEBPA in adults with advanced HCC with cirrhosis, or resulting from non-alcoholic steatohepatitis (NASH) or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose escalation phase (3+3 design).Results: 38 participants have been treated across 6 dose levels (28-160 mg/m2) and 3 dosing schedules. 34 patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatmentrelated adverse events were not associated with dose and no maximum dose was reached across the 3 schedules evaluated. Grade 3 treatment related adverse events occurred in 9 (24%) patients. In 24 HCC patients evaluable for efficacy, an objective tumour response was achieved in 1 patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKI); 3 patients had a complete response with one further PR and two with SD.Conclusions: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging Phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.Funding: This study was funded by MiNA Therapeutics. ClinicalTrials.gov ID: NCT02716012 Statement of translational relevancePreclinical data have emerged suggesting C/EBP-a effects on the tumour microenvironment through myeloid derived suppressor cells could enhance response to sorafenib. The data from this trial provide preliminary validation for targeting C/EBP-a in patients with advanced HCC, particularly in context of sequential administration with TKIs and provide a rationale for combining MTL-CEBPA with TKIs.