Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viralnegative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viralnegative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n ¼ 13) and -negative (n ¼ 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities. Cancer Res; 75(24); 5228-34. Ó2015 AACR.

Cucurbit[n]urils (CB[n]) are potential stabilizing, solubilizing, activating, and delivering agents for drugs. The toxicity of the macrocyclic host molecules cucurbit[7]uril (CB[7]), the most water-soluble homologue, as well as cucurbit[8]uril (CB[8]) has been evaluated. In vitro studies on cell cultures revealed an IC(50) value of 0.53 +/- 0.02 mM for CB[7], corresponding to around 620 mg of CB[7] per kg of cell material. Live-cell imaging studies performed on cells treated with subtoxic amounts of CB[7] showed no detrimental effects on the cellular integrity as assessed by mitochondrial activity. For CB[8], no significant cytotoxicity was observed within its solubility range. The bioadaptability of the compounds was further examined through in vivo studies on mice, where intravenous administration of CB[7] showed a maximum tolerated dosage of 250 mg kg(-1), while oral administration of a CB[7]/CB[8] mixture showed a tolerance of up to 600 mg kg(-1). The combined results indicate a sufficiently low toxicity to encourage further exploration of CB[n] as additives for medicinal and pharmaceutical use.

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.

Deregulated glucose metabolism fulfils the energetic and biosynthetic requirements for tumour growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including HIF1α, c-Myc and MondoA, drives glycolysis downstream of BRAFV600, is critical for responses to BRAF inhibition and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor-resistant melanoma cells. Thus, we provide a proof of principle for treatment of melanoma with combinations of BRAF inhibitors and glycolysis inhibitors.

No abstract

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne).Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.

We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.