Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network.

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.

Epigenetically regulated miR-1247 functions as a novel tumour suppressor via MYCBP2 in methylator colon cancers.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous disease with distinct clinical subsets based on underlying genetic and epigenetic changes. DNA hypermethylation yields a unique CRC subset with a distinct phenotype and clinical behaviour, but this oncogenic pathway is not fully characterised. This study identifies and characterises miR-1247 as a novel tumour suppressor microRNA in methylated human colon cancers. METHOD: Tumour samples from patients with hypermethylated and non-methylated colon cancer and cell lines were evaluated for miR-1247 expression and function. A murine subcutaneous xenograft model was used for in vivo functional studies. RESULTS: miR-1247 was methylated and underexpressed in methylator colon cancers. Overexpression of miR-1247 significantly inhibited cell proliferation, decreased tumour cell motility, induced apoptosis, and mitigated tumour formation capacity both in vivo and in vitro. Pharmacologic demethylation increased miR-1247 expression and produced similar anti-tumour activities. Mechanistic investigations revealed that MYCBP2, a member of the c-myc oncogene family, is a direct functional target of miR-1247. Furthermore, in CRC patients, MYCBP2 protein levels are associated with miR-1247 levels and survival. CONCLUSIONS: miR-1247 acts as a tumour suppressor by inhibiting MYCBP2 in methylator colon cancer. The MYCBP2/c-myc axis may underlie the anti-tumour activities of miR-1247 and is a potential therapeutic target via demethylation agents.

Post-transcriptional regulation of PIAS3 expression by miR-18a in malignant mesothelioma.

Protein inhibitor of activated STAT3 (PIAS3) is an endogenous suppressor of signal transducer and activator of transcription 3 (STAT3) signaling. By directly interacting with phosphorylated STAT3, PIAS3 can block the downstream transcriptional activity of STAT3, which is hyper-activated in various cancers. We previously reported that in malignant mesothelioma (MM), low PIAS3 expression is associated with increased STAT3 activation and correlates with poor patient survival, yet the regulatory mechanism(s) governing PIAS3 expression in MM remain unclear. Here, we demonstrate that PIAS3 protein expression does not correlate with its mRNA level in MM cell lines, indicating that PIAS3 expression is regulated at a post-transcriptional level. Inhibition of proteasomal degradation with MG132 (10 µm) or bortezomib (1 µm), alone and in combination, did not increase PIAS3 protein levels; furthermore, inhibition of protein synthesis by cycloheximide treatment did not decrease PIAS3 levels within 48 h, suggesting that PIAS3 expression is not actively regulated at a post-translational level. To determine whether miRNA (miRs) can translationally regulate PIAS3 expression, we combined miR microarray analysis with bioinformatic screening to identify candidate miRs, in MM cell lines with low PIAS3 expression, followed by luciferase reporter assays to validate miR regulation of the PIAS3 3'UTR. We identified miR-18a as a suppressor of PIAS3 expression that is upregulated in MM cells and whose inhibition can increase PIAS3 expression and suppress STAT3 activity. Moreover, we showed that miR-18a inhibition can decrease MM cell viability and that its expression is negatively correlated with MM patient survival. Taken together, these results suggest that targeting miR-18a may have therapeutic benefit in MM.

Reciprocal expression of INSM1 and YAP1 defines subgroups in small cell lung cancer.

The majority of small cell lung cancer (SCLC) patients demonstrate initial chemo-sensitivity, whereas a distinct subgroup of SCLC patients, termed chemo-refractory, do not respond to treatment. There is little understanding of how to distinguish these patients prior to disease treatment. Here we used gene expression profiling to stratify SCLC into subgroups and characterized a molecular phenotype that may identify, in part, chemo-refractive SCLC patients. Two subgroups of SCLC were identified in both cell lines and tumors by the reciprocal expression of two genes; INSM1, a neuroendocrine transcription factor, and YAP1, a key mediator of the Hippo pathway. The great majority of tumors expressed INSM1, which was prognostic for increased progression-free survival and associated with chemo-sensitivity in cell lines. YAP1 is expressed in a minority of SCLC tumors and was shown in cell lines to be downstream of the retinoblastoma protein (RB1) and associated with decreased drug sensitivity. RB1 expression in SCLC cell lines sensitizes them to CDK4/6 inhibitors. Wild-type RB1 mutation status, used as a surrogate marker of YAP1 expression, was prognostic for decreased patient survival and increased chemo-refractory tumor response. Thus, the reciprocal expression of INSM1 and YAP1 appears to stratify SCLC into distinct subgroups and may be useful, along with RB1 mutation status, to identify chemo-refractory SCLC patients.

RICTOR amplification identifies a subgroup in small cell lung cancer and predicts response to drugs targeting mTOR.

Small cell lung cancer (SCLC) is an aggressive cancer that represents ~15% of all lung cancers. Currently there are no targeted therapies to treat SCLC. Our genomic analysis of a metastatic SCLC cohort identified recurrent RICTOR amplification. Here, we examine the translational potential of this observation. RICTOR was the most frequently amplified gene observed (~14% patients), and co-amplified with FGF10 and IL7R on chromosome 5p13. RICTOR copy number variation correlated with RICTOR protein expression in SCLC cells. In parallel, cells with RICTOR copy number (CN) gain showed increased sensitivity to three mTOR inhibitors, AZD8055, AZD2014 and INK128 in cell growth assays, with AZD2014 demonstrating the best inhibition of downstream signaling. SCLC cells with RICTOR CN gain also migrated more rapidly in chemotaxis and scratch wound assays and were again more sensitive to mTOR inhibitors. The overall survival in SCLC patients with RICTOR amplification was significantly decreased (p = 0.021). Taken together, our results suggest that SCLC patients with RICTOR amplification may constitute a clinically important subgroup because of their potential response to mTORC1/2 inhibitors.