Yvonne Li

Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting.We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized Onco-Genomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/ normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequenc-ing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homo-zygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscapeof an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level. The Oncologist 2014;19:623–630 Implications for Practice: We show that use of next-generation sequencing in clinical settings is practical and can benefit patients because of the ability to define tumors genetically.

The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCa and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1 S102 phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1 S102 and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565) and prostate (PC3, LNCap) cancer cells was inhibited by ,90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics.

designed this study. H.Z. designed and performed biology experiments. J.Q. and J.E.B designed and synthesized JQEZ5, JQEZ6 and JQEZ23.. performed all sequencing and sequencing data analysis. J.P. and J.Q. performed biochemical assays. A.J.F. and J.Q. performed computational modeling.

Substrates and inhibitors of chromatin-modifying enzymes are generated in intermediary metabolism, so changes in nutrient availability and utilization can influence epigenetic regulation 1,2. Importantly, recent studies have indicated that the interplay between metabolism and epigenetics can serve as a programmed switch in cell states. For example, mouse embryonic stem cell differentiation is promoted by succinate-mediated inhibition of histone demethylases (HDMs) and TET DNA demethylases 3 , or by decreased S-adenosyl-methionine (SAM) levels leading to loss of histone H3K4 methylation 4. Moreover, aberrant metabolic activity can produce pathological effects by altering chromatin regulation. Most notably, mutations in the genes encoding the isocitrate dehydrogenase (IDH)1 and IDH2 enzymes lead to the generation of 2-hydroxyglutarate, which inhibits HDMs and TETs and thereby alters DNA and histone methylation—changes that have been implicated in overriding cell differentiation and promoting tumorigenesis 5. Whether this paradigm extends more generally to other oncogenic mutations remains unclear, and this question has implications for understanding cancer pathogenesis and developing improved treatments. Here, we demonstrate that dynamic exchange between metabolism and chromatin regulation contributes to pancreatic tumorigenesis driven by mutation of the LKB1 serine–threonine kinase. LKB1 is mutationally inactivated in a range of sporadic cancers, including pancreatic carcinomas 6–8. Additionally, germline mutations in LKB1 cause Peutz-Jeghers syndrome, which comprises gastrointesti-nal polyps and a high incidence of gastrointestinal tract carcinomas (for example, an approximately 100-fold increase in pancreatic cancer) 9,10. Cancers with LKB1 mutations tend to exhibit aggressive clinical features and different therapeutic sensitivity from cancers without these mutations 11–14. LKB1 directly activates a family of 14 kinases related to AMP-activated protein kinase (AMPK), many of which are coupled to nutrient sensing and broadly reprogram cell metabolism 15. Thus, metabolic rewiring is thought to be a driver of tumorigenesis after LKB1 loss. We now identify an LKB1-regulated program that links metabolic alterations to control of the epigenome and is involved in malignant growth. Our results provide evidence that coupled metabolic and epige-netic states have a more general role in cancer pathogenesis and suggest therapeutic strategies that could target these intersecting processes. Synergy between LKB1 and KRAS mutations LKB1 inactivation frequently coincides with mutations in the RAS–RAF pathway in human cancers and these genetic alterations cooperate to drive tumorigenesis in genetically engineered mouse models (GEMMs) 6,11,14,16. We examined the interactions between oncogenic KRAS G12D and deletion of LKB1 in adult pancreatic ducts using a tamoxifen-inducible GEMM (Extended Data Fig. 1a). The combined alterations resulted in pancreatic cancers by 20–25 weeks, whereas the individual mutations had no pathological effects at this age (Fig. 1a and Extended Data Fig. 1b). To investigate the mechanisms of tumorigenesis, we isolated primary pancreatic ductal epithelial cells from mice with conditional KRAS G12D and LKB1 alleles (n = 2 lines per genotype) and transduced them with adenoviruses expressing Cre and/or Flp recom-binase to generate KRAS G12D/+ , LKB1 −/− and KRAS G12D/+ ;LKB1 −/− cells (K, L and KL cells, respectively) as well as wild-type parental lines (Extended Data Fig. 1c). Only KL cells were tumorigenic following injection into severe combined immunodeficient (SCID) mice or growth in soft agar, and tumorigenicity was blocked by restoration of Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine–glycine– one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.