Genetic studies have identified mutations in key regulators of the Wnt͞␤-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt͞␤-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which ␤-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with ␤-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the ␤-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt͞␤-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.

Methylthioadenosine phosphorylase, (MTAP) is a key enzyme in the adenine and methionine salvage pathways. MTAP is encoded on human chromosome 9p21 in close proximity to the p16INK4a and p14ARF tumor suppressor genes and is frequently co-deleted with p16INK4a in many cancers. Deletion of MTAP has been reported to create a reliance of MTAP-/tumors on de novo purine synthesis to maintain adequate pools of AMP, leading to increased sensitivity to purine synthesis inhibitors, such as L-alanosine. The 'Achilles heel' created by the loss of MTAP in cancer cells provides a unique therapeutic opportunity whereby MTAP-/tumors could be selectively targeted with purine synthesis inhibitors and normal tissues could be preferentially rescued with MTAP substrates, such as MTA. We demonstrate that, in contrast to published literature, MTAP-/cells are not more sensitive to inhibition of de novo purine synthesis than MTAP +/+ cells. Although MTA can preferentially rescue MTAP +/+ cells from purine-synthesis inhibitor toxicity in vitro, MTA protects cells of both genotypes from L-alanosine equivalently in vivo. Our data demonstrate that in vivo, adenine salvaged from plasma and adjacent tissues is sufficient to protect MTAP-/tumors from the effects of purine synthesis inhibitors. These results suggest targeting MTAP-/tumors with de novo purine synthesis inhibitors is unlikely to provide significant benefit over other therapeutic strategies and may explain, at least in part, the lack of efficacy of Lalanosine in clinical trials.

The interpretation of RNAi knockdown experiments can be challenging due to the extensive off-target effects exhibited by siRNAs and shRNAs. This has led to the reporting of results that cannot be independently verified, or to the identification of putative cancer targets that are insufficiently robust to support industrial drug discovery efforts. The problems associated with off target effects are particularly acute in high throughput RNAi screens, whose results often differ wildly between laboratories using different experimental platforms. As a result, the validity of published large-scale RNAi screens has recently been called into question. Here we report results from large scale RNAi screens to identify genes essential for the proliferation or survival of cancer cells. To overcome the inherent liabilities of RNAi screens, we have increased the experimental power by using typically 8, and up to 20 RNAi triggers per gene, and have developed novel statistical algorithms that exploit this increased power. Importantly, this approach allows the calculation of a false discovery rate (FDR) that accurately predicts the ability of results to be confirmed by independent labs using independent reagents, solving a key issue of such screens. Using a stringent FDR cutoff of 5%, known essential genes and driver oncogenes such as KRAS, ERBB2 and BRAF are readily identified, demonstrating the robustness and power of the method. We have also identified a number of novel cancer targets. We present the identification and genetic and pharmacological validation of one of these targets, a kinase present as a fusion partner in multiple fusion oncogenes in primary tumors. Our data demonstrate that large-scale RNAi screens can be a robust and independently verifiable tool to uncover previously unknown cancer vulnerabilities which have yet to be characterized despite extensive genomic sequencing efforts. Citation Format: Astrid A. Ruefli-Brasse, Cynthia Hart, Liza Fajardo, Elissa Swearingen, Linda Wong, Doreen Sakamoto, Kari Hale, Huanying Ge, Jeffery McDowell, Bharath Ramachandran, Elissa Cosgrove, J.E.Vivienne Watson, Seamus Ragan, Seamus Ragan, Paul Kassner, Paul Kassner, Kim Quon. Stringent analysis of large-scale siRNA screens identifies a kinase fusion oncogene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2013-2992

A major goal for cancer immunotherapy is to target pathways that drive optimal anti-tumor efficacy. T cell checkpoint therapy aims to enhance T cell responses during priming and/or within tumor tissue, but the key drivers of efficacy in broad patient populations has remained elusive. Here we first sought to determine whether responsiveness to anti-PD1 therapy in established syngeneic murine tumors depends on recruitment of T cells from lymphoid organs during treatment, or if PD1 blockade on T cells already within tumors is sufficient to drive efficacy. We compared anti-PD1 efficacy on established MC38 tumors when FTY720 (S1PR antagonist that blocks lymphocyte egress from lymphoid organs) or vehicle control was administered from the start of treatment. We found that anti-PD1 efficacy did not require additional T cells migrating from lymphoid organs, suggesting that PD1 blockade can act directly on T cells that were already present within tumor tissue. We next examined the functionality of tumor-infiltrating T cell subsets during anti-PD1 treatment and found that cytokine production is enhanced in a population of PD1intermediate T cells, but not on PD1high T cells. Together, this suggests that PD1 checkpoint therapy is driven by amelioration of a fraction of PD1+ tumor-infiltrated T cells, and that additional pathways within established tumors may need to be co-targeted to drive optimal efficacy. We then developed an in vitro discovery screening platform to identify pathways that co-modulate human T cell effector function against PDL1+ cells under conditions that drive high levels of PD1 on T cells. We are currently screening genes of interest that are expressed in patient tumor tissues and highly associated with a T cell-inflamed signature.