2 SUMMARYWe previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

Introduction: RNF43 is a transmembrane E3 ubiquitin ligase and WNT signaling suppressor that is commonly mutated in colorectal cancer (CRC). A C-terminal RNF43 hotspot mutation, RNF43_G659fs, occurs in approximately 36% (55/151) of microsatellite-instability (MSI)-high CRCs, but its underlying mechanism and function remain poorly understood. This study investigated the functional role of RNF43_G659fs in order to evaluate potential novel therapeutic approaches for tumors harboring this mutation. Methods: Isogenic RNF43117mut and RNF43659mut cell line models were generated using the CRISPR/Cas9 system to evaluate CRC tumorigenesis and WNT dependency. RNF43659mut was screened with a novel high-throughput drug repurposing library that employed a set of 5363 small molecules to identify compounds capable of selectively inhibiting RNF43659mut cell growth. Small molecules that selectively killed the RNF43659mut cells were validated in organoid models. Proteomic analysis, RNA-Seq and gene set enrichment analysis (GSEA) were performed to characterize mechanistic interactions and related signaling pathways of RNF43659mut in CRC. Results: Unlike N-terminal RNF43 frameshift mutations, we observed that RNF43659mut conferred a growth advantage over RNF43WT cells independent of WNT signaling. Furthermore, RNF43659mut and RNF43WT exhibited differential drug responses in the high-throughput drug repurposing screen which revealed that RNF43659mut cells were vulnerable to PI3K/AKT/mTOR inhibitors, including BYL-719 (Alpelisib). Enhanced AKT and mTOR activation was observed in RNF43659mut cell and attenuated by BYL-719 treatment in a dose-dependent manner. These results were subsequently validated in patient-derived organoid models. Furthermore, immunoprecipitation and proteomic analysis revealed interactions between RNF43_G659fs and p85, a negative regulator of PI3K. We also demonstrated that the RNF43_G659fs mutant activated PI3K/AKT/mTOR signaling through binding and degradation of p85. Consistent with the role of PI3K in immunomodulation, our RNA-Seq results showed that the RNF43_G659fs mutation was positively related to NF-kB activation (Normalized Enrichment Score=1.842, p<0.01) and inversely related to Interferon-alpha/Interferon-gamma response pathways (Normalized Enrichment Score= -1.992, p<0.01; Normalized Enrichment Score= -1.577, p<0.01, respectively), indicating its role in tumor microenvironment remodeling. Conclusion: This study confirms that RNF43659mut is an essential driver mutation in CRC and provides evidence that patients harboring RNF43_G659fs-mutant tumors may respond favorably to PI3K inhibition. Citation Format: Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Carino Gurjao, Maximilien Grandclaudon, Steven M. Corsello, Srivatsan Raghavan, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng, Marios Giannakis. RNF43 G659fs is an oncogenic mutation in colorectal cancer and sensitizes tumor cells to PI3K/mTOR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 960.

We sought to develop a strategy to pharmacologically modulate AML1-ETO, the most common gene rearrangement associated with acute myeloblastic leukemia (AML). Like many other oncogenic transcription factors associated with the acute leukemias, AML1-ETO has been considered undruggable. In principle, these well-characterized somatic mutations identified in the acute leukemias represent unique, tumor-specific therapeutic targets. In practice, acute leukemia therapy continues to focus on nonspecific cytotoxic agents. In order to address this challenge, we developed a genomic, signature-based small molecule library screening approach, Gene Expression-based High-throughput Screening (GE-HTS). This approach uses gene expression signatures as surrogates for different biological states in a small molecule library screen. We focused our initial efforts on identifying modulators of AML1-ETO. First, a 25-gene signature for AML1-ETO abrogation was defined by transcriptional profiling of t(8;21) Kasumi-1 cells with and without AML1-ETO-directed RNA interference and with a U937 inducible model of AML1-ETO. The signature was confirmed in microarray data from t(8;21)-containing primary patient leukemias (p < 0.001). Next, the ability of 2,600 FDA-approved drugs and bioactive agents to induce this abrogation signature was evaluated by ligation mediated amplification (LMA) and bead-based fluorescence detection. The screen identified 16 hits confirmed on repeat testing, including seven corticosteroids. Five hits were selected for further study based on their chemical diversity, ability to induce a robust expression signature, and potential for clinical translation: 5-aza-deoxycytidine, floxuridine, methotrexate, methylprednisolone, and pyrimethamine. Next, whole genome effects of these compounds with microarray-based expression profiling were evaluated. Using Gene Set Enrichment Analysis (GSEA), we determined that all five compounds induce both whole genome effects consistent with AML1-ETO knockdown and a whole genome program consistent with neutrophil maturation. In a subset of these compounds, we also see changes in cell surface markers and morphological features consistent with myeloid maturation. Intriguingly, two of the hits are well-characterized DNA methyltransferase inhibitors, and two of the hits are dihydrofolate reductase inhibitors that increase S-adenosylhomocysteine, an inhibitor of methyltransferases. Thus, reversal of AML1-ETO-mediated gene silencing by demethylation may overcome its repressive effects. This application of knockdown-derived expression signatures to small molecule library screening should enable the targeting of nearly any oncogenic transcription factor.