Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the features of this landscape remain poorly understood and cannot be revealed by human cancer genotyping alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. The resulting fitness landscape is rugged (the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context), shows no evidence of diminishing-returns epistasis within variants of the same oncogene, and is inconsistent with expectations of a simple linear signaling relationship among these three oncogenes. Our findings suggest that tumor suppressor effects are strongly context-specific, which limits the set of evolutionary paths that can be taken through the fitness landscape.

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A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.

One strategy to circumvent problems with conventional chemotherapy is to develop drugs against more specific cancer targets. Another is to use molecularly targeted agent (MTA) combinations to circumvent tumor resistance and increase the therapeutic index. Such synergistically lethal (SL) MTA combinations, however, are not easily predicted based on our rudimentary knowledge of cancer biology and drug action mechanisms. In the first strategy, our aim was to identify genes modulating proliferation and survival in leukemic cell lines (K562, Jurkat, and Raji) using a pooled lentiviral library expressing 27,500 shRNA targeting 5,043 human pathway-associated genes. Cells were transduced by the viral library and collected at several time points. Bar-codes were amplified from genomic DNA and sequenced (Illumina GAIIx). In the second strategy, we have adapted the same approach to combinatorially screen shRNA sequences targeting 40 DNA Damage and Repair (DDR) genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype. Human mammary epithelial cancer (HMEC) cells were transduced with a 27K shRNA SL DDR lentiviral library comprised of a redundant set of 16 binary shRNA constructs for each possible gene-gene combination (1,600). The library-transduced HMEC cells were grown for ten days, then bar-codes amplified and sequenced (Illumina HiSeq2000). The viability screen with leukemic cell lines identified more than 250 essential genes for each panel of cells. Subsequent validation using single shRNA-expressing constructs showed that in each screen, about 80% of shRNAs identified did indeed lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed several non-random clusters of interacting proteins that provide some insight into signaling pathways and protein networks specific to these cancers. The SL screen in HMEC cells identified 10 SL shRNA pair candidates including known SL shRNA pair PARP1/BRCA1. Additional analysis of lethal combinations indicated redundant, complementary, and compensatory responses in cancer cells. We believe that newly discovered hematopoietic-specific genes represent potentially novel drug targets. Moreover, they can be used to develop and establish both novel cancer targeted therapies and myeloablative conditioning regimens with decreased toxicity. Based on SL screen results in HMEC cells, we believe that comprehensive experimental annotation of SL gene-gene interactions in a wider range of cancer and normal cells will not only predict the most promising synergistic lethal combinations but also allow the development of a new generation of multi-specific, highly effective anti-cancer therapeutics with unique mechanisms of action. Citation Format: Donato Tedesco, Kyle Bonneau, Mikhail Makhanov, Debbie Deng, Karim Hyder, Paul Diehl, Costas G. Frangou, Alex Chenchik. Viability screens in leukemic and breast cancer cells with pooled lentiviral shRNA libraries identify potential therapeutic targets and synthetic or synergistic lethal interactions. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A12.

Novel single-cell methods for profiling clonal composition and phenotypes of genetically modified cancer cells may improve understanding of tumorigenesis and guide the development of new anti-cancer drugs. To facilitate these studies, we developed a panel of lentiviral barcoded sgRNA libraries to label and monitor cancer cells in time-course experiments in vitro and in mouse xenograft models. The sgRNA effectors and cellular barcodes are transcribed from lentiviral constructs and can be detected by NGS in a single-cell RNA expression profiling assay, allowing characterization of the subpopulation of cells descended from a single, barcoded knockout progenitor cell. To scale up genetic analysis, cell barcodes were incorporated in conjunction with sgRNA effector libraries to identify clonal phenotypic changes induced by specific genetic disruptions in progeny cells derived from a single progenitor cell. However, effective single-cell genetic screening designed for even only a few hundred genes requires expression profiling of one hundred thousand cells (or more) and is not practical using currently available instrumentation. Data will be presented showing how genetic screen technology combined with targeted RNA expression profiling of barcoded knockout cells can be applied for large-scale genetic screens and significantly improve phenotyping of distinct cell populations in several cancer model systems. Preliminary studies demonstrate the assay to have unparalleled throughput, sensitivity, and improved cost-effectiveness for high-throughput drug target discovery applications. Citation Format: Alex Chenchik, Mikhail Makhanov, Russell Darst, Debbie Deng, Donato Tedesco, Paul Diehl, Lester Kobzik. Cell barcoding, genetic screens and expression profiling at a single-cell level [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5868.