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Estrogen-receptor positive (ER+) and human epidermal growth factor receptor positive (HER2+) subtypes of breast cancers feature distinct histopathologies and prognoses. Their molecular profiles inform subtype-specific treatment combinations and possible therapeutic targets. However, the evolutionary trajectory of somatic mutations within each subtype has not been characterized, despite its utility in prioritizing drug development and therapeutic approaches. Quantifying the selective effects of somatic mutations on cancer conveys the likely efficacy of extant targeted therapies between subtypes. Furthermore, knowledge of epistatic interactions involving targetable mutations enables precision therapy at any stage of tumorigenesis. The strength of these interactions differ between receptor subtypes, mediated by differences in the adaptive landscape of each subtype. To investigate differences in subtype etiologies, we quantified selection on single-nucleotide mutations within ER and HER2 receptor subtypes using an aggregated multi-institutional cohort of tumor samples. We observed differential selection acting on mutations: PIK3CA H1047R is a strongly selected variant for which selection varied significantly based on the receptor subtype, experiencing maximal selection in ER+ HER2﹣ breast cancer. On a stage-specific basis, we show that adaptive landscapes change, altering optimal timings for treatment. We then infer epistasis by 1) stage-specific analysis that suggests likely orderings of mutations, 2) calculation of pairwise selective epistasis amongst mutations, which lends mechanistic explanations to these orderings, and 3) phylogenetic reconstruction of the evolution of ten breast cancers, revealing common orderings that further support inferred selective epistatic interactions. Our estimates of differential and stage-specific selection and epistasis between subtypes suggest distinct adaptive landscapes for breast cancer subtypes. These estimates should inform the development of targeted therapeutics and guide treatment schedules based on molecular profiling of the cancer. Citation Format: Krishna Dasari, J. Nick Fisk, Jeffrey Townsend. Somatic mutations of ER and HER2 subtypes exhibit divergent evolutionary trajectories determined by selective epistasis revealed in phylogenetic reconstruction and stage-specific analyses [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-07.

Somatic nucleotide mutations can contribute to cancer cell survival, proliferation, and pathogenesis. Although research has focused on identifying which mutations are “drivers” versus “passengers," quantifying the proliferative effects of specific variants within clinically relevant contexts could reveal novel aspects of cancer biology. To enable researchers to estimate these cancer effects, we developed cancereffectsizeR, an R package that organizes somatic variant data, facilitates mutational signature analysis, calculates site-specific mutation rates, and tests models of selection. Built-in models support effect estimation from single nucleotides to genes. Users can also estimate epistatic effects between paired sets of variants, or design and test custom models. The utility of cancer effect was validated by showing in a pan-cancer dataset that somatic variants classified as likely pathogenic or pathogenic in ClinVar exhibit substantially higher effects than most other variants. Indeed, cancer effect was a better predictor of pathogenic status than variant prevalence or functional impact scores. In addition, the application of this approach toward pairwise epistasis in lung adenocarcinoma showed that driver mutations in BRAF, EGFR, or KRAS typically reduce selection for alterations in the other two genes. Companion reference data packages support analyses using the hg19 or hg38 human genome builds, and a reference data builder enables use with any species or custom genome build with available genomic and transcriptomic data. A reference manual, tutorial, and public source code repository are available at https://townsend-lab-yale.github.io/cancereffectsizeR. Comprehensive estimation of cancer effects of somatic mutations can provide insights into oncogenic trajectories, with implications for cancer prognosis and treatment.Significance:An R package provides streamlined, customizable estimation of underlying nucleotide mutation rates and of the oncogenic and epistatic effects of mutations in cancer cohorts.

The acquisition of resistance by tumors to targeted molecular therapies remains one of the greatest challenges in precision medicine. Evolutionary techniques, such as phylogenetic analysis and the thoughtful calculation of selection intensities, are well suited to offer guiding insights in describing and, ultimately, overcoming therapeutic resistance. Here, we quantify the relative importance of mutations occurring during EGFR targeted therapy on a cohort of Lung Adenocarcinoma (LUAD) patients using clinical records to calibrate phylogenetic chronograms to identify variants occurring on branches of the tumor phylogeny coinciding with targeted EGFR therapy and calculate the evolutionarily-derived cancer effect size for those variants. We then compare these results to other cancer contexts to reveal the strikingly high effect size of the EGFR T790M mutation. To probe and quantify the constraints therapeutically driven selection pressure places on the cohort, we predict the neoantigen composition for each patient for both extant and ancestral tumor states, using a general allele frequency selection model to calculate selection on cancer variants constituting putative neoantigens. We then compare the proportion of negatively and positively selected neoantigens to non-neoantigen variants within the tumor to deduce if neoantigens are subject to additional immune scrutiny in a background of therapeutics. Lastly, we perform ancestral state reconstruction and mutational signature analysis to provide further insight on mutational processes on both individual patient and cohort levels, detailing how these endogenous and exogenous processes shift between etiologically pertinent time-points. Taken together, our results detail the degree of selection on cancer undergoing targeted therapy and highlights unique shifts in mutational processes in response to such therapies, ultimately offering quantitative insights that can inform clinical decision making regarding therapeutic strategies in Lung Adenocarcinoma and provide a framework for analyzing similar cancer systems. Citation Format: J Nicholas Fisk, Stephen Gaffney, Katerina Politi, Scott Gettinger, Fernando de Miguel, Jeffrey Townsend. Extreme selection constrains ability of EGFR-driven lung adenocarcinoma to diversify in response to erlotinib therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 126.

Mutational processes in tumors create distinctive patterns of mutations, composed of neutral “passenger” mutations and oncogenic drivers that have quantifiable effects on the proliferation and survival of cancer cell lineages. Increases in proliferation and survival are mediated by natural selection, which can be quantified by comparing the frequency at which we detect substitutions to the frequency at which we expect to detect substitutions assuming neutrality. Most of the variants detectable with whole-exome sequencing in tumors are neutral or nearly neutral in effect, and thus the processes generating the majority of mutations may not be the primary sources of the tumorigenic mutations. Across 24 cancer types, we identify the contributions of mutational processes to each oncogenic variant and quantify the degree to which each process contributes to tumorigenesis. We demonstrate that the origination of variants driving melanomas and lung cancers is predominantly attributable to the preventable, exogenous mutational processes associated with ultraviolet light and tobacco exposure, respectively, whereas the origination of selected variants in gliomas and prostate adenocarcinomas is largely attributable to endogenous processes associated with aging. Preventable mutations associated with pathogen exposure and apolipoprotein B mRNA-editing enzyme activity account for a large proportion of the cancer effect within head-and-neck, bladder, cervical, and breast cancers. These attributions complement epidemiological approaches—revealing the burden of cancer driven by single-nucleotide variants caused by either endogenous or exogenous, nonpreventable, or preventable processes, and crucially inform public health strategies.

With the aim to identify new second-hit mutations that cause pancreatic ductal adenocarcinoma (PDAC), other than mutations in p53, we performed a genetic interaction analysis based on the concept of mutually exclusive mutations, which suggests that mutations that are exclusive to one another function within the same pathway. Using our unbiased quantitative methods to evaluate the effects of somatic mutations on cancer we analyzed >3000 PDAC cases to prioritize mutations among hundreds that appear at low frequencies (5-10%) and that are mutually exclusive with mutations in p53. We found that mutations in RNA splicing factors SF3B1 and RBM10 (~15% of cases), were among the most significant, and mutually exclusive, to mutant p53. Thus, we hypothesized that aberrant RNA splicing promoted by SF3B1K700E and loss of RBM10 cause an oncogenic program that leads to tumorigenesis and therapy resistance. To test this hypothesis, first, we engineered mouse models to co-express KrasG12D with either Sf3b1K700E or loss of Rbm10 in Mist1 and Pdx1 driven expressing pancreatic cells. Interestingly, mice harboring KrasG12D and Sf3b1 K700E or loss of RBM10—like mutant p53—caused PDACs in mice. To identify the RNA splicing mechanism that leads to PDAC formation, we performed deep RNA sequencing and unbiased transcriptome-wide splicing analyses in isolated PDAC cells bearing wild-type, SF3B1K700E and loss of RBM10, derived from murine and patient PDACs. We found that like the splicing defects induced by p53mut—SF3B1K700E or loss of RBM10—induce splicing defects in sequence-specific coding exons in mRNAs derived from chromosomes 19, 7, 1 and 2 and impacting mainly GTPase activation and lipid metabolism pathways across species models. Consistent with this common mechanism, we found that PDAC cells do not tolerate the forced expression of mutant p53 with either SF3B1K700E or loss of Rbm10, supporting the mutual exclusivity findings from patient samples. To determine a personalized therapy pathway for patients bearing mutant splicing-factor tumors, we found that PDAC cells with SF3B1K700E or RBM10 loss cells are more sensitive to Gemcitabine rather than 5FU, which are the two main chemotherapeutic components of the two standard-of-care therapies. In addition, these mutations sensitize cells to small-molecule splicing inhibitor H3B-8800, which has advanced to phase 2 trial for hematologic malignancies. Lastly, we found that combination of Gemcitabine and H3B-8800 synergize to selective kill mutant splicing cells. We are currently testing these combination therapies in our genetically engineered mouse models and patient derived organoids. We are starting a phase 2 trial for Gemcitabine/Abraxane with escalating doses of H3B-8800 for tumors that have either SF3B1K700E, RBM10 loss or neomorphic p53, all which have aberrant RNA splicing. This work has determined novel drivers and mechanisms of PDAC development and a precision therapeutic strategy for the treatment of 45% pancreatic cancer patients (SF3B1K700E [5%], RBM10 loss [10%] and neomorphic p53 [30%]). Citation Format: Natasha Pinto Medici, Diana Martinez Saucedo, Danny Lee, Li-Ting Ku, Robert Tseng, Vincent Cannataro, Deanne Yugawa, Sumedha Chowdhury, Jeffrey Townsend, Christine A. Iacobuzio-Donahue, Omar Abdel-Wahab, Steven D. Leach, Luisa Escobar-Hoyos. Altered RNA splicing causes pancreatic cancer and exposes a therapeutic vulnerability [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A055.

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.

Among the most common rituals in Japan, the Buddhist post-pregnancy loss ritual mizuko kuyo has come to America. Mizuko kuyo first arrived in Japanese-American Buddhist communities. Temples of various denominations demonstrate a range of attitudes and practices related to mizuko kuyo. The ritual is particularly sought out by new Japanese immigrants, and is largely contextualized by Japanese Buddhist notions of the spirit world and clear distinctions between priests and laypeople. What changes occur in mizuko kuyo are generally the result of indirect forces, such as lack of space. Convert Zen practitioners in America also practice a form of mizuko kuyo, often called "water baby ceremonies." Despite their depiction as unritualistic, since the early 1990s these new Buddhist groups have increasingly taken up mizuko kuyo as a practice to help members deal with pregnancy losses. Women have been the pioneers of this practice, which is buttressed by the rise of bodhisattva-oriented movements. Numerous alterations can be seen in the process of recreating mizuko kuyo as water baby ceremonies. Among the most important is the re-orientation from placating the spirit to psychologically healing the parent. The rise of mizuko kuyo demonstrates that convert Zen has reached a new stage which pays greater attention to ritual and other elements of the tradition left out by earlier American converts. Thus mizuko kuyo pushes these groups closer to Asian models. Buddhists are not the only Americans with an interest in mizuko kuyo. Pro-life and pro-choice proponents rhetorically appropriate mizuko kuyo in ways designed to bolster their political positions. Pro-life adherents use mizuko kuyo to prove that they care about women, and that their convictions arise from objective psychological phenomena. Prochoice proponents use it to show that they care about families, and that they are sensitive to the religious aspects of abortion. More surprising, some American Christians and others seek healing after pregnancy losses through mizuko kuyo. These ritual appropriations come in many forms, from discussing Buddhism in online forums to performing mizuko kuyo. Buddhism may be becoming the specialist religion that ministers to post-pregnancy loss grief for many Americans, regardless of their personal religious affiliation.

doi: 10.1038/nbt1104-1349a; International audience

We report the successful suppression of nopaline synthase (EC 1.5.1.19) enzymatic activity in the leaves of tobacco plants via the overproduction of RNAs complementary to the nopaline synthase (nos) mRNA. Several different regions of the nos gene were fused, in antisense orientation, to the promoter from a strongly expressed petunia chlorophyll a/b-binding protein gene. These constructions were directly introduced into a tobacco line which contained a single copy of the wild-type nos gene and transgenic plants were regenerated. The degree of nopaline synthase suppression in the leaves of the double transformants ranged up to 85% and was dependent on the particular region of the nos gene present in the antisense RNA. The most effective nos antisense sequences were derived from the 3' half of the nos gene transcript. In addition, we report a new sensitive method for the detection and quantitation of nopaline synthase activity in crude plant extracts.

We show here that plant cells are sensitive to the antibiotic hygromycin-B(4). We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.

The 35S promoter of cauliflower mosaic virus and promoters from the nopaline synthase, 1' and 2' genes of Agrobacterium tumefaciens T-DNA were fused to the bacterial octopine synthase and chitinase gene coding regions. These chimaeric gene constructions were introduced into tobacco, sugarbeet and oilseed rape cells and their relative levels of expression measured by primer extension analysis of RNA isolated from pooled populations of stably transformed calli. In tobacco callus, the 35S promoter provided the highest levels of gene expression, followed by the 2', 1' and nopaline synthase promoters. While the ranking of these promoters is conserved in sugarbeet and oilseed rape callus, there is between-species variation in the relative strength of these promoters. In all three species, transcription initiation is conserved for each of the chimaeric gene constructions. Additional constructions in which the 5' untranslated leader of a petunia chlorophyll a/b binding protein gene is substituted for DNA downstream of the 35S transcription start site demonstrates that heterologous 5' leader sequences can be utilized to augment steady-state levels of reporter gene expression.

The technique of genetic transformation of plant cells and regeneration of transgenic plants is possible in many species. The general objectives are to apply this technology in two major areas: the molecular definition of the genetic elements that regulate the expression of genes, and the rapid and directed genetic improvement of existing agricultural, horticultural, and ornamental varieties.

The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide-resistant mutants, C3 and S4-Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro–Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4-Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4-Hra mutant includes a Pro–Ala substitution at amino acid 196 and a Trp–Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.