Anderson, Karen S.

Messenger RNA (mRNA) therapeutics have transformed vaccination and protein replacement strategies, yet efforts to improve their performance have focused largely on sequence engineering, nucleotide modification, and delivery vehicles. Here we show that mRNA function can be controlled by rational design of higher-order RNA architectures. We develop self-assembling mRNA origami (mRNA-OG), a class of unimolecular RNA nanostructures that encode protein-coding sequences within higher-order, programmable, and compact nucleic acid architectures. Using computational design and experimental validation, we demonstrate that mRNA-OG folds into well-defined nanostructures while remaining translationally competent in mammalian cells. Although folded mRNA-OG recruits ribosomes comparably to unfolded constructs, it produces lower protein output, indicating that RNA architecture can directly influence translational efficiency. The compact geometry of mRNA-OG also enhances encapsulation by cationic lipid delivery systems, suggesting a structural route to improved cargo packaging. Beyond its effects on translation, mRNA architecture modulates innate immune recognition. In primary human dendritic cells, folded and unfolded mRNA-OG elicit distinct cytokine programs and differential activation of stress-response pathways, including a modest induction of the integrated stress response that is not fully explained by canonical PKR signaling. Our results establish programmable structure as a new design parameter for mRNA therapeutics that can affect its functionality, delivery properties, and immune sensing.

639 Background: Large cohort studies have shown a survival benefit with the use of GLP1 receptor agonists (GLP1 RA) in cancer patients. The impact of GLP1 RA usage in breast cancer (BC) and the different subtypes is unknown. To investigate the potential influence of GLP1 RA usage on BC survival, a study was conducted on a large institutional database. Methods: A de-identified institutional database identified newly diagnosed Stage I, II, and III BC patients from 2005 to present and use of GLP1 RA. The study compared survival rates for BC patients with and without GLP1 RA and further stratified by BC subtype [ER+HER2-, HER2+, ER-PR-HER2-(triple negative BC (TNBC))], BMI, and age. Kaplan-Meier survival curves were used to estimate overall survival. This study analyzed de-identified data accessed through Mayo Clinic Platform Discover. Per 45 CFR 46.102, this activity did not require IRB review. Data was extracted using an established privacy-preserving protocol and de-identified via expert determination under the HIPAA Privacy Rule. Raw EHR data and other unreported data cannot be shared due to de-identification parameters. Results: 88015 BC patients were identified, of which 3919 (4.5%) received GLP1 RA. The 5-year survival rate was 98.5% with vs 85.5% without GLP1 RA (p-value <0.0001). The survival benefit persisted for different BMI’s and ages: BMI >30 (98.4% vs 85.1%), >25 (98.4% vs 85.4%), and age <50 (99.5% vs 89.3%) and >50 (98.1% vs 84.8%) [all p<0.0001]. 21770 patients with ER+ disease were identified, of which 1412 (6.5%) received GLP1 RA. The 5-year survival rate was 99.0% vs 87.7% (p-value <0.0001). The survival benefit persisted by BMI and age: BMI >30 (99.0% vs 86.2%), >25 (98.9% vs 87.1%) and age <50 (99.6% vs 90.5%) and >50 (98.7% vs 87.2%) [all p<0.0001]. 3599 patients with HER2+ disease were identified, of which 202 (5.6%) received GLP1 RA. The 5-year survival rate was 99.3% vs 80.1% (p-value <0.0001). The survival benefit persisted by stratification: BMI >30 (100.0% vs 80.0%, p-value <0.0004), and <25 (97.7% vs 76.2%, p-value <0.019), and age <50 (100% vs 87.0%, p-value <0.043) and > 50 (99.0% vs 77.2%, p-value <0.0002). 8389 patients with TNBC disease were identified, of which 360 (4.3%) received GLP1 RA. The 5-year survival rate was 91.0% vs 71.9% (p-value <0.0001). The survival benefit persisted by stratification: BMI >30 (96.4% vs 72.9%, p-value <0.0001), >25 (93.2% vs 71.8%, p-value <0.0001), and age <50 (96.0% vs 73.0%, p-value <0.0028) and >50 (88.9% vs 71.8%, p-value <0.0001). For those TNBC patients who received GLP1 RA, having a BMI >30 conferred a protective effect with a 5-year survival rate was 97.9 vs 89.8% (p-value <0.0014). Conclusions: The study suggests that GLP1-RA improves 5-year survival rates of patients with BC and this applies to all subtypes of BC. These results suggest that further study of the impact of GLP1-RA on BC patients is warranted.

Abstract Background: Breast tumors exhibit intratumor heterogeneity resulting in targeted therapy resistance and other challenges in disease management. To address the sources of heterogeneity, we performed a unique, in-depth analysis of clonal architecture in primary chemoradiation-naïve breast cancers. We combined DNA content-based flow cytometry and ploidy analysis with aCGH and whole exome next-generation sequencing (NGS) in multiple biopsies from the tumors and involved lymph nodes (LNs). Material and methods: We sorted nuclei from distinct populations of diploid, tetraploid, and aneuploid cells in surgical tumor samples from three chemoradiation-naïve patients. Each sorted tumor cell population was interrogated with aCGH and whole exome NGS. In Patient #1, we sorted and interrogated the genomes of tumor populations from 12 fresh frozen sections morphologically mapped from within a HER2+, ER+, PR- primary invasive ductal carcinoma (IDC) of histological grade 3 with LN involvement and 2-3 sections from 2 out of 5 LNs. In Patient #2, 11 morphologically mapped fresh frozen sections were analyzed from a grade 2, ER+, PR+, HER2+, BRCA2 mutant LN- IDC. In parallel, matching samples were processed for IHC assays. In Patient #3 biopsies from a grade 2 ER+, PR+ primary tumor were profiled. Results: The 18 primary and LN biopsies from Patient #1 fell into 6 distinct ploidy groups albeit with aberrant but homogenous aCGH profiles, characterized by SARC amplification and homozygous deletions in ROBO1 and ROBO2. In contrast a dominant ploidy was identified throughout 10 biopsies in Patient #2 but with heterogeneous aCGH profiles. Patient #3 had a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Mutation profiles obtained through exome sequencing further confirmed that ploidy was the main driver in Patient #1 whereas copy number aberrations played the key role in Patient #2 with the BRCA2 mutation (R3129X). A dendrogram based on exome variant calls of the aneuploid populations in Patient #1 strongly correlated with ploidy group and further revealed the specific clonal population characterized by a 5N ploidy and homozygous mutations in TP53 and PIK3CA as the progenitor to the ploidies present in the distant LNs. Strikingly, patients #1 and #2 were HER2 wild type across their clonal populations, contradicting IHC staining in a single core biopsy. Conclusions:Rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying clonal populations and then interrogating their genomes, provides an objective method of exploring the sources and clinical significance of tumor heterogeneity. Our approach of clonal analysis has broad implications in the study of tumor heterogeneity and the identification of drivers in breast and other solid tumors that can advance more effective treatment and clinical management of patients with this disease. Citation Format: MARIACARLA ANDREOZZI, Princy Francis, Elizabeth Lenkiewicz, Mia Champion, Brady Laughlin, Karen Anderson, Heather Cunliffe, Ann E. McCullough, Michael T. Barrett, Barbara Pockaj. Deep clonal profiling identifies distinct mechanisms of heterogeneity and evolution in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-175. doi:10.1158/1538-7445.AM2015-LB-175