Abstract There is a critical need to develop novel therapeutic strategies and diagnostic tools to precisely deliver treatments to improve survival for men with prostate cancer. To support this development, improved strategies are needed to better understand heterogenous tumor microenvironments and tumor biology that associate with variable treatment responses. We hypothesized that the tumor immune microenvironment (TIME) plays a critical role in treatment resistance. In this study we aimed to evaluate TIME signatures of treatment response and resistance utilizing a novel, integrated technological tool to identify response patterns and enable precision sampling for comparative cellular and molecular analysis. 30 patients with newly diagnosed, locally advanced, high-risk, primary prostate cancer underwent 18F-DCFPyL PSMA PET/MRI with multiparametric MRI scans followed by 3 cycles of chemohormonal therapy (NCT03358563). Repeat PSMA PET/MRI was performed prior to prostatectomy and scans were used to categorize lesions as complete response (CR), partial response (PR), no response (NR) or normal tissue. MRI scans were used to print a 3D mold of the prostate to allow microdissection of regions of interest from the resected prostate. Cellular infiltrates were analyzed by flow cytometry in 3 to 5 tissue specimens per patient. Statistical analysis was performed with One-way ANOVA with Tukey-correction. The frequency of CD8+ T cells in the total CD45+ infiltrate was highest in normal and CR areas and was significantly reduced in PR vs CR (p<0.01). CXCR3+CD8+ and CD103+CD8+ T cell frequencies were also reduced in PR vs CR foci (p<0.01, p<0.05, respectively). Meanwhile, the frequency of CXCR3+CD8+ T cells was highest and significantly elevated in CR vs normal tissue. A tendency of reduced CCR6+, CXCR5+, and CCR4+CD8+ T cells was observed in PR vs CR foci, while those frequencies remained higher in normal and CR areas. An increase in total CD8+ and CD103+CD8+ T cells associated with longer progression-free survival. Additionally, the analysis of EpCAM+ cells showed a significant increase in B7H3 expression in PR vs CR lesions (p<0.05) and a tendency of reduced HLA I expression in foci that associated with treatment resistance. We are currently integrating analysis of myeloid cells and transcriptomic analysis of sorted CD4+, CD8+ and CD11b/CD14+ cells to further dissect patterns of therapeutic response in our study cohort. In conclusion, PSMA/PET MRI based precision sampling of tumor tissue associated with differential therapeutic response patterns captured differences in the TIME infiltrates and these observations may provide hypothesis to test biological mechanisms to expedite discovery of targetable mechanisms to improve tumor stratification and targeting in high-risk prostate cancer. Citation Format: Erika Heninger, Jamie M. Sperger, Kristin Weinstein, Brian P. Johnson, Peter G. Geiger, Shane Wells, Steve Y. Cho, Wei Huang, Philippos Tsourkas, Sean McIlwain, Irene M. Ong, Sheena C. Kerr, David F. Jarrard, David J. Beebe, Joshua M. Lang. Differential patterns of immune infiltration in the tumor immune microenvironment associate with therapeutic response in primary prostate cancer following chemohormonal therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1175.

Protein kinases integrate cellular signals through complex phosphorylation cascades, yet resolving how chemical perturbations trigger and modulate these cascades in therapeutic targets remains a major challenge. Here, we dissect adenosine 5'-monophosphate-activated protein kinase (AMPK) proteoforms during activation through controlled biochemical reactions with a hybrid mass spectrometry (MS) approach integrating bottom-up MS for site-specific kinetics with top-down proteoform characterization. We reveal that AMPK phosphorylation proceeds through hierarchical cascades rather than binary switching, with dual entry points: canonical calcium- and calmodulin-dependent protein kinase kinase 2 (CaMKK2)-mediated phosphorylation or allosteric activator PF-739 both triggering extensive autophosphorylation with α1-Ser496 (S496) showing the highest kinetic priority. Proteoform-resolved analysis uncovers channeled β1-Ser24/25 (S24/25) + Ser108 (S108) cophosphorylation linking subcellular localization with allosteric responsiveness. Site-directed mutagenesis demonstrates CaMKK2 targets only α1-Thr183 (T183), with all other modifications arising through autophosphorylation. Phosphatase competition reveals asymmetric control where PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected, establishing persistent regulatory states. Resolving AMPK's temporal kinetics and proteoform architecture during activation enables a proteoform-centric understanding on kinase regulation.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer mortality, with a median 5-year survival of 13%. Acquired resistance to single agent targeted inhibitors plays a critical role in progression, however there is a lack of predictive tools on resistance that incorporate functional genomics. Here, we evaluate the resistance mechanisms with early treatment of MRTX1133, a small non-covalent inhibitor of KRASG12D, using a pooled CRISPR-Cas9 lentivirus screen in patient derived cancer organoids (PCO). Methods: PCOs were collected and transduced at a 1:5 cell to lentivirus ratio using a lentiviral-based CRISPR-Cas9 library with 2,292 genes targets from the ‘druggable genome’ (Milipore-Sigma). Transduced PCO’s were expanded in Cultrex matrix and underwent puromycin selection for 6 days. A baseline group was collected post selection to control for basal expression over the course of media treated control. In parallel, PCOs were treated with MRTX1133 (30nM) or control with collection at 6 days post-treatment. Digital PCR was used to normalize lentiviral transduction efficiency to background. PCR libraries were prepared against targets and DNA sequencing was done to assess resistance mechanisms via the Model-based Analysis of the Genome-wide CRISPR/Cas9 Knockout (MAGeCK) analysis. Results from MAGeCK were also used for HALLMARK gene set analysis to evaluate pathway disruption specific to MRTX1133 treatment. Results: Digital PCR showed optimal lentiviral copy number for baseline (0.863 ± 0.025) post puromycin selection. Pearson correlation plot showed increased variance between control groups when treatment was extended from a 6-day collection (PC1 16%, PC2 15%, PC total 31%) to a 9-day collection (PC1 18%, PC2 18%, PC total 36%). MAGeCK analysis revealed a list of 130 significant gene targets from the druggable screen (p<0.05). Top targets from basal PDAC expression included epithelial-mesenchymal transition pathways with knockout of interleukin-6 (p<0.001) and the E2F transcription family pathway with knockout of CDKN1B (p<0.05). HALLMARK analysis revealed 168 expressed pathways expressed in the control against PCO’s treated with MRTX1133 following a 0.05 false discovery rate threshold. These pathways include KRAS signaling (p<0.05), adipogenesis (p<0.05), and E2F targets (p<0.05). Individual gene knockouts observed consistent targets included KCNQT (p<0.05) in KRAS signaling, UQCRC1 (p<0.01) in adipogenesis, and CDKN1B (p< 0.01) in E2F targeting. Conclusions: By scaling a druggable lentiviral based CRISPR-Cas9 screen, we show potential signaling pathways that aid in resistance to tool compound MRTX in PDAC organoids. Thus, addressing the unmet need of predictive resistance modeling in PDAC organoids. Further work includes selective knockout of key targets to confirm synthetic lethality with MRTX1133. Citation Format: Lauryn E. Flannagan, Michela Cadarso, Md Shahadat Hossan, Molly A. Nellen, Sean J. McIlwain, C. Dustin Rubinstein, Sean Ronnekleiv-Kelly, Jeremy D. Kratz. Predicting resistance mechanisms in pancreatic cancer organoids under KRASG12D inhibition via pooled CRISPR-Cas9 druggable library screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1883.

Historically, endocrine cells were perceived to coordinate their output in a uniform manner. Recently however, single-cell technologies have uncovered heterogeneity within these populations, indicating that individual cells may operate as independently regulated units. Using high-resolution tools such as single-molecule fluorescent in situ hybridization (sm-FISH) and single-cell RNA sequencing (scRNA-seq), we investigated the contributions of individual and the collective of fetal Leydig cells to androgen production over time during mouse testis development. Temporal profiles of intratesticular androgens alongside the expression of steroidogenic pathway genes (Star, Cyp11a1, Cyp17a1, and Hsd3b1) from prenatal to perinatal testes demonstrated that the peak in gene expression preceded the peak in androgen production. Spatially, steroidogenic cells were initially observed to be concentrated toward the anterior-posterior poles along the center of the dorsal-ventral axis of the fetal testis at embryonic day (E) 13 and then expanded to a uniform distribution by E16. Next, sm-FISH using probes for individual steroidogenic pathway genes exposed the following findings: gene transcription and processing of individual and combinations of steroidogenic pathway genes are not synchronized among fetal Leydig cells; and some fetal Leydig cells express incomplete sets of genes. Further, sm-FISH and scRNA-seq data corroborated the presence of fetal Leydig and other interstitial cell types harboring incomplete sets of steroidogenic pathway genes throughout developmental stages. Taken together, these findings highlight that fetal steroidogenic gene expression is tightly regulated and that transcript presence among interstitial cell types promotes the possibility that optimal androgen biosynthesis results from a cooperative effort among neighboring steroidogenic cells.

Dynamics of protein phosphorylation are regulated by the interplay of kinases and phosphatases. Current mass spectrometry-based phosphoproteomic approaches are extremely powerful in identifying and quantifying tens of thousands of phosphosites in single biological samples. However, whereas the mapping of phosphosites is successfully automated supporting high sample throughput, the characterization of responsible kinases and phosphatases still largely depends on laborious protein biochemical assays. To show direct (de)phosphorylation events, in vitro kinase or phosphatase assays using single substrates or peptide arrays are often used. Here, we describe the development of an in vitro phosphatase assay using whole proteome under native conditions as input. We employ this approach to study the PP1 and PP2A target repertoire, characterizing thousands of potential target sites. Focusing on PPP2R5E/B56ε-containing complexes, we combine in vitro with in vivo phosphoproteomics to characterize bona fide target sites, which highlight the role of PP2A in regulating stress granule assembly.

Protein phosphorylation creates functionally distinct proteoforms through complex modification cascades, yet capturing their temporal dynamics and combinatorial patterns remains a major analytical challenge. Here, we introduce a hybrid precision mass spectrometry (MS) strategy that integrates intact mass measurements for temporal tracking, bottom-up MS analysis for site-specific kinetics, and top-down MS sequencing for proteoform characterization to resolve phosphorylation dynamics within intact kinase complexes. Using AMP-activated protein kinase (AMPK) as a model system, we uncover coordinated autophosphorylation cascades exhibiting kinetic hierarchies, with α1-S496 showing the highest kinetic efficiency. Allosteric ADaM-site activation bypasses canonical α1-T183 phosphorylation, enabling autophosphorylation even in activation-deficient mutants. Top-down MS sequencing identifies the predominant β1 proteoform as S24/25+S108 double phosphorylation, a pattern linking extranuclear distribution with allosteric responsiveness. Phosphatase competition shows PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected. This integrated strategy uncovers the proteoform dynamics underlying AMPK activation and provides a broadly applicable framework for studying phosphorylation-based regulation in kinases.

Background/Objectives: The current staging of non-small cell lung cancer (NSCLC) relies on conventional imaging, which lacks the sensitivity to detect micrometastatic disease. The functional assessment of NSCLC progression may provide independent information to enhance the prediction of metastatic risk. The objective of this study was to determine if we could identify a metabolomic signature predictive of metastasis in patients with NSCLC treated with definitive radiation. Methods: Plasma samples were collected prospectively from patients enrolled in a clinical trial with non-metastatic NSCLC treated with definitive radiation. Metabolites were extracted, and mass spectrometry-based analysis was performed using a flow injection electrospray (FIE)–Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) method. Early metastasis was defined as metastasis within 1 year of radiation treatment. Results: The study cohort included 28 patients. FIE-FITCR produced highly reproducible profiles in technical replicates. A total of 51 metabolic features were identified to be different in patients with early metastasis compared to patients without early metastasis (all adjusted p-values < 0.05, Welch’s t-test), including glycerophospholipids, sphingolipids, and fatty acyls. In the follow-up samples collected after the initiation of chemotherapy and radiation treatment, a total of 174 metabolic features were significantly altered in patients who developed early metastasis compared to those who did not. Conclusions: We identified several distinct changes in the metabolic profiles of patients with NSCLC who developed metastatic disease within 1 year of definitive radiation. These findings highlight the potential of metabolomic profiling as a predictive tool for assessing metastatic risk in NSCLC.

Tumor heterogeneity is predicted to confer inferior clinical outcomes with precision-based strategies, however, modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain subclonal populations with the potential to recapitulate heterogeneity, although treatment response assessments commonly ignore diversity in the molecular profile or treatment response. Here, we demonstrate the advantage of evaluating individual PCO heterogeneity to enhance the sensitivity of these assays for predicting clinical response. Additionally, organoid subcultures identify subclonal populations with altered treatment response. Finally, dose escalation studies of PCOs to targeted anti-EGFR therapy are utilized which reveal divergent pathway expression when compared to pretreatment cultures. Overall, these studies demonstrate the importance of population-based organoid response assessments, the use of PCOs to identify molecular heterogeneity not observed with bulk tumor sequencing, and PCO heterogeneity for understanding therapeutic resistance mechanisms.

Caloric restriction (CR) is a dietary intervention that delays the onset of age-related diseases and enhances survival in diverse organisms, and although changes in adipose tissues have been implicated in the beneficial effects of CR the molecular details are unknown. Here we show shared and depot-specific adaptations to life-long CR in subcutaneous and visceral adipose depots taken from advanced age male rhesus monkeys. Differential gene expression and pathway analysis identified key differences between the depots in metabolic, immune, and inflammatory pathways. In response to CR, RNA processing and proteostasis-related pathways were enriched in both depots but changes in metabolic, growth, and inflammatory pathways were depot-specific. Commonalities and differences that distinguish adipose depots are shared among monkeys and humans and the response to CR is highly conserved. These data reveal depot-specificity in adipose tissue adaptation that likely reflects differences in function and contribution to age-related disease vulnerability.

Abstract Disclosure: C. Zheng: None. K.O. Allen: None. T. Liu: None. N.M. Solodin: None. K. Salem: None. A.M. Fowler: None. J. Vera: None. P.K. Tsourkas: None. S. McIlwain: None. M.S. Ozers, PhD: Owner/Co-Owner; Self; Co-Founder and CSO of Proteovista LLC. E.T. Alarid: None. Metastasis is governed by an epithelial to mesenchymal transition (EMT) - the ability for an epithelial cell to adopt a mesenchymal phenotype to promote migration and tumor progression. Though EMT can be articulated as a distinctive transition between its binary states, a cell’s capacity to become plastic, and thus, express a hybrid EMT state with both epithelial and mesenchymal markers has re-defined the literature’s understanding of metastasis. To improve our comprehension of EMT, the nuclear transcription factor responsible for driving epithelial cell fate Grainyhead-like protein 2 (GRHL2) became the protein of interest as clinical evidence suggests high GRHL2 expression is indicative of poorer prognosis in breast cancer (BC). A tetracycline-inducible GRHL2 overexpression model in the estrogen receptor (ER) positive BC MCF7 cell line was developed to understand GRHL2’s role in EMT. Surprisingly, overexpression of the EMT suppressor GRHL2 induces EMT plasticity in ER-positive BC with increased E-cadherin and vimentin expression assessed by immunocytochemistry and mRNA expression. This GRHL2 overexpression model was used to further explore the dynamic nature of EMT plasticity. Gene expression analyses was performed to assess representative genes associated with a partial EMT in purified populations of GRHL2 overexpressing cells. Gene set enrichment analysis (GSEA) determined statistically significant hallmark pathways associated with GRHL2 overexpression. Isolated RNA of GRHL2 overexpressing cells revealed increased mRNA expression of p27 and NR2F1 via RT-qPCR, consistent with a dormancy phenotype. Interestingly, mammosphere and flow cytometry further expanded on the dynamic states of EMT plasticity by emphasizing on the enrichment of stem cell characteristics with GRHL2 overexpression. Extensive GRHL2 overexpression provides cells self-renewal capacity, as examined by mammosphere formation efficiency. Flow cytometry for stem cell markers CD44 and ALDH1 expression demonstrates the increased representation of stem cell markers in EMT plastic populations. To evaluate how GRHL2 overexpression influences tumor progression in vivo, immunocompromised mice were injected with GRHL2 overexpressing cells to examine tumor growth and the potential of cells to create micro-metastases using IVIS. Tumor growth responses differed between parental and GRHL2 overexpressing xenografts, with increased GRHL2 expression decreasing tumor growth. mRNA expression of harvested tumors mirrored in vitro expression of p27 and NR2F1. Together, these data indicates that GRHL2 overexpression induces EMT plasticity, in turn stimulating dormancy and stem-cell acquisition in vitro and in vivo, allowing hybrid cells the ability to adjust to its environment. Presentation: 6/3/2024

Breast cancer is often treated with chemotherapy. However, the development of chemoresistance results in treatment failure. Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to contribute to chemoresistance in breast cancer cells. In studying the transcriptional regulation of NEAT1 using multi-omics approaches, we showed that NEAT1 is up-regulated by 5-fluorouracil in breast cancer cells with wild-type cellular tumor antigen p53 but not in mutant-p53-expressing breast cancer cells. The regulation of NEAT1 involves mediator complex subunit 12 (MED12)-mediated repression of histone acetylation marks at the promoter region of NEAT1. Knockdown of MED12 but not coactivator-associated arginine methyltransferase 1 (CARM1) induced histone acetylation at the NEAT1 promoter, leading to elevated NEAT1 mRNAs, resulting in a chemoresistant phenotype. The MED12-dependent regulation of NEAT1 differs between wild-type and mutant p53-expressing cells. MED12 depletion led to increased expression of NEAT1 in a wild-type p53 cell line, but decreased expression in a mutant p53 cell line. Chemoresistance caused by MED12 depletion can be partially rescued by NEAT1 knockdown in p53 wild-type cells. Collectively, our study reveals a novel mechanism of chemoresistance dependent on MED12 transcriptional regulation of NEAT1 in p53 wild-type breast cancer cells.

Yin Yang 1 (YY1) and Structural Maintenance of Chromosomes 3 (SMC3) are two critical chromatin structural factors that mediate long-distance enhancer-promoter interactions and promote developmentally regulated changes in chromatin architecture in hematopoietic stem/progenitor cells (HSPCs). While YY1 plays critical functions in promoting hematopoietic stem cell (HSC) self-renewal and maintaining HSC quiescence, SMC3 is required for proper myeloid lineage differentiation. However, many questions remain unanswered regarding how YY1 and SMC3 interact with each other and impact hematopoiesis. We found that YY1 physically interacts with SMC3 and co-occupies with SMC3 at a large cohort of promoters genome-wide, and YY1 deficiency deregulates the genetic network governing cell metabolism. YY1 occupies the Smc3 promoter and represses SMC3 expression in HSPCs. While deletion of one Smc3 allele partially restores HSC numbers and quiescence in YY1 knockout mice, Yy1-/- Smc3+/- HSCs fail to reconstitute blood after bone marrow transplant. YY1 regulates HSC metabolic pathways and maintains proper intracellular reactive oxygen species levels in HSCs, and this regulation is independent of YY1- SMC3 axis. Our results establish a distinct YY1-SMC3 axis and its impact on HSC quiescence and metabolism.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality with a primary hallmark, including dense collagen matrix in the desmoplastic stroma. Organoid technologies often rely on commercially available EHS mouse sarcoma matrix materials, and it remains uncertain how batch-to-batch variability of this matrix impacts the consistency of cancer signaling. Here, we present a novel photo-activated hydrogel tuned to the properties of the desmoplastic stroma in PDAC organoid cultures. Method: We designed multiple synthetic hydrogel materials with tunable mechanical properties, including SP-139 (Stem Pharm, Inc), a polyethylene glycol-based hydrogel functionalized with norbornene and formed with both degradable and non-degradable crosslinking peptides and a cell adhesion peptide (CRGDS). We used rheometry to measure the elastic modulus of the polymerized hydrogel after visible light photoactivation (395 nm) and expanded multiple cancer organoid cultures. RNAseq was performed from patient-derived xenografts to expanded organoids across matrix designs (Cultrex® v. SP-139) and analyzed by GSVA pathway analyses. Result: We optimized the ratio of non-degradable v. degradable crosslinking peptide to support culture expansion and enzymatic digestion with dispase II (125 µg/mL) and collagenase II (1 mg/mL). Using rheometry, SP-139 at 75% v/v had increased elastic modulus versus Cultrex RGF BME matrix Type 2 at 100% v/v (4992±76.3 v. 68.4±5.6 Pa, p<0.0001). Phenotypic growth was confirmed across serial passages (>8) and a diversity of gastrointestinal cancer types (n=5). Baseline growth rates were identical over 48h between SP-139 (75% v/v) and Cultrex (50% v/v) (+13.4% v. +13.0, p=0.75). Growth remained consistent for SP-139 yet increased with Cultrex at interval of 48-96h (+12.9% SP-139 v +22.4% Cultrex, p<0.005) and interval of 96-144h (+13.6% SP-139 v +24.0% Cultrex®, p<0.0001). Therapeutic response was assessed using 3D CellTiter-Glo® using gemcitabine 100 µM (24h) with comparable inhibition of normalized cell viability in SP-139 v. Cultrex® (35.4±1.9% v. 52.3±6.5%, n.s.) and qualitatively with viability staining using calcein AM and Caspase 3/7FITC. RNAseq revealed Cultrex, when compared to SP-139, yielded down regulation of epoxygenase p450 pathways (padj<0.012) and negative regulation of the p38 MAP kinase cascade (padj<0.048). Conclusions: Functionalized PEG-based synthetic hydrogel matrix materials can be tuned for physical properties to mimic cancer tissue microenvironments more accurately. SP-139, designed to mimic the desmoplastic stroma of the pancreatic cancer microenvironment, has favorable properties including controlled growth rates and compatibility with high content imaging by luminescence and fluorescence. RNAseq pathway analysis shows SP-139 maintains pathways of p450 and MAP kinase signaling of importance with therapeutic resistance. Citation Format: Md Shahadat Hossan, Austin Stram, Ethan Samuel Lin, Sean McIlwain, Connie Lebakken, William Richards, Jeremy D. Kratz. Bioinspired synthetic hydrogel in pancreatic cancer organoid matrix modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 305.

Estrogen receptor (ER)-positive breast cancer is characterized by late recurrences following initial treatment. The epithelial cell fate transcription factor Grainyhead-like protein 2 (GRHL2) is overexpressed in ER-positive breast cancers and is linked to poorer prognosis as compared to ER-negative breast cancers. To understand how GRHL2 contributes to progression, GRHL2 was overexpressed in ER-positive cells. We demonstrated that elevated GRHL2 imparts plasticity with stem cell- and dormancy-associated traits. RNA sequencing and immunocytochemistry revealed that high GRHL2 not only strengthens the epithelial identity but supports a hybrid epithelial to mesenchymal transition (EMT). Proliferation and tumor studies exhibited a decrease in growth and an upregulation of dormancy markers, such as NR2F1 and CDKN1B. Mammosphere assays and flow cytometry revealed enrichment of stem cell markers CD44 and ALDH1, and increased self-renewal capacity. Cistrome analyses revealed a change in transcription factor motifs near GRHL2 sites from developmental factors to those associated with disease progression. Together, these data support the idea that the plasticity and properties induced by elevated GRHL2 may provide a selective advantage to explain the association between GRHL2 and breast cancer progression.

IntroductionBefore they can produce their own antibodies, newborns are protected from infections by transplacental transfer of maternal IgG antibodies and after birth through breast milk IgA antibodies. Rhinovirus (RV) infections are extremely common in early childhood, and while RV infections often result in only mild upper respiratory illnesses, they can also cause severe lower respiratory illnesses such as bronchiolitis and pneumonia.MethodsWe used high-density peptide arrays to profile infant and maternal antibody reactivity to capsid and full proteome sequences of three human RVs - A16, B52, and C11.ResultsNumerous plasma IgG and breast milk IgA RV epitopes were identified that localized to regions of the RV capsid surface and interior, and also to several non-structural proteins. While most epitopes were bound by both IgG and IgA, there were several instances where isotype-specific and RV-specific binding were observed. We also profiled 62 unique RV-C protein loop sequences characteristic of this species’ capsid VP1 protein.DiscussionMany of the RV-C loop sequences were highly bound by IgG from one-year-old infants, indicating recent or ongoing active infections, or alternatively, a level of cross-reactivity among homologous RV-C sites.

Abstract Disclosure: C. Zheng: None. K.O. Allen: None. R. Reese: None. N.M. Solodin: None. J. Vera: None. S. Mcilwain: None. T. Liu: None. E.T. Alarid: None. Metastasis constitutes the permissive migration and growth of cancerous cells from the primary tumor to a distant location in the body. Epithelial to mesenchymal transition (EMT) is implicated in metastasis as it allows for cells to abandon an epithelial identity, adopt a mesenchymal phenotype, become migratory, and promote tumor progression. Grainyhead-like protein 2 (GRHL2) is a nuclear transcription factor responsible for driving epithelial cell fate. By regulating epithelial differentiation and integrity in estrogen receptor (ER) positive breast cancer, GRHL2 suppresses EMT, and as such, it was long thought to act primarily as a tumor suppressor. However, clinical evidence contradicts its tumor suppressive role in that patients expressing high levels of GRHL2 have a significantly lower probability of long-term recurrence free survival compared to low levels of GRHL2, and high levels of GRHL2 are also associated with high grade tumors. To better understand this discrepancy, a tetracycline (tet) inducible GRHL2 overexpression model in the ER-positive breast cancer MCF7 cell line was developed to assess cancer associated phenotypes and the transition between epithelial and mesenchymal states. Dose response and time course studies were performed to determine optimal GRHL2 induction in our tet-inducible model using immunofluorescent microscopy and Western blot analysis. Gene expression analyses was performed to assess representative genes associated with epithelial and mesenchymal states, and the intermediates between them known as hybrid or partial EMT. Interestingly, immunocytochemistry identified that the overexpression of GRHL2 induces the late EMT marker, vimentin, despite the inhibition of migration in the epithelial dominant MCF7 cell line. Together, these data indicates that GRHL2 overexpression leads to a complex phenotype that cannot be defined by a binary epithelial or mesenchymal state. Further analyses are aimed at understanding the pleiotropic roles GRHL2 plays in ER+ tumor progression. Presentation: Thursday, June 15, 2023

Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php . All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

Ultradense peptide binding arrays that can probe millions of linear peptides comprising the entire proteomes or immunomes of human or mouse, or numerous microbes, are powerful tools for studying the abundance of different antibody repertoire in serum samples to understand adaptive immune responses. There are few statistical analysis tools for exploring high-dimensional, significant and reproducible antibody targets for ultradense peptide binding arrays at the linear peptide, epitope (grouping of adjacent peptides), and protein level across multiple samples/subjects (I.e. epitope spread or immunogenic regions within each protein) for understanding the heterogeneity of immune responses. We developed HERON ( H ierarchical antibody binding E pitopes and p RO teins from li N ear peptides), an R package, which allows users to identify immunogenic epitopes using meta-analyses and spatial clustering techniques to explore antibody targets at various resolution and confidence levels, that can be found consistently across a specified number of samples through the entire proteome to study antibody responses for diagnostics or treatment. Our approach estimates significance values at the linear peptide (probe), epitope, and protein level to identify top candidates for validation. We test the performance of predictions on all three levels using correlation between technical replicates and comparison of epitope calls on 2 datasets, which shows HERON’s competitiveness in estimating false discovery rates and finding general and sample-level regions of interest for antibody binding. The code is available as an R package downloadable from http://github.com/Ong-Research/HERON .

Background We utilized a high-density peptide array, to identify linear peptide sequences of protein-targets recognized by anti-tumor antibodies produced in mice cured of melanoma following immunotherapy. Methods Mice bearing B78 melanomas were treated with a combination immunotherapy [local radiation therapy + intratumoral immunocytokine (anti-GD2 mAb linked to IL2)] that induced an 'in situ vaccine' effect (ISV), enabling mice to be cured of their tumors with long-term immune memory.1 Naïve (prior to tumor injection) and immune (post-rechallenge/after cure) sera were collected from these mice. Sera were tested using a whole-proteome peptide-array of stacked 16-mer peptides.2 Specific antibody-binding sites and recognized epitopes were identified, using an algorithm [HERON] that ranks candidates recognized by immune sera but not by sera from naïve mice.3 For proteins highly recognized by immune sera, RNAseq was used to assess differential expression of their genes in tumor vs normal and treated vs untreated tumor tissues. Results We identified many epitopes recognized selectively by sera of immune mice. Among the 100 top epitopes ranked by their binding strength to immune sera from at least 50% of the immune mice tested, we found a 4-amino acid (aa), 'SDTG' motif that was recognized in >60 of these epitopes. However, not all peptides containing this SDTG motif exhibited binding. The aa located just before and the 2 aa just following the SDTG motif play a role in binding. Using an unrelated cohort of mice, we were able to show binding of some additional immune mouse samples to selected peptides containing the SDTG motif. The antibody to the SDTG motif is not caused by the transfection used to induce GD2 expression in B78 cells; as mice cured of the parental B78-H1 cell line (not expressing GD2) also showed reactivity to some SDTG-containing peptides. The immunocytokine does not contain SDTG in its linear sequence. RNAseq analysis revealed upregulation of genes with highly recognized SDTG-containing peptides in radiated vs. untreated B78 cells. Conclusions This SDTG motif might be an important piece in anti-tumor immunity to B78 melanoma. We are further investigating what causes binding to the motif, and if the antibodies against it might have been induced by sensitization to one specific tumor protein, or possibly several proteins. The presence of antibodies against this motif might be a biomarker to predict response to our ISV regimen and this approach might be used for analyses of other effective immunotherapy treatments. References Morris ZS, et al. In Situ Tumor Vaccination by Combining Local Radiation and Tumor-Specific Antibody or Immunocytokine Treatments. Cancer Res, 2016;76(13):3929–41. Hoefges A, et al. Antibody landscape of C57BL/6 mice cured of B78 melanoma via immunotherapy. bioRxiv, 2023:2023.02.24.529012. McIlwain SJ, et al. Ranking Antibody Binding Epitopes and Proteins Across Samples from Whole Proteome Tiled Linear Peptides. bioRxiv, 2023:2023.04.23.536620.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4 + /CD8 + double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.