Adoptive cell transfer of tumor infiltrating CD4+ T cells or T cells genetically modified to express antitumor CD4+ T cell receptors (TCRs) has shown clinical efficacy in patients with advanced solid tumors. We previously defined unique transcriptomic states of antitumor, neoantigen-specific CD8+ and CD4+ tumor infiltrating lymphocytes (TIL) in human cancer. However, optimal cell surface protein markers that identify and would enable viable cell isolation and selective enrichment of antitumor CD4+ T cells have yet to be identified. Here, we define transcriptomic and corresponding cell surface protein profiles of antitumor CD4+ TIL within metastatic colorectal tumors using high dimensional single cell transcriptomic and proteomic analysis. Comprehensive analysis of 45 antitumor CD4+ TCR clonotypes demonstrated uniquely high cell surface protein expression of ADGRG1, CD86, and CD57. In all samples containing tumor neoantigen-reactive CD4+ TIL clonotypes, ADGRG1- and CD86-based cell surface enrichment of known reactive TCR clonotypes were 11-fold and 9-fold higher than bulk CD4+ TIL, respectively. ADGRG1- and CD86-based cell surface enrichment identified >60 validated antitumor CD4+ TCR-clonotypes including tumor organoid-reactive TCRs, as well as neoantigen-specific TCRs targeting private tumor mutations and cancer driver mutations. While both ADGRG1 and CD86 enriched CD4+ TIL displayed an exhausted neoantigen-specific gene expression signature, CD86 also enriched for cytotoxic CD4+ TIL phenotypes. CD4+ TIL isolated based on ADGRG1 expression and expanded in vitro also demonstrated neoantigen reactivity in two samples. Thus, ADGRG1 and CD86 appear to be effective protein markers for isolating human tumor-specific CD4+ TCR clonotypes and studying antitumor CD4+ TIL.

Abstract Background: Breast cancer (BrCa) and post-traumatic stress disorder (PTSD) exhibit a complex, bidirectional relationship. While cancer diagnosis and treatment can induce PTSD, emerging epidemiologic data indicate that pre-existing psychological stress or PTSD may also elevate the risk of developing breast cancer or accelerate tumor progression. The biological mechanisms underlying this interaction remain unclear. We hypothesized that tumor-intrinsic alterations in stress-responsive genes may provide molecular insight into this connection. Methods: We analyzed 17,605 breast tumors using cBioPortal/TCGA datasets and compared them with a 95,474-sample pan-cancer reference. PTSD- and stress-related over sixty genes were curated from published literature and included: TG, CYP11B1, CYP11B2, CRH, NR3C1, SLC6A4, PRKCA, CHRNA6, RGS2, DRD2, BDNF, FKBP5, SLC6A2, TPH2, and others. Copy number alterations, co-amplification patterns, and clinical outcomes (including metastatic progression) were evaluated. Results: Stress-related genes exhibited significantly enriched copy number amplifications in breast cancer compared with a pan-cancer reference cohort. Across 17,605 BrCa tumors, more than sixty PTSD-, mood-, and stress-endocrine-associated genes showed recurrent CNAs. Key alterations included TG (19%), CYP11B1/CYP11B2 (16%), RGS2 (12%), CRH (10%), PRKCA (9%), and CHRNA6 (8%), markedly higher BrCa than their frequency across 95,474 tumors from all cancer types (2-5%). TG-amplified tumors demonstrated coordinated co-amplifications involving 8q24 (MYC/PVT1) and 1q32, along with characteristic 8p22 deletions, genomic features strongly associated with aggressive disease biology. Exploratory clinical annotation further showed that TG- or CYP11B-gated BrCa cohorts exhibited rapid metastatic progression, with a median time to metastasis <10 months. Together, these findings identify a robust stress-tumor genomic axis in breast cancer and highlight Thyroglobulin (TG) and Corticotropin-Releasing Hormone (CRH) as high-priority candidates for development as objective, noninvasive serum biomarkers. Conclusion: Large-scale civilian tumor genomic analyses reveal that stress-responsive and PTSD-linked genes are recurrently amplified in breast cancer and co-cluster with high-risk oncogenic regions. These findings support a biological model in which chronic stress/PTSD and breast cancer aggressiveness intersect through shared neuroendocrine and GPCR-linked molecular pathways. This stress/tumor axis may help explain both PTSD arising after a breast cancer diagnosis and stress-associated increases in breast cancer risk or progression. These insights establish a foundation for developing objective biomarkers and mechanistic studies exploring the PTSD-BrCa interface. Citation Format: Alakesh Bera, Meera Srivastava. Stress-responsive genomic alterations reveal a bi-directional relationship between PTSD biology and breast cancer aggressiveness [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 1429.

Abstract Background: Cytogenetic abnormalities involving multiple loci on the long arm of chromosome 1 are among the most frequent alterations in human breast carcinoma. To define their biological and clinical significance, we conducted a large-scale integrative genomic analysis across diverse breast cancer datasets. Methods: Data from over 13,000 breast tumors (n = 13,146 samples; 12,229 patients across 30 studies) were analyzed using cBioPortal, including The Cancer Genome Atlas (TCGA). Copy number alterations, mRNA/protein expression, and clinical outcomes were evaluated. Comparative analyses with >100,000 samples across 35 other cancers (n = 105,424) determined specificity. Mutual exclusivity and co-occurrence analyses identified genetic interaction patterns relevant to tumor progression. Serum detectability of top candidates was evaluated. Results: Eight genes particularly TRIM67, DISC1, REN, DNM3, ATP1B1, VSIG8, SPTA1, and MUC1 from different loci of 1q chromosome showed recurrent amplifications (12-15%) in breast tumors, significantly exceeding frequencies in pan-cancer datasets (∼3%). These genes displayed low baseline expression in normal breast tissue but were highly expressed in tumors. Co-occurrence analyses revealed significant genetic interplay, including TP53 with MUC1/SPTA1/VSIG8 and PIK3CA with TRIM67/DISC1/REN, suggesting cooperative gain-of-function driving aggressive phenotypes. Among the eight amplified 1q genes, MUC1 and REN showed the strongest clinical relevance, as both encode secreted or shed proteins that were readily detectable in serum and have established associations with aggressive breast cancer biology. In contrast, DNM3 and ATP1B1 were detectable at lower abundance in serum-derived extracellular vesicles, supporting their potential as emerging but less established circulating markers. Conclusions: Multi-locus amplifications contribute collectively to breast cancer progression through distinct gene interaction networks. This study identifies a core set of functionally relevant, serum-detectable gene products that may serve as biomarkers for prognosis, recurrence monitoring, and therapeutic targeting in aggressive breast cancer. Citation Format: Alakesh Bera, Hai Hu, COL Craig D. Shriver, Meera Srivastava. Serum detection of MUC1 and renin identifies circulating biomarkers for breast cancer prognosis [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 3945.

ABSTRACTBackgroundDysbiosis of the bacterial and fungal microbiome has been increasingly implicated in the pathogenesis of chronic rhinosinusitis (CRS). This study explores the relationship between microbiome and mycobiome biodiversity and type 2 (T2) versus non‐type 2 (NT2) inflammation.MethodsMucosal tissues from the ethmoid sinus were collected during endoscopic sinus (CRS) and skull base (controls) surgery between January 2020 and July 2021. Specimens underwent 16S rRNA (bacterial) and internal transcribed spacer (fungal) gene sequencing, along with cytokine analysis using the Luminex assay. Based on cytokine (IL‐4, IL‐5, IL‐13) concentrations and the presence of eosinophils, CRS cases were classified into T2 or NT2 inflammatory profiles. The relationships between CRS endotype and the biodiversity of the microbiome and mycobiome were assessed.ResultsSpecimens from 92 patients (30 control, 31 CRSwNP, 31 CRSsNP) were included in the analyses. Among 62 CRS cases, 20 exhibited T2 inflammation and 42 exhibited NT2 inflammation. Compared with control specimens, NT2 specimens exhibited significantly lower amplicon sequence variants (mean difference −149, 95% CI [−261, −37], p = 0.007), Shannon index (−0.48 [−0.79, −0.16], p = 0.002), and Simpson index (−0.003 [−0.005, −0.001], p = 0.002) for bacterial alpha diversity. However, no significant differences in bacterial alpha diversity were observed between T2 specimens and controls, or between T2 and NT2 specimens. Fungal biodiversity did not differ significantly across endotype and control groups.ConclusionDysbiosis of the sinus bacterial microbiome is more strongly associated with a NT2‐mediated inflammatory profile than with a T2‐mediated inflammatory profile.

Several studies have identified differential expression at various loci on the long arm of chromosome 1 (1q) as the most frequent cytogenetic abnormalities associated with human breast carcinoma. To investigate the impact of genetic alterations on chromosome 1q, we analyzed data from over thirteen thousand human breast tumors (n=13, 146 samples; corresponding to 12, 229 patients across 30 studies) using cBioPortal, which included comprehensive datasets like The Cancer Genome Atlas(TCGA). Our findings revealed that eight genes exhibit significant copy number alterations (12-15%), predominantly amplifications. These genes include TRIM67 (1q42.2, 14%), DISC1 (1q42.2, 14%), REN (1q32.1, 15%), DNM3 (1q24.3, 12%), ATP1B1 (1q24.2, 12%), VSIG8 (1q23.2, 14%), SPTA1 (1q23.1, 14%) and MUC1 (1q22, 14%). In contrast, analysis across more than 100, 000 samples from over 35 different cancers (n=105, 424samples; 101, 597 patients across 226 studies) showed that these genes have much lower alteration frequencies, approximately 3%. Besides, these genes also exhibited notably low expression levels at both RNA and protein levels in normal breast tissues. We further explored the clinical relevance of these genes, examining their roles in breast cancer prognosis and survival outcomes. Our mutual exclusivity and co-occurrence analyses revealed distinct patterns of genetic interplay, indicating a probable collective gain-of-function that drives breast cancer progression. Specifically, TP53 was found to co-occur with MUC1, SPTA1, and VSIG8, while REN exhibited mutual exclusivity. The driver gene PIK3CA co-occurred with DISC1, TRIM67, and REN, whereas ERBB2 showed co-occurrence with all eight genes. These findings suggest a complex network of genetic interactions that may underlie invasive and metastatic breast cancer phenotypes. Additionally, we observed that some of the above mentioned gene products (proteins) including REN (Renin) are detectable in serum, suggesting potential as biomarkers for recurrence or prognosis. Our ongoing studies are focused on validating these proteins as diagnostic and therapeutic targets. Collectively, this study underscores the importance of multi-locus evaluation on chromosome 1q, which could pave the way for novel diagnostic and therapeutic strategies in breast cancer management. Alakesh Bera, Eric Russ, Hai Hu, Craig D. Shriver, Meera Srivastava. Functional role of chromosome 1q loci on breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 191.

Adoptive transfer of unselected autologous tumor-infiltrating lymphocytes (TILs) has mediated meaningful clinical responses in patients with metastatic melanoma but not in cancers of gastrointestinal epithelial origin. In an evolving single-arm phase 2 trial design, TILs were derived from and administered to 91 patients with treatment-refractory mismatch repair proficient metastatic gastrointestinal cancers in a schema with lymphodepleting chemotherapy and high-dose interleukin-2 (three cohorts of an ongoing trial). The primary endpoint of this study was the objective response rate as measured using Response Evaluation Criteria in Solid Tumors 1.0; safety was a descriptive secondary endpoint. In the pilot phase, no clinical responses were observed in 18 patients to bulk, unselected TILs; however, when TILs were screened and selected for neoantigen recognition (SEL-TIL), three responses were seen in 39 patients (7.7% (95% confidence interval (CI): 2.7-20.3)). Based on the high levels of programmed cell death protein 1 in the infused TILs, pembrolizumab was added to the regimen (SEL-TIL + P), and eight objective responses were seen in 34 patients (23.5% (95% CI: 12.4-40.0)). All patients experienced transient severe hematologic toxicities from chemotherapy. Seven (10%) patients required critical care support. Exploratory analyses for laboratory and clinical correlates of response were performed for the SEL-TIL and SEL-TIL + P treatment arms. Response was associated with recognition of an increased number of targeted neoantigens and an increased number of administered CD4+ neoantigen-reactive TILs. The current strategy (SEL-TIL + P) exceeded the parameters of the trial design for patients with colorectal cancer, and an expansion phase is accruing. These results could potentially provide a cell-based treatment in a population not traditionally expected to respond to immunotherapy. ClinicalTrials.gov identifier: NCT01174121 .

Adoptive cell transfer (ACT) using neoantigen reactive T lymphocytes can mediate cancer regression. Here, we developed methods to isolate unique, personalized, neoantigen reactive T cell receptors (TCRs) from tumor infiltrating lymphocytes (TIL) from patients with metastatic gastrointestinal cancers and incorporate the TCR α and β chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes (PBL) using γ-retroviruses and adoptively transferred those cells into patients after lymphodepleting chemotherapy. We treated 7 patients with metastatic, mismatch-repair proficient colorectal cancers who had progressive disease following multiple prior therapies, and three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lung, and lymph nodes. All patients received T cell populations containing ≥50% TCR transduced cells, and all T cell populations were polyfunctional in that they secreted IFNα, GM-CSF, IL-2, and Granzyme B specifically in response to mutant peptides compared to wild type counterparts. TCR transduced cells were detected in the peripheral blood of 5 patients, including the three responders, at levels ≥10% of CD3+ cells approximately one month post-ACT. In one patient with a partial response to therapy, ~20% of CD3+ PBL expressed transduced TCRs more than two years after treatment. These studies demonstrate that ACT with T cells genetically modified to express personalized neoantigen reactive TCRs is safe and can mediate tumor regression in patients with metastatic solid epithelial cancers.

Thyroid cancer is the most common endocrine malignancy in the United States, with an overall favorable prognosis. However, some patients experience poor outcomes due to the development of resistance to conventional therapies. Genetic alterations, including mutations in BRAF, Met, and p53, play critical roles in thyroid cancer progression, with the BRAF V600E mutation detected in over 60% of cases. This study investigates the tumor-suppressive role of Annexin A7 (ANXA7) in thyroid cancer, focusing on its potential impact on tumor behavior and therapeutic response. Our analysis, which included RNA sequencing and protein profiling, revealed reduced ANXA7 expression in thyroid cancer cells, particularly in those harboring the BRAF V600E mutation. Upon treatment with inhibitors targeting BRAF and MEK, ANXA7 expression increased, leading to reduced phosphorylation of ERK and activation of apoptotic pathways. Additionally, we identified the cyclin-dependent kinase inhibitor p21 as a key player in modulating resistance to BRAF inhibitors. Combination therapies aimed at concurrently increasing p21 and ANXA7 levels resulted in a marked enhancement of apoptosis. These findings suggest a previously uncharacterized regulatory network involving the ANXA7/p21/BRAF/MAPK/p53 axis, which may contribute to drug resistance in thyroid cancer. This study provides new insights into overcoming resistance to BRAF and MAPK inhibitors, with implications for treating thyroid cancer and potentially other BRAF-mutant tumors. Future efforts will focus on high-throughput screening approaches to explore ANXA7-targeted therapeutic strategies for thyroid cancer.

Adoptive cell transfer (ACT) with neoantigen-reactive T lymphocytes can mediate cancer regression. Here we isolated unique, personalized, neoantigen-reactive T cell receptors (TCRs) from tumor-infiltrating lymphocytes of patients with metastatic gastrointestinal cancers and incorporated the TCR α and β chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes and adoptively transferred these cells into patients after lymphodepleting chemotherapy. In a phase 2 single-arm study, we treated seven patients with metastatic, mismatch repair-proficient colorectal cancers who had progressive disease following multiple previous therapies. The primary end point of the study was the objective response rate as measured using RECIST 1.1, and the secondary end points were safety and tolerability. There was no prespecified interim analysis defined in this study. Three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lungs and lymph nodes lasting 4 to 7 months. All patients received T cell populations containing ≥50% TCR-transduced cells, and all T cell populations were polyfunctional in that they secreted IFNγ, GM-CSF, IL-2 and granzyme B specifically in response to mutant peptides compared with wild-type counterparts. TCR-transduced cells were detected in the peripheral blood of five patients, including the three responders, at levels ≥10% of CD3+ cells 1 month post-ACT. In one patient who responded to therapy, ~20% of CD3+ peripheral blood lymphocytes expressed transduced TCRs more than 2 years after treatment. This study provides early results suggesting that ACT with T cells genetically modified to express personalized neoantigen-reactive TCRs can be tolerated and can mediate tumor regression in patients with metastatic colorectal cancers. ClinicalTrials.gov registration: NCT03412877 .

Background Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment.Methods We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed “NeoExpand”, was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand.Results We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models.Conclusions Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation.Trial registration number NCT00068003, NCT01174121, and NCT03412877.

Abstract Background and Rational: Transforming growth factor β (TGFβ) signaling is pivotal in cellular proliferation and can exhibit either tumor-suppressive or promoting effects depending on cellular context and genetic alterations. TGFβ receptor type-2 (TGFβR2), the ligand-binding receptor, is implicated in various cancers. This study explores the functional role of TGFβR2 in breast cancer (BrCa) progression. Materials and Method: Genomic alterations in TGFβR2 were assessed in TCGA samples. TGFβR2 mRNA and protein expression were examined in a panel of BrCa cell lines, including MCF7, MCF12A, MDA-MB-231, and MDA-MB-468. Additionally, serum samples from BrCa patients, meticulously categorized into subtypes such as triple-negative (TN), human epidermal growth factor receptor 2-positive (Her2+), Luminal A (LA), Luminal B1 (LB1), and Luminal B2 (LB2), were analyzed. BrCa cell lines were treated with TGFβ ligand to investigate receptor expression modulation. Results: Analysis of genomic alterations in TGFβR2 from The Cancer Genome Atlas (TCGA, n=1084, BrCa samples) revealed TGFβ copy number amplification in 25% of samples, while less than 1% showed TGFβR2 amplification. Elevated TGFβR2 levels were identified in aggressive triple-negative MDA-MB-231 cells compared to other tested cell lines. Serum samples from BrCa patients (n=240) demonstrated higher TGFβR2 levels, particularly in Luminal B1 subtype and African American patients. Treatment of BrCa cell lines with TGFβ ligand induced TGFβR2 expression, suggesting ligand-induced receptor upregulation. Discussion: The findings suggest TGFβR2 as a potential biomarker for BrCa, with higher levels associated with aggressiveness and racial disparities. Ligand-induced receptor upregulation implies a potential oncogenic role. These results contribute to understanding TGFβR2's functional significance and its implications in breast cancer progression, offering insights into potential biomarkers and therapeutic targets. Citation Format: Meera Srivastava, Alakesh Bera, Harvey B. Pollard, Hai Hu, Craig D. Shriver. Functional role of TGF-beta receptor type-2 in breast cancer progression and survival [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 779.

Supplementary Figure from Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor–Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors

Pancreatic cancer remains a major health concern, being among the deadliest forms of cancer with over 80% of the patients presenting with metastatic disease. According to the American Cancer Society, for all stages of pancreatic cancer combined, the 5-year survival rate is less than 10%. Genetic research on pancreatic cancer has generally been focused on familial pancreatic cancer, which is only 10% of all pancreatic cancer patients. This study focuses on finding genes that impact the survival of pancreatic cancer patients which can be used as biomarkers and potential targets to develop personalized treatment options. We used cBioPortal platform using NCI-initiated The Cancer Genome Atlas (TCGA) dataset to find genes that were altered differently in different ethnic groups which can serve as potential biomarkers and analyzed the genes' impact on patient survival. MD Anderson Cell Lines Project (MCLP) and genecards.org were also utilized to identify potential drug candidates that can target the proteins encoded by the genes. The results showed that there are unique genes that are associated with each race category which may influence the survival outcomes of patients, and their potential drug candidates were identified.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.

We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.

Annexin A7/ANXA7 is a calcium-dependent membrane fusion protein with tumor suppressor gene (TSG) properties, which is located on chromosome 10q21 and is thought to function in the regulation of calcium homeostasis and tumorigenesis. However, whether the molecular mechanisms for tumor suppression are also involved in the calcium- and phospholipid-binding properties of ANXA7 remain to be elucidated. We hypothesized that the 4 C-terminal endonexin-fold repeats in ANXA7 (GX(X)GT), which are contained within each of the 4 annexin repeats with 70 amino acids, are responsible for both calcium- and GTP-dependent membrane fusion and the tumor suppressor function. Here, we identified a dominant-negative triple mutant (DNTM/DN-ANXA7J) that dramatically suppressed the ability of ANXA7 to fuse with artificial membranes while also inhibiting tumor cell proliferation and sensitizing cells to cell death. We also found that the [DNTM]ANA7 mutation altered the membrane fusion rate and the ability to bind calcium and phospholipids. In addition, in prostate cancer cells, our data revealed that variations in phosphatidylserine exposure, membrane permeabilization, and cellular apoptosis were associated with differential IP3 receptor expression and PI3K/AKT/mTOR modulation. In conclusion, we discovered a triple mutant of ANXA7, associated with calcium and phospholipid binding, which leads to the loss of several essential functions of ANXA7 pertinent to tumor protection and highlights the importance of the calcium signaling and membrane fusion functions of ANXA7 for preventing tumorigenesis.

It is known that inflammatory cytokines exacerbate the persistence and severity of various disease states. Breast cancer is the most frequently detected cancer among women worldwide and our recent studies suggest that the inflammatory state of breast (BrCa) cancer, a byproduct of elevated cytokine expression, induces epigenetic modifications leading to increased recurrence. Ongoing NCI clinical trial data (ClinicalTrials.gov, CCC19, NCT04354701) indicates that among patients with cancer and COVID-19, the mortality is high, and the most prevalent malignancies are of breast [21%] and prostate [16%] origin. Due to the risk of cytokine storm during SARS-CoV-2 infection, it is crucial to identify potential mechanisms of hyperinflammation in BrCa patients. In this study, we have evaluated the level of copy number alteration (CNA) of different inflammatory cytokines including IL-8, IL-1b, IL6, IL-8, GM-CSF, TNF-alpha and many others using cBioportal platform which includes over sixty-nine thousand tumor samples (n>69,000 from 213 different studies) from over 33 different cancers. We found that IL-8 has the highest level of amplification in different breast cancers subtypes. Besides, we also analyzed serum samples from BrCa patients, both recurrent and non-recurrent, by different proteomics methods to identify serum cytokines involved in prognosis and recurrence. Comparative data analysis between non-recurrent BrCa against recurrent BrCa patients identified several proteins with very high significance, mostly proteins associated with epigenetic pathways including HDAC9 (P = 0.0035), HDAC5 (P = 0.013), and HDAC7 (P = 0.020). Besides, we identified differential expression of several pro-inflammatory cytokines and immune regulators (IL-8, IL-4, IL-18, IL-12p70) that were present only in recurrent BrCa patient serum. Our data indicate that inflammatory processes contribute to epigenetic modifications that ultimately play a critical role in breast cancer recurrence. In terms of COVID-19 associated co-morbidity, the already dysregulated inflammatory state of BrCa patients may increase their susceptibility to cytokine-storm, leading to increased severity of COVID-related complications and increased mortality rate. Specifically, we hypothesize that the identified elevated level of IL-8 in BrCa patients may lead to a higher basal level of inflammation and contribute to the risk of attaining cytokine-storm during SARS-CoV-2 infection, making it a valuable target for future studies. Citation Format: Alakesh Bera, Eric Russ, Surya Radhakrishnan, Hai Hu, COL Craig D. Shriver, Meera Srivastava. Inflammatory biomarkers: Breast cancer survival and possible role in COVID-19 associated comorbidity [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 807.

BackgroundCellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive T-cell receptors (TCRs) comes to the forefront of clinical investigation, the rapid, scalable, and cost-effective detection of patient-specific neoantigen-reactive TIL remains a top priority.MethodsWe analyzed the single-cell transcriptomic states of 31 neoantigen-specific T-cell clonotypes to identify cell surface dysfunction markers that best identified the metastatic transcriptional states enriched with antitumor TIL. We developed an efficient method to capture neoantigen-reactive TCRs directly from resected human tumors based on cell surface co-expression of CD39, programmed cell death protein-1, and TIGIT dysfunction markers (CD8(+) TILTP).ResultsTIL(TP) TCR isolation achieved a high degree of correlation with single-cell transcriptomic signatures that identify neoantigen-reactive TCRs, making it a cost-effective strategy using widely available resources. Reconstruction of additional TILTP TCRs from tumors identified known and novel antitumor TCRs, showing that at least 39.5% of TILTP TCRs are neoantigen-reactive or tumor-reactive. Despite their substantial enrichment for neoantigen-reactive TCR clonotypes, clonal dynamics of 24 unique antitumor TILTP clonotypes from four patients indicated that most in vitro expanded TILTP populations failed to demonstrate neoantigen reactivity, either by loss of neoantigen-reactive clones during TIL expansion, or through functional impairment during cognate neoantigen recognition.ConclusionsWhile direct usage of in vitro-expanded CD8(+) TILTP as a source for cellular therapy might be precluded by profound TIL dysfunction, isolating TILTP represents a streamlined effective approach to rapidly identify neoantigen-reactive TCRs to design engineered cellular immunotherapies against cancer.

Pancreatic cancer remains a major health concern, being among the most deadly forms of cancer. It is the seventh most common cancer, yet it is the third leading cause of cancer deaths in the United States. It is estimated that in 2021, 48,220 patients will die from pancreatic cancer. Risk factors for pancreatic cancer include smoking, diabetes, obesity, chronic pancreatitis, and family history. Over 80% of the patients present with metastatic disease. Despite advances in chemotherapy, the average survival remains less than 5 years even after surgery. Genetic research on pancreatic cancer has generally only focused on familial pancreatic cancer, which is only 10% of all pancreatic cancer patients. This study focuses on finding genes that impact the survival of pancreatic cancer patients based on race, with the goal of finding genes that are race specific and can be used as biomarkers and potential targets to develop personalized treatment options. All datasets for pancreatic cancer in cBioPortal were used for this study. The data were then stratified based on race information for the patients. Three (3) racial categories, 1) White 2) African American and Black, and 3) Asian were chosen. Our analysis finds that White, African Americans and Asians have distinct copy number alterations, more specifically amplifications in specific genes, with GATA6, RECQL4, and MIB1 being only altered in White patients, PKD1L1, GARS1, and NEUROD6 only altered in Black & African American patients, and the entire 4p16.3 cytoband of genes (39 in total), only altered in Asian patients. Patients with alterations in these unique genes also had poor survival. Using the Protein Atlas, MD Anderson Cell Lines Project (MCLP) dataset and genecards.org data, we identified potential drug candidates that target the proteins encoded by these genes. Our study clearly demonstrates that race specific genetic alterations exist in pancreatic cancer and provides a foundation for the development of race specific diagnostic/prognostic biomarkers and targeted therapeutic options with better survival outcomes. Citation Format: Digonto Chatterjee, Jack Hester, Meera Srivastava, Alakesh Bera. Racial disparities in pancreatic cancer: A quantitative proteo-genomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5862.