Human Cancer Patients Research Articles

Proteomic Analysis Identifies FNDC1, A1BG, and Antigen Processing Proteins Associated with Tumor Heterogeneity and Malignancy in a Canine Model of Breast Cancer.

Simple SummaryComparative oncology is centered around the study of naturally occurring tumors in animals as a parallel and complementary model for human cancer research. Canine mammary tumors pose as excellent models since they share similarities in their spontaneous nature, histological subtypes, genetic background, and clinical course, which would be impossible to reproduce in murine models. Our study aimed to investigate cancer heterogeneity in primary tumors and metastasis, by applying bottom-up proteomics and mass spectrometry imaging to identify potential disease-state markers. We have demonstrated that the malignant phenotype may have arisen as a consequence of alterations in the expression of proteins involved in immune evasion facilitating metastatic events. To our knowledge, this is the first study to use mass spectrometry imaging in a dog model of breast cancer, that have demonstrated that poorly described proteins might play important roles in cancer spreading and should be further validated as potential early-stage tumor biomarkers.New insights into the underlying biological processes of breast cancer are needed for the development of improved markers and treatments. The complex nature of mammary cancer in dogs makes it a great model to study cancer biology since they present a high degree of tumor heterogeneity. In search of disease-state biomarkers candidates, we applied proteomic mass spectrometry imaging in order to simultaneously detect histopathological and molecular alterations whilst preserving morphological integrity, comparing peptide expression between intratumor populations in distinct levels of differentiation. Peptides assigned to FNDC1, A1BG, and double-matching keratins 18 and 19 presented a higher intensity in poorly differentiated regions. In contrast, we observed a lower intensity of peptides matching calnexin, PDIA3, and HSPA5 in poorly differentiated cells, which enriched for protein folding in the endoplasmic reticulum and antigen processing, assembly, and loading of class I MHC. Over-representation of collagen metabolism, coagulation cascade, extracellular matrix components, cadherin-binding and cell adhesion pathways also distinguished cell populations. Finally, an independent validation showed FNDC1, A1BG, PDIA3, HSPA5, and calnexin as significant prognostic markers for human breast cancer patients. Thus, through a spatially correlated characterization of spontaneous carcinomas, we described key proteins which can be further validated as potential prognostic biomarkers.

Determinants and Dynamic Changes of Generic Quality of Life in Human Bladder Cancer Patients.

We measured and determined the factors associated with long-term generic quality-of-life (QOL) changes in human bladder cancer patients. We utilized the World Health Organization QOL-Brief questionnaire to assess consecutive patients’ QOL at outpatient clinics of our hospital. A mixed-effects model was constructed to investigate the determinants of QOL changes according to each domain and individual item after controlling for demographic and clinical factors, as well as the effect of radical cystectomy. We also applied a kernel smoothing method to describe the long-term dynamic changes after the first definite treatment. In total, 1185 repeated measurements were collected from 343 bladder cancer patients. The mixed-effects models demonstrated that marital status, monthly income, and comorbidity with heart disease and diabetes were significant determinants among all the study participants. Regardless of the urinary diversion type, radical cystectomy contributed to lower scores for all four domains, mainly from 4–5 years after cystectomy, which declined significantly in patients who were older than 60 years. As for non-muscle-invasive bladder cancer (NMIBC) patients with preserved bladders, tumor recurrence was a major predictor for lower scores for sexual activity in the social domain. In summary, generic QOL can be independently influenced by many factors, not only cystectomy and tumor recurrence, which should be discussed with patients before treatment.

Tolerability of palbociclib in younger and older patients with advanced breast cancer.

Palbociclib is a small-molecule cyclin-dependent kinase 4/6 inhibitor used to treat hormone receptor-positive, human epidermal growth factor receptor-2 negative advanced breast cancer. Patient-specific factors impacting dose reductions or discontinuations are unknown. The primary objective was to evaluate the association of age (<60 vs. ≥60 years) with palbociclib dose reductions or discontinuations secondary to neutropenia. This single-center, retrospective chart review included hormone receptor-positive, human epidermal growth factor receptor-2 negative advanced breast cancer patients ≥18 years treated with palbociclib between April 2015 and May 2020. Patients <60 years at the time of palbociclib initiation were in the younger group and patients ≥60 years were in the older group. Among the 107 patients included, younger patients were less likely than older patients to have a palbociclib starting dose <125 mg (0% vs. 11.9%, p = 0.02). Differences in palbociclib dose reductions or treatment discontinuations secondary to neutropenia were not detected (35.4% vs. 42.4%, p = 0.55). Neither the total number of palbociclib dose reductions (none: 54.2% vs. 49.1%, one: 33.3% vs. 42.4%, two: 12.5% vs. 8.5%, p = 0.61), nor the final dose of palbociclib (125 mg: 54.2% vs. 40.7%, 100 mg: 29.2% vs. 27.1%, 75 mg: 16.7% vs. 32.2%, p = 0.17) differed between younger and older patients. Age (<60 vs. ≥60 years) was not associated with the rate of palbociclib dose reductions or discontinuations secondary to neutropenia. Older (≥60 years) patients were more likely to start palbociclib at lower doses which may impact neutropenia and non-neutropenic intolerance.

Identification of a novel cancer microbiome signature for predicting prognosis of human breast cancer patients.

Prognosis of breast cancer (BC) patients differs considerably and identifying reliable prognostic biomarker(s) is imperative. With evidence that the microbiome plays a critical role in the response to cancer therapies, we aimed to identify a cancer microbiome signature for predicting the prognosis of BC patients. The TCGA BC microbiome data (TCGA-BRCA-microbiome) was downloaded from cBioPortal. Univariate and multivariate Cox regression analyses were used to examine association of microbial abundance with overall survival (OS) and to identify a microbial signature for creating a prognostic scoring model. The performance of the scoring model was assessed by the area under the ROC curve (AUC). Nomograms using the microbial signature, clinical factors, and molecular subtypes were established to predict OS and progression-free survival (PFS). Among 1406 genera, the abundances of 94 genera were significantly associated with BC patient OS in TCGA-BRCA-microbiome dataset. From that set we identified a 15-microbe prognostic signature and developed a 15-microbial abundance prognostic scoring (MAPS) model. Patients in low-risk group significantly prolong OS and PFS as compared to those in high-risk group. The time-dependent ROC curves with MAPS showed good predictive efficacy both in OS and PFS. Moreover, MAPS is an independent prognostic factor for OS and PFS over clinical factors and PAM50-based molecular subtypes and superior to the previously published 12-gene signature. The integration of MAPS into nomograms significantly improved prognosis prediction. MAPS was successfully established to have independent prognostic value, and our study provides a new avenue for developing prognostic biomarkers by microbiome profiling.

Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation.

From a mouse triple‐negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G‐protein–coupled receptor (ADGR) family, G‐protein–coupled receptor 56 (GPR56)/adhesion G‐protein–coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1‐transduced 4T1, TS/A (mouse breast cancer cell line), or MDA‐MB‐231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1‐induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

Target-Based Radiosensitization Strategies: Concepts and Companion Animal Model Outlook.

External beam radiotherapy is indicated in approximately 50-60% of human cancer patients. The prescribed dose of ionizing radiation that can be delivered to a tumor is determined by the sensitivity of the normal surrounding tissues. Despite dose intensification provided by highly conformal radiotherapy, durable locoregional tumor control remains a clinical barrier for recalcitrant tumor histologies, and contributes to cancer morbidity and mortality. Development of target-based radiosensitization strategies that selectively sensitizes tumor tissue to ionizing radiation is expected to improve radiotherapy efficacy. While exploration of radiosensitization strategies has vastly expanded with technological advances permitting the precise and conformal delivery of radiation, maximal clinical benefit derived from radiotherapy will require complementary discoveries that exploit molecularly-based vulnerabilities of tumor cells, as well as the assessment of investigational radiotherapy strategies in animal models that faithfully recapitulate radiobiologic responses of human cancers. To address these requirements, the purpose of this review is to underscore current and emerging concepts of molecularly targeted radiosensitizing strategies and highlight the utility of companion animal models for improving the predictive value of radiotherapy investigations.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Comparative Cancer Cell Signaling in Muscle-Invasive Urothelial Carcinoma of the Bladder in Dogs and Humans.

Muscle-invasive urothelial carcinoma (MIUC) is the most common type of bladder malignancy in humans, but also in dogs that represent a naturally occurring model for this disease. Dogs are immunocompetent animals that share risk factors, pathophysiological features, clinical signs and response to chemotherapeutics with human cancer patients. This review summarizes the fundamental pathways for canine MIUC initiation, progression, and metastasis, emerging therapeutic targets and mechanisms of drug resistance, and proposes new opportunities for potential prognostic and diagnostic biomarkers and therapeutics. Identifying similarities and differences between cancer signaling in dogs and humans is of utmost importance for the efficient translation of in vitro research to successful clinical trials for both species.

A Novel Mechanism of Endoplasmic Reticulum Stress- and c-Myc-Degradation-Mediated Therapeutic Benefits of Antineurokinin-1 Receptor Drugs in Colorectal Cancer.

The neurokinin‐1 receptor (NK‐1R) antagonists are approved as treatment for chemotherapy‐associated nausea and vomiting in cancer patients. The emerging role of the substance P‐NK‐1R system in oncogenesis raises the possibility of repurposing well‐tolerated NK‐1R antagonists for cancer treatment. This study reports that human colorectal cancer (CRC) patients with high NK‐1R expression have poor survival, and NK‐1R antagonists SR140333 and aprepitant induce apoptotic cell death in CRC cells and inhibit CRC xenograft growth. This cytotoxicity induced by treatment with NK‐1R antagonists is mediated by induction of endoplasmic reticulum (ER) stress. ER stress triggers calcium release, resulting in the suppression of prosurvival extracellular signal‐regulated kinase (ERK)‐c‐Myc signaling. Along with ER calcium release, one ER stress pathway mediated by protein kinase RNA‐like ER kinase (PERK) is specifically activated, leading to increased expression of proapoptotic C/EBP‐homologous protein (CHOP). Moreover, NK‐1R antagonists enhance the efficacy of chemotherapy by increasing the sensitivity and overcoming resistance to 5‐fluorouracil in CRC cells through the induction of sustained ER stress and the consequent suppression of ERK‐c‐Myc signaling both in vitro and in vivo. Collectively, the findings provide novel mechanistic insights into the efficacy of NK‐1R antagonists either as a single agent or in combination with chemotherapy for cancer treatment.

STAT3-mediated upregulation of the AIM2 DNA sensor links innate immunity with cell migration to promote epithelial tumourigenesis

ObjectiveThe absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of...

Simple and effective bacterial-based intratumoral cancer immunotherapy

BackgroundWe describe intratumoral injection of a slow-release emulsion of killed mycobacteria (complete Freund’s adjuvant (CFA)) in three preclinical species and in human cancer patients.MethodsEfficacy and safety were tested in mammary...

Up-regulation of Aquaporin 5 Defines Spasmolytic Polypeptide-Expressing Metaplasia and Progression to Incomplete Intestinal Metaplasia.

Background & AimsMetaplasia in the stomach is highly associated with development of intestinal-type gastric cancer. Two types of metaplasias, spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia (IM), are considered precancerous lesions. However, it remains unclear how SPEM and IM are related. Here we investigated a new lineage-specific marker for SPEM cells, aquaporin 5 (AQP5), to assist in the identification of these 2 metaplasias.MethodsDrug- or Helicobacter felis (H felis) infection-induced mouse models were used to identify the expression pattern of AQP5 in acute or chronic SPEM. Gene-manipulated mice treated with or without drug were used to investigate how AQP5 expression is regulated in metaplastic lesions. Metaplastic samples from transgenic mice and human gastric cancer patients were evaluated for AQP5 expression. Immunostaining with lineage-specific markers was used to differentiate metaplastic gland characteristics.ResultsOur results revealed that AQP5 is a novel lineage-specific marker for SPEM cells that are localized at the base of metaplastic glands initially and expand to dominate glands after chronic H felis infection. In addition, AQP5 expression was up-regulated early in chief cell reprogramming and was promoted by interleukin 13. In humans, metaplastic corpus showed highly branched structures with AQP5-positive SPEM. Human SPEM cells strongly expressing AQP5 were present at the bases of incomplete IM glands marked by TROP2 but were absent from complete IM glands.ConclusionsAQP5-expressing SPEM cells are present in pyloric metaplasia and TROP2-positive incomplete IM and may be an important component of metaplasia that can predict a higher risk for gastric cancer development.

Increased incidence of gastrointestinal toxicity in canine cancer patients treated with concurrent abdominal radiation therapy and toceranib phosphate.

Receptor tyrosine kinase inhibitors (TKIs) are used to treat human and canine cancers and may be combined with radiation therapy (RT) to enhance tumor control due their anticancer and antiangiogenic effects; however, recent case reports have emerged describing incidences of gastrointestinal toxicity when antiangiogenic therapies are combined with hypofractionated radiotherapy in human cancer patients. We evaluated the incidence of gastrointestinal (GI) toxicity in dogs receiving concurrent hypofractionated abdominal RT and the TKI toceranib (TOC) compared to those receiving abdominal RT alone, TOC alone, or concurrent non-abdominal RT and TOC. Medical records of canine cancer patients were retrospectively reviewed and identified dogs were included in the following treatment categories: dogs which received RT to a portion of the abdomen and concurrent TOC (n = 19), abdominal RT alone (n-29), TOC alone (n = 20), or non-abdominal RT plus TOC (n = 9). Toxicities were graded using the Veterinary Cooperative Oncology Group - Common Terminology Criteria for Adverse Events criteria and compared to published data on TOC-associated GI toxicity. Patients receiving TOC while undergoing abdominal RT had significantly increased rates of any grade of diarrhea (p = 0.002), hyporexia (p = 0.0045), and vomiting (p = 0.003), as well as severe hyporexia (p = 0.003) when compared across the treatment groups. This retrospective study reveals significantly increased incidences of GI toxicity when abdominal RT is combined with TOC in canine patients. These findings are in-line with the clinical concerns reported for increased normal tissue toxicity in human patients when antiangiogenics are combined with RT.

Implementation of a Low-Carbohydrate Diet Improves the Quality of Life of Cancer Patients - An Online Survey.

Background: The ketogenic diet (KD), a high-fat low-carbohydrate diet, has gained in popularity in recent years, which is reflected by an increasing number of scientific articles, books, websites, and other publications related to low carbohydrate (LC) diets and KDs. Numerous preclinical studies in different animal models of cancer have examined the effect of KDs on cancer growth, but no large randomized controlled studies or prospective cohort studies are available for human cancer patients. Evidence supporting the use of KDs as an adjunct to traditional cancer therapy has come predominantly from anecdotes and case reports. The first KD clinical trials in patients with glioblastoma revealed good acceptance and a possible anti-tumor effect. Metabolic therapy options such as the KD are not yet part of the standard of care in cancer patients. However, many cancer patients have begun implementing a KD or LC diet on their own. The aim of the present study was to gather information, via an online questionnaire, about how cancer patients go about implementing a KD or LC diet, what resources they rely on, whether they perceive benefits from the diet on quality of life (QoL), and what factors influence feasibility and adherence to the diet.Method: Recruitment of participants was carried out via social media platforms, forums and cooperating physicians (April 2018 through November 2018). To be eligible for the study, participants had to be diagnosed with cancer and on a KD or LC diet at the time of participating in the study or been on a KD or LC diet during cancer treatment. Study participants were asked to fill out an online questionnaire. The questionnaire was divided into four parts and contained a total of 64 questions. The questions were focused on the current health status of the participant, type of cancer, time since diagnosis, and treatment regimen. In addition, questions addressed social support, extent of professional counseling, food preferences and QoL.Results: A total of 96 participants (77 F, 17 M) submitted the questionnaire, of which 94 were included in the final data analysis. Ages ranged between 24 and 79 years (mean 50.1 ± 12.1 years). In 73.4% of the participants, the tumor had not formed metastases at the time of initial diagnosis. Twenty-four (26%) participants had a PET-positive tumor, 8 (9%) a PET-negative tumor, and the remainder (66.0%) did not report a PET scan. Eighty seven percent had undergone surgery in the course of their cancer treatment. The most frequent tumor type was breast cancer, followed by cervical cancer, prostate cancer, colorectal cancer and melanoma. Fifty nine percent of the study participants stated that they followed a KD during cancer therapy, 21% followed a low carbohydrate/high fat (LCHF) diet and 12% followed a low glycemic index (LOGI) diet. Sixty nine percent reported an improvement of QoL because of the diet. Almost half of the study participants sourced their initial information on KDs and LC diets from the internet. We found a significant correlation between weight loss upon implementation of a KD and the extent of overweight (p < 0.001). Weight loss in already lean participants was not reported. Overall, 67% of the participants found long-term adherence to the diet to be “easy” and 10.6% described it as being “very easy.” Participants who like fatty foods tended to perceive the diet as being easier to follow (p = 0.063).Conclusion: The KD or LC diet improved self-reported QoL in more than two-thirds of study participants. The KD had a normalizing effect on body weight. The majority of the participants rated the diet as easy or very easy to follow long term. There was an obvious gap between patients' desire for professional dietary counseling and what is currently offered by health care providers. In the future, efforts should be made to invest in nutrition experts who are trained in the KD to support cancer patients with implementation of a KD.

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for invivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

Risk evaluation of early-stage hormone receptor-positive and human epidermal growth factor receptor 2-negative breast cancer patients: a population-based study from Taiwan

To assess the prognostic value of the Dutch criteria for patients with early-stage hormone receptor-positive and human epidermal growth factor receptor 2-negative breast cancer from the Taiwan Cancer RegistryDatabase. We included 8295 patients with early-stage node-negative breast cancer who underwent surgery during January 2008-December 2012. Patients were stratified into low- and high-risk groups based on the Dutch criteria. The Kaplan-Meier method and log-rank test were used to estimate the difference in breast cancer-specific survival (BCSS) and overall survival (OS) between groups. Multivariable analysis was used to evaluate the prognostic value of the Dutch criteria. Overall, the low-risk and high-risk groups comprised 5375 and 2920 patients, respectively. In the low- and high-risk groups, the 5-year BCSS rate was 99.6% and 98.2% (P < 0.0001) and the 5-year OS rate was 98.3% and 96.8% (P < 0.0001), respectively. The hazard ratio for BCSS was 4.18 (95% confidence interval [CI] 2.63-6.63, P < 0.0001), and the hazard ratio for OS was 1.94 (95% CI 1.48-2.55); both were significantly poorer in the high-risk group than in the low-risk group. In the low-risk group, the 5-year BCSS and OS of patients who did and did not receive adjuvant chemotherapy were similar (99.5% versus 99.6% [P = 0.927] and 98.8% and 98.1% [P = 0.0683], respectively). The prognosis of low-risk patients as classified using the Dutch criteria is excellent with or without adjuvant chemotherapy. The benefit of multi-gene testing for chemotherapy decision-making might be minimal in these patients.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1590: An experimental tumor model resistant to anti-PD-1 is sensitive to local mRNA immunotherapy

Abstract Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or β2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFβ and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFβ and Notch in combination with PD-1 blockade decelerated tumor growth, local administration of a mixture of mRNAs encoding immune-stimulatory cytokines (IL12sc, GM-CSF, IL15s and IFNα) potently induced regression of resistant tumors and resulted in complete tumor remission. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade. Citation Format: Marie Bernardo, Tatiana Tolstykh, Yu-an Zhang, Dinesh S. Bangari, Hui Cao, Kerstin A. Heyl, Joon Sang Lee, Natalia Malkova, Jack Pollard, Hui Qu, Fangxian Sun, Dmitri Wiederschain, Timothy R. Wagenaar. An experimental tumor model resistant to anti-PD-1 is sensitive to local mRNA immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1590.

Abstract 1736: Antigen-specific targeting of tissue-resident gamma delta T cells with recombinant butyrophilin heterodimeric fusion proteins

Abstract A major mechanism of acquired resistance to immune checkpoint inhibition involves downregulation of antigen presentation, including the MHC I complex itself. Downregulation of antigen presentation on MHC I can render tumor cells invisible to any α/β T cell directed therapy. γδ T cells (γδT) are a small subset of the overall T cell compartment, but are characterized by increased cytolytic capacity relative to α/β T cells. Rather than MHC I, γδT recognize target cells via a complex of heterodimerized butyrophilin (BTN) proteins. Thus, display of BTN heterodimers on the surface of tumor cells may enhance immunity to tumors that have downregulated MHC I, or which express low abundance or low affinity antigens. We have previously reported the generation of a heterodimeric BTN protein targeting the CD19 antigen, referred to as BTN2A1/3A1-Fc-CD19scFv. While the BTN2A1/3A1 complex is a potent activator of γδT expressing the Vγ9δ2 T cell receptor (TCR), which is the major γδT population in human peripheral blood, Vγ9δ2 T cells are not the predominant γδT in many tissues. The murine equivalent of BTN2A1/3A1-Fc-CD19scFv, BTNL1/6-Fc-CD19scFv, stimulated specific proliferation and increased the cytolytic capacity of peripheral blood γδT in mice, but not other tissue-restricted GDT populations. These data suggested that distinct BTN heterodimers may preferentially activate tissue-restricted subsets of GDT. To characterize potential differences between peripheral blood and tissue-restricted γδT , we performed a multi-layered analysis, including single-cell sequencing of γδ TCR from paired peripheral blood and tumor tissues from human cancer patients such as melanoma, prostate and colon cancer. These data identified tissue-specific preferences for individual γδ TCRs, with corresponding tissue-specific preferences for individual BTN proteins. Based on this information, we generated a panel of distinct heterodimeric BTN proteins, and show that specific γδT populations are preferentially activated by specific BTN heterodimers in a lock-and-key fashion. These data are a necessary pre-requisite for designing γδT specific therapeutics that may target both immune neglected and acquired resistant tumors that have limited visibility to α/β T cell directed approaches. Citation Format: Suresh de Silva, George Fromm, Anne Lai, Louis Gonzalez, Arpita Patel, Kyung Jin Yoo, Kellsey Johannes, Kinsley Evans, Keith Wilson, Taylor H. Schreiber. Antigen-specific targeting of tissue-resident gamma delta T cells with recombinant butyrophilin heterodimeric fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1736.

Abstract 2140: Delineating a functional link between a phosphoserine aminotransferase (PSAT1)-associated gene signature and EGFR-mutant NSCLC

Abstract Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second enzymatic step within the serine synthetic pathway and its expression is elevated in numerous human cancers, such as NSCLC. While PSAT1 suppression decreases SSP activity and cell proliferation, non-canonical activities have recently been suggested that may contribute to its pro-tumorigenic function, as is seen with other metabolic enzymes such as pyruvate kinase. Thus, we conducted a genome wide expression screen to uncover potential unknown cellular processes impacted by PSAT1 and to identify a PSAT1-associated gene signature that might inform patient outcomes in NSCLC. Loss of PSAT1 in EGFR-mutant PC9 lung cancer cells led to 491 differentially expressed genes (279-down; 212-up). KEGG and gene ontology analysis identified biological pathways such as metabolic processes, cell cycle regulation, cell adhesion, and cell motility. Transcript analysis confirmed inhibitory effects on the Rb/E2F1 and β-catenin pathways as well as expression of effectors on the actin cytoskeleton upon loss of PSAT1. Importantly, these could be rescued by PSAT1 restoration, including the loss of F-actin stress fibers as observed with phalloidin staining. Lastly, a bioinformatics approach utilizing transcriptomic data from human lung cancer patients was used to define genes altered between EGFR-mutant lung cancers and normal lung and that were differentially expressed in our PSAT1 depletion RNA-seq study. From this, we identified a PSAT1-associated gene signature that was then used to assign risk survival scores to these NSCLC patients. Subsequent, Kaplan Meier analysis demonstrated the prognostic ability of this gene signature for both relapse free and overall survival in EGFR-mutant NSCLC but was unable to predict risk groups in other NSCLC subtypes. Together, transcriptomic analysis under PSAT1 silencing identified its impact on various biological processes, including actin cytoskeleton rearrangement, and led to identification of a gene signature that has predictive ability towards survival outcomes in EGFR-mutant lung cancer. Citation Format: Rumeysa Biyik-Sit, Brian F. Clem. Delineating a functional link between a phosphoserine aminotransferase (PSAT1)-associated gene signature and EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2140.