Therapeutic Strategies Research Articles

High level non-carbapenemase carbapenem resistance by overlaying mutations of mexR , oprD, and ftsI in Pseudomonas aeruginosa

ABSTRACT Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the mechanism of non-carbapenemase carbapenem resistance is still unclear. In the current study, we investigated the contributions of point mutations in mexR , oprD , and ftsI to carbapenem resistance in P. aeruginosa during in vivo evolution studies with consecutive clinical isolates. Real-time qPCR and Electrophoretic Mobility Shift Assay demonstrated that MexR (Gln55Pro) mutation increased MexAB efflux pump genes expression by altering MexR’s binding capacity, leading to a four- to eight-fold increase in meropenem MIC in the Pae d1 Green ∆ mexR and PAO1∆ mexR mutants. The OprD (Trp415*) truncation affected porin structure, and the constructed mutant Pae d1 Green oprD Trp415* increased meropenem MIC by 16-fold (from 0.25 to 4 µg/mL). The contribution of ftsI mutation to meropenem resistance was confirmed by clinical linkage analysis and was estimated to cause a two-fold increase in meropenem MIC by comparing the resistant clinical isolate with the Pae d1 Green oprD Trp415*∆ mexR double mutant. The study found that the oprD Trp415* allele alone accounts for the imipenem MIC in clinical isolates, while the ∆ mexR and ftsI Arg504Cys alleles do not contribute to imipenem resistance. In conclusion, we identified and explored the contributions of mexR , oprD, and ftsI mutations to high level non-carbapenemase carbapenem resistance in P. aeruginosa . These findings highlight the interplay of different mutations in causing non-carbapenemase carbapenem-resistance in P. aeruginosa . IMPORTANCE The emergence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant global health threat, complicating treatment options for infections caused by this pathogen. Understanding the mechanisms behind non-carbapenemase carbapenem resistance is critical for developing effective therapeutic strategies. This study provides crucial insights into how specific point mutations in key genes- mexR , oprD , and ftsI -contribute to carbapenem resistance, particularly the MexR (Gln55Pro) mutation’s effect on efflux pump expression and the OprD (Trp415*) truncation’s impact on porin structure. The findings elucidate the complex interplay of these mutations, highlighting their roles in conferring high-level resistance, and underscore the imperative for continued research to inform therapeutic strategies against CRPA infections.

Metabolomics reveals soluble epoxide hydrolase as a therapeutic target for high-sucrose diet-mediated gut barrier dysfunction

Highsucrose diet (HSD) was reported as a causative factor for multiorgan injuries. The underlying mechanisms and therapeutic strategies remain largely uncharted. In the present study, by using a metabolomics approach, we identified the soluble epoxide hydrolase (sEH) as a therapeutic target for HSD-mediated gut barrier dysfunction. Specifically, 16-week feeding on an HSD caused gut barrier dysfunction, such as colon inflammation and tight junction impairment in a murine model. A metabolomics analysis of mouse colon tissue showed a decrease in the 5(6)-epoxyeicosatrienoic acid [5(6)-EET] level and an increase in soluble epoxide hydrolase, which is related to HSD-mediated injuries to the gut barrier. The mice treated with a chemical inhibitor of sEH and the mice with genetic intervention by intestinal-specific knockout of the sEH gene significantly attenuated HSD-caused intestinal injuries by reducing HSD-mediated colon inflammation and improving the impaired tight junction caused by an HSD. Further, in vitro studies showed that treatment with 5(6)-EET, but not its hydrolytic product 5,6-dihydroxyeicosatrienoic acid (5,6-DiHET), significantly ablated high sucrose-caused intestinal epithelial inflammation and impaired tight junction. Additionally, 5(6)-EET is anti-inflammatory and improves gut epithelial tight junction while 5,6-DiHET cannot do so. This study presents an underlying mechanism of and a therapeutic strategy for the gut barrier dysfunction caused by an HSD.

Roles of posttranslational modifications in lipid metabolism and cancer progression

AbstractLipid metabolism reprogramming has emerged as a hallmark of malignant tumors. Lipids represent a complex group of biomolecules that not only compose the essential components of biological membranes and act as an energy source, but also function as messengers to integrate various signaling pathways. In tumor cells, de novo lipogenesis plays a crucial role in acquiring lipids to meet the demands of rapid growth. Increasing evidence has suggested that dysregulated lipid metabolism serves as a driver of cancer progression. Posttranslational modifications (PTMs), which occurs in most eukaryotic proteins throughout their lifetimes, affect the activity, abundance, function, localization, and interactions of target proteins. PTMs of crucial molecules are potential intervention sites and are emerging as promising strategies for the cancer treatment. However, there is limited information available regarding the PTMs that occur in cancer lipid metabolism and the potential treatment strategies associated with these PTMs. Herein, we summarize current knowledge of the roles and regulatory mechanisms of PTMs in lipid metabolism. Understanding the roles of PTMs in lipid metabolism in cancer could provide valuable insights into tumorigenesis and progression. Moreover, targeting PTMs in cancer lipid metabolism might represent a promising novel therapeutic strategy.

Impact of nodal status evaluation on therapeutic strategy for clinically unifocal T1b/small T2 node negative papillary thyroid carcinoma

Impact of nodal status evaluation on therapeutic strategy for clinically unifocal T1b/small T2 node negative papillary thyroid carcinoma

Renal remodeling by CXCL10-CXCR3 axis-recruited mesenchymal stem cells and subsequent IL4I1 secretion in lupus nephritis

AbstractHuman umbilical cord mesenchymal stem cells (hUC-MSCs) have shown potential as a therapeutic option for lupus nephritis (LN), particularly in patients refractory to conventional treatments. Despite extensive translational research on MSCs, the precise mechanisms by which MSCs migrate to the kidney and restore renal function remain incompletely understood. Here, we aim to clarify the spatiotemporal characteristics of hUC-MSC migration into LN kidneys and their interactions with host cells in microenvironment. This study elucidates that the migration of hUC-MSCs to the LN kidney is driven by elevated levels of CXCL10, predominantly produced by glomerular vascular endothelial cells through the IFN-γ/IRF1-KPNA4 pathway. Interestingly, the blockade of CXCL10-CXCR3 axis impedes the migration of hUC-MSCs to LN kidney and negatively impacts therapeutic outcomes. Single cell-RNA sequencing analysis underscores the importance of this axis in mediating the regulatory effects of hUC-MSCs on the renal immune environment. Furthermore, hUC-MSCs have been observed to induce and secrete interleukin 4 inducible gene 1 (IL4I1) in response to the microenvironment of LN kidney, thereby suppressing Th1 cells. Genetically ablating IL4I1 in hUC-MSCs abolishes their therapeutic effects and prevents the inhibition of CXCR3+ Th1 cell infiltration into LN kidneys. This study provides valuable insights into the significant involvement of CXCL10-CXCR3 axis in hUC-MSC migration to the LN kidneys and the subsequent remodeling of renal immune microenvironment. Regulating the CXCL10-CXCR3 axis and IL4I1 secretion may be developed as a novel therapeutic strategy to improve treatment outcomes of LN.

Identification of a New Role of miR-199a-5p as Factor Implied in Neuronal Damage: Decreasing the Expression of Its Target X-Linked Anti-Apoptotic Protein (XIAP) After SCI

Spinal cord injury (SCI) results in a cascade of primary and secondary damage, with apoptosis being a prominent cause of neuronal cell death. The X-linked inhibitor of apoptosis (XIAP) plays a critical role in inhibiting apoptosis, but its expression is reduced following SCI, contributing to increased neuronal vulnerability. This study investigates the regulatory role of miR-199a-5p on XIAP expression in the context of SCI. Using bioinformatic tools, luciferase reporter assays, and in vitro and in vivo models of SCI, we identified miR-199a-5p as a post-transcriptional regulator of XIAP. Overexpression of miR-199a-5p significantly reduced XIAP protein levels, although no changes were observed at the mRNA level, suggesting translational repression. In vivo, miR-199a-5p expression was upregulated at 3 and 7 days post-injury, while XIAP expression inversely decreased in both neurons and oligodendrocytes, being particularly significant in the latter at 7 dpi. These findings suggest that miR-199a-5p contributes to the downregulation of XIAP and may exacerbate neuronal apoptosis after SCI. Targeting miR-199a-5p could offer a potential therapeutic strategy to modulate XIAP levels and reduce apoptotic cell death in SCI.

Oxidative Stress and Cancer Therapy: Controlling Cancer Cells Using Reactive Oxygen Species

Cancer is a multifaceted disease influenced by various mechanisms, including the generation of reactive oxygen species (ROS), which have a paradoxical role in both promoting cancer progression and serving as targets for therapeutic interventions. At low concentrations, ROS serve as signaling agents that enhance cancer cell proliferation, migration, and resistance to drugs. However, at elevated levels, ROS induce oxidative stress, causing damage to biomolecules and leading to cell death. Cancer cells have developed mechanisms to manage ROS levels, including activating pathways such as NRF2, NF-κB, and PI3K/Akt. This review explores the relationship between ROS and cancer, focusing on cell death mechanisms like apoptosis, ferroptosis, and autophagy, highlighting the potential therapeutic strategies that exploit ROS to target cancer cells.

Interest and Expectations for a Herpes Vaccine Among People Diagnosed with Genital HSV 1-2 Infection: Results from an Italian Survey

Genital herpes simplex virus (HSV) is associated with a reduction in quality of life and adverse outcomes. The aim of this study is to assess the interest and expectations for a therapeutic HSV vaccine among individuals diagnosed with genital herpes in Italy. A retrospective survey was conducted at the Infectious Diseases Unit of the IRCCS San Raffaele Scientific Institute, Milan, Italy. The study collected data on demographics, clinical history and interest in HSV vaccination. The results showed that 87.5% of participants were interested in a therapeutic vaccine, with interest higher among younger people and those with frequent genital herpes recurrences. Participants most expected the vaccine to reduce the pain associated with outbreaks, followed by a reduction in the frequency and duration of recurrences. These findings underscore the strong demand for a therapeutic HSV vaccine, especially among those who experience recurrent outbreaks, and highlight the importance of considering patient expectations when developing preventive and therapeutic strategies for genital herpes.

Cutaneous T cell lymphoma atlas reveals malignant TH2 cells supported by a B cell-rich tumor microenvironment

AbstractCutaneous T cell lymphoma (CTCL) is a potentially fatal clonal malignancy of T cells primarily affecting the skin. The most common form of CTCL, mycosis fungoides, can be difficult to diagnose, resulting in treatment delay. We performed single-cell and spatial transcriptomics analysis of skin from patients with mycosis fungoides-type CTCL and an integrated comparative analysis with human skin cell atlas datasets from healthy and inflamed skin. We revealed the co-optation of T helper 2 (TH2) cell-immune gene programs by malignant CTCL cells and modeling of the tumor microenvironment to support their survival. We identified MHC-II+ fibroblasts and dendritic cells that can maintain TH2 cell-like tumor cells. CTCL tumor cells are spatially associated with B cells, forming tertiary lymphoid structure-like aggregates. Finally, we validated the enrichment of B cells in CTCL and its association with disease progression across three independent patient cohorts. Our findings provide diagnostic aids, potential biomarkers for disease staging and therapeutic strategies for CTCL.

A special focus on polyadenylation and alternative polyadenylation in neurodegenerative diseases: A systematic review

AbstractNeurodegenerative diseases (NDDs) are one of the prevailing conditions characterized by progressive neuronal loss. Polyadenylation (PA) and alternative polyadenylation (APA) are the two main post‐transcriptional events that regulate neuronal gene expression and protein production. This systematic review analyzed the available literature on the role of PA and APA in NDDs, with an emphasis on their contributions to disease development. A comprehensive literature search was performed using the PubMed, Scopus, Cochrane, Google Scholar, Embase, Web of Science, and ProQuest databases. The search strategy was developed based on the framework introduced by Arksey and O'Malley and supplemented by the inclusion and exclusion criteria. The study selection was performed by two independent reviewers. Extraction and data organization were performed in accordance with the predefined variables. Subsequently, quantitative and qualitative analyses were performed. Forty‐seven studies were included, related to a variety of NDDs, namely Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Disease induction was performed using different models, including human tissues, animal models, and cultured cells. Most investigations were related to PA, although some were related to APA or both. Amyloid precursor protein (APP), Tau, SNCA, and STMN2 were the major genes identified; most of the altered PA patterns were related to mRNA stability and translation efficiency. This review particularly underscores the key roles of PA and APA in the pathogenesis of NDDs through their mechanisms that contribute to gene expression dysregulation, protein aggregation, and neuronal dysfunction. Insights into these mechanisms may lead to new therapeutic strategies focused on the modulation of PA and APA activities. Further research is required to investigate the translational potential of targeting these pathways for NDD treatment. image

Machine learning insights into scapular stabilization for alleviating shoulder pain in college students

Abstract Non-specific shoulder pain is a common musculoskeletal condition, especially among college students, and it can have a negative impact on the patient’s life. Therapists have used scapular stabilization exercises (SSE) to enhance scapular control and mobility. This study investigates the prediction of the impact of scapular stability exercises in treating non-specific shoulder pain, leveraging advanced machine learning techniques for comprehensive evaluation and analysis. Using a diverse range of regression models, including Gamma Regressor, Tweedie Regressor, Poisson Regressor, and others, the study examines the relationship between the effectiveness of various exercises and their impact on shoulder pain management. Furthermore, the study employs optimization techniques, such as Hyperopt, scikit-optimize, optunity, GPyOpt, and Optuna, to fine-tune the exercise protocols for optimal outcomes. The results reveal that scapular stabilization exercises, when optimized using machine learning algorithms, significantly contribute to reducing shoulder pain in college students. Among the optimization techniques, scikit-optimize demonstrated the best performance, resulting in a mean squared error of 0.0085, a mean absolute error of 0.0712, and an impressive R2 score of 0.8501. This indicates that the scikit-optimize approach yielded the most accurate predictions and effectively captured the relationship between the exercises and shoulder pain management. The findings highlight the critical role of scapular stabilization exercise interventions in ameliorating non-specific shoulder pain and underscore the potential of machine learning techniques in optimizing therapeutic strategies for musculoskeletal health management. The utilization of scikit-optimize, in particular, showcases its effectiveness in fine-tuning the exercise protocols for optimal outcomes. The study’s results serve as a crucial stepping stone in developing personalized rehabilitation programs for non-specific shoulder pain, emphasizing the importance of integrating machine learning methodologies in the assessment and treatment of musculoskeletal disorders among college students.

Abstract A040: A novel stroma-mediated positive interaction between resistant and sensitive cells in non-small cell lung cancer facilitates drug resistance

Abstract Resistance to targeted therapies, particularly those inhibiting oncogenic tyrosine kinases (TKIs), presents a significant challenge in the treatment of advanced non-small cell lung cancer (NSCLC). Despite initial robust clinical responses, tumors often relapse due to the emergence of resistant cell populations. The origins of this resistance—whether it is pre-existing in a subset of tumor cells or arises de novo during treatment—remains a critical question in understanding and combating therapeutic failure. To investigate this, we developed in silico mathematical models that integrate both pre-existing and de novo resistance mechanisms within a framework that explicitly integrates the stromal niche. Our study focused on the evolutionary dynamics of NSCLC in response to therapy, using a hybrid agent-based spatial modeling framework (HAL) to simulate tumor behavior under treatment. These models were designed to simulate the complex interactions between resistant and sensitive tumor cells, mediated by the stroma, and to predict the timing of relapse based on these interactions. Our findings suggest that stroma-mediated interactions play a crucial role in the evolutionary dynamics of therapy responses, influencing the fitness and survival of both resistant and sensitive cells. Specifically, our models predict that pre-existing resistance is unlikely to be compatible with the long remission periods observed in a substantial subset of patients treated with advanced anaplastic lymphoma kinase (ALK) inhibitors. This conclusion was supported by the observed relapse times and volumetric tumor growth data from subsequent animal models. Moreover, our analyses highlight the importance of ecological factors within the tumor microenvironment in shaping the response to therapy. The spatial models we developed reveal that the stroma can foster positive interactions between resistant and sensitive cells, thereby accelerating the emergence of resistance. These insights underscore the need to consider the stromal niche when developing new therapeutic strategies and when interpreting the dynamics of drug resistance. In conclusion, our study provides new insights into the role of stroma in mediating resistance to TKIs in NSCLC. By combining mathematical modeling with experimental data, we have elucidated how stroma-mediated interactions between tumor cells can influence the evolution of resistance, challenging the assumption that pre-existing resistance alone drives relapses in TKI-treated patients. Our findings emphasize the need for adaptive therapeutic approaches that anticipate and disrupt these microenvironmental interactions to improve patient outcomes. Citation Format: Rishi M. Shah, Sagnik Yarlagadda, Mark Robertson-Tessi, Bina Desai, Tatiana Miti, Daria Miroshnychenko, David Basanta, Andriy Marusyk, Alexander Anderson. A novel stroma-mediated positive interaction between resistant and sensitive cells in non-small cell lung cancer facilitates drug resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A040.

Abstract IA019: Regulation of EMT tumor states by stromal cells

Abstract Epithelial-to-mesenchymal transition (EMT) is a reversible process during which epithelial cells lose their epithelial characteristics and acquire mesenchymal features, that are important for tumor initiation, progression, stemness, metastasis, and therapy resistance. Different EMT states, each with distinct functional properties, have been identified across various tumor models. These EMT states reside within specific physical niches, characterized by unique stromal cell populations. Understanding the cellular interactions within the tumor microenvironment (TME) and their influence on EMT could lead to novel therapeutic strategies. To understand how the TME regulates the distinct tumor states, we performed single-cell RNA sequencing of the tumor cells and their TME in mouse cancer models presenting EMT. We identified distinct subsets of cancer-associated fibroblasts (CAFs), macrophages, neutrophils, endothelial cells, pericytes, adipocytes, dendritic cells, mast cells, Schwann cells, and T cells in different tumor types. To unravel how TME-tumor cell interactions regulates EMT, we used different bioinformatic approaches to identify ligand-receptor pairs that are likely to organize the tumor niche and to regulate tumor transition states. In vitro treatment of tumor cells with some of these ligands demonstrated their EMT-promoting effects. These findings provide novel mechanistic insights on the crosstalk between tumor cells and the surrounding TME, that may have important implications for anti-cancer therapy. Citation Format: Cédric Blanpain. Regulation of EMT tumor states by stromal cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr IA019.

Abstract C050: Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs

Abstract Ewing sarcoma (EwS) is an aggressive bone and soft tissue tumor and EwS patients often succumb to their disease years after initial treatment due to relapses at metastatic sites. Novel therapeutic strategies are needed that can successfully eliminate microscopic residual disease foci that persist at the end of primary therapy. Extracellular matrix (ECM) proteins in the tumor microenvironment (TME) provide critical pro-survival and pro-invasion signals to tumor cells. Our published and unpublished data show that the glycoprotein tenascin-C (TNC) is enriched in EwS metastases and that TNC and other pro-tumorigenic ECM proteins are deposited by subpopulations of CAF-like tumor cells that are activated in response to tumor and TME-derived TGF-beta ligands. TNC is abundantly produced by multiple human tumors of epithelial and non-epithelial lineage but is otherwise rarely expressed outside of development and wound healing. Importantly, TNC is specifically enriched in metastatic lesions where it has been implicated as a mediator of metastatic competence and treatment resistance. In the current work we have tested whether the TNC-rich TME of disseminated EwS tumor foci could be leveraged to direct therapies to sites of residual micrometastatic disease. To achieve this, we generated monovalent and bivalent anti-human TNC VHHs (hTNC-VHH) using a mammalian expression platform. Camelid-derived hTNC-VHH sequences were sourced from published literature. VHHs are single domain heavy chain only antibody fragments with higher stability and tissue penetration than conventional monoclonal antibodies. Using EwS tumor spheroids in collagen-rich 3D culture, hTNC-VHH penetrated dense ECM matrices and bound through multiple cell layers in under 10 minutes. hTNC-VHH accumulated in TNC-positive but not TNC-knockout EwS spheroids and were retained beyond 96 hours. To assess their potential to drive protein and immune therapies to TNC-rich TMEs, we fused hTNC-VHH to immune modulating CD3- and CD28-activating VHHs. These hTNC-VHH-CD3/CD28-conjugates promoted immune cell activation and tumor cell death in PBMC-tumor co-culture assays as determined by CD25 flow cytometry and fluorescence microscopy, respectively. To assess in vivo localization, fluorescently-tagged hTNC-VHH were intravenously injected into mice that had been xenografted with EwS cells. Tissue microscopy confirmed selective binding of hTNC-VHH to small lung and liver EwS micrometastases in less than 4 hours. VHHs were not retained in non-tumor bearing organs including the brain and heart or in tumor-free lung and liver regions. Experiments are ongoing to assess anti-tumor efficacy and pharmacodynamics of hTNC-VHH conjugates in EwS xenografts. Together these findings suggest that the ECM-remodeling properties of CAF-like EwS cells can be exploited to recruit novel ECM-targeting protein therapeutics to micrometastases. If successful, this innovative approach could eradicate microscopic residual disease and prevent metastatic EwS recurrence. Citation Format: Emma D. Wrenn, Jason P. Price, Raymond O. Ruff, Nicolas M. Garcia, James M. Olson, Elizabeth R. Lawlor. Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C050.

Abstract A021: HRI inhibition by hemin as a novel targeted therapy for glioblastoma via the integrated stress response

Abstract The treatment of glioblastoma (GBM), a highly malignant brain tumor, is critically hindered by the ineffectiveness of current modalities such as surgery, radiation, and chemotherapy. These traditional methods fail to completely remove the tumor mass and lack the ability to discriminate between cancerous and normal brain cells, often resulting in damage to healthy tissue and recurrence of the disease. This underscores an urgent necessity to develop novel therapeutic strategies that can target tumor cells with precision, offering hope for improved survival rates and quality of life for GBM patients. This study investigates targeted therapy, focusing on the Integrated Stress Response (ISR) that cancer cells harness to survive hypoxic stress. Specifically, it demonstrates that EIF2AK1, which encodes Heme-regulated eIF2α kinase (HRI), is activated under hypoxia and co-expressed with the glioma stem cell marker SOX2, which specifically happens in glioma cells, increasing the targeted accuracy of the repurposing drug. This correlation, indicating hypoxia-driven stemness, is confirmed at both the genetic level and through Gene Set Enrichment Analysis (GSEA). Furthermore, GSEA in Spatial Transcriptomics shows hypoxia-induced glycolysis, disrupting the tumor microenvironment and causing necrotic cell death. Stemness phenotype is induced in the peripheral cells due to the unfavorable hypoxic environment. Hemin, an HRI inhibitor, has been repurposed to inhibit ISR and mitigate hypoxia. Treatment with Hemin on the U87 cell line resulted in IC50 values of 23.50 µM and 52.46 µM at 24 and 48 hours, respectively, surpassing Temozolomide's efficacy. A decrease in HRI expression after the Hemin treatment suggests the and ISR activity and, potentially, hypoxia. This would reverse the unfavorable microenvironment so that the stemness phenotype doesn’t spread. Potentially, invasiveness and recurrences of GBM in clinic situation would decrease, thus potentially improving patient prognosis. The therapeutic potential of Hemin is enhanced by its ability to kill glioma cells directly and accurately in the glioma cell in original TME when cells are proliferating with adequate oxygen. Therefore, this study demonstrates the therapeutic potential of repurposing Hemin, an HRI inhibitor, to precisely target hypoxia-induced glioma stem cells in glioblastomas, disrupting the aggressive tumor microenvironment to potentially improve patient prognosis. Citation Format: Xingchuan Ma. HRI inhibition by hemin as a novel targeted therapy for glioblastoma via the integrated stress response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A021.

Abstract A042: Desmosome mutations in the microenvironment regulate melanoma proliferation

Abstract Desmosomes are transmembrane protein complexes critical for cell-cell adhesion in epithelial tissues and other cell types. In skin cancers like melanoma, epithelial cells called keratinocytes are the predominant cells in the primary melanoma microenvironment, yet it remains largely unknown whether this cell type is subject to genetic alterations during melanoma development and its role in early melanoma progression. In this study, using an integrative analysis of tumor mutations and protein biophysical interactions, we identify a high frequency of genetic alterations in desmosome-related genes in human cancers, with the highest occurrences in cutaneous melanoma. Notably, over 70% of cutaneous melanoma cases exhibit mutations in desmosome genes and desmosome gene mutations are associated with lowered expression of the desmosome complex in melanoma. Using spatial transcriptomics and immunohistochemistry analyses, we find that the decrease in desmosome gene expression predominantly occurs in keratinocytes within the tumor microenvironment rather than in the melanoma cells themselves. To study the functional significance of this alteration in melanoma development, we used a human melanoma/keratinocyte co-culture system using primary melanoma cell lines. Our studies show that knockdown of desmosome genes in keratinocytes markedly increased proliferation of adjacent melanoma cells in melanoma/keratinocyte co-cultures. Additionally, melanoma cell proliferation is enhanced when exposed to media preconditioned by desmosome-deficient keratinocytes. These observations suggest that the gradual accumulation of mutations in desmosome genes within neighboring keratinocytes may create a conducive environment that primes melanoma cells for neoplastic transformation. This study underscores the critical role of genetic alterations in the tumor microenvironment in melanoma progression and highlights the potential for targeting desmosome-related pathways in therapeutic strategies against melanoma. Citation Format: Mohita Tagore. Desmosome mutations in the microenvironment regulate melanoma proliferation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A042.

Abstract C040: Spatial transcriptomics unveils disparities in tumor microenvironment dynamics between breast cancer responders and non-responders

Abstract Spatial transcriptomics is revolutionizing our understanding of cellular dynamics and interactions in the tumor microenvironment (TME). By mapping distinct cellular populations and their interactions, we can gain valuable insights into tumor behavior. To investigate these dynamics in breast cancer, we conducted Xenium In-situ spatial transcriptomics on twenty formalin-fixed paraffin-embedded (FFPE) samples taken from eight patients undergoing neoadjuvant chemotherapy. These samples have been taken pre-treatment, mid-treatment and post-treatment from four cases with pathological complete response (pCR), and four cases with residual disease (RD) at time of surgery. Our analyses have found distinct cellular composition changes between pCR and RD cases. Patients with RD demonstrated an increase in endothelial, perivascular-like (PVL) and epithelial cells from pre- to post-treatment. This was accompanied by a decrease in myeloid and malignant epithelial cells at mid-treatment. In contrast, patients with pCR demonstrated increases in cancer-associated fibroblasts (CAFs) and myeloid cells, with reductions in plasmablasts and cancer epithelial cells at mid-treatment. This suggests that an increase in CAF and myeloid cells observed between pre-treatment and mid-treatment samples may be predictive of response. We performed ligand-receptor (LR) analyses, which demonstrated diverse myeloid interactions in RD cases and diverse CAF interactions in pCR cases in pre-treatment samples. Mid-treatment samples exhibited a decrease in interactions for CAFs, myeloid and T cells in pCR and RD cases. The most frequently identified CAF LR interactions, as senders, were between TGFB1/TGFB3 and TGFBR1/TGFBR2, while the most frequently identified myeloid LR interactions were between PDCD1LG2 - PDCD1 (encoding PDL2 & PD1), CCL8 - ACKR1 (encoding Atypical Chemokine Receptor 1) and CCL2 - ACKR1. Our findings indicate that the dynamic changes in cellular composition and interactions within the TME differs between responders and non-responders. To further explore these differences, we plan to perform spatially constrained analyses to identify unique cellular niches that may serve as potential biomarkers for informing novel therapeutic strategies. Citation Format: Jeremy Mo, Hanyun Zhang, Kate Harvey, Alexander Swarbrick. Spatial transcriptomics unveils disparities in tumor microenvironment dynamics between breast cancer responders and non-responders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C040.

Abstract C034: Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors

Abstract Although various effective anti-cancer treatments have become available over the last decades, resistance to all available therapies remains the major cause of death of cancer patients with disseminated tumors. Striking examples are patients with triple-negative breast cancer (TNBC), which are frequently defective in the repair of DNA double strand breaks, e.g. due to loss of BRCA1 function. Because of this defect, the patients initially respond very well to DNA damage-inducing chemotherapy. Unfortunately, disseminated tumors are usually not eradicated and resistant tumor cells are eventually selected from residual primary or metastatic tumor sites. It is therefore crucial to understand the molecular mechanisms underlying the drug tolerance of the residual tumor cells. To study residual disease, we used the K14cre;Brca1 F/F ;p53 F/F (KB1P) mouse model for hereditary breast cancer, which provides the unique opportunity to explore and target those mechanisms in a fully immunocompetent model. The mammary tumors that spontaneously develop highly resemble their human counterparts, both morphologically and in their therapy response. For example, tumors shrink in response to poly(ADP-ribose) polymerase inhibition, platinum-based treatment or the commonly used doxorubicin, docetaxel and cyclophosphamide (TAC) combination therapy. But despite repeated drug sensitivity, the KB1P mammary tumors are not eradicated, not even by a frequent dosing schedule. By combining single-cell RNA sequencing, spatial transcriptomics and imaging mass cytometry, we dissected the intratumoral heterogeneity and alterations in the tumor microenvironment of residual disease. We identified specific subpopulations of tumor cells that have a survival benefit after treatment, and these occurred together with an altered microenvironment characterized by a highly reactive stroma infiltrated with both anti-inflammatory and pro-tumoral immune cells. Interestingly, those structural and transcriptional changes are reversed in the relapsed tumors, highlighting the plasticity of drug tolerance. To investigate the mechanisms of the altered tumor-stroma interactions that are relevant for drug tolerance, we have designed a custom-made CRISPR/Cas9 library based on differential gene expression of the residual tumor cells. This library enables us to functionally test relevant mechanisms of drug tolerance in vivo and we expect that these analyses will provide useful insights into the tumor-stroma interactions that contribute to residual disease. Taken together, our detailed analyses demonstrate the substantial remodeling of tumor cells in their microenvironment upon treatment. To develop new therapeutic approaches to eradicate drug-tolerant tumor cells and thereby circumvent tumor relapse, it is essential to better understand the heterogeneous cellular composition of residual tumors in its spatial context. With this project, we hope to provide comprehensive data to develop better therapeutic strategies that target the tumor-stroma interaction and eradicate residual tumors. Citation Format: Morgane Decollogny, Demeter Túrós, Astrid Chanfon, Myriam Siffert, Ismar Klebic, Sven Rottenberg. Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C034.

Abstract A017: HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by altering energy metabolism in the tissue microenvironment

Abstract Breast cancer is the most common cancer globally, with approximately 70% of pre-malignant breast tumors, known as ductal carcinoma in situ (DCIS), and 20% of invasive breast tumors overexpressing human epidermal growth factor receptor 2 (HER2). While the biological role of HER2 in invasive breast cancer has been extensively studied, its impact on breast cancer stem cells (BCSCs) and early-stage tumorigenesis, particularly within the tumor microenvironment, remains unclear. Here, we explore the role of HER2 overexpression in driving cellular events within the tumor microenvironment during the premalignant stage of breast tumorigenesis, using in vitro, in vivo, and ex vivo models. We conducted multi-omics analyses on mammary ducts from a HER2+ breast cancer mouse model at the pre-cancerous stage. Our results showed that HER2/Neu overexpression alters glucose and lipid metabolism and pentose phosphate pathway in mammary epithelial cells. Ex vivo organ culture of mammary ducts confirmed that these metabolic changes lead to increased reactive oxygen species (ROS) levels within the tumor microenvironment suggesting that oxidative stress and subsequent DNA damage are early events in HER2-driven tumorigenesis. Immunostaining of histological sections from mouse mammary glands further revealed that elevated oxidative stress and DNA damage not only in HER2+ cells but also in neighboring HER2- cells. Moreover, whole genome sequencing of tumor cells confirmed that both HER2+ and HER2- tumor cells have similar mutational burden. This suggests a non-cell-autonomous effect, where HER2 overexpression in one cell population affects the tumor microenvironment and adjacent cells, contributing to early-stage cancer development. To investigate the involvement of BCSCs during tumor initiation, we performed mammosphere assays on primary cells from HER2+ mouse models and clinical DCIS samples. Our findings showed that neither BCSCs nor their cells of origin express HER2 in both DCIS samples and murine mammary glands and tumors. This suggests that HER2 overexpression may not directly drive BCSC formation but rather creates a genotoxic microenvironment that facilitates the transformation of HER2- cell populations through a non-cell-autonomous mechanism. In summary, our findings indicate that HER2 overexpression contributes to the creation of a genotoxic tumor microenvironment by altering cellular energy metabolism and increasing ROS levels. This, in turn, may lead to the transformation of BCSCs in HER2- cell populations during early breast tumorigenesis. Understanding the role of HER2 overexpression in shaping the tumor microenvironment could provide new insights into therapeutic and preventive strategies against breast cancer. Citation Format: Sevim B. Gurler, Oliver Wagstaff, Lili Dimitrova, Fuhui Chen, Robert Pedley, William Weston, Ian J. Donaldson, Brian A. Telfer, David Novo, Kyriaki Pavlou, George Taylor, Yaqing Ou, Kaye Williams, Andrew Gilmore, Keith Brennan, Ahmet Ucar. HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by altering energy metabolism in the tissue microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A017.

Abstract A006: Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer death in the United States with a 5-year survival rate of only 13%. Early diagnosis is very difficult and thus 53% of patients are diagnosed after metastasis has already occurred, with the liver being the most frequently affected site. Both primary tumors and metastases have dense desmoplastic stroma due to a high percentage of fibroblasts within the tumor microenvironment (TME). Previously, we developed a tunable 3D biomimetic model of the liver metastatic niche (LMN) to investigate how local fibroblasts affect tumor growth and chemoresistance in PDAC liver metastasis. This study aims to investigate the contributions of fibroblasts at the primary versus metastatic sites. We used matched primary PDAC and liver metastatic (LM) organoids derived from the Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre (“KPC”) genetically engineered mouse model. This allows us to compare the effects of primary PDAC cancer associated fibroblasts (CAFs) and liver metastasis associated fibroblasts (MAFs) on tumor cell growth and chemoresistance. Rheometry analyses were conducted to study the impact of local fibroblasts on the extracellular matrix (ECM) stiffness. Our results showed that both storage and loss moduli were enhanced only when PDAC CAFs were present in the co-cultured gels compared to gels with MAFs. Additionally, the 3D biomimetic model showed fibroblasts increased tumor growth and chemoresistance to gemcitabine, only when they were derived from the same site as the tumor cells. This highlighted the importance of studying the organ-specific microenvironment to develop better treatment options for patients with metastatic PDAC. To delve deeper into the molecular basis of these observations, we conducted RNA-sequencing to identify and compare the gene expression profiles in primary and metastatic tumor organoids exposed to conditioned media from CAFs and MAFs using a transwell membrane co-culture model. Our results showed significant differences in gene expression based on the type of fibroblasts co-cultured with the organoids. Moreover, we investigated the role of MAF-derived exosomes on LM growth and chemoresistance using exosome depletion and direct exosome addition to LM organoids. Lastly, we performed exosomes size and distribution characterizations using dynamic light scattering (DLS) analysis. Taken together, these findings highlight the critical role of the organ-specific microenvironment in tumor progression and the necessity of targeted therapies to improve patient outcomes in metastatic PDAC. Our results underscore the importance of developing a tunable 3D biomimetic model of the LMN, as it enables the testing of novel therapeutic strategies designed to disrupt interactions between MAFs and tumor cells, and potentially advancing treatments for metastatic PDAC. Citation Format: Mahsa Pahlavanneshan, Weikun Xiao, Eileen Fung, Vaidhyanathan Mahaganapathy, Chae Young Eun, Chang-Il Hwang, Shannon Mumenthaler, Reginald Hill. Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A006.