Myeloid Cell Populations Research Articles

CSF-1R inhibitor PLX3397 attenuates peripheral and brain chronic GVHD and improves functional outcomes in mice

Graft-versus-host disease (GVHD) is a serious complication of otherwise curative allogeneic haematopoietic stem cell transplants. Chronic GVHD induces pathological changes in peripheral organs as well as the brain and is a frequent cause of late morbidity and death after bone-marrow transplantation. In the periphery, bone-marrow-derived macrophages are key drivers of pathology, but recent evidence suggests that these cells also infiltrate into cGVHD-affected brains. Microglia are also persistently activated in the cGVHD-affected brain. To understand the involvement of these myeloid cell populations in the development and/or progression of cGVHD pathology, we here utilized the blood–brain-barrier permeable colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX3397 (pexidartinib) at varying doses to pharmacologically deplete both cell types. We demonstrate that PLX3397 treatment during the development of cGVHD (i.e., 30 days post-transplant) improves disease symptoms, reducing both the clinical scores and histopathology of multiple cGVHD target organs, including the sequestration of T cells in cGVHD-affected skin tissue. Cognitive impairments associated with cGVHD and neuroinflammation were also attenuated by PLX3397 treatment. PLX3397 treatment prior to the onset of cGVHD (i.e., immediately post-transplant) did not change in clinical scores or histopathology. Overall, our data demonstrate significant benefits of using PLX3397 for the treatment of cGVHD and associated organ pathologies in both the periphery and brain, highlighting the therapeutic potential of pexidartinib for this condition.

A Peripheral Blood Signature of Increased Th1 and Myeloid Cells Combined with Serum Inflammatory Mediators Is Associated with Response to Abatacept in Rheumatoid Arthritis Patients.

Abatacept (CTLA4-Ig)-a monoclonal antibody which restricts T cell activation-is an effective treatment for rheumatoid arthritis (RA). Nevertheless, only 50% of RA patients attain clinical responses, while predictors of response are rather limited. Herein, we aimed to investigate for early biomarkers of response to abatacept, based on a detailed immunological profiling of peripheral blood (PB) cells and serum proteins. We applied flow cytometry and proteomics analysis on PB immune cells and serum respectively, of RA patients starting abatacept as the first biologic agent. After 6 months of treatment, 34.5% of patients attained response. At baseline, Th1 and FoxP3+ T cell populations were positively correlated with tender joint counts (p-value = 0.047 and p-value = 0.022, respectively). Upon treatment, CTLA4-Ig effectively reduced the percentages of Th1 and Th17 only in responders (p-value = 0.0277 and p-value = 0.0042, respectively). Notably, baseline levels of Th1 and myeloid cell populations were significantly increased in PB of responders compared to non-responders (p-value = 0.009 and p-value = 0.03, respectively). Proteomics analysis revealed that several inflammatory mediators were present in serum of responders before therapy initiation and strikingly 10 amongst 303 serum proteins were associated with clinical responses. Finally, a composite index based on selected baseline cellular and proteomics' analysis could predict response to abatacept with a high sensitivity (90%) and specificity (88.24%).

Abstract C040: Single cell analysis of circulating peripheral blood mononuclear cells in African American men with prostate cancer and their association with lethal disease

Abstract Prostate cancer is a leading cause of cancer-related deaths among men in the United States and affects African American (AA) men more so than other men. We and others previously described a distinct tumor immunobiology in AA prostate cancer patients. Furthermore, we discovered a serum proteome-defined suppression of tumor immunity signature that is prevalent among these patients and associated with lethal prostate cancer. In the current study, we sought to investigate the composition of circulating peripheral blood mononuclear cells (PBMCs) in AA and European American (EA) prostate cancer patients and their association with patient group and lethal prostate cancer. We obtained high-quality RNA-sequencing data for 277642 single cells representing circulating PBMCs from 59 AA and EA men. In the analysis, being an AA patients associated with an increased abundance of exhausted CD8+T cells and higher numbers of macrophages that classified as tumor-associated macrophages, whereas metallothionine-expressing macrophages were decreased in these men. Gene signatures indicative of T cell exhaustion were generally elevated in AA PBMCs while T cell effector function features were decreased. Within the myeloid cell population, cytokine and NFKB signatures were more prevalent in AA than EA patients. This gene signature pattern that we observed in AA patients also associated with lethal prostate cancer, namely the T cell exhaustion and elevated cytokine gene signatures, pointing to potential clinical implications. Lastly, the interferon-induced transmembrane protein (IFITM3), interferon alpha inducible protein (IFI6), cysteine rich protein-1 (CRIP1), and the RAC family small GTPase2 (RAC2) were notably upregulated in AA patient-derived immune cells. In summary, we show that circulating immune cell populations and the associated gene signatures differ between men of African and European descent. Those elevated in AA patients correlated with lethal prostate cancer. Citation Format: Md Shakir U. Ahmed, Tiffany Dorsey, Stefan Ambs. Single cell analysis of circulating peripheral blood mononuclear cells in African American men with prostate cancer and their association with lethal disease [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C040.

Tristetraprolin regulates the skeletal phenotype and osteoclastogenic potential through monocytic myeloid-derived suppressor cells.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, invitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.

Abstract C048: Single-cell RNA sequencing analysis in prostate cancer patients from African ancestry

Abstract Prostate cancer is the second most common malignancy in men worldwide. Men of African ancestry (AA) have a higher incidence rate and increased prostate cancer mortality, present with more advanced disease at earlier ages, than men of European ancestry (EA). Aside from socioeconomic factors driving this disparity, genomic factors also greatly contribute to the differences in the incidence and mortality rates. Despite recent advancements in comparisons of genomic alterations, a comprehensive transcriptomic and epigenomic landscape of tumors from AA prostate cancer patients has yet to be developed. Droplet-based single-cell technologies have evolved and now enabled the profiling of transcriptome, epigenome, and chromatin organization from complex tissues at single-cell resolution. To identify cell populations with distinctive molecular features and cell-type specific transcriptional programs and cell states from AA prostate cancer patients, we performed single-cell RNA sequencing for 11 EA and 6 AA prostate cancer patients. By investigating the differentially expressed genes in a cell-type-specific manner between AA and EA patients, we found that endothelial and myeloid cell populations showed significant enrichment of inflammatory pathways in AA patients. In the analysis, we also identified a group of heat shock protein family genes that were upregulated in AA patients in multiple cell types. Then, ligand-receptor interactions between tumor cells and immune microenvironments from AA and EA prostate cancer patients identified significant interactions involving frizzled receptors (FZD) that were enriched in AA patients. Overall, the transcriptomic differences may reveal oncogenic mechanisms in AA prostate cancer patients compared to EA patients. Citation Format: Hanbing Song, Yih-An Chen, William Yue, Hannah N.W. Weinstein, Paul Allegakoen, Heiko Yang, Emory Kim, Julia Pham, Keliana Hui, Matthew R. Cooperberg, Franklin W. Huang. Single-cell RNA sequencing analysis in prostate cancer patients from African ancestry [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C048.

Development of a Machine Learning Decision Support System for Clinical Flow Cytometry

Abstract High parameter flow cytometry is a highly utilized tool in both research and clinical settings for accurate immunophenotyping in immunology, oncology, and many other disciplines. With increasing dimensionality, manual analysis of data becomes more complex and time consuming. Over the past decade, several automated methods have been developed for cell population clustering and dimensionality reduction of high parameter flow cytometry data which have sped up and simplified the discovery of cell populations not observed by manual gating strategies. However, the input and output of such tools are stochastic in nature, thus making their results difficult to reuse with novel samples such as diagnostic patient samples. Several groups have recently attempted to utilize machine learning algorithms to analyze clinical flow cytometry data. These studies focused on training machine learning models with a goal of predicting a specific diagnosis for each sample. This approach can be limiting when the diagnostic features are not perfectly met to predict a specific diagnosis as well as the absence of the patient's clinical data. To our knowledge, no studies have utilized machine learning to identify and classify individual cells based on their raw fluorescence signature to various cellular populations, whether normal or abnormal, to aid a diagnostician to interpret their significance in a clinical context. In this study, we utilized a data set of ~240 million individual cells from ~9,500 flow cytometry files from 2,300 patients to train a machine learning model to predict cell population classifications as annotated by expert hematopathologists. These annotations were implemented into an end-to-end machine learning based decision support system allowing for rapid, automated analysis of high dimensional flow cytometry data with an output aiding the diagnostician in the generation of a diagnosis. A clinical validation highlighted the robustness of this methodology with 100% sensitivity and 94% specificity in the identification of abnormal B-cell populations on a cohort of 1,500 patients. This decision support system also identifies abnormal T-cell and myeloid cell populations with 100% sensitivity. Overall, the decision support system saves significant time as it removes the need for manual gating and analysis, and aids the diagnostician in identifying discrete cell populations and offering descriptive information about any abnormal populations identified.

High Dimensional Clustering and Identification of Immune Cell Hybrid Populations in B-ALL across the Disease Continuum

Introduction: Nearly half of adults with acute lymphoblastic leukemia (ALL) will experience relapse of their disease, and those with relapsed ALL carry a worse prognosis than those who remain in remission. The influence of the tumor immune microenvironment (TME) on relapse and survival outcomes in ALL is not well understood. Mass cytometry (CyTOF) enables immune profiling based on single cell protein expression and can identify phenotypic diversity of cells in the TME that cannot reliably be detected by standard flow cytometry. Recently, T-cell/monocyte hybrid cells have been discovered in patients with uveal melanoma and latent tuberculosis infections. However, cells expressing CD19, a surface antigen expressed on B-ALL cells, and markers of other cells in the TME including T cells and tumor-associated macrophages (TAMs), have not been described in ALL or other hematologic malignancies. The presence of CD3/CD163 hybrid cells in the TME may have predictive and prognostic implications for adults with ALL, as TAMs play a role in suppressing antitumor immunity which can lead to T-cell exhaustion. In this study, we identify the presence of multilineage hybrid cells in peripheral blood (PB) and bone marrow (BM) samples of patients with B-ALL alongside accompanying evolution of the TME at each disease stage. Methods: PB (N=16) and BM aspirate (N=16) isolates were collected from patients with B-ALL at diagnosis, relapse, or remission, as well as from healthy donors (HDs, N=7) for comparison of cellular profiles. A 28-marker CYTOF panel was designed to evaluate cell surface and intracellular markers of interest in the myeloid compartment. Antibodies were purchased pre-conjugated or conjugated in house based on manufacturer guidelines (Standard BioTools). Samples were barcoded with palladium isotopes prior to staining to reduce the impact of batch effect, and were then analyzed on a first generation CyTOF system (Standard BioTools). Following background attenuation and singlet gating in FlowJo (BD), the CATALYST R package enabled deconvolution and visualization of single cell data, including dimensional reduction techniques (tSNE) and clustering of all cell populations. An iterative approach was taken to determine a stable configuration of clusters across sample populations to protect from over-fragmentation of data, further informed by K-means clustering using the elbow method. Heatmaps were then generated based on median scaled expression of markers contained in each cluster. Results: The CyTOF panel identified several different myeloid cell populations in newly diagnosed, relapsed, and remission samples in both PB and BM compared to HDs. Most interestingly, quantifiable levels of a cluster of cells co-expressing CD19, CD3 and CD163 as well as a second cluster of cells expressing CD19 and CD163 were detected in newly diagnosed and relapsed patients, but not in HDs. A single patient with the largest proportion of these cell populations in their PB at the time of relapse presented with an aggressive leukemia variant and relapsed multiple times prior to succumbing to illness. On investigation of BM aspirates, the triple positive hybrid population was found in a similar distribution amongst the newly diagnosed and relapsed patients, as well as affecting one patient who achieved remission. This patient also had a highly aggressive leukemia that was refractory to chemotherapy and immunotherapy, and only achieved this remission following CAR-T cell therapy. She subsequently underwent stem cell transplant and is in remission. Conclusions: Our findings add to the literature highlighting the utility of high parameter single cell capture techniques in detecting rare cell populations in the leukemic TME. Further, we identified two unique hybrid cell populations that are reliably detected across different disease stages in patients with B-ALL. Importantly, these populations persisted following stringent gating protocols to rule out doublets and debris. Although our sample size was too small to demonstrate a prognostic impact of these hybrid cell populations in patients with B-ALL, the identification of these populations alone is a significant finding. Further investigation and patient sample accrual are warranted to better understand the role of these cell types in tumor biology, which may pave the way towards new prognostic models that can leverage well-established immune monitoring techniques.

CD22 TCR-Engineered T Cells Exert Anti-Leukemia Cytotoxicity without Causing Inflammatory Responses

Chimeric antigen receptor (CAR) T cells are remarkably effective, especially against B-cell malignancies. However, CAR-T cell therapy is also associated with cytokine-driven inflammatory toxicities such as cytokine release syndrome. Similar cytokine-related toxicity can occur after bispecific T-cell engager (BiTE) administration. It is unknown whether these inflammatory responses are specifically related to T-cell-mediated therapies that are activated through artificial CAR or BiTE signals. Cytokine-driven inflammatory toxicities were not commonly encountered prior to the wide-spread use of CAR-T cells in the clinic–neither from the adoptive transfer of ex vivo-expanded autologous polyclonal antigen-specific T cells, tumor-infiltrating lymphocytes, nor TCR-engineered T cells (TCR-T cells). However, it is challenging to compare these diverse cell products, utilized in different clinical contexts and against different antigens. Preclinical comparisons of CARs and TCRs consistently showed lower sensitivity of CARs for antigen as compared to that of TCRs, at least partly due to inefficient downstream signaling of the former (Salter AI et al. Science Signaling. 2021; Gudipati V et al. Nat Immunol. 2020; Harris DT et al. J Immunol. 2018, Oren R et al. J Immunol. 2014). Nonetheless, it is not known whether these differences in signaling and T cell effector functions affect the preponderance of eliciting inflammatory responses. Here, we discovered a TCR targeting the CD22 antigen expressed by various B-cell malignancies in order to evaluate whether anti-leukemia/lymphoma T-cell cytotoxicity could be achieved without causing inflammatory responses by using TCR-activated T cells rather than CAR-activated T cells for the same antigen, CD22. A TCR specific to HLA-A*02:01-restricted epitope of human CD22 was isolated from an HLA-A2 negative healthy donor T cells through allogeneic in vitro stimulations. TCR alpha/beta-paired single cell sequencing was performed, and the sequence of the most enriched TCR clonotype was reconstructed in a retroviral expression vector. Primary human T cells transduced with this CD22 TCR recognized malignant B-cell lines only when they expressed both HLA-A*02:01 and CD22, confirming the intended specificity of the TCR. The TCR did not cross-react with other human proteins. CD22 TCR-T cells were then compared with CD22 CAR-T cells that are successful in the treatment of patients with CD22+ leukemia/lymphoma but associated with dose-limiting inflammatory toxicities (Shah NN et al. J Clin Oncol. 2020, Frank MJ et al. Blood. 2021). CD22 TCR-T cells mediated efficient in vitro cytotoxicity against CD22-dim lymphoma cells whereas CD22 CAR-T cells exerted suboptimal cytotoxicity. Importantly, TCR-T cells (1e7-3e7 cells/mouse) also cleared leukemia in xenograft models without inducing systemic proinflammatory cytokine elevation (Figure). In contrast, CD22 CAR-T cells, given at the dose necessary to control in vivo leukemia growth (5e6 cells/mouse), led to significantly increased levels of circulating proinflammatory cytokines such as IFNγ (Figure), TNFα, GM-CSF, MIP-1α and MIP-1β. Systemic cytokine elevation after CAR-T cell infusion exacerbated when NSG xenograft mice were pre-infused (24 hours prior to adoptive T cell transfer) with PBMC containing myeloid cell populations that were autologous to T cells. TCR-T cell therapy was not associated with proinflammatory cytokine elevation even in the presence of myeloid cell populations. Consistent with these in vivo observations, in vitro cytokine production by CAR-T cells were higher compared to TCR-T cells in response to CD22+ leukemia cells. Moreover, T cells activated through the CD22 TCR and CD22 CAR by the identical leukemia cell line demonstrated differential transcriptional signatures; CAR-T cells exhibited a disproportionate and significant upregulation of inflammatory pathways compared to TCR-T cells while effector response pathways were either equivalent or enriched in TCR-T cells. Together, these results highlight the opportunity for TCR-T cells to mediate anti-leukemia responses with a potentially safer toxicity profile compared to CAR-T cells. Our work provides the basis for incorporating TCR-based immunotherapy into the treatment paradigm for patients with relapsed/refractory B-cell malignancies, improving the safety of adoptive cell therapy.

Multimodal Single-Cell Transcriptomic and Proteomic Correlatives of Patients Outcomes Following Anti-BCMA Cellular Therapy with Ciltacabtagene Autoleucel (Cilta-cel) in Relapsed Multiple Myeloma

Introduction Multimodal single cell technologies allow us to dissect the mechanisms of therapeutic resistance by integrating transcriptomics and proteomics through the combination of antibody labeling and next-generation sequencing. Using CITE-seq, mass cytometry (CyTOF) and quantitative multiplexed proteomics (Olink), we sought to understand the determinants of chimeric antigen receptor (CAR) T cell response and the overall survival of patients with relapsed or refractory multiple myeloma (RRMM) receiving ciltacabtagene autoleucel (Cilta-Cel) cellular therapy. Methods Twenty five patients received Cilta-Cel and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and after CAR T infusion. We isolated 262,520 BM cells and 241,657 PB cells, which were later sequenced using CITE-seq. Additionally, we submitted PB samples for CyTOF analysis and acquired 10,162,426 cells used for downstream analysis. We analyzed 92 cytokines using Olink immuno-oncology panel in PB samples. Downstream analysis was performed using the R packages Seurat, CATALYST, FlowSOM and Olink Analyze. Results Our cohort had a median progression-free survival (PFS) of 732 days. To focus our correlative analyses on patients experiencing early relapse, the initial cohort was divided into 2 groups: PFS <18 months (n = 10) and PFS >18 months (n = 15). CAR-T cell expansion was observed in week 2 post-infusion and continued up to week 4. The percentage of CD4 and CD8 CAR-T cells significantly increased between weeks 1-3 and weeks 4-6 weeks (p<0.001) and significantly decreased after week seven (p<0.05). We detected novel and significant differences among four cell populations associated with PFS longer than 18 months. These patients had a higher percentage of activated CD4 Central Memory (CM) and CD4 cytotoxic cells (p<0.05) relative to the total percentage of CAR-T cells in weeks 4-6, suggesting a key role for CD4 cells in cross priming or direct cytotoxicity in this context. In patients with a PFS longer than 18 months, the CD8 CM CART cell population had a significantly higher percentage in weeks 1-3 and 4-6 (p<0.05) when compared to their counterparts, suggesting that these were the cardinal effector population in these patients. Myeloid-derived suppressor cells (MDSCs) have been shown to be a central component of the tumor microenvironment in myeloma, with subsets being capable to mount potent suppressive activity at the tumor site. In the BM CITE-seq myeloid compartment, MDSCs were significantly increased in month 1 (p<0.05) in patients with a shorter PFS. Our PB CyTOF data confirmed this finding as the percentage of MDSCs was significantly higher in weeks 1-3 (p<0.05) in the shorter PFS group when compared to their counterparts. Using a mixed linear regression model on Olink data, we detected 26 cytokines significantly different (p<0.05) between the shorter and longer PFS groups. A pseudobulk analysis of the BM CITE-seq samples for differentially expressed genes encoding the 26 cytokines revealed 22 genes differentially expressed (p<0.05) between patients with a PFS shorter than 18 months and patients with a PFS longer than 18 months in CAR-T, T-cell, NK cell and myeloid cell populations. In the shorter PFS group, VEGFA was significantly higher in CD8 TEMRA CAR-T cells when compared to their counterparts. In the longer PFS group, we observed significantly higher levels of genes involved in T cell activation, such as CD27 and CD28, and pro-inflammatory cytokines such as TNF and IL-15 in the T cell and Myeloid cell compartments. This pattern suggests that higher production of cytotoxic and pro-inflammatory cytokines, combined with enhanced T cell activation, plays an important role in prolonging the response to CAR-T therapy. Conclusions Single cell immune profiling and transcriptomic sequencing identified subpopulations of CD4 and CD8 cells which in concert may influence long term CAR-T outcomes. Our findings demonstrate an early expansion of CART, with very few CART cells surviving after 3 months, suggesting that the efficacy of this therapy is related to early dynamics of these populations. We also provide additional evidence associating immunosuppressive MDSC populations in BM and PB patients with a shorter PFS. Ongoing studies will further analyze the role of the immune microenvironment and clonal T cell dynamics in relation to patient outcomes.

Targeted Inhibition of Myeloperoxidase (MPO): A New Therapeutic Strategy for the Treatment of Multiple Myeloma

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

Drug Repurposing in a Zebrafish AML Model

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy. AML arises from the stepwise accumulation of mutations in hematopoietic stem cells (HSCs). The t(9;11)(p22;q23) translocation is a translocation detected in about 5 to 7% of AML patients, which gives rise to the KMT2A/MLLT3 or MLL/AF9 ( MA9) fusion gene. Zebrafish (ZF) are well-established model organisms for investigating human hematopoiesis. Approximately 70% of human genes have corresponding orthologues in zebrafish, and over 80% of disease-related genes are conserved between the two species. Previously, we established a transgenic MA9 zebrafish AML model (Runx1+23:MA9), in which the AML was serially transplantable. Our investigations revealed that MA9 expression alters hematopoiesis in transgenic embryos before the onset of AML, leading to myeloid expansion. Whole-mount in situ hybridization (WISH) of embryos showed increased expression of granulocyte markers such as lyz 24 and 48 hours after fertilization (hpf). Based on these findings, we developed a Runx1+23:MA9/lyz-EGFP zebrafish leukemia line, in which lyz-expressing myeloid cells are EGFP positive. This model provides a phenotype-based platform to evaluate the therapeutic potential of any drug that can affect the myeloid cell population. We used this ZF model to investigate the therapeutic potential of nine drugs in MA9-driven AML. MA9 transgenic fish (Runx1+23:MA9) were crossed with lyz-EGFP fish to generate a MA9/lyz-EFGP line. In this line, myeloid cells can be visualized with a fluorescent microscope. The expression of lyz in the MA9/lyz-EFGP transgenic embryos was compared to that in the lyz-EGFP line. In our previous study on MA9 transgenic leukemic fish, RNA-seq analysis had revealed higher expression of genes acting in the EGFR, MEK, RAF, KRAS pathways and downregulation of genes in TP53. Based on these results, we selected nine drugs that are known to affect some of these pathways as well as drugs that are used to treat leukemia for screening in our model. Control and MA9/lyz-EFGP ZF 48 hpf embryos were treated with 10 µM of each drug. 24 hrs after drug treatment, the number of lyz expressing cells was analyzed by visualizing the embryos under a fluorescent microscope, and lyz transcript levels were measured by qPCR. Treatment with erlotinib, lapatinib, cytarabine, and gefitinib led to a visible reduction in the number of lyz-expressing cells in the MA9 transgenic embryos but not in the controls (Figure 1). In comparison, treatment with decitabine resulted in a decrease in the number of lyz expressing cells in both the MA9/lyz-EGFP and the control embryos. Treatment with doxorubicin, zoledronic and arsenic trioxide did not have an effect on the number of lyz-expressing population in the transgenic nor in the control embryos. These results were confirmed by qPCR, which showed that expression of lyz was reduced by 33% to 51% in the erlotinib, lapatinib, cytarabine, and gefitinib treated MA9 transgenic embryos (Figure 2). Treatment with these drugs did not significantly change the lyz expression in lyz-EFGP control embryos. Our results provide strong evidence for the successful establishment of a zebrafish model of KMT2A-rearranged AML that can be used for low to medium-throughput drug screening by employing the expression level of an endogenous gene (lyz) as a read-out. This model is a valuable tool to assess the drug's specificity in targeting oncogene-expressing cells and normal hematopoietic cells and, therefore, to identify novel therapeutic agents that can target the cancer cells with fewer side effects on normal hematopoietic cells.

Imaging mass cytometry reveals tissue-specific cellular immune phenotypes in the mouse knee following ACL injury

ObjectiveTo develop an imaging mass cytometry method for identifying complex cell phenotypes, inter-cellular interactions, and population changes in the synovium and infrapatellar fat pad (IFP) of the mouse knee following a non-invasive compression injury. DesignFifteen male C57BL/6 mice were fed a high-fat diet for 8 weeks prior to random assignment to sham, 0.88 ​mm, or 1.7 ​mm knee compression displacement at 24 weeks of age. 2-weeks after loading, limbs were prepared for histologic and imaging mass cytometry analysis, focusing on myeloid immune cell populations in the synovium and IFP. Results1.7 ​mm compression caused anterior cruciate ligament (ACL) rupture, development of post-traumatic osteoarthritis, and a 2- to 3-fold increase in cellularity of synovium and IFP tissues compared to sham or 0.88 ​mm compression. Imaging mass cytometry identified 11 myeloid cell subpopulations in synovium and 7 in IFP, of which approximately half were elevated 2 weeks after ACL injury in association with the vasculature. Notably, two monocyte/macrophage subpopulations and an MHC IIhi population were elevated 2-weeks post-injury in the synovium but not IFP. Vascular and immune cell interactions were particularly diverse in the synovium, incorporating 8 unique combinations of 5 myeloid cell populations, including a monocyte/macrophage population, an MHC IIhi population, and 3 different undefined F4/80+ myeloid populations. ConclusionsDeveloping an imaging mass cytometry method for the mouse enabled us to identify a diverse array of synovial and IFP vascular-associated myeloid cell subpopulations. These subpopulations were differentially elevated in synovial and IFP tissues 2-weeks post injury, providing new details on tissue-specific immune regulation.

Integrative Analysis of Clinical and Bioinformatics Databases to Reveal the Role of Peripheral Innate Immunity in Kawasaki Disease

Kawasaki disease (KD) is an immune-response disorder with unknown etiology. KD is an acute systemic immune vasculitis caused by infectious factors that can be complicated by coronary artery lesions. Innate immune cells are closely associated with KD onset, but we know little regarding the expression of immunity-related genes (IRGs) and the possible immune regulatory mechanisms involved in KD. In this study, we analyzed public single-cell RNA sequencing (scRNA-seq) and microarray data of peripheral blood mononuclear cells from normal controls and KD patients. The results of scRNA-seq revealed myeloid cells, T cells, B cells, NK cells, erythrocytes, platelets, plasma cells, hematopoietic stem cells, and progenitor cells in the peripheral blood of patients with KD. In particular, myeloid cells were expanded and heterogeneous. Further analysis of the myeloid cell population revealed that monocytes in KD exhibited higher expression of the inflammatory genes S100A8, S100A9, and S100A12; furthermore, CD14+CD16+ monocyte clusters were associated with inflammatory responses. Microarray data revealed that activation of the innate immune response contributed to KD development and progression. Differential expression and weighted gene coexpression network analysis identified 48 differentially expressed IRGs associated with response to intravenous immunoglobulin, currently the most effective treatment of KD, although numerous patients are resistant. Protein–protein interaction analysis identified ten hub genes (IL1R1, SOCS3, IL1R2, TLR8, IL1RN, CCR1, IL1B, IL4R, IL10RB, and IFNGR1) among the IRGs. In addition, the expressions of IL1R1, SOCS3, CCR1, IL1B, and IL10RB were validated in Chinese KD patients using the real-time reverse transcriptase-polymerase chain reaction. Finally, we found that the neutrophil/lymphocyte ratio could be used as a biomarker to predict responsiveness to intravenous immunoglobulin in KD. In conclusion, our data highlight the importance of innate immunity in KD pathogenesis and its potential in predicting treatment response.

IMMU-25. ANALYSIS OF IMMUNOSUPPRESSIVE MYELOID CELLS IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT REVEALS THERAPEUTICALLY TARGETABLE IMMUNOMODULATORY SIGNALS ACROSS POPULATIONS

Abstract Immunosuppression in glioblastoma (GBM) remains a critical and poorly understood barrier to the development of effective novel therapies. Increasing evidence points to the critical role of immunosuppressive myeloid cells in the immunosuppressive tumor microenvironment and an intriguing target for ameliorating tumor-mediated immune suppression. Efforts to characterize the myeloid milieu in the tumor microenvironment have revealed a highly heterogenous population of cells with the capacity to exert immunosuppressive effects. In order to maximize the efficacy of potential therapeutic targets, we sought to identify immune checkpoint markers expressed across a range of immunomodulatory myeloid cell populations in the tumor microenvironment of patients undergoing resection for GBM. Freshly collected ultrasonic aspirate obtained during the primary surgery for GBM resection (based on preoperative imaging and intraoperative frozen section, later confirmed on final pathology) was prepared for high dimensional flow cytometry to quantify the immune checkpoint marker expression profile of a number of described immunosuppressive myeloid cell populations, including both monocytic and granulocytic myeloid-derived suppressor cells (mMDSC and gMDSC), and CD16+ non-classical monocytes (NCM). mMDSCs expressed particularly high levels of TIGIT (53.9±19.27%), which was also highly expressed in the NCM population (25.03±5.44%). NCMs also demonstrated high levels of B7H3 expression (39.85±18.05%), which has recently been implicated as an important immune checkpoint protein in GBM. gMDSCs demonstrated a rather different expression profile, with significantly higher levels of VISTA expression than other immunomodulatory markers (p< 0.05). These findings underscore the diversity of immunomodulatory signaling in the tumor-associated myeloid cell compartment, emphasizing the challenges in developing single-target immune checkpoint inhibitor therapy in GBM. Nevertheless, this data points to some common patterns of expression across major immunomodulatory cell populations that remain understudied in GBM and could herald more promising avenues of immunotherapy development.

IMMU-58. CHARACTERIZATION OF MYELOID CELL POPULATIONS IN PEDIATRIC HIGH-GRADE BRAIN TUMORS: THE MIMIC PROGRAM

Abstract BACKGROUND Pediatric high-grade brain tumors (pHGBT) pose treatment challenges with limited options. Myeloid cells, particularly tumor-associated macrophages (TAMs) in the tumor-microenvironment (TME), exhibit potent immunosuppressive features inhibiting effective anti-tumor T-cell responses. Targeting these cells holds promise to improve outcomes, however the underlying mechanisms of myeloid cell dysfunction remain poorly understood. This study aimed to comprehensively characterize myeloid cell populations in pHGBT and investigate their functional phenotype. METHODS Spectral flow cytometry was performed on tumor tissue (biopsies or resections) and tumor fragments obtained by cavitron ultrasonic surgical aspiration (CUSA) in pHGBT patients (High-grade glioma n=2, Ependymoma n=1, Medulloblastoma n=6, Pilocytic Astrocytoma n=2) enrolled in the MIMIC program. This allowed quantification and comprehensive characterization of myeloid cell populations within the TME. RESULTS The pHGBT TME showed a low abundance of immune cells across different entities, with myeloid cells comprising the predominant proportion. CD49d was used to identify invading cells derived from bone marrow (CD49+) and resident microglia (CD49-). The total TAMs/microglia population was detected in both tumor tissue and CUSA tumor fragments in similar proportions, however resident CD49d- microglia were notably increased in CUSA tumor fragments. Additionally, invading TAMs displayed a mixed "M1" and "M2" polarization phenotype and exhibited higher expression of the chemokine receptor CCR2 and HLA-DR, and of the immune-checkpoint receptors SIRP1A/B, CD33, and TIM3, compared to resident microglia. CONCLUSIONS TAMs play a multifaceted role in pHGBT. The high expression of immune-checkpoint receptors by invading TAMs suggests an immunosuppressive phenotype, potentially facilitating tumor growth, infiltration and (immune)therapy escape. Although, differences between disease entities could not yet be analyzed, disparities were observed in the TME between tumor and CUSA samples, likely related to spatial cellular organization. Understanding TAM dynamics and functional differences between invading and resident subsets, as well as their underlying mechanisms, is crucial for advancing therapeutic efficacy in pHGBT.

TMIC-34. SKULL-DERIVED MYELOID CELLS AUGMENT T CELL RESPONSES AND SUPPRESS GLIOBLASTOMA GROWTH

Abstract While recent studies have implicated the skull marrow as a functional immune reservoir in CNS pathology, its contribution to malignancy remains unexplored. To that end, we transplanted fluorescently-labeled calvarial flaps into syngeneic glioblastoma-bearing mice and cytometrically characterized tumor-infiltrative immune cells, observing a robust population of skull-derived myeloid cells (SDMCs; 40.3% of all intratumoral myeloid cells) in a Balb/C mouse tumor model (CMFDA-stained flaps, 72h). These SDMCs were robust, remaining viable for 3 weeks in an alternative longitudinal model (C57BL/6 mice, GFP+ flaps). As MHCII+ leukocytes were disproportionately enriched among SDMCs (87.9% vs 67.3%, n=3, p=0.012), we interrogated the immunogenicity of these cells through targeted skull ablation, achieved via 13 Gy irradiation of body-shielded mice prior to orthotopic glioblastoma implantation. Indeed, skull-ablated mice had reduced infiltration by antigen-presenting cells than controls (cDCs: 1.1% vs 4.5%, p=0.002; macrophages: 14.4% vs 36.6%, n=10/group, p< 0.001) and 6-fold greater necrosis (67.7% vs 13.5% apoptotic, p< 0.001), suggesting an impaired ability to mount a controlled, adaptive immune response. To clarify the role of SDMCs in mediating secondary immune function, we administered the CXCR4 antagonist AMD3100 between the inner and outer tables of the skull, inhibiting marrow homing signals and promoting myeloid precursor egress (0.26% vs 0.02% of intratumoral myeloid cells, n=10/group, p< 0.001). This yielded cDC2 expansion (10.4% vs 3.0%, p=0.003) and consequent CD4+ T effector memory differentiation (92.3% vs 60.0%, p=0.007). These differences were clinically meaningful, as skull irradiation accelerated tumor growth and attenuated survival compared to controls, while AMD3100 treatment accomplished the opposite (median OS: 17d vs 22d, p< 0.001; 26.5d vs 22d, p=0.006). Concurrent αCD4/CD8/Thy1.2 administration abrogated the therapeutic effect of AMD3100 entirely, confirming the dependency of SDMC-mediated tumor suppression on T cells. Together, our work clarifies the antitumoral role of skull marrow and represents the first-ever therapeutic modulation of this space.

TMIC-58. ALTERATIONS IN GLIOBLASTOMA TUMOR MICROENVIRONMENT TRANSCRIPTOME AFTER TREATMENT WITH PEMBROLIZUMAB

Abstract BACKGROUND Immune checkpoint inhibitors (CPI) have failed to show survival benefit in glioblastoma. Understanding the alterations in the tumor microenvironment after treatment with CPI is crucial for development of immunotherapy in glioblastoma. We previously reported our window-of-opportunity trial of pembrolizumab in GBM (NCT02337686). In the current study, we obtained 8 paired glioblastoma tumors pre- and post-treatment with pembrolizumab and conducted RNA sequencing analysis to understand the alterations in the tumor microenvironment transcriptome in response to pembrolizumab. Method: We conducted bulk RNA-sequencing profiling and differential gene expression analysis to compare the expression levels of patient-derived glioblastoma pre- and post-treatment with pembrolizumab, while controlling for paired information. We performed gene enrichment analysis on the differentially expressed genes. We employed the single sample mww-GST method to assess the enrichment of various immune cell types that were previously identified, across the two groups. RESULTS Our analysis revealed 294 down-regulated and 259 up-regulated genes in the post treatment samples. The gene enrichment analysis demonstrated that the up-regulated genes were enriched in pathways related to the immune system. The single sample mwwGST analysis indicated a consistent overall up-regulation of expression in immune cell infiltration. Notably, T cell-related signatures, such as CD4, CD8, and NKT exhibited significantly higher infiltration in the post-treatment group. Interestingly, gene ontology analyses showed upregulation of phagocytosis and antigen presentation gene modules in the post-treatment samples. Additionally, we found neuronal components and genes related to hyperexcitability and synaptic connectivity with an overrepresentation of AMPA and DLG4 pathways in post-treatment tissues. CONCLUSION Our data show that in addition to the anticipated infiltration of myeloid and lymphoid cell populations, checkpoint blockade can also unexpectedly influence glioma-neuron interactions, which may represent additional immunotherapy targets in GBM. Further proteomic validation of infiltration of different cell types and their interactome in pre- and post-CPI treated tissue is warranted.

TMIC-51. THE RELEASE OF INFLAMMATORY CYTOKINES ARE REGULATED BY PYK2 SIGNALING IN GBM CELLS

Abstract Glioblastoma (GBM) is a highly aggressive brain cancer that is typically fatal within a year of diagnosis. Previous research has shown a positive relationship between the activation of a protein called proline-rich non-receptor tyrosine kinase (Pyk2) in GBM cells in addition to the expression of cytokines by tumor-infiltrating myeloid (TIM cells). Leading to a sustained increase in the expression levels of monocyte chemoattractant protein 1 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL10, IL12, and vascular endothelial growth factor (VEGF), as well as an increase in the CD86+/CD206+ TIM population. We hypothesize that Pyk2 signaling in GBM cells is involved in the regulation of the release of inflammatory cytokines, which in turn suppresses the immune response in the tumor microenvironment. We used CRISPR/Cas9 knockout Pyk2 (Pyk2KO) and wild type (Pyk2WT) GL261 mouse glioma cells in this study. Analysis of the cytokines present in the cell culture medium conditioned by Pyk2KO and Pyk2WT cells identified a decrease in the release of CCL2 and CCL5 in the Pyk2KO cells compared to the Pyk2WT cells. RT-PCR analysis of myeloid cell population, isolated from Pyk2WT and Pyk2KO tumors in the GL261-C57Bl/6 GBM model, identified a significant down-regulation of key pro-tumorigenic factors such as CCL2, CCL12, CCL5, tumor necrosis factor (TNF), VEGF and epidermal growth factor (EGF) in the Pyk2KO tumors compared to the Pyk2WT tumors. Additionally, flow cytometry analysis revealed a reduction in myeloid-derived suppressor cells (MDSCs), an increase in lymphoid dendritic cells (DC), and up-regulation in TNF/IFNg expressing and antigen-specific CD8+ T cells in Pyk2KO tumors. Furthermore, a significant decrease in pro-tumorigenic Ly6C+/CD206+ and an up-regulation of inflammatory Ly6C+/CD86+ myeloid cells were found in the Pyk2KO tumors. These findings suggest that Pyk2KO tumors display an immunocompetent microenvironment with enhanced phagocytic and cytotoxic function compared to Pyk2WT tumors. NIH Grant 1SC1GM122691 and PRSTRT2022 supported this study

CTIM-23. SAFETY RUN-IN RESULTS OF A PHASE I/II STUDY TO EVALUATE ATEZOLIZUMAB IN COMBINATION WITH CABOZANTINIB IN PATIENTS WITH RECURRENT GLIOBLASTOMA

Abstract BACKGROUND Immune checkpoint inhibitor therapy has shown limited efficacy in the treatment of glioblastoma (GBM) in both newly diagnosed and recurrent disease which underscores the need for rational combinatorial strategies. Atezolizumab is a humanized monoclonal antibody that targets PD-L1 and has been shown to enhance the magnitude of tumor-specific T cell responses. Combining PD-L1/PD-1 axis inhibition with targeted therapy like cabozantinib which has both anti-tumoral and immunomodulatory properties may result in synergistic effects. Cabozantinib may modulate the myeloid cell population to overcome resistance to anti-PD-1/PD-L1 therapy. METHODS Eligibility criteria were those with recurrent GBM, age >18 yrs with 1st or 2nd recurrence who were naïve to antiangiogenic and receptor tyrosine kinase inhibitor treatment. The primary objective of the phase I run-in was to evaluate the safety of atezolizumab in combination with cabozantinib. RESULTS 1 dose-limiting toxicity (DLT) of a grade 3 ALT elevation was observed in the first 6 DLT evaluable patients treated during the safety run-in phase. Regarding immune related adverse events, 1 of 6 patients developed atezolizumab-related grade 1 hypothyroidism. 1 of 6 patients developed study treatment-related grade 2 diarrhea. 2 patients after completion of 2 cycles of treatment had evidence of a partial radiographic response ( > 50% decrease). 1 patient had a near partial radiographic response (45% decrease). 2 patients had stable disease after completion of 2 cycles of the combinatorial treatment. 1 patient had only completed the first 28-day cycle, DLT period, and had not yet had imaging to reassess disease status. CONCLUSIONS The combination of atezolizumab with cabozantinib was tolerable, and no new safety signals were noted. In this small cohort of recurrent GBM patients, radiographic responses were observed. The phase II component of the trial is recruiting patients (n = 45) to evaluate clinical efficacy. Multi-omic correlative studies and fecal microbiome analyses are planned.

Single-nucleus RNA sequencing of TMEM43 induced arrhythmogenic cardiomyopathy reveals altered cell compositions and transcriptional states in mice

Abstract Introduction and purpose TMEM43 induced Arrhythmogenic cardiomyopathy, also referred to as arrhythmogenic cardiomyopathy type 5 or ACM5, is caused by a point mutation in TMEM43 (p.S358L). The disease is characterised by chamber dilation and a progressive dystrophy of the ventricular myocardium causing life-threatening ventricular arrhythmias and sudden cardiac death. Loss of cardiomyocytes and fibrofatty replacement of the myocardium is clearly observed in our transgenic mouse model of ACM5 overexpressing TMEM43-S358L in postnatal cardiomyocytes. However, the mechanisms underlying cardiomyocyte death and fibroblast activation remain elusive, precluding the development of effective therapeutic options. Here we applied the state-of-the-art analysis on single-nucleus RNA sequencing (snRNA-seq) to characterise the cellular compositions and molecular states in ACM5 mouse hearts. Methods Left ventricular heart tissues of severely affected ACM5 mice and their corresponding transgenic controls overexpressing wild-type TMEM43 were used for snRNA-seq analysis. We identified 10 major cell types, which aggregated into 23 distinct clusters on the basis of transcriptional similarity. Results ACM5 nuclei showed significant depletion of cardiomyocytes and expanded populations of fibroblasts and myeloid cells. Fibroblasts from mutant mice acquired an activated phenotype related to remodelling of the extracellular matrix with a large number of genes like LOX, POSTN, NOX4 or COL1A1 significantly upregulated. In addition, the mutant cardiomyocytes showed altered transcriptional states characterized by cardiac stress and heart failure markers such as NPPB or MYH7. Interestingly, we were able to identify a cluster of mutant cardiomyocytes with a highly apoptotic profile. This cluster is also enriched in genes related to the unfolding protein response (UPR) stress sensors and the DNA damage response (DDR), elucidating a new potential mechanism of myocyte death in ACM5. Furthermore, heterogeneity of the myeloid populations is also observed. The number of tissue resident macrophages is increased in mutant samples and myeloid cells show additional disease-associated transcriptional states that are almost entirely absent in control samples. Conclusion Together these data illuminated shared and distinct cell and molecular architectures of ACM5 in mouse hearts. Further confirmation on diverse gene expression profiles like the UPR and DDR pathways related to cardiomyocyte death will establish a valuable resource to explore novel therapeutic targets and personalized therapies for ACM5.