Distinct Transcriptomic Changes Research Articles

DIPG-35. OVERCOMING THE BLOOD-BRAIN BARRIER CHALLENGE IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are aggressive paediatric brainstem tumours with no known cure. The development of effective treatments is greatly impeded by the failure of most anti-tumour agents to penetrate the blood-brain barrier (BBB). Our research, along with that of others, has revealed that the BBB remains intact in DMG. We employed DMG orthotopic models to explore potential region-specific variations in response to the therapeutic agent temsirolimus as well as confocal microscopy and single-cell RNA sequencing (scRNAseq) to elucidate the impact of DMGs on brain endothelial morphology and pathways governing BBB integrity. We first assessed the effectiveness of the mTOR inhibitor temsirolimus when administered in cortex-injected DMG models compared to brainstem-injected DMG. We found significantly increased survival rates, elevated temsirolimus levels, and reduced tumour cell proliferation in the cortex versus the brainstem. Confocal imaging analysis indicated no structural differences between DMG and Matrigel-injected animals. However, scRNAseq unveiled distinct transcriptomic changes: brainstem-injected DMG exhibited downregulation of genes related to inflammatory and apoptotic pathways, whereas cortex-injected DMG showed downregulation of interferon pathways. To overcome the tightened barrier in the brainstem, we explored three MCL1 inhibitors and SNGR-TNFa (targeting CD13) as potential blood-brain barrier modulators. These inhibitors significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro DMG BBB model, and decreased the protein expression of claudin-5. Moreover, in vivo, we found that administration of a single dose of either SNGR-TNFa or the MCL1 inhibitor S63845 improved penetration of Texas-Red (3KDa) in DMG-engrafted animals, indicating the effective opening of the BBB. In summary, our study revealed that DMG directly affects BBB pathways in endothelial cells, tightening the barrier and reducing treatment efficacy. MCL1 inhibitors and SNGR-TNFa show promise in modulating the BBB and have the potential to enhance therapeutic activity.

Hypoxia drives shared and distinct transcriptomic changes in two invasive glioma stem cell lines

Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.

Abstract B084: Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

Abstract Background: Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 30 years, highlighting the need for therapy innovation. Although immunotherapy has revolutionized cancer treatment, efforts to harness endogenous patient immune responses have yielded limited therapeutic activity in ovarian cancer patients. T cells engineered to express a T cell receptor (TCR) targeting proteins uniquely overexpressed in tumors have the potential to control tumor growth without toxicity. Mesothelin (Msln) is over-expressed in ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill ovarian cancer. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies alone or in combination, ultimately targeting up to three inhibitory receptors simultaneously. Single-cell RNA-sequencing (scRNAseq) was used to profile the impact of combination checkpoint blockade on engineered T cells and the tumor microenvironment (TME). Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated in established tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, elements in the TME limited engineered T cell persistence and cytolytic function. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased antitumor function by intratumoral TCRMsln T cells. scRNAseq of tumor-infiltrating cells revealed distinct transcriptome changes in engineered and endogenous T cells and myeloid-derived cells. Engineered T cells, when combined with triple checkpoint blockade, increased expression of genes associated with effector and memory gene signatures, including proliferation and metabolic function, and reduced expression of genes associated with exhaustion. Moreover, combining adoptive immunotherapy with triple checkpoint blockade significantly prolonged survival in the cohort of treated tumor-bearing mice, relative to mice that received TCRMsln T cells alone or with anti-PD1 or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the TME can dramatically reduce T cell function, suggesting tumor cells may upregulate the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In an advanced ovarian cancer model, triple checkpoint blockade significantly improved engineered T cell function and outcomes in mice in a setting where single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory pathways simultaneously, which can be more safely pursed in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Valentin Voillet, Raphael Gottardo, Philip D. Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B084.

Transcriptional Changes in Patients with CLL Starting Venetoclax Therapy Identifies Inflammatory and Adaptive Stress Responses

Venetoclax is a selective BCL2 inhibitor that was recently approved for CLL treatment. Venetoclax inhibits the pro-survival protein BCL2 and induces deep and durable responses in a significant proportion of CLL patients. Herein, we aimed to study gene expression changes in leukemic cells from CLL patients during the ramp-up period of venetoclax treatment and to assess the transcriptional effect of escalating doses of BCL2 inhibition on tumor biology. We performed RNA sequencing on CD19+ selected tumor cells from 29 patients, ramped-up according to the standard Venetoclax schedule, with weekly increments from 20 mg to 400mg. Four samples were collected per patient, at baseline, and after completing one week on 50mg,100mg, and 400mg. Twelve (41%) patients switched from BTK inhibitor therapy to venetoclax due to disease progression on the BTKi. Those patients discontinued BTKi the day before they started Venetoclax ramp-up. 19 (59%) patients were either treatment-naïve or relapsed with at least months of treatment free-intervals before starting venetoclax. Circulating CD19+ cell counts, typically composed of >95% CLL cells, were enumerated using flow-cytometry. during the ramp-up period, revealed gradual reduction in CLL burden with escalating doses. In the off-BTKi group, median CLL cell counts started to decline on the 100mg dose and were reduced to 98% of baseline at the end of the ramp-up. In the other patients, reductions in median counts were already apparent after one week on 20mg and reached 99% at the end of the ramp-up. Principal component analysis (PCA) of transcriptome data grouped samples by patient, as we have commonly observed in such unsupervised analysis. After batch correcting the patient effect, PCA clearly separated on treatment timepoints from baseline (Figure). Interestingly, patients who recently stopped BTKi had distinct distribution on PCA plots in comparison to the other patients (Others). Indicating distinct transcriptomic changes in patients based on their recent treatment histories. To identify the impact of venetoclax on the CLL transcriptome, we carried out Gene set enrichment analysis using the Hallmark and C3 gene sets from the molecular signature database. Gene sets were identified using a false discovery rate (FDR) of < 0.05, and a normalized enrichment score (NES) of ≥ |1.8|. Gene sets were ranked by NES. In the off-BTKi patient group, 27 gene sets were identified as statistically significantly enriched at 100mg and 400 mg doses. In the “other” patients group, 15 gene sets were found significantly enriched at the same doses. Eight gene sets relating to inflammatory, interferon, and reactive oxygen species responses, cytokine signaling, and fatty acid metabolism. In the off-BTKi we prominently found enriched gene sets regulated by TP53 and related to DNA repair, cell cycle checkpoints, hypoxia and apoptosis. Consistent with an oxidative stress response we identified upregulation of NRF2, NFE2, TCF11, and BACH motif gene sets. The apparent more pronounced transcriptional response to venetoclax in the off-BTKi group prompted us to look more closely at changes in gene. At baseline, expression of BCL2L2 and NOXA was significantly lower in the off-BTKi patients in comparison to the “others” patient group. Surprisingly, on the 50mg dose expression of both the pro-apoptotic and anti-apoptotic BCL2 family members was significantly increased in the off-BTKi group in comparison to the others group; at the 100mg dose only pro-apoptotic genes remained significantly overexpressed in the off-BTKi group. In conclusion, venetoclax induced a distinct and prominent transcriptional response in CLL patients indicating broad impact on tumor biology, even at doses below 400mg. Intriguingly, CLL cells from patients who had progressed on BTKi therapy and switched to venetoclax showed a more extensive stress response that included activation of DNA damage and anti-oxidant pathways. It will be interesting to correlate the identified transcriptional changes with the depth and durability of the venetoclax response.

Functional Pdgfra fibroblast heterogeneity in normal and fibrotic mouse lung.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α-positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast-supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

High-Throughput Screens Identify NEDD8 Inhibition As a Strategy to Augment Natural Killer Cell Cytotoxicity Against Blood Cancers

Natural killer (NK) cell-based therapies are amongst the emerging immunotherapeutic approaches that aim to target malignant cells refractory to standard therapies. Currently used strategies to augment the NK cell anti-cancer function of NK cells include cytokine treatments and the use of feeder expansion protocols for culturing NK cells. Recent studies have indicated that NK cell cytotoxicity and function may also be enhanced by certain anti-cancer drugs. However, the effects of the hundreds of available oncology drugs on NK cell function have not been systematically evaluated. Here, we perform large-scale high-throughput testing of NK cell-drug combinations using co-culture models with diverse blood cancer cells, identifying the NEDD8-activating enzyme inhibitor pevonedistat as a promising candidate for augmenting NK cell anti-cancer responses. To identify oncology drugs synergizing with NK cell-based immunotherapy, we established a high-throughput drug sensitivity and resistance testing (DSRT) platform to investigate effects of 527 approved drugs and investigational compounds on NK cell cytotoxicity against blood cancer cell lines. Overall, 10 cell lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM), were screened in combination with primary expanded NK cells. Target cells were treated for 24 hours with drugs at five different concentrations both with and without NK cells. In NK cell co-culture, an effector-target (ET) cell ratio at which half of the target cells were killed was selected for experiments. Most effective compounds were then selected for further testing using multiplexed single-cell RNA sequencing (scRNA-seq) and analysis of secreted cytokines (Figure 1). Overall, the results from our DSRT platform demonstrated varying effects of different drug classes on NK cell cytotoxicity against blood cancer cell lines. In general, 5-10% of drugs had NK cell cytotoxicity-enhancing effects, 30-40% inhibited NK function and 35-50% of drugs had no effect. Similarities in results between cell lines highlighted the dependence of NK cell-drug synergistic effects on disease pathobiology. Across multiple disease models, pevonedistat (MLN4924), a selective NEDD8 activating enzyme inhibitor that has been studied as treatment for relapsed/refractory AML, showed strong synergistic effects with NK cells. For example, in OCI-AML-3 (AML), pevonedistat alone showed no effect on target cell viability, but in combination with NK cells at a 1:4 ET ratio, it resulted in 98% target cell killing at 100 nM. Similar effects were also seen in MM1.S (MM), although a higher concentration (10000 nM) of pevonedistat was required (Figure 2). Results from scRNA-seq after combination treatment with NK cells and pevonedistat revealed distinct transcriptomic changes in both NK cells and target cells when compared to controls. In both NK cells and target cells, pevonedistat caused upregulation of TXN, TALDO1, TXNRD1, NQO1, SRXN1 and SLC7A11,collectively indicating effects on the NRF2 signaling pathway. The upregulation of these genes correlated with the tested concentration of pevonedistat, with notably higher expression when treated with 1000 nM pevonedistat compared to 100 nM. Changes were also observed in the secretion of cytokines detected from the cell culture media, where concentrations of IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α all increased up to 300% compared to controls. In HEL (AML) and LAMA-84 (CML), where pevonedistat had only minor effects on NK cell cytotoxicity, the expression TNF and TNFRSF1B at baseline and after pevonedistat treatment were notably lower, when compared to cell lines where the combination therapy led to stronger responses. This suggests a possible mechanism for pevonedistat-NK cell synergy, given that previous reports have demonstrated a synergy between pevonedistat and TNF-α to activate apoptosis. In conclusion, we discovered a cell context-specific synergy between pevonedistat and NK cells, which increased NK cell cytotoxicity against malignant cells in vitro. In addition, our results demonstrate the potential of using a high-throughput platform for studying NK cell-drug interactions against malignant cells, while aiming to improve treatment for hematological malignancies through combination immunotherapies.

Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

The APOEε4 allele exacerbates the transcriptomic responses to Aβ plaques and neurofibrillary tangles in Alzheimer’s disease

AbstractBackgroundMicroglia, astrocytes, and neurons undergo profound functional alterations in response to amyloid‐β (Aβ) plaques and neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD). Recent studies using human induced pluripotent stem cell‐derived glial cells and neurons, APOE knock‐in mice, and human brain bulk RNA‐seq have implicated the APOE genotype in these changes, but lack spatial information with respect to Aβ plaques and NFTs. Here we tested the hypotheses that (1) transcriptomic differences between AD and control subjects are maximum within and near Aβ plaques and NFT‐bearing neurons; (2) Aβ plaques and NFTs are associated with distinct transcriptomic changes; and (3) the APOE genotype differentially impacts the transcriptomic changes associated with Aβ plaques and NFTs in the AD brain.MethodLaser capture microdissection (LCM) in cryostat sections was performed from superior temporal gyrus (BA22) cortex of human AD (n = 10, including n = 5 APOEε4/ε4, n = 4 APOEε3/ε3, and n = 1 APOEε3/ε4) and age‐ and sex‐matched controls (n = 8). Thioflavin‐S‐positive Aβ plaques, the 50 µm halo around them, NFTs with the 50 µm halo around them, and areas far (>50 µm) from plaques and NFTs were laser‐capture microdissected and subjected to RNA‐sequencing to identify differentially expressed genes (DEGs).ResultAβ plaques had a greater impact on the transcriptome than NFTs (i.e., higher number of DEGs, both upregulated [logFC>0, unadj. P‐value<0.05: 2,623 vs. 1,230 genes; adj. P‐value<0.05: 1,152 vs. 0 genes] and downregulated [logFC>0, unadj. P‐value<0.05: 2,563 vs. 1,056 genes; adj. P‐value<0.05: 827 vs. 0 genes]) relative to control cortex. Aβ plaques were characterized by upregulated microglial and downregulated neuronal genes, whereas NFTs had primarily downregulated neuronal genes. A gradient of Aβ plaques > plaque halo > NFTs > far areas was evident, with upregulation of neuroinflammation and downregulation of synaptic neurotransmission and energy metabolism‐related gene sets. Comparing APOEε4 and APOEε3 age‐ and sex‐matched homozygotes revealed greater changes in APOEε4 homozygotes across locations.ConclusionAβ plaques and, to a lesser extent, NFTs, concentrate the bulk of transcriptomic changes in the AD cortex. The APOEε4 allele is associated with greater microglial and neuronal transcriptomic responses to Aβ plaques and NFTs, compared to APOEε3 carriers. These findings will inform future spatial transcriptomics studies.

Human enteroids as a tool to study conventional and ultra-high dose rate radiation.

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

Full-length IL-33 augments pulmonary fibrosis in an ST2- and Th2-independent, non-transcriptomic fashion

Mature IL-33 (MIL33) acting through its receptor, ST2, is known to regulate fibrosis. The precursor, full-length IL-33 (FLIL33), may function differently from MIL33 and independently of ST2. Here we report that genetic deletion of either IL-33 or ST2 attenuates pulmonary fibrosis in the bleomycin model, as does Cre-induced IL-33 deficiency in response to either acute or chronic bleomycin challenge. However, adenovirus-mediated gene delivery of FLIL33, but not MIL33, to the lungs of either wild-type or ST2-deficient mice potentiates the profibrotic effect of bleomycin without inducing a Th2 phenotype. In cultured mouse lung cells, FLIL33 overexpression induces moderate and distinct transcriptomic changes compared with a robust response induced by MIL33, whereas ST2 deletion abrogates the effects of both IL-33 forms. Thus, FLIL33 may contribute to fibrosis in an ST2-independent, Th2-independent, non-transcriptomic fashion, suggesting that pharmacological targeting of both FLIL33 and MIL33 may prove efficacious in patients with pulmonary fibrosis.

Treatment response in acute myeloid leukaemia-Clues in the biopsy core.

In their paper the authors describe distinct transcriptomic changes associated to treatment response in core bone marrow biopsies from patients with acute myeloid leukaemia. This finding raises the possibility that stratifying patients for treatment according to their transcriptomic profiles could improve patients' response and prognosis. Commentary on: Treaba et al. Transcriptomics of AML core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles. Br J Haematol 2023;200:740-754.

Obesity alters the mouse endometrial transcriptome in a cell context-dependent manner

Obesity impacts fertility and is positively correlated with endometrial hyperplasia and endometrial cancer occurrence. Endometrial epithelia often harbor disease driver-mutations, while endometrial stroma are highly regulative of neighboring epithelia. Here, we sought to determine distinct transcriptome changes occurring in individual cell types in the obese mouse uterus. Outbred CD-1 mice were fed high-fat or control diets for 18 weeks, estrous cycle staged, and endometrial epithelia, macrophages, and stroma isolated for transcriptomic analysis. High-fat diet mice displayed increased body mass and developed glucose intolerance, hyperinsulinemia, and fatty liver. Obese mouse epithelia displayed differential gene expression for genes related to innate immunity and leukocyte chemotaxis. The obese mouse stroma differentially expressed factors related to circadian rhythm, and expression of these genes correlated with glucose tolerance or body mass. We observed correlations between F4/80 + macrophage numbers, Cleaved Caspase 3 (CC3) apoptosis marker staining and glucose intolerance among obese mice, including a subgroup of obese mice with high CC3 + luminal epithelia. This subgroup displayed differential gene expression among all cell types, with pathways related to immune escape in epithelia and macrophages, while the stroma dysregulated pathways related to regulation of epithelia. These results suggest an important role for differential response of both the epithelia and stroma in their response to obesity, while macrophages are dysregulated in the context of apoptotic epithelia. The obesity-related gene expression programs in cells within the uterine microenvironment may influence the ability of the endometrium to function during pregnancy and influence disease pathogenesis.

RNA-sequencing reveals significant differences in the inflammatory response of iPSC-derived endothelial cells from ACS patients and healthy controls

Abstract Background Coronary artery disease (CAD) and cardiovascular diseases (CVD) in general still represent the leading cause of death worldwide. As a multifactorial disease entity, genetic predisposition and lifestyle strongly impact disease development and progression. Here, induced pluripotent stem cells (iPSC) are an ideal tool to differentiate (epi-)genetic from lifestyle disease risk modulation and identify the underlying molecular mechanisms. Aim This study aims to determine differences in iPSC-derived endothelial cell (iPSC-EC) characteristics and functionality between healthy donors and high-risk CAD patients. Methods IPSC from participants with similar lifestyle but different disease status (healthy >65 y/o, n=5, acute coronary syndrome (ACS) <65y/o, n=6) were differentiated into iPSC-EC. Cells were exposed to inflammatory stimulation with tumour necrosis factor alpha (TNF-a) followed by bulk RNA-seq. Monocyte (THP-1) recruitment was assessed under protective and atherogenic flow conditions. Mitotic age was calculated based on DNA-methylation via Hannum estimate (1) for each donor at the iPSC and iPSC-EC (passage 4) stage. Results ACS-iPSC-EC had significantly higher upregulation of E-selectin upon TNF-a stimulation on mRNA level (ACS: 532.1-fold±48.7 vs healthy: 322.3-fold±55.7 with p=0.02). Similarly, ICAM1 protein upregulation was higher in ACS-iPSC-EC compared to healthy iPSC-EC as determined by flow cytometry (ACS: 1.4-fold±0.01 vs healthy: 1.1-fold±0.0007; p<0.001). ACS-iPSC-EC recruited significantly more THP-1 per view field under protective (high shear stress, laminar) flow conditions (ACS: 78.4±20.7 vs healthy: 33.25±12.3 with p=0.04) compared to healthy iPSC-EC. While both groups recruited significantly more THP-1 under atherogenic than protective conditions (ACS: 302±38.9 with p=0.0055 vs healthy: 340±70.4 with p=0.0005), there was no difference between the donor groups (p=0.43). Analysis of bulk RNA-seq data showed significant differences in TNF-a-induced transcriptome changes. Gene set enrichment analysis identified differences in signalling pathways related to leukocyte and lymphocyte-mediated immunity, adaptive immune response, cytokine activity, and many more as shown in Figure 1 (p.adj ≤1e-10). IPSC of both groups had a negative calculated mitotic age (in years) which significantly increased after endothelial differentiation and four population doublings (ACS-iPSC: −14.1±0.7 vs ACS-EC: −0.1±1.1, p<0.0001 and healthy-iPSC: −13.7±0.8 vs healthy-EC: 2.5±1.5, p=0.0001). Conclusion The higher rate of basal THP-1 recruitment and upregulation of adhesion molecules on mRNA and protein level and the distinct transcriptome changes suggest intrinsic differences between patient and healthy iPSC-EC both in the basal state and in inflammatory response. Furthermore, this demonstrates iPSC as a viable in vitro model also for polygenetic diseases such as CAD. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Cardiac Society, Research Scholarship

Abstract P2015: Metabolic-epigenetic Coupling In High Fat Diet And Heart Failure With Reduced Ejection Fraction

Given rising obesity rates, understanding how metabolic changes exacerbate other cardiovascular disease (CVD) risk factors will yield novel therapeutic targets for associated comorbidities, such as heart failure. We hypothesize that obesity-related metabolic syndrome induces transcriptional programs that engender cardiac pathology via mechanisms involving chromatin regulators, thereby perpetuating the pathology by epigenetic memory. C57BL/6J adult male mice were put on a high fat diet (HFD) for 24 weeks. After HFD, hearts were in hypertrophy, as defined by an increase in heart weight to tibia length ratio (24.67 ± 5.14 mg/mm in HFD versus 17.06 ± 0.98 mg/mm in regular chow controls, p=0.026). Examination of blood plasma revealed significant increase in LDL-C (74.37 ± 22.45 mg/dL in HFD versus 21.91 ± 5.98 mg/dL in controls, p=0.0005) and insulin (452.81 ± 224.77 uIU/mL in HFD versus 89.66 ± 45.60 uIU/mL in controls, p=0.0003), indicative of metabolic disorder. RNA-seq of isolated cardiomyocytes revealed distinct transcriptomic changes at 1 week, 6 weeks, and 24 weeks of HFD. At 1 week, HFD stimulus induced promotion of lipid metabolism ( Pdk4, Per2 ) and suppression of genes related to development and proliferation ( Sox4, Epha4 ). By 6 weeks, the cardiomyocytes underwent metabolic reprogramming (downregulation of Lpl and Irs2 ) and structural changes ( Acta2, Col8a1). At 24 weeks, we observed suppression of ketolytic machinery (downregulation of Abat, Bdh1, and Oxct1 ) in response to obesity-driven hyperglycemia, suggesting that cardiomyocytes transitioned to a pre-diabetic state. To test whether this metabolic disease state compounds with other CVD risk factors, we devised a two-hit model, where mice were fed HFD for 6 weeks and then underwent transverse aortic constriction as a model of pressure overload. Cardiomyocytes from this two-hit model exhibited an enhanced hypertrophic response ( Myh7, Nppa, Col1a1 mRNA levels) compared to pressure overload with regular chow, sham surgery with HFD, or sham surgery with chow. Thus, phenotypic and transcriptional data suggest that HFD reprograms cardiomyocytes to a pre-diabetic state characterized by insulin resistance which exacerbates the effects of other CVD risk factors like hypertrophy.

Hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin induces distinct transcriptomic changes in ovarian tumor and normal tissues

ObjectiveTo determine the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin on the transcriptomic profiles of normal and ovarian cancer (OC) tissues. MethodsNormal and tumor samples from four OCs were prospectively collected pre- and immediately post-HIPEC treatment and subjected to RNA-sequencing. Differential gene expression, gene ontology enrichment and pathway analyses were performed. Heat shock protein and immune-response protein expression was assessed using protein arrays and western blotting. ResultsRNA-sequencing revealed 4231 and 322 genes significantly differentially expressed between pre- and post-treatment normal and OC tissues, respectively (both adjusted p-value <0.05). Gene enrichment analyses demonstrated that the most significantly upregulated genes in normal tissues played a role in immune as well as heat shock response (both adjusted p < 0.001). In contrast, HIPEC induced an increased expression of primarily heat shock response and protein folding-related genes in tumor tissues (both adjusted p < 0.001). HIPEC-induced heat shock protein (HSP) expression changes, including in HSP90, HSP40, HSP60, and HSP70, were also observed at the protein level in both normal and tumor tissues. ConclusionsHIPEC with carboplatin resulted in an upregulation of heat shock-related genes in both normal and tumor tissue, with an additional immune response gene induction in normal and protein folding in tumor tissue. The findings of our exploratory study provide evidence to suggest that HIPEC administration may suffice to induce gene expression changes in residual tumor cells and raises a biological basis for the consideration of combinatorial treatments with HSP inhibitors.

Maternal cold exposure induces distinct transcriptome changes in the placenta and fetal brown adipose tissue in mice

BackgroundBrown adipose tissue (BAT) is specialized to dissipate energy in the form of heat. BAT-mediated heat production in rodents and humans is critical for effective temperature adaptation of newborns to the extrauterine environment immediately after birth. However, very little is known about whether and how fetal BAT development is modulated in-utero in response to changes in maternal thermal environment during pregnancy. Using BL6 mice, we evaluated the impact of different maternal environmental temperatures (28 °C and 18 °C) on the transcriptome of the placenta and fetal BAT to test if maternal cold exposure influences fetal BAT development via placental remodeling.ResultsMaternal weight gain during pregnancy, the average number of fetuses per pregnancy, and placental weight did not differ between the groups at 28 °C and 18 °C. However, the average fetal weight at E18.5 was 6% lower in the 18 °C-group compared to the 28 °C-group. In fetal BATs, cold exposure during pregnancy induced increased expression of genes involved in de novo lipogenesis and lipid metabolism while decreasing the expression of genes associated with muscle cell differentiation, thus suggesting that maternal cold exposure may promote fetal brown adipogenesis by suppressing the myogenic lineage in bidirectional progenitors. In placental tissues, maternal cold exposure was associated with upregulation of genes involved in complement activation and downregulation of genes related to muscle contraction and actin-myosin filament sliding. These changes may coordinate placental adaptation to maternal cold exposure, potentially by protecting against cold stress-induced inflammatory damage and modulating the vascular and extravascular contractile system in the placenta.ConclusionsThese findings provide evidence that environmental cold temperature sensed by the mother can modulate the transcriptome of placental and fetal BAT tissues. The ramifications of the observed gene expression changes warrant future investigation.

A miR-375/YAP axis regulates neuroendocrine differentiation and tumorigenesis in lung carcinoid cells

Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. We also observed a significant decrease in neuroendocrine differentiation and substantial reductions in cell proliferation, transformation, and tumor growth in cell culture and xenograft mouse disease models. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Transient YAP knockdown in miR-375-depleted cells reversed the effects of miR-375 on neuroendocrine differentiation and cell proliferation. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated target genes indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

Iron deficiency and the loss of chloroplast iron-sulfur cluster assembly trigger distinct transcriptome changes in Arabidopsis rosettes.

Regulation of mRNA abundance revealed a genetic program for plant leaf acclimation to iron (Fe) limitation. The transcript for SUFB, a key component of the plastid iron-sulfur (Fe-S) assembly pathway is down-regulated early after Fe deficiency, and prior to down-regulation of mRNAs encoding abundant chloroplast Fe containing proteins, which should economize the use of Fe. What controls this system is unclear. We utilized RNA-seq. aimed to identify differentially expressed transcripts that are co-regulated with SUFB after Fe deficiency in leaves. To distinguish if lack of Fe or lack of Fe-S cofactors and associated loss of enzymatic and photosynthetic activity trigger transcriptome reprogramming, WT plants on low Fe were compared with an inducible sufb-RNAi knockdown. Fe deficiency targeted a limited set of genes and predominantly affected transcripts for chloroplast localized proteins. A set of glutaredoxin transcripts was concertedly down-regulated early after Fe deficiency, however when these same genes were down-regulated by RNAi the effect on known chloroplast Fe deficiency marker proteins was minimal. In promoters of differentially expressed genes, binding motifs for AP2/ERF transcription factors were most abundant and three AP2/ERF transcription factors were also differentially expressed early after low Fe treatment. Surprisingly, Fe deficiency in a WT on low Fe and a sufb-RNAi knockdown presented very little overlap in differentially expressed genes. sufb-RNAi produced expression patterns expected for Fe excess and up-regulation of a transcript for another Fe-S assembly component not affected by low Fe. These findings indicate that Fe scarcity, not Fe utilization, triggers reprogramming of the transcriptome in leaves.

Promoter anchored interaction landscape of THP-1 macrophages captures early immune response processes

Macrophages are highly plastic immune cells with temporally distinct transcriptome changes upon lipopolysaccride (LPS) activation. However, to what extent transcriptome reprogramming is mediated via spatial chromatin looping is not well studied. We generated high resolution chromatin interaction maps for LPS-stimulated THP-1 macrophages (0 and 2 h) using capture Hi-C. Success of LPS stimulation was validated with transcriptome sequencing. Circa 2900 genes changed their interaction profile upon LPS stimulation and those gaining interactions were enriched for LPS response relevant processes, suggesting a substantial role for distal regulation. Immune and cardiovascular risk variants were enriched within the interacting regions, thereby providing insights into macrophage biology.

Douglas-Fir (Pseudotsuga menziesii (Mirb.) Franco) Transcriptome Profile Changes Induced by Diesel Emissions Generated with CeO2 Nanoparticle Fuel Borne Catalyst

It is important to understand molecular effects on plants exposed to compounds released from use of products containing engineered nanomaterials. Here, we present mRNA sequencing data on transcriptome impacts to Douglas-fir following 2 weeks of sublethal exposure to 30:1 diluted airborne emissions released from combustion of diesel fuel containing engineered CeO2 nanoparticle catalysts (DECe). Our hypothesis was that chamber exposure to DECe would induce distinct transcriptome changes in seedling needles compared with responses to conventional diesel exhaust (DE) or filtered DECe Gas Phase. Significantly increased uptake/binding of Ce in needles of DECe treated seedlings was 2.7X above background levels and was associated with altered gene expression patterns. All 225 Blast2GO gene ontologies (GOs) enriched by up-regulated DECe transcripts were nested within GOs for DE, however, 29 of 31 enriched GOs for down-regulated DECe transcripts were unique. MapMan analysis also identified three pathways enriched with DECe down-regulated transcripts. There was prominent representation of genes with attenuated expression in transferase, transporter, RNA regulation and protein degradation GOs and pathways. CeO2 nanoparticle additive decreased and shifted molecular impact of diesel emissions. Wide-spread use of such products and chronic environmental exposure to DECe may adversely affect plant physiology and development.