Conditional Mouse Model Research Articles

O19 Improvement of a junctional epidermolysis bullosa mouse model

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is the most severe of the epidermolysis bullosa (EB) disorders and is caused by mutations in genes encoding the skin basement membrane proteins laminin 332, type XVII collagen or integrin α6β4. Affected individuals experience blistering from birth leading to scarring, granulation tissue and susceptibility to infection. In JEB, generalized severe, there is 50% mortality in the first 2 years of life, often owing to failure to thrive or overwhelming infection. A number of animal models for JEB exist; however, because of severe blistering on the entire surface of the skin, these mice die soon after birth, reducing their experimental utility. In 2017, the first adult inducible knockout mouse model of JEB was published. In this mouse model, the disruption of the Lama3 gene is specifically induced in epidermal basal keratinocytes after birth. These mice remain viable, which allows us to study the longer-term consequences of the loss of laminin α3. However, the mice did not develop the severe phenotype observed in patients with JEB. Methods We have optimized this previously published methodology of a conditional knockout JEB mouse model (Lama3flx/flxK14CreERT) to now include more regular tamoxifen injections, which Results in a more controlled and severe phenotype of JEB. Results Widespread skin blistering develops after 12 weeks of tamoxifen treatment with an impaired skin barrier (transepidermal water loss) and a loss of epidermal lipids. Immunofluorescent staining was conducted to assess skin differentiation (K14, transglutaminase 1, loricrin), proliferation (Ki67), basement membrane proteins (laminin α3, laminin β3, laminin γ2, collagen VII) and immune markers (F4/80, Ly6-G/C, CD3, CD79). Conclusions These mice recapitulate all key features of human JEB with loss of laminin α3 at the basement membrane, epidermal thickening and visible blisters. This allows us to delve deeper into the functional and downstream pathways affected by the loss of laminin α3 and discover why the prognosis of patients with JEB is so severe.

Defining the In Vivo Role of mTORC1 in Thyrocytes by Studying the TSC2 Conditional Knockout Mouse Model.

Background: The thyroid gland is susceptible to abnormal epithelial cell growth, often resulting in thyroid dysfunction. The serine-threonine protein kinase mechanistic target of rapamycin (mTOR) regulates cellular metabolism, proliferation, and growth through two different protein complexes, mTORC1 and mTORC2. The PI3K-Akt-mTORC1 pathway's overactivity is well associated with heightened aggressiveness in thyroid cancer, but recent studies indicate the involvement of mTORC2 as well. Methods: To elucidate mTORC1's role in thyrocytes, we developed a novel mouse model with mTORC1 gain of function in thyrocytes by deleting tuberous sclerosis complex 2 (TSC2), an intracellular inhibitor of mTORC1. Results: The resulting TPO-TSC2KO mice exhibited a 70-80% reduction in TSC2 levels, leading to a sixfold increase in mTORC1 activity. Thyroid glands of both male and female TPO-TSC2KO mice displayed rapid enlargement and continued growth throughout life, with larger follicles and increased colloid and epithelium areas. We observed elevated thyrocyte proliferation as indicated by Ki67 staining and elevated cyclin D3 expression in the TPO-TSC2KO mice. mTORC1 activation resulted in a progressive downregulation of key genes involved in thyroid hormone biosynthesis, including thyroglobulin (Tg), thyroid peroxidase (Tpo), and sodium-iodide symporter (Nis), while Tff1, Pax8, and Mct8 mRNA levels remained unaffected. NIS protein expression was also diminished in TPO-TSC2KO mice. Treatment with the mTORC1 inhibitor rapamycin prevented thyroid mass expansion and restored the gene expression alterations in TPO-TSC2KO mice. Although total thyroxine (T4), total triiodothyronine (T3), and TSH plasma levels were normal at 2 months of age, a slight decrease in T4 and an increase in TSH levels were observed at 6 and 12 months of age while T3 remained similar in TPO-TSC2KO compared with littermate control mice. Conclusions: Our thyrocyte-specific mouse model reveals that mTORC1 activation inhibits thyroid hormone (TH) biosynthesis, suppresses thyrocyte gene expression, and promotes growth and proliferation.

Single-cell sequencing reveals the impact of endothelial cell PIEZO1 expression on thoracic aortic aneurysm

IntroductionThoracic aortic aneurysm (TAA) is a severe vascular disease that threatens human life, characterized by focal dilatation of the entire aortic wall, with a diameter 1.5 times larger than normal. PIEZO1, a mechanosensitive cationic channel, monitors mechanical stimulations in the environment, transduces mechanical signals into electrical signals, and converts them into biological signals to activate intracellular signaling pathways. However, the role of PIEZO1 in TAA is still unclear. MethodsWe analyzed a single-cell database to investigate the expression level of PIEZO1 in TAA. We constructed a conditional knockout mouse model of Piezo1 and used the PIEZO1 agonist Yoda1 to intervene in the TAA model mice established by co-administration of BAPN and ANG-II. Finally, we explored the effect of Yoda1 on TAA in vitro. Results and discussionWe observed decreased PIEZO1 expression in TAA at both RNA and protein levels. Single-cell sequencing identified a specific reduction in Piezo1 expression in endothelial cells. Administration of PIEZO1 agonist Yoda1 prevented the formation of TAA. In PIEZO1 endothelial cell conditional knockout mice, Yoda1 inhibited TAA formation by interfering with PIEZO1. In vivo and in vitro experiments demonstrated that the effect of Yoda1 on endothelial cells involved macrophage infiltration, extracellular matrix degradation, and neovascularization. This study highlights the role of PIEZO1 in TAA and its potential as a therapeutic target, providing opportunities for clinical translation.

TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Concurrent, dual perturbations of the 5-HT and NA systems reveal dynamic interplay in regulating the neonate autoresuscitation reflex

Sudden Infant Death Syndrome (SIDS) is a leading cause of infant mortality worldwide. Failure of the neonate auto-resuscitation reflex is hypothesized to be a common endpoint in many SIDS cases. Although SIDS is idiopathic, postmortem studies indicate abnormalities in the serotonergic (5-HT) and noradrenergic (NA) systems of the respiratory network. To better understand the contributions and interactions of these two systems in autoresuscitation, we combined a novel series of Cre- and FLPo- recombinase driver mouse lines and conditional inhibitory and excitatory DREADD lines for singular excitation or inhibition of either the whole 5-HT or whole NA systems. These models were then uniquely combined to further enable 1) concurrent excitation or inhibition of both systems in the same neonate or 2) inhibition of one system while exciting the other system in the same neonate. In total, we developed 8 distinct sets of conditional genetic mouse models for singular and combined 5-HT and NA system perturbation paradigms. The 8 experimental groups and 2 control groups were repeatedly assayed for the autoresuscitation reflex using a robotic neonate cardiorespiratory assessment platform, Looper. Neonates are repeatedly tested until they succumb. Resulting data were analyzed with our recently published waveform analysis software, Breathe Easy, to calculate operant outcomes. In 320 mice total, we found 4 groups with decreased autoresuscitation success: NA activation, NA activation + 5-HT activation, NA activation + 5-HT inhibition, and 5-HT activation. There was no change in survival in any of the other groups including 5-HT inhibition. Preliminary results suggest a potential cardiovascular mechanism of autoresuscitation failure independent of respiratory outcomes. At baseline, all 4 groups with decreased survival showed increased heart rate (HR) whereas the other groups either had the same HR or a decreased HR. During early recovery, defined as beginning at the 10th breath following the primary gasp until recovery, the 4 groups with decreased survival show higher HR than the other groups following the 1st bout of autoresuscitation. As recovery progressed in these groups, HR continued to increase as well until the next autoresuscitation test. Ventilatory frequency, tidal volume, and ventilation all increased in the 3 groups where NA was activated either in isolation or in combination, but there were decreases in all other groups, including 5-HT activation. As the underlying genetic and environmental mechanisms driving SIDS are likely to have pleiotropic effects across multiple neural systems, our first-of-its-kind dual 5-HT and NA system modulations set the stage to more comprehensively model key aspects of SIDS neuropathology that reveal a previously uncharacterized dynamic functional interplay between the NA and 5-HT systems in protective respiratory reflexes. NIH: 1F32HL160073-01A1, R01HL130249 44617-S4. BCM McNair Scholar Program, March of Dimes Basil O'Connor Research Award, Parker B. Francis Fellowship, CJ Foundation for SIDS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2077: The Epigenetic Regulator PRDM16 Controls Smooth Muscle Phenotypic Modulation And Atherosclerosis Risk

Despite decades of progress, coronary artery disease (CAD) remains the top cause of death worldwide. Additionally, trends in outcomes have worsened recently, highlighting the critical need for additional treatments. Human genetics has identified over 300 loci associated with CAD, but understanding the molecular mechanisms leading to disease remains a huge barrier to developing new therapies. These CAD-associated loci are enriched in smooth muscle cells (SMC) of the vascular wall, but no current therapies target these cells. Since insulin resistance is an important risk factor for CAD, understanding the molecular functions of genes associated with both insulin resistance and CAD will prioritize therapeutic candidates, especially if expressed in SMCs. One promising candidate is the gene PRDM16 (PR domain containing 16), which is highly expressed in vascular tissue. PRDM16 regulates cell fate decisions and insulin resistance in adipose tissue, but its role in atherosclerosis and SMC function is unknown. Recent advances in lineage tracing and conditional SMC knockout mouse models in conjunction with single cell technologies have demonstrated the cellular trajectories of SMC into several cellular states, including fibromyocytes (FMC) and chondromyocytes (CMC). Here, we employ this innovative approach to understand the role of Prdm16 on SMC phenotypic modulation in vivo and its role in the development of FMC and CMC. We also demonstrate the impact of Prdm16 in atherosclerosis and other important lesion characteristics relating to disease risk. We validate these effects in vitro and employ epigenetic analysis to identify the gene regulatory mechanisms whereby PRDM16 mediates its effects on SMC phenotypic modulation. Collectively these data demonstrate that PRDM16 is a causal factor that promotes risk of CAD.

Roles of mitofusin-2 in mouse neutrophil adhesion and development

Neutrophils play a critical role in inflammation and innate immunity. A crucial step in neutrophil recruitment is their adhesion to vascular endothelium. Previous research demonstrated the necessity of mitofusin-2 (MFN2) for integrin activation, adhesion, and the development of neutrophil-like HL60 cells. However, it remains unknown whether this holds true for primary neutrophils. Additionally, neutrophil adhesion is closely associated with their development, making it challenging to independently investigate the roles of MFN2 in regulating mouse neutrophil adhesion and development. In this study, we successfully addressed these challenges by utilizing two distinct conditional knockout mouse models. Our findings revealed that blood neutrophils from MFN2flox/flox LysM-cre mice exhibited approximately a 50% reduction in MFN2 expression. Remarkably, these MFN2 knockdown (KD) mice did not display any defects in neutrophil development. In contrast, blood neutrophils from MFN2flox/flox Vav-iCre mice showed a substantial (>90%) reduction in MFN2 expression. These MFN2 knockout mice exhibited a notable defect in the maturation of band cells into polymorphonuclear neutrophils. We further investigated the neutrophil adhesion using MFN2 knockdown mice and identified the neutrophil adhesion defect using microfluidics, the integrin activation defect using soluble ligand binding, and the Ca2+ flux defect using time-lapse flow cytometry. These findings provide valuable insights into the role of MFN2 in leukocyte adhesion and development and offer potential new perspectives for understanding and treating MFN2-deficiency-related diseases. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454), National Institute of General Medical Sciences (R35GM119787), National Institute on Aging (P30AG024832), a Career re-entry fellowship from the American Association of Immunologists (G602182), and a startup fund from UConn Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of age-associated alpha-synuclein aggregation in a conditional transgenic mouse model of Parkinson's disease: Implications for Lewy body formation.

Parkinson's disease (PD) is a common neurodegenerative disorder that is affecting an increasing number of older adults. Although PD is mostly sporadic, genetic mutations have been found in cohorts of families with a history of familial PD (FPD). The first such mutation linked to FPD causes a point mutation (A53T) in α-synuclein (α-syn), a major component of Lewy bodies, which are a classical pathological hallmark of PD. These findings suggest that α-syn is an important contributor to the development of PD. In our previous study, we developed an adenoviral mouse model of PD and showed that the expression of wild-type (WT) α-syn or a mutant form with an increased propensity to aggregate, designated as WT-CL1 α-syn, could be used to study how α-syn aggregation contributes to PD. In this study, we established a transgenic mouse model that conditionally expresses WT or WT-CL1 α-syn in dopaminergic (DA) neurons and found that the expression of either WT or WT-CL1 α-syn was associated with an age-dependent degeneration of DA neurons and movement dysfunction. Using this model, we were able to monitor the process of α-syn aggregate formation and found a correlation between age and the number and sizes of α-syn aggregates formed. These results provide a potential mechanism by which age-dependent α-syn aggregation may lead to the formation of Lewy bodies in PD pathogenesis.

UBE2N Is Essential for Maintenance of Skin Homeostasis and Suppression of Inflammation

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n-knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant Ccl27a. Consistently, the infiltrating immune cells were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that expressed high levels of inflammatory cytokines such as Il1β and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated inflammation, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity, and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Inactivation of the Tumor Suppressor CYLD Sensitizes Mice to Breast Cancer Development.

Breast cancer is a leading cause of cancer-related deaths among women. Down-regulation of the tumor suppressor gene Cyld in breast cancer has been linked to a poor prognosis. This study investigated the role of Cyld in breast cancer using conditional mutant mouse models carrying a Cyld mutation, which inactivates the deubiquitinating activity of its protein product CYLD in mammary epithelial cells. We examined the potential of CYLD inactivation to induce mammary tumors spontaneously or modify the susceptibility of mice to mammary tumorigenesis by DMBA treatment or ErbB2 over-expression. CYLD inactivation significantly increased susceptibility to breast cancer induced by either DMBA treatment or ErbB2 over-expression. Moreover, while CYLD inactivation alone did not lead to spontaneous mammary tumorigenesis, it did contribute to the formation of multifocal hyperplastic lesions in virgin mice of predominantly FVB/NJ background. Our study demonstrates the tumor enhancing potential of CYLD inactivation in mammary tumorigenesis in vivo and establishes novel relevant mouse models that can be exploited for developing prognostic and therapeutic protocols.

Serine protease inhibitor, SerpinA3n, regulates cardiac remodelling after myocardial infarction.

Following myocardial infarction (MI), the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix (ECM) by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure, and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors, and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human MI as well as non-ischaemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodelling is poorly understood. The aim of this study was to understand the role of Serpins in regulating scar formation after MI. Using a SerpinA3n conditional knockout mice model, we observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homologue of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix, and significantly worse post-infarct cardiac function. Single-cell transcriptomics complemented with histology in SerpinA3n-deficient animals demonstrated increased inflammation, adverse myocyte hypertrophy, and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross-linking of ECM peptides consistent with increased proteolysis in SerpinA3n-deficient animals. Our study demonstrates a hitherto unappreciated causal role of Serpins in regulating matrix function and post-infarct cardiac remodelling.

The homeostatic effects of the RE-1 silencing transcription factor on cortical networks are altered under ictogenic conditions in the mouse.

The Repressor Element-1 Silencing Transcription Factor (REST) is an epigenetic master regulator playing a crucial role in the nervous system. In early developmental stages, REST downregulation promotes neuronal differentiation and the acquisition of the neuronal phenotype. In addition, postnatal fluctuations in REST expression contribute to shaping neuronal networks and maintaining network homeostasis. Here we investigate the role of the early postnatal deletion of neuronal REST in the assembly and strength of excitatory and inhibitory synaptic connections. We investigated excitatory and inhibitory synaptic transmission by patch-clamp recordings in acute neocortical slices in a conditional knockout mouse model (RestGTi) in which Rest was deleted by delivering PHP.eB adeno-associated viruses encoding CRE recombinase under the control of the human synapsin I promoter in the lateral ventricles of P0-P1 pups. We show that, under physiological conditions, Rest deletion increased the intrinsic excitability of principal cortical neurons in the primary visual cortex and the density and strength of excitatory synaptic connections impinging on them, without affecting inhibitory transmission. Conversely, in the presence of a pathological excitation/inhibition imbalance induced by pentylenetetrazol, Rest deletion prevented the increase in synaptic excitation and decreased seizure severity. The data indicate that REST exerts distinct effects on the excitability of cortical circuits depending on whether it acts under physiological conditions or in the presence of pathologic network hyperexcitability. In the former case, REST preserves a correct excitatory/inhibitory balance in cortical circuits, while in the latter REST loses its homeostatic activity and may become pro-epileptogenic.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

Characterization of an ornithine transcarbamylase conditional knockout mouse model: A novel model for understanding the effect of MRNA therapies for treatment of ornithine transcarbamylase deficiency

Characterization of an ornithine transcarbamylase conditional knockout mouse model: A novel model for understanding the effect of MRNA therapies for treatment of ornithine transcarbamylase deficiency

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P<0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p<0.001) and hyperproliferative Ki67+ GCB cells (p<0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value <0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p<0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p<0.01), and vs. these cell lines cultured in vitro (all p<0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p<0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p<0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p<0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7586.

Abstract 1490: Elucidating the effect of glutamine metabolism in breast to bone metastasis

Abstract Bone-metastatic lesions will develop in approximately 65-75% of patients with metastatic breast cancer and are associated with high morbidity and mortality. Despite our limited understanding of how breast-to-bone metastases form, the recent emergence of metabolic adaptation as a critical factor in metastasis is a promising development. Breast cancer cells are particularly reliant on glutamine, a key amino acid that is metabolized by the enzyme glutaminase (GLS) to facilitate many downstream metabolic processes. Bone-tropic breast cancer cell lines significantly enhance their glutamine uptake, suggesting that glutamine metabolism may be a significant factor in bone metastasis. In addition to tumor cells, glutamine metabolism is critical for osteoblasts, important bone-resident cells that contribute to the “vicious cycle” of bone destruction during metastatic outgrowth in the bone niche. In this study, we sought to evaluate the role of glutamine metabolism in breast cancer bone metastasis by examining the effect of GLS function in metastasizing tumor cells and bone-native osteoblasts. Using an intracardiac inoculation model of bone metastasis, we found that breast cancer cells lacking GLS (GLS KO) failed to effectively form bone metastatic tumors. We also observed that GLS KO bone tumors show a reduction in osteolysis using a direct intratibial injection model. Mechanistically, we have found that GLS KO breast cancer cells develop a “senescence-like” phenotype that may limit metastatic outgrowth within the bone. To investigate the importance of glutamine metabolism in the bone niche, we developed a conditional genetic mouse model to delete GLS in osterix-expressing in osteoblast progenitors and mature osteoblasts (GLSOBKO). Remarkably, we observed reduced bone destruction in GLSOBKO mice after intratibial injection of syngeneic breast cancer cells. We also report a reduction in the number of osteoclasts at the bone/tumor interface in these mice, suggesting that loss of osteoblast GLS could temporarily halt the “vicious cycle” of bone metastasis. Together, this work suggests that glutamine metabolism may promote breast-to-bone metastasis via both intrinsic and extrinsic mechanisms. These findings raise the possibility of using pre-existing cancer therapies targeting glutamine metabolism to ameliorate bone metastatic burden in breast cancer. Citation Format: Breelyn A. Karno, Yoonha Hwang, Rachelle Johnson, Jin Chen, Deanna Edwards. Elucidating the effect of glutamine metabolism in breast to bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1490.

Abstract 1358: Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity

Abstract Immune checkpoint receptors (ICRs) negatively regulate antitumor immune responses. While significant advances have been made in understanding the mechanisms of well-established ICRs such as PD-1 and CTLA-4, other checkpoint proteins remain incompletely characterized and may be targeted to synergistically boost antitumor immunity. One such protein is V-domain immunoglobulin suppressor of T-cell activation (VISTA), an ICR whose expression curtails the function of several lymphoid and myeloid lineages, including tumor-reactive cytotoxic T cells. Globally blocking VISTA elicits potent T cell-mediated antitumor immunity and systemic myeloid cell-mediated inflammatory responses. Mechanistically, VISTA has been shown to inhibit T cell activation by acting as an inhibitory ligand on antigen-presenting cells. However, the impact of T cell-intrinsic VISTA on modulating tumor-reactive T cell responses has not been thoroughly investigated, and it remains unclear whether blocking T cell-specific VISTA is adequate for revitalizing T cell-mediated tumor control. In this study, we utilized VISTA conditional knockout mice models to examine the relative contribution of T cell-intrinsic VISTA versus myeloid-derived VISTA in the regulation of antitumor immunity. We present evidence that blocking VISTA on T cells alone is insufficient to induce strong antitumor immune responses, whereas blocking VISTA on myeloid cells counteracts its dominant role in orchestrating T cell suppression. Citation Format: Sachin Patnaik, Elizabeth Delaney, Cassandra Gilmour, Keman Zhang, Amin Zakeri, Hieu M. Ta, Prerana B. Parthasarathy, Tyler Alban, Timothy Chan, Li Lily Wang. Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1358.

Abstract 1496: The role of endothelial ACKR1 in breast cancer metastasis

Abstract Background: Tumor cells can co-opt mechanisms of leukocyte extravasation. Atypical Chemokine Receptor 1 (ACKR1) expression in endothelial cells has an established role in promoting leukocyte extravasation; however, the potential for endothelial ACKR1 to facilitate the metastatic process through the extravasation of tumor cells has not been studied. Methods: To understand ACKR1 function, we investigated endothelial ACKR1 expression at the distant metastatic site of the lung, a common site of breast cancer metastasis. We analyzed lung endothelial ACKR1 expression using immunofluorescent staining of lung vasculature during tumor progression in immunocompetent mice bearing orthotopically implanted E0771.LMB breast tumor cells. We assessed the timing of metastatic tumor cell arrival in the lung and evaluated the spatial relationship between disseminated tumor cells and ACKR1-positive vessels. To investigate secreted tumor factors that may mediate ACKR1 regulation in the lung, we performed intratracheal injections of TNF-alpha. We developed an endothelial cell specific ACKR1 conditional knockout mouse model (ACKR1 ECKO) to understand the impact of endothelial ACKR1 on primary tumor growth and subsequent lung metastasis in breast cancer. Results: Our results reveal a significant upregulation of ACKR1 in the lung endothelium of mice implanted with breast cancer cells in the mammary fat pad. Tumor-dependent ACKR1 expression was specifically localized to pulmonary venule endothelium, a common site of leukocyte and tumor cell extravasation. Lung endothelial ACKR1 expression became evident by day 13 after implantation, persisting through endpoint at day 28. Notably, ACKR1 upregulation occurred prior to tumor cell arrival in lungs of tumor bearing mice. We observed spatial clustering of disseminated tumor cells in proximity to ACKR1-positive vessels, implying a preferential site of tumor cell extravasation. In the absence of primary tumors, intratracheal delivery of TNF-alpha induced ACKR1 expression within lungs. When tumors were orthotopically implanted into our ACKR1 ECKO mouse model, we observed a marked decrease in lung metastases compared to control mice. Conclusion/Future Directions: We have determined that pulmonary ACKR1 expression is induced by breast tumors, and vessels expressing ACKR1 appear to act as entry points for lung metastasis. Loss of endothelial ACKR1 reduced metastasis, suggesting that endothelial ACKR1 regulates tumor cell extravasation at distant metastatic sites. We will upregulate lung endothelial ACKR1 expression and directly test its requirement for extravasation using an intravenous tumor cell injection metastasis model. Citation Format: Samuel Tanner Roach, Rishi Patel, Serena Thomas, Chinwe Ewenighi-Amankwah, Joseph Dufraine, L A. Naiche, Jan Kitajewski. The role of endothelial ACKR1 in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1496.

Abstract 6234: Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML)

Abstract AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. Using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre) to mimic the somatic acquisition of fusion gene CBFB-MYH11 (CM), created in the recurrent chromosome translocation inv(16)(p13q22) of AML, we previously reported that the blood transcriptome undergoes state-transition from health to leukemia. In this study, we analyze the transcriptomic alterations and leukemic cell heterogeneities in a leukemia state-space using single-cell RNA sequencing (scRNA-seq). CM expression was induced in Cbfb56M/+/Mx1-Cre mice by poly (I:C) injection, and scRNA-seq analysis was performed on peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from leukemic mice and age-matched wild-type (WT) controls. More than 105,000 cells were subjected to the following dimensionality reduction and cell type annotation. By performing principal component analysis (PCA), we observed a Y-shape cell distribution with emergence of a divergent branch occupied by leukemic cells which can be mapped to a leukemic state. The principal components 1 and 2 (PC1 and PC2) clearly distinguish the leukemic cells from normal cell types such as myeloid cells and lymphocytes. By accessing the eigenvalue of genes for PC1 and PC2, we quantified the crucial genes and pathways related to leukemia progression at a single-cell level. We characterized cKit+ leukemic cell clusters by transcriptome-based correlation and gene expression pattern and found that they resembled stem cells and megakaryocytic-erythroid progenitors (MEP) with dysregulated pathways, including upregulated "inflammatory response" and downregulated pathways related to Myc targets, oxidative phosphorylation, mitochondrial protein translation, and ribosomal biogenesis/assembly. Examining the 31 clusters (C0-C30) seen in cKit+ BM cells, we identified 9 leukemic clusters exhibiting distinct features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). In conclusion, our study unveils state-transition from health to leukemia at single-cell resolution, and uncovers diverse biological heterogeneities associated with the inv(16) AML state. Further refinement of transcriptome state-transition and single-cell analysis will provide an analytical framework for modeling the dynamic changes during disease evolution at the single-cell level. Citation Format: Yu-Hsuan Fu, Lianjun Zhang, Ying-Chieh Chen, David E. Frankhouser, Lisa Uechi, Denis O'Meally, Sergio Branciamore, Guido Marcucci, Russell Rockne, Ya-Huei Kuo. Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6234.