Non-cell Autonomous Mechanisms Research Articles

Metabolic regulation of the tumour and its microenvironment: The role of Epstein-Barr virus.

The Epstein-Barr virus (EBV), the first identified human tumour virus, infects over 95% of the individuals globally and has the potential to induce different types of cancers. It is increasingly recognised that EBV infection not only alters cellular metabolism, contributing to neoplastic transformation, but also utilises several non-cell autonomous mechanisms to shape the metabolic milieu in the tumour microenvironment (TME) and its constituent stromal and immune cells. In this review, we explore how EBV modulates metabolism to shape the interactions between cancer cells, stromal cells, and immune cells within a hypoxic and acidic TME. We highlight how metabolites resulting from EBV infection act as paracrine factors to regulate the TME, and how targeting them can disrupt barriers to immunotherapy.

Huntington’s disease cellular phenotypes are rescued non-cell autonomously by healthy cells in mosaic telencephalic organoids

Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.

Schwann cell-secreted PGE2 promotes sensory neuron excitability during development

Electrical excitability-the ability to fire and propagate action potentials-is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage-gated sodium channels, and to fire action potential trains. Inactivating this signaling pathway in Schwann cells impairs somatosensory neuron maturation, causing multimodal sensory defects that persist into adulthood. Collectively, our studies uncover a neurodevelopmental role for prostaglandin E2 distinct from its established role in inflammation, revealing a cell non-autonomous mechanism by which glia regulate neuronal excitability to enable the development of normal sensory functions.

Astrocytic autophagy plasticity modulates Aβ clearance and cognitive function in Alzheimer’s disease

BackgroundAstrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer’s disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD.MethodsWhole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR).ResultsHere, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and GFAP-positive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Aβ aggregates in the brain of APP/PS1 mice. An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients.ConclusionsTaken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice.

Chronic β3 adrenergic agonist treatment improves brain microvascular endothelial function and cognition in aged mice.

Microvascular endothelial dysfunction, characterized by impaired neurovascular coupling, reduced glucose uptake, blood-brain barrier disruption, and microvascular rarefaction, plays a critical role in the pathogenesis of age-related vascular cognitive impairment (VCI). Emerging evidence points to non-cell autonomous mechanisms mediated by adverse circulating milieu (an increased ratio of pro-geronic to anti-geronic circulating factors) in the pathogenesis of endothelial dysfunction leading to impaired cerebral blood flow and cognitive decline in the aging population. In particular, age-related adipose dysfunction contributes, at least in part, to an unfavorable systemic milieu characterized by chronic hyperglycemia, hyperinsulinemia, dyslipidemia, and altered adipokine profile, which together contribute to microvascular endothelial dysfunction. Hence, in the present study, we aimed to test whether thermogenic stimulation, an intervention known to improve adipose and systemic metabolism by increasing cellular energy expenditure, could mitigate brain endothelial dysfunction and improve cognition in the aging population. Eighteen-month-old old C57BL/6J mice were treated with saline or CL (β3-adrenergic agonist) for 6 weeks followed by functional analysis to assess endothelial function and cognition. CL treatment improved neurovascular coupling responses and rescued brain glucose uptake in aged animals. In addition, CL treatment also attenuated blood-brain barrier leakage and associated neuroinflammation in the cortex of aged animals. More importantly, these beneficial changes in microvascular function translated to improved cognitive performance in radial arm water maze and Y-maze tests. Our results suggest that β3-adrenergic agonist treatment improves multiple aspects of brain microvascular endothelial function and can be potentially repurposed for treating age-associated cognitive decline.

Neuroinflammation in amyotrophic lateral sclerosis: pathogenic insights and therapeutic implications.

Neuroinflammation appears to be an important pathogenic process in amyotrophic lateral sclerosis (ALS). Dysfunction of central immune pathways, including activation of microglia and astrocytes, and peripherally derived immune cells, initiate noncell autonomous inflammatory mechanisms leading to degeneration. Cell autonomous pathways linked to ALS genetic mutations have been recently identified as contributing mechanism for neurodegeneration. The current review provides insights into the pathogenic importance of central and peripheral inflammatory processes in ALS pathogenesis and appraises their potential as therapeutic targets. ALS is a multistep process mediated by a complex interaction of genetic, epigenetic, and environmental factors. Noncell autonomous inflammatory pathways contribute to neurodegeneration in ALS. Activation of microglia and astrocytes, along with central nervous system infiltration of peripherally derived pro-inflammatory innate (NK-cells/monocytes) and adaptive (cell-mediated/humoral) immune cells, are characteristic of ALS. Dysfunction of regulatory T-cells, elevation of pro-inflammatory cytokines and dysbiosis of gut microbiome towards a pro-inflammatory phenotype, have been reported as pathogenic mechanisms in ALS. Dysregulation of adaptive and innate immunity is pathogenic in ALS, being associated with greater disease burden, more rapid disease course and reduced survival. Strategies aimed at modulating the pro-inflammatory immune components could be of therapeutic utility.

Adult aberrant astrocytes submitted to late passage cultivation lost differentiation markers and decreased their pro-inflammatory profile

In amyotrophic lateral sclerosis (ALS), astrocytes are considered key players in some non-cell non-neuronal autonomous mechanisms that underlie motor neuron death. However, it is unknown how much of these deleterious features were permanently acquired. To assess this point, we evaluated if the most remarkable features of neurotoxic aberrant glial phenotypes (AbAs) isolated from paralytic rats of the ALS model G93A Cu/Zn superoxide dismutase 1 (SOD1) could remain upon long lasting cultivation. Real time PCR, immunolabelling and zymography analysis showed that upon many passages, AbAs preserved the cell proliferation capacity, mitochondrial function and response to different compounds that inhibit some key astrocyte functions but decreased the expression of parameters associated to cell lineage, homeostasis and inflammation. As these results are contrary to the sustained inflammatory status observed along disease progression in SOD1G93A rats, we propose that the most AbAs remarkable features related to homeostasis and neurotoxicity were not permanently acquired and might depend on the signaling coming from the injuring microenvironment present in the degenerating spinal cord of terminal rats.

Enhanced levels of fractalkine and HSP60 in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis patients

Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neurodegenerative disorder with a significant contribution of non-cell autonomous mechanisms to motor neuronal degeneration. Amongst a plethora of molecules, fractalkine (C-X3-C motif chemokine ligand 1), and Heat Shock Protein 60 (HSP60), are key modulators of microglial activation. The contribution of these molecules in Sporadic ALS (SALS) remains unexplored. To investigate this, fractalkine levels were estimated in Cerebrospinal fluid (CSF) of SALS patients (ALS-CSF; n = 44) by Enzyme-linked Immunosorbent Assay (ELISA) and correlated with clinical parameters including disease severity and duration. CSF HSP60 levels were estimated by Western blotting (ALS-CSF; n = 19). Also, CSF levels of Chitotriosidase-1 (CHIT-1), a microglia-specific neuroinflammatory molecule, were measured and its association, if any, with fractalkine and HSP60 was investigated. Both fractalkine and HSP60 levels were significantly elevated in ALS-CSF. Similar to our earlier observation, CHIT-1 levels were also upregulated. Fractalkine showed a moderate negative correlation with the ALS-Functional Rating Scale (ALSFRS) score indicating its significant rise in mild cases which plateaued in cases with high disease severity. However, no obvious correlation was found between fractalkine, HSP60, and CHIT-1. Our study hints that high fractalkine levels in mild cases might be conferring neuroprotection by combating microglial activation and highlights its importance as a novel therapeutic target for SALS. On the other hand, significantly enhanced levels of HSP60, a pro-inflammatory molecule, hint towards its role in accentuating microgliosis, although, it doesn’t act synergistically with CHIT-1. Our study suggests that fractalkine and HSP60 act independently of CHIT-1 to suppress and accentuate neuroinflammation, respectively.

Mecp2 knock-out astrocytes affect synaptogenesis by interleukin 6 dependent mechanisms

Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.

Loss of Neogenin alters branchial arch development and leads to craniofacial skeletal defects.

The formation of complex structures, such as the craniofacial skeleton, requires precise and intricate two-way signalling between populations of cells of different embryonic origins. For example, the lower jaw, or mandible, arises from cranial neural crest cells (CNCCs) in the mandibular portion of the first branchial arch (mdBA1) of the embryo, and its development is regulated by signals from the ectoderm and cranial mesoderm (CM) within this structure. The molecular mechanisms underlying CM cell influence on CNCC development in the mdBA1 remain poorly defined. Herein we identified the receptor Neogenin as a key regulator of craniofacial development. We found that ablation of Neogenin expression via gene-targeting resulted in several craniofacial skeletal defects, including reduced size of the CNCC-derived mandible. Loss of Neogenin did not affect the formation of the mdBA1 CM core but resulted in altered Bmp4 and Fgf8 expression, increased apoptosis, and reduced osteoblast differentiation in the mdBA1 mesenchyme. Reduced BMP signalling in the mdBA1 of Neogenin mutant embryos was associated with alterations in the gene regulatory network, including decreased expression of transcription factors of the Hand, Msx, and Alx families, which play key roles in the patterning and outgrowth of the mdBA1. Tissue-specific Neogenin loss-of-function studies revealed that Neogenin expression in mesodermal cells contributes to mandible formation. Thus, our results identify Neogenin as a novel regulator of craniofacial skeletal formation and demonstrates it impinges on CNCC development via a non-cell autonomous mechanism.

Intervertebral disc-intrinsic Hedgehog signaling maintains disc cell phenotypes and prevents disc degeneration through both cell autonomous and non-autonomous mechanisms

Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.

Abstract A037: Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS)

Abstract Heterozygous germline variants in DDX41 are identified in ~5% of patients with Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML). Recognition of the hereditary nature of such cancers and a better understanding of the factors that affect progression to malignancy has deep implications for health surveillance of patients and relatives that share a pathogenic DNA variant. DDX41 has a role in ribosome biogenesis and in opposing R-loop accumulation at promoters, but the mechanisms through which DDX41 loss drives cancer development are poorly understood. In a collaboration between Cambridge, UK and Mayo Clinic, USA we identified 85 patients with germline DDX41 variants [73/85 with germline pathogenic variants (GPV) and 12 with variants of uncertain significance (VUS)]. The majority were identified through targeted myeloid NGS upon presentation with AML (30/85) or MDS (44/85). Average age at diagnosis was 65 (35-93) with male predominance (57/85). The most common GPV was the NM_016222.4(DDX41):c.415_418dup; p.D140Gfs*2 (15/85). The majority (53.4% 39/73), also acquired a somatic variants in the other DDX41 allele (referred to as GPV+Som). The most common somatic variant (82% 32/39) was the R525H, a variant predicted to reduce the ATPase function of the helicase domain. We hypothesise that the markedly reduced DDX41 activity in cells with biallelic variants, is a key cause of progression to overt malignancy. In our cohort, we observed that somatic variants occurred at a similar frequency in association with start loss, truncating, splice, in-frame deletion, and missense GPV. However, whereas MDS was the predominant diagnosis within GPV (67.6% 23/34, AML 26.4% 9/34), AML was the most frequent diagnosis within GPV+Som (AML 51%.2 20/39, MDS 38.4% 15/39). Overall, there was a significantly higher likelihood of AML in the presence of a somatic variant (p<0.05), suggesting that there might be a dosage effect on the phenotype. This pattern was observed across all the GPV, with the exception of missense, suggesting that many might be benign polymorphysms. In addition, although somatic variants are typically subclonal (overall mean variant allele fraction VAF=9.87%, 3-27.8%), we observed a higher somatic VAF in AML over MDS cases (12.4% vs 7.7% respectively p<0.05). A lower bone marrow cellularity was also characteristic of GPV+Som vs GPV alone (mean observed/expected cellularity for age 0.9 vs 1.32 respectively p<0.05). In two cases, serial bone marrow morphology and NGS data were available, and we observed marked reduction in bone marrow cellularity upon acquisition of the R525H clone. The mechanisms of AML evolution appear different in DDX41 HMMS compared to sporadic cases. Interestingly, over half of our patients (21/34 GPV and 23/39 GPV+Som) did not exhibit additional somatic drivers. Our work suggests that acquisition of a somatic variant is a critical event in disease progression. Furthermore, the near universally subclonal nature of somatic variants suggest that they may affect disease phenotype through non-cell autonomous mechanisms. Citation Format: Ludovica Marando, Talha Badar, Yael Kusne, Audrey Morris, Terra Lasho, Abhishek Mangaonkar, Christy Finke, Carles Crawley, James M. Foran, Aref Al-Kali, Andrew King, Hassan Alkhateeb, Naseema Gangat, Chi Wong, Rong He, David Viswanatha, Faisal Basheer, Mark Litzow, Alejandro Ferrer, George Vassiliou, Mrinal Patnaik. Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A037.

Second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP): the cell autonomous and non-autonomous roles in cancer progression.

Cytosolic double-stranded DNA (dsDNA) is frequently accumulated in cancer cells due to chromosomal instability or exogenous stimulation. Cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor, which is activated upon binding to dsDNA to synthesize the crucial second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) that in turn triggers stimulator of interferon genes (STING) signaling. The canonical role of cGAS-cGAMP-STING pathway is essential for innate immunity and viral defense. Recent emerging evidence indicates that 2'3'-cGAMP plays an important role in cancer progression via cell autonomous and non-autonomous mechanisms. Beyond its role as an intracellular messenger to activate STING signaling in tumor cells, 2'3'-cGAMP also serves as an immunotransmitter produced by cancer cells to modulate the functions of non-tumor cells especially immune cells in the tumor microenvironment by activating STING signaling. In this review, we summarize the synthesis, transmission, and degradation of 2'3'-cGAMP as well as the dual functions of 2'3'-cGAMP in a STING-dependent manner. Additionally, we discuss the potential therapeutic strategies that harness the cGAMP-mediated antitumor response for cancer therapy.

Potential contribution of spinal interneurons to the etiopathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) consists of a group of adult-onset fatal and incurable neurodegenerative disorders characterized by the progressive death of motor neurons (MNs) throughout the central nervous system (CNS). At first, ALS was considered to be an MN disease, caused by cell-autonomous mechanisms acting specifically in MNs. Accordingly, data from ALS patients and ALS animal models revealed alterations in excitability in multiple neuronal populations, including MNs, which were associated with a variety of cellular perturbations such as protein aggregation, ribonucleic acid (RNA) metabolism defects, calcium dyshomeostasis, modified electrophysiological properties, and autophagy malfunctions. However, experimental evidence rapidly demonstrated the involvement of other types of cells, including glial cells, in the etiopathogenesis of ALS through non-cell autonomous mechanisms. Surprisingly, the contribution of pre-motor interneurons (INs), which regulate MN activity and could therefore critically modulate their excitability at the onset or during the progression of the disease, has to date been severely underestimated. In this article, we review in detail how spinal pre-motor INs are affected in ALS and their possible involvement in the etiopathogenesis of the disease.

P53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.

Intra-Leukemic IFNγ Signaling Mediates Cell Cycle Suppression and Chemoresistance in AML

Introduction: The acute myeloid leukemia stem cell (LSC) model proposes that LSCs are a quiescent and functionally distinct cellular fraction that can escape chemotherapy and produce disease during relapse. Here, we reevaluated the relationship between quiescence and LSCs, and explored cell non-autonomous mechanisms contributing to chemotherapy escape. Methods: Using the definition that LSCs can engraft in immunodeficient mice, we evaluated the serial engraftment capability and cell cycle status of xenotransplanted AML patient samples and AML cells derived from AML-iPSC (iAML). To functionally interrogate regulators of cell cycle and chemotherapy escape, we used molecular profiling and perturbation studies to identify and validate mechanisms of cell cycle suppression. Results: To test the paradigm that quiescent LSCs are responsible for relapse after chemotherapy, we xenotransplanted both G0 and cycling cells from fresh primary AML cells or iAML. Contrary to previous reports, both G0 and cycling cells were transplantable, indicating that LSCs can exist in any cell cycle status. Having demonstrated that cell cycle status is heterogeneous in primary AML and not linked to cell-autonomous LSC identity, we speculated that cell cycle can be regulated by cell-extrinsic mechanisms. In particular, we hypothesized that leukemia cells themselves can interact to affect cell cycle, engraftment, and response to chemotherapy. To investigate this hypothesis, we established primary patient co-injection xenograft models in which the co-injected samples could be identified using two different HLA antibodies (Figure). Each sample expanded when transplanted by itself. Surprisingly, in all three patient pair models, one of the two co-injected patient samples (“Suppressed”) displayed very limited and decreased engraftment relative to the other sample (“Dominant”). Importantly, the Suppressed samples displayed less active cycling in Co-injection mice than in Alone mice, suggesting that the presence of Dominant samples inhibited the cycling of Suppressed samples. However, when isolated from primary recipients for secondary transplantation, Suppressed samples from Co-injection and Alone mice demonstrated similar engraftment capacity and caused similar mortality. Similar results were obtained in an iAML syngeneic co-injection model, demonstrating that suppression also occurs in cells derived from the same patient. These findings support our hypothesis that AML cell cycle can be suppressed by other AML cells without affecting the Suppressed cells' ability to initiate disease. Next, to identify mechanisms regulating cell cycle suppression, we evaluated RNA-seq of Dominant and Suppressed samples from our AML co-injection xenograft models and single-cell RNA-seq data from 12 AML patient specimens described in the literature. Suppressed samples exhibited enrichment of IFNγ signaling, which was also observed in G1 cells compared to S/G2/M cells in patient samples, suggesting IFNγ involvement in regulating cell cycle. To validate that IFNγ signaling mediates suppression, we disrupted expression of the IFNγ receptor IFNGR1 using CRIPSR/Cas9 gene editing in the Suppressed samples in the co-injection model. IFNGR1 loss increased cell cycle in the Suppressed samples compared to controls. Thus, IFNγ signaling contributes to AML cell cycle suppression. Finally, we investigated the role of IFNγ signaling in modulating cell cycle and chemosensitivity by treating our co-injection model with chemotherapy with and without anti-IFNGR1 blocking antibody. RNA-seq of both Suppressed and Dominant samples before and after chemotherapy revealed that Dominant samples and samples that escape suppression after chemotherapy downregulate IFNγ receptor expression and signaling. Furthermore, in samples enriched for IFNγ signaling, blocking IFNGR1 before and during chemotherapy treatment increased cell cycling before chemotherapy and decreased leukemia burden after chemotherapy. Conclusion: Here, we show that AML cell cycle can be suppressed in a cell non-autonomous manner that is mediated by IFNγ signaling. Moreover, blocking IFNγ signaling can increase chemosensitivity through activating the cell cycle. These findings provide novel insights into the mechanisms of chemotherapy escape and highlight IFNγ signaling as a potential therapeutic target to counter chemoresistance.

Gamma Secretase Inhibition Sensitizes Pancreatic Adenocarcinoma Tumors to RT In Vivo

Pancreatic adenocarcinoma (PDAC) is an extremely aggressive cancer that lacks curative treatment options. Almost half of patients present with unresectable disease limiting treatment to non-curative options. Patients treated with neoadjuvant radiation therapy (RT) exhibit increases in fibrosis and epithelial-to-mesenchymal transition (EMT). ADAM10, an extracellular sheddase, can stimulate stromal fibrosis, EMT, and radioresistance. ADAM10 also mediates EMT through Notch signaling by cleaving its extracellular domain. Further cleavage by gamma secretase produces the Notch intracellular domain (NICD), which translocates to the nucleus and activates downstream transcriptional targets. Here, we explore whether inhibition of Notch cleavage by gamma secretase radiosensitizes PDAC tumors. Bilateral flank subcutaneous PDAC isografts were produced in 40 mice using PK5L1940 KPC cells. Intraperitoneal injections of the gamma secretase inhibitor, DAPT (5 mg/kg), were delivered daily for 7 days, starting 3 days prior to RT. A single dose of 20 Gy was administered to each flank tumor, and volumes were measured twice weekly. Colony formation assays of KPC cells were performed after RT, in the presence or absence of DAPT. Since stromal fibrosis can mediate radio-resistance in the tumor microenvironment (TME), the effect of tumor cells on Notch pathway activation in mouse fibroblasts (3T3 cells) was investigated using a luciferase reporter assay. Thus, 3T3 cells transfected with a Notch pathway luciferase reporter were incubated with PDAC cells for 48 h, followed by measurement of luciferase activity. In vivo, the combination of DAPT and RT significantly delayed tumor growth, and some tumors were completely eradicated. Mean tumor size for the combination at 21 days was 21 mm3 (range = 0-53, p = 0.005), while tumor size was 577 mm3 (range = 217-955, p = 0.69) for DAPT alone, 435 mm3 for RT alone (range = 51-932, p = 0.79), and 367 mm3 for untreated vehicle (range = 97-1144). Surprisingly, DAPT did not reduce clonogenic survival in vitro. Both ADAM10 knockout and DAPT decreased NICD cleavage and transcription of the downstream target Hes1 in vivo and in vitro. Co-culture with PDAC cells increased Notch luciferase reporter activity in fibroblasts. This effect was not mimicked by PDAC-conditioned media, suggesting a requirement for intercellular contact. Notch pathway inhibition sensitizes PDAC tumors to RT in vivo, but not in vitro, suggesting involvement of the TME. Indeed, co-culture with PDAC cells stimulates notch signaling in fibroblasts, suggesting non-cell autonomous mechanisms mediating fibrosis in the TME driving radioresistance. Future studies will determine if ADAM10 inhibition targeting PDAC cells and/or gamma secretase inhibition targeting the TME enhances radiation sensitivity in vivo by blocking fibroblast Notch signaling.

Delayed skeletal development and IGF-1 deficiency in a mouse model of lysinuric protein intolerance.

SLC7A7 deficiency, or lysinuric protein intolerance (LPI), causes loss of function of the y+LAT1 transporter critical for efflux of arginine, lysine and ornithine in certain cells. LPI is characterized by urea cycle dysfunction, renal disease, immune dysregulation, growth failure, delayed bone age, and osteoporosis. We previously reported that Slc7a7 knockout mice (C57BL/6 x 129/SvEv F2) recapitulate LPI phenotypes, including growth failure. Our main objective was to characterize the skeletal phenotype in these mice. Compared to wild type littermates, juvenile Slc7a7 knockout mice demonstrated 70% lower body weights, 87% lower plasma IGF-1 concentrations, and delayed skeletal development. Because poor survival prevents evaluation of mature knockout mice, we generated a conditional Slc7a7 deletion in mature osteoblasts or mesenchymal cells of the osteo-chondroprogenitor lineage, butno differences in bone architecture were observed. Overall, global Slc7a7 deficiency caused growth failure with low plasma IGF-1 concentrations and delayed skeletal development, but Slc7a7 deficiency in the osteoblastic lineage was not a major contributor to these phenotypes. Future studies utilizing additional tissue-specific Slc7a7 knockout models may help dissect cell autonomous and non-cell autonomous mechanisms underlying phenotypes in LPI.

Exploiting inter-tissue stress signaling mechanisms to preserve organismal proteostasis during aging.

Aging results in a decline of cellular proteostasis capacity which culminates in the accumulation of phototoxic material, causing the onset of age-related maladies and ultimately cell death. Mechanisms that regulate proteostasis such as cellular stress response pathways sense disturbances in the proteome. They are activated to increase the expression of protein quality control components that counteract cellular damage. Utilizing invertebrate model organisms such as Caenorhabditis elegans, it has become increasingly evident that the regulation of proteostasis and the activation of cellular stress responses is not a cell autonomous process. In animals, stress responses are orchestrated by signals coming from other tissues, including the nervous system, the intestine and the germline that have a profound impact on determining the aging process. Genetic pathways discovered in C. elegans that facilitate cell nonautonomous regulation of stress responses are providing an exciting feeding ground for new interventions. In this review I will discuss cell nonautonomous proteostasis mechanisms and their impact on aging as well as ongoing research and clinical trials that can increase organismal proteostasis to lengthen health- and lifespan.

Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila.

Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3'-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. In this study, we used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. We found that this modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms.