Although certain human genetic variants are conspicuously loss-of-function, decoding the impact of many variants is challenging. Previously, we described a leukemia predisposition syndrome (GATA2-deficiency) patient with a germline GATA2 variant that inserts nine amino acids between the two zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies and a genetic rescue system with Gata2 enhancer-mutant hematopoietic progenitor cells to compare how GATA2 and 9aa-Ins function genome-wide. Despite nuclear localization, 9aa-Ins was severely defective in occupying and remodeling chromatin and regulating transcription. Variation of the inter-zinc finger spacer length revealed that insertions were more deleterious to activation than repression. GATA2-deficiency generated a lineage-diverting gene expression program and a hematopoiesis-disrupting signaling network in progenitors with reduced Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and elevated Interleukin-6 (IL-6) signaling. As insufficient GM-CSF signaling causes pulmonary alveolar proteinosis and excessive IL-6 signaling promotes bone marrow failure, GATA2-deficiency patient phenotypes, these results inform mechanisms underlying GATA2-linked pathologies.
Mabel Minji Jung, Siqi Shen, Giovanni A. Botten, Thomas Olender, Koichi R. Katsumura, Kirby D. Johnson, Alexandra A. Soukup, Peng Liu, Qingzhou Zhang, Zena D. Jensvold, Peter W. Lewis, Robert A. Beagrie, Jason K.K. Low, Lihua Yang, Joel P. Mackay, Lucy A. Godley, Marjorie Brand, Jian Xu, Sunduz Keles, Emery H. Bresnick
Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we show that deleting c-Src abrogates the activity of Forkhead Box M1 (FOXM1), a master transcriptional regulator of the cell cycle, in a genetically engineered model mimicking the Luminal B molecular subtype of breast cancer. By phosphorylating it on two tyrosine residues, c-Src stimulates the nuclear localization of FOXM1 and the expression of its target genes, including key regulators of G2-M cell cycle progression as well as c-Src itself. This positive feedback loop drives proliferation in genetically engineered and patient-derived models of Luminal B-like breast cancer. Targeting this mechanism, including through novel compounds that destabilize the FOXM1 protein, induces G2-M cell cycle arrest and apoptosis, blocking tumor progression and impairing metastasis. We identify a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the Luminal B subtype, which responds poorly to approved therapies. These findings indicate that a regulatory network centered on c-Src and FOXM1 is a targetable vulnerability in aggressive luminal breast cancers.
Ipshita Nandi, Harvey W. Smith, Virginie Sanguin-Gendreau, Linjia Ji, Alain Pacis, Vasilios Papavasiliou, Dongmei Zuo, Stella Nam, Sherif S. Attalla, Sung Hoon Kim, Sierra Lusson, Hellen Kuasne, Anne-Marie Fortier, Paul Savage, Constanza Martinez Ramirez, Morag Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen, William J. Muller
Lysosomal inhibition elicited by palmitoyl protein transferase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC), but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12, was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naïve T cells, and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells, engendered adaptive immunity and tumor rejection in “immune hot” tumors but not in “immune cold” tumors. These findings demonstrate LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.
Monika Bhardwaj, Jennifer J. Lee, Amanda M. Versace, Sandra L. Harper, Aaron R. Goldman, Mary Ann S. Crissey, Vaibhav Jain, Mahendra Pal Singh, Megane Vernon, Andrew E. Aplin, Seokwoo Lee, Masao Morita, Jeffrey D. Winkler, Qin Liu, David W. Speicher, Ravi K. Amaravadi
Although protein hydroxylation is a relatively poorly characterized post-translational modification, it has received significant recent attention following seminal work uncovering its role in oxygen sensing and hypoxia biology. Although the fundamental importance of protein hydroxylases in biology is becoming clear, the biochemical targets and cellular functions often remain enigmatic. JMJD5 is a ‘JmjC-only’ protein hydroxylase that is essential for murine embryonic development and viability. However, no germline variants in JmjC-only hydroxylases, including JMJD5, have yet been described that are associated with any human pathology. Here we demonstrate that biallelic germline JMJD5 pathogenic variants are deleterious to JMJD5 mRNA splicing, protein stability, and hydroxylase activity, resulting in a human developmental disorder characterised by severe failure to thrive, intellectual disability, and facial dysmorphism. We show that the underlying cellular phenotype is associated with increased DNA replication stress and that this is critically dependent on the protein hydroxylase activity of JMJD5. This work contributes to our growing understanding of the role and importance of protein hydroxylases in human development and disease.
Sally C. Fletcher, Charlotte L. Hall, Tristan J. Kennedy, Sander Pajusalu, Monica H. Wojcik, Uncaar Boora, Chan Li, Kaisa Teele Oja, Eline Hendrix, Christian A.E. Westrip, Regina Andrijes, Sonia K. Piasecka, Mansi Singh, Mohammed E. El-Asrag, Anetta Ptasinska, Vallo Tillmann, Martin R. Higgs, Deanna Alexis Carere, Andrew D. Beggs, John Pappas, Rachel Rabin, Stephen J. Smerdon, Grant S. Stewart, Katrin Õunap, Mathew L. Coleman
Glucose homeostasis can be improved after bariatric surgery that alters bile flow and stimulate gut hormone secretion, in particular FGF15/19. FGFR1 expression in AGRP expressing cells is required for bile acids's ability to improve glucose control. We show that the mouse Agrp gene has 3 promoter/enhancer regions that direct transcription of each of their own AGRP transcripts. One of these Agrp promoters/enhancers, Agrp B, is regulated by bile acids. We generated an Agrp B knock-in FLP/knockout allele. AGRP B expressing cells are found in endocrine cells the pars tuberalis (PT) and co-expressDAGLB (an endocannabinoid biosynthetic enzyme), distinct from PT thyrotropes. AGRP B expression is also found in the folliculostellate cells of the pituitary's anterior lobe. Mice without AGRP B are protected from high fat feeding induced glucose intolerance but not excess weight gain. Chemogenetic inhibition of AGRP B cells improves glucose tolerance by enhancing glucose stimulated insulin secretion. Inhibition of the AGRP B cells also caused weight loss. The improved glucose tolerance and reduced body weight persisted up to 6 weeks after cessation of the DREADD mediated inhibition, suggesting the presence of a biological switch for glucose homeostasis that is regulated by long term stability of food availability.
Shun-Mei Liu, Bruno Ifebi, Fred Johnson III, Alison Xu, Jacquelin Ho, Yunlei Yang, Gary J. Schwartz, Young-Hwan Jo, Streamson Chua
STING-Type I interferon (IFN) signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonist as monotherapy has shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that Protein phosphatase 2A (PP2A) with its specific B regulatory subunit STRN4 negatively regulated STING-Type I IFN in macrophages. Mice with macrophages PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that hippo kinase MST1/2 was required for STING activation. STING agonist induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human GBM patients, YAP/TAZ was highly expressed in tumor-associated macrophages but not in non-tumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ is a previously unappreciated mechanism that mediate immunosuppression in tumor-associated macrophages and targeting PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy.
Winson S. Ho, Isha Mondal, Beisi Xu, Oishika Das, Raymond C. Sun, Pochin Chiou, Xiaomin Cai, Foozhan Tahmasebinia, Caren Yu-Ju Wu, Zhihao Wu, William Matsui, Michael Lim, Zhipeng Meng, Rongze Olivia Lu
Inflammatory mediators released by cancer cells promote the induction of immune suppression and tolerance in myeloid cells. Interleukin-1 receptor-associated kinase-3 (IRAK3) is a pseudokinase that inhibits IL-1/TLR signaling but its role in patients treated with immune checkpoint blockade (ICB) therapy remains unclear. Using RNAseq data from the IMvigor210 trial, we found that tumors with high IRAK3 expressions showed enriched anti-inflammatory pathways and worse clinical response to ICB therapy. Upon IRAK3 protein deletion with CRISPR/Cas9, primary human monocytes displayed altered global protein expression and phosphorylation in quantitative proteomics and released more pro-inflammatory cytokines in response to stimulation. Bone-marrow derived macrophages from an IRAK3 CRISPR knockout (KO) mouse model demonstrated a pro-inflammatory phenotype and enhanced sensitivity to TLR agonists, compared to wild-type cells. IRAK3 deficiency delayed the growth of carcinogen-induced and oncogene-driven murine cancer cells and induced enhanced activation in myeloid cells and T cells. Upon ICB treatment, IRAK3 KO mice showed enrichment of TCF1+PD-1+ stem-like memory CD8+ T cells and resulted in superior growth inhibition of immunologically cold tumors in vivo. Altogether, our study demonstrated a novel cancer-driven immune tolerance program controlled by IRAK3 in humans and mice and proposed its suitability as an immunotherapy target.
Gürcan Tunalı, Marta Rúbies Bedós, Divya Nagarajan, Patrik Fridh, Irineos Papakyriacou, Yumeng Mao
Hypersecretory malignant cells underlie therapeutic resistance, metastasis, and poor clinical outcomes. However, the molecular basis for malignant hypersecretion remains obscure. Here, we showed that epithelial-to-mesenchymal transition (EMT) initiates exocytic and endocytic vesicular trafficking programs in lung cancer. The EMT-activating transcription factor ZEB1 executed a PI4KIIIβ-to-PI4KIIα (PI4K2A)-dependency switch that drove PI4P synthesis in Golgi and endosomes. EMT enhanced the vulnerability of lung cancer cells to PI4K2A small molecule antagonists. PI4K2A formed a MYOIIA-containing protein complex that facilitated secretory vesicle biogenesis in the Golgi, thereby establishing a hypersecretory state involving osteopontin (SPP1) and other pro-metastatic ligands. In the endosomal compartment, PI4K2A accelerated recycling of SPP1 receptors to complete an SPP1-dependent autocrine loop and interacted with HSP90 to prevent lysosomal degradation of AXL receptor tyrosine kinase, a driver of cell migration. These results show that EMT coordinates exocytic and endocytic vesicular trafficking to establish a therapeutically actionable hypersecretory state that drives lung cancer progression.
Xiaochao Tan, Guan-Yu Xiao, Shike Wang, Lei Shi, Yanbin Zhao, Xin Liu, Jiang Yu, William K. Russell, Chad J. Creighton, Jonathan M. Kurie
Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves transformation of infected phagolysomes from a site of killing into a nutrient-rich replicative niche. Here we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis (Mtb), 1-tuberculosinyladenosine (1-TbAd), causes lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing Mtb, caused intralysosomal and peribacillary lipid storage patterns that match both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters, and 1-TbAd increased Mtb growth under conditions of restricted lipid access in macrophages. Further, lipidomics dentified 1-TbAd induced lipid substrates that define Gaucher's disease, Wolman's disease and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of Mtb-induced lysosomal failure, leading to successful testing of an gonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.
Melissa Bedard, Sanne van der Niet, Elliott M. Bernard, Gregory H. Babunovic, Tan-Yun Cheng, Beren Aylan, Anita E. Grootemaat, Sahadevan Raman, Laure Botella, Eri Ishikawa, Mary P. O'Sullivan, Seonadh O'Leary, Jacob A. Mayfield, Jeffrey Buter, Adriaan J. Minnaard, Sarah M. Fortune, Leon O. Murphy, Daniel S. Ory, Joseph Keane, Sho Yamasaki, Maximiliano Gabriel Gutierrez, Nicole van der Wel, D. Branch Moody
Sepsis pathogenesis is complex and heterogeneous; hence, a precision medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial reactive oxygen species (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15, a chemical uncoupler, dissipates mitochondrial proton gradients without generating mtROS. We injected BAM15 into mice at 0, 6, or 12 hours after cecal ligation and puncture (CLP) treated with fluids and antibiotics. BAM15 reduced mortality, even after 12 hours, when mice were ill, and BAM15 reduced kidney damage and splenic apoptosis. Serial plasma and urinary mitochondrial DNA (mtDNA) levels increased post-CLP and decreased after BAM15 administration (at 0 or 6 hours). In vitro septic serum proportionately increased mtROS overproduction and mtDNA release from kidney tubule cells, which BAM15 prevented. BAM15 decreased neutrophil apoptosis, mtDNA release; neutrophil depletion counteracted BAM15 benefits. Further, mtDNA injection in vivo replicated inflammation and kidney injury, which was prevented by BAM15. A large dose of exogenous mtDNA reversed protection by BAM15. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis, and that BAM15 and mtDNA, a potential drug-companion diagnostic/drug efficacy pair for clinical sepsis, are mechanistically linked via mtROS.
Naoko Tsuji, Takayuki Tsuji, Tetsushi Yamashita, Naoki Hayase, Xuzhen Hu, Peter S.T. Yuen, Robert A. Star
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