Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model

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Abstract

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modelled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/- and MPLW515K providing 20 different trisomy or disomy 21 iPSC clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility and GATA1 binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally-involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides a unique array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Authors

Brahim Arkoun, Elie Robert, Fabien Boudia, Stefania Mazzi, Virginie Dufour, Aurelie Siret, Yasmine Mammasse, Zakia Aid, Mathieu Vieira, Aygun Imanci, Marine Aglave, Marie Cambot, Rachel Petermann, Sylvie Souquere, Philippe Rameau, Cyril Catelain, Romain Diot, Gerard Tachdjian, Olivier Hermine, Nathalie Droin, Najet Debili, Isabelle Plo, Sebastien Malinge, Eric Soler, Hana Raslova, Thomas Mercher, William Vainchenker

Erythroid lineage Jak2^V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis

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Abstract

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis (CH). Jak2VF mice with elevated white blood cells, platelets and red blood cells (RBCs) display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic Erythropoietin Receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis and ferroptosis. Selective induction of erythrocytosis with low dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor Liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating CH, but had no impact in controls. Erythroid lineage Jak2VF expression leads to qualitative and quantitative defects in RBCs that exacerbate atherosclerosis. Phagocytosis of RBCs by plaque macrophages promotes ferroptosis, suggesting a new therapeutic target to reduce RBC-mediated cardiovascular risk.

Authors

Wenli Liu, Nataliya K. Östberg, Mustafa Yalcinkaya, Huijuan Dou, Kaori Endo-Umeda, Yang Tang, Xintong Hou, Tong Xiao, Trevor Filder, Sandra Abramowicz, Yong-Guang Yang, Oliver Soehnlein, Alan R. Tall, Nan Wang

Repeated exposure to heterologous hepatitis C viruses associates with enhanced neutralizing antibody breadth and potency

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Abstract

A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically-related, antibody sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody sensitive viruses is a promising approach to induce potent bNAbs in humans.

Authors

Nicole Frumento, Alexis Figueroa, Tingchang Wang, Muhammad Nauman Zahid, Shuyi Wang, Guido Massaccesi, Georgia Stavrakis, James E. Crowe, Jr., Andrew I. Flyak, Hongkai Ji, Stuart C. Ray, George Shaw, Andrea L Cox, Justin R. Bailey

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naïve B cells

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Abstract

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T-cell compartments by supporting homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B-cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B-cells into Rag2-/- mouse recipients. Highly purified follicular B-cells transdifferentiated into marginal zone-like B-cells when transferred into Rag2-/- lymphopenic hosts, but not into wild-type hosts. In lymphopenic spleens, transferred B-cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B-cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B-cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B-cells and expression of Delta-like1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B-cells. Thus, naïve mature B-cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Authors

Daniela Gómez Atria, Brian T. Gaudette, Jennifer Londregan, Samantha Kelly, Eric Perkey, Anneka Allman, Bhaskar Srivastava, Ute Koch, Freddy Radtke, Burkhard Ludewig, Christian W. Siebel, Russell J.H. Ryan, Tanner F. Robertson, Janis K. Burkhardt, Warren S. Pear, David Allman, Ivan Maillard

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice

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Abstract

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Over-expression of wild-type PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using novel artificial microRNAs targeting human and mouse PMP22/Pmp22 mRNAs. Our lead therapeutic microRNA, miR871, was packaged into an AAV9 vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22/Pmp22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. Treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871-driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

Authors

Marina Stavrou, Alexia Kagiava, Sarah G. Choudury, Matthew J. Jennings, Lindsay M. Wallace, Allison M. Fowler, Amanda Heslegrave, Jan Richter, Christina Tryfonos, Christina Christodoulou, Henrik Zetterberg, Rita Horvath, Scott Q. Harper, Kleopas A. Kleopa

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR mutant lung cancer

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Abstract

Molecularly targeted cancer therapy has improved outcomes for cancer patients with targetable oncoproteins, such as mutant epidermal growth factor receptor (EGFR) in lung cancer. Yet, long-term patient survival remains limited because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations in the EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alteration of the mRNA splicing factor RBM10 that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of Bcl-xS-(pro-apoptotic)-to-Bcl-xL(anti-apoptotic) Bcl-x isoforms. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Co-inhibition of Bcl-xL and mutant EGFR overcame resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations, and on the impact of splicing factor deficiency in the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Authors

Shigeki Nanjo, Wei Wu, Niki Karachaliou, Collin M. Blakely, Junji Suzuki, Yu-Ting Chou, Siraj M. Ali, D. Lucas Kerr, Victor R. Olivas, Jonathan Shue, Julia Rotow, Manasi K. Mayekar, Franziska Haderk, Nilanjana Chatterjee, Anatoly Urisman, Jia Chi Yeo, Anders J. Skanderup, Aaron C. Tan, Wai Leong Tam, Oscar Arrieta, Kazuyoshi Hosomichi, Akihiro Nishiyama, Seiji Yano, Yuriy Kirichok, Daniel S.W. Tan, Rafael Rosell, Ross A. Okimoto, Trever G. Bivona

mTORC2 mediates structural plasticity in distal nociceptive endings that contributes to pain hypersensitivity following inflammation

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Abstract

The encoding of noxious stimuli into action potential firing is largely mediated by nociceptive free nerve endings. Tissue inflammation, by changing the intrinsic properties of the nociceptive endings, leads to nociceptive hyperexcitability, and thus to the development of inflammatory pain. Here, we showed that tissue inflammation-induced activation of the mammalian target of rapamycin complex 2 (mTORC2) triggers changes in the architecture of nociceptive terminals and leads to inflammatory pain. Pharmacological activation of mTORC2 induced elongation and branching of nociceptor peripheral endings and caused long-lasting pain hypersensitivity. Conversely, nociceptor-specific deletion of the mTORC2 regulatory protein, Rictor, prevented inflammation-induced elongation and branching of cutaneous nociceptive fibers and attenuated inflammatory pain hypersensitivity. Computational modelling demonstrated that mTORC2-mediated structural changes in the nociceptive terminal tree are sufficient to increase the excitability of nociceptors. Targeting mTORC2 using a single injection of antisense oligonucleotide against Rictor provided long-lasting alleviation of inflammatory pain hypersensitivity. Collectively, we showed that tissue inflammation-induced activation of mTORC2 causes structural plasticity of nociceptive free nerve endings in the epidermis and inflammatory hyperalgesia, representing a therapeutic target for inflammatory pain.

Authors

Calvin Wong, Omer Barkai, Feng Wang, Carolina Thörn Pérez, Shaya Lev, Weihua Cai, Shannon Tansley, Noosha Yousefpour, Mehdi Hooshmandi, Kevin C. Lister, Mariam Latif, A. Claudio Cuello, Masha Prager-Khoutorsky, Jeffrey S. Mogil, Philippe Séguéla, Yves De Koninck, Alfredo Ribeiro-da-Silva, Alexander M. Binshtok, Arkady Khoutorsky

KRAS mutant-driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer

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Abstract

Lymph node (LN) metastasis occurs frequently in pancreatic ductal adenocarcinoma (PDAC) and predicts poor prognosis for patients. The KRASG12D mutation confers an aggressive PDAC phenotype that is susceptible to lymphatic dissemination. However, the regulatory mechanism underlying KRASG12D mutation-driven LN metastasis in PDAC remains unclear. Herein, we identified that PDAC with KRASG12D mutation (KRASG12D PDAC) sustained extracellular vesicle (EV)-mediated transmission of hnRNPA1 in a SUMOylation-dependent manner and promoted lymphangiogenesis and LN metastasis in vitro and in vivo. Mechanistically, hnRNPA1 bound with SUMO2 at the lysine 113 residue via KRASG12D-induced hyperactivation of SUMOylation, which enabled its interaction with TSG101 to enhance hnRNPA1 packaging and transmission via EVs. Subsequently, SUMOylation induced EV-packaged hnRNPA1 anchoring to the adenylate and uridylate-rich elements of PROX1 in lymphatic endothelial cells, thus stabilizing PROX1 mRNA. Importantly, impeding SUMOylation of EV-packaged hnRNPA1 dramatically inhibited LN metastasis of KRASG12D PDAC in a genetically engineered KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mice model. Our findings highlight the mechanism by which KRAS mutant-driven SUMOylation triggers EV-packaged hnRNPA1 transmission to promote lymphangiogenesis and LN metastasis, shedding light on the potential application of hnRNPA1 as a therapeutic target in patients with KRASG12D PDAC.

Authors

Yuming Luo, Zhihua Li, Yao Kong, Wang He, Hanhao Zheng, Mingjie An, Yan Lin, Dingwen Zhang, Jiabin Yang, Yue Zhao, Changhao Chen, Rufu Chen

LFA-1 activation enriches tumor-specific T cells in a cold tumor model and synergizes with CTLA-4 blockade

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Abstract

The inability of CD8+ T effectors (Teff) to reach tumor cells is an important mechanism of tumor resistance to cancer immunotherapy. The recruitment of these cells to the tumor microenvironment (TME) is regulated by integrins, a family of adhesion molecules that is expressed on T cells. Here we show that 7HP349, a small molecule activator of Lymphocyte function–associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrin-cell-adhesion receptors, facilitated the preferential localization of tumor-specific T cells to the tumor and improve antitumor response. 7HP349 monotherapy had modest effects on anti- programmed death 1 (PD-1)–resistant tumors, whereas combinatorial treatment with anti- T-lymphocyte-associated protein 4 (CTLA-4) therapy increased CD8+ Teff intratumoral sequestration and synergized in inducing cancer regression, in cooperation with neutrophils. 7HP349 intratumoral CD8+ Teff enrichment activity depended on CXCL12. We analyzed gene expression profiles using RNA from baseline and on treatment tumor samples of 14 melanoma patients. We identified baseline CXCL12 gene expression may improve response likelihood to anti-CTLA-4 therapies. Our results provided a proof-of-principle demonstration that LFA-1 activation could convert a T cell-exclusionary TME to a T-cell enriched TME through mechanisms involving cooperation with innate immune cells.

Authors

Amber Hickman, Joost Koetsier, Trevin Kurtanich, Michael C. Nielsen, Glenn Winn, Yunfei Wang, Salah-Eddine Bentebibel, Leilei Shi, Simone Punt, Leila Williams, Cara Haymaker, Charles B. Chesson, Faisal Fa'ak, Ana Dominguez, Richard Jones, Isere Kuiatse, Amy R. Caivano, Sayadeth Khounlo, Navin D. Warier, Upendra Marathi, Robert V. Market, Ronald J. Biediger, John W. Craft Jr, Patrick Hwu, Michael A. Davies, Darren G. Woodside, Peter Vanderslice, Adi Diab, Willem W. Overwijk, Yared Hailemichael

Factors influencing maternal microchimerism throughout infancy and its impact on infant T cell immunity

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Abstract

Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors which influence MMc detection and level across infancy and the effect of MMc on T cell responses to BCG vaccination in a cohort of HIV exposed, uninfected and HIV unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day one and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy with levels ranging from 0-1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal-infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc levels in HIV exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence MMc, which may subsequently impact infant T cell responses.

Authors

Christina Balle, Blair Armistead, Agano Kiravu, Xiaochang Song, Anna-Ursula Happel, Angela A. Hoffmann, Sami B. Kanaan, J. Lee Nelson, Clive M. Gray, Heather B. Jaspan, Whitney E. Harrington