TNFα impairs platelet function by inhibiting autophagy and disrupting metabolism via Syntaxin-17 downregulation

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Abstract

Platelets play a dual role in hemostasis and inflammation-associated thrombosis and hemorrhage. While the mechanisms linking inflammation to platelet dysfunction remain poorly understood, our previous work demonstrated that TNFα alters mitochondrial mass, platelet activation, and autophagy-related pathways in megakaryocytes. Here, we hypothesized that TNFα impairs platelet function by disrupting autophagy, a process critical for mitochondrial health and cellular metabolism. Using human and murine models of TNFα-driven diseases, including myeloproliferative neoplasms and rheumatoid arthritis, we found that TNFα downregulates STX17, a key mediator of autophagosome–lysosome fusion. This disruption inhibited autophagy, leading to the accumulation of dysfunctional mitochondria and reduced mitochondrial respiration. These metabolic alterations compromised platelet-driven clot contraction, a process linked to thrombotic and hemorrhagic complications. Our findings reveal a mechanism by which TNFα disrupts hemostasis through autophagy inhibition, highlighting TNFα as a critical regulator of platelet metabolism and function. This study provides new insights into inflammation-associated pathologies and suggests autophagy-targeting strategies as potential therapeutic avenues to restore hemostatic balance.

Authors

Guadalupe Rojas-Sanchez, Jorge Calzada-Martinez, Brandon McMahon, Aaron C. Petrey, Gabriela Dveksler, Gerardo P. Espino-Solis, Orlando Esparza, Giovanny Hernandez, Dennis Le, Eric P. Wartchow, Ken Jones, Lucas H. Ting, Catherine Jankowski, Marguerite R. Kelher, Marilyn Manco-Johnson, Marie L. Feser, Kevin D. Deane, Travis Nemkov, Angelo D'Alessandro, Andrew Thorburn, Paola Maycotte, José A. López, Pavel Davizon-Castillo

Polygenic modifiers impact penetrance and expressivity in telomere biology disorders

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Abstract

BACKGROUND. Telomere biology disorders (TBDs) exhibit incomplete penetrance and variable expressivity, even among individuals harboring the same pathogenic variant. We assessed whether common genetic variants associated with telomere length combine with large-effect variants to impact penetrance and expressivity in TBDs. METHODS. We constructed polygenic scores (PGS) for telomere length in the UK Biobank to quantify common variant burden, and assessed the PGS distribution across patient cohorts and biobanks to determine whether individuals with severe TBD presentations have increased polygenic burden causing short telomeres. We also characterized rare TBD variant carriers in the UK Biobank. RESULTS. Individuals with TBDs in cohorts enriched for severe pediatric presentations have polygenic scores predictive of short telomeres. In the UK Biobank, we identify carriers of pathogenic TBD variants who are enriched for adult-onset manifestations of TBDs. Unlike individuals in disease cohorts, the PGS of adult carriers do not show a common variant burden for shorter telomeres, consistent with the absence of childhood-onset disease. Notably, TBD variant carriers are enriched for idiopathic pulmonary fibrosis diagnoses, and telomere length PGS stratifies pulmonary fibrosis risk. Finally, common variants affecting telomere length were enriched in enhancers regulating known TBD genes. CONCLUSION. Common genetic variants combine with large-effect causal variants to impact clinical manifestations in rare TBDs. These findings offer a framework for understanding phenotypic variability in other presumed monogenic disorders. FUNDING. This work was supported by National Institutes of Health grants R01DK103794, R01HL146500, R01CA265726, R01CA292941, and the Howard Hughes Medical Institute.

Authors

Michael Poeschla, Uma P. Arora, Amanda Walne, Lisa J. McReynolds, Marena R. Niewisch, Neelam Giri, Logan P. Zeigler, Alexander Gusev, Mitchell J. Machiela, Hemanth Tummala, Sharon A. Savage, Vijay G. Sankaran

Splicing of erythroid transcription factor is associated with therapeutic response in myelodysplastic syndromes

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Abstract

Anemia is the primary clinical manifestation of myelodysplastic syndromes (MDS), but the molecular pathogenesis of ineffective erythropoiesis remains incompletely understood. Luspatercept, an activin receptor 2B (ACVRIIB-Fc) ligand trap, has been approved to treat anemia, however its molecular mechanism of action is unclear. We found that the ACVR2B, its ligand GDF11, and effector, SMAD2, are upregulated in MDS patient samples. GDF11 inhibited human erythropoiesis in vitro and caused anemia in zebrafish, effects that were abrogated by luspatercept. Upon GDF11 stimulation of human erythroid progenitors, SMAD2 binding occurred in the erythroid regulatory regions, including at GATA1 intron. Intronic SMAD2 binding led to skipping of exon 2 of GATA1, resulting in a shorter, hypomorphic isoform (GATA1s). CRISPR deletion of the SMAD2 binding intronic region decreased GATA1s production upon GDF11 stimulation. Expression of gata1s in a mouse model led to anemia, rescued by a murine ActRIIB-Fc (RAP-536). Finally, RNA-seq analysis of samples from the Phase 3 MEDALIST trial revealed that responders to Luspatercept had a higher proportion of GATA1s compared to non-responders. Moreover, the increase RBCs post-treatment was linked to a relative decrease in GATA1s isoform. Our study indicates that GDF11-mediated SMAD2 activation results in an increase in functionally impaired GATA1 isoforms, consequently contributing to anemia and influencing responses to Luspatercept in MDS.

Authors

Srinivas Aluri, Te Ling, Ellen Fraint, Samarpana Chakraborty, Kevin Zhang, Aarif Ahsan, Leah Kravets, Gowri Poigaialwar, Rongbao Zhao, Kith Pradhan, Anitria Cotton, Kimo Bachiashvili, Jung-In Yang, Anjali Budhathoki, Beamon Agarwal, Shanisha Gordon-Mitchell, Milagros Carbajal, Srabani Sahu, Jacqueline Boultwood, Andrea Pellagatti, Ulrich Steidl, Amittha Wickrema, Satish Nandakumar, Aditi Shastri, Rajasekhar N.V.S. Suragani, Teresa V. Bowman, John D. Crispino, Sadanand Vodala, Amit Verma

Ubiquitin-conjugating enzyme UBE2N modulates proteostasis in immunoproteasome-positive acute myeloid leukemia

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Abstract

Altered protein homeostasis through proteasomal degradation of ubiquitinated proteins is a hallmark of many cancers. Ubiquitination, coordinated by E1, E2, and E3 enzymes, involves up to 40 E2-conjugating enzymes in humans to specify substrates and ubiquitin linkages. In a screen for E2 dependencies in acute myeloid leukemia (AML), ubiquitin conjugating enzyme E2 N (UBE2N) emerged as the top candidate. To investigate UBE2N’s role in AML, we characterized an enzymatically defective mouse model of UBE2N, revealing UBE2N’s requirement in AML without an impact on normal hematopoiesis. Unlike other E2s, which mediate lysine-48 (K48) polyubiquitination and degradation of proteins, UBE2N primarily synthesizes K63-linked chains, stabilizing or altering protein function. Proteomic analyses and a whole-genome CRISPR-activation screen in pharmacologically and genetically UBE2N-inhibited AML cells unveiled a network of UBE2N-regulated proteins, many of which are implicated in cancer. UBE2N inhibition reduced their protein levels, leading to increased K48-linked ubiquitination and degradation through the immunoproteasome and revealing UBE2N activity is enriched in immunoproteasome-positive AML. Furthermore, an interactome screen identified tripartite motif–containing protein 21 (TRIM21) as the E3 ligase partnering with activated UBE2N in AML to modulate UBE2N-dependent proteostasis. In conclusion, UBE2N maintains proteostasis in AML by stabilizing target proteins through K63-linked ubiquitination and prevention of K48 ubiquitin–mediated degradation by the immunoproteasome. Thus, inhibition of UBE2N catalytic function suppresses leukemic cells through selective degradation of critical proteins in immunoproteasome-positive AML.

Authors

Chiharu Ishikawa, Laura Barreyro, Avery M. Sampson, Kathleen M. Hueneman, Kwangmin Choi, Sophia Y. Philbrook, Issac Choi, Lyndsey C. Bolanos, Mark Wunderlich, Andrew G. Volk, Stephanie S. Watowich, Kenneth D. Greis, Daniel T. Starczynowski

The tumor suppressor HNRNPK induces p53-dependent nucleolar stress to drive ribosomopathies

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Abstract

The nucleolus is a membraneless organelle and an excellent stress sensor. Any changes in its architecture or composition lead to nucleolar stress, resulting in cell cycle arrest and interruption of ribosomal activity, critical factors in aging and cancer. In this study, we identified and described the pivotal role of the RNA-binding protein (RBP) HNRNPK in ribosome and nucleolar dynamics. We developed an in vitro model of endogenous HNRNPK overexpression and an in vivo mouse model of ubiquitous HNRNPK overexpression. These models showed disruptions in translation and caused alterations in the nucleolar structure, resulting in p53-dependent nucleolar stress, cell cycle arrest, senescence, and bone marrow failure phenotype, similar to what is observed in patients with ribosomopathies. Together, our findings identify HNRNPK as a master regulator of ribosome biogenesis (RiBi) and nucleolar homeostasis through p53, providing a new perspective on the orchestration of nucleolar integrity, ribosome function and cellular senescence.

Authors

Pedro Aguilar-Garrido, María Velasco-Estévez, Miguel Ángel Navarro-Aguadero, Alvaro Otero-Sobrino, Marta Ibañez-Navarro, Miguel Ángel Marugal, María Hernández-Sánchez, Prerna Malaney, Ashley Rodriguez, Oscar Benitez, Xiaorui Zhang, Marisa J.L. Aitken, Alejandra Ortiz-Ruiz, Diego Megias, Manuel Pérez-Martínez, Gadea Mata, Jesús Gomez, Miguel Lafarga, Orlando Dominguez, Osvaldo Graña-Castro, Eduardo Caleiras, Pilar Ximenez-Embun, Marta Isasa, Paloma J. de Andrés, Sandra Rodriguez-Perales, Raul Torres-Ruiz, Enrique Revilla, Rosa María García-Martín, Daniel Azorín, Josune Zubicaray, Julian Sevilla, Oleksandra Sirozh, Vanesa Lafarga, Joaquín Martinez-Lopez, Sean M. Post, Miguel Gallardo

PPIL2 is a target of the JAK2/STAT5 pathway and promotes myeloproliferation via p53-mediated degradation

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Abstract

The activated JAK2/STAT pathway is characteristic of myeloproliferative neoplasms (MPNs). Pleckstrin-2 (PLEK2) signalosome is downstream of the JAK2/STAT5 pathway and plays an important role in MPN development. The detailed molecular composition of this signalosome is unclear. Here, we revealed peptidylprolyl isomerase-like 2 (PPIL2) as a critical component of the complex in regulating human and murine erythropoiesis. PPIL2 was a direct target of STAT5 and was upregulated in MPN patients and a Jak2V617F MPN mouse model. Mechanistically, PPIL2 interacted with and catalyzed p53 polyubiquitination and proteasome-mediated degradation to promote cell growth. Ppil2 deficiency, or inhibition by cyclosporin A, led to a marked upregulation of p53 in vivo and ameliorated myeloproliferative phenotypes in Jak2V617F mice. Cyclosporin A also markedly reduced JAK2 mutated erythroid and myeloid proliferation in an induced pluripotent stem cell-derived human bone marrow organoid model. Our findings revealed PPIL2 as a critical component of the PLEK2 signalosome in driving MPN pathogenesis through negatively regulating p53, thus providing a target and an opportunity for drug repurposing by using cyclosporin A to treat MPNs.

Authors

Pan Wang, Xu Han, Kehan Ren, Ermin Li, Honghao Bi, Inci Aydemir, Madina Sukhanova, Yijie Liu, Jing Yang, Peng Ji

Inborn errors of immunity underlie clonal T cell expansions in large granular lymphocyte leukemia

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Abstract

BACKGROUND T cell large granular lymphocyte leukemia (T-LGLL) is a lymphoproliferative disorder of cytotoxic T lymphocytes (CTLs), often with gain-of-function STAT3 mutations. T-LGLL represents a unique model for the study of persistent CTL expansions. Albeit autoimmunity is implied, various paradoxical observations led us to investigate whether immunodeficiency traits underpin T-LGLL.METHODS This is a comprehensive immunogenomic study of 92 consecutive patients from a large T-LGLL cohort with full laboratory-clinical characterization (n = 271). Whole-exome profiling of variants associated with inborn errors of immunity (IEI) and somatic mutations in T cell lymphoid drivers was analyzed. Single-cell RNA-Seq and TCR-Seq in T-LGLL samples and RNA-Seq in T cell cancer cell lines were utilized to establish biological correlations.RESULTS Lymphocytopenia and/or hypogammaglobulinemia were identified in 186 of 241 (77%) T-LGLL patients. Genetic screening for IEI revealed 43 rare heterozygous variants in 38 different immune genes in 34 of 92 (36%) patients (vs. 167/63,026 [0.26%] in controls). High-confidence deleterious variants associated with dominant, adult-onset IEIs were detected in 15 of 92 (16%) patients. Carriers showed atypical features otherwise tied to the cryptic IEI, such as earlier onset, lower lymphocyte counts, lower STAT3 mutational rate, and higher proportions of hypogammaglobulinemia and immune cytopenia/bone marrow failure than noncarriers. Somatic mutational landscape, RNA-Seq, and TCR-Seq analyses supported immune imbalance caused by the IEI variants and interactions with somatic mutations in T cell lymphoid drivers.CONCLUSIONS Our findings in T-LGLL reveal that maladaptive CTL expansions may stem from cryptic immunodeficiency traits and open the horizon of IEIs to clonal hematopoiesis and bone marrow failure.FUNDING NIH; Aplastic Anemia and MDS International Foundation; VeloSano; Edward P. Evans Foundation; Instituto de Salud Carlos III; European Research Council; European Research Area Network on Personalised Medicine; Academy Finland; Cancer Foundation Finland.

Authors

Carlos Bravo-Perez, Carmelo Gurnari, Jani Huuhtanen, Naomi Kawashima, Luca Guarnera, Aashray Mandala, Nakisha D. Williams, Christopher Haddad, Michaela Witt, Serhan Unlu, Zachary Brady, Olisaemeka Ogbue, Mark Orland, Arooj Ahmed, Yasuo Kubota, Simona Pagliuca, Arda Durmaz, Satu Mustjoki, Valeria Visconte, Jaroslaw P. Maciejewski

Biallelic OSM deficiency presents with juvenile myelodysplastic syndrome and response to treatment

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Abstract

Authors

Abdullah H. Alfalah, Alfadil Haroon, Ahmed Alfares, Syed Osman Ahmed, Sateesh Maddirevula

Identification of CD84 as a potent survival factor in acute myeloid leukemia

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Abstract

Acute myeloid leukemia (AML) is an aggressive and often deadly malignancy associated with proliferative immature myeloid blasts. Here, we identified CD84 as a critical survival regulator in AML. High levels of CD84 expression provided a survival advantage to leukemia cells, whereas CD84 downregulation disrupted their proliferation, clonogenicity and engraftment capabilities in both human cell lines and patient derived xenograft cells. Critically, loss of CD84 also markedly blocked leukemia engraftment and clonogenicity in MLL-AF9 and inv(16) AML mouse models, highlighting its pivotal role as survival factor across species. Mechanistically, CD84 regulated leukemia cells’ energy metabolism and mitochondrial dynamics. Depletion of CD84 altered mitochondrial ultra-structure and function of leukemia cells, and it caused down-modulation of both oxidative phosphorylation and fatty acid oxidation pathways. CD84 knockdown induced a block of Akt phosphorylation and down-modulation of nuclear factor erythroid 2-related factor 2 (NRF2), impairing AML antioxidant defense. Conversely, CD84 over-expression stabilized NRF2 and promoted its transcriptional activation, thereby supporting redox homeostasis and mitochondrial function in AML. Collectively, our findings indicated that AML cells depend on CD84 to support antioxidant pro-survival pathways, highlighting a therapeutic vulnerability of leukemia cells.

Authors

Yinghui Zhu, Mariam Murtadha, Miaomiao Liu, Enrico Caserta, Ottavio Napolitano, Le Xuan Truong Nguyen, Huafeng Wang, Milad Moloudizargari, Lokesh Nigam, Theophilus Tandoh, Xuemei Wang, Alex Pozhitkov, Rui Su, Xiangjie Lin, Marc Denisse Estepa, Raju Pillai, Joo Song, James F. Sanchez, Yu-Hsuan Fu, Lianjun Zhang, Man Li, Bin Zhang, Ling Li, Ya-Huei Kuo, Steven Rosen, Guido Marcucci, John C. Williams, Flavia Pichiorri

ATM-dependent DNA damage response constrains cell growth and drives clonal hematopoiesis in telomere biology disorders

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Abstract

Telomere biology disorders (TBD) are genetic diseases caused by defective telomere maintenance. TBD patients often develop bone marrow failure and have an increased risk of myeloid neoplasms. To better understand the factors underlying hematopoietic outcomes in TBD, we comprehensively evaluated acquired genetic alterations in hematopoietic cells from 166 pediatric and adult TBD patients. 47.6% of patients (28.8% of children, 56.1% of adults) had clonal hematopoiesis. Recurrent somatic alterations involved telomere maintenance genes (7.6%), spliceosome genes (10.4%, mainly U2AF1 p.S34), and chromosomal alterations (20.2%), including 1q gain (5.9%). Somatic variants affecting the DNA damage response (DDR) were identified in 21.5% of patients, including 20 presumed loss-of-function variants in ATM. Using multimodal approaches, including single-cell sequencing, assays of ATM activation, telomere dysfunction-induced foci analysis, and cell growth assays, we demonstrate telomere dysfunction-induced activation of ATM-dependent DDR pathway with increased senescence and apoptosis in TBD patient cells. Pharmacologic ATM inhibition, modeling the effects of somatic ATM variants, selectively improved TBD cell fitness by allowing cells to bypass DDR-mediated senescence without detectably inducing chromosomal instability. Our results indicate that ATM-dependent DDR induced by telomere dysfunction is a key contributor to TBD pathogenesis and suggest dampening hyperactive ATM-dependent DDR as a potential therapeutic intervention.

Authors

Christopher M. Sande, Stone Chen, Dana V. Mitchell, Ping Lin, Diana M. Abraham, Jessie M. Cheng, Talia Gebhard, Rujul J. Deolikar, Colby Freeman, Mary Zhou, Sushant Kumar, Michael Bowman, Robert L. Bowman, Shannon Zheng, Bolormaa Munkhbileg, Qijun Chen, Natasha L. Stanley, Kathy Guo, Ajibike Lapite, Ryan Hausler, Deanne M. Taylor, James Corines, Jennifer J.D. Morrissette, David B. Lieberman, Guang Yang, Olga Shestova, Saar Gill, Jiayin Zheng, Kelcy Smith-Simmer, Lauren G. Banaszak, Kyle N. Shoger, Erica F. Reinig, Madilynn Peterson, Peter Nicholas, Amanda J. Walne, Inderjeet Dokal, Justin P. Rosenheck, Karolyn A. Oetjen, Daniel C. Link, Andrew E. Gelman, Christopher R. Reilly, Ritika Dutta, R. Coleman Lindsley, Karyn J. Brundige, Suneet Agarwal, Alison A. Bertuch, Jane E. Churpek, Laneshia K. Tague, F. Brad Johnson, Timothy S. Olson, Daria V. Babushok