Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR-Cas9 screens implicated decreased translation initiation as protective to GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to GSPT1 degradation. We defined two Crbn amino acids that prevent Gspt1 degradation in mice, generated a knock-in mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant AML.
Rob S. Sellar, Adam S. Sperling, Mikołaj Słabicki, Jessica A. Gasser, Marie E. McConkey, Katherine A. Donovan, Nada Mageed, Dylan N. Adams, Charles Zou, Peter G. Miller, Ravi Kumar Dutta, Steffen Boettcher, Amy E. Lin, Brittany E. Sandoval, Vanessa A. Quevedo Barrios, Veronica Shkolnik, Jonas Koeppel, Elizabeth K. Henderson, Emma C. Fink, Lu Yang, Anthony K.N. Chan, Sheela Pangeni Pokharel, Erik J. Bergstrom, Rajan Burt, Namrata D. Udeshi, Steven A. Carr, Eric S. Fischer, Chun-Wei Chen, Benjamin L. Ebert
Human cytomegalovirus (HCMV) is the most common congenital infection and a leading cause of stillbirth, neurodevelopmental impairment, and pediatric hearing loss worldwide. Development of a maternal vaccine or therapeutic to prevent congenital HCMV has been hindered by limited knowledge of the immune responses that protect against HCMV transmission in utero. To identify protective antibody responses, we measured HCMV-specific IgG binding and anti-viral functions in paired maternal and cord blood sera from HCMV seropositive transmitting (n=41) and non-transmitting (n=40) mother-infant dyads identified via a large U.S.-based public cord blood bank. We found that high avidity IgG binding to HCMV and antibody-dependent cellular phagocytosis (ADCP) were associated with reduced risk of congenital HCMV infection. We also determined that HCMV-specific IgG activation of FcγRI and FcγRII was enhanced in non-transmitting dyads and that increased ADCP responses were mediated through both FcγRI and FcγRIIA expressed on human monocytes. These findings suggest that engagement of FcγRI/FcγRIIA and Fc effector functions including ADCP may protect against congenital HCMV infection. Taken together, these data can guide future prospective studies on immune correlates against cCMV transmission and inform HCMV vaccine and immunotherapeutic development.
Eleanor C. Semmes, Itzayana G. Miller, Courtney E. Wimberly, Caroline T. Phan, Jennifer A. Jenks, Melissa J. Harnois, Stella J. Berendam, Helen Webster, Jillian H. Hurst, Joanne Kurtzberg, Genevieve G. Fouda, Kyle M. Walsh, Sallie R. Permar
Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor-3 (VEGFR3), encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here we used gene deletion, blocking antibodies, transgene induction and gene transfer to study how Ang2, its Tie2 receptor and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) is involved in full Akt activation downstream of phosphoinositide-3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of Ang2 blocking antibody decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of PI3K catalytic p110α subunit or with small molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2-Tie-PI3K signaling.
Emilia A. Korhonen, Aino Murtomäki, Sawan Kumar Jha, Andrey Anisimov, Anne Pink, Yan Zhang, Simon Stritt, Inam Liaqat, Lukas Stanczuk, Laura Alderfer, Zhiliang Sun, Emmi Kapiainen, Abhishek Singh, Ibrahim Sultan, Anni Lantta, Veli-Matti Leppänen, Lauri Eklund, Yulong He, Hellmut G. Augustin, Kari Vaahtomeri, Pipsa Saharinen, Taija Mäkinen, Kari Alitalo
Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1β, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free wild type mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
Michael J. Grey, Heidi De Luca, Doyle V. Ward, Irini A.M. Kreulen, Katlynn Bugda Gwilt, Sage E. Foley, Jay R. Thiagarajah, Beth A. McCormick, Jerrold R. Turner, Wayne I. Lencer
Acquired resistance is inevitable in non-small cell lung cancers (NSCLCs) treated with osimertinib (OSI), and the mechanisms are not well defined. The MERTK ligand GAS6 promoted downstream oncogenic signaling in EGFR-mutated (EGFRMT) NSCLC cells treated with OSI, suggesting a role for MERTK activation in OSI resistance. Indeed, treatment with MRX-2843, a first-in-class MERTK kinase inhibitor, re-sensitized GAS6-treated NSCLC cells to OSI. Both GAS6 and EGF stimulated downstream PI3K-AKT and MAPK-ERK signaling in parental cells, but only GAS6 activated these pathways in OSI resistant (OSIR) derivative cell lines. Functionally, OSIR cells were more sensitive to MRX-2843 than parental cells, suggesting acquired dependence on MERTK signaling. Furthermore, MERTK and/or its ligands were dramatically upregulated in EGFRMT tumors after treatment with OSI in both xenograft models and patient samples, consistent with induction of autocrine/paracrine MERTK activation. Moreover, treatment with MRX-2843 in combination with OSI, but not OSI alone, provided durable suppression of tumor growth in vivo, even after treatment was stopped. These data identify MERTK as a driver of bypass signaling in treatment-naïve and EGFRMT-OSIR NSCLC cells and predict that MRX-2843 and OSI combination therapy will provide clinical benefit in patients with EGFRMT NSCLC.
Dan Yan, Justus M. Huelse, Dmitri Kireev, Zikang Tan, Luxiao Chen, Subir Goyal, Xiaodong Wang, Stephen V. Frye, Madhusmita Behera, Frank Schneider, Suresh S. Ramalingam, Taofeek K. Owonikoko, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham
Aberrant expression of viral-like repeat elements is a common feature in epithelial cancers, but the significant diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering that is independent of tissue of origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that HSATII satellite repeat expression was highly associated with epithelial mesenchymal transition (EMT) and anti-correlated with interferon (IFN) response genes indicative of a more aggressive phenotype. This relationship of HSATII with high EMT and low IFN response genes was also found in RNA-seq of primary ovarian cancers and associated with significantly shorter survival in a second independent cohort of ovarian cancer patients. Repeat RNAs were also found enriched in tumor derived extracellular vesicles that were capable of stimulating monocyte derived macrophages demonstrating a mechanism of altering the tumor microenvironment with these viral-like sequences. Targeting of HSATII with anti-sense locked nucleic acids (LNAs) stimulated IFN response and induced MHC I expression in ovarian cancer cells lines, highlighting a potential strategy of modulating the repeatome to re-establish anti-tumor cell immune surveillance.
Rebecca L. Porter, Siyu Sun, Micayla N. Flores, Emily Berzolla, Eunae You, Ildiko E. Phillips, Neelima KC, Niyati Desai, Eric C. Tai, Annamaria Szabolcs, Evan R. Lang, Amaya Pankaj, Michael J. Raabe, Vishal Thapar, Katherine H. Xu, Linda T. Nieman, Daniel C. Rabe, David L. Kolin, Elizabeth H. Stover, David Pepin, Shannon L. Stott, Vikram Deshpande, Joyce F. Liu, Alexander Solovyov, Ursula A. Matulonis, Benjamin D. Greenbaum, David T. Ting
A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens and T cell repertoire size is believed to decline with age. However, the precise size of human TCR repertoire in total and subsets of T cells, and their changes with age are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRβ repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier (UMI) based RNAseq method. Thorough analysis of 1.9 x 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 x 108. Alterations of TCR repertoire with age were observed in all four subsets of T cells. The greatest reduction was observed in naïve CD8+ T cells; the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting aging has a more profound impact on the cytotoxic than on the helper T cell functions. This may explain the increased susceptibility of older adults to the novel infections.
Xiaoping Sun, Thomas Nguyen, Achouak Achour, Annette Ko, Jeffrey Cifello, Chen Ling, Jay Sharma, Toyoko Hiroi, Yongqing Zhang, Chee W. Chia, William Wood III, Wells W. Wu, Linda Zukley, Je-Nie Phue, Kevin G. Becker, Rong-Fong Shen, Luigi Ferrucci, Nan-ping Weng
Mitochondrial stress triggers a response in the cell’s mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DELE1. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 is critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.
Mario K. Shammas, Xiaoping Huang, Beverly P. Wu, Evelyn Fessler, Insung Song, Nicholas P. Randolph, Yan Li, Christopher K.E. Bleck, Danielle A. Springer, Carl Fratter, Ines A. Barbosa, Andrew F. Powers, Pedro M. Quirós, Carlos Lopez-Otin, Lucas T. Jae, Joanna Poulton, Derek P. Narendra
Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
Peibo Xu, Hui He, Qinqin Gao, Yingying Zhou, Ziyan Wu, Xiao Zhang, Linyu Sun, Gang Hu, Qian Guan, Zhiwen You, Xinyue Zhang, Wenping Zheng, Man Xiong, Yuejun Chen
Resistance to regeneration of insulin-producing pancreatic beta cells is a fundamental challenge for Type 1 and Type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human beta cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and beta cell transcriptomic databases seeking to understand why beta cells in insulinomas proliferate, while normal beta cells do not. This search suggested the DREAM complex as a central regulator of quiescence in human beta cells. DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here we demonstrate that small molecule DYRK1A inhibitors induce human beta cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.
Peng Wang, Esra Karakose, Carmen Argmann, Huan Wang, Metodi Balev, Rachel I. Brody, Hembly G. Rivas, Xinyue Liu, Olivia Wood, Hongtao Liu, Lauryn Choleva, Dan Hasson, Emily Bernstein, Joao A. Paulo, Donald K. Scott, Luca Lambertini, James A. DeCaprio, Andrew F. Stewart.
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