Molecular profiling of clear cell RCC (ccRCC) tumors of clinical trial patients has identified distinct transcriptomic signatures with predictive value, yet data in non-clear cell variants (nccRCC) are lacking. We examined the transcriptional profiles of RCC tumors representing key molecular pathways, from a multi-institutional, real-world patient cohort, including ccRCC (n = 508) and centrally-reviewed nccRCC (n = 149) samples. ccRCC had increased angiogenesis signature scores compared to the heterogeneous group of nccRCC tumors (mean z-score 0.37 vs –0.99, P < 0.001), while cell cycle, fatty acid oxidation (FAO)/AMPK signaling, fatty acid synthesis (FAS)/pentose phosphate signature scores were increased in one or more nccRCC subtypes. Among both ccRCC and nccRCC tumors, T-effector scores statistically correlated with increased immune cell infiltration and were more commonly associated with immunotherapy-related markers (PD-L1+/TMB-High/MSI-High). In conclusion, this study provides evidence of differential gene transcriptional profiles among ccRCC vs nccRCC tumors, providing new insights for optimizing personalized and histology-specific therapeutic strategies for patients with advanced RCC.
Pedro Barata, Shuchi Gulati, Andrew Elliott, Hans J. Hammers, Earle F. Burgess, Benjamin A. Gartrell, Sourat Darabi, Mehmet A. Bilen, Arnab Basu, Daniel M. Geynisman, Nancy A. Dawson, Matthew R. Zibelman, Tian Zhang, Shuanzeng Wei, Charles J. Ryan, Elisabeth I. Heath, Kelsey A. Poorman, Chadi Nabhan, Rana R. McKay
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor–ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.
Tobias Suske, Helena Sorger, Gabriele Manhart, Frank Ruge, Nicole Prutsch, Mark W. Zimmerman, Thomas Eder, Diaaeldin I. Abdallah, Barbara Maurer, Christina Wagner, Susann Schönefeldt, Katrin Spirk, Alexander Pichler, Tea Pemovska, Carmen Schweicker, Daniel Pölöske, Emina Hubanic, Dennis Jungherz, Tony Andreas Müller, Myint Myat Khine Aung, Anna Orlova, Ha Thi Thanh Pham, Kerstin Zimmel, Thomas Krausgruber, Christoph Bock, Mathias Müller, Maik Dahlhoff, Auke Boersma, Thomas Rülicke, Roman Fleck, Elvin Dominic de Araujo, Patrick Thomas Gunning, Tero Aittokallio, Satu Mustjoki, Takaomi Sanda, Sylvia Hartmann, Florian Grebien, Gregor Hoermann, Torsten Haferlach, Philipp Bernhard Staber, Heidi Anne Neubauer, Alfred Thomas Look, Marco Herling, Richard Moriggl
Mutations in genes encoding chromatin modifiers are enriched among mutations causing intellectual disability. The continuing development of the brain postnatally, coupled with the inherent reversibility of chromatin modifications, may afford an opportunity for therapeutic intervention following a genetic diagnosis. Development of treatments requires an understanding of protein function and models of the disease. Here, we provide a mouse model of Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) (OMIM 603736) and demonstrate proof-of-principle efficacy of postnatal treatment. SBBYSS results from heterozygous mutations in the KAT6B (MYST4/MORF/QFK) gene and is characterized by intellectual disability and autism-like behaviors. Using human cells carrying SBBYSS-specific KAT6B mutations and Kat6b heterozygous mice (Kat6b+/–), we showed that KAT6B deficiency caused a reduction in histone H3 lysine 9 acetylation. Kat6b+/– mice displayed learning, memory, and social deficits, mirroring SBBYSS individuals. Treatment with a histone deacetylase inhibitor, valproic acid, or an acetyl donor, acetyl-carnitine (ALCAR), elevated histone acetylation levels in the human cells with SBBYSS mutations and in brain and blood cells of Kat6b+/– mice and partially reversed gene expression changes in Kat6b+/– cortical neurons. Both compounds improved sociability in Kat6b+/– mice, and ALCAR treatment restored learning and memory. These data suggest that a subset of SBBYSS individuals may benefit from postnatal therapeutic interventions.
Maria I. Bergamasco, Hannah K. Vanyai, Alexandra L. Garnham, Niall D. Geoghegan, Adam P. Vogel, Samantha Eccles, Kelly L. Rogers, Gordon K. Smyth, Marnie E. Blewitt, Anthony J. Hannan, Tim Thomas, Anne K. Voss
The mammalian SUMO-targeted E3 Ubiquitin Ligase, Rnf4, has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4 conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi Anemia gene family and the helicases, PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anti-cancer therapy, especially in tumors expressing c-myc.
Joonyoung Her, Haiyan Zheng, Samuel F. Bunting
BACKGROUND. Weakly virulent environmental mycobacteria (EM) can cause severe disease in HLA-DRB1*15:02 or 16:02 adult individuals harboring neutralizing anti-IFN-γ autoantibodies (nAIGAs). The overall prevalence of nAIGA in a general population are unknown as is the the penetrance of nAIGA in HLA-DRB1*15:02 or 16:02 individuals, and the proportion of patients with unexplained, adult-onset EM infections carrying nAIGAs. METHODS. This study analyzed the detection and neutralization of anti-IFN-γ autoantibodies (auto-Abs) from 8,430 healthy individuals of the general population, 257 HLA-DRB1*15:02 or 16:02 carriers, 1,063 patients with autoimmune disease, and 497 patients with unexplained severe disease due to EM. RESULTS. We find that anti-IFN-γ autoantibodies detected in 4,148 of 8,430 healthy individuals (49.2%) from the general population of an unknown HLA-DRB1 genotype are not neutralizing. Moreover, we do not find nAIGAs in 257 individuals carrying HLA-DRB1* 15:02 or 16:02, including 71 individuals with detectable anti-IFN-g autoantibodies (27.6%). Additionally, nAIGA are absent in 1,063 patients with an autoimmune disease. Furthermore, we find only a few other autoantibodies in seven patients with nAIGAs tested. Finally, seven of 497 patients (1.4%) with unexplained severe disease due to EM harbored nAIGA. Yet, nAIGA are absent in the remaining 41 patients who are HLA-DRB1*15:02 or 16:02, the 45 patients with IFN-g autoantibodies, and the five patients with HLA-DRB1*15:02 or 16:02 and IFN-g autoantibodies . CONCLUSION. These findings suggest that nAIGAs are isolated and that their penetrance in HLA-DRB1*15:02 or 16:02 individuals is low, implying that they may be triggered by rare germline or somatic variants. In contrast, the risk of mycobacterial disease in patients with nAIGAs is high, confirming that these nAIGAs are causal of EM disease. FUNDING. The Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI095983), the National Center for Advancing Translational Sciences (NCATS), the NIH Clinical and Translational Science Award (CTSA) program (UL1 TR001866), and partly by French National Research Agency (ANR).
Jessica N. Peel, Rui Yang, Tom Le Voyer, Adrian Gervais, Jérémie Rosain, Paul Bastard, Anish Behere, Axel Cederholm, Aaron Bodansky, Yoann Seeleuthner, Clément Conil, Jing-Ya Ding, Wei-Te Lei, Lucy Bizien, Camille Soudee, Mélanie Migaud, Masato Ogishi, Ahmad Yatim, Danyel Lee, Jonathan Bohlen, Thomas Perpoint, Laura Perez, Fernando Messina, Roxana Genet, Ludovic Karkowski, Mathieu Blot, Emmanuel Lafont, Laurie Toullec, Claire Goulvestre, Souad Mehlal-Sedkaoui, Jérôme Sallette, Fernando Martin, Anne Puel, Emmanuelle Jouanguy, Mark S. Anderson, Nils Landegren, Pierre Tiberghien, Laurent Abel, Stéphanie Boisson-Dupuis, Jacinta Bustamante, Cheng-Lung Ku, Jean-Laurent Casanova
Loss of BRCA2 (BReast CAncer 2) is lethal for normal cells. Yet, it remains poorly understood how in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene, MLH1 as a genetic interactor of BRCA2 whose over-expression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed the genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described that estrogen induces MLH1 expression through estrogen receptor alpha (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and how it may contribute to the establishment of BRCA2-deficient breast tumors.
Satheesh K. Sengodan, Xiaoju Hu, Vaishnavi Peddibhotla, Kuppusamy Balamurugan, Alexander Y. Mitrophanov, Lois McKennett, Suhas S. Kharat, Rahul Sanawar, Vinod Kumar Singh, Mary E. Albaugh, Sandra S. Burkett, Yongmei Zhao, Bao Tran, Tyler Malys, Esta Sterneck, Subhajyoti De, Shyam K. Sharan
Aneuploidy, a deviation from the normal chromosome copy number, is common in human embryos and is considered a primary cause of implantation failure and early pregnancy loss. Meiotic errors lead to uniformly abnormal karyotypes, while mitotic errors lead to chromosomal mosaicism: the presence of cells with at least two different karyotypes within an embryo. Knowledge about mosaicism in blastocysts mainly derives from bulk DNA sequencing of multicellular trophectoderm (TE) and/or inner cell mass (ICM) samples. However, this can only detect an average net gain or loss of DNA above a detection threshold of 20-30%. To accurately assess mosaicism, we separated the TE and ICM of 55 good quality surplus blastocysts and successfully applied single-cell whole genome sequencing (scKaryo-seq) on 1057 cells. Mosaicism involving numerical and structural chromosome abnormalities was detected in 82% of the embryos, where most abnormalities affected less than 20% of the cells. Structural abnormalities, potentially caused by replication stress and DNA damage, were observed in 69% of the embryos. In conclusion, our findings indicated that mosaicism is prevalent in good-quality blastocysts, while these blastocysts would likely be identified as normal with current bulk DNA sequencing techniques used for preimplantation genetic testing for aneuploidy (PGT-A).
Effrosyni A. Chavli, Sjoerd J. Klaasen, Diane Van Opstal, Joop S.E. Laven, Geert J.P.L. Kops, Esther B. Baart
Manale El Kharbili, Sarah K. Sasse, Lynn Sanford, Sean Jacobson, Katja Aviszus, Arnav Gupta, Claire J. Guo, Susan M. Majka, Robin D. Dowell, Anthony N. Gerber, Russell P. Bowler, Fabienne Gally
Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we employed kidney-specific expression of quantitative traits and single-nuclear open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2 knock-out mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulates de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.
Dhanunjay Mukhi, Lingzhi Li, Hongbo Liu, Tomohito Doke, Lakshmi P. Kolligundla, Eunji Ha, Konstantin A. Klötzer, Amin Abedini, Sarmistha Mukherjee, Junnan Wu, Poonam Dhillon, Hailong Hu, Dongyin Guan, Katsuhiko Funai, Kahealani Uehara, Paul M. Titchenell, Joseph A. Baur, Kathryn E. Wellen, Katalin Susztak
BACKGROUND. Systemic administration of Adeno-associated virus (AAV) can trigger life-threatening inflammatory responses including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome (aHUS)-like complement activation, immune-mediated myocardial inflammation and hepatic toxicity. METHODS. We describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 subjects following two distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation. RESULTS. Group 1 subjects had a rapid increase of immunoglobulin M (IgM) and immunoglobulin G (IgG). Increase in D-dimer, decline in platelet count and complement activation are indicative of TMA. All Group 1 subjects demonstrated activation of both classical and alternative complement pathways indicated by depletion of C4, elevated SC5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation. CONCLUSIONS. This study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune mediated events which impact the safety and efficacy of systemic gene therapy.
Stephanie M. Salabarria, Manuela Corti, Kirsten E. Coleman, Megan B. Wichman, Julie A. Berthy, Precilla D'Souza, Cynthia J. Tifft, Roland W. Herzog, Melissa E. Elder, Lawrence R. Shoemaker, Carmen Leon-Astudillo, Fatemeh Tavakkoli, David H. Kirn, Jonathan D. Schwartz, Barry J. Byrne