Research
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI169993.
Abstract
Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity towards increased purine synthesis in MLL3/4 knockout (KO) mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA sequencing identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag (TMT) proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4 KO cells. Mechanistically, compensation by MLL1/COMPASS underlied these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.
Authors
Zibo Zhao, Kaixiang Cao, Jun Watanabe, Cassandra N. Philips, Jacob M. Zeidner, Yukitomo Ishi, Qixuan Wang, Sarah R. Gold, Katherine Junkins, Elizabeth T. Bartom, Feng Yue, Navdeep S. Chandel, Rintaro Hashizume, Issam Ben-Sahra, Ali Shilatifard
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI170191.
Abstract
Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single cell RNA transcriptomic and TCRα/β sequencing on rBx from patients with ACR under differing IS: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within clonally expanded cells (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCRα/β cDNAs from CD8EXP into Jurkat76 cells (TCR–/–) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful anti-rejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps to improve detection, assessment, and treatment of rejection.
Authors
Tiffany Shi, Ashley R. Burg, J. Timothy Caldwell, Krishna M. Roskin, Cyd M. Castro-Rojas, P. Chukwunalu Chukwuma, George I. Gray, Sara G. Foote, Jesus A. Alonso, Carla M. Cuda, David A. Allman, James S. Rush, Catherine H. Regnier, Grazyna Wieczorek, Rita R. Alloway, Adele R. Shields, Brian M. Baker, E. Steve Woodle, David A. Hildeman
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI163290.
Abstract
Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by hypoxia-inducible factor (HIF)-1 and -2 (HIFs) remain elevated in diabetic eyes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF-inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.
Authors
Jing Zhang, Deepti Sharma, Aumreetam Dinabandhu, Jaron Sanchez, Brooks Applewhite, Kathleen Jee, Monika Deshpande, Miguel Flores-Bellver, Ming-Wen Hu, Chuanyu Guo, Shaima Salman, Yousang Hwang, Nicole M. Anders, Michelle A. Rudek, Jiang Qian, Valeria Canto-Soler, Gregg L. Semenza, Silvia Montaner, Akrit Sodhi
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI166070.
Abstract
Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of lymph node (LN) fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identify the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network, expressing elevated fibroblast activated protein (FAP). RNA-sequencing analyses reveal that exposure to DLBCL reprograms key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen presentation molecules. Functional assays show that DLBCL-activated FRCs (DLBCL-FRCs) hinder optimal TIL and chimeric antigen receptor T cell (CAR-T) migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrate the potential to target inhibitory FRCs to rejuvenate interacting TILs. Co-treating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented anti-lymphoma TIL cytotoxicity. Together, our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis and optimizing immunotherapy for patients.
Authors
Benedetta Apollonio, Filomena Spada, Nedyalko Petrov, Domenico Cozzetto, Despoina Papazoglou, Peter Jarvis, Shichina Kannambath, Manuela Terranova-Barberio, Rose-Marie Amini, Gunilla Enblad, Charlotte E. Graham, Reuben Benjamin, Elizabeth H. Phillips, Richard J. Ellis, Rosamond Nuamah, Mansoor Saqi, Dinis P. Calado, Richard Rosenquist, Lesley A. Sutton, Jonathan R. Salisbury, Georgios Zacharioudakis, Anna Vardi, Patrick R. Hagner, Anita K. Gandhi, Marina Bacac, Christina Claus, Pablo Umana, Ruth F. Jarrett, Christian Klein, Alexander J.A. Deutsch, Alan G. Ramsay
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI164637.
Abstract
Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system (CNS). The individual course is highly variable with complete remission in some patients and relentless courses in others. We generated induced pluripotent stem cells (iPSCs) to investigate possible mechanisms in benign MS (BMS), compared to progressive MS (PMS). We differentiated neurons and astrocytes that were then stressed with inflammatory cytokines typically associated with MS. TNFα/IL-17A treatment increased neurite damage in MS neurons irrespective of clinical phenotypes. In contrast, TNFα/IL-17A-reactive BMS astrocytes cultured with healthy control (HC) neurons exhibited significantly decreased axonal damage, compared to PMS astrocytes. Accordingly, single cell transcriptomic analysis of BMS-astrocyte co-cultured neurons demonstrated upregulated pathways of neuronal resilience, namely these astrocytes revealed differential growth factor expression. Moreover, supernatants from BMS astrocyte-neuron co-cultures rescued TNFα/IL-17-induced neurite damage. This process was associated with the unique expression of the growth factors, LIF and TGF-β1, as induced by TNFα/IL-17 and JAK-STAT activation. Our findings highlight a potential therapeutic role of modulating astrocyte phenotypes that generate a neuroprotective milieu preventing permanent neuronal damage.
Authors
Janis Kerkering, Bakhrom Muinjonov, Kamil Sebastian Rosiewicz, Sebastian Diecke, Charlotte Biese, Juliane Schiweck, Claudia Chien, Dario Zocholl, Thomas Conrad, Friedemann Paul, Marlen Alisch, Volker Siffrin
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI158630.
Abstract
The ADP ribosyl transferases (PARPs 1–17) regulate diverse cellular processes, including DNA damage repair. PARPs are classified based on their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). While PARP9 mRNA expression is significantly increased in progressive human tuberculosis (TB), its participation in host immunity to TB is unknown. Here, we show that PARP9 mRNA encoding the MARylating PARP9 enzyme is upregulated during TB in humans and mice and provide evidence of a critical modulatory role for PARP9 in DNA damage, cGAS and type I IFN production during TB. Thus, Parp9-deficient mice are susceptible to Mtb infection and exhibit increased TB disease, cGAS expression, cGAMP and type I IFN production along with upregulation of complement and coagulation pathways. Enhanced Mtb susceptibility is type I IFN-dependent, as blockade of IFNAR signaling reversed the enhanced susceptibility of Parp9-/- mice. Thus, in sharp contrast with PARP9 enhancement of type I IFN production in viral infections, this member of the MAR family plays a protective role by limiting type I IFN responses during TB.
Authors
Shyamala Thirunavukkarasu, Mushtaq Ahmed, Bruce A. Rosa, Mark Boothby, Sung Hoon Cho, Javier Rangel-Moreno, Stanley K. Mbandi, Valérie Schreiber, Ananya Gupta, Joaquin Zúñiga, Makedonka Mitreva, Deepak Kaushal, Thomas J. Scriba, Shabaana A. Khader
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI167762.
Abstract
Heterogeneity in human immune responses is difficult to model in standard laboratory mice. To understand how host variation affects BCG-induced immunity against Mycobacterium tuberculosis, we studied 24 unique Collaborative Cross (CC) mouse strains, which differ primarily in the genes and alleles they inherit from founder strains. The CC strains were vaccinated with or without BCG, and then challenged with aerosolized M. tuberculosis. As BCG protects only half of the CC strains tested, we conclude that host genetics has a major influence on BCG-induced immunity against M. tuberculosis infection, making it an important barrier to vaccine-mediated protection. Importantly, BCG efficacy is dissociable from inherent susceptibility to TB. T cell immunity was extensively characterized to identify components associated with protection that were stimulated by BCG and recalled after Mtb infection. Although considerable diversity is observed, BCG has little impact on the composition of T cells in the lung after infection. Instead, variability is largely shaped by host genetics. BCG-elicited protection against TB correlated with changes in immune function. Thus, CC mice can be used to define correlates of protection and to identify vaccine strategies that protect a larger fraction of genetically diverse individuals instead of optimizing protection for a single genotype.
Authors
Rocky Lai, Diana N. Gong, Travis Williams, Abiola F. Ogunsola, Kelly Cavallo, Cecilia S. Lindestam Arlehamn, Sarah Acolatse, Gillian Beamer, Martin T. Ferris, Christopher M. Sassetti, Douglas A. Lauffenburger, Samuel M. Behar
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI164660.
Abstract
Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young individuals. Due to heterogeneity in the clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.
Authors
Qianqian Zhuang, Fengfeng Guo, Lei Fu, Yufei Dong, Shaofang Xie, Xue Ding, Shuangyi Hu, Xuanhao D. Zhou, Yangwei Jiang, Hui Zhou, Yue Qiu, Zhaoying Lei, Mengyao Li, Huajian Cai, Mingjie Fan, Lingjie Sang, Yong Fu, Dong Zhang, Aifu Lin, Xu Li, Tilo Kunath, Ruhong Zhou, Ping Liang, Zhong Liu, Qingfeng Yan
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI158419.
Abstract
During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation which can cause life-threatening diseases and cancer. We have identified a novel role of Dpf2 in inflammation. Dpf2 is a defining subunit of the hematopoietic-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncover that hematopoietic-specific Dpf2 knock-out mice develop leukopenia, severe anemia and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration, resembling a clinical hyper-inflammatory state. Dpf2 loss impairs the polarization of macrophages responsible for tissue repair, induces unrestrained activation of T helper cells, and generates an emergency-like state of HSC hyperproliferation and myeloid-biased differentiation. Mechanistically, Dpf2 deficiency results in the loss of the BAF catalytic subunit Brg1 from Nrf2-controlled enhancers, impairing the anti-oxidant and anti-inflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of Nrf2 can suppress the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes the essential role of the Dpf2/BAF complex in licensing Nrf2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.
Authors
Gloria Mas, Na Man, Yuichiro Nakata, Concepcion Martinez-Caja, Daniel L. Karl, Felipe Beckedorff, Francesco Tamiro, Chuan Chen, Stephanie Duffort, Hidehiro Itonaga, Adnan K. Mookhtiar, Kranthi Kunkalla, Alfredo M. Valencia, Clayton K. Collings, Cigall Kadoch, Francisco Vega, Scott C. Kogan, Lluis Morey, Daniel Bilbao, Stephen D. Nimer
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI149310.
Abstract
ATRX is one of the most frequently altered genes in solid tumors, and mutation is especially frequent in soft tissue sarcomas. However, the role of ATRX in tumor development and response to cancer therapies remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and showed that Atrx deleted tumors were more sensitive to radiation therapy and to oncolytic herpesvirus. In the absence of Atrx, irradiated sarcomas had increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. Our work also showed that Atrx deletion resulted in downregulation of the CGAS/STING signaling pathway at multiple points in the pathway, and was not driven by mutations or by transcriptional downregulation of the CGAS/STING pathway components. We found that both human and mouse models of Atrx deleted sarcoma had a reduced adaptive immune response, markedly impaired CGAS/STING signaling, and increased sensitivity to TVEC, an oncolytic herpesvirus that is currently FDA approved for the treatment of aggressive melanomas. Translation of these results to patients with ATRX mutant cancers could enable genomically-guided cancer therapeutic approaches that improve patient outcomes.
Authors
Warren Floyd, Matthew Pierpoint, Chang Su, Rutulkumar Patel, Lixia Luo, Katherine Deland, Amy J. Wisdom, Daniel Zhu, Yan Ma, Suzanne Bartholf DeWitt, Nerissa T. Williams, Alexander L. Lazarides, Jason A. Somarelli, David L. Corcoran, William C. Eward, Diana M. Cardona, David G. Kirsch
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