In-Press Preview
Abstract
Authors
Jianfei Ji, Wenjing You, Xiaoli Sun, Peng Zhao
Abstract
Cell metabolic rewiring is associated with resistance to venetoclax-azacitidine (Ven-Aza) combination therapy and relapse in acute myeloid leukemia (AML) patients. Drug-resistant cells exhibit an enhanced reliance on oxidative phosphorylation (OXPHOS) for energy production. Therefore, impairing mitochondrial metabolism represents an exciting strategy to face this unmet clinical need. We recently demonstrated that the specific activation of the phosphatase PP2A-B56α enhances the pro-apoptotic efficacy of venetoclax in AML. Here, through leveraging unbiased multi-omics-based approaches and using both genetic and pharmacological tools, we define key roles for the tumor suppressor PP2A-B56α complex in OXPHOS regulation and treatment response in disease-relevant AML models. From a translational perspective, the specific stabilization of PP2A-B56α heterocomplex with the novel PP2A molecular glue activator, RPT04402, reduces OXPHOS levels in treatment-resistant AML cells and improves treatment response in both Ven-Aza-sensitive and -resistant AML cell lines, primary cells, and in vivo models. Together, our work supports further research on targeted combination therapy approaches based on PP2A-B56α stabilization to counteract OXPHOS-related treatment resistance and improve AML responses in a patient population with historically poor outcomes.
Authors
Silvia Romero-Murillo, Irene Peris, Anna Maria Lucianò, Nerea Marcotegui, Carmen Vicente, Brian Tran, Kelsey Barrie, Caitlin M. O'Connor, Andrea Torres-López, Maria C. Mateos, Maria L. Cayuela, Victoriano Mulero, Joaquín Fernández-Irigoyen, Enrique Santamaría, Maria D. Odero, Goutham Narla
Abstract
Despite advances in treatment approaches for lung cancer, the morbidity and survival of lung cancer patients with malignant pleural effusions (MPE) remain poor. This is in part due to gaps in understanding the role of immune cells in the pleural fluid microenvironment. We performed single cell analysis with flow cytometry validation of CD45+ cells in eight malignant and five benign pleural fluid (BPE) specimens to identify changes in the transcriptomic landscape of immune cells across disease states. We found upregulation of pro-inflammatory signaling pathways, including interferon and TNF signaling, in T cells, B cells, and macrophages in benign compared to malignant pleural effusions. Pro-inflammatory HLA-DR+ macrophages were associated with good survival outcomes while pro-tumorigenic HLA-DR- macrophages with upregulation of angiogenesis, TGFβ, and fibronectin signaling were associated with poor survival outcomes in patients with MPE. We also validated these findings with macrophage cell surface expression markers using flow cytometry in 14 MPE and 7 BPE specimens. Finally, we performed multiplex cytokine analysis which showed enrichment of the type 3 inflammatory cytokine, IL17A, in MPE as a putative mechanism for macrophage reprogramming. These data provide a rich resource for interrogating the immune cell types and states present across the spectrum of pleural disease. They offer not only prognostic value for patient outcomes at the time of pleural fluid collection, but also insights into novel immunotherapy targets.
Authors
Aaditya Khatri, Huimin Wang, Zhicheng Ji, Prekshaben Patel, Smita K. Nair, Javid P. Mohammed, Beth H. Shaz, Andrew B. Nixon, Scott M. Palmer, Kamran Mahmood
Abstract
Activation of the mechanistic target of rapamycin (mTOR) complex1 (mTORC1) promotes muscle protein synthesis, mass, and function. Muscle mTORC1 can be activated by feeding and contraction. Here, muscle mTORC1 signaling, protein synthesis, mass, and function are characterized in a genetic mouse model that separates these two major modes of muscle mTORC1 regulation. AKT signaling is required for feeding-induced muscle mTORC1 signaling and protein synthesis, and mice expressing a mutant of tuberous sclerosis complex 2 (TSC2) that cannot be phosphorylated by AKT specifically in skeletal muscle (SkM-TSC2-5A) attenuate these effects of feeding. Despite this loss of postprandial protein synthesis, SkM-TSC2-5A mice have similar muscle and myofiber size compared to SkM-TSC2-WT mice. SkM-TSC2-5A mice maintain normal muscle mTORC1 activation in response to contraction and exhibit no differences in atrophy-related gene expression or ribosomal content. SkM-TSC2-5A mice exhibit improved maximal endurance capacity without changes in muscle contractile function. This phenotype occurs without alterations in muscle glycogen content or myofiber type but does coincide with a modest increase in muscle mitochondrial content. Therefore, AKT-mediated phosphorylation of TSC2 is required for postprandial mTORC1 activation and the induction of protein synthesis; however, these are dispensable for the development and maintenance of muscle mass in sedentary mice.
Authors
Samuel C. Lapp, Krystle C. Kalafut, Madi Y. Cissé, Khaled Tighanimine, Dean M. Rosenthal, Will Doxsey, Sheng Hui, Karen E. Inouye, Claire E. Morrow, Yann Cormerais, Brendan D. Manning
Endothelial cell cycle inhibition enables blood vessel maturation to normalize the tumor vasculature
Abstract
Dysfunctional tumor vessels promote disease progression, whereas improved function enhances therapeutic delivery. However, current approaches to normalize tumor vasculature have limited efficacy. In vascular malformations, vessels are similarly dysfunctional, with endothelial cell (EC) hyperproliferation impairing arterial-venous specification. These defects are corrected with palbociclib, a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) that has beneficial effects on tumor and immune cells, but the effects on tumor vasculature are not well characterized. In our studies, murine mammary tumor ECs (TECs) exhibited disrupted cell cycle and specification, and CDK4/6i promoted TEC cycle control, enabling improved tumor vascular function. To investigate transcriptomic changes, we performed single-cell RNA sequencing (scRNAseq) of treated and untreated tumors, and healthy tissues. CDK4/6i-mediated TEC cycle arrest promoted arterial-venous specification, cellular junctions, and pericyte association, and suppressed glycolytic and immunosuppressive gene expression. These effects were associated with increased vessel perfusion, decreased tumor hypoxia, and a more favorable immune landscape with immunotherapy. In scRNAseq datasets from patients treated long-term with CDK4/6i, TECs exhibited similar transcriptomic changes associated with arterial-venous specification, pericyte recruitment, and immune signaling. Thus, in contrast to current strategies, CDK4/6i-mediated vascular changes may be maintained with continued treatment, highlighting the relevance of modulating TEC cycle to improve vessel maturation/function.
Authors
Shelby R. Cain, Gael Genet, Nafiisha Genet, Jordon W. Aragon, Madeline G. Jackson, Victoria M. Milosek, Mark R. Schwartz, Umadevi Paila, Aleksandra Cwiek, Zaneta Markowska, Nicholas W. Chavkin, Richard J. Price, Andrew C. Dudley, Karen K. Hirschi
Abstract
Wnt signaling drives tumorigenesis in multiple cancers, in part through complex interactions with other oncogenic pathways including the MAPK cascade. In Wnt-addicted cancers, pharmacologic and genetic inhibition of Wnt signaling activates multiple receptor tyrosine kinases (RTKs), increases ERK phosphorylation and induces MAPK target gene expression, but the specific RTKs responsible for this MAPK hyperactivation are not known. Here we performed phosphotyrosine-targeted mass spectrometry, which revealed robust phosphorylation of EPHA2 and EGFR upon Wnt inhibition. Unexpectedly, we find that in xenografts, EPHA2 suppresses EGFR and ERK activation. Most notably, the increased ERK phosphorylation observed in EPHA2 KO tumors is transcriptionally inert, as there is no concomitant increase in MAPK target gene expression until concomitant Wnt inhibition. This suggests a Wnt-activated transcriptional repressor such as GATA3 that gates MAPK signaling in Wnt-high cancers. While Wnt-high KRAS-mutant cancers are resistant to erlotinib alone, adding Wnt inhibitor mitigates this resistance. Additionally, loss of EPHA2 enhances their sensitivity to both erlotinib and Wnt inhibitors. These studies therefore identify therapeutic vulnerabilities in Wnt-high tumors, even within traditionally EGFR inhibitor-resistant, RAS-mutant contexts.
Authors
Shawn R. Wadia, Changyuan Hu, Siddhi Patnaik, Shreya Sridharan, Roger J. Daly, David M. Virshup, Babita Madan
Abstract
Autophagy is a critical host defense mechanism that restricts intracellular pathogens such as Mycobacterium tuberculosis (Mtb). A key step in this process is the ubiquitination of Mtb or Mtb-associated structures. The E3 ligase SMURF1 catalyzes K48-linked ubiquitination, promoting bacterial clearance. However, the function of its homolog, SMURF2, in host defense remains undefined. Here, we demonstrate that Smurf2 deletion in murine macrophages increases SMURF1 levels, enhances LC3B lipidation, augments K48 ubiquitination of Mtb-associated structures, and reduces intracellular Mtb replication. These effects are reversed by Smurf1 deletion, supporting a role for SMURF1 in SMURF2-dependent control of Mtb. Mice with myeloid-specific Smurf2 deletion exhibit modestly prolonged survival following aerosol Mtb infection. In human macrophages, SMURF2 knockdown or its pharmacological inhibition with the HECT E3-ligase inhibitor Heclin reduces Mtb replication. Together, our findings identify SMURF2 as a negative regulator of macrophage control of Mtb and support further investigation of SMURF2 as a potential target for host-directed therapy in tuberculosis.
Authors
Priscila C. Campos, Kathryn C. Rahlwes, Victoria A. Ektnitphong, Beatriz R.S. Dias, Kubra F. Naqvi, Samuel Alvarez-Arguedas, Michael U. Shiloh
Abstract
Fractures heal by rapid formation of mineralized callus, a process requiring periosteal cell proliferation and differentiation. Our objective was to dissect the contribution of proliferating osteoblast lineage cells to fracture callus formation. First, mice expressing thymidine kinase (TK) in 3.6Col1a1-lineage cells were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. Immunostaining demonstrated that this approach specifically depleted TK+ proliferating cells in the bony regions of the callus, while sparing other proliferating cells. Single-cell RNA-seq of callus cells revealed that GCV-treated Col1-TK mice had fewer osteoblasts and chondrocytes than controls, with more myofibroblasts and immune cells, consistent with fibrous nonunion. In controls, 15-30% of callus cells expressing the early osteoblast markers osterix (Sp7) and the late marker osteocalcin (Bglap) were in the cell cycle. Next, we targeted proliferating osteolineage cells at different stages of differentiation by crossing Osx-CreERT2, Ocn-Cre and Dmp1-CreERT2 mice with ROSA-TK mice. Following fracture, each Cre;ROSA-TK mouse line exhibited decreased callus bone volume and a shift from callus bone to fibrous tissue. Therefore, during fracture repair, proliferation of callus cells at early and mature stages of osteoblast differentiation is critical to the formation of a mineralized callus that is essential for healing.
Authors
Nicole R. Gould, Andre F. Coello, Jennifer A. McKenzie, Mariam Obaji, Tiandao Li, Katherine R. Hixon, Leyi Chen, Kristen Barwick, Tiffany Lee, Bo A. Zhang, David Ornitz, Matthew J. Silva
Abstract
Mutations in mitochondrial DNA (mtDNA) cause various mitochondrial diseases that are currently incurable. Allotopic expression of nuclear-recoded mitochondrial genes represents a promising therapeutic strategy, given its demonstrated capacity to restore mitochondrial function in human cell models harboring mtDNA mutations. However, the in vivo evaluation of allotopic gene therapy has been hindered by optimization challenges and the lack of appropriate animal models. Here, we overcome these limitations by utilizing an optimized AAV2-ND6 construct with codon optimization and mitochondrial targeting sequence in a mouse model bearing the homoplasmic ND6P25L mutation, which recapitulates Leber hereditary optic neuropathy (LHON). High-dose administration of the AAV2-ND6 construct resulted in robust, sustained expression within the retina and optic nerve without apparent systemic toxicity. Strikingly, We compared the therapeutic efficacy in mutant mice at different ages and pre-symptomatic intervention with AAV2-ND6 effectively attenuated disease progression, mitigated retinal cellular deficiencies and optic nerve damage, and restored visual function in ND6P25L mice. Mechanistically, allotopic ND6 expression markedly rescued the mitochondrial dysfunction, corrected dysregulated retinol metabolism and phototransduction pathways, and suppressed apoptotic processes in the mutant retina. Our study validates the safety and therapeutic potential of allotopic expression in vivo and provide critical mechanistic insights into its role in treating LHON and other mitochondrial diseases.
Authors
Cheng Ai, Huiying Li, Jing Wu, Tianwei Zhou, Jing Wang, Shao-Hui Pan, Jun Yu, Douglas C. Wallace, Min-Xin Guan
Abstract
Inflammatory cytokines reprogram keratinocyte metabolism, but the metabolic pathways that couple immune signals to pathological epidermal growth remain incompletely defined. Here, we identify GLS1-mediated glutaminolysis as a metabolic program preferentially induced in keratinocytes under type 3 inflammatory conditions. Integrated transcriptomic, metabolomic, genetic, and functional analyses showed that IL-17A induced GLS1 expression and glutaminolysis in keratinocytes. Keratinocyte-specific Gls1 deletion reduced the intracellular availability of arginine, proline, and methionine, impaired amino acid-dependent mTORC1 activation, disrupted redox homeostasis, and limited keratinocyte proliferation. Amino acid or antioxidant supplementation partially rescued these defects, whereas rapamycin blocked the amino acid-mediated proliferative rescue. Gls1 deletion did not impair steady-state skin development or homeostasis and did not alter MC903-induced type 2 dermatitis, but it delayed wound re-epithelialization and attenuated IMQ-induced psoriasiform inflammation. Loss of keratinocyte GLS1 also reduced epidermal chemokine expression and the accumulation of neutrophils and IL-17A-producing γδ T cells, revealing a role for glutaminolysis in amplifying epithelial-immune crosstalk. These findings define GLS1-mediated glutaminolysis as a context-specific metabolic checkpoint linking type 3 inflammation to keratinocyte proliferation and cutaneous immune amplification, and support locally or temporally controlled GLS1 inhibition as a potential therapeutic strategy for psoriasis.
Authors
Yongfei Hu, Hai Yu, Kaiyu Liang, Liyan Yuan, Wenjun Zhang, Zhe Zhuang, Suyun Ji, Xichun Xia
No posts were found with this tag.
