ASXL1 mutation frequently occurs in all forms of myeloid malignancies and is associated with aggressive disease and poor prognosis. ASXL1 recruits Polycomb Repressive Complex 2 (PRC2) to specific gene loci to repress transcription through tri-methylation of histone H3 on lysine 27 (H3K27me3). ASXL1 alterations reduce H3K27me3 levels, which results in leukemogenic gene expression and the development of myeloid malignancies. Standard therapies for myeloid malignancies have limited efficacy when mutated ASXL1 is present. We discovered up-regulation of lysine demethylase 6B (KDM6B), a demethylase for H3K27me3, in ASXL1-mutant leukemic cells, which further reduces H3K27me3 levels and facilitates myeloid transformation. Here, we demonstrated that heterozygous deletion of Kdm6b restored H3K27me3 levels and normalized dysregulated gene expression in Asxl1Y588XTg hematopoietic stem/progenitor cells (HSPCs). Furthermore, heterozygous deletion of Kdm6b decreased the HSPC pool, restored their self-renewal capacity, prevented biased myeloid differentiation, and abrogated progression to myeloid malignancies in Asxl1Y588XTg mice. Importantly, administration of GSK-J4, a KDM6B inhibitor, not only restored H3K27me3 levels but also reduced the disease burden in NSG mice xenografted with human ASXL1 mutant leukemic cells in vivo. This preclinical finding provides compelling evidence that targeting KDM6B may be a therapeutic strategy for myeloid malignancies with ASXL1 mutations.
Guo Ge, Peng Zhang, Pinpin Sui, Shi Chen, Hui Yang, Ying Guo, Ivan P. Rubalcava, Asra Noor, Caroline R. Delma, Joel Agosto-Peña, Hui Geng, Edward A. Medina, Ying Liang, Stephen D. Nimer, Ruben Mesa, Omar Abdel-Wahab, Mingjiang Xu, Feng-Chun Yang
Hormone-receptor-positive breast cancer (HR+) is immunologically cold and has not benefited from advances in immunotherapy. In contrast, subsets of triple-negative breast cancer (TNBC) display high leukocytic infiltration and respond to checkpoint blockade. CD8+T cells, the main effectors of anti-cancer responses, recognize MHC I-associated peptides (MAPs). Our work aimed to characterize the repertoire of MAPs presented by HR+ and TNBC tumors. Using mass spectrometry, we identified 57,094 unique MAPs in 26 primary breast cancer samples. MAP source genes highly overlapped between both subtypes (>70%). We identified 25 tumor-specific antigens (TSAs) mainly deriving from aberrantly expressed regions. TSAs were most frequently identified in TNBC samples (70%) and were more shared among TCGA TNBC than HR+ samples. In the TNBC cohort, the predicted number of TSAs positively correlated with leukocytic infiltration (p<0.05) and overall survival (p<0.05), supporting their immunogenicity in vivo. We detected 49 tumor-associated antigens, some of which derived from cancer-associated fibroblasts. Functional expansion of specific T cell assays confirmed the in vitro immunogenicity of several TSAs and TAAs. Our study identified attractive targets for cancer immunotherapy in both breast cancer subtypes. The higher prevalence of TSAs in TNBC tumors provides a rationale for their responsiveness to checkpoint blockade.
Eralda Kina, Jean-Philippe Laverdure, Chantal Durette, Joël Lanoix, Mathieu Courcelles, Qingchuan Zhao, Anca Apavaloaei, Jean-David Larouche, Marie-Pierre Hardy, Krystel Vincent, Patrick Gendron, Leslie Hesnard, Catherine Thériault, Maria Virginia Ruiz Cuevas, Grégory Ehx, Pierre Thibault, Claude Perreault
Mineralocorticoid excess commonly leads to hypertension and kidney disease. In our study, we employed single-cell expression and chromatin accessibility tools to characterize the mineralocorticoid target genes and cell types. We demonstrated that mineralocorticoid effects are established through open chromatin and target gene expression, primarily in principal and connecting tubule cells, and to a lesser extent, in segments of the distal convoluted tubule cells. We examined the kidney-protective effects of steroidal and non-steroidal mineralocorticoid antagonists (MRAs), as well as amiloride, an epithelial sodium channel inhibitor, in a rat model of deoxycorticosterone acetate, unilateral nephrectomy, and high salt consumption-induced hypertension and cardiorenal damage. All antihypertensive therapies protected from cardiorenal damage. However, finerenone was particularly effective in reducing albuminuria and improving gene expression changes in podocytes and proximal tubule cells, even with equivalent blood pressure reduction. There was a strong correlation between the accumulation of injured/profibrotic tubule cells expressing Spp1, Il34, and Pdgfb and the degree of fibrosis in rat kidneys. This gene signature also showed potential for classifying human kidney samples. Our multi-omics approach provides fresh insights into the possible mechanisms underlying hypertension associated kidney disease, the target cell types, and the protective effects of steroidal, non-steroidal MRAs, and amiloride.
Amin Abedini, Andrea Sanchez-Navarro, Junnan Wu, Konstantin A. Klötzer, Ziyuan Ma, Bibek Poudel, Tomohito Doke, Michael S. Balzer, Julia Frederick, Hana Cernecka, Hongbo Liu, Xiujie Liang, Steven Vitale, Peter Kolkhof, Katalin Susztak
Several poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are approved by FDA treat cancer with BRCA mutations. BRCA mutation is considered to fuel PARPi killing effect by inducing apoptosis. However, resistance to PARPi is frequently observed in clinic due to incomplete understanding on the molecular basis of PARPi function and lack of good markers to predict response in addition to BRCA mutations. Here we show that gasdermin C (GSDMC) sensitized tumor cells to PARPi in vitro and in immunocompetent mice and caused durable tumor regression in an immune-dependent manner. High expression level of GSDMC predicted better response to PARPi treatment in triple-negative breast cancer (TNBC) patients. PARPi treatment triggered GSDMC/caspase-8-mediated cancer cell pyroptosis (CCP) that enhanced PARPi killing of tumor cells. GSDMC-mediated CCP increased memory CD8+ T cell population in lymph node (LN), spleen, tumor, and thus promoted cytotoxic CD8+ T cell infiltration in tumor microenvironment. T cell-derived granzyme B (GZMB) activated caspase-6, which subsequently cleaved GSDMC to induce pyroptosis. Interestingly, IFN-γ induced GSDMC expression, which in turn enhanced the cytotoxicity of PARPi and T cell. Importantly, GSDMC promoted tumor clearance independent of BRCA deficiency in multiple cancer types with PARPi treatment. This study identifies a general marker and target for PARPi therapy and offers new insights into the mechanism of PARPi function.
Shuanglian Wang, Chiung-Wen Chang, Juan Huang, Shan Zeng, Xin Zhang, Mien-Chie Hung, Junwei Hou
Physiologic activation of the estrogen receptor alpha (ERα) is mediated by estradiol (E2) binding in the ligand binding pocket of the receptor, repositioning helix 12 (H12) to facilitate binding of coactivator proteins in the unoccupied coactivator binding groove. In breast cancer, activation of ERα is often observed through point mutations that lead to the same H12 repositioning in the absence of E2. Through expanded genetic sequencing of breast cancer patients, we identified a collection of mutations located far from H12 but nonetheless capable of promoting E2-independent transcription and breast cancer cell growth. Using machine learning and computational structure analyses, this set of mutants was inferred to act distinctly from the H12-repositioning mutants and instead was associated with conformational changes across the ERα dimer interface. Through both in vitro and in cell assays of full length ERα protein and isolated ligand binding domain (LBD), we found that these mutants promoted ERα dimerization, stability, and nuclear localization. Point mutations that selectively disrupted dimerization abrogated E2-independent transcriptional activity of these dimer-promoting mutants. The results revealed a distinct mechanism for activation of ERα function through enforced receptor dimerization and suggested dimer disruption as a potential therapeutic strategy to treat ER-dependent cancers.
Seema Irani, Wuwei Tan, Qing Li, Weiyi Toy, Catherine Jones, Mayur Gadiya, Antonio Marra, John A. Katzenellenbogen, Kathryn E. Carlson, Benita S. Katzenellenbogen, Mostafa Karimi, Ramya Segu Rajappachetty, Isabella S. Del Priore, Jorge S. Reis-Filho, Yang Shen, Sarat Chandarlapaty
We have previously demonstrated that cystatin E/M (CST6), elevated in a subset of multiple myeloma (MM) patients lacking osteolytic lesions (OL), suppresses MM bone disease by blocking osteoclast differentiation and function. CST6 is a secreted type 2 cystatin, a cysteine protease inhibitor that regulates lysosomal cysteine proteases and the asparaginyl endopeptidase legumain. We have developed B cell maturation antigen (BCMA)-CST6-chimeric antigen receptor (CAR) T cells, which lyse MM cells and release CST6 proteins. Our in vitro studies show that these CAR-T cells suppress differentiation and formation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. Using xenografted MM mice, bioluminescence images show that both BCMA-CAR-T and BCMA-CST6-CAR-T cells inhibit MM growth to a similar extent. Reconstructed micro-computed tomography (µCT) images reveal that BCMA-CST6-CAR-T cells, but not BCMA-CAR-T cells, prevent MM-induced bone damage and decrease osteoclast numbers. Our results provide a CAR-T strategy that targets tumor cells directly and delivers an inhibitor of bone resorption.
Fumou Sun, Yan Cheng, Jin-Ran Chen, Visanu Wanchai, David E. Mery, Hongwei Xu, Dongzheng Gai, Samer Al Hadidi, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr., Fenghuang Zhan
Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A by Zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages towards an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that Zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon response uniformly across models. The induction of an interferon response is partially due to the inhibition of Sox4 translation by Zotatifin. A similar induction of interferon-stimulated genes was observed in breast cancer patient biopsies following Zotatifin treatment. Surprisingly, Zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened interferon response resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for Zotatifin, and provide a rationale for new combination regimens comprising Zotatifin and chemotherapy or immunotherapy as treatments for TNBC.
Na Zhao, Elena B. Kabotyanski, Alexander B. Saltzman, Anna Malovannaya, Xueying Yuan, Lucas C. Reineke, Nadia Lieu, Yang Gao, Diego A. Pedroza, Sebastian J. Calderon, Alex J. Smith, Clark W. Hamor, Kazem Safari, Sara R. Savage, Bing Zhang, Jianling Zhou, Luisa M. Solis Soto, Susan G. Hilsenbeck, Cheng Fan, Charles M. Perou, Jeffrey M. Rosen
Reactivation and dysregulation of the mTOR signaling pathway is a hallmark of aging and chronic lung disease, however the impact on microvascular progenitor cells (MVPC), capillary angiostasis and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue resident MVPC present in both adult mouse and human lungs using functional, lineage and transcriptomic analyses. These studies link human and mouse MVPC specific mTORC1 activation to decreased stemness, angiogenic potential, disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation these MVPC adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function, microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.
Emma C. Mason, Swapna Menon, Benjamin R. Schneider, Christa F. Gaskill, Maggie M. Dawson, Camille M. Moore, Laura Craig Armstrong, Okyong J. Cho, Bradley W. Richmond, Jonathan A. Kropski, James D. West, Patrick Geraghty, Brigitte N. Gomperts, Kevin C. Ess, Fabienne Gally, Susan M. Majka
Heart Failure with Preserved Ejection Fraction (HFpEF) is a widespread syndrome with limited therapeutic options and poorly understood immune-pathophysiology. Using a two-hit preclinical model of cardiometabolic HFpEF that induces obesity and hypertension, we found that cardiac T cell infiltration and lymphoid expansion occur concomitantly with cardiac pathology, and that diastolic dysfunction, cardiomyocyte hypertrophy and cardiac phospholamban phosphorylation are T cell-dependent. Heart-infiltrating T cells were not restricted to cardiac antigens and were uniquely characterized by impaired activation of the Inositol-requiring enzyme-1α (IRE1α)-X-box binding protein 1 (XBP1) arm of the unfolded protein response. Notably, selective ablation of XBP1 in T cells enhanced their persistence in the heart and lymphoid organs of mice with preclinical HFpEF. Furthermore, T cell IRE1α-XBP1 activation was restored after withdrawal of the two comorbidities inducing HFpEF, resulting in partial improvement of cardiac pathology. Our results demonstrate that diastolic dysfunction and cardiomyocyte hypertrophy in preclinical HFpEF are T cell-dependent, and that reversible dysregulation of the T cell IRE1α-XBP1 axis is a T cell signature of HFpEF.
Sasha Smolgovsky, Abraham L. Bayer, Kuljeet Kaur, Erin Sanders, Mark Aronovitz, Mallory E. Filipp, Edward B. Thorp, Gabriele G. Schiattarella, Joseph A. Hill, Robert M. Blanton, Juan R. Cubillos-Ruiz, Pilar Alcaide
Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by two oxidation-sensitive methionine residues within the regulatory domain. We previously showed that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model, in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice post-IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.
Simon Lebek, Xurde M. Caravia, Leon G. Straub, Damir Alzhanov, Wei Tan, Hui Li, John R. McAnally, Kenian Chen, Lin Xu, Philipp E. Scherer, Ning Liu, Rhonda Bassel-Duby, Eric N. Olson
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