Targeting tumor-associated blood vessels to increase immune infiltration may enhance treatment effectiveness, yet limited data exists regarding anti-angiogenesis effects on the tumor microenvironment. We hypothesized that dual targeting of angiogenesis with immune checkpoints will improve both intracranial and extracranial disease. We used subcutaneous and left ventricle melanoma models to evaluate anti-PD-1/anti-vascular endothelial growth factor (VEGF) and anti-PD-1/lenvatinib (pan-VEGF receptor inhibitor) combinations. Cytokine/chemokine profiling and flow cytometry were performed to assess signaling and immune infiltrating populations. An in vitro blood-brain barrier model was utilized to study intracranial treatment effects on endothelial integrity and leukocyte transmigration. Anti-PD-1 with either anti-VEGF or lenvatinib improved survival and decreased tumor growth in systemic melanoma murine models; treatment increased TH1 cytokine/chemokine signaling. Lenvatinib decreased tumor-associated macrophages but increased plasmacytoid dendritic cells early in treatment; this effect was not evident with anti-VEGF. Both lenvatinib and anti-VEGF resulted in decreased intratumoral blood vessels. Although anti-VEGF promoted endothelial stabilization in an in vitro blood-brain barrier model while lenvatinib did not, both regimens enabled leukocyte transmigration. Combined targeting of PD-1 and VEGF or its receptors promotes enhanced melanoma anti-tumor activity, yet their effects on the tumor microenvironment are quite different. These studies provide insights into dual anti-PD-1 and anti-angiogenesis combinations.
Thuy T. Tran, Jasmine I. Caulfield, Lin Zhang, David A. Schoenfeld, Dijana Djureinovic, Veronica L. Chiang, Victor O. Oria, Sarah A. Weiss, Kelly Olino, Lucia B. Jilaveanu, Harriet M. Kluger
SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers.
Yuchen Xie, Merve Sahin, Toru Wakamatsu, Akane Inoue-Yamauchi, Wanming Zhao, Song Han, Amrita M. Nargund, Shaoyuan Yang, Yang Lyu, James J. Hsieh, Christina S. Leslie, Emily H. Cheng
Inhibitors of the DNA damage signaling kinase ATR increase tumor cell killing by chemotherapies that target DNA replication forks but also kill rapidly proliferating immune cells including activated T cells. Nevertheless, ATR inhibitor (ATRi) and radiotherapy (RT) can be combined to generate CD8+ T cell–dependent antitumor responses in mouse models. To determine the optimal schedule of ATRi and RT, we determined the impact of short-course versus prolonged daily treatment with AZD6738 (ATRi) on responses to RT (days 1–2). Short-course ATRi (days 1–3) plus RT caused expansion of tumor antigen–specific, effector CD8+ T cells in the tumor-draining lymph node (DLN) at 1 week after RT. This was preceded by acute decreases in proliferating tumor-infiltrating and peripheral T cells and a rapid proliferative rebound after ATRi cessation, increased inflammatory signaling (IFN-β, chemokines, particularly CXCL10) in tumors, and an accumulation of inflammatory cells in the DLN. In contrast, prolonged ATRi (days 1–9) prevented the expansion of tumor antigen–specific, effector CD8+ T cells in the DLN, and entirely abolished the therapeutic benefit of short-course ATRi with RT and anti–PD-L1. Our data argue that ATRi cessation is essential to allow CD8+ T cell responses to both RT and immune checkpoint inhibitors.
Frank P. Vendetti, Pinakin Pandya, David A. Clump, Sandra Schamus-Haynes, Meysam Tavakoli, Maria diMayorca, Naveed M. Islam, Jina Chang, Greg M. Delgoffe, Jan H. Beumer, Christopher J. Bakkenist
Cancer stem-like cells (CSCs) are critically involved in cancer metastasis and chemoresistance, acting as one major obstacle in clinical practice. While accumulating studies have implicated the metabolic reprogramming of CSCs, mitochondrial dynamics in such cells remain poorly understood. Here we pinpointed OPA1high with mitochondrial fusion as a metabolic feature of human lung CSCs, licensing their stem-like properties. Specifically, human lung CSCs exerted enhanced lipogenesis, inducing OPA1 expression via transcription factor SPDEF. In consequence, OPA1high promoted mitochondrial fusion and stemness of CSCs. Such lipogenesishigh, SPDEFhigh, and OPA1high metabolic adaptions were verified with primary CSCs from lung cancer patients. Accordingly, blocking lipogenesis and mitochondrial fusion efficiently impeded CSC expansion and growth of lung cancer patient-derived organoids. Together, lipogenesis regulates mitochondrial dynamics via OPA1 for controlling CSCs in human lung cancer.
Zhen Liu, Jiaxin Lei, Tong Wu, Weijie Hu, Ming Zheng, Ying Wang, Jingdong Song, Hang Ruan, Lin Xu, Tao Ren, Wei Xu, Zhenke Wen
Ovarian cancer (OvCa) preferentially metastasizes in association with mesothelial cell-lined surfaces. We sought to determine if mesothelial cells are required for OvCa metastasis and detect alterations in mesothelial cell gene expression and cytokine secretion upon interaction with OvCa cells. Using omental samples from patients with high-grade serous OvCa and mouse models with Wt1-driven GFP-expressing mesothelial cells, we validated the intratumoral localization of mesothelial cells during human and mouse OvCa omental metastasis. Removing mesothelial cells ex vivo from human and mouse omenta or in vivo using diphtheria toxin mediated ablation in Msln-Cre mice, significantly inhibited OvCa cell adhesion and colonization. Human ascites induced angiopoietin-like 4 (ANGPTL4) and stanniocalcin 1 (STC1) expression and secretion by mesothelial cells. Inhibition of STC1 or ANGPTL4 via RNAi, obstructed OvCa cell-induced mesothelial cell to mesenchymal transition while inhibition of ANGPTL4 alone obstructed OvCa cell-induced mesothelial cell migration and glycolysis. Inhibition of mesothelial cell ANGPTL4 secretion via RNAi prevented mesothelial cell induced monocyte migration, endothelial cell vessel formation and OvCa cell adhesion, migration, and proliferation. In contrast, inhibition of mesothelial cell STC1 secretion via RNAi prevented mesothelial cell induced endothelial cell vessel formation and OvCa cell adhesion, migration, proliferation, and invasion. Additionally, blocking ANPTL4 function with antibodies reduced the ex vivo colonization of three different OvCa cell lines on human omental tissue explants and in vivo colonization of ID8p53-/-Brca2-/- cells on mouse omenta. These findings indicate that mesothelial cells are important to the initial stages of OvCa metastasis, and that the crosstalk between mesothelial cells and the tumor microenvironment, promotes OvCa metastasis through the secretion of ANGPTL4.
Preety Bajwa, Kasjusz Kordylewicz, Agnes Bilecz, Ricardo R. Lastra, Kristen Wroblewski, Yuval Rinkevich, Ernst Lengyel, Hilary A. Kenny
Glioblastoma is the most malignant primary brain tumor for which the prognosis remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared to NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor β (PDGFRβ) proliferative and stem cell signaling pathways through post-transcriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.
Ryan C. Gimple, Guoxin Zhang, Shuai Wang, Tengfei Huang, Jina Lee, Suchet Taori, Deguan Lv, Deobrat Dixit, Matthew E. Halbert, Andrew R. Morton, Reilly L. Kidwell, Zhen Dong, Briana C. Prager, Leo Kim, Zhixin Qiu, Linjie Zhao, Qi Xie, Qiulian Wu, Sameer Agnihotri, Jeremy N. Rich
Metastatic progression of epithelial cancers can be associated with epithelial-mesenchymal-transition (EMT) including transcriptional inhibition of E-cadherin (CDH1) expression. Recently, EM plasticity (EMP) and E-cadherin-mediated, cluster-based metastasis and treatment resistance have become more appreciated. However, the mechanisms that maintain E-cadherin expression in this context are less understood. Through studies of inflammatory breast cancer (IBC) and a 3D tumor cell “emboli” culture paradigm, we discovered that COX-2 (PTGS2), a target gene of C/EBPd (CEBPD), or its metabolite prostaglandin E2 (PGE2) promotes protein stability of E-cadherin, β-catenin and p120 catenin through inhibition of GSK3β. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcome and, along with inhibitory GSK3β phosphorylation, in patient-derived xenografts (PDX) including triple negative breast cancer (TNBC). Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, though CDH1 mRNA levels increased, and reduced circulating tumor cell (CTC) clusters, and in combination with paclitaxel attenuated or regressed lung metastases. This study uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-cell adhesions and cell survival, suggesting that some patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.
Kuppusamy Balamurugan, Dipak K. Poria, Saadiya W. Sehareen, Savitri Krishnamurthy, Wei Tang, Lois McKennett, Veena Padmanaban, Kelli Czarra, Andrew J. Ewald, Naoto T. Ueno, Stefan Ambs, Shikha Sharan, Esta Sterneck
To better characterize the heterogeneity of multiple myeloma (MM), we profiled plasma cells (PCs) and their B cell lymphopoiesis in the BM samples from patients with monoclonal gammopathy of undetermined significance, smoldering MM, and active MM by mass cytometry (CyTOF) analysis. Characterization of intra- and interneoplastic heterogeneity of malignant plasmablasts and PCs revealed overexpression of the MM SET domain (MMSET), Notch-1, and CD47. Variations in upregulation of B cell signaling regulators (IFN regulatory factor 4 [IRF-4], CXCR4, B cell lymphoma 6 [Bcl-6], c-Myc, myeloid differentiation primary response protein 88 [MYD88], and spliced X box-binding protein 1 [sXBP-1]) and aberrant markers (CD319, CD269, CD200, CD117, CD56, and CD28) were associated with different clinical outcomes in clonal PC subsets. In addition, prognosis was related to heterogeneity in subclonal expression of stemness markers, including neuroepithelial stem cell protein (Nestin), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (KLF-4), and Nanog. Furthermore, we have defined significantly elevated levels of MMSET, MYD88, c-Myc, CD243, Notch-1, and CD47 from hematopoietic stem cells to PCs in myeloma B cell lymphopoiesis, noted even in premalignant conditions, with variably modulated expression of B cell development regulators, including IRF-4, Bcl-2, Bcl-6, and sXBP-1; aberrant PC markers (such as CD52, CD44, CD200, CD81, CD269, CD117, and CXCR4); and stemness-controlling regulators, including Nanog, KLF-4, octamer-binding transcription factor 3/4 (Oct3/4), Sox2, and retinoic acid receptor α2 (RARα2). This study provides the rationale for precise molecular profiling of patients with MM by CyTOF technology to define disease heterogeneity and prognosis.
Jana Jakubikova, Danka Cholujova, Gabor Beke, Teru Hideshima, Lubos Klucar, Merav Leiba, Krzysztof Jamroziak, Paul G. Richardson, Efstathios Kastritis, David M. Dorfman, Kenneth C. Anderson
Gastroesophageal adenocarcinomas (GEAs) harbor recurrent amplification of KRAS, leading to marked overexpression of WT KRAS protein. We previously demonstrated that SHP2 phosphatase, which acts to promote KRAS and downstream MAPK pathway activation, is a target in these tumors when combined with MEK inhibition. We hypothesized that SHP2 inhibitors may serve as a foundation for developing novel combination inhibitor strategies for therapy of KRAS-amplified GEA, including with targets outside the MAPK pathway. Here, we explore potential targets to effectively augment the efficacy of SHP2 inhibition, starting with genome-wide CRISPR screens in KRAS-amplified GEA cell lines with and without SHP2 inhibition. We identify candidate targets within the MAPK pathway and among upstream RTKs that may enhance SHP2 efficacy in KRAS-amplified GEA. Additional in vitro and in vivo experiments demonstrated the potent cytotoxicity of pan-ERBB kinase inhibitions in vitro and in vivo. Furthermore, beyond targets within the MAPK pathway, we demonstrate that inhibition of CDK4/6 combines potently with SHP2 inhibition in KRAS-amplified GEA, with greater efficacy of this combination in KRAS-amplified, compared with KRAS-mutant, tumors. These results suggest therapeutic combinations for clinical study in KRAS-amplified GEAs.
Tianxia Li, Osamu Kikuchi, Jin Zhou, Yichen Wang, Babita Pokharel, Klavdija Bastl, Prafulla Gokhale, Aine Knott, Yanxi Zhang, John G. Doench, Zandra V. Ho, Daniel V.T. Catenacci, Adam J. Bass
We assessed vaccine-induced antibody responses to the SARS-CoV2 ancestral virus and Omicron variant before and after booster immunization in 57 patients with B-cell malignancies. Over one third of vaccinated patients at the pre-booster timepoint were seronegative, and these patients were predominantly on active cancer therapies such as anti-CD20 monoclonal antibody. While booster immunization was able to induce detectable antibodies in a small fraction of seronegative patients, the overall booster benefit was disproportionately evident in patients already seropositive and not receiving active therapy. While ancestral and Omicron-reactive antibody levels among individual patients were largely concordant, neutralizing antibodies against Omicron tended to be reduced. Interestingly, in all patients, including those unable to generate detectable antibodies against SARS-CoV2 spike, we observed comparable levels of EBV and influenza reactive antibodies demonstrating that B cell-targeting therapies primarily impair de novo but not pre-existing antibody levels. These findings support rationale for vaccination prior to cancer treatment.
Joseph H. Azar, John P. Evans, Madison H. Sikorski, Karthik B. Chakravarthy, Selah McKenney, Ian Carmody, Cong Zeng, Rachael Teodorescu, No-Joon Song, Jamie L. Hamon, Donna Bucci, Maria Velegraki, Chelsea Bolyard, Kevin P. Weller, Sarah A. Reisinger, Seema A. Bhat, Kami J. Maddocks, Nathan Denlinger, Narendranath Epperla, Richard Gumina, Anastasia N. Vlasova, Eugene Oltz, Linda Saif, Dongjun Chung, Jennifer A. Woyach, Peter G. Shields, Shan-Lu Liu, Zihai Li, Mark P. Rubinstein
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