Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
Beom-Jun Kim, Young-Sun Lee, Sun-Young Lee, Wook-Young Baek, Young Jin Choi, Sung Ah Moon, Seung Hun Lee, Jung-Eun Kim, Eun-Ju Chang, Eun-Young Kim, Jin Yoon, Seung-Whan Kim, Sung Ho Ryu, Sun-Kyeong Lee, Joseph A. Lorenzo, Seong Hee Ahn, Hyeonmok Kim, Ki-Up Lee, Ghi Su Kim, Jung-Min Koh
Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber (PF)-forming cerebellar granule cells (Cgc). Postnatal Pkn1–/– animals showed a defective PF-Purkinje cell (PC) synapse formation. In vitro, Pkn1–/– Cgc exhibited deregulated axonal outgrowth, elevated AKT phosphorylation and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly Pkn1–/– Cgc had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels and the reduced density of presynaptic specifications in Pkn1–/– Cgc. In line with our in vitro data, Pkn1–/– mice showed AKT hyperactivation, elevated NeuroD2 levels and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgc and subsequently the correct PF-PC synapse formation.
Stephanie zur Nedden, Rafaela Eith, Christoph Schwarzer, Lucia Zanetti, Hartwig Seitter, Friedrich Fresser, Alexandra Koschak, Angus J.M. Cameron, Peter J. Parker, Gottfried Baier, Gabriele Baier-Bitterlich
Non-antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalised neo-epitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen-anti-CLEC-9A mAb conjugates target cross-presenting DCs, adjuvant must be co-delivered for cytotoxic T-cell (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating ovalbumin (OVA) antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation, CTL and antibody responses. OVA-Clec9A-TNE-induced DC activation required CD4 and CD8 epitopes, CD40 and IFN-α. Clec9A-TNE encapsulating human papillomavirus (HPV) E6-E7 significantly suppressed HPV-associated tumor growth while E6-E7-CpG did not. Clec9A-TNE loaded with pooled B16F10 melanoma neo-epitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neo-epitopes. Clec9A-TNE encapsulating six neo-epitopes significantly suppressed B16-F10 melanoma growth in a CD4 T cell-dependent manner. Thus, cross-presenting DCs targeted with antigen-Clec9A-TNE stimulate therapeutically-effective tumor-specific immunity, dependent on T cell help.
Bijun Zeng, Anton P.J. Middelberg, Adrian Gemiarto, Kelli MacDonald, Alan G. Baxter, Meghna Talekar, Davide Moi, Kirsteen M. Tullett, Irina Caminschi, Mireille H. Lahoud, Roberta Mazzieri, Riccardo Dolcetti, Ranjeny Thomas
A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that re-directing T cells to specialized niches in the bone marrow (BM) that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration towards vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis and a heightened capacity to generate poly-functional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, re-directed homing of T cells to the BM confers increased memory differentiation and anti-tumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.
Anjum B. Khan, Ben Carpenter, Pedro Santos e Sousa, Constandina Pospori, Reema Khorshed, James Griffin, Pedro Veliça, Mathias Zech, Sara Ghorashian, Calum Forrest, Sharyn Thomas, Sara Gonzalez Anton, Maryam Ahmadi, Angelika Holler, Barry Flutter, Zaida Ramirez-Ortiz, Terry K. Means, Clare L. Bennett, Hans Stauss, Emma Morris, Cristina Lo Celso, Ronjon Chakraverty
Metastatic breast cancers are still incurable. Characterizing the evolutionary landscape of these cancers, including the role of metastatic axillary lymph nodes (ALNs) in seeding distant organ metastasis, can provide a rational basis for effective treatments. Here, we have described the genomic analyses of the primary tumors and metastatic lesions from 99 samples obtained from 20 patients with breast cancer. Our evolutionary analyses revealed diverse spreading and seeding patterns that govern tumor progression. Although linear evolution to successive metastatic sites was common, parallel evolution from the primary tumor to multiple distant sites was also evident. Metastatic spreading was frequently coupled with polyclonal seeding, in which multiple metastatic subclones originated from the primary tumor and/or other distant metastases. Synchronous ALN metastasis, a well-established prognosticator of breast cancer, was not involved in seeding the distant metastasis, suggesting a hematogenous route for cancer dissemination. Clonal evolution coincided frequently with emerging driver alterations and evolving mutational processes, notably an increase in apolipoprotein B mRNA–editing enzyme, catalytic polypeptide-like–associated (APOBEC-associated) mutagenesis. Our data provide genomic evidence for a role of ALN metastasis in seeding distant organ metastasis and elucidate the evolving mutational landscape during cancer progression.
Ikram Ullah, Govindasamy-Muralidharan Karthik, Amjad Alkodsi, Una Kjällquist, Gustav Stålhammar, John Lövrot, Nelson-Fuentes Martinez, Jens Lagergren, Sampsa Hautaniemi, Johan Hartman, Jonas Bergh
Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti–CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund’s adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti–CTLA-4–induced, non-gp100–specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti–CTLA-4–induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti–CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.
Yared Hailemichael, Amber Woods, Tihui Fu, Qiuming He, Michael C. Nielsen, Farah Hasan, Jason Roszik, Zhilan Xiao, Christina Vianden, Hiep Khong, Manisha Singh, Meenu Sharma, Faisal Faak, Derek Moore, Zhimin Dai, Scott M. Anthony, Kimberly S. Schluns, Padmanee Sharma, Victor H. Engelhard, Willem W. Overwijk
The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.
Na Zhao, Jin Cao, Longyong Xu, Qianzi Tang, Lacey E. Dobrolecki, Xiangdong Lv, Manisha Talukdar, Yang Lu, Xiaoran Wang, Dorothy Z. Hu, Qing Shi, Yu Xiang, Yunfei Wang, Xia Liu, Wen Bu, Yi Jiang, Mingzhou Li, Yingyun Gong, Zheng Sun, Haoqiang Ying, Bo Yuan, Xia Lin, Xin-Hua Feng, Sean M. Hartig, Feng Li, Haifa Shen, Yiwen Chen, Leng Han, Qingping Zeng, John B. Patterson, Benny Abraham Kaipparettu, Nagireddy Putluri, Frank Sicheri, Jeffrey M. Rosen, Michael T. Lewis, Xi Chen
Breast cancer metastasis remains a clinical challenge, even within a single patient across multiple sites of the disease. Genome-wide comparisons of both the DNA and gene expression of primary tumors and metastases in multiple patients could help elucidate the underlying mechanisms that cause breast cancer metastasis. To address this issue, we performed DNA exome and RNA sequencing of matched primary tumors and multiple metastases from 16 patients, totaling 83 distinct specimens. We identified tumor-specific drivers by integrating known protein-protein network information with RNA expression and somatic DNA alterations and found that genetic drivers were predominantly established in the primary tumor and maintained through metastatic spreading. In addition, our analyses revealed that most genetic drivers were DNA copy number changes, the TP53 mutation was a recurrent founding mutation regardless of subtype, and that multiclonal seeding of metastases was frequent and occurred in multiple subtypes. Genetic drivers unique to metastasis were identified as somatic mutations in the estrogen and androgen receptor genes. These results highlight the complexity of metastatic spreading, be it monoclonal or multiclonal, and suggest that most metastatic drivers are established in the primary tumor, despite the substantial heterogeneity seen in the metastases.
Marni B. Siegel, Xiaping He, Katherine A. Hoadley, Alan Hoyle, Julia B. Pearce, Amy L. Garrett, Sunil Kumar, Vincent J. Moylan, Claudia M. Brady, Amanda E.D. Van Swearingen, David Marron, Gaorav P. Gupta, Leigh B. Thorne, Niamh Kieran, Chad Livasy, Elaine R. Mardis, Joel S. Parker, Mengjie Chen, Carey K. Anders, Lisa A. Carey, Charles M. Perou
Chuvash polycythemia is an inherited disease caused by a homozygous germline VHLR200W mutation, which leads to impaired degradation of HIF2α, elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous VhlR200W mutation. We previously showed that iron-regulatory protein 1–knockout (Irp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2α, suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed VhlR200W mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2α expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in VhlR200W mice in which Irp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2α expression and markedly reduced life-threatening erythrocytosis/polycythemia in the VhlR200W mice.
Manik C. Ghosh, De-Liang Zhang, Hayden Ollivierre, Michael A. Eckhaus, Tracey A. Rouault
The lack of defined correlates of protection hampers development of vaccines against tuberculosis (TB). In vitro mycobacterial outgrowth assays are thought to better capture the complexity of the human host/Mycobacterium tuberculosis (Mtb) interaction. We used a PBMC-based “mycobacterial-growth-inhibition-assay” (MGIA) to investigate the capacity to control outgrowth of Bacille Calmette-Guérin (BCG). Interestingly, strong control of BCG outgrowth was observed almost exclusively in individuals with recent exposure to Mtb, but not in (long-term) latent TB infection, and only modestly in BCG vaccinees. Mechanistically, control of mycobacterial outgrowth strongly correlated with the presence of a CD14dim monocyte population, but also required the presence of T cells. The nonclassical monocytes produced CXCL10, and CXCR3-receptor blockade inhibited the capacity to control BCG outgrowth. Expression of CXCR3 splice variants was altered in recently Mtb exposed individuals. Cytokines previously associated with trained immunity were detected in MGIA supernatants, and CXCL9, CXCL10, and CXCL11 represent new markers of trained immunity. These data indicate that CXCR3-ligands are associated with trained immunity and critical factors in controlling mycobacterial outgrowth.In conclusion, control of mycobacterial outgrowth early after exposure to Mtb is the result of trained immunity mediated by a CXCL10-producing non-classical CD14dim monocyte subset.
Simone A. Joosten, Krista E. van Meijgaarden, Sandra M. Arend, Corine Prins, Fredrik Oftung, Gro Ellen Korsvold, Sandra V. Kik, Rob J.W. Arts, Reinout van Crevel, Mihai G. Netea, Tom H.M. Ottenhoff