Notch signaling critically controls cell fate decisions in mammals, both during embryogenesis and in adults. In the skeleton, Notch suppresses osteoblast differentiation and sustains bone marrow mesenchymal progenitors during postnatal life. Stabilizing mutations of Notch2 cause Hajdu-Cheney syndrome, which is characterized by early-onset osteoporosis in humans, but the mechanism whereby Notch inhibits bone accretion is not fully understood. Here, we report that activation of Notch signaling by either Jagged1 or the Notch2 intracellular domain suppresses glucose metabolism and osteoblast differentiation in primary cultures of bone marrow mesenchymal progenitors. Importantly, deletion of Notch2 in the limb mesenchyme increases both glycolysis and bone formation in the long bones of postnatal mice, whereas pharmacological reduction of glycolysis abrogates excessive bone formation. Mechanistically, Notch reduces the expression of glycolytic and mitochondrial complex I genes, resulting in a decrease in mitochondrial respiration, superoxide production, and AMPK activity. Forced activation of AMPK restores glycolysis in the face of Notch signaling. Thus, suppression of glucose metabolism contributes to the mechanism, whereby Notch restricts osteoblastogenesis from bone marrow mesenchymal progenitors.
Seung-Yon Lee, Fanxin Long
Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide–gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3–/–) mice to obtain triallelic Cnga3+/– Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
Markus Burkard, Susanne Kohl, Timm Krätzig, Naoyuki Tanimoto, Christina Brennenstuhl, Anne E. Bausch, Katrin Junger, Peggy Reuter, Vithiyanjali Sothilingam, Susanne C. Beck, Gesine Huber, Xi-Qin Ding, Anja K. Mayer, Britta Baumann, Nicole Weisschuh, Ditta Zobor, Gesa-Astrid Hahn, Ulrich Kellner, Sascha Venturelli, Elvir Becirovic, Peter Charbel Issa, Robert K. Koenekoop, Günther Rudolph, John Heckenlively, Paul Sieving, Richard G. Weleber, Christian Hamel, Xiangang Zong, Martin Biel, Robert Lukowski, Matthias W. Seeliger, Stylianos Michalakis, Bernd Wissinger, Peter Ruth
The agonistic/antagonistic biocharacter of selective estrogen receptor modulators (SERMs) can have therapeutic advantages, particularly in the case of premenopausal breast cancers. Although the contradictory effects of these modulators have been studied in terms of crosstalk between the estrogen receptor α (ER) and coactivator dynamics and growth factor signaling, the molecular basis of these mechanisms is still obscure. We identify a series of regulatory mechanisms controlling cofactor dynamics on ER and SERM function, whose activities require F-box protein 22 (Fbxo22). Skp1, Cullin1, F-box–containing complex (SCFFbxo22) ubiquitylated lysine demethylase 4B (KDM4B) complexed with tamoxifen-bound (TAM-bound) ER, whose degradation released steroid receptor coactivator (SRC) from ER. Depletion of Fbxo22 resulted in ER-dependent transcriptional activation via transactivation function 1 (AF1) function, even in the presence of SERMs. In living cells, TAM released SRC and KDM4B from ER in a Fbxo22-dependent manner. SRC release by TAM required Fbxo22 on almost all ER-SRC–bound enhancers and promoters. TAM failed to prevent the growth of Fbxo22-depleted, ER-positive breast cancers both in vitro and in vivo. Clinically, a low level of Fbxo22 in tumor tissues predicted a poorer outcome in ER-positive/human epidermal growth factor receptor type 2–negative (HER2-negative) breast cancers with high hazard ratios, independently of other markers such as Ki-67 and node status. We propose that the level of Fbxo22 in tumor tissues defines a new subclass of ER-positive breast cancers for which SCFFbxo22-mediated KDM4B degradation in patients can be a therapeutic target for the next generation of SERMs.
Yoshikazu Johmura, Ichiro Maeda, Narumi Suzuki, Wenwen Wu, Atsushi Goda, Mariko Morita, Kiyoshi Yamaguchi, Mizuki Yamamoto, Satoi Nagasawa, Yasuyuki Kojima, Koichiro Tsugawa, Natsuko Inoue, Yasuo Miyoshi, Tomo Osako, Futoshi Akiyama, Reo Maruyama, Jun-ichiro Inoue, Yoichi Furukawa, Tomohiko Ohta, Makoto Nakanishi
People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney’s innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney’s collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell–specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor–mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.
Matthew J. Murtha, Tad Eichler, Kristin Bender, Jackie Metheny, Birong Li, Andrew L. Schwaderer, Claudia Mosquera, Cindy James, Laura Schwartz, Brian Becknell, John David Spencer
Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.
Hadas Lewinsky, Avital F. Barak, Victoria Huber, Matthias P. Kramer, Lihi Radomir, Lital Sever, Irit Orr, Vita Mirkin, Nili Dezorella, Mika Shapiro, Yosef Cohen, Lev Shvidel, Martina Seiffert, Yair Herishanu, Shirly Becker-Herman, Idit Shachar
Concordant activation of MYC and BCL-2 oncoproteins in double-hit lymphoma (DHL) results in aggressive disease that is refractory to treatment. By integrating activity-based proteomic profiling and drug screens, polo-like kinase-1 (PLK1) was identified as an essential regulator of the MYC-dependent kinome in DHL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression, and connoted poor outcome. Further, PLK1 signaling augmented MYC protein stability, and in turn, MYC directly induced PLK1 transcription, establishing a feed-forward MYC-PLK1 circuit in DHL. Finally, inhibition of PLK1 triggered degradation of MYC and of the antiapoptotic protein MCL-1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL cell growth, survival, and tumorigenicity, supporting clinical targeting of PLK1 in DHL.
Yuan Ren, Chengfeng Bi, Xiaohong Zhao, Tint Lwin, Cheng Wang, Ji Yuan, Ariosto S. Silva, Bijal D. Shah, Bin Fang, Tao Li, John M. Koomen, Huijuan Jiang, Julio C. Chavez, Lan V. Pham, Praneeth R. Sudalagunta, Lixin Wan, Xuefeng Wang, William S. Dalton, Lynn C. Moscinski, Kenneth H. Shain, Julie Vose, John L. Cleveland, Eduardo M. Sotomayor, Kai Fu, Jianguo Tao
The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14+HLA-DRneg cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14+ monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.
Veronica Huber, Viviana Vallacchi, Viktor Fleming, Xiaoying Hu, Agata Cova, Matteo Dugo, Eriomina Shahaj, Roberta Sulsenti, Elisabetta Vergani, Paola Filipazzi, Angela De Laurentiis, Luca Lalli, Lorenza Di Guardo, Roberto Patuzzo, Barbara Vergani, Elena Casiraghi, Mara Cossa, Ambra Gualeni, Valentina Bollati, Flavio Arienti, Filippo De Braud, Luigi Mariani, Antonello Villa, Peter Altevogt, Viktor Umansky, Monica Rodolfo, Licia Rivoltini
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.
Aditi Shastri, Gaurav Choudhary, Margarida Teixeira, Shanisha Gordon-Mitchell, Nandini Ramachandra, Lumie Bernard, Sanchari Bhattacharyya, Robert Lopez, Kith Pradhan, Orsolya Giricz, Goutham Ravipati, Li-Fan Wong, Sally Cole, Tushar D. Bhagat, Jonathan Feld, Yosman Dhar, Matthias Bartenstein, Victor J. Thiruthuvanathan, Amittha Wickrema, B. Hilda Ye, David A. Frank, Andrea Pellagatti, Jacqueline Boultwood, Tianyuan Zhou, Youngsoo Kim, A. Robert MacLeod, P.K. Epling-Burnette, Minwei Ye, Patricia McCoon, Richard Woessner, Ulrich Steidl, Britta Will, Amit Verma
Itch (pruritis) and pain represent two distinct sensory modalities; yet both have evolved to alert us to potentially harmful external stimuli. Compared with pain, our understanding of itch is still nascent. Here, we report a new clinical case of debilitating itch and altered pain perception resulting from the heterozygous de novo p.L811P gain-of-function mutation in NaV1.9, a voltage-gated sodium (NaV) channel subtype that relays sensory information from the periphery to the spine. To investigate the role of NaV1.9 in itch, we developed a mouse line in which the channel is N-terminally tagged with a fluorescent protein, thereby enabling the reliable identification and biophysical characterization of NaV1.9-expressing neurons. We also assessed NaV1.9 involvement in itch by using a newly created NaV1.9–/– and NaV1.9L799P/WT mouse model. We found that NaV1.9 is expressed in a subset of nonmyelinated, nonpeptidergic small-diameter dorsal root ganglia (DRGs). In WT DRGs, but not those of NaV1.9–/– mice, pruritogens altered action potential parameters and NaV channel gating properties. Additionally, NaV1.9–/– mice exhibited a strong reduction in acute scratching behavior in response to pruritogens, whereas NaV1.9L799P/WT mice displayed increased spontaneous scratching. Altogether, our data suggest an important contribution of NaV1.9 to itch signaling.
Juan Salvatierra, Marcelo Diaz-Bustamante, James Meixiong, Elaine Tierney, Xinzhong Dong, Frank Bosmans
Despite breakthroughs in immune checkpoint inhibitor (ICI) immunotherapy, not all human cancers respond to ICI immunotherapy and a large fraction of patients with the responsive types of cancers do not respond to current ICI immunotherapy. This clinical conundrum suggests that additional immune checkpoints exist. We report here that interferon regulatory factor 8 (IRF8) deficiency led to impairment of cytotoxic T lymphocyte (CTL) activation and allograft tumor tolerance. However, analysis of chimera mice with competitive reconstitution of WT and IRF8-KO bone marrow cells as well as mice with IRF8 deficiency only in T cells indicated that IRF8 plays no intrinsic role in CTL activation. Instead, IRF8 functioned as a repressor of osteopontin (OPN), the physiological ligand for CD44 on T cells, in CD11b+Ly6CloLy6G+ myeloid cells and OPN acted as a potent T cell suppressor. IRF8 bound to the Spp1 promoter to repress OPN expression in colon epithelial cells, and colon carcinoma exhibited decreased IRF8 and increased OPN expression. The elevated expression of OPN in human colon carcinoma was correlated with decreased patient survival. Our data indicate that myeloid and tumor cell–expressed OPN acts as an immune checkpoint to suppress T cell activation and confer host tumor immune tolerance.
John D. Klement, Amy V. Paschall, Priscilla S. Redd, Mohammed L. Ibrahim, Chunwan Lu, Dafeng Yang, Esteban Celis, Scott I. Abrams, Keiko Ozato, Kebin Liu