Background: Proglucagon can be processed to Glucagon-Like Peptide-1 (GLP-1) within the islet but its contribution to islet function in humans remains unknown. We sought to understand whether ‘pancreatic’ GLP-1 alters islet function in humans and whether this is affected by type 2 diabetes.Methods: We therefore studied individuals with and without type 2 diabetes on 2 occasions in random order. On one occasion exendin 9-39, a competitive antagonist of the GLP-1 Receptor (GLP1R), was infused, while on the other saline was infused. The tracer dilution technique ([3-3H] glucose) was used to measure glucose turnover during fasting and during a hyperglycemic clamp.Results: Exendin 9-39 increased fasting glucose concentrations; fasting islet hormone concentrations were unchanged, but inappropriate for the higher fasting glucose observed. In people with type 2 diabetes fasting glucagon concentrations were markedly elevated and persisted despite hyperglycemia. This impaired suppression of endogenous glucose production by hyperglycemia. These data show that GLP1R blockade impairs islet function, implying that intra-islet GLP1R activation alters islet responses to glucose and does so to a greater degree in people with type 2 diabetes.
Andrew A. Welch, Rahele A. Farahani, Aoife M. Egan, Marcello C. Laurenti, Maya Zeini, Max Vella, Kent R. Bailey, Claudio Cobelli, Chiara Dalla Dalla Man, Aleksey Matveyenko, Adrian Vella
The BCL-2 inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B-cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL-2 mutations have been described, this likely only explains a subset of resistant cases. Using two complementary functional precision medicine techniques -- BH3-profiling and high throughput-kinase activity mapping -- we found that hyperphosphorylation of BCL-2 family proteins, including anti-apoptotic MCL-1 and BCL-2 and pro-apoptotic BAD and BAX, underlies functional mechanisms of both intrinsic and acquired resistance of venetoclax in CLL and DLBCL. Additionally, we provide evidence that anti-apoptotic BCL-2 family protein phosphorylation alters the apoptotic protein interactome, thereby changing the profile of functional dependence on these pro-survival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs re-wired these dependences, thus restoring sensitivity to venetoclax in a panel of venetoclax resistant lymphoid cell lines, resistant mouse model, and paired patient samples pre-venetoclax and at time of progression.
Stephen Jun Fei Chong, Fen Zhu, Olga Dashevsky, Rin Mizuno, Jolin X.H. Lai, Liam Hackett, Christine E. Ryan, Mary C. Collins, J. Bryan Iorgulescu, Romain Guièze, Johany Penailillo, Ruben Carrasco, Yeonjoo C. Hwang, Denise P. Muñoz, Mehdi Bouhaddou, Yaw Chyn Lim, Catherine J. Wu, John N. Allan, Richard R. Furman, Boon Cher Goh, Shazib Pervaiz, Jean-Philippe Coppé, Constantine S. Mitsiades, Matthew S. Davids
Monocytes and monocyte-derived macrophages (MDM) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here we show that PDGFB-driven GBM cells induce the expression of the potent pro-inflammatory cytokine IL-1β in MDM, which engages IL-1R1 in tumor cells, activates the NF-kB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1β/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1β/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, reduced exhausted CD8+ T cells, and thereby extends the survival of tumor-bearing mice. In contrast to IL-1β, IL-1α exhibits anti-tumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss of interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively-active NF-kB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1β could be considered as an effective therapy specifically for proneural GBM.
Zhihong Chen, Bruno Giotti, Milota Kaluzova, Montserrat Puigdelloses Vallcorba, Kavita Rawat, Gabrielle Price, Cameron J. Herting, Gonzalo Piñero, Simona Cristea, James L. Ross, James Ackley, Victor Maximov, Frank Szulzewsky, Wes Thomason, Mar Marquez-Ropero, Angelo Angione, Noah Nichols, Nadejda M. Tsankova, Franziska Michor, Dmitry M. Shayakhmetov, David H. Gutmann, Alexander M. Tsankov, Dolores Hambardzumyan
Systemic autoimmune and autoinflammatory diseases are characterized by genetic and cellular heterogeneity. While current single-cell genomics methods provide insights into known disease subtypes, these analysis methods do not readily reveal novel cell-type perturbation programs shared amongst distinct patient subsets. Here, we performed single-cell RNA-Seq of PBMCs of systemic juvenile idiopathic arthritis (SJIA) patients with diverse clinical manifestations, including macrophage activation syndrome (MAS) and lung disease (LD). We introduced two new computational frameworks called UDON and SATAY-UDON which define new patient subtypes based on their underlying disrupted cellular programs as well as associated biomarkers or clinical features. Among twelve independently identified subtypes, this analysis uncovered a novel complement and interferon activation program identified in SJIA-LD monocytes. Extending these analyses to adult and pediatric lupus patients found new but also shared disease programs with SJIA, including interferon and complement activation. Finally, supervised comparison of these programs in a compiled single-cell pan-immune atlas of over 1,000 healthy donors found a handful of normal healthy donors with evidence of early inflammatory activation in subsets of monocytes and platelets, nominating new possible biomarkers for early disease detection. Thus, integrative pan-immune single-cell analysis resolved new conserved gene programs underlying inflammatory disease pathogenesis and associated complications.
Emely L. Verweyen, Kairavee Thakkar, Sanjeev Dhakal, Elizabeth J. Baker, Kashish Chetal, Daniel Schnell, Scott W. Canna, Alexei A. Grom, Nathan Salomonis, Grant S. Schulert
Productively infected cells are generally thought to arise by HIV infection of activated CD4+ T cells, and these infected activated cells are also thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate by direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues restricted to resting CD4+ T cells and expression of pTEFb in these resting cells to enable productive infection, and we documented persistence of HIV producing resting T cells during ART. We thus provide evidence of a mechanism by which direct infection of resting T cell populations in lymphoid tissues to generate productively and latently infected cells could continually replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.
Stephen W. Wietgrefe, Jodi Anderson, Lijie Duan, Peter J. Southern, Paul Zuck, Guoxin Wu, Bonnie J. Howell, Cavan Reilly, Eugène Kroon, Suthat Chottanapund, Supranee Buranapraditkun, Carlo Sacdalan, Nicha Tulmethakaan, Donn J. Colby, Nitiya Chomchey, Peeriya Prueksakaew, Suteeraporn Pinyakorn, Rapee Trichavaroj, Julie L. Mitchell, Lydie Trautmann, Denise C. Hsu, Sandhya Vasan, Sopark Manasnayakorn, Mark de Souza, Sodsai Tovanabutra, Alexandra Schuetz, Merlin L. Robb, Nittaya Phanuphak, Jintanat Ananworanich, Timothy W. Schacker, Ashley T. Haase
Antibody-drug conjugates(ADCs) are promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer(mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, conferred sensitivity and were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive(ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 wild-type ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 non-expressors governed response. Importantly, wild-type TP53 predicted non-responsiveness (7/8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.
Supreet Agarwal, Lei Fang, Kerry McGowen, JuanJuan Yin, Joel Bowman, Anson T. Ku, Aian Neil Alilin, Eva Corey, Martine P. Roudier, Lawrence D. True, Ruth F. Dumpit, Ilsa Coleman, John K. Lee, Peter S. Nelson, Brian J. Capaldo, Aida Mariani, Clare E. Hoover, Ilya S. Senatorov, Michael Beshiri, Adam G. Sowalsky, Elaine M. Hurt, Kathleen Kelly
The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow derived macrophages. Alternatively activated macrophages from the skin of atopic dermatitis patients showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and co-staining, and GLUT3 expression was significantly decreased in non-healing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independent from its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL4Ra subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain (ICH), and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.
Dong-Min Yu, Jiawei Zhao, Eunice E. Lee, Dohun Kim, Ruchika Mahapatra, Elysha K. Rose, Zhiwei Zhou, Calvin R. Hosler, Abdullah El-Kurdi, Jun-yong Choe, E. Dale Abel, Gerta Hoxhaj, Kenneth D. Westover, Raymond J. Cho, Jeffrey B. Cheng, Richard C. Wang
The metastasis of cancer cells is the main cause of death for patients with gastric cancer (GC). Mounting evidence has demonstrated the vital importance of tumor-associated macrophages in promoting tumor invasion and metastasis; however, the interaction between tumor cells and macrophages in GC is largely unknown. In this study, we demonstrated that cyclase-associated protein 2 (CAP2) was upregulated in GC, especially in cases with lymph node metastasis, and was correlated with a poorer prognosis. The transcription factor JUN directly bound to the promoter region of CAP2 and activated CAP2 transcription. The N-terminal domain of CAP2 bound to the WD5-7 domain of receptor for activated C kinase 1 (RACK1) and induced M2 macrophage polarization by activating the SRC/focal adhesion kinase (FAK)/ ERK signaling pathway, which resulted in interleukin-4 (IL4) and IL10 secretion. Polarized M2 macrophages induced premetastatic niche formation and promoted GC metastasis by secreting transforming growth factor beta (TGFB1), which created a TGFB1/JUN/CAP2-positive feedback loop to activate CAP2 expression continuously. Furthermore, we identified Salvianolic acid B as an inhibitor of CAP2, which effectively inhibited GC cell invasion capabilities by suppressing the SRC/FAK/ERK signaling pathway. Our data suggest that CAP2, a key molecule mediating the interaction between GC cells and tumor-associated macrophages, may be a promising therapeutic target for suppressing tumor metastasis in GC.
Guohao Zhang, Zhaoxin Gao, Xiangyu Guo, Ranran Ma, Xiaojie Wang, Pan Zhou, Chunlan Li, Zhiyuan Tang, Ruinan Zhao, Peng Gao
Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat-derived RNAs in a non-coding and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases. Here, we show that a CWG repeat DNA-targeting compound, cyclic pyrrole¬–imidazole polyamide (CWG-cPIP), suppresses the pathogenesis of coding and non-coding CWG repeat diseases. CWG-cPIP binds to the hairpin form of mismatched CWG DNA, interfering with transcription elongation by RNA polymerase through a preferential activity towards repeat-expanded DNA. We found that CWG-cPIP selectively inhibits pathogenic mRNA transcripts from expanded CWG repeats, reducing CUG RNA foci and polyglutamine accumulation in cells from patients with myotonic dystrophy type-1 (DM1) and Huntington’s disease (HD). Treatment with CWG-cPIP ameliorated behavioral deficits in adeno-associated virus-mediated CWG repeat-expressing mice and a genetic mouse model of HD, without cytotoxicity or off-target effects. Together, we present a novel candidate compound that targets expanded CWG repeat DNA independent of its genomic location and reduces both pathogenic RNA and protein levels. CWG-cPIP may be used for the treatment of CWG repeat diseases and for improving clinical outcomes.
Susumu Ikenoshita, Kazuya Matsuo, Yasushi Yabuki, Kosuke Kawakubo, Sefan Asamitsu, Karin Hori, Shingo Usuki, Yuki Hirose, Toshikazu Bando, Kimi Araki, Mitsuharu Ueda, Hiroshi Sugiyama, Norifumi Shioda
BACKGROUND. HIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (EC) and people on long-term therapy suggests that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing. METHODS. In this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells. RESULTS. The proviral landscape was dominated by two large clones with replication-competent proviruses integrated into Zinc Finger genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-folds less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene. CONCLUSION. We provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.
Filippo Dragoni, Abena Kwaa, Caroline C.G. Traut, Rebecca T. Veenhuis, Bezawit A. Woldemeskel, Angelica Camilo-Contreras, Hayley E. Raymond, Arbor G. Dykema, Eileen P. Scully, Amanda M. Rosecrans, Kellie N. Smith, Frederic D. Bushman, Francesco R. Simonetti, Joel N. Blankson
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