Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia

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Abstract

The protein-protein interaction between menin and Mixed Lineage Leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the Nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates for the first time profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1-rearrangements or NPM1 mutations

Authors

Szymon Klossowski, Hongzhi Miao, Katarzyna Kempinska, Tao Wu, Trupta Purohit, EunGi Kim, Brian M. Linhares, Dong Chen, Gloria Jih, Eric Perkey, Huang Huang, Miao He, Bo Wen, Yi Wang, Ke Yu, Stanley Chun-Wei Lee, Gwenn Danet-Desnoyers, Winifred Trotman, Malathi Kandarpa, Anitria Cotton, Omar Abdel-Wahab, Hongwei Lei, Yali Dou, Monica Guzman, Luke Peterson, Tanja A. Gruber, Sarah M. Choi, Duxin Sun, Pingda Ren, Lian-Sheng Li, Yi Liu, Francis J. Burrows, Ivan Maillard, Tomasz Cierpicki, Jolanta Grembecka

Graft-versus-host disease of the CNS is mediated by TNF upregulation in microglia

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Abstract

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes, MHC-II- and CD80-upregulation occurred upon GVHD induction. RNA-sequencing-based analysis of purified microglial obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER:Tnffl/-mice reduced MHC-II-expression, decreased CNS T-cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-β-activated kinase-1 (TAK1) activation and NF-κB/p38-MAPK-signaling. Selective Tak1-deletion in microglia using Cx3cr1creER:Tak1fl/fl-mice resulted in reduced TNF-production, microglial MHC-II, and improved neurocognitive-activity. Pharmacological TAK1-inhibition reduced TNF-production and MHC-II-expression by microglia, Th1 and Th17 T-cell infiltrates, VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF-production of microglia in the CNS of GVHD-patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as mediator of CNS-GVHD and provide a novel TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.

Authors

Nimitha R. Mathew, Janaki M. Vinnakota, Petya Apostolova, Daniel Erny, Shaima’a Hamarsheh, Geoffroy Andrieux, Jung-Seok Kim, Kathrin Hanke, Tobias Goldmann, Louise Chappell-Maor, Nadia El-Khawanky, Gabriele Ihorst, Dominik Schmidt, Justus Duyster, Jürgen Finke, Thomas Blank, Melanie Boerries, Bruce R. Blazar, Steffen Jung, Marco Prinz, Robert Zeiser

Myalgic encephalomyelitis/chronic fatigue syndrome patients exhibit altered T cell metabolism and cytokine associations

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Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease with no known cause or mechanism. There is an increasing appreciation for the role of immune and metabolic dysfunction in the disease. ME/CFS has historically presented in outbreaks, often has a flu-like onset, and results in inflammatory symptoms. Patients suffer from severe fatigue and post-exertional malaise. There is little known about the metabolism of specific immune cells in ME/CFS patients. To investigate immune metabolism in ME/CFS, we isolated CD4+ and CD8+ T cells from 53 ME/CFS patients and 45 healthy controls. We analyzed glycolysis and mitochondrial respiration in resting and activated T cells, along with markers related to cellular metabolism, and plasma cytokines. We found that ME/CFS CD8+ T cells have reduced mitochondrial membrane potential compared to healthy controls. Both CD4+ and CD8+ T cells from ME/CFS patients had reduced glycolysis at rest, while CD8+ T cells also had reduced glycolysis following activation. ME/CFS patients had significant correlations between measures of T cell metabolism and plasma cytokine abundance that differed from healthy control subjects. Our data indicate that patients have impaired T cell metabolism consistent with ongoing immune alterations in ME/CFS that may illuminate the mechanism behind this disease.

Authors

Alexandra H. Mandarano, Jessica Maya, Ludovic Giloteaux, Daniel L. Peterson, Marco Maynard, C. Gunnar Gottschalk, Maureen R. Hanson

IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

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Abstract

Epidermal growth factor receptor (EGFR) and MEK inhibitors (EGFR/MEKi) are beneficial for the treatment of solid cancers but are frequently associated with severe therapy-limiting acneiform skin toxicities. The underlying molecular mechanisms are poorly understood. Using gene expression profiling we identified IL-36γ and IL-8 as candidate drivers of EGFR/MEKi skin toxicity. We provide molecular and translational evidence that EGFR/MEKi in concert with the skin commensal bacterium Cutibacterium acnes act synergistically to induce IL-36γ in keratinocytes and subsequently IL-8, leading to cutaneous neutrophilia. IL-36γ expression was the combined result of C. acnes-induced NF-κB activation and EGFR/MEKi-mediated expression of the transcription factor Krüppel-like factor 4 (KLF4), due to the presence of both NF-κB- and KLF4-binding sites in the human IL-36γ gene promoter. EGFR/MEKi increased KLF4 expression by blockade of the EGFR-MEK-ERK pathway. These results provide an insight into understanding the pathological mechanism of the acneiform skin toxicities induced by EGFR/MEKi and identify IL-36γ and the transcription factor KLF4 as potential therapeutic targets.

Authors

Takashi K. Satoh, Mark Mellett, Barbara Meier-Schiesser, Gabriele Fenini, Atsushi Otsuka, Hans-Dietmar Beer, Tamara Rordorf, Julia-Tatjana Maul, Jürg Hafner, Alexander A. Navarini, Emmanuel Contassot, Lars E. French

Chikungunya virus replication in skeletal muscle cells is required for disease development

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Abstract

Chikungunya virus (CHIKV) is an arbovirus capable of causing a severe and often debilitating rheumatic syndrome in humans. CHIKV replicates in a wide variety of cell types in mammals, which has made attributing pathologic outcomes to replication at specific sites difficult. To assess the contribution of CHIKV replication in skeletal muscle cells to pathogenesis, we engineered a CHIKV strain exhibiting restricted replication in these cells via incorporation of target sequences for skeletal muscle cell-specific miR-206. This virus, which we term SKE, displayed diminished replication in skeletal muscle cells in a mouse model of CHIKV disease. Mice infected with SKE developed less severe disease signs, including diminished swelling in the inoculated foot and less necrosis and inflammation in the interosseous muscles. SKE infection was associated with diminished infiltration of T cells into the interosseous muscle as well as decreased production of IL-1b, IL-6, IP-10, and TNFa. Importantly, blockade of the IL-6 receptor led to diminished swelling of a control CHIKV strain capable of replication in skeletal muscle, reducing swelling to levels observed in mice infected with SKE. These data implicate replication in skeletal muscle cells and release of IL-6 as important mediators of CHIKV disease.

Authors

Anthony J. Lentscher, Mary K. McCarthy, Nicholas A. May, Bennett J. Davenport, Stephanie A. Montgomery, Krishnan Raghunathan, Nicole McAllister, Laurie A. Silva, Thomas E. Morrison, Terence S. Dermody

Lysine acetyltransferase 8 is involved in cerebral development and syndromic intellectual disability

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Abstract

Epigenetic integrity is critical for many eukaryotic cellular processes. An important question is how different epigenetic regulators control development and impact disease. Lysine acetyltransferase 8 (KAT8) is critical for acetylation of histone H4 at lysine 16 (H4K16), an evolutionarily conserved epigenetic mark. It is unclear what roles KAT8 plays in cerebral development and human disease. Here, we report that cerebrum-specific knockout mice displayed cerebral hypoplasia in the neocortex and hippocampus, along with improper neural stem and progenitor cell (NSPC) development. Mutant cerebrocortical neuroepithelia exhibited faulty proliferation, aberrant neurogenesis, massive apoptosis and scant H4K16 propionylation. Mutant NSPCs formed poor neurospheres, and pharmacological KAT8 inhibition abolished neurosphere formation. Moreover, we describe KAT8 variants in nine patients with intellectual disability, seizures, autism, dysmorphisms and other anomalies. The variants altered chromobarrel and catalytic domains of KAT8, thereby impairing nucleosomal H4K16 acetylation. Valproate was effective for treating epilepsy in at least two of the individuals. This study uncovers a critical role of KAT8 in cerebral and NSPC development, identifies nine individuals with KAT8 variants, and links deficient H4K16 acylation directly to intellectual disability, epilepsy and other developmental anomalies.

Authors

Lin Li, Mohammad Ghorbani, Monika Weisz-Hubshman, Justine Rousseau, Isabelle Thiffault, Rhonda E. Schnur, Catherine Breen, Renske Oegema, Marjan M.M. Weiss, Quinten Waisfisz, Sara Welner, Helen Kingston, Jordan A. Hills, Elles M.J. Boon, Lina Basel-Salmon, Osnat Konen, Hadassa Goldberg-Stern, Lily Bazak, Shay Tzur, Jianliang Jin, Xiuli Bi, Michael Bruccoleri, Kirsty McWalter, Megan T. Cho, Maria Scarano, G. Bradley Schaefer, Susan S. Brooks, Susan Starling Hughes, K.L.I. van Gassen, Johanna M. van Hagen, Tej K. Pandita, Pankaj B. Agrawal, Philippe M. Campeau, Xiang-Jiao Yang

CD73 Immune Checkpoint Defines Regulatory NK-cells within the Tumor Microenvironment

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Abstract

High levels of ecto-5'-nucleotidase (CD73) have been implicated in immune suppression and tumor progression, and have also been observed in cancer patients who progress on anti-PD-1 immunotherapy. While regulatory T cells can express CD73 and inhibit T cell responses via the production of adenosine, less is known about CD73 expression in other immune cell populations. We found that tumor-infiltrating NK cells upregulate CD73 expression and the frequency of these CD73+ NK cells correlated with larger tumor size in breast cancer patients. In addition, the expression of multiple alternative immune checkpoint receptors including LAG-3, VISTA, PD-1, and PD-L1 was significantly higher in CD73 positive NK cells than in CD73 negative NK cells. Mechanistically, NK cells transport CD73 in intracellular vesicles to the cell surface and the extracellular space via actin polymerization-dependent exocytosis upon engagement of 4-1BBL on tumor cells. These CD73 positive NK cells undergo transcriptional reprogramming and upregulate IL10 production via STAT3 transcriptional activity, suppressing CD4 T cell proliferation and IFN-ɣ production. Taken together, our results support that tumors can hijack NK cells as a means to escape immunity and that CD73 expression defines an inducible population of NK cells with immune regulatory properties within the tumor microenvironment.

Authors

Shi Yong Neo, Ying Yang, Record Julien, Ran Ma, Xinsong Chen, Ziqing Chen, Nicholas P. Tobin, Emily Blake, Christina Seitz, Ron Thomas, Arnika Kathleen Wagner, John Andersson, Jana de Boniface, Jonas Bergh, Shannon Murray, Evren Alici, Richard Childs, Martin Johansson, Lisa S. Westerberg, Felix Haglund, Johan Hartman, Andreas Lundqvist

A stress-responsive enhancer induces dynamic drug resistance in acute myeloid leukemia

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Abstract

The drug efflux pump ABCB1 is a key driver of chemoresistance, and high expression predicts for treatment failure in acute myeloid leukemia (AML). In this study, we identified and functionally validated the network of enhancers that controls expression of ABCB1. We show that exposure of leukemia cells to daunorubicin activated an integrated stress response-like transcriptional program to induce ABCB1 through remodeling and activation of an ATF4-bound, stress-responsive enhancer. Protracted stress primed enhancers for rapid increases in activity following re-exposure of cells to daunorubicin, providing an epigenetic memory of prior drug treatment. In primary human AML, exposure of fresh blast cells to daunorubicin activated the stress-responsive enhancer and led to dose-dependent induction of ABCB1. Dynamic induction of ABCB1 by diverse stressors, including chemotherapy, facilitated escape of leukemia cells from targeted third-generation ABCB1 inhibition, providing an explanation for the failure of ABCB1 inhibitors in clinical trials. Stress-induced up regulation of ABCB1 was mitigated by combined use of pharmacologic inhibitors U0126 and ISRIB, which inhibit stress signalling and have potential for use as adjuvants to enhance the activity of ABCB1 inhibitors.

Authors

Mark S. Williams, Fabio M.R. Amaral, Fabrizio Simeoni, Tim C.P. Somervaille

Oral immunotherapy tolerizes mice to enzyme replacement therapy for Morquio A syndrome

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Abstract

Immune response to therapeutic enzymes poses a detriment to patient safety and treatment outcome. Enzyme replacement therapy (ERT) is a standard therapeutic option for some types of Mucopolysaccharidoses including Morquio A syndrome caused by GALNS deficiency. Current protocols tolerize patients using cytotoxic immunosuppressives which can cause adverse effects. Here we show development of tolerance in Morquio A mice via oral delivery of peptide or GALNS during ten days prior to ERT. Our results show that using an immunodominant peptide (I10) or the complete enzyme (GALNS) to orally induce tolerance to GALNS prior to ERT, resulted in several improvements to ERT in mice: i) decreased splenocyte proliferation after in-vitro GALNS stimulation; ii) modulation of cytokine secretion profile; iii) decline in GALNS-specific IgG or IgE plasma; iv) decreased GAG storage in liver; and v) fewer circulating immune-complexes in plasma. This model could be extrapolated to other lysosomal storage disorders where immune response hinders ERT.

Authors

Angela C. Sosa, Barbara Kariuki, Qi Gan, Alan P. Knutsen, Clifford J. Bellone, Miguel A. Guzmán, Luis A. Barrera, Shunji Tomatsu, Anil K. Chauhan, Eric Armbrecht, Adriana M. Montaño

GABA interneurons are the cellular trigger for ketamine’s rapid antidepressant actions

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Abstract

A single sub-anesthetic dose of ketamine, an NMDA receptor (NMDAR) antagonist, produces rapid and sustained antidepressant actions in depressed patients, addressing a major unmet need for the treatment of mood disorders. Ketamine produces a rapid increase in extracellular glutamate and synaptic formation in the prefrontal cortex, but the initial cellular trigger that initiates these and its behavioral actions has not been identified. To address this question, we used a combination of viral shRNA and conditional mutation to produce cell specific knockdown or deletion of a key NMDAR subunit, GluN2B, implicated in the actions of ketamine. The results demonstrate that the antidepressant actions of ketamine were blocked by GluN2B-NMDAR knockdown on GABA (Gad1) interneurons, as well as subtypes expressing somatostatin (Sst), or parvalbumin (Pvalb), but not glutamate principle neurons in the mPFC. Further analysis of GABA subtypes showed that cell specific knockdown or deletion of GluN2B in Sst interneurons blocked or occluded the antidepressant actions of ketamine and revealed sex-specific differences that are associated with excitatory postsynaptic currents on mPFC principle neurons. These findings demonstrate that GluN2B-NMDARs on GABA interneurons are the initial cellular trigger for the rapid antidepressant actions of ketamine and show sex-specific adaptive mechanisms to GluN2B modulation.

Authors

Danielle M. Gerhard, Santosh Pothula, Rong-Jian Liu, Min Wu, Xiao-Yuan Li, Matthew J. Girgenti, Seth R. Taylor, Catharine H. Duman, Eric Delpire, Marina Picciotto, Eric S. Wohleb, Ronald S. Duman