Activation Of Innate Immune Pathways Research Articles

Platelet TLR9 Promotes Pulmonary Thrombo-Inflammation in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder, which affects millions of people worldwide. Erythrocyte sickling, chronic hemolysis and ischemia-reperfusion injury promote sterile inflammation in SCD.Recent evidence suggests that activation of innate immune pathways in platelets contribute to development of acute chest syndrome (ACS), a type of acute lung injury in SCD by promoting occlusion of lung arterioles by neutrophil-platelet-erythrocyte aggregates (pulmonary thrombo-inflammation). Although, TLR9 is functionally expressed on the surface of activated platelets and may bind to circulating double stranded DNA, the role of platelet TLR-9 in promoting pulmonary thrombo-inflammation in SCD remains elusive. Our initial findings using intravenous lipopolysaccharide (0.1 μg/kg LPS) or oxy-hemoglobin (10 μmole/kg oxy-Hb) triggered model of vaso-occlusive crisis in transgenic-humanized SCD mice reveal that pulmonary vaso-occlusion is associated with elevated surface-expression of platelet TLR-9 in SCD mice. Unlike SCD mice, surface-expression of TLR9 was undetectable in the platelets of control mice challenged with either IV LPS or oxy-Hb. These findings suggest a potential role for platelet TLR9 in promoting lung vaso-occlusion and injury in SCD. Currently, studies are in progress to identify the innate immune signaling downstream to platelet TLR9, contributing to pulmonary thrombo-inflammation in SCD.

Novel Evidence That Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) By Inhibiting Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) - Implications to Understand Better Diurnal Trafficking of HSPCs and Anti-Inflammatory Effects of Melatonin

Background. Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and pick at early morning hours. We have reported that the level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) (Stem Cell Rev Rep. 2018;14(5):677-685) that drives activation of Nlrp3 inflammasome (Stem Cell Rev Rep. 2020;16(2):335-343). Therefore, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Cell migration is also inhibited by the anti-inflammatory enzyme heme oxygenase-1 (HO-1), which inhibits Nlrp3 inflammasome (Leukemia. 2022;36(1):23-32). It is well known that circadian rhythm is regulated by the release of melatonin from the pineal gland, which also has some anti-inflammatory effects (J. Pineal Res. 2018;65:e12525). Aim of the study.Since stem cell trafficking is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from bone marrow (BM) into PB by inhibiting Nlrp3 inflammasome activation. Materials and Methods. To test this hypothesis, first, we tested the expression of melatonin receptors in murine and human HSPCs. We employed Transwell system to see the effect of melatonin on the migration of human and murine HSPCs to the SDF-1 gradient. Next, we performed pharmacological mobilization of normal mice with G-CSF or AMD3100 in the presence of melatonin. Finally, employing Glow assay, we evaluated the effect of melatonin on activation of Nlrp3 inflammasome in HSPCs stimulated by BM chemotactic factors and the effect of melatonin on expression of migration inhibitory HO-1. Since membrane lipid rafts (MLRs) are crucial for the proper response of cells to chemoattractants, we also evaluated the effect of melatonin on the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in the synthesis of cholesterol and sphingolipids that are major constituents of MLRs. Results. We noticed that HSPCs express melatonin receptors, and melatonin inhibits in vitro Transwell assays migration of HSPCs and negatively affects mobilization of these cells into PB in response to G-CSF and AMD3100 administration. We also observed that melatonin inhibits Nlrp3 inflammasome activation and the formation of MLRs. This is explained by a decrease in the expression of enzymes such as SREBP2, HMGCs, and HMGCR involved in cholesterol synthesis leading to impaired formation of MLRs. Moreover, melatonin increases in HSPCs expression of heme oxygenase-1 (HO-1), that is a known inhibitor of Nlrp3 inflammasome and a negative regulator of cell migration. Conclusions. We provide for the first-time evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs in vitro assays and in vivo their release from BM in response to G-CSF and AMD3100. This is correlated with inhibition of cholesterol synthesis, which results in defective assembly of MLRs, as well as a decrease in Nlrp3 inflammasome activity and an increase in expression of HO-1. Since melatonin is a commonly used shelf drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more light on molecular mechanisms of melatonin involvement in the diurnal circulation of HSPCs.

IRAK1 Contributes to IRAK4 Inhibitor Resistance Via Non-Canonical Signaling Mechanisms in MDS/AML

IRAKs are a family of related kinases that operate at the nexus of multiple innate immune and inflammatory pathways implicated in myeloid malignancies. IRAK4 inhibitors have advanced into clinical trials for MDS and AML validating IRAK4 as a therapeutic target. Early data from these trials has been encouraging; however, the overall responses remain modest. Herein, we explored mechanisms of resistance to IRAK4-selective inhibitors in MDS/AML. Our findings uncovered non-canonical IRAK1/4 signaling paradigms driving leukemic stem and progenitor cells (LSPC) and yielded novel therapeutic strategies for MDS/AML. Consistent with the initial observations from the clinical trials, inhibition or deletion of IRAK4 in a panel of MDS/AML cell lines and patient samples resulted in incomplete suppression of LSPCs and a corresponding activation of innate immune pathways. Given the evolutionary conserved redundancy of IRAK-dependent pathways, we first examined the expression of IRAK paralogs. In IRAK4 knockout (KO) MDS/AML cells, we only observed overexpression and activation of the IRAK4 paralog, IRAK1. IRAK4 kinase inhibitors or PROTACs resulted in a similar compensatory activation of IRAK1 in MDS/AML. To validate the compensation of IRAK1, we deleted IRAK1 in MDS/AML cells and found that concomitant suppression of IRAK1 led to a significant reduction of LSPC function in IRAK4-KO MDS/AML cells. Co-deletion of IRAK1 and IRAK4 similarly reduced leukemic engraftment and extended survival in an AML xenograft model. Hence, IRAK1 mitigates the efficacy of IRAK4-inhibitors in MDS/AML. Canonical IRAK signaling depends on recruitment of MyD88 and IRAK4 to activated receptors, which results in the subsequent recruitment and activation of IRAK1. Based on this canonical model, IRAK1 and IRAK4 are thus independently essential for signaling. Since dual inhibition of IRAK1/4 is obligatory for suppression of LSPCs, we posited that conventional IRAK signaling is not sufficient nor essential in MDS/AML. Therefore, we first examined the role of proximal upstream (MyD88) and downstream (TRAF6) effectors of IRAK1/4. Deletion of TRAF6 resulted in suppression of LSPCs as observed upon inhibition of IRAK1/4. In contrast, MyD88-KO AML cells did not exhibit a functional defect but did retain sensitivity to deletion of IRAK1 and IRAK4, indicating canonical MyD88-dependent signaling is not operational in MDS/AML. To investigate non-canonical IRAK1/4 signaling we performed RNA- and ATAC-seq and identified gene expression programs dependent on IRAK1 and/or IRAK4, yet independent of MyD88. IRAK1/4 deletion correlated with dysregulation of transcription factors involved in stem cell maintenance and myeloid differentiation. The requirement for IRAK1/4 in preserving an undifferentiated LSPC state was corroborated by morphological assessment of IRAK1/4-KO MDS/AML cells. To delineate the signaling mechanisms by which IRAK1/4 governs stem programs, we performed mass spectrometry proteomics and identified unique IRAK4- and IRAK1-interacting proteins. Integration of the proteomic and transcriptomic studies identified pathways (HIF1A, STAT3/IRFs, E2F4) implicated in stem cell and undifferentiated states as effectors of IRAK1 and IRAK4. To extend these studies to improve the efficacy of IRAK4-targeted therapy in MDS/AML, we developed a novel series of structurally related inhibitors targeting IRAK1 and IRAK4 (KME-2780: IRAK1 IC50 = 32 nM; IRAK4 IC50 = 0.9 nM) or IRAK4 alone (KME-3859: IRAK1 IC50 = >500 nM; IRAK4 IC50 = 5 nM). In human AML cell lines and patient samples, the dual IRAK1/4 inhibitor was significantly more effective at inducing cell death and differentiation, and attenuating LSPC function as compared to the IRAK4 inhibitor. Thus, the mandate for targeting both IRAK1 and IRAK4 established in our genetic studies translates to pharmacologic interventions. Overall, we demonstrate that compensation by IRAK1 is a barrier to IRAK4-directed therapy and reveal that dual IRAK1/4 inhibitors are needed for achieving optimal clinical response in MDS/AML. In the process, we also uncovered novel and non-canonical signaling paradigms governing the oncogenic role of IRAK1/4.

Unraveling the Tomaralimab Epitope on the Toll-like Receptor 2 via Molecular Dynamics and Deep Learning.

Tomaralimab (OPN-305) is the first humanized immunoglobulin G4 monoclonal antibody against TLR2 and is designed to prevent inflammation that is driven by inappropriate or excessive activation of innate immune pathways. Here, we constructed a homology model of Tomaralimab and its complex with TLR2 at different mapped epitopes and unraveled their behavior at the atomistic level. Furthermore, we predicted a novel epitope (leucine-rich region 9–12) near the lipopeptide-binding site that can be targeted and studied for the utility of therapeutic antibodies. A geometric deep learning algorithm was used to envisage Tomaralimab binding affinity changes upon mutation. There was a significant difference in binding affinity for Tomaralimab following epitope-mutated alanine substitutions of Val266, Pro294, Arg295, Asn319, Pro326, and His372. Using deep learning-based ΔΔG prediction, we computationally contrasted human TLR2–TLR2, TLR2–TLR1, and TLR2–TLR6 dimerization. These results reveal the mechanism that underlies Tomaralimab binding to TLR2 and should help to design structure-based mimics or bispecific antibodies that can be used to inhibit both lipopeptide-binding and TLR2 dimerization.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways—interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)—activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung–derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Testing the role of mab‐5/Hoxin C. elegansInnate Immunity

The innate immune system acts as an organism’s first line of defense against invading pathogens. It accomplishes this by using pathogen‐associated molecular patterns (PAMPs) or damage‐associated molecular patterns (DAMPs) to recognize and destroy invaders. While the mechanisms for innate immunity in response to pathogens have been studied, the genetic and environmental influences that govern innate immune development are unknown. In the Ackley lab we use Caenorhabditis elegans,a soil‐dwelling bacterivore nematode, to study the genetics of innate immunity. My project focuses on how mutations in the mab‐5gene affect resistance to bacterial pathogens. mab‐5 is a Hox‐family transcription factor that has primarily been studied in early organismal development, but transcriptome analyses of both gain and loss‐of‐function mutations identified mab‐5 regulation of innate immune genes. I have tested the resistance of four mab‐5mutant strains following exposure to a bacterial pathogen, Staphylococcus epidermidis,and compared survival to animals fed a normal diet of non‐pathogenic Escherichia coli. I have tested the wild‐type strain, N2, two loss‐of‐function mutations, mab‐5(gk670) and mab‐5(e1239), and one gain‐of‐ function mutant, mab‐5(e1750). I have found that the mab‐5(e1750)strain has increased resistance compared to the loss‐of‐function and wild‐type strains during the first week following exposure to S. epidermidis. This is consistent with our observation that increased mab‐5activity leads to increased expression of innate immune genes. To corroborate this observation I am testing another potential gain‐of‐function line, wgIs77,from the ModEncode project, which has an integrated MAB‐5::GFP fusion protein. I am also using a transcriptional mab‐5gfp reporter, muIs16, to monitor when and where mab‐5expression might be occurring in animals after bacterial infection. Together, the results of this project will help illuminate the mechanisms of innate immune pathway activation and the role that transcriptional regulation plays in organismal health.

Investigating the mechanisms of brain innate immune activation following bacterial infection in Drosophila

AbstractBackgroundCentral Nervous System (CNS) infections caused by bacteria and resulting immune responses could underlie neurodegenerative diseases such as Alzheimer’s disease (AD). Neuroinflammation is a well‐known component of AD furthering disease pathology and progression. However, the mechanisms by which infectious agents contribute to neuroinflammatory reactions and neurodegeneration are not fully understood. The model organism Drosophila melanogaster shares conserved mechanisms of innate immune activation and neural development with mammals, and thus represents an excellent system for the investigation of pathogen‐associated neurodegeneration. Previous work in flies has shown that bacterial brain injection results in age‐dependent neurodegeneration and impaired locomotor activity. Furthermore, these phenotypes depend on NF‐κB in both glia and neurons, suggesting that long‐term inflammatory processes take place in both cell types. However, a detailed picture of innate immune pathway activation in the Drosophila brain following bacterial infection is lacking. Here, we investigate the activation in the brain of the two Drosophila NF‐ κB pathways, Toll and Immune deficiency (IMD), in response to infection with Gram‐positive and Gram‐negative bacteria, respectively.MethodFly brains are infected by pricking heads with a thin, metal needle, dipped into concentrated pellets of overnight cultured bacterial solutions. Activation of NF‐κB pathways in brain cell types is assayed using immunohistochemistry and confocal microscopy of flies carrying fluorescent NF‐κB reporters. The dependence of canonical NF‐κB pathway components is assayed using quantitative reverse‐transcription PCR (RT‐qPCR) using RNA isolated from dissected brains of control‐injected or bacterially injected flies.ResultOur preliminary results show that brain bacterial injection leads to the activation of GFP reporters for both Toll and IMD pathways in multiple glial subtypes in the fly brain 24h following infection. We have optimized a protocol for brain‐specific gene expression of NF‐κB targets and are currently examining the response to bacterial challenge of multiple mutants for components of the Toll and IMD pathways.ConclusionWe have established a Drosophila model of bacterial brain infection that allows studying the long‐term consequences of pathogen‐mediated neuroinflammatory reactions associated with neurodegeneration. Our system could be used to identify the processes of immediate protection as well as the long‐term effects of brain infection.

Prolonged activation of innate immune pathways by a polyvalent STING agonist

The stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that is a target of therapeutics for infectious diseases and cancer. However, early-phase clinical trials of small-molecule STING agonists have shown limited antitumour efficacy and dose-limiting toxicity. Here, we show that a polyvalent STING agonist—a pH-sensitive polymer bearing a seven-membered ring with a tertiary amine (PC7A)—activates innate-immunity pathways through the polymer-induced formation of STING–PC7A condensates. In contrast to the natural STING ligand 2′,3′-cyclic-GMP-AMP (cGAMP), PC7A stimulates the prolonged production of pro-inflammatory cytokines by binding to a non-competitive STING surface site that is distinct from the cGAMP binding pocket. PC7A induces antitumour responses that are dependent on STING expression and CD8+ T-cell activity, and the combination of PC7A and cGAMP led to synergistic therapeutic outcomes (including the activation of cGAMP-resistant STING variants) in mice bearing subcutaneous tumours and in resected human tumours and lymph nodes. The activation of the STING pathway through polymer-induced STING condensation may offer new therapeutic opportunities.

Innate Immune Interference Attenuates Inflammation In Bacillus Endophthalmitis.

PurposeTo explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis.Methods Bacillus endophthalmitis was induced in mice. Innate immune pathway activation was interfered by injecting S layer protein-deficient (∆slpA) B. thuringiensis or by treating wild-type (WT)–infected mice with a TLR2/4 inhibitor (WT+OxPAPC). At 10 hours postinfection, eyes were harvested and RNA was purified. A NanoString murine inflammation panel was used to compare gene expression in WT-infected, WT+OxPAPC, ∆slpA-infected, and uninfected eyes.ResultsIn WT-infected eyes, 56% of genes were significantly upregulated compared to uninfected controls. Compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ∆slpA-infected eyes, respectively. Expression of 61 genes that were upregulated in WT-infected eyes was decreased in WT+OxPAPC and ∆slpA-infected eyes. Innate interference resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate pathway genes (TLRs 2, 4, 6, and 8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2). Innate interference also reduced the expression of several inflammatory cytokines (CSF2, CSF3, IL-6, IL-1β, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β) and chemokines (CCL2, CCL3, and CXCLs 1, 2, 3, 5, 9, and 10). All of the aforementioned genes were significantly upregulated in WT-infected eyes.ConclusionsThese results suggest that interfering with innate activation significantly reduced the intraocular inflammatory response in Bacillus endophthalmitis. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.

The Commensal Microbiota Enhances ADP-Triggered Integrin αIIbβ3 Activation and von Willebrand Factor-Mediated Platelet Deposition to Type I Collagen.

The commensal microbiota is a recognized enhancer of arterial thrombus growth. While several studies have demonstrated the prothrombotic role of the gut microbiota, the molecular mechanisms promoting arterial thrombus growth are still under debate. Here, we demonstrate that germ-free (GF) mice, which from birth lack colonization with a gut microbiota, show diminished static deposition of washed platelets to type I collagen compared with their conventionally raised (CONV-R) counterparts. Flow cytometry experiments revealed that platelets from GF mice show diminished activation of the integrin αIIbβ3 (glycoprotein IIbIIIa) when activated by the platelet agonist adenosine diphosphate (ADP). Furthermore, washed platelets from Toll-like receptor-2 (Tlr2)-deficient mice likewise showed impaired static deposition to the subendothelial matrix component type I collagen compared with wild-type (WT) controls, a process that was unaffected by GPIbα-blockade but influenced by von Willebrand factor (VWF) plasma levels. Collectively, our results indicate that microbiota-triggered steady-state activation of innate immune pathways via TLR2 enhances platelet deposition to subendothelial matrix molecules. Our results link host colonization status with the ADP-triggered activation of integrin αIIbβ3, a pathway promoting platelet deposition to the growing thrombus.

Transcriptome Profiling of Human Monocyte-Derived Macrophages Upon CCL2 Neutralization Reveals an Association Between Activation of Innate Immune Pathways and Restriction of HIV-1 Gene Expression.

Macrophages are key targets of human immunodeficiency virus type 1 (HIV-1) infection and main producers of the proinflammatory chemokine CC chemokine ligand 2 (CCL2), whose expression is induced by HIV-1 both in vitro and in vivo. We previously found that CCL2 neutralization in monocyte-derived macrophages (MDMs) strongly inhibited HIV-1 replication affecting post-entry steps of the viral life cycle. Here, we used RNA-sequencing to deeply characterize the cellular factors and pathways modulated by CCL2 blocking in MDMs and involved in HIV-1 replication restriction. We report that exposure to CCL2 neutralizing antibody profoundly affected the MDM transcriptome. Functional annotation clustering of up-regulated genes identified two clusters enriched for antiviral defense and immune response pathways, comprising several interferon-stimulated, and restriction factor coding genes. Transcripts in the clusters were enriched for RELA and NFKB1 targets, suggesting the activation of the canonical nuclear factor κB pathway as part of a regulatory network involving miR-155 up-regulation. Furthermore, while HIV-1 infection caused small changes to the MDM transcriptome, with no evidence of host defense gene expression and type I interferon signature, CCL2 blocking enabled the activation of a strong host innate response in infected macrophage cultures, and potently inhibited viral genes expression. Notably, an inverse correlation was found between levels of viral transcripts and of the restriction factors APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 A), ISG15, and MX1. These findings highlight an association between activation of innate immune pathways and HIV-1 restriction upon CCL2 blocking and identify this chemokine as an endogenous factor contributing to the defective macrophage response to HIV-1. Therapeutic targeting of CCL2 may thus strengthen host innate immunity and restrict HIV-1 replication.

The first case of COVID-19 treated with the complement C3 inhibitor AMY-101

Acute respiratory distress syndrome (ARDS) is a devastating clinical manifestation of COVID-19 pneumonia and is mainly based on an immune-driven pathology. Mounting evidence suggests that COVID-19 is fueled by a maladaptive host inflammatory response that involves excessive activation of innate immune pathways. While a “cytokine storm” involving IL-6 and other cytokines has been documented, complement C3 activation has been implicated as an initial effector mechanism that exacerbates lung injury in preclinical models of SARS-CoV infection. C3-targeted intervention may provide broader therapeutic control of complement-mediated inflammatory damage in COVID-19 patients. Herein, we report the clinical course of a patient with severe ARDS due to COVID-19 pneumonia who was safely and successfully treated with the compstatin-based complement C3 inhibitor AMY-101.

Proteomic and Transcriptional Profiles of Human Stem Cell-Derived β Cells Following Enteroviral Challenge.

Enteroviral infections are implicated in islet autoimmunity and type 1 diabetes (T1D) pathogenesis. Significant β-cell stress and damage occur with viral infection, leading to cells that are dysfunctional and vulnerable to destruction. Human stem cell-derived β (SC-β) cells are insulin-producing cell clusters that closely resemble native β cells. To better understand the events precipitated by enteroviral infection of β cells, we investigated transcriptional and proteomic changes in SC-β cells challenged with coxsackie B virus (CVB). We confirmed infection by demonstrating that viral protein colocalized with insulin-positive SC-β cells by immunostaining. Transcriptome analysis showed a decrease in insulin gene expression following infection, and combined transcriptional and proteomic analysis revealed activation of innate immune pathways, including type I interferon (IFN), IFN-stimulated genes, nuclear factor-kappa B (NF-κB) and downstream inflammatory cytokines, and major histocompatibility complex (MHC) class I. Finally, insulin release by CVB4-infected SC-β cells was impaired. These transcriptional, proteomic, and functional findings are in agreement with responses in primary human islets infected with CVB ex vivo. Human SC-β cells may serve as a surrogate for primary human islets in virus-induced diabetes models. Because human SC-β cells are more genetically tractable and accessible than primary islets, they may provide a preferred platform for investigating T1D pathogenesis and developing new treatments.

Overcoming Adaptive Therapy Resistance in AML By Targeting Immune Response Pathways

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

Overcoming adaptive therapy resistance in AML by targeting immune response pathways.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course; however, most patients will relapse because of target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. Here, we describe mechanisms of adaptive resistance in FMS-like receptor tyrosine kinase (FLT3)-mutant acute myeloid leukemia (AML) by examining integrative in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). We identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i and showed that innate immune pathway activation via the interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) complex contributes to adaptive resistance in FLT3-mutant AML cells. To overcome this adaptive resistance mechanism, we developed a small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The multikinase FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cells in vitro and in vivo and displayed superior efficacy as compared to current targeted FLT3 therapies. These findings uncover a polypharmacologic strategy for overcoming adaptive resistance to therapy in AML by targeting immune stress response pathways.

DNA Repair Deficiency in Breast Cancer: Opportunities for Immunotherapy.

Historically the development of anticancer treatments has been focused on their effect on tumor cells alone. However, newer treatments have shifted attention to targets on immune cells, resulting in dramatic responses. The effect of DNA repair deficiency on the microenvironment remains an area of key interest. Moreover, established therapies such as DNA damaging treatments such as chemotherapy and PARP inhibitors further modify the tumor microenvironment. Here we describe DNA repair pathways in breast cancer and activation of innate immune pathways in DNA repair deficiency, in particular, the STING (STimulator of INterferon Genes) pathway. Breast tumors with DNA repair deficiency are associated with upregulation of immune checkpoints including PD-L1 (Programmed Death Ligand-1) and may represent a target population for single agent or combination immunotherapy treatment.

Loss of Mitochondrial Transcription Factor A (TFAM) in Tubule Cells Causes Renal Failure Via Activating the cGAS-STING Innate Immune Pathway

Kidney fibrosis is characterized by tubule atrophy, compromised bioenergetics and proinflammatory gene expression. The relationship between these events are poorly understood. In patient samples, the expression of TFAM and mitochondrial genes correlates with kidney fibrosis. Mice with tubule specific deletion of TFAM (Ksp-Cre/Tfam flox/flox) developed severe mitochondrial loss and decline of ATP content by 6 weeks of age. Progressive azotemia, kidney fibrosis and death of the animals was only observed after 12 weeks of age. Mechanistic studies demonstrated that aberrant mtDNA packaging upon TFAM deficiency in tubule cells resulted in escape of mtDNA into the cytosol, activation of the cytosolic DNA sensing pathway, the Stimulator of interferon genes (STING), resulting in cytokine expression and immune cell recruitment. Genetic deletion or pharmacological inhibition of STING ameliorated TFAM-loss induced kidney fibrosis, demonstrating, that in addition to its essential role in metabolism, TFAM sequesters mtDNA to prevent the activation of innate immune pathways and fibrosis.

Chronic Innate Immune Signaling in Myelodysplasia

Chronic inflammatory diseases associated with activated innate immune signaling pathways often precede Myelodysplastic Syndromes (MDS), thus a role for chronic innate immune signaling in hematopoietic cells is suspected in the development of MDS. The innate immune system recognizes pathogens and host cellular by-products by pattern recognition receptors (PRR). Among the first PRRs to be identified were the Toll-like receptors (TLRs), which consist of 10 human members. Upon binding to ligand, TLRs recruit intracellular adaptors, kinases, and effector molecules which results in pathway activation. Chronic TLR signaling impairs HSC function, creates an inflammatory environment, and alters normal hematopoiesis, which suggests a causal role in MDS. Despite growing evidence linking dysregulation of innate immune signaling in MDS, how sustained activation of innate immune signaling downstream of various receptor families contributes to MDS remains an open question. This talk will highlight the recent evidence that implicates a cell-intrinsic role of innate immune pathway activation in MDS HSC and environmental inflammatory factors that are involved in the pathogenesis of MDS. DisclosuresNo relevant conflicts of interest to declare.

Immuno-PET of Innate Immune Markers CD11b and IL-1β Detects Inflammation in Murine Colitis

Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory disease of the gastrointestinal tract. The diagnosis and monitoring of IBD are reliant on endoscopy, which is invasive and does not provide information on specific mediators. Symptom flare in IBD is associated with increased activation of innate immune pathways. Immuno-PET approaches have previously demonstrated the ability to detect colitis; however, a direct comparison of antibodies targeted to innate immune mediators and cells has not been done. We aimed to compare immuno-PET of antibodies to IL-1β and CD11b against standard 18F-FDG and MRI approaches to detect colonic inflammation. Methods: Colonic concentrations of IL-1β and myeloperoxidase were determined by ELISA, and colonic infiltration by CD11b-positive CD3-negative innate immune cells was determined by flow cytometry and compared between healthy and dextran sodium sulphate-treated colitic mice. PET of 89Zr-lα-IL-1β, 89Zr-α-CD11b, and 18F-FDG was compared by volume-of-interest analysis and with MRI by region-of-interest analysis. Imaging results were confirmed by ex vivo biodistribution analysis. Results: Colonic inflammation was associated with impaired colonic epithelial barrier permeability, increased colonic IL-1β and myeloperoxidase concentrations, and increased CD11b-positive CD3-negative innate immune cell infiltration into the colon. 89Zr-α-IL-1β and 89Zr-α-CD11b immuno-PET detected colonic inflammation, as did 18F-FDG, and all PET tracers were more sensitive than MRI. Although 18F-FDG volumes of interest correlated with colitis severity and a strong trend was observed with 89Zr-α-IL-1β, no correlation was observed for 89Zr-α-CD11b or MRI. 89Zr-α-IL-1β was distributed mainly to the gastrointestinal tract, whereas 89Zr-α-CD11b was distributed to more tissue types. Conclusion: Immuno-PET using antibodies directed to innate immune markers detected colonic inflammation, with 89Zr-α-IL-1β providing a more tissue-specific signal than 89Zr-α-CD11b. Development of these technologies for human subjects will potentially provide a less invasive approach than endoscopy for diagnosing and monitoring IBD.

Targeting STING with covalent small-molecule inhibitors.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.