Myeloid Cell Function Research Articles

Nod1 promotes colorectal carcinogenesis by regulating the immunosuppressive functions of tumor-infiltrating myeloid cells

Pioneering studies from the early 1980s suggested that bacterial peptidoglycan-derived muramyl peptides (MPs) could exert either stimulatory or immunosuppressive functions depending, in part, on chronicity of exposure. However, this Janus-faced property of MPs remains largely unexplored. Here, we demonstrate the immunosuppressive potential of Nod1, the bacterial sensor of diaminopimelic acid (DAP)-containing MPs. Using a model of self-limiting peritonitis, we show that systemic Nod1 activation promotes an autophagy-dependent reprogramming of macrophages toward an alternative phenotype. Moreover, Nod1 stimulation induces the expansion of myeloid-derived suppressor cells (MDSCs) and maintains their immunosuppressive potential via arginase-1 activity. Supporting the role of MDSCs and tumor-associated macrophages in cancer, we demonstrate that myeloid-intrinsic Nod1 expression sustains intra-tumoral arginase-1 levels to foster an immunosuppressive and tumor-permissive microenvironment during colorectal cancer (CRC) development. Our findings support the notion that bacterial products, via Nod1 detection, modulate the immunosuppressive activity of myeloid cells and fuel tumor progression in CRC.

Blocking NHE1 stimulates glioma tumor immunity by restoring OXPHOS function of myeloid cells.

Background: Immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) is one of the contributing factors for failed immunotherapies. Therefore, there is an urgent need to better understand TME and to identify novel modulators of TME for more effective GBM therapies. We hypothesized that H+ extrusion protein Na/H exchanger 1 (NHE1) plays a role in dysregulation of glucose metabolism and immunosuppression of GBM. We investigated the efficacy of blockade of NHE1 activity in combination with temozolomide (TMZ) therapy in increasing anti-tumor immunity.Methods: Mouse syngeneic intracranial glioma model was used to test four treatment regimens: DMSO (Vehicle-control), TMZ, NHE1 specific inhibitor HOE642, or TMZ+HOE642 (T+H) combination. Ex vivo 1H/19Fluorine magnetic resonance imaging (MRI) with cell tracking agent Vsense was performed to monitor the infiltration of glioma-associated microglia/myeloid cells (GAMs). Glucose metabolism and transcriptome profiles were analyzed by Seahorse analyzer and bulk RNA-sequencing. The impact of selective Nhe1 deletion in GAMs on sensitivity to anti-PD-1 therapy was evaluated in transgenic NHE1 knockout (KO) mice.Results: Among the tested treatment regimens, the T+H combination therapy significantly stimulated the infiltration of GAMs and T-cells; up-regulated Th1 activation, and mitochondrial oxidative phosphorylation (OXPHOS) pathway genes, increased glucose uptake and mitochondrial mass, and decreased aerobic glycolysis in GAMs. Selective deletion of Nhe1 in Cx3cr1+ Nhe1 KO mice increased anti-tumor immunity and sensitivity to TMZ plus anti-PD-1 combinatorial therapy.Conclusions: NHE1 plays a role in developing glioma immunosuppressive TME in part by dysregulating glucose metabolism of GAMs and emerges as a therapeutic target for improving glioma immunity.

Redox regulation of immunometabolism.

Metabolic pathways and redox reactions are at the core of life. In the past decade(s), numerous discoveries have shed light on how metabolic pathways determine the cellular fate and function of lymphoid and myeloid cells, giving rise to an area of research referred to as immunometabolism. Upon activation, however, immune cells not only engage specific metabolic pathways but also rearrange their oxidation-reduction (redox) system, which in turn supports metabolic reprogramming. In fact, studies addressing the redox metabolism of immune cells are an emerging field in immunology. Here, we summarize recent insights revealing the role of reactive oxygen species (ROS) and the differential requirement of the main cellular antioxidant pathways, including the components of the thioredoxin (TRX) and glutathione (GSH) pathways, as well as their transcriptional regulator NF-E2-related factor 2 (NRF2), for proliferation, survival and function of T cells, B cells and macrophages.

Apigenin Increases SHIP-1 Expression, Promotes Tumoricidal Macrophages and Anti-Tumor Immune Responses in Murine Pancreatic Cancer.

Simple SummarySrc Homology 2 (SH2) domain-containing Inositol 5’-Phosphatase-1 (SHIP-1) is an essential protein and the master regulator of myeloid cell development and function that impacts tumor immunity. We previously published that SHIP-1 regulates the expansion and function of immunosuppressive myeloid cells, which correlated with pancreatic cancer progression in mice. Here, we show that the bioflavonoid Apigenin restored SHIP-1 expression, significantly increased tumoricidal Tumor-Associated Macrophages (TAM) while significantly decreased immunosuppressive TAM percentages and improved anti-tumor immune responses in the tumor microenvironment using different pancreatic cancer models. Our research findings suggest that SHIP-1 may be a potential novel therapeutic target to promote the development of tumoricidal TAM that can assist in the treatment of pancreatic cancer.Pancreatic cancer (PC) has an extremely poor prognosis due to the expansion of immunosuppressive myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in the inflammatory tumor microenvironment (TME), which halts the recruitment of effector immune cells and renders immunotherapy ineffective. Thus, the identification of new molecular targets that can modulate the immunosuppressive TME is warranted for PC intervention. Src Homology-2 (SH2) domain-containing Inositol 5′-Phosphatase-1 (SHIP-1) is a lipid signaling protein and a regulator of myeloid cell development and function. Herein, we used the bioflavonoid apigenin (API) to reduce inflammation in different PC models. Wild type mice harboring heterotopic or orthotopic PC were treated with API, which induced SHIP-1 expression, reduced inflammatory tumor-derived factors (TDF), increased the proportion of tumoricidal macrophages and enhanced anti-tumor immune responses, resulting in a reduction in tumor burden compared to vehicle-treated PC mice. In contrast, SHIP-1-deficient mice exhibited an increased tumor burden and displayed augmented proportions of pro-tumor macrophages. These results provide further support for the importance of SHIP-1 expression in promoting pro-tumor macrophage development in the pancreatic TME. Our findings suggest that agents augmenting SHIP-1 expression may provide novel therapeutic options for the treatment of PC.

Haematopoetic TREM2 deficiency modulates atherosclerosis and lipid metabolism

Background and Aims: Triggering receptor expressed on myeloid cells 2 (TREM2) is a lipid-sensing receptor involved in the regulation of several key myeloid cell functions, including proliferation, survival and phagocytosis. TREM2 is highly expressed on a subset of atherosclerosis-associated aortic macrophages, which express genes associated with lipid metabolism, oxidative stress and calcification while downregulating pro-inflammatory genes. These macrophages correspond to foamy macrophages, which are important players in atherogenesis. Therefore, we sought to investigate the effect of TREM2 deficiency on atherosclerosis.

Interaction of microglia with infiltrating immune cells in the different phases of stroke.

Stroke, in association with its complications, is one of the leading causes of mortality and morbidity worldwide. Cerebral ischemia triggers an inflammatory response in the brain that is controlled by the activation of resident microglia as well as the infiltration of peripheral myeloid and lymphoid cells into the brain parenchyma. This inflammation has been shown to have both beneficial and detrimental effects on stroke outcome. The focus of this review lies on the functions of myeloid cells and their interaction with infiltrating lymphocytes in different phases of stroke. A detailed and time‐specific understanding of the contribution of different immune cell subsets during the course of cerebral ischemia is crucial to specifically promote beneficial and inhibit detrimental effects of inflammation on stroke outcome.

The Role of Metabolic Enzymes in the Regulation of Inflammation.

Immune cells undergo dramatic metabolic reprogramming in response to external stimuli. These metabolic pathways, long considered as simple housekeeping functions, are increasingly understood to critically regulate the immune response, determining the activation, differentiation, and downstream effector functions of both lymphoid and myeloid cells. Within the complex metabolic networks associated with immune activation, several enzymes play key roles in regulating inflammation and represent potential therapeutic targets in human disease. In some cases, these enzymes control flux through pathways required to meet specific energetic or metabolic demands of the immune response. In other cases, key enzymes control the concentrations of immunoactive metabolites with direct roles in signaling. Finally, and perhaps most interestingly, several metabolic enzymes have evolved moonlighting functions, with roles in the immune response that are entirely independent of their conventional enzyme activities. Here, we review key metabolic enzymes that critically regulate inflammation, highlighting mechanistic insights and opportunities for clinical intervention.

Hypoxia-inducible factors not only regulate but also are myeloid-cell treatment targets.

Hypoxia describes limited oxygen availability at the cellular level. Myeloid cells are exposed to hypoxia at various bodily sites and even contribute to hypoxia by consuming large amounts of oxygen during respiratory burst. Hypoxia-inducible factors (HIFs) are ubiquitously expressed heterodimeric transcription factors, composed of an oxygen-dependent α and a constitutive β subunit. The stability of HIF-1α and HIF-2α is regulated by oxygen-sensing prolyl-hydroxylases (PHD). HIF-1αand HIF-2α modify the innate immune response and are context dependent. We provide a historic perspective of HIF discovery, discuss the molecular components of the HIF pathway, and how HIF-dependent mechanisms modify myeloid cell functions. HIFs enable myeloid-cell adaptation to hypoxia by up-regulating anaerobic glycolysis. In addition to effects on metabolism, HIFs control chemotaxis, phagocytosis, degranulation, oxidative burst, and apoptosis. HIF-1α enables efficient infection defense by myeloid cells. HIF-2αdelays inflammation resolution and decreases antitumor effects by promoting tumor-associated myeloid-cell hibernation. PHDs not only control HIF degradation, but also regulate the crosstalk between innate and adaptive immune cells thereby suppressing autoimmunity. HIF-modifying pharmacologic compounds are entering clinical practice. Current indications include renal anemia and certain cancers. Beneficial and adverse effects on myeloid cells should be considered and could possibly lead to drug repurposing for inflammatory disorders.

Analysis of Trans-Ancestral SLE Risk Loci Identifies Unique Biologic Networks and Drug Targets in African and European Ancestries

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disorder with a prominent genetic component. Individuals of African ancestry (AA) experience the disease more severely and with an increased co-morbidity burden compared to European ancestry (EA) populations. We hypothesize that the disparities in disease prevalence, activity, and response to standard medications between AA and EA populations is partially conferred by genomic influences on biological pathways. To address this, we applied a comprehensive approach to identify all genes predicted from SNP-associated risk loci detected with the Immunochip. By combining genes predicted via eQTL analysis, as well as those predicted from base-pair changes in intergenic enhancer sites, coding-region variants, and SNP-gene proximity, we were able to identify 1,731 potential ancestry-specific and trans-ancestry genetic drivers of SLE. Gene associations were linked to upstream and downstream regulators using connectivity mapping, and predicted biological pathways were mined for candidate drug targets. Examination of trans-ancestral pathways reflect the well-defined role for interferons in SLE and revealed pathways associated with tissue repair and remodeling. EA-dominant genetic drivers were more often associated with innate immune and myeloid cell function pathways, whereas AA-dominant pathways mirror clinical findings in AA subjects, suggesting disease progression is driven by aberrant B cell activity accompanied by ER stress and metabolic dysfunction. Finally, potential ancestry-specific and non-specific drug candidates were identified. The integration of all SLE SNP-predicted genes into functional pathways revealed critical molecular pathways representative of each population, underscoring the influence of ancestry on disease mechanism and also providing key insight for therapeutic selection.

Expression of TAM-R in Human Immune Cells and Unique Regulatory Function of MerTK in IL-10 Production by Tolerogenic DC.

Tumor-infiltrating myeloid cells are a key component of the immune infiltrate often correlated with a poor prognosis due to their capacities to sustain an immunosuppressive environment. Among membrane receptors implicated in myeloid cell functions, Tyro3, Axl, and MerTK, which are a family of tyrosine kinase receptors (TAM-R), have been described in the regulation of innate cell functions. Here, we have identified MerTK among TAM-R as the major marker of both human M2 macrophages and tolerogenic dendritic cells (DC). In situ, MerTK expression was found within the immune infiltrate in multiple solid tumors, highlighting its potential role in cancer immunity. TAM-R ligands Gas6 and PROS1 were found to be constitutively produced by myeloid cells in vitro. Importantly, we describe a novel function of MerTK/PROS1 axis in the regulation of IL-10 production by tolerogenic DC. Finally, the analysis of TAM-R expression within the lymphoid compartment following activation revealed that MerTK, but not Axl or Tyro3, is expressed on activated B lymphocytes and regulatory T cells, as well as CD4+ and CD8+ T cells. Thus, our findings deepen the implication of MerTK in the regulation of myeloid cell-mediated immunosuppression and identified new cellular targets expressing MerTK that could participate in the antitumor immune response.

Ageing and atherosclerosis: vascular intrinsic and extrinsic factors and potential role of IL-6.

The number of old people is rising worldwide, and advancing age is a major risk factor for atherosclerotic cardiovascular disease. However, the mechanisms underlying this phenomenon remain unclear. In this Review, we discuss vascular intrinsic and extrinsic mechanisms of how ageing influences the pathology of atherosclerosis. First, we focus on factors that are extrinsic to the vasculature. We discuss how ageing affects the development of myeloid cells leading to the expansion of certain myeloid cell clones and induces changes in myeloid cell functions that promote atherosclerosis via inflammation, including a potential role for IL-6. Next, we describe vascular intrinsic factors by which ageing promotes atherogenesis — in particular, the effects on mitochondrial function. Studies in mice and humans have shown that ageing leads to a decline in vascular mitochondrial function and impaired mitophagy. In mice, ageing is associated with an elevation in the levels of the inflammatory cytokine IL-6 in the aorta, which participates in a positive feedback loop with the impaired vascular mitochondrial function to accelerate atherogenesis. We speculate that vascular and myeloid cell ageing synergize, via IL-6 signalling, to accelerate atherosclerosis. Finally, we propose future avenues of clinical investigation and potential therapeutic approaches to reduce the burden of atherosclerosis in old people.

Interleukin-6-mediated resistance to immunotherapy is linked to impaired myeloid cell function.

High serum levels of interleukin‐6 (IL‐6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)‐associated tumor model expressing IL‐6 to investigate the impact of tumor‐expressed IL‐6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor‐produced IL‐6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor‐produced IL‐6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T‐cell‐mediated killing. Furthermore, no overt differences in the frequencies of tumor‐infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL‐6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL‐6‐mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL‐6‐mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer.

Enzalutamide, an Androgen Receptor Antagonist, Enhances Myeloid Cell-Mediated Immune Suppression and Tumor Progression.

Androgen receptor (AR) antagonism increases overall survival in prostate cancer; however, treatment failure leads to tumor progression and patient mortality. The effect of AR modulation on AR+ nontumor cells that participate in the resistance to AR antagonism is poorly understood. Tumor-infiltrating myeloid cells, including macrophages and myeloid-derived suppressor cells (MDSC), express AR and promote prostate cancer progression. We investigated how AR antagonism affects myeloid cell function and metabolism in an AR-independent murine colon tumor model. Systemic blockade of AR with enzalutamide resulted in increased MC-38 tumor growth in vivo even when AR was knocked out of MC-38 tumor cells. MC-38 tumor growth was also increased when immunocompetent, but not immunodeficient, mice were coinjected with tumor cells and MDSCs treated with enzalutamide or lacking AR, suggesting that AR regulated the ability of MDSCs to suppress adaptive immunity. Myeloid AR-knockout male mice also displayed increased growth of TRAMP C2 prostate tumors when compared with wild type. Inhibition of AR signaling suppressed mitochondrial respiration in myeloid cells via MPC/AMPK signaling pathways; suppression of mitochondrial respiration increased MDSC tumor-promoting functions. Our work showed that AR regulates a tumor-promoting myeloid cell phenotype and influences myeloid cell metabolism. These findings suggest that tumor resistance to AR antagonism is due, in part, to changes in myeloid cell function and metabolism.

Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma

BackgroundNeuroinflammation is a major component of secondary damage after traumatic brain injury (TBI). We recently reported that pharmacological inhibition of Pannexin-1 (Panx1) channels markedly reduced the inflammatory response after TBI. Panx1 channels have been shown to be important conduits for adenosine 5′-triphosphate (ATP) release and are associated with leukocyte infiltration and pyroptosis. Because Panx1 blockers significantly decrease ATP release and migration of activated microglia and other myeloid cells (such as monocyte-derived macrophages and dendritic cells) in vitro, we hypothesized that myeloid Panx1 channels play a specific role in immune cell infiltration promoting tissue damage following TBI.MethodsThe murine-controlled cortical impact (CCI) model was used on myeloid-specific Panx1 conditional knockout (Cx3cr1-Cre::Panx1fl/fl) mice to determine whether myeloid Panx1 mediates neuroinflammation and brain damage. Immune cell infiltration was measured using flow cytometry. Locomotor and memory functions were measured using the rotarod and Barnes maze test, respectively. The levels of biomarkers for tissue damage and blood–brain barrier leakage were measured using western blot and magnetic resonance imaging. Panx1 channel activity was measured with ex vivo dye uptake assays, using flow cytometry and confocal microscopy.ResultsCCI-injured Cx3cr1-Cre::Panx1fl/fl mice showed markedly reduced immune cell infiltration to the brain parenchyma compared with Panx1fl/fl mice. As expected, Panx1 dependent activity, assessed by dye uptake, was markedly reduced only in myeloid cells from Cx3cr1-Cre::Panx1fl/fl mice. The expression of biomarkers of tissue damage was significantly reduced in the CCI-injured Cx3cr1-Cre::Panx1fl/fl mice compared with Panx1fl/fl mice. In line with this, magnetic resonance imaging showed reduced blood–brain barrier leakage in CCI-injured Cx3cr1-Cre::Panx1fl/fl mice. There was also a significant improvement in motor and memory function in Cx3cr1-Cre::Panx1fl/fl mice when compared with Panx1fl/fl mice within a week post-CCI injury.ConclusionOur data demonstrate that CCI-related outcomes correlate with Panx1 channel function in myeloid cells, indicating that activation of Panx1 channels in myeloid cells is a major contributor to acute brain inflammation following TBI. Importantly, our data indicate myeloid Panx1 channels could serve as an effective therapeutic target to improve outcome after TBI.

Myeloid Cell-Intrinsic IRF5 Promotes T Cell Responses through Multiple Distinct Checkpoints In Vivo, and IRF5 Immune-Mediated Disease Risk Variants Modulate These Myeloid Cell Functions.

Common IRF5 genetic risk variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern-recognition receptor (PRR)-induced cytokines in myeloid cells. However, how myeloid cell-intrinsic IRF5 regulates the multiple distinct checkpoints mediating T cell outcomes in vivo and IRF5-dependent mechanisms contributing to these distinct checkpoints are not well defined. Using an in vivo Ag-specific adoptive T cell transfer approach into Irf5-/- mice, we found that T cell-extrinsic IRF5 regulated T cell outcomes at multiple critical checkpoints, including chemokine-mediated T cell trafficking into lymph nodes and PDK1-dependent soluble Ag uptake, costimulatory molecule upregulation, and secretion of Th1 (IL-12)- and Th17 (IL-23, IL-1β, and IL-6)-conditioning cytokines by myeloid cells, which then cross-regulated Th2 and regulatory T cells. IRF5 was required for PRR-induced MAPK and NF-κB activation, which, in turn, regulated these key outcomes in myeloid cells. Importantly, mice with IRF5 deleted from myeloid cells demonstrated T cell outcomes similar to those observed in Irf5-/- mice. Complementation of IL-12 and IL-23 was able to restore T cell differentiation both in vitro and in vivo in the context of myeloid cell-deficient IRF5. Finally, human monocyte-derived dendritic cells from IRF5 disease-associated genetic risk carriers leading to increased IRF5 expression demonstrated increased Ag uptake and increased PRR-induced costimulatory molecule expression and chemokine and cytokine secretion compared with monocyte-derived dendritic cells from low-expressing IRF5 genetic variant carriers. These data establish that myeloid cell-intrinsic IRF5 regulates multiple distinct checkpoints in T cell activation and differentiation and that these are modulated by IRF5 disease risk variants.

A characterization of the molecular phenotype and inflammatory response of schizophrenia patient-derived microglia-like cells

Different lines of evidence support a causal role for microglia in the pathogenesis of schizophrenia. However, how schizophrenia patient-derived microglia are affected at the phenotypic and functional level is still largely unknown. We used a recently described model to induce patient-derived microglia-like cells and used this to analyze changes in the molecular phenotype and function of myeloid cells in schizophrenia. We isolated monocytes from twenty recent-onset schizophrenia patients and twenty non-psychiatric controls. We cultured the cells towards an induced microglia-like phenotype (iMG), analyzed the phenotype of the cells by RNA sequencing and mass cytometry, and their response to LPS. Mass cytometry showed a high heterogeneity of iMG in cells derived from patients as well as controls. The prevalence of two iMG clusters was significantly higher in schizophrenia patients (adjusted p-value < 0.001). These subsets are characterized by expression of ApoE, Ccr2, CD18, CD44, and CD95, as well as IRF8, P2Y12, Cx3cr1 and HLA-DR. In addition, we found that patient-derived iMG show an enhanced response to LPS, with increased secretion of TNF-α. Further studies are needed to replicate these findings, to determine whether similar subclusters are present in schizophrenia patients in vivo, and to address how these subclusters are related to the increased response to LPS, as well as other microglial functions.

Abstract 3414: Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis

Abstract Myeloid derived suppressor cells (MDSCs) antagonize antitumor immune responses, and limit the efficacy of immune based therapies for cancer. As a result, MDSCs have garnered attention as therapeutic targets. Unfortunately, there has been limited success in translating agents targeting MDSCs to the clinic. Brd4 is an epigenetic reader and is itself a therapeutic target in oncology due to its ability to regulate the expression of oncogenes such as Myc. In addition, Brd4 is known to regulate inflammatory cytokine production and innate immune responses including myeloid cell function. As a result, it was hypothesized that inhibition of Brd4 would impact MDSC function or expansion. In multiple tumor models (EMT6, 4T1, LLC, and C26) Brd4 inhibitors, both experimental (JQ1) and those in clinical development (PLX51107 and PLX2853) significantly reduced the abundance of total, PMN, and M-MDSC subsets within the tumor and spleen as measured by flow cytometry and IHC (p&amp;lt; 0.05). Furthermore, Nanostring gene expression analysis of EMT6 tumors identified a significant reduction in the abundance signature of neutrophils and macrophages with PLX51107 further confirming a reduction of myeloid cells in the presence of a Brd4 inhibitor (p&amp;lt; 0.01). The Nanostring analysis also showed a significant reduction of IL6 mRNA in PLX51107 treated tumors, and this was confirmed at the protein level by ELISA in the serum of EMT6 and 4T1 tumor bearing mice (p&amp;lt; 0.05). However, exogenous IL6 could not rescue MDSC levels in vivo or in vitro suggesting Brd4 inhibition might be directly affecting MDSCs. This was confirmed by showing that PLX51107 significantly inhibited the expansion of MDSCs from mouse bone marrow cells or healthy donor PBMCs cultured in IL6 and GMCSF as measured by flow cytometry (p&amp;lt;0.05). Furthermore, mouse and FACS sorted MDSCs from patients with melanoma or bladder cancer were found to undergo apoptosis when treated with PLX51107 as measured by flow cytometry (annexin and caspase3, p&amp;lt; 0.05), IF, and western blot. In addition, a pancaspase inhibitor rescued both mouse and patient derived MDSCs from apoptosis in the presence of PLX51107. Further investigation with specific caspase inhibitors showed this effect relied mostly on activation of the intrinsic apoptosis pathway and caspase9. The importance of Brd4 to MDSC survival was further confirmed using a LysMCre Brd4 floxed mouse model. Finally, depletion of MDSCs in vivo with PLX51107 significantly enhanced the efficacy of anti-PDL1 therapy as compared to either agent alone (p&amp;lt; 0.05 in EMT6, 4T1, and LLC tumor models). These results identify Brd4 and the TXNIP/ASK1 apoptosis pathway as novel regulators of MDSC survival, and provide evidence to further investigate Brd4 inhibitors in combination with immune based therapies for solid tumors. Citation Format: Andrew Stiff, Himanshu Savardekar, Robert Wesolowski, Megan Duggan, Luke Scarberry, Brooke Benner, Darren Wethington, Gabby Lapurga, Steven Sun, Jack Hedberg, Logan Good, William E. Carson. Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3414.

3106 – DIRECT INDUCTION OF HEMOGENIC ENDOTHELIUM AND MYELOID CELLS FROM HUMAN IPSCS USING ETV2 MODIFIED RNA

Previously, we demonstrated that overexpressing transcription factors ETV2 and GATA2 is sufficient to induce a pan-myeloid program in human induced pluripotent stem cells (hiPSCs), which proceeds through a hemogenic endothelium (HE) stage (Elcheva et al., 2014). Although we have found that constitutive overexpression of ETV2 using lentiviral vectors induces predominantly non-hemogenic endothelium. We also noted that ETV2 induces GATA2 expression in human pluripotent stem cells (hPSCs) and very few HE with macrophage potential. In addition, our recent studies suggest that molecular mechanisms upstream of GATA2 are sufficient to specify hematoendothelial program in hPSCs, while GATA2 is required for endothelial-to-hematopoietic transition (EHT) (Kang et al., 2018). Given these findings and studies demonstrating the critical role of ETV2 threshold for hematoendothelial commitment (Zhao and Choi, 2017) and obligatory downregulation of ETV2 during subsequent stages of hematopoietic development in the embryo (Hayashi et al., 2012), we explored whether transitional expression of ETV2 with modified mRNA (mmRNA) alone is sufficient for hematoendothelial programming in hiPSCs. We found that overexpression of modified ETV2 mmRNA (mmETV2) following culture of transfected hiPSCs in StemLine II serum-free medium with FGF2, rapidly induces CD144+ expressing endothelial cells that, upon addition of GM-CSF, form floating CD43+ blood cells most of which co-express CD45. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2 and UM171 led to production of CD34+CD33+ myeloid progenitors which could be harvested every 8-10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into functional neutrophils in the presence of G-CSF and the retinoic acid agonist Am580 or macrophages in presence of M-CSF and IL3 or IL-1β and M-CSF. Overall, this technology is suitable for generating functional neutrophils and macrophages from iPSCs to interrogate the role of genes in a myeloid cell development and function when coupled with genetic engineering technologies.

Denatonium as a Bitter Taste Receptor Agonist Modifies Transcriptomic Profile and Functions of Acute Myeloid Leukemia Cells.

The contribution of cell-extrinsic factors in Acute Myeloid Leukemia (AML) generation and persistence has gained interest. Bitter taste receptors (TAS2Rs) are G protein-coupled receptors known for their primary role as a central warning signal to induce aversion toward noxious or harmful substances. Nevertheless, the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. In this study, we found that AML cells express functional TAS2Rs. We also highlighted a significant association between the modulation of some TAS2Rs and the poor-prognosis AML groups, i.e., TP53- and TET2-mutated, supporting a potential role of TAS2Rs in AML cell biology. Gene expression profile analysis showed that TAS2R activation with the prototypical agonist, denatonium benzoate, significantly modulated a number of genes involved in relevant AML cellular processes. Functional assay substantiated molecular data and indicated that denatonium reduced AML cell proliferation by inducing cell cycle arrest in G0/G1 phase or induced apoptosis via caspase cascade activation. Moreover, denatonium exposure impaired AML cell motility and migratory capacity, and inhibited cellular respiration by decreasing glucose uptake and oxidative phosphorylation. In conclusion, our results in AML cells expand the observation of cancer TAS2R expression to the setting of hematological neoplasms and shed light on a role of TAS2Rs in the extrinsic regulation of leukemia cell functions.

Advances in Molecular Mechanisms for Traditional Chinese Medicine Actions in Regulating Tumor Immune Responses.

Traditional Chinese medicine (TCM) has been developed for thousands of years with its various biological activities. The interest in TCM in tumor prevention and treatment is rising with its synergistic effect on tumor cells and tumor immunosuppressive microenvironment (TIM). Characteristic of TCM fits well within the whole system and multi-target cancer treatment. Herein we discuss the underlying mechanisms of TCM actions in TIM via regulating immunosuppressive cells, including restoring the antigen presentation function of dendritic cells, enhancing NK cells-mediated killing activity, restraining the functions of myeloid cell-derived suppressor cells, and inhibiting cancer-associated fibroblasts. TCM also regulates tumor progression through enhancing immune response, preventing immune escape and inducing cell death of tumor cells, which triggers immune response in nearby cells. In addition, we discuss TCM in clinical applications and the advantages and disadvantages of TCM in cancer prevention and treatment, as well as current therapeutic challenges and strategies. It might be helpful for understanding the therapeutic potential of TCM for cancer in clinic.