Inhibition Of Myeloid-derived Suppressor Cells Research Articles

Prototypical innate immune mechanism hijacked by leukemia-initiating mutant stem cells for selective advantage and immune evasion in Ptpn11-associated juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML), a clonal hematologic malignancy, originates from mutated hematopoietic stem cells (HSCs). The mechanism sustaining the persistence of mutant stem cells, leading to leukemia development, remains elusive. In this study, we conducted comprehensive examination of gene expression profiles, transcriptional factor regulons, and cell compositions/interactions throughout various stages of tumor cell development in Ptpn11 mutation-associated JMML. Our analyses revealed that leukemia-initiating Ptpn11 E76K/+ mutant stem cells exhibited de novo activation of the myeloid transcriptional program and aberrant developmental trajectories. These mutant stem cells displayed significantly elevated expression of innate immunity-associated anti-microbial peptides and pro-inflammatory proteins, particularly S100a9 and S100a8. Biological experiments confirmed that S100a9/S100a8 conferred a selective advantage to the leukemia-initiating cells through autocrine effects and facilitated immune evasion by recruiting and promoting immune suppressive myeloid-derived suppressor cells (MDSCs) in the microenvironment. Importantly, pharmacological inhibition of S100a9/S100a8 signaling effectively impeded leukemia development from Ptpn11 E76K/+ mutant stem cells. These findings collectively suggest that JMML tumor-initiating cells exploit evolutionarily conserved innate immune and inflammatory mechanisms to establish clonal dominance.

Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade

Immune exhaustion corresponds to a loss of effector function of T cells that associates with cancer or chronic infection. Here, our objective was to decipher the mechanisms involved in the immune suppression of myeloid-derived suppressor cells (MDSCs) and to explore the potential to target these cells for immunotherapy to enhance checkpoint blockade efficacy in a chronic parasite infection. We demonstrated that programmed cell-death-1 (PD-1) expression was significantly upregulated and associated with T-cell dysfunction in advanced alveolar echinococcosis (AE) patients and in Echinococcus multilocularis-infected mice. PD-1 blockade ex vivo failed to reverse AE patients’ peripheral blood T-cell dysfunction. PD-1/PD-L1 blockade or PD-1 deficiency had no significant effects on metacestode in mouse model. This was due to the inhibitory capacities of immunosuppressive granulocytic MDSCs (G-MDSCs), especially in the liver surrounding the parasite pseudotumor. MDSCs suppressed T-cell function in vitro in an indoleamine 2, 3 dioxygenase 1 (IDO1)-dependent manner. Although depleting MDSCs alone restored T-cell effector functions and led to some limitation of disease progression in E. multilocularis-infected mice, combination with PD-1 blockade was better to induce antiparasitic efficacy. Our findings provide preclinical evidence in support of targeting MDSC or combining such an approach with checkpoint blockade in patients with advanced AE.

Abstract 7553: Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast cancer

Abstract Epigenetics plays a central role in the initiation, establishment, and progression of cancer. In this capacity, epigenetics regulates several abnormal nuclear processes in the cancer cell including DNA replication, DNA damage repair and most prominently transcription. Because of their reversibility, epigenetic modifications have been aggressively pursued pharmacologically to correct abnormal cancer biology in the hopes of reversing cancer phenotypes. To develop novel epigenetic based therapeutic strategies for triple negative breast cancer (TNBC) we combined the DNA methyltransferase inhibitor (DNMTi) guadecitabine (Gua) with a genetic CRISPR/Cas9 mediated knockout (KO) of the chromatin remolding protein Bromodomain PHD-finger Transcription Factor (BPTF). Using the E0771 and 4T1 mouse TNBC models we show that BPTF depleted tumor cells are more susceptible to the antitumor effects of Gua. These antitumor effects are not observed in culture, or in immune compromised NSG mice, suggesting that their effects on cancer cell growth are not cancer cell intrinsic. RNA-Seq, ATAC-Seq and MBD-Seq analysis from FACS purified cancer cells shows that the combination of BPTF inhibition and Gua alters immune regulatory cytokine expression (BMP4 and CSF3) in the cancer cell, and it is theorized that these changes alter the tumor microenvironment to become less immune suppressive and permissive to the antitumor effects of immune cells. This hypothesis is supported by Cytek phenotyping of tumor resident and peripheral immune cells from treated mice showing reduced levels of immune suppressive myeloid derived suppressor cells (MDSC). Inhibiting BPTF pharmacologically using the bromodomain inhibitor AU1 does not recapitulate the effects of the genetic inhibition suggesting that the benefits of BPTF depletion to sensitization to Gua are not captured by inhibition of its bromodomain. To model the effects of pharmacologic BPTF inhibition we created a BPTF mouse KO genetically modified mouse model (GEMM). We show using this GEMM that BPTF KO in the adult mouse (a tamoxifen inducible Cre) is not lethal and has beneficial effects to the control of E0771 tumors, possibly through depleting tumor resident naïve T cells, and that transplanting BPTF KO E0771 tumors into BPTF KO adult mice further improves tumor growth control. These benefits in tumor growth control are prevented with CD8 depletion. These results in total suggests that a combination therapy regimen including BPTF inhibition (a PROTAC degradation approach) with a DNMTi could provide clinical benefits to patients with TNBC through both tumor cell intrinsic and extrinsic mechanisms. Citation Format: Joseph W. Landry, Jubin Kang, Christiane Morecock, Harry Bear, Rebecca Martin. Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7553.

Abstract 5306: PTPN22 targeting partners with 41BB/OX40 costimulation to overcome anti-PD1 resistance in pancreatic ductal adenocarcinoma

Abstract Background: The pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) is notable for a scarcity of cytotoxic T cells and an overabundance of immunosuppressive myeloid and regulatory T cells. Checkpoint immunotherapies targeting PD/L1 or CTLA4 have shown efficacy in immunogenic cancers but remain ineffective in PDAC, highlighting a need for novel approaches. Abrogating protein tyrosine phosphatase non-receptor type 22 (PTPN22), a phosphatase in the TCR signaling cascade, has been shown to modulate multiple mechanisms of immune resistance. Furthermore, 41BB costimulatory pathway has recently been investigated as a potential strategy to enhance immunotherapy against PDAC. Using immune-resistant murine models of PDAC, we investigated PTPN22 as an immunotherapeutic target in combination with 41BB co-stimulation and PD1 inhibition against PDAC. Methods: We employed a well-characterized murine PDAC cell line expressing mutant KrasG12D and Trp53R172H under the Pdx1-cre promoter (KPCY 6419c5). This tumor line is resistant to combination immuno- and chemotherapies. To determine treatment efficacy, PTPN22 deficient mice (KO) were inoculated with KPCY6419c5 cells either subcutaneously or orthotopically into the pancreas. To extend the translatability of PTPN22 targeting, we also injected PTPN22 competent mice with the same tumor cells and treated them with our novel PTPN22 inhibitor L1. Harvested tumors were processed for FFPE and mass cytometry (CyTOF) analysis to track changes in the immune compartment. Results: When PTPN22 was abrogated, treatment with anti-PD1 and 41BB agonism led to a 10-day delay in substantial tumor formation and roughly 75% reduction in tumor volume by 21-days post treatment. These tumors showed elevated levels of OX40 and OX40L primarily in the setting of PTPN22 KO/PD1/41BB therapy. In a subsequent in vivo experiment using an OX40L neutralizing antibody, we found that OX40 signaling is essential for the efficacy of PTPN22 KO/PD1/41BB therapy. Furthermore, combining OX40 agonist with anti-PD1 in PTPN22 KO sufficiently mimicked the efficacy seen with the anti-PD-1 and 41BB combination. CyTOF analysis indicated that OX40 expression is enriched in Tregs and OX40L is enriched in myeloid derived suppressor cells (MDSCs). Moreover, combination therapy efficacy was reproduced using the PTPN22 inhibitor L1. Conclusions: PTPN22 abrogation enhances the success of PD-1 inhibition given with either OX-40 or 41BB agonist therapies. In addition, PTPN22 abrogation together with PD1 inhibition and 41BB costimulation reduces MDSC and Treg suppression of effector T cells via the OX40L-OX40 axis. Finally, PTPN22 can be blocked by a small molecule inhibitor strengthening its candidacy as a therapeutic target. Citation Format: Soren Charmsaz, Nicole E. Gross, Jae W. Lee, Jianping Lin, Alexei G. Hernandez, Erin M. Coyne, Sarah M. Shin, Courtney Cannon, Xuan Yuan, Zhong-Yin Zhang, Elizabeth M. Jaffee, Won Jin Ho. PTPN22 targeting partners with 41BB/OX40 costimulation to overcome anti-PD1 resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5306.

Abstract 6522: Epigenetic modulation decreases myeloid suppression to improve response to immune checkpoint inhibition in breast cancer

Abstract Immune checkpoint inhibitors (ICIs) promote a durable anti-tumor immune response. Unfortunately, many patients with breast cancer are thought to be intrinsically resistant. One potential mechanism of resistance is a myeloid suppressed tumor microenvironment (TME). Targeting myeloid suppression is under investigation as a therapeutic strategy to sensitize tumors to ICI. In our trial, NCI-9844, we demonstrated that the combination of the epigenetic modulator, entinostat with ICIs—nivolumab (anti-PD-1) + ipilimumab (anti-CTLA-4), leads to a 25% objective response rate in patients with advanced breast cancer. Work in our preclinical models showed entinostat decreases suppressive function by myeloid derived suppressor cells (MDSCs). We hypothesized that entinostat decreases intra-tumoral MDSC suppression via the STAT3/AP-1 axis and drives cellular shifts within the TME to improve response to ICI. Here, we examined expression of upstream, downstream, and interacting factors within the STAT3 pathway in MDSCs treated with entinostat. Macro-dissected lung metastases from entinostat treated NeuN mice revealed differential gene expression of AP-1 subunits JunB and FOSL1 in MDSCs identified by single cell RNA sequencing (scRNAseq). Western blot of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. In an entinostat treated MDSC-like cell line, J774M, JunB phosphorylation and FOSL1 were both decreased. scRNAseq also revealed decreased gene expression of STAT3 upstream receptors IL4RA and IFNGR1 and increased expression of STAT3 negative regulator SOCS3. Chromatin immunoprecipitation sequencing is planned to further narrow the molecular mechanism of action. Cell-cell communication analysis of scRNAseq data was performed to evaluate the contribution of entinostat treated macrophages, dendritic cells (DCs), and natural killer (NK) cells on MDSC function. We observed increased frequency of DC populations (classic DC2s and Ccr7 DCs). Following combined treatment with entinostat + ICIs, we also found increased DC to T cell signaling strength for receptor-ligand pairs involved in T cell activation, including Tnfsf9__Tnfrsf9, Cd70_Cd27, H2-dmb2_Cd4, and Cd80_Cd28. Analysis of macrophage-T cell interactions revealed a synergistic decrease in signaling associated with immunosuppression after combined treatment, such as Cd80_Ctla4, Tnf_Tnfrsf1b, and Entpd1_Adora2a. Suppressive function of treated sub-populations of macrophages is planned to determine functional effect of findings. Finally, we are using imaging mass cytometry and bulk RNA sequencing to evaluate proposed mechanisms in patient specimens (NCI-9844). The goal of this work is to identify the molecular and cellular mechanisms driving myeloid suppression of the TME thereby identifying potential therapeutic targets. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Jesse Kreger, Yingtong Liu, Batul Al-Zubeidy, Ludmila Danilova, Sarah M. Shin, Valerie H. Narumi, Sofi Castanon, Julie Jang, Elana J. Fertig, Ashley Cimino-Mathews, Elizabeth M. Jaffee, Vered Stearns, Roisin M. Connolly, Won Jin Ho, Adam MacLean, Evanthia T. Roussos Torres. Epigenetic modulation decreases myeloid suppression to improve response to immune checkpoint inhibition in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6522.

Abstract 4989: Targeting IL-1β mitigates radiation induced MDSC expansion & improves survival in glioblastoma

Abstract Background: Our previous research demonstrated that radiation therapy (RT) for glioblastoma (GBM) induces aberrant myelopoiesis, increasing production of myeloid derived suppressor cells (MDSCs) and exacerbating T-cell suppression. However, the precise biological mechanisms underlying this phenomenon remain unclear. This study aims to elucidate the role of the pro-inflammatory cytokine interleukin-1beta (IL-1β) in RT-induced myelopoiesis and its correlation with poor prognosis in GBM patients. Methods: We conducted an extensive analysis of circulatory immune cells and pro-inflammatory cytokines in GBM patients treated with chemoradiotherapy. Using an orthotopic mouse model of GBM, we investigated the impact of IL-1β on myelopoiesis and survival. Functional assays were employed to examine MDSC expansion. Multi-color flow cytometry was used for immune profiling. Results: We found that RT elevated the plasma IL-1β concentration in both GBM patients and GBM-bearing mice, which also corresponded with an increase of MDSC in blood. In the murine GBM model, RT appeared to promote hematopoietic stem (HSPC) differentiation towards myelopoiesis. This led to increased MDSC precursor cells in the bone marrow, elevated circulating MDSCs in blood, and more infiltration of MDSCs in the tumor microenvironment (TME). Similar effects were observed in GBM-bearing mice following the administration of exogenous IL-1β. In contrast, inhibition of IL-1β using anti-IL-1β antibody during RT led to reduced myelopoiesis. When anti-IL-1β antibody was combined with functional inhibition of MDSCs using phosphodiesterase-5 inhibitor-tadalafil during RT, we observed decreased MDSC infiltration and increased CD8+ T cell infiltration in the TME. The combination approach also extended the survival of GBM-bearing mice after RT longer than either drug alone. Conclusions: Irradiation of GBM appears to induce IL-1β to promote aberrant myelopoiesis and MDSC expansion. A multi-pronged approach to inhibit MDSC production using IL-1β inhibitor and MDSC function using tadalafil during RT may be a promising approach to overcome the immune-suppressive TME of GBM. Citation Format: Subhajit Ghosh, Jiayi Huang, David DeNardo, Dennis Hallahan, Jerry Jaboin, Dinesh Thotala. Targeting IL-1β mitigates radiation induced MDSC expansion & improves survival in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4989.

Abstract B087: Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer

Abstract Introduction: High grade serous ovarian, fallopian tube and primary peritoneal carcinoma (HGSC) has an immunosuppressive and tumor promoting immune microenvironment. In this study, we have targeted the migration of myeloid derived suppressor cells (MDSC) through chemical inhibition of the CXCR2 chemotactic receptor to reverse the tumor-induced immunosuppression.Methods: ID8 cells with a p53 mutation (ID8-P53null) were used as a murine model of HGSC. ID8-p53null cells were injected intraperitoneally into immune intact C57/Bl6 mice, with 10 mice per group. After 7 days, mice were treated with cisplatin (cis, 1mg/kg, i.p., weekly), a CXCR2 inhibitor (CXCR2i, 4mg/kg, i.p., daily) or a combination of the 2 treatments. Necropsy was performed on day 29. The tumors were weighed, and the number of metastatic sites was quantified. The spleen and the tumor were resected, digested and flow cytometry was performed to evaluate the effects of treatment on the tumor immune microenvironment (TIME) as well as any systemic changes (evaluated by immune cells in the spleen). Granulocytic MDSC (G-MDSC) and monocytic MDSCs (M-MDSC) were defined as CD11b+Ly6G+,Ly6Clo and CD11b+Ly6G−Ly6Chi, respectively. One-way ANOVA with Tukey multiple comparisons was used for statistics with p<0.05 as the cut off for significance. Results: Tumor weights were significantly less in cis treated vs control mice (0.064g vs 0.220g, p <0.001) and CXCR2i treated vs control mice (0.171g vs 0.220g, p=.003). There was no additive effect of the CXCR2i to the cis treatment (cis 0.064g vs cis + CXCR2i 0.049g, p=0.62). The number of metastatic sites as a measurement of dissemination also decreased with cis or CXCR2i (number of sites: control: 47 vs cis: 12.7 vs CXCR2i: 32.5 vs cis + CXCR2i: 10.2). When the TIME was evaluated by flow cytometry, there were no significant differences in the immune populations including T-cells, NK cells, myeloid cells, and macrophages. However, there was a trend for cis to increase both the G-MDSC and M-MDSC populations and this effect was inhibited with the combination treatment of cis and CXCR2i. For the G-MDSC population control: 0.6% vs cis: 0.9% vs cis+CXCR2i: 0.5% and M-MDSC population control 3.4% vs cis: 6.8% vs cis+CXCR2i: 2.6%. Conclusion: CXCR2 is a chemotactic receptor present on MDSCs. We targeted this receptor to decrease the migration and infiltration of MDSCs into the ovarian cancer TIME. The CXCR2i decreased tumor progression in the ID8-p53null model of ovarian cancer. The TIME was evaluated by flow cytometry, and there was a trend for cis to increase the suppressive MDSC populations and this increase was inhibited by the CXCR2i. These exciting data suggest that traditional chemotherapy may lead to a more immunosuppressive TIME, which could have implications for future recurrence and treatment. Blocking the migration of immunosuppressive cells into the TIME may have a long-term impact on survival in HGSC. Survival studies in this murine model of HGSC are currently underway. Citation Format: Nicole Marjon, Railey Mikeska, Elizabeth R. Woodruff, Ritsuko Iwanaga, Tomomi Yamamoto, Benjamin G. Bitler. Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B087.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging

ObjectivesCurrently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence.Materials and methodsIn a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function.ResultsThe number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow.ConclusionsMultiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

Intratumoral Delivery of Interleukin 9 via Oncolytic Vaccinia Virus Elicits Potent Antitumor Effects in Tumor Models.

The success of cancer immunotherapy is largely associated with immunologically hot tumors. Approaches that promote the infiltration of immune cells into tumor beds are urgently needed to transform cold tumors into hot tumors. Oncolytic viruses can transform the tumor microenvironment (TME), resulting in immunologically hot tumors. Cytokines are good candidates for arming oncolytic viruses to enhance their function in this transformation. Here, we used the oncolytic vaccinia virus (oVV) to deliver interleukin-9 (IL-9) into the tumor bed and explored its antitumor effects in colon and lung tumor models. Our data show that IL-9 prolongs viral persistence, which is probably mediated by the up-regulation of IL-10. The vvDD-IL-9 treatment elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, granzyme B, and perforin. IL-9 expression increased the percentages of CD4+ and CD8+ T cells in the TME and decreased the percentage of oVV-induced immune suppressive myeloid-derived suppressor cells (MDSC), leading to potent antitumor effects compared with parental virus treatment. The vvDD-IL-9 treatment also increased the percentage of regulatory T cells (Tregs) in the TME and elevated the expression of immune checkpoint molecules such as PD-1, PD-L1, and CTLA-4, but not GITR. The combination therapy of vvDD-IL-9 and the anti-CTLA-4 antibody, but not the anti-GITR antibody, induced systemic tumor-specific antitumor immunity and significantly extended the overall survival of mice, indicating a potential translation of the IL-9-expressing oncolytic virus into a clinical trial to enhance the antitumor effects elicited by an immune checkpoint blockade for cancer immunotherapy.

Global stability and parameter analysis reinforce therapeutic targets of PD-L1-PD-1 and MDSCs for glioblastoma

Glioblastoma (GBM) is an aggressive primary brain cancer that currently has minimally effective treatments. Like other cancers, immunosuppression by the PD-L1-PD-1 immune checkpoint complex is a prominent axis by which glioma cells evade the immune system. Myeloid-derived suppressor cells (MDSCs), which are recruited to the glioma microenviroment, also contribute to the immunosuppressed GBM microenvironment by suppressing T cell functions. In this paper, we propose a GBM-specific tumor-immune ordinary differential equations model of glioma cells, T cells, and MDSCs to provide theoretical insights into the interactions between these cells. Equilibrium and stability analysis indicates that there are unique tumorous and tumor-free equilibria which are locally stable under certain conditions. Further, the tumor-free equilibrium is globally stable when T cell activation and the tumor kill rate by T cells overcome tumor growth, T cell inhibition by PD-L1-PD-1 and MDSCs, and the T cell death rate. Bifurcation analysis suggests that a treatment plan that includes surgical resection and therapeutics targeting immune suppression caused by the PD-L1-PD1 complex and MDSCs results in the system tending to the tumor-free equilibrium. Using a set of preclinical experimental data, we implement the approximate Bayesian computation (ABC) rejection method to construct probability density distributions that estimate model parameters. These distributions inform an appropriate search curve for global sensitivity analysis using the extended fourier amplitude sensitivity test. Sensitivity results combined with the ABC method suggest that parameter interaction is occurring between the drivers of tumor burden, which are the tumor growth rate and carrying capacity as well as the tumor kill rate by T cells, and the two modeled forms of immunosuppression, PD-L1-PD-1 immune checkpoint and MDSC suppression of T cells. Thus, treatment with an immune checkpoint inhibitor in combination with a therapeutic targeting the inhibitory mechanisms of MDSCs should be explored.

ABCB1 Regulates Immune Genes in Breast Cancer.

Resistance to standard chemotherapy is a critical problem for breast cancer patients. The ATP-binding cassette (ABC) superfamily transporters actively pump out drugs and play an important role in chemoresistance. ABCB1 (ABC subfamily B, member 1, also named as multidrug resistance protein 1, MDR1) and suppressive myeloid-derived suppressor cells (MDSCs) potentially involve in chemoresistance of breast cancer. The relationship between ABCB1 and immune genes in breast cancer has not been widely studied. Microarray and RNA sequencing data were obtained from The Cancer Genome Atlas Breast Invasive Carcinoma in Genomic Data Commons Data Portal and Gene Expression Omnibus database. A patient-derived xenograft (PDX) model of HER2+ breast cancer was established to investigate the association between ABCB1 and immune genes in breast cancer. Expression of ABCB1 increased in doxorubicin-selected MCF-7/ADR cells. High expression of ABCB1 mRNA is correlated with lymph-node metastasis and worse overall survival in patients with breast cancer. ABCB1 is positively correlated with IL6, CSF1, CSF3, and PTGS2. In the HER2+ stage IIA breast cancer PDX model, both doxorubicin and paclitaxel suppressed growth of P2 tumors. IL6, CSF1, CSF3, and PTGS2 expression were suppressed by paclitaxel but not doxorubicin. Intrasplenic MDSCs, including CD11b+Ly6G+ and CD11b+Ly6C+ cells, were more abundant than intratumor MDSCs in PDX-carrying nude mice. Clinically, the patient developed cancer recurrence after adjuvant chemotherapy with doxorubicin-based regimen and was well controlled after paclitaxel-trastuzumab combined therapy. ABCB1 was a poor predictor of HER2+ LN- breast cancer. Regulation of immune genes by ABCB1 contributed to cancer recurrence and treatment effect. The PDX model was suitable for investigation the expression of target genes and expansion of immune cells.

MDSCs-derived GPR84 induces CD8+ T-cell senescence via p53 activation to suppress the antitumor response

BackgroundsG-protein-coupled receptor 84 (GPR84) marks a subset of myeloid-derived suppressor cells (MDSCs) with stronger immunosuppression in the tumor microenvironment. Yet, how GPR84 endowed the stronger inhibition of MDSCs to CD8+...

E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

BackgroundAlterations in several tripartite motif-containing (TRIM) family proteins have been implicated in the pathogenesis of lung cancer. TRIM28, a member of the TRIM E3 ligase family, has been associated with tumorigenesis, cell proliferation, and inflammation. However, little is known about TRIM28 expression and its role in the immune microenvironment of non-small cell lung cancer (NSCLC).MethodsWe assessed the clinical significance of TRIM28 in tissue microarrays and TCGA cohorts. We investigated the function of TRIM28 in syngeneic mouse tumor models, the KrasLSL−G12D/+; Tp53fl/fl (KP) mouse model, and humanized mice. Immune cell composition was analyzed using flow cytometry and immunohistochemistry.ResultsOur findings revealed a positive correlation between TRIM28 expression and the infiltration of suppressive myeloid-derived suppressor cells (MDSCs) in NSCLC. Moreover, silencing TRIM28 enhanced the efficacy of anti-PD-1 immunotherapy by reshaping the inflamed tumor microenvironment. Mechanistically, we demonstrated that TRIM28 could physically interact with receptor-interacting protein kinase 1 (RIPK1) and promote K63-linked ubiquitination of RIPK1, which is crucial for sustaining activation of the NF-κB pathway. Mutagenesis of the E3 ligase domain corroborated the essential role of E3 ligase activity in TRIM28-mediated NF-κB activation. Further experiments revealed that TRIM28 could upregulate the expression of CXCL1 by activating NF-κB signaling. CXCL1 could bind to CXCR2 on MDSCs and promote their migration to the tumor microenvironment. TRIM28 knockdown increased responsiveness to anti-PD-1 therapy in immunocompetent mice, characterized by increased CD8+T tumor-infiltrating lymphocytes and decreased MDSCs.ConclusionThe present study identified TRIM28 as a promoter of chemokine-driven recruitment of MDSCs through RIPK1-mediated NF-κB activation, leading to the suppression of infiltrating activated CD8+T cells and the development of anti-PD-1 resistance. Understanding the regulation of MDSC recruitment and function by TRIM28 provides crucial insights into the association between TRIM28 signaling and the development of an immunosuppressive tumor microenvironment. These insights may inform the development of combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in NSCLC.

Size-Tunable Nanoregulator-Based Radiofrequency Ablation Suppresses MDSCs and Their Compensatory Immune Evasion in Hepatocellular Carcinoma.

Radiofrequency ablation (RFA) is a widely used therapy for hepatocellular carcinoma (HCC). However, in cases of insufficient RFA (iRFA), nonlethal temperatures in the transition zone increase the risk of postoperative relapse. The pathological analysis of HCC tissues shows that iRFA-induced upregulation of myeloid-derived suppressor cells (MDSCs) in residual tumors is critical for postoperative recurrence. Furthermore, this study demonstrates, for the first time, that combining MDSCs suppression strategy during iRFA can unexpectedly lead to a compensatory increase in PD-L1 expression on the residual MDSCs, attributed to relapse due to immune evasion. To address this issue, a novel size-tunable hybrid nano-microliposome is designed to co-deliver MDSCs inhibitors (IPI549) and αPDL1 antibodies (LPIP) for multipathway activation of immune responses. The LPIP is triggered to release immune regulators by the mild heat in the transition zone of iRFA, selectively inhibiting MDSCs and blocking the compensatory upregulation of PD-L1 on surviving MDSCs. The combined strategy of LPIP + iRFA effectively ablates the primary tumor by activating immune responses in the transition zone while suppressing the compensatory immune evasion of surviving MDSCs. This approach avoids the relapse of the residual tumor in a post-iRFA incomplete ablation model and appears to be a promising strategy in RFA for the eradication of HCC.

BTK inhibition potentiates anti-PD-L1 treatment in murine melanoma: potential role for MDSC modulation in immunotherapy.

Myeloid-derived suppressor cells (MDSC) have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species and the production of inhibitory cytokines. Our group has shown that signaling through Bruton's tyrosine kinase (BTK) is important for MDSC function. Ibrutinib is an orally administered targeted agent that inhibits BTK activation and is currently used for the treatment of B cell malignancies. Using a syngeneic murine model of melanoma, the effect of BTK inhibition with ibrutinib on the therapeutic response to systemic PD-L1 blockade was studied. BTK was expressed by murine MDSC and their activation was inhibited by ibrutinib. Ibrutinib was not directly cytotoxic to cancer cells in vitro, but it inhibited BTK activation in MDSC and reduced expression of inducible nitric oxide synthase (NOS2) and production of nitric oxide. Ibrutinib treatments decreased the levels of circulating MDSC in vivo and increased the therapeutic efficacy of anti-PD-L1 antibody treatment. Gene expression profiling showed that ibrutinib decreased Cybb (NOX2) signaling, and increased IL-17 signaling (upregulating downstream targets Mmp9, Ptgs2, and S100a8). These results suggest that further exploration of MDSC inhibition could enhance the immunotherapy of advanced melanoma.PrécisInhibition of Bruton's tyrosine kinase, a key enzyme in myeloid cellular function, improves therapeutic response to an anti-PD-L1 antibody in an otherwise fairly resistant murine melanoma model.

Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

Staphylococcus aureus is an important cause of chronic infections resulting from the failure of the host to eliminate the pathogen. Effective S. aureus clearance requires CD4+ T cell-mediated immunity. We previously showed that myeloid-derived suppressor cells (MDSC) expand during staphylococcal infections and support infection chronicity by inhibiting CD4+ T cell responses. The aim of this study was to elucidate the mechanisms underlying the suppressive effect exerted by MDSC on CD4+ T cells during chronic S. aureus infection. It is well known that activated CD4+ T cells undergo metabolic reprogramming from oxidative metabolism to aerobic glycolysis to meet their increased bioenergetic requirements. In this process, pyruvate is largely transformed into lactate by lactate dehydrogenase with the concomitant regeneration of NAD+, which is necessary for continued glycolysis. The by-product lactate needs to be excreted to maintain the glycolytic flux. Using SCENITH (single-cell energetic metabolism by profiling translation inhibition), we demonstrated here that MDSC inhibit CD4+ T cell responses by interfering with their metabolic activity. MDSC are highly glycolytic and excrete large amount of lactate in the local environment that alters the transmembrane concentration gradient and prevent removal of lactate by activated CD4+ T. Accumulation of endogenous lactate impedes the regeneration of NAD+, inhibit NAD-dependent glycolytic enzymes and stop glycolysis. Together, the results of this study have uncovered a role for metabolism on MDSC suppression of CD4+ T cell responses. Thus, reestablishment of their metabolic activity may represent a mean to improve the functionality of CD4+ T cells during chronic S. aureus infection.

A biomarker study of atezolizumab (atezo) + bevacizumab (bev) + carboplatin (carbo) + paclitaxel (pac) (ABCP) for patients with NSCLC harboring EGFR mutations (EGFRm) after failure of TKI therapy: NEJ043.

9079 Background: EGFRm NSCLC patients (pts) have limited treatment options after EGFR-TKI failure. We previously showed that the NEJ043 study investigating ABCP in EGFRm NSCLC pts after EGFR-TKI failure had a clinically meaningful efficacy (Furuya et al, ASCO 2022). Here we report the results of the biomarker study of NEJ043. Methods: NEJ043 study is a phase II study to evaluate the efficacy of ABCP in sensitizing EGFRm nonsquamous NSCLC pts after TKI failure. Pts received atezo (1200 mg), bev (15 mg/kg), carbo (AUC 6 mg/mL/min), and pac (175 mg/m2) every 3 weeks up to 4 cycles followed by atezo plus bev until loss of clinical benefit. Before treatment initiation and three cycles of ABCP, peripheral blood mononuclear cells (PBMCs) were collected, and immunophenotypic data at each time point was obtained by flow cytometry. Results: 60 pts were included in the NEJ043 study and PBMCs were obtained from 31 pts. Pts with a higher percentage of CD11b+DRlowCD3-CD14+ myeloid-derived suppressor cells (MDSCs) before ABCP had significantly longer PFS (median, 9.7 months (95% CI, 5.3-17.7) vs 5.6 months (95% CI, 3.9-8.1); HR 0.38; P = 0.0313). Further analysis of the immune cell phenotypes that changed between pre-treatment and the third course showed that pts with increased CD62LlowCD8+ cells (median, 12.8 months (95% CI, 5.6-20.3) vs 5.7 months (95% CI, 4.4-8.1); HR 0.25; P = 0.003) and PD-1+CCR7-CD45RA-CD8+ cells (median, 8.5 months (95% CI, 5.6-13.8) vs 5.7 months (95% CI, 2.6-8.1); HR 0.37; P = 0.0324) had significantly longer PFS. Conclusions: Inhibition of MDSCs by ABCP, especially bev, may be important for EGFRm pts whose antitumor immunity is suppressed by MDSCs before treatment. Better PFS may be expected in pts in whom ABCP therapy induced CD62LlowCD8+ effector T cells and PD-1+CCR7-CD45RA-CD8+ effector memory T cells. Clinical trial information: jRCTs031190066 .

Abstract 1259: Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment

Abstract Background: Overexpression of sialic acids on glycans, called hypersialylation is a common alteration in cancer that drives immune evasion via interaction with Sialic acid-binding immunoglobulin-like lectin (Siglec) immunoregulatory receptors on tumor-infiltrating immune cells. Myeloid derived suppressor cells (MDSCs) generated by cancer-induced aberrant myelopoiesis are highly immunosuppressive cells inhibiting immune cells via direct interaction or secretion of suppressive cytokines. Targeting the complex suppressive tumor microenvironment (TME) remains a challenge. Methods: Siglec expression of murine and human MDSCs in healthy conditions and tumor setting was assessed by flow cytometry. Functional analysis of Siglec-E knockout on MDSCs in mice was evaluated using SigE-LysMCre mice and suppressive capacity was tested in vitro in combination with SiglecE blocking and cleavage of Sialoglycans using Sialidase. Results were confirmed in the human setting using an in vitro assay to generate MDSC-like cells including RNA-Sequencing and a MDSC suppression assay with cancer-derived MDSCs. Results: MDSCs express multiple inhibitory Siglec receptors in humans and mice. Knockout of SiglecE in the myeloid compartment in vivo resulted in prolonged survival and increased T cell infiltration compared to litter mates expressing Siglec-E. Of note, desialylation of MDSCs and Siglec-E blocking in vitro reduced MDSCs suppressive function against T cells. Similarly, reducing Sialoglycans by Sialidase in human MDSC generated in vitro resulted in increased suppressive capacity and downregulation of functional MDSC markers on RNA level. Findings were validated with lung cancer patient derived MDSCs. Conclusion: Our results provide first insights into the importance of the Siglec-Sialoglycans axis to modulate MDSCs in the TME. Targeting glycosylation of MDSCs with Sialidase reduces their suppressive capacity making it a powerful approach to improve cancer immunotherapy. Further studies are needed to reveal the underlying mechanisms. Citation Format: Ronja Wieboldt, Andreas Zingg, Emanuele Carlini, Anastasiya Börsch, Heinz Läubli, Natalia Rodrigues Manutano. Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1259.

Abstract 5167: Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling

Abstract Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5167.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.