Downregulating PD-L1 Expression Research Articles

OSCC cell-secreted exosomal CMTM6 induced M2-like macrophages polarization via ERK1/2 signaling pathway.

CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) is a critical regulator of tumor immunology among various cancers. However, the role and underlying molecular mechanism of CMTM6 in oral squamous cell carcinoma (OSCC) progression remains unclear. The expression of CMTM6, PD-L1 and CD163 in OSCC tissues were detected by immunohistochemistry on tissue microarray. The effect of CMTM6 knockdown on OSCC cells and macrophage polarization were analyzed by CCK-8 assay, apoptotic assay, would-healing assay, transwell assay and qPCR. OSCC cell derived exosomes were obtained by ultracentrifugation and the mechanistic studies were conducted by qPCR and Western Blot. 4-Nitroquinoline N-oxide (4NQO) induced OSCC mice were used for verifying the effect of CMTM6 downregulation on M2 macrophage infiltration and tumor growth. In OSCC samples, higher CMTM6 expression has been obviously associated with higher pathological stage of OSCC patients, CD163 + macrophages infiltration and PD-L1 expression. CMTM6 knockdown of OSCC cells inhibited proliferative, migrative and invasive abilities of OSCC cells, as well as inhibited M2 macrophage polarization in vitro with downregulating PD-L1 expression. Importantly, exosomes from OSCC cells shuttled CMTM6 to macrophages and promoted M2-like macrophage polarization through activating ERK1/2 signaling. In addition, in 4NQO-induced OSCC mice, CMTM6 level was positively associated with CD163, CD206 and PD-L1 as well as M2-like macrophage infiltration. OSCC cell-secreted exosomal CMTM6 induces M2-like macrophages polarization to promote malignant progression via ERK1/2 signaling pathway, revealing a novel crosstalk between cancer cells and immune cells in OSCC microenvironment.

Manipulating immune system using nanoparticles for an effective cancer treatment: Combination of targeted therapy and checkpoint blockage miRNA

Accumulating clinical data shows that less than half of patients are beneficial from PD-1/PD-L1 blockage therapy owing to the limited infiltration of effector immune cells into the tumor and abundant of the immunosuppressive factors in the tumor microenvironment. In this study, PD-L1 inhibition therapy and BRAF-targeted therapy, which showed clinical benefit, were combined in a CXCR4-targeted nanoparticle co-delivering dabrafenib (Dab), a BRAF inhibitor, and miR-200c which can down-regulate PD-L1 expression. The cationic PCL-PEI core containing Dab- and miR-200c- were coated with poly-L-glutamic acid conjugated with LY2510924, a CXCR-4 antagonist peptide, (PGA-pep) to obtain miR@PCL-PEI/Dab@PGA-pep nanoformulation. The stimulus pH- and redox- reactive of PGA-pep was ascribed to exhibit an enhanced release of drug in the tumor microenvironment as well as improve the stability of miR-200c during the blood circulation. In addition, the presence of LY2510924 peptide would enhance the binding affinity of miR@PCL-PEI/Dab@PGA-pep NPs to cancer cells, leading to improved cellular uptake, cytotoxicity, and in vivo accumulation into tumor area. The in vivo results indicated that both, the immunogenic cell death (ICD) and the inhibition of PD-L1 expression, induced by treatment with CXCR-4 targeted nanoparticles, enables to improve the DC maturation in lymph node and CD8+ T cell activation in the spleen. More importantly, effector T cells were increasingly infiltrated into the tumor, whereas the immunosuppressive factors like PD-L1 expression and regulatory T cells were significantly reduced. They, all together, promote the immune responses against the tumor, indicating the therapeutic efficiency of the current strategy in cancer treatment.

MiR-502-5p affects gastric cancer progression by targeting PD-L1

BackgroundStudies have shown that miR-502-5p functions as a tumor suppressor and is associated with tumor growth and metastasis. This study intends to uncover the potential mechanism of miR-502-5p functioning as a tumor suppressor in gastric cancer.MethodsExpression levels of miR-502-5p and PD-L1 were measured by using qRT-PCR. Cell proliferation abilities were examined by EDU incorporation assay. Cell migration, invasion and cell cycle analysis of cells were determined by transwell assay, transwell-matrigel assay and flow cytometry, respectively. The relationship between miR-502-5p expression and the overall survival of xenograft tumor mice was statistically analyzed. Bioinformatics analysis and luciferase reporter assays were applied to analyze the relationship between miR-502-5p and CD40, STAT3 or PD-L1. Expressions of CD40, STAT3 and PD-L1 at protein level were detected by western blot.ResultsThe results showed that miR-502-5p was significantly downregulated in gastric cancer tumor tissues compared with adjacent normal tissues. Overexpression of miR-502-5p significantly attenuated the proliferation, migration/invasion and induced the G1 phase arrest of gastric cancer cells. Consistently, miR-502-5p suppressed tumor growth and metastasis in vivo. Mechanically, we demonstrated that miR-502-5p had inhibited the malignant behaviour of gastric cancer by down-regulating PD-L1 expression at transcriptional level and post-transcriptional levels.ConclusionsThese findings suggest that miR-502-5p acts as a tumor suppressor in gastric cancer (GC). MiR-502-5p/PD-L1 may be a novel therapeutic target in GC treatment.

Abstract 5178: MEDI7526: a novel bispecific antibody that activates the CD40 pathway and down-regulates cell surface PD-L1 expression

Abstract CD40 stimulation on antigen-presenting cells (APCs) promotes activation of CD8(+) cytotoxic T cells and several CD40 agonistic antibodies have shown promising anti-tumor activities in initial clinical trials. However, agonistic CD40 stimulation also leads to PD-L1 upregulation on APCs, thus creating a negative feedback loop to dampen the anti-tumor immune response. We hypothesized that the combination of PD-L1 antagonism with CD40 agonists would achieve stronger antitumor responses than with anti-CD40 alone. Here we report a novel bispecific antibody MEDI7526, which is composed of an anti-PD-L1 antibody, two trimer forms of CD40 ligand (CD40L) fusion protein (FP) and a human IgG4p Fc. MEDI7526 has been designed to stimulate the CD40 pathway and suppress PD-L1 function to improve T cell activation and potentially overcome the limitations associated with conventional CD40 agonists. The binding characteristics and functional activity of MEDI7526 were analyzed in vitro using flow cytometry and cell-based reporter assays. Internalization of MEDI7526, CD40, and PD-L1 were examined by flow cytometry and immunofluorescence microscopy. Effects of MEDI7526 on cytokine production from human PBMC were analyzed with MSD multiplex assay. Anti-tumor efficacy was determined by a murine surrogate version of MEDI7526 in a B16F10 syngeneic model. We confirmed that MEDI7526 can bind CD40 and PD-L1 simultaneously. MEDI7526 induced robust activation of CD40 signaling and triggered fast internalization of CD40. Interestingly, MEDI7526 also triggered rapid and robust down-regulation of PD-L1 on the cell surface of multiple types of cells that express CD40, including THP1, MDA-MB-231 and human monocytes, whereas PD-L1 expression was not affected by the treatment of either anti-PD-L1 antibody or CD40L FP. Prolonged treatment of MEDI7526 on MDA-MB-231 cells induced sustained down-regulation of PD-L1 for up to 4 days. When THP1 cells were transiently treated with MEDI7526, cell surface CD40 returned after 24 hours but levels of PD-L1 on the cell membrane remained low, indicating re-expression of cell surface CD40 and PD-L1 following distinct pathways. We demonstrated that internalized PD-L1 undergoes lysosome-dependent, proteasome-independent degradation, resulting in sustained down-regulation of PD-L1 from cell surface. Moreover, MEDI7526 induced robust IFNγ production in antigen recall assays, and the murine surrogate of MEDI7526 effectively inhibited tumor growth in the B16F10 syngeneic model. These studies demonstrate that MEDI7526 drives the internalization and degradation of PD-L1 and exhibits robust in vitro and in vivo activities. Building this novel bispecific molecule brings two molecules together, resulting in novel mechanisms of action that are different from combinations of parental reagents. MEDI7526, through its unique function of simultaneously stimulating the CD40 pathway and down-regulating PD-L1 expression, represents a promising therapeutic approach against cancer. Citation Format: Yaya Wang, Sean Turman, Deepali Malhotra, Ryan Gilbreth, Yue Wang. MEDI7526: a novel bispecific antibody that activates the CD40 pathway and down-regulates cell surface PD-L1 expression [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 5178.

Abstract 4454: TUSC2 immunogene therapy enhances efficacy of immunotherapy and targeted drugs in human non-small cell lung cancer (NSCLC) in humanized mouse models

Abstract TUSC2 is a tumor suppressor gene, whose expression is reduced in almost all NSCLC. Systemic nanovesicle delivery of TUSC2 inhibits cancer cell growth through inhibition of a broad spectrum of kinases and mTOR downregulation as well as stimulation of the immune system through innate activation. We previously reported that TUSC2 downregulates PD-L1 expression in NSCLC and synergizes with anti-PD1 in inhibiting tumor growth in Kras mutant syngeneic mouse models through upregulation of NK and cytotoxic T cells. We developed an improved CD34-derived humanized mouse model (Hu-mice), with faster and higher human immune reconstitution than other available humanized mice, to evaluate immune responses in lung cancer. In this study, we tested whether TUSC2 immunogene therapy would enhance response to standard checkpoint blockade immunotherapy, chemotherapy and targeted therapies in humanized NSG mice implanted with highly metastatic Krasmt/LKB1− A549 cells. A significantly increased antitumor effect was found when TUSC2 was combined with pembrolizumab. Pembrolizumab alone reduced tumor burden as compared with an untreated control, whereas no antitumor effect was observed in non-Hu-mice implanted with A549 cells. The observed antitumor effect correlated with increased levels of CD8+ T and CD8+CD69+ active T, and decreased levels of MDSC and regulatory T cells in the combination group. A significantly higher percentages of CD56+ NK and CD56+CD69+ active NK cells were found in the TUSC2 alone and combination groups indicating TUSC2 related NK activation. Next, we tested whether TUSC2 enhances efficacy to carboplatin+pembrolizumab. The level of antitumor effect of carboplatin+pembrolizumab was similar to that of TUSC2 alone. However, when TUSC2 was combined with carboplatin+pembrolizumab, metastases regression was significantly greater than either TUSC2 alone or carboplatin+pembrolizumab treatments. Significantly fewer or no visible tumor nodules were found in dissected lungs in the TUSC2 combination as compared with other groups. Immune analysis of the triple combination in CMT167 syngeneic mice showed increased infiltration of CD3+ T, CD8+ T, NK cells and significantly less Treg cells into tumor, which was associated with significant tumor inhibition by the treatments. A higher percentage of CD3+CD44+ and CD8+CD44+ memory T cells were found in tumors after carbo+aPD1+TUSC2 treatment, as compared with either Carbo+aPD1 or control groups. The antitumor activity of Carbo+aPD1+TUSC2 was further enhanced when MEKi (Trametinib) was added. Moreover, we also combined TUSC2 with the anti-angiogenic agent, bevacizumab (anti-VEGF) to enhance efficacy in the highly angiogenic 786-O renal cell carcinoma. Synergistic antitumor activity was found with the combination, which was significantly stronger than either single agent. In conclusion, the addition of TUSC2 immunogene therapy with checkpoint blockade, chemotherapy, and targeted therapies showed enhanced antitumor efficacy. Citation Format: Ismail M. Meraz, Mourad Majidi, Meng Feng, RuPing Shao, Min Jin Ha, Elizabeth J. Shpall, Jack A. Roth. TUSC2 immunogene therapy enhances efficacy of immunotherapy and targeted drugs in human non-small cell lung cancer (NSCLC) in humanized mouse models [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 4454.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn’t inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

Abstract B18: YAP1 and TAZ mediate BET inhibitor-dependent immune regulation in NSCLC

Abstract Bromodomain and extra-terminal domain proteins inhibitors (BETi) are promising immunomodulatory agents, able to regulate gene expression in tumor microenvironment (TME), influencing immune cell differentiation and activation. However, immune-related molecular mechanisms underlying BETi activity need to be clarified. We demonstrated that BET protein BRD4 is an important regulator of YAP1/TAZ oncogene expression in several cancer cell lines, including non-small cell lung cancer (NSCLC) cells. YAP1 and TAZ are paralogue genes involved in tumor cell proliferation, migration, and survival. BETi affect YAP1/TAZ expression and impair their transcriptional program. As previously reported, YAP1 and TAZ oncogenes are also involved in tumor immune escape, through the impairment of lymphocyte activation and enhanced PD-L1 expression. We investigated whether BETi-dependent immune regulation depends on YAP1/TAZ activity in NSCLC patients. To this end, we isolated NSCLC patient-derived tissue-originated spheroids (CTOS) and tumor-infiltrating lymphocytes (TILs) and treated both populations with BETi (JQ1 or OTX-015). BETi treatment significantly increased the activation of TILs, which was also associated with decreased expression of the immune checkpoint receptors PD-1, LAG-3, TIM-3, BTLA, and CD160. Notably, BETi treatment also downregulated the expression of corresponding immune checkpoint ligands (PD-L1, LGALS9, and TNFRSF14). Intriguingly, patients displaying higher expression of immune checkpoint molecules exhibited also higher expression of YAP1 and TAZ, suggesting that these transcription factors may be involved in this regulatory network. Accordingly, YAP1 and TAZ were significantly downregulated after BETi treatment in both CTOS and TILs. To confirm specific YAP/TAZ activity in immune regulation, the knockout (KO) of YAP1 or TAZ was achieved through CRISPR/Cas9 approach in NSCLC cell lines. Of interest, YAP1 and TAZ display distinct regulatory effects, suggesting a not overlapping function. The KO of YAP1, but not of TAZ, was sufficient to downregulate PD-L1 expression. Conversely, KO of TAZ alone efficiently downregulated LGALS9, which was not affected by YAP1 KO. TNFRSF14 was significantly reduced in both TAZ and YAP1 KO. These preliminary data suggest that BETi exert a YAP/TAZ-dependent immunomodulatory activity, acting on both tumor cells and TME immune landscape. In this context, BETi may reactivate immune response against tumor cells, acting on both immune checkpoint inhibitor expression and TIL-specific activation. These findings further support the use of BETi as immunomodulatory agents in NSCLC patients. Citation Format: Francesca Reggiani, Giulia Gobbi, Filippo Lococo, Massimiliano Paci, Eleonora Zznetti, Simonetta Piana, Alessia Ciarrocchi, Valentina Sancisi. YAP1 and TAZ mediate BET inhibitor-dependent immune regulation in NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B18.

Silibinin down-regulates PD-L1 expression in nasopharyngeal carcinoma by interfering with tumor cell glycolytic metabolism

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

Anlotinib alters tumor immune microenvironment by downregulating PD-L1 expression on vascular endothelial cells

Aberrant vascular network is a hallmark of cancer. However, the role of vascular endothelial cells (VECs)-expressing PD-L1 in tumor immune microenvironment and antiangiogenic therapy remains unclear. In this study, we used the specimens of cancer patients for immunohistochemical staining to observe the number of PD-L1+ CD34+ VECs and infiltrated immune cells inside tumor specimens. Immunofluorescence staining and flow cytometry were performed to observe the infiltration of CD8+ T cells and FoxP3+ T cells in tumor tissues. Here, we found that PD-L1 expression on VECs determined CD8+ T cells’, FoxP3+ T cells’ infiltration, and the prognosis of patients with lung adenocarcinoma. Anlotinib downregulated PD-L1 expression on VECs through the inactivation of AKT pathway, thereby improving the ratio of CD8/FoxP3 inside tumor and remolding the immune microenvironment. In conclusion, our results demonstrate that PD-L1 high expression on VECs inhibits the infiltration of CD8+ T cells, whereas promotes the aggregation of FoxP3+ T cells into tumor tissues, thus becoming an “immunosuppressive barrier”. Anlotinib can ameliorate the immuno-microenvironment by downregulating PD-L1 expression on VECs to inhibit tumor growth.

Apigenin modulates immune checkpoint molecules in pancreatic cancer enhancing anti-tumor immunity

Abstract Pancreatic cancer (PC) remains as a very aggressive malignancy with an extremely poor prognosis and an overall 5-year survival rate of only 8%. Immunotherapies are promising against solid tumors with exception of PC. This poor prognosis is partly due to PC’s inflammatory tumor microenvironment, resulting in an increased expression of both Programmed Death-Ligand (PD-L1) in tumors and Programmed cell death protein 1 (PD-1) on effector CD8+ T cells. Upon PD-L1/PD-1 interaction, CD8+ T cells exhibit anergy, allowing PC evasion and tumor progression. We hypothesize that reducing PC-induced inflammation will increase anti-tumor immune responses and promote tumor regression. Therefore, we examined the expression of checkpoint molecules (PD-L1, PD-L2, PD-1, Major Histocompatibility Complex-I, etc.) in different murine PC cell lines (i.e. Panc02, UN-KPC-960, UN-KC-6141) and modulation by an anti-cancer natural bioflavonoid called apigenin (API). To do this, we treated the PC cell lines, in vitro, with and without API along with interferon-γ (potent inducer of PD-L1) to detect the expression profiles of these checkpoint molecules using flow cytometry. Subsequently, we tested T cell proliferation in response to untreated/treated PC cells. Our results show that API down-regulated PD-L1 expression on PC cell lines, which coincides with increased T cell responses. Our results suggest that API may reduce PC-induced immunosuppression allowing increased anti-tumor immunity. Therefore, clinical application of API as an adjuvant may potentiate the efficacy of current PC immunotherapies.

Abstract A75: Efficacy of novel immunogene combinations for Kras and LKB1 mutant NSCLC in a humanized mouse model

Abstract Due to lack of suitability of current preclinical models for immunotherapy research, we recently developed an improved humanized mouse by reconstituting a human immune system in NSG mice by transplanting fresh human cord blood-derived CD34+ stem cells (Hu-mice). The Hu-mice show functional representation of human T, B, natural killer (NK), dendritic cells (DC), myeloid-derived suppressor cells (MDSC), and responsiveness to checkpoint blockade. TUSC2 has recently been recognized as a novel immunogene that induces apoptosis in tumor cells and promotes a wide spectrum of tumor-specific innate and adaptive immune responses. We previously reported that TUSC2 delivered systemically by nanovesicles downregulates PD-L1 expression in NSCLC and synergizes with anti-PD1 in inhibiting tumor growth in Kras-mutant syngeneic mouse models through upregulating NK and cytotoxic T cells. In this study, we aimed to evaluate the antitumor efficacy of TUSC2 in combination with standard immunotherapy on highly metastatic Kras and LKB1 mutant human lung cancer in Hu-mice. Hu-mice were challenged with A549 cells (Krasmt/LKB1-) and lung metastases were treated with TUSC2, nivolumab, or the combination. The results showed a synergistic antitumor effect with the combination. A significantly increased antitumor effect was found when TUSC2 was combined with pembrolizumab in Hu-mice. Pembrolizumab alone significantly reduced tumor burden as compared with an untreated control, whereas no antitumor effect was observed in non-Hu-mice implanted with A549 cells. The antitumor effect was correlated with significantly higher levels of CD8+ T and CD8+CD69+ active T and significantly lower levels of MDSC and regulatory T cells in the combination group. A significantly higher percentage of CD56+ NK and CD56+CD59+ active NK cells was found in the TUSC2 alone and combination groups, indicating TUSC2 related NK activation. We tested whether TUSC2 enhances efficacy to carboplatin+pembrolizumab in Hu-mice implanted with A549-luc metastatic cells. The results showed that the level of antitumor effect of carboplatin+pembrolizumab was similar to that of TUSC2 alone, but when TUSC2 was combined with carboplatin+pembrolizumab, metastases regression was significantly greater than either TUSC2 alone or carboplatin+pembrolizumab treatments. Significantly fewer or no visible tumor nodules were found in dissected lungs in the TUSC2 combination as compared with other groups. In conclusion, TUSC2 immunogene therapy in combination with pembrolizumab and carboplatin+pembrolizumab showed strong antitumor efficacy in metastatic human NSCLC in a clinically relevant humanized mouse model, supporting a clinical trial. Citation Format: Ismail M. Meraz, Mourad Majidi, Meng Feng, RuPing Shao, Min Jin Ha, Jeffrey Morris, Elizabeth J. Shpall, Jack A. Roth. Efficacy of novel immunogene combinations for Kras and LKB1 mutant NSCLC in a humanized mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A75.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

Glutamine Deprivation Induces PD-L1 Expression via Activation of EGFR/ERK/c-Jun Signaling in Renal Cancer.

The programmed death-ligand 1/programmed death-1 (PD-L1/PD-1) pathway plays a pivotal role in the immune escape of tumors. Many tumor cells show "glutamine dependence." However, the relationship between glutamine metabolism and PD-L1 expression has not been reported. In this study, changes in PD-L1 expression in renal carcinoma cells were evaluated during glutamine deprivation and recovery. Although PD-L1 expression differed in two renal cancer cell lines, both cell lines upregulated PD-L1 during glutamine deprivation, and the upregulated PD-L1 was restored to normal after glutamine recovery. Mechanistically, glutamine deprivation resulted in activation of EGFR signaling via ERKs 1 and 2 (ERK1/2) and c-Jun. In addition, treatment of renal cancer cells with EGF also induced PD-L1 expression and ERK1/2 phosphorylation. Finally, inhibitors of EGFR, ERK, and c-Jun all inhibited phosphorylation of c-Jun and downregulated PD-L1 expression induced by glutamine deprivation. Taken together, the data suggest that glutamine regulates the expression of PD-L1 through the EGFR/ERK/c-Jun pathway in renal cancer. IMPLICATIONS: This study reveals glutamine deprivation induces PD-L1 expression via activation of EGFR/ERK/c-Jun signaling in renal cancer and provides novel markers for the treatment of renal cancer.

IL-2 enhanced MHC class I expression in papillary thyroid cancer with Hashimoto's thyroiditis overcomes immune escape in vitro.

The impact of Hashimoto's thyroiditis (HT) on the progression of papillary thyroid cancer (PTC) is still unclear. Interleukin-2 (IL-2) is a growth factor and crucial for HT development. This study aimed at investigating the effect of IL-2 on MHC class I expression in PTC cells and immune activation with experimental treatment for PTC using PTC cell lines. We assessed the expression of IL-2, HLA class I, PD-L1, CD3, CD8 and CD16 molecules in paired PTC tissues and HLA-ABC and PD-L1 expression in IL-2 pre-treated K1, TPC-1 and BCPAP cells by immunohistochemistry, qPCR, flow cytometry and Western blotting. The effect of IL-2 on immunogenicity of PTC cells to stimulate activated human T cells was determined for the percentages of activated CD8+ T cells and their cytokine production as well as PD-1 and PD-L1 expression. Compared with non-tumor tissues, we found that IL-2 expression was up-regulated in PTC tissues, particularly in PTC+HT tissues and correlated positively with HLA-class I, CD3 and CD8 expression in PTC+HT tissues. Conversely, PD-L1 expression decreased in PTC+HT tissues. Treatment with IL-2 significantly up-regulated HLA-class I expression, but down-regulated PD-L1 expression in PTC cells. Co-culture with IL-2-pre-treated PTC cells significantly promoted the proliferation of activated CD8+ T cells and their IL-2 secretion, but decreased their PD-1 expression, accompanied by decreased PD-L1 expression in IL-2-treated PTC cells in vitro. In conclusion, IL-2 up-regulated HLA-class I expression and enhanced anti-tumor T cell immunity during the development of PTC and HT. IL-2 may be a promising immunotherapy for PTC.

The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration.

Cigarette smoking is associated with an increased risk of melanoma metastasis. Smokers show higher PD-L1 expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Here, we investigate whether nicotine, a primary constituent of tobacco, induces PD-L1 expression and promotes melanoma cell proliferation and migration, which is mediated by the α9 nicotinic acetylcholine receptor (α9-nAChR). α9-nAChR overexpression in melanoma using melanoma cell lines, human melanoma tissues, and assessment of publicly available databases. α9-nAChR expression was significantly correlated with PD-L1 expression, clinical stage, lymph node status, and overall survival (OS). Overexpressing or knocking down α9-nAChR in melanoma cells up- or downregulated PD-L1 expression, respectively, and affected melanoma cell proliferation and migration. Nicotine-induced α9-nAChR activity promoted melanoma cell proliferation through stimulation of the α9-nAChR-mediated AKT and ERK signaling pathways. In addition, nicotine-induced α9-nAchR activity promoted melanoma cell migration via activation of epithelial-mesenchymal transition (EMT). Moreover, PD-L1 expression was upregulated in melanoma cells after nicotine treatment via the transcription factor STAT3 binding to the PD-L1 promoter. These results highlight that nicotine-induced α9-nAChR activity promotes melanoma cell proliferation, migration, and PD-L1 upregulation. This study may reveal important insights into the mechanisms underlying nicotine-induced melanoma growth and metastasis through α9-nAChR-mediated carcinogenic signals and PD-L1 expression.

Inhibition of DCLK1 down-regulates PD-L1 expression through Hippo pathway in human pancreatic cancer

Immunotherapy is one of the most promising strategies for cancer, compared with traditional treatments. As one of the key emerging immunotherapies, anti-PD-1/PD-L1 treatment has brought survival benefits to many advanced cancer patients. However, in pancreatic cancer, immunotherapy-based approaches have not achieved a favorable clinical effect because of mismatch repair deficiencies. Therefore, the majority of pancreatic tumors are regarded as immune-quiescent tumors and non-responsive to single-checkpoint blockade therapies. Many preclinical and clinical studies suggest that it is still important to clarify the regulatory mechanism of the PD-1/PD-L1 pathway in pancreatic cancer. As a marker of cancer stem cells, DCLK1 has been found to play an important role in the occurrence and development of a plethora of human cancers. Recent researches have revealed that DCLK1 is closely related to EMT process of tumor cells, meanwhile, it could also be used as a biomarker in gastrointestinal tumors to predict the prognoses of patients. However, the role that DCLK1 plays in the immune regulation of tumor microenvironments remains unknown. Therefore, we sought to understand if DCLK1 could positively regulate the expression of PD-L1 in pancreatic cancer cells. Furthermore, we examined if DCLK1 highly correlated with the Hippo pathway through TCGA database analysis. We found that DCLK1 helped regulate the level of PD-L1 expression by affecting the corresponding expression level of yes-associated protein in the Hippo pathway. Collectively, our study identifies DCLK1 as an important regulator of PD-L1 expression in pancreatic tumor and highlights a central role of DCLK1 in the regulation of tumor immunity.

Fatty acid synthase inhibitor orlistat impairs cell growth and down-regulates PD-L1 expression of a human T-cell leukemia line

Fatty Acid Synthase (FASN) is responsible for the de novo synthesis of fatty acids, which are involved in the preservation of biological membrane structure, energy storage and assembly of factors involved in signal transduction. FASN plays a critical role in supporting tumor cell growth, thus representing a potential target for anti-cancer therapies. Moreover, this enzyme has been recently associated with increased PD-L1 expression, suggesting a role for fatty acids in the impairment of the immune response in the tumor microenvironment. Orlistat, a tetrahydrolipstatin used for the treatment of obesity, has been reported to reduce FASN activity, while inducing a sensible reduction of the growth potential in different cancer models. We have analyzed the effect of orlistat on different features involved in the tumor cell biology of the T-ALL Jurkat cell line. In particular, we have observed that orlistat inhibits Jurkat cell growth and induces a perturbation of cell cycle along with a decline of FASN activity and protein levels. Moreover, the drug produces a remarkable impairment of PD-L1 expression. These findings suggest that orlistat interferes with different mechanisms involved in the control of tumor cell growth and can potentially contribute to decrease the tumor-associated immune-pathogenesis.

γ-Glutamyl cyclotransferase contributes to endometrial carcinoma malignant progression and upregulation of PD-L1 expression during activation of epithelial-mesenchymal transition

Recent increases in the incidence of endometrial carcinoma represent a significant risk to women’s health. We found that γ-glutamyl cyclotransferase (GGCT) was significantly up-regulated in endometrial carcinoma tissues and cells, which suggested that it may be a potential target for treatment of endometrial carcinoma. Furthermore, the impact of GGCT on proliferation, migration, and invasion of endometrial carcinoma has been demonstrated in vitro and in vivo using GGCT silencing and overexpression techniques. In addition, the epithelial-mesenchymal transition (EMT) was significantly inhibited in response to GGCT knockdown, which indicated that GGCT may contribute endometrial carcinoma malignancy during activation of the EMT. We also found that GGCT regulated PD-L1 expression during EMT activation. Furthermore, co-culture of endometrial carcinoma cells with CD8+ T lymphocytes showed that downregulation of PD-L1 expression following GGCT knockdown contributed to the killing activity of CD8+ T lymphocytes on endometrial carcinoma cells. In conclusion, our study showed that GGCT contributed to malignant progression and upregulation of PD-L1 expression of endometrial carcinoma, and may be a potential target for treatment of endometrial carcinoma.

Screening of kinase inhibitors downregulating PD-L1 expression via on/in cell quantitative immunoblots.

The interaction of programmed death-1 (PD-1) and it's ligands (PD-L1) is an important immune checkpoint and blockade of PD-1/PD-L1 axis with antibodies against PD-L1 showed promising anti-tumor activity in clinical practice. However, only a small percentage of patients can benefit from PD-L1 mAbs. Small molecular kinase inhibitors have been widely used as antitumor drugs for many years, and several kinase inhibitors were recently reported to inhibit the expression of PD-L1. However, the connections between PD-L1 expression and kinase inhibitors were not thoroughly elucidated. Herein, we set up a novel and robust screening system to identify small molecular compounds which downregulate the PD-L1 level of tumor cell based on Odyssey on/in cell quantitative immunoblots technology. A collected kinase inhibitor library was screened and 14 hits were further confirmed by western blot and flow cytometry. System biological analysis and further bio-assay identified a synergy combination between KU-60019 and Vacquinol-1 in downregulation of PD-L1. Taken together, the work established a novel method to screen the PD-L1 down-regulators using kinase inhibitors library, thus providing new clues for the application of kinase inhibitors in cancer immunotherapy.

Adipocytes as Anticancer Drug Delivery Depot

Summary Tumor-associated adipocytes promote tumor growth by providing energy and causing chronic inflammation. Here, we have exploited the lipid metabolism to engineer adipocytes that serve as a depot to deliver cancer therapeutics at the tumor site. Rumenic acid (RA), as an anticancer fatty acid, and a doxorubicin prodrug (pDox) with a reactive oxygen species (ROS)-cleavable linker, are encapsulated in adipocytes to deliver therapeutics in a tumor-specific bioresponsive manner. After intratumoral or postsurgical administration, lipolysis releases the RA and pDox that is activated by intracellular ROS-responsive conversion, subsequently promoting antitumor efficacy. Furthermore, downregulation of PD-L1 expression is observed in tumor cells, favoring the emergence of CD4+ and CD8+ T cell-mediated immune responses.