Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.

Mechanisms Controlling PD-L1 Expression in Cancer.

The engagement of programmed cell death protein 1 (PD-1; encoded by the PDCD1 gene) receptor expressed on activated T cells and its ligand, programmed death-ligand 1 (PD-L1; encoded by the CD274 gene), is a major co-inhibitory checkpoint signaling that controls T cell activities. Various types of cancers express high levels of PD-L1 and exploit PD-L1/PD-1 signaling to evade T cell immunity. Blocking the PD-L1/PD-1 pathway has consistently shown remarkable anti-tumor effects in patients with advanced cancers and is recognized as the gold standard for developing new immune checkpoint blockade (ICB) and combination therapies. However, the response rates of anti-PD-L1 have been limited in several solid tumors. Therefore, furthering our understanding of the regulatory mechanisms of PD-L1 can bring substantial benefits to patients with cancer by improving the efficacy of current PD-L1/PD-1 blockade or other ICBs. In this review, we provide current knowledge of PD-L1 regulatory mechanisms at the transcriptional, posttranscriptional, post-translational, and extracellular levels, and discuss the implications of these findings in cancer diagnosis and immunotherapy.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer.

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

Ribonuclease 7-driven activation of ROS1 is a potential therapeutic target in hepatocellular carcinoma.

BACKGROUND & AIMS: There are currently limited therapeutic options for hepatocellular carcinoma (HCC), particularly when it is diagnosed at advanced stages. Herein, we examined the pathophysiological role of ROS1 and assessed the utility of ROS1-targeted therapy for the treatment of HCC. METHODS: Recombinant ribonucleases (RNases) were purified, and the ligand-receptor relationship between RNase7 and ROS1 was validated in HCC cell lines by Duolink, immunofluorescence, and immunoprecipitation assays. Potential interacting residues between ROS1 and RNase7 were predicted using a protein-protein docking approach. The oncogenic function of RNase7 was analyzed by cell proliferation, migration and invasion assays, and a xenograft mouse model. The efficacy of anti-ROS1 inhibitor treatment was evaluated in patient-derived xenograft (PDX) and orthotopic models. Two independent patient cohorts were analyzed to evaluate the pathological relevance of RNase7/ROS1. RESULTS: RNase7 associated with ROS1's N3-P2 domain and promoted ROS1-mediated oncogenic transformation. Patients with HCC exhibited elevated plasma RNase7 levels compared with healthy individuals. High ROS1 and RNase7 expression were strongly associated with poor prognosis in patients with HCC. In both HCC PDX and orthotopic mouse models, ROS1 inhibitor treatment markedly suppressed RNase7-induced tumorigenesis, leading to decreased plasma RNase7 levels and tumor shrinkage in mice. CONCLUSIONS: RNase7 serves as a high-affinity ligand for ROS1. Plasma RNase7 could be used as a biomarker to identify patients with HCC who may benefit from anti-ROS1 treatment. LAY SUMMARY: Receptor tyrosine kinases are known to be involved in tumorigenesis and have been targeted therapeutically for a number of cancers, including hepatocellular carcinoma. ROS1 is the only such receptor with kinase activity whose ligand has not been identified. Herein, we show that RNase7 acts as a ligand to activate ROS1 signaling. This has important pathophysiological and therapeutic implications. Anti-ROS1 inhibitors could be used to treatment patients with hepatocellular carcinoma and high RNase7 levels.

Thymosin beta-4 knockdown in IEC-6 normal intestinal epithelial cells induces DNA re-replication via downregulating Emi1.

Thymosin ß4 (Tß4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tß4 alteration influences normal intestinal epithelial cells because Tß4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tß4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tß4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tß4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3ß (GSK-3ß). To our best knowledge, this is the first report showing that inhibiting Tß4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tß4 .

Reshaping the Tumor Microenvironment of KRASG12D Pancreatic Ductal Adenocarcinoma with Combined SOS1 and MEK Inhibition for Improved Immunotherapy Response.

KRAS inhibitors have demonstrated exciting preclinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KrasG12D, p53-mutant, murine pancreatic ductal adenocarcinoma-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intratumoral CD8+ T cells without durable responses. Single-cell RNA sequencing revealed an increase in inflammatory cancer-associated fibroblasts (iCAF), M2 macrophages, and a decreased dendritic cell (DC) quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen-presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor-free mice with established immune memory. Our data suggest that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong antitumor effects. SIGNIFICANCE: Combination of SOS1 and MEK inhibitors increase T cell infiltration while blunting pro-immune myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.

Ribonuclease 1 Enhances Antitumor Immunity against Breast Cancer by Boosting T cell Activation.

The secretory enzyme human ribonuclease 1 (RNase1) is involved in innate immunity and anti-inflammation, achieving host defense and anti-cancer effects; however, whether RNase1 contributes to adaptive immune response in the tumor microenvironment (TME) remains unclear. Here, we established a syngeneic immunocompetent mouse model in breast cancer and demonstrated that ectopic RNase1 expression significantly inhibited tumor progression. Overall changes in immunological profiles in the mouse tumors were analyzed by mass cytometry and showed that the RNase1-expressing tumor cells significantly induced CD4+ Th1 and Th17 cells and natural killer cells and reduced granulocytic myeloid-derived suppressor cells, supporting that RNase1 favors an antitumor TME. Specifically, RNase1 increased expression of T cell activation marker CD69 in a CD4+ T cell subset. Notably, analysis of cancer-killing potential revealed that T cell-mediated antitumor immunity was enhanced by RNase1, which further collaborated with an EGFR-CD3 bispecific antibody to protect against breast cancer cells across molecular subtypes. Our results uncover a tumor-suppressive role of RNase1 through adaptive immune response in breast cancer in vivo and in vitro, providing a potential treatment strategy of combining RNase1 with cancer immunotherapies for immunocompetent patients.

Inhibition of Galectin-9 sensitizes tumors to anthracycline treatment via inducing antitumor immunity.

Anthracyclines are a class of conventionally and routinely used first-line chemotherapy drugs for cancer treatment. In addition to the direct cytotoxic effects, increasing evidence indicates that the efficacy of the drugs also depends on immunomodulatory effects with unknown mechanisms. Galectin-9 (Gal-9), a member of the ß-galactoside-binding protein family, has been demonstrated to induce T-cell death and promote immunosuppression in the tumor microenvironment. Here, we asked whether anthracycline-mediated immunomodulatory activity might be related to Gal-9. We found that combining doxorubicin with anti-Gal-9 therapy significantly inhibited tumor growth and prolonged overall survival in immune-competent syngeneic mouse models. Moreover, Gal-9 expression was increased in response to doxorubicin in various human and murine cancer cell lines. Mechanistically, doxorubicin induced tumoral Gal-9 by activating the STING/interferon ß pathway. Clinically, Gal-9 and p-STING levels were elevated in the tumor tissues of breast cancer patients treated with anthracyclines. Our study demonstrates Gal-9 upregulation in response to anthracyclines as a novel mechanism mediating immune escape and suggests targeting Gal-9 in combination with anthracyclines as a promising therapeutic strategy for cancer treatment.

Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

Phosphorylation and Stabilization of PD-L1 by CK2 Suppresses Dendritic Cell Function.

Targeting immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has transformed cancer treatment, with durable clinical responses across a wide range of tumor types. However, a high percentage of patients fail to respond to anti-PD-1/PD-L1 treatment. A greater understanding of PD-L1 regulation is critical to improving the clinical response rate of PD-1/PD-L1 blockade. Here, we demonstrate that PD-L1 is phosphorylated and stabilized by casein kinase 2 (CK2) in cancer and dendritic cells (DC). Phosphorylation of PD-L1 at Thr285 and Thr290 by CK2 disrupted PD-L1 binding with speckle-type POZ protein, an adaptor protein of the cullin 3 (CUL3) ubiquitin E3 ligase complex, protecting PD-L1 from CUL3-mediated proteasomal degradation. Inhibition of CK2 decreased PD-L1 protein levels by promoting its degradation and resulted in the release of CD80 from DC to reactivate T-cell function. In a syngeneic mouse model, combined treatment with a CK2 inhibitor and an antibody against T-cell immunoglobulin mucin-3 (Tim-3) suppressed tumor growth and prolonged survival. These findings uncover a mechanism by which PD-L1 is regulated and suggest a potential antitumor treatment option to activate DC function by blocking the CK2-PD-L1 pathway and inhibiting Tim-3. SIGNIFICANCE: This work identifies a role for CK2 in immunosuppression by phosphorylation and stabilization of PD-L1, identifying CK2 inhibition as an immunotherapeutic approach for treating cancer.

ATR-mediated CD47 and PD-L1 up-regulation restricts radiotherapy-induced immune priming and abscopal responses in colorectal cancer.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

Targeting the ALK-CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex.

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK-p-Tyr19-CDK9 expression.

TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis.

Immune checkpoint blockade therapy has demonstrated promising clinical outcomes for multiple cancer types. However, the emergence of resistance as well as inadequate biomarkers for patient stratification have largely limited the clinical benefits. Here, we showed that tumors with high TYRO3 expression exhibited anti-programmed cell death protein 1/programmed death ligand 1 (anti-PD-1/PD-L1) resistance in a syngeneic mouse model and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, TYRO3 inhibited tumor cell ferroptosis triggered by anti-PD-1/PD-L1 and facilitated the development of a protumor microenvironment by reducing the M1/M2 macrophage ratio, resulting in resistance to anti-PD-1/PD-L1 therapy. Inhibition of TYRO3 promoted tumor ferroptosis and sensitized resistant tumors to anti-PD-1 therapy. Collectively, our findings suggest that TYRO3 could serve as a predictive biomarker for patient selection and a promising therapeutic target to overcome anti-PD-1/PD-L1 resistance.

Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation.

Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis.

New Approaches on Cancer Immunotherapy.

Metastasis, which occurs when cancer cells disseminate from the primary tumor site to other parts of the body, is the primary cause of mortality in patients, and the recurrence of multiple metastatic tumors is an obstacle to eliminating cancer. Recent clinical studies demonstrated that patients who respond to immunotherapy have longer survival rates with lower metastatic relapse, suggesting that immunotherapy may be one of the solutions to overcome cancer metastasis. Indeed, various host immune cells not only shape the tumor microenvironment but also participate in multiple stages of metastasis. Therefore, to improve clinical outcome, it is critical to understand the immunological events associated with tumor development and progression. In this article, we summarize those events that are involved in tumor progression and discuss immunotherapies that can potentially target cancer metastasis.

The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

The immune checkpoint blockade therapy has emerged as encouraging treatment strategies in various cancer types. Anti-PD-L1 (programmed death-ligand 1) antibodies have been approved for triple-negative breast cancer, however the response rate yet to be optimized. It would be imperative to further understand and investigate the molecular mechanisms of PD-L1 regulation. Here, we identified glucose regulatory protein 78 (GRP78), a major endoplasmic reticulum (ER) stress responding protein, as a novel binding partner of PD-L1. GRP78 interacts with PD-L1 at the ER region and increases PD-L1 levels via regulating its stability. ER stress, triggered by different stimuli such as conventional chemotherapy, leads to the induction of PD-L1 in a GRP78-dependent manner. We showed that GRP78 modulates the response to chemotherapy, and dual-high levels of GRP78 and PD-L1 correlates with poor relapse-free survival in triple-negative breast cancer. Altogether, our study provides novel molecular insights into the regulatory mechanism of PD-L1 by revealing its interaction with GRP78, and offers a rationale to target GRP78 as a potential therapeutic strategy to enhance anti-tumor immunity.

Erratum: The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

[This corrects the article on p. 2621 in vol. 10, PMID: 32905506.].

Inhibition of c-MET upregulates PD-L1 expression in lung adenocarcinoma.

Non-small cell lung cancer (NSCLC) patients with c-MET dysregulation may benefit from c-MET inhibitors therapy as inhibition of c-MET activity has emerged as a therapeutic approach against this disease. Although several c-MET inhibitors have been evaluated in multiple clinical trials in lung cancer, their benefits so far have been modest. Thus, furthering our understanding of the mechanisms contributing to the lack of success of c-MET inhibitors in clinical trials is essential toward the development of rational and effective combination strategies. Here we show that exposure of NCSLC cell lines to c-MET inhibitor tivantinib increases their expression of PD-L1, which in turn causes cells to become more resistant to T-cell killing. Mechanistically, inhibition of c-MET suppresses p-GSK3ß, leading to the stabilization of PD-L1 similar to that observed in liver cancer cells. Collectively, our findings suggest a potential crosstalk between c-MET inhibition and immune escape and provide a rationale for the combination therapy of c-MET inhibitors and immune checkpoint blockade in NSCLC.