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Purpose: The addition of immune checkpoint blockade (ICB) to platinum/etoposide chemotherapy changed the standard of care for small cell lung cancer (SCLC) treatment. However, ICB addition only modestly improved clinical outcomes, likely reflecting the high prevalence of an immunologically “cold” tumor microenvironment in SCLC, despite high mutational burden. Nevertheless, some patients clearly benefit from ICB and recent reports have associated clinical responses to ICB in SCLC with (i) decreased neuroendocrine characteristics and (ii) activation of NOTCH signaling. We previously showed that inhibition of the lysine-specific demethylase 1a (LSD1) demethylase activates NOTCH and suppresses neuroendocrine features of SCLC, leading us to investigate whether LSD1 inhibition would enhance the response to PD-1 inhibition in SCLC. Experimental Design: We employed a syngeneic immunocompetent model of SCLC, derived from a genetically engineered mouse model harboring Rb1/Trp53 inactivation, to investigate combining the LSD1 inhibitor bomedemstat with anti-PD-1 therapy. In vivo experiments were complemented by cell-based studies in murine and human models. Results: Bomedemstat potentiated responses to PD-1 inhibition in a syngeneic model of SCLC, resulting in increased CD8+ T-cell infiltration and strong tumor growth inhibition. Bomedemstat increased MHC class I expression in mouse SCLC tumor cells in vivo and augmented MHC-I induction by IFNγ and increased killing by tumor-specific T cells in cell culture. Conclusions: LSD1 inhibition increased MHC-I expression and enhanced responses to PD-1 inhibition in vivo, supporting a new clinical trial to combine bomedemstat with standard-of-care PD-1 axis inhibition in SCLC. more...

Purpose:The addition of immune checkpoint blockade (ICB) to platinum/etoposide chemotherapy changed the standard of care for small cell lung cancer (SCLC) treatment. However, ICB addition only modestly improved clinical outcomes, likely reflecting the high prevalence of an immunologically “cold” tumor microenvironment in SCLC, despite high mutational burden. Nevertheless, some patients clearly benefit from ICB and recent reports have associated clinical responses to ICB in SCLC with (i) decreased neuroendocrine characteristics and (ii) activation of NOTCH signaling. We previously showed that inhibition of the lysine-specific demethylase 1a (LSD1) demethylase activates NOTCH and suppresses neuroendocrine features of SCLC, leading us to investigate whether LSD1 inhibition would enhance the response to PD-1 inhibition in SCLC.Experimental Design:We employed a syngeneic immunocompetent model of SCLC, derived from a genetically engineered mouse model harboring Rb1/Trp53 inactivation, to investigate combining the LSD1 inhibitor bomedemstat with anti-PD-1 therapy. In vivo experiments were complemented by cell-based studies in murine and human models.Results:Bomedemstat potentiated responses to PD-1 inhibition in a syngeneic model of SCLC, resulting in increased CD8+ T-cell infiltration and strong tumor growth inhibition. Bomedemstat increased MHC class I expression in mouse SCLC tumor cells in vivo and augmented MHC-I induction by IFNγ and increased killing by tumor-specific T cells in cell culture.Conclusions:LSD1 inhibition increased MHC-I expression and enhanced responses to PD-1 inhibition in vivo, supporting a new clinical trial to combine bomedemstat with standard-of-care PD-1 axis inhibition in SCLC. more...

Supplementary Figure from Inhibition of LSD1 with Bomedemstat Sensitizes Small Cell Lung Cancer to Immune Checkpoint Blockade and T-Cell Killing

Angiogenesis enhances cancer metastasis and progression, however, the roles of transcription regulation in angiogenesis are not fully defined. ZNF322A is an oncogenic zinc-finger transcription factor. Here, we demonstrate a new mechanism of Kras mutation-driven ZNF322A transcriptional activation and elucidate the interplay between ZNF322A and its upstream transcriptional regulators and downstream transcriptional targets in promoting neo-angiogenesis. Methods: Luciferase activity, RT-qPCR and ChIP-qPCR assays were used to examine transcription regulation in cell models. In vitro and in vivo angiogenesis assays were conducted. Immunohistochemistry, Kaplan-Meier method and multivariate Cox regression assays were performed to examine the clinical correlation in tumor specimens from lung cancer patients. Results: We validated that Yin Yang 1 (YY1) upregulated ZNF322A expression through targeting its promoter in the context of Kras mutation. Reconstitution experiments by knocking down YY1 under KrasG13V activation decreased KrasG13V-promoted cancer cell migration, proliferation and ZNF322A promoter activity. Knockdown of YY1 or ZNF322A attenuated angiogenesis in vitro and in vivo. Notably, we validated that ZNF322A upregulated the expression of sonic hedgehog (Shh) gene which encodes a secreted factor that activates pro-angiogenic responses in endothelial cells. Clinically, ZNF322A protein expression positively correlated with Shh and CD31, an endothelial cell marker, in 133 lung cancer patient samples determined using immunohistochemistry analysis. Notably, patients with concordantly high expression of ZNF322A, Shh and CD31 correlated with poor prognosis. Conclusions: These findings highlight the mechanism by which dysregulation of Kras/YY1/ZNF322/Shh transcriptional axis enhances neo-angiogenesis and cancer progression in lung cancer. Therapeutic strategies that target Kras/YY1/ZNF322A/Shh signaling axis may provide new insight on targeted therapy for lung cancer patients. more...

Single‑cell RNA sequencing (scRNA‑seq) of bone marrow or peripheral blood samples from patients with acute myeloid leukemia (AML) enables the characterization of heterogeneous malignant cells. A total of 87 cells from two patients with t(8;21) AML were analyzed using scRNA‑seq. Clustering methods were used to separate leukemia cells into different sub‑populations, and the expression patterns of specific marker genes were used to annotate these populations. Among the 31 differentially expressed genes in the cells of a patient who relapsed after hematopoietic stem cell transplantation, 13 genes were identified to be associated with leukemia. Furthermore, three genes, namely AT‑rich interaction domain 2, lysine methyltransferase 2A and synaptotagmin binding cytoplasmic RNA interacting protein were validated as possible prognostic biomarkers using two bulk expression datasets. Taking advantage of scRNA‑seq, the results of the present study may provide clinicians with several possible biomarkers to predict the prognostic outcomes of t(8;21) AML. more...

Development of new inhibitors targeting histone deacetylases (HDACs) with improved efficacy for solid tumor therapy is urgently needed. Here, we report the development of a novel HDAC inhibitor TMU-35435 and verify it as a single agent and in combination treatment with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza-dC) in lung cancer preclinical models. TMU-35435 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. Expression microarrays revealed a unique TMU-35435-induced gene networks enriched in biological processes, including "negative regulation of cell proliferation" and "Wnt receptor signaling pathway" compared to FDA-approved HDAC inhibitor SAHA. TMU-35435 inhibited tumor growth with good pharmacokinetic properties and safety features in lung orthotopic and subcutaneously implanted xenograft models. TMU-35435 and 5-aza-dC showed synergistic antitumor effects through reactivation of tumor suppressor genes and those genes encoding negative regulators of Wnt signaling pathway in vitro and in vivo. Some genes showed additive inhibition of DNA methylation upon TMU-35435 and 5-aza-dC combined treatment. Our findings suggested that TMU-35435 is a potential HDAC inhibitor for lung cancer treatment as a single agent and in combination with 5-aza-dC. more...

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models. more...

Growth-arrest-specific 7 (GAS7) belongs to a group of adaptor proteins that coordinate the actin cytoskeleton. Among human GAS7 isoforms, only GAS7C possesses a Src homology 3 domain. We report here that GAS7C acts as a migration suppressor and can serve as a prognostic biomarker in lung cancer. GAS7C overexpression reduces lung cancer migration, whereas GAS7C knockdown enhances cancer cell migration. Importantly, ectopically overexpressed GAS7C binds tightly with N-WASP thus inactivates the fibronectin/integrin/FAK pathway, which in turn leads to the suppression of F-actin dynamics. In addition, overexpression of GAS7C sequesters hnRNP U and thus decreases the level of β-catenin protein via the β-TrCP ubiquitin-degradation pathway. The anti-metastatic effect of GAS7C overexpression was also confirmed using lung cancer xenografts. Our clinical data indicated that 23.6% (25/106) of lung cancer patients showed low expression of GAS7C mRNA which correlated with a poorer overall survival. In addition, low GAS7C mRNA expression was detected in 60.0% of metastatic lung cancer patients, indicating an association between low GAS7C expression and cancer progression. A significant inverse correlation between mRNA expression and promoter hypermethylation was also found, which suggests that the low level of GAS7C expression was partly due to promoter hypermethylation. Our results provide novel evidence that low GAS7C correlates with poor prognosis and promotes metastasis in lung cancer. Low GAS7C increases cancer cell motility by promoting N-WASP/FAK/F-actin cytoskeleton dynamics. It also enhances β-catenin stability via hnRNP U/β-TrCP complex formation. Therefore, GAS7C acts as a metastasis suppressor in lung cancer. more...

e14152 Background: Immune checkpoint blockade (ICB) targeted to CTLA-4, PD-1, and PD-L1 can promote antitumor T cell immunity and clinical responses. Tracking the dynamic changes of the T Cell Receptor (TCR) Immune Repertoire can reflect the evolution of the immune system in tumor immunotherapy patients, predict the immune response to ICB, and provide high-value information for clinical monitoring of immune function status of cancer patients to make effective clinical decisions. The purpose of this study was to explore the correlation between the peripheral blood TCR repertoire dynamic changes and the immune response of patients with anti-PD-1/PD-L1, and to accurately guide the individualized treatment of immunotherapy. Methods: We sequenced the TCRβ genes from T cells contained in peripheral blood mononuclear cells (PBMCs) from 24 advanced solid tumors patients (PD/7, PR/4, SD/13) treated with immune checkpoint blockade. Peripheral blood samples were collected from baseline, 8-week, and 16-week after treatment for each patient. bioinformatic analysis was used to detect the diversity and clonality changes of TCRβ-CDR3 sequence before and after treatment with ICB in patients, and provide auxiliary technology for further guiding clinical tumor immunotherapy. Results: The analysis result of Chao index showed that the TCR-CDR3 diversity in the PR group was significantly increased after treatment than SD or PD group. (pvalue = 0.001), and the number of CDR3 aa clonotypes also increased significantly (pvalue = 0.012). A total of 61 Vβ gene fragments and 15 Jβ gene fragments were uniquely identified during immunotherapy . There was a significant difference in the number of Vβ gene fragments between baseline samples and after treatment in PR group (pvalue = 0.042). Conclusions: In this project, PBMC samples was used to evaluate the changes of TCR repertoire between before and after treatment with immune checkpoint blockade in advanced solid tumor patients. The immune function status was significantly changed before and after treatment. Patients with PR had significantly increased diversity in TCR repertoire than patients with SD or PD. The results provide T cell receptor β-chain repertoire sequencing and analysis has potential to assess whether immune checkpoint blockade promotes global T cell expansion and/or loss of T cell clonotype diversity, and to identify predictive biomarker for the use of immune checkpoint blockade. more...

Development of new inhibitors targeting histone deacetylases (HDACs) with improved efficacy for solid tumor therapy is urgently needed. Here, we report the development of a novel HDAC inhibitor TMU-35435 and verify it as a single agent and in combination treatment with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza-dC) in lung cancer preclinical models. TMU-35435 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. Expression microarrays revealed a unique TMU-35435-induced gene networks enriched in biological processes, including "negative regulation of cell proliferation" and "Wnt receptor signaling pathway" compared to FDA-approved HDAC inhibitor SAHA. TMU-35435 inhibited tumor growth with good pharmacokinetic properties and safety features in lung orthotopic and subcutaneously implanted xenograft models. TMU-35435 and 5-aza-dC showed synergistic antitumor effects through reactivation of tumor suppressor genes and those genes encoding negative regulators of Wnt signaling pathway in vitro and in vivo. Some genes showed additive inhibition of DNA methylation upon TMU-35435 and 5-aza-dC combined treatment. Our findings suggested that TMU-35435 is a potential HDAC inhibitor for lung cancer treatment as a single agent and in combination with 5-aza-dC. more...

Overexpression of Cys2His2 zinc-finger 322A (ZNF322A) oncogenic transcription factor is associated with lung tumorigenesis. However, the mechanism of ZNF322A overexpression remains poorly understood. Here, we discover that protein stability of ZNF322A is regulated by coordinated phosphorylation and ubiquitination through the CK1δ/GSK3β/FBXW7α axis. CK1δ and GSK3β kinases sequentially phosphorylate ZNF322A at serine-396 and then serine-391. Moreover, the doubly phosphorylated ZNF322A protein creates a destruction motif for the ubiquitin ligase FBXW7α leading to ZNF322A protein destruction. Overexpression of FBXW7α induces ZNF322A protein degradation, thereby blocks ZNF322A transcription activity and suppresses ZNF322A-induced tumor growth and metastasis in vitro and in vivo. Clinically, overexpression of ZNF322A correlates with low FBXW7α or defective CK1δ/GSK3β-mediated phosphorylation in lung cancer patients. Multivariate Cox regression analysis indicates that patients with ZNF322A high/FBXW7 low expression profile can be used as an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of ZNF322A oncoprotein destruction regulated by the CK1δ/GSK3β/FBXW7α axis. Deregulation of this signaling axis results in ZNF322A overexpression and promotes cancer progression. more...

An alternative approach for robust electromagnetic pulse (EMP) protection circuit was proposed by using a parallel coupled band-pass fllter (BPF) with high thermal conductivity alumina nitride (AlN) substrate coupled with a traditional gas discharge tube (GDT) and fast response metal oxide varistor (MOV). This proposed conflguration can suppress slow as well as fast voltage surges. The fabricated BPF with a center frequency of 2.5GHz on the high thermal conductive (180 » 200W/m¢K) AlN substrate could e-ciently suppress high power over voltage surge. Through the purposed cascade protection conflguration, it is observed that 6kV electrostatic discharge (ESD) pulse (5ns/50ns) and 4kV lightning surge pulse (1"s/50"s) were attenuated to 511V and 396V, respectively, and that is capable to be applied to an EMP protection circuit in the front end of a linear amplifler applications. more...

Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGS cells' invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer. more...

Polyphenols are natural antioxidants that are thought to contribute to prevention of cardiovascular disease and malignancy. Although many studies have been carried out to investigate the chemopreventive role of flavonoids, less attention has been focused on phenolic acids. In this study, the aim was to investigate the effect of phenolic acids found abundantly in vegetables, i.e. gallic acid (GA), caffeic acid (CA) and protocatechuic acid (PCA), on the inhibition of gastric adenocarcinoma (AGS) cell metastasis. The results showed 0.01 mM GA induced the same level of cell toxicity as 4.0mM PCA. Using wound-healing assay and Boyden chamber assay, GA had potent inhibitory effects on AGS cell migration. The expression of MMP-2/9 of AGS cells was inhibited by 2.0 microM of GA. It is possible that the suppressive effect of GA on MMP-2/9 might involve the inhibition of NF-kappaB activity. Multiple proteins involved in metastasis and the cytoskeletal reorganization signal pathway, including Ras, Cdc42, Rac1, RhoA, RhoB, PI3K and p38MAPK, were also inhibited by GA. Furthermore, immunoreactivity assay of cytoskeletal F-actin demonstrated a significant inhibitory effect of GA treatment. In conclusion, GA may have the potential to be an effective agent for prevention and treatment of gastric cancer metastasis. more...