KDM6A Regulates Immune Response Genes in Multiple Myeloma.

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration.

KDM6A Regulates Immune Response Genes in Multiple Myeloma.

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration. Statement of significance: We show that KDM6A participates in immune recognition of myeloma tumor cells by directly regulating the expression of the master regulators of MHC-I and II, NLRC5 and CIITA. The expression of these regulators can by rescued by the HDAC3 inhibitors in KDM6A-null cell lines.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

MicroRNA-940 as a Potential Serum Biomarker for Prostate Cancer.

Prostate cancer is one of the leading causes of death despite an astoundingly high survival rate for localized tumors. Though prostate specific antigen (PSA) test, performed in conjunction with digital rectal examinations, is reasonably accurate, there are major caveats requiring a thorough assessment of risks and benefits prior to conducting the test. MicroRNAs, a class of small non-coding RNAs, are stable molecules that can be detected in circulation by non-invasive methods and have gained importance in cancer prognosis and diagnosis in the recent years. Here, we investigate circulating miR-940, a miRNA known to play a role in prostate cancer progression, in both cell culture supernatants as well as patient serum and urine samples to determine the utility of miR-940 as a new molecular marker for prostate cancer detection. We found that miR-940 was significantly higher in serum from cancer patients, specifically those with clinically significant tumors (GS ≥ 7). Analysis of receiver operating characteristic curve demonstrated that miR-940 in combination with PSA had a higher area under curve value (AUC: 0.818) than the miR-940 alone (AUC: 0.75) for the diagnosis of prostate cancer. This study provides promising results suggesting the use of miR-940 for prostate cancer diagnosis.

Next-generation CAR T cells to overcome current drawbacks.

As a rapidly emerging treatment in the oncology field, adoptive transfer of autologous, genetically modified chimeric antigen receptor (CAR) T cells has shown striking efficacy and is curative in certain relapsed/refractory patients with hematologic malignancy. This treatment modality of using a "living drug" offers many tantalizing and novel therapeutic strategies for cancer patients whose remaining treatment options may have otherwise been limited. Despite the early success of CAR T cells in hematologic malignancies, many barriers remain for widespread adoption. General barriers include cellular manufacturing limitations, baseline quality of the T cells, adverse events post-infusion such as cytokine release syndrome (CRS) and neurotoxicity, and host rejection of non-human CARs. Additionally, each hematologic disease presents unique mechanisms of relapse which have to be addressed in future clinical trials if we are to augment the efficacy of CAR T treatment. In this review, we will describe current barriers to hindering efficacy of CAR T-cell treatment for hematologic malignancies in a disease-specific manner and review recent innovations aimed at enhancing the potency and applicability of CAR T cells, with the overall goal of building a framework to begin incorporating this form of therapy into the standard medical management of blood cancers.

Clinical Significance of Annexin A2 Expression in Breast Cancer Patients.

Increasing evidence suggests that AnxA2 contributes to invasion and metastasis of breast cancer. However, the clinical significance of AnxA2 expression in breast cancer has not been reported. The expression of AnxA2 in cell lines, tumor tissues, and serum samples of breast cancer patients were analyzed by immunoblotting, immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. We found that AnxA2 was significantly upregulated in tumor tissues and serum samples of breast cancer patients compared with normal controls. The high expression of serum AnxA2 was significantly associated with tumor grades and poor survival of the breast cancer patients. Based on molecular subtypes, AnxA2 expression was significantly elevated in tumor tissues and serum samples of triple-negative breast cancer (TNBC) patients compared with other breast cancer subtypes. Our analyses on breast cancer cell lines demonstrated that secretion of AnxA2 is associated with its tyrosine 23 (Tyr23) phosphorylation in cells. The expression of non-phosphomimetic mutant of AnxA2 in HCC1395 cells inhibits its secretion from cells compared to wild-type AnxA2, which further suggest that Tyr23 phosphorylation is a critical step for AnxA2 secretion from TNBC cells. Our analysis of AnxA2 phosphorylation in clinical samples further confirmed that the phosphorylation of AnxA2 at Tyr23 was high in tumor tissues of TNBC patients compared to matched adjacent non-tumorigenic breast tissues. Furthermore, we observed that the diagnostic value of serum AnxA2 was significantly high in TNBC compared with other breast cancer subtypes. These findings suggest that serum AnxA2 concentration could be a potential diagnostic biomarker for TNBC patients.

Correction to: Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis.

After publication of the original article [1], we were notified that the wrong version of Fig. 2b has been published.

Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis.

BACKGROUND: Limited information is available on biomarker(s) for triple-negative breast cancer (TNBC) that can address the higher incidence and aggressiveness of TNBC in African-American (AA) women. Our previous studies have demonstrated annexin A2 (AnxA2) association with exosomes which promotes angiogenesis and metastasis. Therefore, our goal was to examine the expression and function of exosomal-annexin A2 (exo-AnxA2) derived from the serum samples of breast cancer patients. METHODS: The expression of serum exo-AnxA2 and its association with clinicopathological features of the breast cancer patients were determined. The role of serum exo-AnxA2 to promote angiogenesis was determined by an in vivo Matrigel plug assay. RESULTS: Our results show that the expression of serum exo-AnxA2 in breast cancer patients (n = 169; 83.33 ± 2.040 ng/mL, P < 0.0001) is high compared to non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). High expression of exo-AnxA2 levels in breast cancer was significantly associated with tumor grade (P < 0.0001), poor overall survival (hazard ratio (HR) 2.802; 95% confidence intervals (CI) = 1.030-7.620; P = 0.0353), and poor disease-free survival (HR 7.934; 95% CI = 1.778-35.398; P = 0.0301). The expression of serum exo-AnxA2 levels was significantly elevated in TNBC (n = 68; 109.1 ± 2.905 ng/mL; P < 0.0001) in comparison to ER+ (n = 50; 57.35 ± 1.545 ng/mL), HER2+ (n = 59; 78.25 ± 1.146 ng/mL), and non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). Exo-AnxA2 showed diagnostic values with a maximum AUC as 1.000 for TNBC, 0.8304 for ER+, and 0.9958 for HER2+ compared to non-cancer females. The expression of serum exo-AnxA2 was significantly elevated in AA women with TNBC (n = 29; 118.9 ± 4.086 ng/mL, P < 0.0001) in comparison to Caucasian-American TNBC (n = 27; 97.60 ± 3.298 ng/mL) patients. Our in vivo results suggest a role of serum exo-AnxA2 in angiogenesis and its association with aggressiveness of TNBC in AA women. CONCLUSIONS: Our results demonstrated that the expression of serum exo-AnxA2 is high in AA women with TNBC and promotes angiogenesis. These findings suggest that exo-AnxA2 holds promise as a potential prognosticator of TNBC and may lead to an effective therapeutic option.

UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition.

Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Extracellular vesicles in liver diseases.

Extracellular vesicles (EVs) are membrane-bound vesicles that are released by cells into their extracellular environment, have selective enrichment of specific proteins and RNA, and can mediate intercellular communication. In this review we highlight recent observations of the role of EVs in liver injury, viral hepatitis, alcoholic or nonalcoholic liver disease, biliary tract disease, and liver cancers. Potential applications as markers of diseases or for therapeutic applications are outlined to emphasize the new opportunities that are arising from the study of EVs.

BAP1 dependent expression of long non-coding RNA NEAT-1 contributes to sensitivity to gemcitabine in cholangiocarcinoma.

BACKGROUND: Genetic alterations in chromatin modulators such as BRCA-1 associated protein-1 (BAP1) are the most frequent genetic alteration in intrahepatic cholangiocarcinomas (CCA). We evaluated the contribution of BAP1 expression on tumor cell behavior and therapeutic sensitivity to identify rationale therapeutic strategies. METHODS: The impact of BAP1 expression on sensitivity to therapeutic agents was evaluated in CCA cells with a 7-fold difference in BAP1 expression (KMBC-low, HuCCT1-high) and genetically engineered haplo-insufficient BAP1 knockout cells. We also identified long non-coding RNA genes associated with loss of BAP1 and their role in therapeutic sensitivity. RESULTS: Sensitivity to gemcitabine was greater in low BAP1 expressing or BAP1 knockout cells compared with the high BAP1 expressing cells or control haplo-insufficient cells respectively. Similar results were observed with TSA, olaparib, b-AP15 but not with GSK126. A differential synergistic effect was observed in combinations of gemcitabine with olaparib or GSK126 in KMBC cells and TSA or bAP15 in HuCCT1 cells, indicating BAP1 dependent target-specific synergism and sensitivity to gemcitabine. A BAP1 dependent alteration in expression of lncRNA NEAT-1 was identified by RT-PCR based lncRNA expression profiling, and an inverse relationship between this lncRNA and BAP1 was observed in analysis of the Tumor Cancer Genome Atlas cholangiocarcinoma dataset. Exogenous modulation of NEAT-1 and/or BAP1 expression altered tumor cell phenotype and modulated sensitivity to gemcitabine. CONCLUSIONS: NEAT-1 is a downstream effector of gemcitabine sensitivity in CCA. The expression of BAP1 is a determinant of sensitivity to therapeutic drugs that can be exploited to enhance responses through combination strategies.

In vitro toxicology studies of extracellular vesicles.

Extracellular vesicles (EVs) are membrane-bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC-EV) or from bovine milk (BM-EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC-EVs nor BM-EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM-EVs but not with MSC-EVs. MSC-EVs did not have any significant effect on either spontaneous or collagen-induced platelet aggregation. In contrast, BM-EVs were noted to increase collagen-induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM-EV. Neither MSC-EVs nor BM-EVs induced HL-60 phagocytosis, although BM-EVs decreased zymosan-induced phagocytosis. Furthermore, neither MSC-EVs nor BM-EVs induced nitric oxide production. Unlike MSC-EVs, BM-EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC-EVs and BM-EVs that may reflect variations in techniques for EV isolation, EV content or cross-species differences. The safety of MSC-EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM-EVs. Copyright © 2016 John Wiley & Sons, Ltd.

Exosomal Annexin II Promotes Angiogenesis and Breast Cancer Metastasis.

Tumor-derived exosomes are emerging mediators of tumorigenesis and tissue-specific metastasis. Proteomic profiling has identified Annexin II as one of the most highly expressed proteins in exosomes; however, studies focused on the biological role of exosomal Annexin II (exo-Anx II) are still lacking. In this study, mechanistic insight was sought regarding exo-Anx II and its function in angiogenesis and breast cancer metastasis. Multiple in vitro and in vivo techniques were used to study the role of exo-Anx II in angiogenesis. Using atomic force microscopy and Western blotting, exo-Anx II expression was characterized in normal and breast cancer cells. In addition, organ-specific metastatic breast cancer cells and animal models were used to define the role exo-Anx II in breast cancer metastasis. Results revealed that exo-Anx II expression is significantly higher in malignant cells than normal and premetastatic breast cancer cells. In vitro and in vivo studies demonstrated that exo-Anx II promotes tPA-dependent angiogenesis. Furthermore, in vivo analysis indicated that metastatic exosomes create a favorable microenvironment for metastasis, and exo-Anx II plays an important role in this process, as priming with Anx II-depleted exosomes reduces brain (∼4-fold) and lung (∼2-fold) metastasis. Upon delineating the mechanism, it was discovered that exo-Anx II causes macrophage-mediated activation of the p38MAPK, NF-κB, and STAT3 pathways and increased secretion of IL6 and TNFα. These data demonstrate an important role for exo-Anx II in breast cancer pathogenesis. IMPLICATIONS: Exosome-associated Annexin II plays an important role in angiogenesis and breast cancer metastasis, which can be exploited as a potential biomarker as well as a therapeutic target for diagnosis and treatment of metastatic breast cancer. Mol Cancer Res; 15(1); 93-105. ©2016 AACR.

Long non-coding RNAs as novel targets for therapy in hepatocellular carcinoma.

The recognition of functional roles for transcribed long non-coding RNA (lncRNA) has provided a new dimension to our understanding of cellular physiology and disease pathogenesis. LncRNAs are a large group of structurally complex RNA genes that can interact with DNA, RNA, or protein molecules to modulate gene expression and to exert cellular effects through diverse mechanisms. The emerging knowledge regarding their functional roles and their aberrant expression in disease states emphasizes the potential for lncRNA to serve as targets for therapeutic intervention. In this concise review, we outline the mechanisms of action of lncRNAs, their functional cellular roles, and their involvement in disease. Using liver cancer as an example, we provide an overview of the emerging opportunities and potential approaches to target lncRNA-dependent mechanisms for therapeutic purposes.

Rationalizing the use of functionalized poly-lactic-co-glycolic acid nanoparticles for dendritic cell-based targeted anticancer therapy.

BACKGROUND: Delivery of PLGA (poly [D, L-lactide-co-glycolide])-based biodegradable nanoparticles (NPs) to antigen presenting cells, particularly dendritic cells, has potential for cancer immunotherapy. MATERIALS & METHODS: Using a PLGA NP vaccine construct CpG-NP-Tag (CpG-ODN-coated tumor antigen [Tag] encapsulating NP) prepared using solvent evaporation technique we tested the efficacy of ex vivo and in vivo use of this construct as a feasible platform for immune-based therapy. RESULTS: CpG-NP-Tag NPs were avidly endocytosed and localized in the endosomal compartment of bone marrow-derived dendritic cells. Bone marrow-derived dendritic cells exposed to CpG-NP-Tag NPs exhibited an increased maturation (higher CD80/86 expression) and activation status (enhanced IL-12 secretion levels). In vivo results demonstrated attenuation of tumor growth and angiogenesis as well as induction of potent cytotoxic T-lymphocyte responses. CONCLUSION: Collectively, results validate dendritic cells stimulatory response to CpG-NP-Tag NPs (ex vivo) and CpG-NP-Tag NPs' tumor inhibitory potential (in vivo) for therapeutic applications, respectively.

MIEN1 promotes oral cancer progression and implicates poor overall survival.

Oral squamous cell carcinoma is a highly malignant tumor with the potential to invade local and distant sites and promote lymph node metastasis. Major players underlying the molecular mechanisms behind tumor progression are yet to be fully explored. Migration and invasion enhancer 1 (MIEN1), a novel protein overexpressed in various cancers, facilitates cell migration and invasion. In the present study we investigated the expression and role of MIEN1 in oral cancer progression using an in vitro model, patient derived oral tissues and existing TCGA data. Expression analysis using immortalized normal and cancer cells demonstrated increased expression of MIEN1 in cancer. Assays performed after MIEN1 knockdown in OSC-2 cells showed decreased migration, invasion and filopodia formation; while MIEN1 overexpression in DOK cells increased these characteristics and also up-regulated some Akt/NF-κB effectors, thereby suggesting an important role for MIEN1 in oral cancer progression. Immunohistochemical staining and analyses of oral tissue specimens, collected from patients over multiple visits, revealed significantly more staining in severe dysplasia and squamous cell carcinoma compared to mildly dysplastic or hyperplastic tissues. Finally, this was corroborated with the TCGA dataset, where MIEN1 expression was not only higher in intermediate and high grade cancer with significantly lower survival but also correlated with smoking. In summary, we demonstrate that MIEN1 expression not only positively correlates with oral cancer progression but also seems to be a critical molecular determinant in migration and invasion of oral cancer cells, thereby, playing a possible role in their metastatic dissemination.

Cell surface translocation of annexin A2 facilitates glutamate-induced extracellular proteolysis.

Glutamate-induced elevation in intracellular Ca(2+) has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca(2+)-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca(2+) imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca(2+) influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes.