National Cancer Institute Collaborative Workshop on Shaping the Landscape of Brain Metastases Research: challenges and recommended priorities.

Brain metastases are an increasing global public health concern, even as survival rates improve for patients with metastatic disease. Both metastases and the sequelae of their treatment are key determinants of the inter-related priorities of patient survival, function, and quality of life, mandating a multidimensional approach to clinical care and research. At a virtual National Cancer Institute Workshop in September, 2022, key stakeholders convened to define research priorities to address the crucial areas of unmet need for patients with brain metastases to achieve meaningful advances in patient outcomes. This Policy Review outlines existing knowledge gaps, collaborative opportunities, and specific recommendations regarding consensus priorities and future directions in brain metastases research. Achieving major advances in research will require enhanced coordination between the ongoing efforts of individual organisations and consortia. Importantly, the continual and active engagement of patients and patient advocates will be necessary to ensure that the directionality of all efforts reflects what is most meaningful in the context of patient care.

The State of Preclinical Modeling for Early Phase Cancer Trials Using Molecularly Targeted Agents with Radiation.

Preclinical studies inform and guide the development of novel treatment combination strategies that bridge the laboratory with the clinic. We aimed to evaluate approaches cancer researchers used to justify advancing new combinations of molecularly targeted agents and radiation treatment into early-phase human clinical trials. Unsolicited early phase clinical trial proposals submitted to the National Cancer Institute's Cancer Therapy Evaluation Program between January 2016 and July 2020 were curated to quantify key characteristics and proportion of preclinical data provided by trialists seeking to conduct molecularly targeted agent-radiation combination studies in cancer patients. These data elucidate the current landscape for how the rationale for a molecularly targeted agent-radiation combination therapy is supported by preclinical research and illustrate unique challenges faced in translation at the intersection of precision medicine and radiation oncology.

Moving Forward in the Next Decade: Radiation Oncology Sciences for Patient-Centered Cancer Care.

In a time of rapid advances in science and technology, the opportunities for radiation oncology are undergoing transformational change. The linkage between and understanding of the physical dose and induced biological perturbations are opening entirely new areas of application. The ability to define anatomic extent of disease and the elucidation of the biology of metastases has brought a key role for radiation oncology for treating metastatic disease. That radiation can stimulate and suppress subpopulations of the immune response makes radiation a key participant in cancer immunotherapy. Targeted radiopharmaceutical therapy delivers radiation systemically with radionuclides and carrier molecules selected for their physical, chemical, and biochemical properties. Radiation oncology usage of "big data" and machine learning and artificial intelligence adds the opportunity to markedly change the workflow for clinical practice while physically targeting and adapting radiation fields in real time. Future precision targeting requires multidimensional understanding of the imaging, underlying biology, and anatomical relationship among tissues for radiation as spatial and temporal "focused biology." Other means of energy delivery are available as are agents that can be activated by radiation with increasing ability to target treatments. With broad applicability of radiation in cancer treatment, radiation therapy is a necessity for effective cancer care, opening a career path for global health serving the medically underserved in geographically isolated populations as a substantial societal contribution addressing health disparities. Understanding risk and mitigation of radiation injury make it an important discipline for and beyond cancer care including energy policy, space exploration, national security, and global partnerships.

National Cancer Institute support for targeted alpha-emitter therapy.

BACKGROUND: Radiopharmaceutical targeted therapy (RPT) has been studied for decades; however, recent clinical trials demonstrating efficacy have helped renewed interest in the modality. METHODS: This article reviews National Cancer Institute (NCI)'s support of RPT through communication via workshops and interest groups, through funding extramural programs in academia and small business, and through intramural research, including preclinical and clinical studies. RESULTS: NCI has co-organized workshops and organized interest groups on RPT and RPT dosimetry to encourage the community and facilitate rigorous preclinical and clinical studies. NCI has been supporting RPT research through various mechanisms. Research has been funded through peer-reviewed NCI Research and Program Grants (RPG) and NCI Small Business Innovation Research (SBIR) Development Center, which funds small business-initiated projects, some of which have led to clinical trials. The NCI Cancer Therapy Evaluation Program (CTEP)'s Radiopharmaceutical Development Initiative supports RPT in NCI-funded clinical trials, including Imaging and Radiation Oncology Core (IROC) expertise in imaging QA and dosimetry procedures. Preclinical targeted a-emitter therapy (TAT) research at the NCI's intramural program is ongoing, building on foundational work dating back to the 1980s. Ongoing "bench-to-bedside" efforts leverage the unique infrastructure of the National Institutes of Health's (NIH) Clinical Center. CONCLUSION: Given the great potential of RPT, our goal is to continue to encourage its development that will generate the high-quality evidence needed to bring this multidisciplinary treatment to patients.

Randomized phase II trial of a first-in-human cancer cell lysate vaccine in patients with thoracic malignancies.

BACKGROUND: Although most malignancies express cancer-testis antigens (CTA), immune responses to these proteins are limited in thoracic oncology patients. This trial was undertaken to examine if a cancer cell lysate vaccine could induce immunity to CTA, and to ascertain if metronomic cyclophosphamide and celecoxib enhances vaccine-induced immune responses. METHODS: Eleven patients with primary thoracic malignancies and 10 patients with extrathoracic neoplasms metastatic to the chest rendered NED by conventional therapies were randomized to receive H1299 lung cancer cell lysates (10 mg protein/vaccine) with Iscomatrix™ adjuvant via deep intradermal injection q 4 weeks ×6 with or without daily oral metronomic cyclophosphamide/celecoxib. The primary endpoint was serologic response to purified CTA assessed 1 month after the 6th vaccination. Secondary endpoints included assessment of the effects of cyclophosphamide and celecoxib on frequency and magnitude of vaccine-induced immune responses to CTA. Exploratory endpoints included evaluation of the effects of the vaccine regimens on peripheral immune subsets. Standard of care imaging studies were obtained at baseline and 1 month after the 3rd and 6th vaccinations. RESULTS: All patients exhibited local and systemic inflammatory responses lasting 72-96 hours following vaccinations. There were no dose limiting treatment related toxicities. Fourteen patients (67%) completed all six vaccinations. Eight of 14 patients (57%) exhibited serologic responses to NY-ESO-1. One patient developed antibodies to GAGE7; several patients exhibited reactivity to XAGE and MAGE-C2. Vaccine therapy decreased the percent of Tregs (P=0.0068), PD-1 expression on Tregs (P=0.0027), PD-L1 expression on CD14+ monocytes (P=0.0089), PD-L1 expression on classical monocytes (P=0.016), and PD-L1 expression on intermediate monocytes (P=0.0031). Cyclophosphamide/celecoxib did not appear to increase immune responses or enhance vaccine-induced alterations in peripheral immune subsets. CONCLUSIONS: H1299 lysate vaccines with Iscomatrix™ induce immune responses to CTA and modulate peripheral immune subsets in a manner that may enhance antitumor immunity in patients with thoracic malignancies.

UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural Mesothelioma.

INTRODUCTION: Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM. METHODS: Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression. RESULTS: UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM. CONCLUSIONS: UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

Severe Hepatotoxicity of Mithramycin Therapy Caused by Altered Expression of Hepatocellular Bile Transporters.

Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P < 0.05) and were associated with ≥grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day ×7, every 28 days; P < 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter α/ß) in several cell lines [Huh7, HepaRG, HepaRG BSEP (-/-)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P < 0.0001), and mithramycin inhibited chenodeoxycholic acid- and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P < 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (-/-) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P < 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT: The present study characterizes a novel mechanism of drug-induced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel single-nucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.

CD38 knockout suppresses tumorigenesis in mice and clonogenic growth of human lung cancer cells.

The ectodomain of the plasma membrane ectoenzyme CD38 functions as both an NAD glycohydrolase and an ADP-ribosyl cyclase by catalyzing, respectively, the conversion of NAD to nicotinamide and ADP-ribose or cyclic ADP-ribose. CD38 is attracting particular attention in cancer therapy. An anti-CD38 monoclonal antibody (daratumumab) was approved for treatment of patients with multiple myeloma. However, the role of CD38 in non-hematological malignancies has not been explored. Previously, we reported that ADP-ribose-acceptor hydrolase (ARH)-1 deficiency in mice was associated with tumor development. In the present study, we found that in wild-type and ARH1-deficient mice deletion of the CD38 gene reduced tumor formation. Significant reductions in tumor number were observed in lymphomas, adenocarcinomas and hemangio/histolytic sarcomas. Consistent with a role for CD38 in tumorigenesis, CRISPR/Cas9-based knockout of CD38 in A549 human adenocarcinoma cells inhibited anchorage-independent cell growth, cell invasion and xenograft growth in nude mice. CD38 mRNA and protein expression were evaluated in human lung cancer cell lines and in human lung cancer specimens. CD38 overexpression in tumor cells was identified in 11 of 27 patient samples. In addition, some human lung cancer cell lines had dramatically higher CD38 mRNA and protein expression than normal cells. Consistent with these observations, search of the Oncomine database showed that some human lung adenocarcinomas had higher CD38 mRNA levels compared to normal lung tissues. In total, our data are consistent with the conclusion that CD38 plays a role in murine and human lung tumorigenesis and that anti-CD38 treatment may have therapeutic potential in lung cancer.

ASXL3 Is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267-81. ©2017 AACR.

Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells.

PURPOSE: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms. EXPERIMENTAL DESIGN: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, ß-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters. RESULTS: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression. CONCLUSIONS: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma.

Pulmonary Metastases Exhibit Epigenetic Clonality: Implications for Precision Cancer Therapy.

BACKGROUND: Development of effective cancer therapies may be limited by intratumoral heterogeneity, which facilitates outgrowth and organ-specific dissemination of treatment resistant clones. At present, limited information is available regarding epigenetic landscapes of pulmonary metastases. This study was undertaken to characterize epigenetic signatures of pulmonary metastases and to identify potential therapeutic targets. METHODS: RNA and DNA were extracted from 65 pulmonary metastases resected from 12 patients (5 with sarcoma, 7 with adrenocortical carcinoma). Quantitative reverse transcription polymerase chain reaction techniques were used to evaluate expression levels of cancer-testis (CT) genes (NY-ESO-1, MAGE-A3, MAGE-A9, MAGE-A12, GAGE1, CT-45, SSX-1, and SSX-2), tumor suppressor (TS) genes (p16 and RASSF1A), and genes encoding epigenetic modifiers (DNMT1, DNMT3A, DNMT3B, EZH2, EED, and SUZ12), aberrantly expressed in human malignant diseases. Pyrosequencing techniques were used to quantitate DNA methylation levels in LINE1, NBL2, and D4Z4 repetitive sequences and promoter methylation status of differentially regulated genes. Results of these analyses were compared with a standardized panel of normal lung tissues. RESULTS: Pulmonary metastases exhibited histologically related and patient-specific global DNA demethylation. Significant interpatient heterogeneity of gene expression was observed even among patients with similar tumor histologic features. Epigenetic signatures appeared consistent among metastases from the same patient, irrespective of the time of resection (synchronous/metachronous) or the anatomic location. EZH2, EED, and SUZ12 (core components of Polycomb repressive complex-2 [PRC-2]) were upregulated in the majority of metastases. CONCLUSIONS: Pulmonary metastases exhibit patient-specific epigenetic clonality, which may be exploited for precision therapies targeting aberrant CT or TS gene expression. PRC-2 may be a shared target for epigenetic therapy of pulmonary metastases.

Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas.

BACKGROUND: Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. METHODS: Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and ß-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. RESULTS: Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-ß-catenin complex, markedly depleted ß-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. CONCLUSIONS: A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.

Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis.

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.

Mithramycin represses basal and cigarette smoke-induced expression of ABCG2 and inhibits stem cell signaling in lung and esophageal cancer cells.

Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.

Polycomb repressor complex-2 is a novel target for mesothelioma therapy.

PURPOSE: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM). EXPERIMENTAL DESIGN: Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep. RESULTS: EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment. CONCLUSIONS: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy.

Inhibition of histone lysine methylation enhances cancer-testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer.

Cancer-testis antigens (CTA), such as NY-ESO-1, MAGE-A1, and MAGE-A3, are immunogenic proteins encoded by genes, which are normally expressed only in male germ cells but are activated by ill-defined epigenetic mechanisms in human tumors, including lung cancers. Previously, we reported induction of these CTAs in cancer cells, but not normal cells, by DNA-demethylating agents and histone deacetylase inhibitors using clinically achievable exposure conditions. In the present study, we evaluated chromatin alterations associated with repression/activation of cancer-testis genes in lung cancer cells to further develop gene-induction regimens for cancer immunotherapy. Repression of NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment, and binding of polycomb-group proteins, and histone heterochromatin modifications within the promoters of these genes. Derepression coincided with DNA demethylation, dissociation of polycomb proteins, and presence of euchromatin marks within the respective promoters. Short hairpin RNAs were used to inhibit several histone methyltransferases (KMT) and histone demethylases (KDM) that mediate histone methylation and repress gene expression. Knockdown of KMT6, KDM1, or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated activation of these cancer-testis genes in lung cancer cells. DZNep, a pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6 knockdown. Following DAC-DZNep exposure, lung cancer cells were specifically recognized and lysed by allogeneic lymphocytes expressing recombinant T-cell receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA-demethylating agents with compounds, such as DZNep, that modulate histone lysine methylation may provide a novel epigenetic strategy to augment cancer-testis gene expression as an adjunct to adoptive cancer immunotherapy.