Tom Liu

CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth

Metastasis of solid tumors is associated with poor prognosis and bleak survival rates. Tumor-infiltrating myeloid cells (TIMs) are known to promote metastasis, but the mechanisms underlying their collaboration with tumor cells remain unknown. Here, we report an oncogenic role for microRNA (miR) in driving M2 reprogramming in TIMs, characterized by the acquisition of pro-tumor and pro-angiogenic properties. The expression of miR-21, miR-29a, miR-142-3p and miR-223 increased in myeloid cells during tumor progression in mouse models of breast cancer and melanoma metastasis. Further, we show that these miRs are regulated by the CSF1-ETS2 pathway in macrophages. A loss-of-function approach utilizing selective depletion of the miR-processing enzyme Dicer in mature myeloid cells blocks angiogenesis and metastatic tumor growth. Ectopic expression of miR-21 and miR-29a promotes angiogenesis and tumor cell proliferation through the downregulation of anti-angiogenic genes such as Col4a2, Spry1 and Timp3, whereas knockdown of the miRs impedes these processes. miR-21 and miR-29a are expressed in Csf1r+ myeloid cells associated with human metastatic breast cancer, and levels of these miRs in CD115+ non-classical monocytes correlates with metastatic tumor burden in patients. Taken together, our results suggest that miR-21 and miR-29a are essential for the pro-tumor functions of myeloid cells and the CSF1-ETS2 pathway upstream of the miRs serves as an attractive therapeutic target for the inhibition of M2 remodeling of macrophages during malignancy. In addition, miR-21 and miR-29a in circulating myeloid cells may potentially serve as biomarkers to measure therapeutic efficacy of targeted therapies for CSF1 signaling.

Genomic Characterization of Non–Small-Cell Lung Cancer in African Americans by Targeted Massively Parallel Sequencing

PURPOSE Technologic advances have enabled the comprehensive analysis of genetic perturbations in non-small-cell lung cancer (NSCLC); however, African Americans have often been underrepresented in these studies. This ethnic group has higher lung cancer incidence and mortality rates, and some studies have suggested a lower incidence of epidermal growth factor receptor mutations. Herein, we report the most in-depth molecular profile of NSCLC in African Americans to date. METHODS A custom panel was designed to cover the coding regions of 81 NSCLC-related genes and 40 ancestry-informative markers. Clinical samples were sequenced on a massively parallel sequencing instrument, and anaplastic lymphoma kinase translocation was evaluated by fluorescent in situ hybridization. RESULTS The study cohort included 99 patients (61% males, 94% smokers) comprising 31 squamous and 68 nonsquamous cell carcinomas. We detected 227 nonsilent variants in the coding sequence, including 24 samples with nonoverlapping, classic driver alterations. The frequency of driver mutations was not significantly different from that of whites, and no association was found between genetic ancestry and the presence of somatic mutations. Copy number alteration analysis disclosed distinguishable amplifications in the 3q chromosome arm in squamous cell carcinomas and pointed toward a handful of targetable alterations. We also found frequent SMARCA4 mutations and protein loss, mostly in driver-negative tumors. CONCLUSION Our data suggest that African American ancestry may not be significantly different from European/white background for the presence of somatic driver mutations in NSCLC. Furthermore, we demonstrated that using a comprehensive genotyping approach could identify numerous targetable alterations, with potential impact on therapeutic decisions.

Somatic Mutation Spectrum of Non–Small-Cell Lung Cancer in African Americans: A Pooled Analysis

Introduction: The mutational profile of non–small-cell lung cancer (NSCLC) has become an important tool in tailoring therapy to patients, with clear differences according to the population of origin. African Americans (AAs) have higher lung cancer incidence and mortality than Caucasians, yet discrepant results have been reported regarding the frequency of somatic driver mutations. We hypothesized that NSCLC has a distinct mutational profile in this group. Methods: We collected NSCLC samples resected from self-reported AAs in five sites from Tennessee, Michigan, and Ohio. Gene mutations were assessed by either SNaPshot or next generation sequencing, and ALK translocations were evaluated by fluorescence in situ hybridization. Results: Two hundred sixty patients were included, mostly males (62.3%) and smokers (86.6%). Eighty-one samples (31.2%) were squamous cell carcinomas. The most frequently mutated genes were KRAS (15.4%), epidermal growth factor receptor (EGFR, 5.0%), PIK3CA (0.8%), BRAF, NRAS, ERBB2, and AKT1 (0.4% each). ALK translocations were detected in two nonsquamous tumors (1.7%), totaling 61 cases (23.5%) with driver oncogenic alterations. Among 179 nonsquamous samples, 54 (30.2%) presented a driver alteration. The frequency of driver alterations altogether was lower than that reported in Caucasians, whereas no difference was detected in either EGFR or KRAS mutations. Overall survival was longer among patients with EGFR mutations. Conclusions: We demonstrated that NSCLC from AAs has a different pattern of somatic driver mutations than from Caucasians. The majority of driver alterations in this group are yet to be described, which will require more comprehensive panels and assessment of noncanonical alterations.

The Epstein-Barr virus lytic protein BZLF1 as a candidate target antigen for vaccine development

Hartlage, Liu, and colleagues show that vaccination of hu-PBL-SCID mice with EBV protein BZLF1-pulsed dendritic cells induced specific cellular immunity and prolonged survival from fatal EBV-driven lymphoproliferative disease, identifying BZLF1 as a prophylactic vaccine candidate for EBV-associated diseases. The Epstein–Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. Although most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing life-threatening diseases, such as Hodgkin lymphoma and posttransplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV infection in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1 as a potential target antigen for vaccine development. Primary tumors from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) express BZLF1 protein. Pulsing human dendritic cells (DC) with recombinant BZLF1 followed by incubation with autologous mononuclear cells led to expansion of BZLF1-specific CD8+ T cells in vitro and primed BZLF1-specific T-cell responses in vivo. In addition, vaccination of hu-PBL-SCID mice with BZLF1-transduced DCs induced specific cellular immunity and significantly prolonged survival from fatal EBV-LPD. These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide a rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases. Cancer Immunol Res; 3(7); 787–94. ©2015 AACR.

Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency.

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized.

Abstract C30: The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression

Purpose of Study: Research over the past decade has established a critical role for the tumor microenvironment in facilitating tumor growth and promoting invasiveness. Cells of the myeloid lineage, including macrophages, have been known to be key mediators of tumor progression and facilitate metastasis. Recent work from our lab highlights the Ets transcription factors, PU.1 and ETS2, as important players in tumor associated macrophages (TAMs) during breast cancer progression. Both transcription factors are downstream effectors of the Colony Stimulating Factor 1 (CSF1) signaling pathway, which is not only important for myeloid cell survival, but also has a well-established role within the tumor microenvironment. Here, we investigated the requirement of PU.1 in TAMs in a mouse mammary tumor model and have begun to address the role of PU.1 during metastasis. Research Method: We employed cre-loxP technology to conditionally delete PU.1 in the myeloid cell compartment. Syngeneic mice were orthotopically injected with mammary tumor cells directly into the fat pad. To model breast cancer brain metastasis in the mouse, we employed an intracranial injection model which recapitulates the biology of the human disease. We were able to isolate tumor associated macrophages from either site (mammary or brain tumor) for downstream applications. Novel Findings: Macrophage-specific deletion of PU.1 resulted in a significant reduction in mammary tumor growth as well as tumor angiogenesis. Our results suggest that ETS2 and PU.1, acting downstream of the CSF1 signaling pathway, cooperatively regulate the expression of pro-tumor genes along with ‘oncogenic’ microRNA. Conventional ChIP assays show that both transcription factors occupy enhancer regions adjacent to a set of selected genes and cooperatively regulate expression in tumor infiltrating macrophages. We focused on miR-21 and miR-29a for a more detailed investigation as both are expressed in CSF1R+ myeloid cells within the brain metastatic microenvironment. In our intracranial injection model, macrophages (F4/80 positive cells) are recruited to the growing tumor, and both miR-21 and miR-29a are upregulated in TAMs isolated by FACS from tumor-bearing mice as compared to controls. Conclusions and Implications: Given our present data, we believe that the Ets factors, PU.1 and Ets2, regulate a transcriptional program, including microRNAs, that is critical for CSF1 action in tumor infiltrating macrophages. Currently, clinical trials are underway using CSF1R inhibitors for several solid tumor types [clinicaltrials.gov]. The macrophage-specific microRNA, including miR-21 and miR-29a, may serve as indicators for to the efficacy of CSF1R inhibition. Citation Format: Katie Thies, David A. Taffany, Haritha Mathsyaraja, Sudarshana M. Sharma, Walter Hans Meisen, Tom Liu, Cynthia Timmers, Jose Otero, Balveen Kaur, Michael C. Ostrowski. The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C30.

Abstract 1514: Oncolytic viral therapy for glioblastoma induces robust natural killer cell activation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Oncolytic viral (OV) therapy is a promising treatment being tested for safety and efficacy. Natural killer cells (NK) limit viral infections, and previous work suggests they may similarly attenuate virotherapy. We used flow cytometry to evaluate the temporal pattern of NK recruitment in intracranial tumors (U87DEGFR glioma) treated with the oncolytic herpes virus, rQnestin 34.5, or PBS. Quantification of NK recruitment into intracranial tumors treated with rQnestin34.5 or PBS revealed significant enrichment of these cells into the tumor hemisphere at early timepoints and continuing through 72 hours (2.2-fold, p=0.02) following infection. Moreover, NK cells recruited to the site of inoculation exhibited an activated phenotype, including enhanced CD69, CD62L, CD27, NKG2D, and CD127 staining compared to PBS treated mice. This robust NK response was demonstrated to be detrimental to OV efficacy through the enhanced survival of NK depleted mice treated with OV compared to OV treated mice containing NK cells (median survival 31.6 days compared to median survival 23.6 days, p=0.04). Since NK cells are recruited into the tumor microenvironment following infection, we postulated that OV infection of glioma cells was also activating NK cells. We measured granzyme B and IFNg production by NK cells in the presence of infected or uninfected glioma. Our results indicate that glioma mediated activation of NK cells was augmented by OV infection. Additionally, these findings were corroborated with increased NK killing of OV infected glioma compared to uninfected glioma. Due to the enhanced NK cytotoxic properties induced by OV infection, we proceeded to investigate the expression of NK activating ligands on the surface of infected glioma cells that could potentially mediate this effect. Focusing on ligands for NKG2D and the natural cytotoxicity receptors (NKp30, NKp44, and NKp46), viral infection of cultured gliomas was shown to upregulate ligand expression for NKG2D (24%, p=0.004), NKp46 (41%, p=0.01), and NKp30 (2.75-fold, p=0.001) while selectively downregulating ligand expression for NKp44 (2.8-fold, p=0.005). These findings suggest that viral infection of gliomas is altering the ligand signature for NK activation. Since NK cells possess both antiviral and antitumor properties, further work will investigate if virus induced NK activating signals can be selectively blocked in order to enhance viral efficacy while maintaining NK antitumor efficacy. This is the first study investigating the changes in NK cell activation and recruitment in gliomas upon oncolytic HSV-1 treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1514.