First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes.

PURPOSE: Fluorescence molecular imaging, using cancer-targeted near infrared (NIR) fluorescent probes, offers the promise of accurate tumor delineation during surgeries and the detection of cancer specific molecular expression in vivo. However, nonspecific probe accumulation in normal tissue results in poor tumor fluorescence contrast, precluding widespread clinical adoption of novel imaging agents. Here we present the first clinical evidence that fluorescence lifetime (FLT) imaging can provide tumor specificity at the cellular level in patients systemically injected with panitumumab-IRDye800CW, an EGFR-targeted NIR fluorescent probe. EXPERIMENTAL DESIGN: We performed wide-field and microscopic FLT imaging of resection specimens from patients injected with panitumumab-IRDye800CW under an FDA directed clinical trial. RESULTS: We show that the FLT within EGFR-overexpressing cancer cells is significantly longer than the FLT of normal tissue, providing high sensitivity (>98%) and specificity (>98%) for tumor versus normal tissue classification, despite the presence of significant nonspecific probe accumulation. We further show microscopic evidence that the mean tissue FLT is spatially correlated (r > 0.85) with tumor-specific EGFR expression in tissue and is consistent across multiple patients. These tumor cell-specific FLT changes can be detected through thick biological tissue, allowing highly specific tumor detection and noninvasive monitoring of tumor EFGR expression in vivo. CONCLUSIONS: Our data indicate that FLT imaging is a promising approach for enhancing tumor contrast using an antibody-targeted NIR probe with a proven safety profile in humans, suggesting a strong potential for clinical applications in image guided surgery, cancer diagnostics, and staging.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.

Inhibitors of the Transcription Factor STAT3 Decrease Growth and Induce Immune Response Genes in Models of Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive cancer defined by loss-of-function mutations with few therapeutic options. We examined the contribution of the transcription factor Signal transducer and activator of transcription 3 (STAT3) to cell growth and gene expression in preclinical models of MPM. STAT3 is activated in a variety of tumors and is thought to be required for the maintenance of cancer stem cells. Targeting STAT3 using specific small hairpin RNAs (shRNAs) or with the pharmacologic inhibitors atovaquone or pyrimethamine efficiently reduced cell growth in established cell lines and primary-derived lines while showing minimal effects in nontransformed LP9 mesothelial cells. Moreover, atovaquone significantly reduced viability and tumor growth in microfluidic cultures of primary MPM as well as in an in vivo xenotransplant model. Biological changes were linked to modulation of gene expression associated with STAT3 signaling, including cell cycle progression and altered p53 response. Reflecting the role of STAT3 in inducing localized immune suppression, using both atovaquone and pyrimethamine resulted in the modulation of immunoregulatory genes predicted to enhance an immune response, including upregulation of ICOSLG (Inducible T-Cell Costimulator Ligand or B7H2). Thus, our data strongly support a role for STAT3 inhibitors as anti-MPM therapeutics.

BRG1 promotes survival of UV-irradiated melanoma cells by cooperating with MITF to activate the melanoma inhibitor of apoptosis gene.

Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.