Genomic analysis of human brain metastases treated with stereotactic radiosurgery reveals unique signature based on treatment failure.

Stereotactic radiosurgery (SRS) has been shown to be efficacious for the treatment of limited brain metastasis (BM); however, the effects of SRS on human brain metastases have yet to be studied. We performed genomic analysis on resected brain metastases from patients whose resected lesion was previously treated with SRS. Our analyses demonstrated for the first time that patients possess a distinct genomic signature based on type of treatment failure including local failure, leptomeningeal spread, and radio-necrosis. Examination of the center and peripheral edge of the tumors treated with SRS indicated differential DNA damage distribution and an enrichment for tumor suppressor mutations and DNA damage repair pathways along the peripheral edge. Furthermore, the two clinical modalities used to deliver SRS, LINAC and GK, demonstrated differential effects on the tumor landscape even between controlled primary sites. Our study provides, in human, biological evidence of differential effects of SRS across BM's.

Targeted inhibition of BET proteins in HPV-16 associated head and neck squamous cell carcinoma reveals heterogeneous transcription response.

Integrated human papillomavirus (HPV-16) associated head and neck squamous cell carcinoma (HNSCC) tumors have worse survival outcomes compared to episomal HPV-16 HNSCC tumors. Therefore, there is a need to differentiate treatment for HPV-16 integrated HNSCC from other viral forms. We analyzed TCGA data and found that HPV+ HNSCC expressed higher transcript levels of the bromodomain and extra terminal domain (BET) family of transcriptional coregulators. However, the mechanism of BET protein-mediated transcription of viral-cellular genes in the integrated viral-HNSCC genomes needs to be better understood. We show that BET inhibition downregulates E6 significantly independent of the viral transcription factor, E2, and there was overall heterogeneity in the downregulation of viral transcription in response to the effects of BET inhibition across HPV-associated cell lines. Chemical BET inhibition was phenocopied with the knockdown of BRD4 and mirrored downregulation of viral E6 and E7 expression. Strikingly, there was heterogeneity in the reactivation of p53 levels despite E6 downregulation, while E7 downregulation did not alter Rb levels significantly. We identified that BET inhibition directly downregulated c-Myc and E2F expression and induced CDKN1A expression. Overall, our studies show that BET inhibition provokes a G1-cell cycle arrest with apoptotic activity and suggests that BET inhibition regulates both viral and cellular gene expression in HPV-associated HNSCC.

Genomic Analysis of Human Brain Metastases Treated with Stereotactic Radiosurgery Under the Phase-II Clinical Trial (NCT03398694) Reveals DNA Damage Repair at the Peripheral Tumor Edge.

Stereotactic Radiosurgery (SRS) is one of the leading treatment modalities for oligo brain metastasis (BM), however no comprehensive genomic data assessing the effect of radiation on BM in humans exist. Leveraging a unique opportunity, as part of the clinical trial (NCT03398694), we collected post-SRS, delivered via Gamma-knife or LINAC, tumor samples from core and peripheral-edges of the resected tumor to characterize the genomic effects of overall SRS as well as the SRS delivery modality. Using these rare patient samples, we show that SRS results in significant genomic changes at DNA and RNA levels throughout the tumor. Mutations and expression profiles of peripheral tumor samples indicated interaction with surrounding brain tissue as well as elevated DNA damage repair. Central samples show GSEA enrichment for cellular apoptosis while peripheral samples carried an increase in tumor suppressor mutations. There are significant differences in the transcriptomic profile at the periphery between Gamma-knife vs LINAC.

Development and characterization of patient-derived xenografts from non-small cell lung cancer brain metastases.

Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.

FGFR Inhibition Enhances Sensitivity to Radiation in Non-Small Cell Lung Cancer.

FGFRs are commonly altered in non-small cell lung cancer (NSCLC). FGFRs activate multiple pathways including RAS/RAF/MAPK, PI3K/AKT, and STAT, which may play a role in the cellular response to radiation. We investigated the effects of combining the selective FGFR 1-3 tyrosine kinase inhibitor AZD4547 with radiation in cell line and xenograft models of NSCLC. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, autophagy, cell cycle, and DNA damage signaling and repair assays. In vivo xenografts and IHC were used to confirm in vitro results. NSCLC cell lines demonstrated varying degrees of FGFR protein and mRNA expression. In vitro clonogenic survival assays showed radiosensitization with AZD4547 in two NSCLC cell lines. In these two cell lines, an increase in apoptosis and autophagy was observed with combined radiation and AZD4547. The addition of AZD4547 to radiation did not significantly affect γH2AX foci formation. Enhanced xenograft tumor growth delay was observed with the combination of radiation and AZD4547 compared with radiation or drug alone. IHC results revealed inhibition of pMAPK and pS6 and demonstrated an increase in apoptosis in the radiation plus AZD4547 group. This study demonstrates that FGFR inhibition by AZD4547 enhances the response of radiation in FGFR-expressing NSCLC in vitro and in vivo model systems. These results support further investigation of combining FGFR inhibition with radiation as a clinical therapeutic strategy.

Fibroblast Growth Factor Receptors as Targets for Radiosensitization in Head and Neck Squamous Cell Carcinomas.

PURPOSE: We examined the capacity of the pan-fibroblast growth factor receptor (FGFR) inhibitor AZD4547 to augment radiation response across a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts. METHODS AND MATERIALS: FGFR1, FGFR2, and FGFR3 RNA in situ hybridization expression was assessed in a cohort of HNSCC patient samples, cell lines, and patient-derived xenografts (PDXs). In vitro effects of AZD4547 and radiation on cell survival, FGFR signaling, apoptosis, autophagy, cell cycle, and DNA damage repair were evaluated. Reverse phase protein array was used to identify differentially phosphorylated proteins in cells treated with AZD4547. In vivo tumor responses were evaluated in cell lines and PDX models. RESULTS: FGFR1, FGFR2, and FGFR3 RNA in situ hybridization were expressed in 41%, 81%, and 89% of 107 oropharynx patient samples. Sensitivity to AZD4547 did not directly correlate with FGFR protein or RNA expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time-dependent manner. Significant radiosensitization with AZD4547 was observed in cell lines that were sensitive to AZD4547. The mechanism underlying these effects appears to be multifactorial, involving inhibition of the MTOR pathway and subsequent enhancement of autophagy and activation of apoptotic pathways. Significant tumor growth delay was observed when AZD4547 was combined with radiation compared with radiation or drug alone in an FGFR-expressing HNSCC cell line xenograft and PDX. CONCLUSIONS: These findings suggest that AZD4547 can augment the response of radiation in FGFR-expressing HNSCC in vivo model systems. FGFR1 and FGFR2 may prove worthy targets for radiosensitization in HNSCC clinical investigations.

177Lu-NM600 Targeted Radionuclide Therapy Extends Survival in Syngeneic Murine Models of Triple-Negative Breast Cancer.

There is a clinically unmet need for effective treatments for triple-negative breast cancer (TNBC), as it remains the most aggressive subtype of breast cancer. Herein, we demonstrate a promising strategy using a tumor-targeting alkylphosphocholine (NM600) for targeted radionuclide therapy of TNBC. Methods: NM600 was radiolabeled with 86Y for PET imaging and 177Lu for targeted radionuclide therapy. 86Y-NM600 PET imaging was performed on female BALB/C mice bearing syngeneic 4T07 (nonmetastatic) and 4T1 (metastatic) TNBC tumor grafts (n = 3-5). Quantitative data derived from a PET-image region-of-interest analysis, which was corroborated by ex vivo biodistribution, were used to estimate the dosimetry of 177Lu-NM600 treatments. Weight measurement, complete blood counts, and histopathology analysis were performed to determine 177Lu-NM600 toxicity in naïve BALB/C mice administered 9.25 or 18.5 MBq. Groups of mice bearing 4T07 or 4T1 grafts (n = 5-6) received excipient or 9.25 or 18.5 MBq of 177Lu-NM600 as a single or fractionated schedule, and tumor growth and overall survival were monitored. Results: Excellent tumor targeting and rapid normal-tissue clearance of 86Y-NM600 were noted in both 4T07 and 4T1 murine models. Ex vivo biodistribution corroborated the accuracy of the PET data and validated 86Y-NM600 as a surrogate for 177Lu-NM600. 177Lu-NM600 dosimetry showed absorbed doses of 2.04 ± 0.32 and 1.68 ± 0.06 Gy/MBq to 4T07 and 4T1 tumors, respectively, which were larger than those delivered to liver (1.28 ± 0.09 Gy/MBq) and to bone marrow (0.31 ± 0.05 Gy/MBq). The 177Lu-NM600 injected activities used for treatment were well tolerated and resulted in significant tumor growth inhibition and prolonged overall survival in both tested TNBC models. A complete response was attained in 60% of treated mice bearing 4T07 grafts. Conclusion: Overall, our results suggest that 177Lu-NM600 targeted radionuclide therapy has potential for TNBC and merits further exploration in a clinical setting.

Enhanced Radiosensitivity in Solid Tumors using a Tumor-selective Alkyl Phospholipid Ether Analog.

Antitumor alkyl phospholipid (APL) analogs comprise a group of structurally related molecules with remarkable tumor selectivity. Some of these compounds have shown radiosensitizing capabilities. CLR127 is a novel, clinical-grade antitumor APL ether analog, a subtype of synthetic APL broadly targeting cancer cells with limited uptake in normal tissues. The purpose of this study was to investigate the effect of CLR127 to modulate radiation response across several adult and pediatric cancer types in vitro as well as in murine xenograft models of human prostate adenocarcinoma, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma. In vitro, CLR127 demonstrated selective uptake in cancer cells compared to normal cells. In cancer cells, CLR127 treatment prior to radiation significantly decreased clonogenic survival in vitro, and led to increased radiation-induced double-stranded DNA (dsDNA) breakage compared with radiation alone, which was not observed in normal controls. In animal models, CLR127 effectively increased the antitumor response to fractionated radiotherapy and led to delayed tumor regrowth at potentially clinically achievable doses. In conclusion, our study highlights the ability of CLR127 to increase radiation response in several cancer types. Given almost universal uptake of CLR127 in malignant cells, future research should test whether the observed effects can be extended to other tumor types. Our data provide a strong rationale for clinical testing of CLR127 as a tumor-targeted radiosensitizing agent. Mol Cancer Ther; 17(11); 2320-8. ©2018 AACR.

Identification of stable housekeeping genes in response to ionizing radiation in cancer research.

Housekeeping genes (HKGs) are essential for basic maintenance of a variety of cellular processes. They ideally maintain uniform expression independent of experimental conditions. However, the effects of ionizing radiation (IR) on HKG expression is unclear. Statistical algorithms, geNorm and Normfinder were used for estimating the stability of HKGs as raw quantification cycle (Cq) values were not a reliable factor for normalization. Head and neck, non-small lung and pancreas cells were exposed to 2, 4 and 6 Gy IR doses and expression of fourteen HKGs was measured at 5 min to 48 h post-irradiation within a given tissue. Paired and single cell line analyses under these experimental conditions identified TATA-Box Binding Protein (TBP) and Importin 8 (IPO8) to be stable in non-small cell lung cancer. In addition to these two genes, Ubiquitin C (UBC) in head and neck cancer and Transferrin receptor (TFRC) and ß-Glucuronidase (GUSB) in pancreatic cancer were identified to be stable as well. In summary we present a resource for top ranked five stable HKGs and their transcriptional behavior in commonly used cancer model cell lines and suggest the use of multiple HKGs under radiation treatment conditions is a reliable metric for quantifying gene expression.

Insulin-like growth factor 1 receptor mediated tyrosine 845 phosphorylation of epidermal growth factor receptor in the presence of monoclonal antibody cetuximab.

BACKGROUND: The epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and several other human cancers. Monoclonal antibodies, such as cetuximab that block EGFR signaling, have emerged as valuable molecular targeting agents in clinical cancer therapy. Prolonged exposure to cetuximab can result in cells acquiring resistance by a process that remains incompletely understood. METHODS: In this study, we analyzed the immediate early molecular response of cetuximab on physical interactions between EGFR and Insulin growth factor 1 like receptor (IGF-1R) in head and neck cancer cells that are resistant to cetuximab. Co-immunoprecipitation, small molecule inhibitors against phospho-Src and IGF-1R, quantitative western blot of EGFR and Src phosphorylation, cell proliferation assays were used to suggest the role of IGF-1R mediated phosphorylation of specific tyrosine Y845 on EGFR via increased heterodimerization of EGFR and IGF-1R in cetuximab resistant cells. RESULTS: Heterodimerization of EGFR with IGF-1R was increased in cetuximab resistant HNSCC cell line UMSCC6. Basal levels of phosphorylated EGFR Y845 showed significant increase in the presence of cetuximab. Surprisingly, this activated Y845 level was not inhibited in the presence of Src inhibitor PP1. Instead, inhibition of IGF-1R by picropodophyllin (PPP) reduced the EGFR Y845 levels. Taken together, these results suggest that heterodimerization of EGFR with IGF-1R can lead to increased activity of EGFR and may be an important platform for cetuximab mediated signaling in head and neck tumors that have become resistant to anti-EGFR therapy. CONCLUSIONS: EGFR-IGF-1R interaction has a functional consequence of phosphorylation of EGFR Y845 in cetuximab resistant HNSCC cells and dual targeting of EGFR and IGF-1R is a promising therapeutic strategy.

Microfluidic enrichment for the single cell analysis of circulating tumor cells.

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

Small Molecule Inhibition of MDM2-p53 Interaction Augments Radiation Response in Human Tumors.

MDM2-p53 interaction and downstream signaling affect cellular response to DNA damage. AMG 232 is a potent small molecule inhibitor that blocks the interaction of MDM2 and p53. We examined the capacity of AMG 232 to augment radiation response across a spectrum of human tumor cell lines and xenografts. AMG 232 effectively inhibited proliferation and enhanced radiosensitivity via inhibition of damage repair signaling. Combined AMG 232 and radiation treatment resulted in the accumulation of γH2AX-related DNA damage and induction of senescence with promotion of apoptotic and/or autophagic cell death. Several molecules involved in senescence, autophagy, and apoptosis were specifically modulated following the combined AMG 232/radiation treatment, including FoxM1, ULK-1, DRAM, and BAX. In vivo xenograft studies confirmed more potent antitumor and antiangiogenesis efficacy with combined AMG 232/radiation treatment than treatment with drug or radiation alone. Taken together, these data identify the capacity of AMG 232 to augment radiation response across a variety of tumor types harboring functional p53.

Single-step conjugation of antibodies to quantum dots for labeling cell surface receptors in mammalian cells.

Labeling of cell surface receptors in living cells can be achieved using antibody-conjugated semiconductor quantum dots (QDs). The inherent photostable property of QDs can be exploited for understanding the arrangement and distribution of receptors in the plasma membrane. We describe herein methods that allow conjugation of antibodies to QDs in a single step without the formation of side products. This protocol can be adapted universally for any type of QD structure with a coating of free amino groups.

Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging.

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

Particle size, surface coating, and PEGylation influence the biodistribution of quantum dots in living mice.

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

Peptide coated quantum dots for biological applications.

Quantum dots (QDOTs) have been widely recognized by the scientific community and the biotechnology industry, as witnessed by the exponential growth of this field in the past several years. We describe the synthesis and characterization of visible and near infrared QDots--a critical step for engineering organic molecules like proteins and peptides for building nanocomposite materials with multifunctional properties suitable for biological applications.

Nonself recognition is mediated by HET-C heterocomplex formation during vegetative incompatibility.

Nonself recognition during vegetative growth in filamentous fungi is mediated by heterokaryon incompatibility (het) loci. In Neurospora crassa, het-c is one of 11 het loci. Three allelic specificity groups, termed het-c(OR), het-c(PA) and het-c(GR), exist in natural populations. Heterokaryons or partial diploids that contain het-c alleles of alternative specificity show severe growth inhibition, repression of conidiation and hyphal compartmentation and death (HCD). Using epitope-tagged HET-C, we show that nonself recognition is mediated by the presence of a heterocomplex composed of polypeptides encoded by het-c alleles of alternative specificity. The HET-C heterocomplex localized to the plasma membrane (PM); PM-bound HET-C heterocomplexes occurred in all three het-c incompatible allelic interactions. Strains containing het-c constructs deleted for a predicted signal peptide sequence formed HET-C heterocomplexes in the cytoplasm and showed a growth arrest phenotype. Our finding is a step towards understanding nonself recognition mechanisms that operate during vegetative growth in filamentous fungi, and provides a model for investigating relationships between recognition mechanisms and cell death.