Targeting Riboswitches with Beta-Axial-Substituted Cobalamins.

RNA-targeting small-molecule therapeutics is an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch is a particularly useful model system, as similar sequences show highly specialized binding preferences for different biological forms of cobalamin. This riboswitch is also widely dispersed across bacteria and therefore holds strong potential as an antibiotic target. Many synthetic cobalamin forms have been developed for various purposes including therapeutics, but their interaction with cobalamin riboswitches is yet to be explored. In this study, we characterize the interactions of 11 cobalamin derivatives with three representative cobalamin riboswitches using in vitro binding experiments (both chemical footprinting and a fluorescence-based assay) and a cell-based reporter assay. The derivatives show productive interactions with two of the three riboswitches, demonstrating simultaneous plasticity and selectivity within these RNAs. The observed plasticity is partially achieved through a novel structural rearrangement within the ligand binding pocket, providing insight into how similar RNA structures can be targeted. As the derivatives also show in vivo functionality, they serve as several potential lead compounds for further drug development.

Regulation of Gene Expression Through Effector-dependent Conformational Switching by Cobalamin Riboswitches.

Riboswitches are an outstanding example of genetic regulation mediated by RNA conformational switching. In these non-coding RNA elements, the occupancy status of a ligand-binding domain governs the mRNA's decision to form one of two mutually exclusive structures in the downstream expression platform. Temporal constraints upon the function of many riboswitches, requiring folding of complex architectures and conformational switching in a limited co-transcriptional timeframe, make them ideal model systems for studying these processes. In this review, we focus on the mechanism of ligand-directed conformational changes in one of the most widely distributed riboswitches in bacteria: the cobalamin family. We describe the architectural features of cobalamin riboswitches whose structures have been determined by x-ray crystallography, which suggest a direct physical role of cobalamin in effecting the regulatory switch. Next, we discuss a series of experimental approaches applied to several model cobalamin riboswitches that interrogate these structural models. As folding is central to riboswitch function, we consider the differences in folding landscapes experienced by RNAs that are produced in vitro and those that are allowed to fold co-transcriptionally. Finally, we highlight a set of studies that reveal the difficulties of studying cobalamin riboswitches outside the context of transcription and that co-transcriptional approaches are essential for developing a more accurate picture of their structure-function relationships in these switches. This understanding will be essential for future advancements in the use of small-molecule guided RNA switches in a range of applications such as biosensors, RNA imaging tools, and nucleic acid-based therapies.

Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

Pancreatic Tumor Microenvironment Modulation by EphB4-ephrinB2 Inhibition and Radiation Combination.

PURPOSE: A driving factor in pancreatic ductal adenocarcinoma (PDAC) treatment resistance is the tumor microenvironment, which is highly immunosuppressive. One potent immunologic adjuvant is radiotherapy. Radiation, however, has also been shown to induce immunosuppressive factors, which can contribute to tumor progression and formation of fibrotic tumor stroma. To capitalize on the immunogenic effects of radiation and obtain a durable tumor response, radiation must be rationally combined with targeted therapies to mitigate the influx of immunosuppressive cells and fibrosis. One such target is ephrinB2, which is overexpressed in PDAC and correlates negatively with prognosis.Experimental Design: On the basis of previous studies of ephrinB2 ligand-EphB4 receptor signaling, we hypothesized that inhibition of ephrinB2-EphB4 combined with radiation can regulate the microenvironment response postradiation, leading to increased tumor control in PDAC. This hypothesis was explored using both cell lines and in vivo human and mouse tumor models. RESULTS: Our data show this treatment regimen significantly reduces regulatory T-cell, macrophage, and neutrophil infiltration and stromal fibrosis, enhances effector T-cell activation, and decreases tumor growth. Furthermore, our data show that depletion of regulatory T cells in combination with radiation reduces tumor growth and fibrosis. CONCLUSIONS: These are the first findings to suggest that in PDAC, ephrinB2-EphB4 interaction has a profibrotic, protumorigenic role, presenting a novel and promising therapeutic target.

Inhibition of EphB4-Ephrin-B2 Signaling Reprograms the Tumor Immune Microenvironment in Head and Neck Cancers.

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact, resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.

Role of EphB3 Receptor in Mediating Head and Neck Tumor Growth, Cell Migration, and Response to PI3K Inhibitor.

Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Our The Cancer Genome Atlas (TCGA) data analysis showed that EphB3, a receptor tyrosine kinase, is frequently coamplified with PIK3CA in head and neck squamous cell carcinoma (HNSCC). We therefore hypothesized that EphB3 amplification plays a protumorigenic role in HNSCC and that EphB3 and PIK3CA are cooperating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported, because EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is correlated with an increase in prosurvival proteins, S6 and BcL-XL, in the EphB3 shRNA tumors treated with BKM120 compared with controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. Although total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2-Fc fusion protein resulted in decreased HNSCC migration and cell growth, and enhanced response to BKM120 in vitro These data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K signaling. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC. Mol Cancer Ther; 17(9); 2049-59. ©2018 AACR.

Resistance to Radiotherapy and PD-L1 Blockade Is Mediated by TIM-3 Upregulation and Regulatory T-Cell Infiltration.

Purpose: Radiotherapy (RT) can transform the immune landscape and render poorly immunogenic tumors sensitive to PD-L1 inhibition. Here, we established that the response to combined RT and PD-L1 inhibition is transient and investigated mechanisms of resistance.Experimental Design: Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine head and neck squamous cell carcinoma (HNSCC) tumors using mass cytometry and whole-genome sequencing. Mice were treated with anti-PD-L1 or anti-TIM-3 alone and in combination with and without RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Depletion of regulatory T cells (Treg) was performed using anti-CD25 antibody.Results: We show that the immune checkpoint receptor, TIM-3, is upregulated on CD8 T cells and Tregs in tumors treated with RT and PD-L1 blockade. Treatment with anti-TIM-3 concurrently with anti-PD-L1 and RT led to significant tumor growth delay, enhanced T-cell cytotoxicity, decreased Tregs, and improved survival in orthotopic models of HNSCC. Despite this treatment combination, the response was not durable, and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored antitumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory.Conclusions: These data reveal multiple layers of immune regulation that can promote tumorigenesis and the therapeutic potential of sequential targeting to overcome tumor resistance mechanisms. We propose that targeted Treg inhibitors may be critical for achieving durable tumor response with combined radiotherapy and immunotherapy. Clin Cancer Res; 24(21); 5368-80. ©2018 AACR.

Inhibition of EphB4-Ephrin-B2 Signaling Enhances Response to Cetuximab-Radiation Therapy in Head and Neck Cancers.

Purpose: The clinical success of targeted therapies such as cetuximab and radiotherapy (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4-ephrin-B2 protumorigenic signaling in mediating resistance to EGFR inhibition and RT in head and neck cancers.Experimental Design: We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and Western blotting to detect changes in EphB4-ephrin-B2 targets. mRNA sequencing and microarray data analysis were performed on PDX tumors and HNSCC cell lines, respectively, to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation.Results: Our data from locally advanced HNSCC patients treated with standard-of-care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response toward cetuximab and RT following EphB4-ephrin-B2 inhibition, resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by a decrease in the levels of proliferation and prosurvival molecules and increased apoptosis.Conclusions: Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT with EphB4-ephrin-B2 blockade has the potential to translate into the clinic to benefit this patient population. Clin Cancer Res; 24(18); 4539-50. ©2018 AACR.

Hypofractionated Radiotherapy Is Superior to Conventional Fractionation in an Orthotopic Model of Anaplastic Thyroid Cancer.

BACKGROUND: Anaplastic thyroid cancer (ATC) is an aggressive and highly lethal disease with poor outcomes and resistance to therapy. Despite multimodality treatment, including radiation therapy and chemotherapy, response rates remain <15%, with a median time to progression of less than three months. Recent advances in radiotherapy (RT) delivery and gene-expression profiling may help guide patient selection for personalized therapy. The purpose of this study was to characterize the response to radiation in a panel of ATC cell lines and to test alternative RT fractionation schedules for overcoming radioresistance. MATERIALS AND METHODS: The cellular response to radiation was characterized based on clonogenic assays. Radiation response was correlated with microarray gene-expression data. Hypofractionated and conventional RT was tested in an orthotopic ATC tumor model, and tumor growth was assayed locally and distantly with in vivo and ex vivo bioluminescence imaging. RESULTS: A spectrum of radiosensitivities was observed in ATC cell lines. Radioresistant cell lines had higher levels of CXCR4 compared to radiosensitive cell lines. Compared to conventionally fractionated RT, hypofractionated RT resulted in significantly improved tumor growth delay, decreased regional and distant metastases, and improved overall survival. CONCLUSIONS: The findings demonstrate the heterogeneity of response to radiation in ATC tumors and the superiority of hypofractionated RT in improving local control, metastatic spread, and survival in preclinical models. These data support the design of clinical trials targeting radioresistant pathways in combination with hypofractionated RT.

Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma.

Immunotherapy clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that most head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. We investigated whether local radiation to the tumor can transform the immune landscape and render poorly immunogenic HNSCC tumors sensitive to PD-L1 inhibition. We used the first novel orthotopic model of HNSCC with genetically distinct murine cell lines. Tumors were resistant to PD-L1 checkpoint blockade, harbored minimal PD-L1 expression and tumor infiltrating lymphocytes at baseline, and were resistant to radiotherapy. The combination of radiation and PD-L1 inhibition significantly enhanced tumor control and improved survival. This was mediated in part through upregulation of PD-L1 on tumor cells and increased T-cell infiltration after RT, resulting in a highly inflamed tumor. Depletion of both CD4 and CD8 T-cells completely abrogated the effect of anti PD-L1 with radiation on tumor growth. Our findings provide evidence that radiation to the tumor can induce sensitivity to PD-L1 checkpoint blockade in orthotopic models of HNSCC. These findings have direct relevance to high risk HNSCC patients with poorly immunogenic tumors and who may benefit from combined radiation and checkpoint blockade.