Tyler Sheetz

Human anti-nucleolin recombinant immunoagent for cancer therapy

Significance Because of its selective expression on the surface of a variety of different cancer cells, but not on their normal counterparts, nucleolin (NCL) represents an attractive target for antineoplastic treatments. However, previously described NCL-targeting molecules, although promising, still suffer from intrinsic limitations. Here, we describe the identification of the first fully human anti-NCL immune-based agent displaying antineoplastic activity against solid tumors, both in vitro and in vivo. This molecule could represent the prototype of a novel class of NCL-targeting drugs with enormous clinical potential as tools for the diagnosis and therapy of a wide range of human cancers. Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.

Quaking and miR-155 interactions in inflammation and leukemogenesis

Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein, whose expression is downregulated in several solid tumors. Here we report that QKI plays an important role in the immune response and suppression of leukemogenesis. We show that the expression of Qki is reduced in lipopolysaccharide (LPS)-challenged macrophages, suggesting that Qki is a key regulator of LPS signaling pathway. Furthermore, LPS-induced downregulation of Qki expression is miR-155-dependent. Qki overexpression impairs LPS-induced phosphorylation of JNK and particularly p38 MAPKs, in addition to increasing the production of anti-inflammatory cytokine IL-10. In contrast, Qki ablation decreases Fas expression and the rate of Caspase3/7 activity, while increasing the levels of IL-1α, IL-1β and IL-6, and p38 phosphorylation. Similarly, the p38 pathway is also a target of QKI activity in chronic lymphocytic leukemia (CLL)-derived MEC2 cells. Finally, B-CLL patients show lower levels of QKI expression compared with B cells from healthy donor, and Qki is similarily downregulated with the progression of leukemia in Eμ-miR-155 transgenic mice. Altogether, these data implicate QKI in the pathophysiology of inflammation and oncogenesis where miR-155 is involved.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

Genetic ablation of interacting with Spt6 (Iws1) causes early embryonic lethality

IWS1 is an RNA-polymerase II (RNAPII)-associated transcription elongation factor whose biological functions are poorly characterized. To shed some light on the function of this protein at the organismal level, we performed a systematic tissue analysis of its expression and generated Iws1-deficient mice. A thorough immunohistochemical characterization shows that IWS1 protein is present in the nucleus of all cells in most of the examined tissues, with few notable exceptions. We also report that ablation of Iws1 consistently causes lethality at the pre-implantation stage with high expression of the gene in fertilized oocytes. In summary, we are providing evidence that Iws1 is expressed in all adult organs and it is an essential gene for mouse embryonic development.

Abstract 476: A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs

Trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNA) have recently been recognized as prime regulators of gene expression during tumorigenesis. Nucleolin (NCL) is the most abundant nucleolar RNA-binding protein, controlling critical cellular processes such as chromatin remodeling, ribosome biogenesis and miRNA biogenesis. In the cytoplasm, NCL regulates gene expression post-transcriptionally by controlling mRNA stability and/or translation via ARE- and miRNA-mediated pathways. In contrast, NCL at the cell surface plays pivotal roles in cancer progression and metastasis in many types of tumors. Therefore, surface-NCL is an appealing target for cancer therapy. Earlier our group described 4LB5, a human-derived single chain fragment variable (scFv) that specifically targets surface-NCL exerting cytotoxic effects, both in vitro and in vivo. We have identified the central RNA-binding domains (RBD1-4) of NCL as 4LB5-interacting sites. 4LB5-mediated blocking of NCL-RBDs significantly impairs the biogenesis of a subset of oncogenic miRNAs, such as miR-21, -221 and -222, and their binding to the microprocessor complex. The structural fold of all the 4 RBDs is highly conserved, but there are significant differences in their sequences. RBD1-2 have been well studied in their mechanism and capability to form high-affinity RNA-binding complexes, but RBD 3-4 have not been explored to the same extent. We hypothesize that consecutive or selective combinations of RBDs in NCL might have distinct roles in RNA biology. NCL RBD1-2 domains are well documented in the literature for their high affinity for mRNAs and rRNAs. However, RNA-specificity on NCL-RBDs has yet to be elucidated. In this study, we used protein-antibody and protein-RNA docking analyses to identify potential differential binding affinities between multiple NCL modules (RDB1-2, RBD3-4 and RBD1-4) and either 4LB5 or miRNAs. In silico results were also evaluated by multiple biochemical assays (ELISA, Surface Plasmon Resonance, Pull-Down and Electrophoretic Mobility Shift Assays) using NCL Modules, 4LB5 and RNA molecules. Finally, competition assays were performed to assess ability of our scFv to impair miRNA binding to NCL. Our results indicate the importance of the NCL region targeted by 4LB5 in microRNA maturation. Functional studies will help us to better elucidate the biologic role of NCL RBDs, and their involvement in cancer pathogenesis. Citation Format: Valerio Embrione, Michael Scarpati, Zachary D. Uzzel, Rahimah Ahmad, Fortuna A. De Sury, Tyler J. Sheetz, Vincenzo Coppola, Shaneen M. Singh, Anjana D. Saxena, Carlo M. Croce, Dario Palmieri. A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 476.

MP83-15 A NOVEL THERAPY FOR CASTRATION-RESISTANT PROSTATE CANCER THROUGH INHIBITION OF ONCOGENIC MICRORNAS

INTRODUCTION AND OBJECTIVES: Management of castration-resistant prostate cancer (CPRC) remains challenging due to the inevitable emergence of resistance to treatments including radiotherapy (RT) and chemotherapy (CT). We previously reported that zoledronic acid (ZOL) clinically potentiates the antitumor effects of RT in patients with renal cell carcinoma (Kijima et al, BJU Int 2009) and that this radiosensitization could occur through the osteoclast-independent inhibition of signal transducer and activator of transcription 1 (STAT1) (Kijima et al, PLoS One 2013). As the association between STAT1 overexpression and treatment resistance has been reported in several cancers, we investigated whether STAT1 is associated with resistance to RT and CT in CRPC cells and whether ZOL could overcome this resistance by downregulating STAT1. METHODS: Baseline expression of STAT1 was compared between androgen-dependent LNCaP cells and androgen-independent LNCaP (LNCaP-CR), PC3, and DU145 cells. The effect of ZOL on STAT1 expression was evaluated by Western blot and real-time PCR. The sensitizing effects of ZOL on RT and CT (docetaxel) were examined by clonogenic assay and MTS assay with combination index analysis. To confirm the importance of STAT1 on radioand chemosensitization by ZOL, both siRNA knockdown and forced expression by cDNA transfection were performed. RESULTS: STAT1 levels were higher in androgen-independent cell lines (PC3, DU145) than in LNCaP cells (Figure A). STAT1 was gradually upregulated in LNCaP as these cells acquired androgen independency through continuous androgen ablation (Figure B). ZOL decreased STAT1 at the protein level (Figure C) through proteasomemediated degradation and sensitized PC3 and DU145 to both RT and CT. Functional siRNA knockdown of STAT1 resulted in the sensitization of DU145 to RT and CT. Forced expression of STAT1 in LNCaP cells rendered them resistant to those therapies. CONCLUSIONS: ZOL sensitizes CRPC cells to RT and CT by downregulating STAT1.

Abstract C171: Human anti-Nucleolin recombinant immunoagents as new potential tools for melanoma treatment

Immunotherapy and immune-based anti-cancer molecules represent a valid strategy to fight cancer. However, the choice of tumor-specific surface molecules for the selective targeting of cancer cells still represents a critical step in the study design for the development of new therapeutic approaches. Notably, the development of phage-display technology for the selection of fully human single chain antibody fragments (scFvs) and complete antibodies directed toward tumor-associated antigens has represented a significant advancement for immunotherapy. Nucleolin (NCL) is one of the most abundant non-ribosomal proteins in the nucleolus. NCL is frequently up-regulated in cancer and in cancer-associated endothelial cells compared to normal tissues, where it is also present on the cell surface. Altered NCL expression and localization results in oncogenic effects such as stabilization of oncogenic mRNAs and microRNAs (miRNAs). Particularly, we demonstrated that NCL enhances the maturation of specific miRNAs (including miR-21, miR-221 and miR-222) causally involved in cancer pathogenesis, aggressiveness, metastatic potential and resistance to several anti-neoplastic treatments. Because of its oncogenic role and specific expression on cancer cell surface, NCL represents an attractive target for anti-neoplastic therapies. To produce a new anti-NCL molecule with significant potential for clinical applications, we took advantage of phage-display technology to isolate a fully human single chain Fragment variable (scFv), named 4LB5, which binds with high affinity to the RNA binding domain (RBD) of NCL. In our previous study we demonstrated that 4LB5 binds NCL on the surface of aggressive breast cancer cells and inhibits their proliferation both in vitro and in vivo, representing the prototype of a new class of immune-based anti-NCL compounds. Since NCL expression has been previously reported on the cell surface of skin cancer cell lines and up-regulation of NCL-dependent microRNAs was described in human melanomas, the objective of this project was the assessment of 4LB5 as a potential tool for melanoma therapy. To this aim, the recombinant scFv was expressed as His6-fusion protein in E.Coli and purified by affinity chromatography, as previously described. By using Enzyme-Linked Immunosorbent Assays (ELISA), we demonstrated a significant binding of 4LB5 to the cell surface of different melanoma cell lines of both human and mouse origins. Notably, inhibition of NCL expression by siRNA transfection reduced the binding of 4LB5 to the cell surface of these cell lines, further supporting its specificity against NCL. Then, we assessed the potential effects of 4LB5 treatment on cell proliferation. Colony formation assays demonstrated that 4LB5 significantly affected cell proliferation of both human and mouse melanoma cell lines. Our results, in agreement with previously reported data, further support the potential activity of 4LB5 as a tool for cancer therapy, paving the way for additional investigations aimed to fully elucidate the molecular mechanisms affected by this scFv and resulting in its anti-neoplastic therapy in human melanomas. Furthermore, this study supports the idea that anti-NCL immunoagents might represent a class of new anti-cancer compounds with a strong clinical relevance for a wide range of human tumors. Citation Format: Ashley Braddom, Timothy Richmond, Tyler Sheetz, Erika Reese, Anna Tessari, Kathleen Tober, Christin E. Burd, Claudia De Lorenzo, Edward W. Martin, Jr., Vincenzo Coppola, Michael F. Tweedle, Tatiana Oberyszyn, Carlo M. Croce, Dario Palmieri. Human anti-Nucleolin recombinant immunoagents as new potential tools for melanoma treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C171.

Abstract C17: Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients

Background: MicroRNAs are a class of small non-coding RNAs that modulate gene expression at post-transcriptional level, resulting crucial in many physiological and pathological processes. Their involvement in cancer initiation and tumor progression has been clearly established, as well as their linkage with chemoresistance. Gemcitabine is a nucleoside analog used in the treatment of metastatic breast cancer (MBC), characterized by a favorable safety profile. Only a small percentage of pts show a strong and prolonged response to this drug, thus suggesting the need of predictive biomarkers. Here, we aimed to identify microRNAs whose expression could be related to gemcitabine sensitivity in MBC. Methods: 24 MBC pts were treated with gemcitabine as single-agent therapy from 1999 to 2011 at Fondazione IRCCS Istituto Nazionale Tumori (Milan). They were selected and classified by the response to the treatment (11 gemcitabine sensitive, stable or partial response after >24 weeks of treatment, and 13 gemcitabine resistant, progressive disease within 16 weeks from the first drug administration). MicroRNA expression profiling was performed by Nanostring nCounter Technology using RNA from FFPE metastatic lesions. Biological normalization was executed to correct for differences in sample abundances. Each sample was normalized to the geometric mean of the top 100 most highly expressed microRNAs. Student9s t test was used to calculate statistical significances of pairwise comparisons. The nCounter analysis was validated on a wider population of 50 MBC pts (17 sensitive and 33 resistant) by qRT-PCR. The correlation between microRNA expression levels and gemcitabine half maximal inhibitory concentration (IC50) was evaluated using different BC cell lines. Finally, gemcitabine sensitivity was assessed on these cell lines by cell survival assays following exogenous modulation of microRNA expression levels. Results: Median age at diagnosis was 47 yrs (range 21-74 yrs). Hormone receptors were positive in thirty-one pts (62%), while Her2 in 15 cases (30%). Soft tissues were the most frequent site of metastasis (84%), followed by bone (64%), liver (36%), lung (34%) and CNS (16%). Sixteen pts (32%) had local recurrences. Median number of chemotherapies and endocrine therapies received prior to gemcitabine was 3 (range 1-6) and 2 (range 1-5) respectively. From the nCounter analysis, a subset of 20 microRNAs was significantly deregulated (p Conclusions: We identified miR-135b, miR-146b and miR-155 as potentially predictive for gemcitabine sensitivity in MBC pts. In particular, miR-135b appears to be causally involved in BC cells response to gemcitabine. These results pave the way to a better understanding of the molecular mechanisms underlying gemcitabine resistance in BC, and may have a clinical impact to identify those patients expected to obtain strong and prolonged benefits from this well-tolerated treatment. Citation Format: Anna Tessari, Dario Palmieri, Giovanni Nigita, Dario Veneziano, Sara Cresta, Biagio Paolini, Maria Silvia Cona, Taylor Vargo, Erika Reese, Tyler Sheetz, Vincenzo Coppola, Filippo De Braud, Carlo M. Croce. Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C17.

Abstract LB-289: Interaction between miR-155 and Quaking in the innate immune response and leukemia

Rationale: miR-155 is a pro-inflammatory microRNA upregulated in human and mouse macrophages exposed to lipopolysaccharide (LPS) that is required to mount an effective immune response. High levels of miR-155 are observed in different solid tumors as well as leukemias, including chronic lymphocytic leukemia (CLL). Quaking ( QKI ) is a tumor-suppressor gene encoding a conserved RNA-binding protein. In silico analyses predict that the QKI transcript is a target of miR-155 , and we hypothesized that miR-155 might carry out its pro-inflammatory and oncogenic signals at least in part by targeting QKI . Methods: Mouse RAW-264.7 macrophages were stimulated with LPS or mock PBS three times over a period of 6 days. qRT-PCR was used to monitor the expression of QKI , miR-155 and Tnf (tumor necrosis factor alpha), and Quaking protein (Qki) expression was analyzed by Western blot. Expression of miR-155 and QKI in Burkitt9s lymphoma cell lines and CLL cell lines was measured by qRT-PCR; QKI expression was also determined by Western blot. Cell lines were transfected with miR-155 or miR-control, or 155-I , and Western blot analysis of QKI was performed after 48 hours. To study the in vivo effects of the cooperation between miR-155 and QKI , we analyzed Qki expression in splenic B cells from wild type C57B1/6 mice, miR-155 −/- mice, and Eμ-miR-155 transgenic mice (which develop aggressive CLL), with and without LPS challenge. Finally, expression of Qki was determined in samples from patients with B-cell CLL. Results: After 8 hours of LPS challenge there was a 2-fold decrease in QKI expression, while miR-155 and Tnf both increased approximately 10-fold (p QKI returned to pre-treatment levels at 2 days, while miR-155 remained high for 3 days. LPS re-stimulation at 3 days (mimicking chronic inflammation) reduced QKI expression 2.3-fold (p miR-155 , and Western blotting confirmed the above observed changes for Qki. CLL-derived cell lines showed significantly higher expression of miR-155 , and lower expression of QKI , compared with Burkitt9s lymphoma cell lines (p miR-155 led to decreased Qki expression in Burkitt9s lymphoma lines, while 155-I transfection of MEC2 CLL cells caused Qki upregulation at 48 hours. Splenic B cells from leukemic Eμ-miR-155 mice had decreased Qki levels compared with C57B1/6 mice and miR-155 −/− mice, and LPS challenge of cultured B cells resulted in a pronounced downregulation of Qki expression only in B cells coming from Eμ-miR-155 mice. Finally, reduced expression of Qki was observed in B cells coming from patients with CLL compared with healthy patients. Conclusions: The inverse relationship between miR-155 and Qki is specific, and may represent a regulatory mechanism for the immune response mounted by macrophages and B cells. This relationship also sheds light on the pro-inflammatory and oncogenic properties of miR-155 in leukemia. Citation Format: Timothy K. Richmond, Esmerina Tili, Melissa Brown, Marcela Chiabai, Dario Palmieri, Ri Cui, Tyler Sheetz, Carlo M. Croce. Interaction between miR-155 and Quaking in the innate immune response and leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2015-LB-289