Browsing by Author "Pollok, Karen"

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    ARF6 is a Novel Target for Immunotherapy in Triple Negative Breast Cancer
    (2024-07) Moulana, Fathima Ishara; Lu, Xiongbin; Pollok, Karen; Hopewell, Emily; Liu, Jing
    Triple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes with poor clinical outcomes due to lack of effective treatments owing to its hormone receptor negative status. Immune checkpoint blockade (ICB) therapy, which prevents the exhaustion of CD8+ T cells, has shown promise in treating these patients. However, only a small proportion respond, possibly due to resistance and immune evasion mechanisms by the tumor cells. A primary mechanism by which tumor cells evade immune surveillance is by reduced tumor antigen presentation, as indicated by a decreased level of antigen-MHC-I (major histocompatibility complex-I) on the surface of tumor cells. The dynamics of tumor antigens on the cell surface and how cell endocytosis contributes to antigen presentation and their recycling is little known. Here we sought to study the roles of two proteins: clathrin and ADP-Ribosylation Factor 6 (ARF6) which are essential for clathrin-mediated endocytosis and clathrin-independent endocytosis respectively, on the surface turnover of fluorophore-conjugated antigenic peptide bound to MHC-I. We employed Total Internal Reflection Fluorescence Microscopy (TIRFM) and Single Molecule Tracking (SMT) to determine the dynamics of tumor antigen endocytosis on the surface of EO771 murine TNBC cells. We found that the inhibition of ARF6 remarkably impaired the endocytosis of the antigen-MHC-I foci, leading to extended stay of the foci on the cell membrane, while inhibition of clathrin did not, suggesting that clathrin-independent endocytosis is the primary route for MHC-I-mediated antigen endocytosis. Consistent with this finding, reduced ARF6 levels promoted in vitro tumor cell killing by CD8+ T cells and suppressed tumor growth in mice when combined with ICB therapy. We further investigated the effect of pharmacological inhibition of ARF6 in murine TNBC cells and splenic CD8+ T cells using a commercially available ARF6 inhibitor NAV-2729. Treatment with NAV-2729 increased surface MHC-I levels and enhanced the secretion of T cell functional markers such as IFN-, TNF- and IL-2, suggesting the possibility of in vivo administration of ARF6 inhibitors in combination with ICB therapy. Collectively, these data suggest that ARF6 is a novel target for the combined treatment with ICB therapy to improve its efficacy in TNBC patients.
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    Dissecting the Role of Novel O-GlcNAcylation of NF-κB in Pancreatic Cancer
    (2024-06) Motolani, Aishat Abiola; Lu, Tao; Safa, Ahmad; Dong, Charlie; Pollok, Karen; Corson, Timothy
    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited PDAC cellular proliferation, migration, and anchorage-independent growth compared to WT-p65. We also show that inhibition of NF-κB O-GlcNAcylation may mitigate gemcitabine resistance and enhance its efficacy in PDAC cells. Collectively, our study uncovers a novel aspect of NF-κB regulation, which could aid future therapeutic development by targeting O-GlcNAc transferase (OGT) in pancreatic cancer.
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    Exploring a structural protein-drug interactome for new therapeutics in lung cancer
    (Royal Society of Chemistry, 2014-03-04) Peng, Xiaodong; Wang, Fang; Li, Liwei; Bum-Erdene, Khuchtumur; Xu, David; Wang, Bo; Sinn, Tony; Pollok, Karen; Sandusky, George; Li, Lang; Turchi, John; Jalal, Shadia I.; Meroueh, Samy; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The pharmacology of drugs is often defined by more than one protein target. This property can be exploited to use approved drugs to uncover new targets and signaling pathways in cancer. Towards enabling a rational approach to uncover new targets, we expand a structural protein-ligand interactome () by scoring the interaction among 1000 FDA-approved drugs docked to 2500 pockets on protein structures of the human genome. This afforded a drug-target network whose properties compared favorably with previous networks constructed using experimental data. Among drugs with the highest degree and betweenness two are cancer drugs and one is currently used for treatment of lung cancer. Comparison of predicted cancer and non-cancer targets reveals that the most cancer-specific compounds were also the most selective compounds. Analysis of compound flexibility, hydrophobicity, and size showed that the most selective compounds were low molecular weight fragment-like heterocycles. We use a previously-developed screening approach using the cancer drug erlotinib as a template to screen other approved drugs that mimic its properties. Among the top 12 ranking candidates, four are cancer drugs, two of them kinase inhibitors (like erlotinib). Cellular studies using non-small cell lung cancer (NSCLC) cells revealed that several drugs inhibited lung cancer cell proliferation. We mined patient records at the Regenstrief Medical Record System to explore the possible association of exposure to three of these drugs with occurrence of lung cancer. Preliminary in vivo studies using the non-small cell lung cancer (NCLSC) xenograft model showed that losartan- and astemizole-treated mice had tumors that weighed 50 (p < 0.01) and 15 (p < 0.01) percent less than the treated controls. These results set the stage for further exploration of these drugs and to uncover new drugs for lung cancer therapy.
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    EXTH-43. Targeting the DNA Damage Response Through Combination MDM2 and AKT Inhibitor Therapy Improves Temozolomide Effectiveness in Chemo-Resistant Glioblastoma
    (Oxford University Press, 2023-11-10) Koenig, Jenna; Bailey, Barbara; Alfonso, Anthony; Saadatzadeh, M. Reza; Bijangi-Vishehsaraei, Khadijeh; Pandya, Pankita; Damayanti, Nur; Dobrota, Erika; Young, Courtney; Shannon, Harlan; Pollok, Karen; Graduate Medical Education, School of Medicine
    Temozolomide remains the lone pharmacotherapeutic option for glioblastoma (GBM), yet the development of resistance to temozolomide has been a major challenge contributing to the persistent median < 2-year survival for patients after diagnosis. Tumor heterogeneity and induction of treatment response networks, such as the DNA damage response (DDR), are major contributors to temozolomide resistance in GBM. Targeting DDR treatment response networks, such as the MDM2/p53/p73 and PI3K/AKT/mTOR networks, with small-molecule inhibitors (SMIs) presents an opportunity to disrupt resistance mechanisms and enhance temozolomide efficacy. We utilized a triple drug combination of clinically relevant concentrations of the blood-brain-barrier penetrant SMIs of AKT (ipatasertib; GDC-0068) and MDM2 (idasanutlin; RG7388) with temozolomide to evaluate this targeted strategy using the recurrent, temozolomide-resistant, p53wt GBM10 xenoline. Proliferation studies demonstrated dose-related additive to synergistic inhibition of proliferation at clinically relevant concentrations of ipatasertib and idasanutlin. Further, IncuCyte live-cell imaging demonstrated dose-and time-related growth inhibition of these GBM cells and apoptosis marked by increased cleaved caspase 3 expression following the temozolomide+idasanutlin+ipatasertib triple combination treatment. Cells treated with temozolomide+idasanutlin+ipatasertib also displayed senescence phenotypes, with increased cell cycle arrest and elevated expression of SPiDER β-Gal expression and cell-cycle inhibitors such as p53 and p21. Experiments are in progress to determine the extent to which the effects of temozolomide+idasanutlin+ipatasertib combination therapy are dependent on p53 using siRNA knockdown of p53. In the present study, targeting the temzolomide-induced DNA damage response with idasanutlin+ipatasertib increased the effectiveness of temozolomide. These results indicate that this triple combination may be a promising approach to improving patient outcomes in temozolomide-resistant GBM.
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    G2 Cell Cycle Arrest and Cyclophilin A in Lentiviral Gene Transfer
    (Elsevier, 2006-10-01) Zhang, Shangming; Joseph, Guiandre; Pollok, Karen; Berthoux, Lionel; Sastry, Lakshmi; Luban, Jeremy; Cornetta, Kenneth; Medical and Molecular Genetics, School of Medicine
    Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34+ peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, rather than PTK inhibition, appears to be the major factor responsible for increased gene transfer. Genistein also increases cyclophilin A (CypA) protein, a cellular protein important for efficient HIV-1 infection. While we show that CypA−/− Jurkat cells transduce poorly with lentiviral vectors, genistein does increase gene transfer in CypA-deficient cells. CypA and G2 cell cycle arrest appear to be two independent factors important for efficient lentiviral gene transfer. The role of genistein and other G2-arresting agents may be useful for improving the efficiency of lentiviral gene therapy.
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    Genetic Approach to Discover ARMC4 as a Novel NF-κB Negative Regulator and Tumor Suppressor in Colorectal Cancer
    (2020-04) Martin, Matthew Peter; Lu, Tao; Safa, Ahmad; Corson, Tim; Jerde, Travis; Pollok, Karen
    The nuclear factor κB (NF-κB) plays pivotal roles in inflammatory and immune responses and in cancer. Therefore, understanding its regulation holds great promise for disease therapy. Using validation-based insertional mutagenesis (VBIM), a powerful technique established by us, we discovered armadillo repeat containing protein 4 (ARMC4) as a novel negative regulator of NF-κB in colorectal cancer (CRC). ARMC4 is a rarely studied protein only known to date for its role in primary ciliary dyskinesia (PCD) and mouse spermatogenesis. Thus, my work reveals a completely new facet of ARMC4 function that has never been reported before. We showed that ARMC4 overexpression downregulated the expression of NF-κB-dependent genes, many of which are related to cancer. Additionally, compared to the vector control group, overexpression of ARMC4 in HEK293 cells or CRC HT29, DLD1, and HCT116 cells dramatically reduced NF-κB activity, cellular proliferation, anchorage-independent growth, and migratory ability in vitro, and unsurprisingly, significantly decreased xenograft tumor growth in vivo. In contrast, shARMC4 knockdown cells showed quite opposite effect. Furthermore, co-immunoprecipitation (Co-IP) experiment confirmed that ARMC4 may form a complex with the p65 subunit of NF-κB. Importantly, immunohistochemistry (IHC) data exhibited much lower ARMC4 expression level in CRC patient tumor tissues compared to normal tissues, indicating that ARMC4 may function as a tumor suppressor in CRC. To conclude, my important findings for the first time uncovered the negative regulatory function of ARMC4 in NF-κB signaling, and present ARMC4 as an innovative therapeutic target in CRC treatment.
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    Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium
    (American Thoracic Society, 2016-12) Pandya, Pankita H.; Fisher, Amanda J.; Mickler, Elizabeth A.; Temm, Constance J.; Lipking, Kelsey P.; Gracon, Adam; Rothhaar, Katia; Sandusky, George E.; Murray, Mary; Pollok, Karen; Renbarger, Jamie; Blum, Janice S.; Lahm, Tim; Wilkes, David S.; Microbiology and Immunology, School of Medicine
    Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.
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    Lentiviral vectors pseudotyped with glycoproteins from Ross River and vesicular stomatitis viruses: variable transduction related to cell type and culture conditions
    (Elsevier, 2005-03-01) Kahl, Christoph A.; Pollok, Karen; Haneline, Laura S.; Cornetta, Kenneth; Medicine, School of Medicine
    HIV-1-derived lentiviral vectors have been pseudotyped with various envelope glycoproteins to alter their host range. Previously, we found that envelope glycoproteins derived from the alphavirus Ross River virus (RRV) can pseudotype lentiviral vectors and mediate efficient transduction of a variety of epithelial and fibroblast-derived cell lines. In this study, we have investigated transduction of hematopoietic cells using RRV-pseudotyped vectors encoding the enhanced green fluorescent protein (EGFP). RRV-mediated transduction of human CD34+ cord blood cells and progenitors was very inefficient, even at multiplicities of infection of 100 (0.4% EGFP-positive progenitor colonies). Inefficient transduction was also observed in a variety of hematopoietic cell lines. However, two erythroleukemia-derived cell lines and monocytic cells that were driven to macrophage-like differentiation were moderately transduced. Transduction of hematopoietic cells with a control VSV-G-pseudotyped lentiviral vector was generally efficient, but unexpectedly decreased up to threefold upon stimulation of lymphocytic cell lines or primary murine bone marrow cells. Also, the tested hematopoietic cell lines were essentially nonpermissive for adeno-associated type 2 (AAV) vectors, and this was not affected by lineage, activity, or differentiation. Treatment of permissive 293 cells with proteases revealed that transduction with both the RRV- and the VSV-G-pseudotyped vectors in part depends on the presence of cell surface proteins. These results show a severely restricted ability of RRV glycoproteins to mediate transduction in hematopoietic cells that is likely due to specific receptor requirements that differ from those of VSV-G and AAV. Conversely, transduction with the VSV glycoprotein is affected by cellular activation more than widely believed. Our findings suggest that the envelope glycoproteins and culture conditions employed need to be carefully evaluated for each application. Furthermore, the uniquely restricted host range of RRV-pseudotyped vectors may aid in the design of novel cell-selective transduction strategies.
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    Pharmacological Depletion of Fibrinogen Suppresses the Growth of Primary Tumors and Metastasis of Pancreatic Ductal Adenocarcinoma (PDAC)
    (2024-08) Chowdhury, Nayela Nabiha; Fishel, Melissa L.; Yeh, Elizabeth; Pollok, Karen; Manchanda, Naveen; Jerde, Travis J.
    PDAC is a highly metastatic disease often linked to the dysregulated activation of both coagulation and fibrinolytic systems. Clinically, patients show elevated plasma fibrinogen levels, particularly in those with distant metastasis. The presence of systemic fibrinogen plays a crucial role in shaping the complex tumor microenvironment characteristic of PDAC, evidenced by the excessive deposition of its active substrate, fibrin, in PDAC tumors. To investigate its contribution to disease progression, fibrinogen was significantly depleted from the TME in multiple PDAC patient-derived xenograft (PDX) models, and the impact on tumor growth and metastasis was followed. In an aggressive, metastatic orthotopic Pa03C model, Fib depletion using antisense oligonucleotide (ASO) treatment markedly decreased the size of primary pancreatic tumors and subsequent spontaneous metastasis to the liver. Following implantation of tumor chunks (PDX21) from a patient with high Fib staining, Fib ASO treatment significantly diminished growth of primary tumors. Using a third orthotopic model (PDX33) and lipid nanoparticle (LNP)-encapsulated-siRNA to deplete the fibrinogen--chain, tumor growth was significantly reduced compared to control. This confirms that Fib deposition within the tumor microenvironment (TME) is an important driver of disease progression. Mechanistically, global proteomics revealed a remarkable upregulation of matrisome and extracellular matrix (ECM)-associated proteins, indicating that the reduction in primary orthotopic Pa03C tumors was associated with fibrin-mediated TME remodeling. Loss of fibrin matrices led to enhanced collagen, laminin, fibronectin and emilin1 deposition, and increased recruitment of collagen-producing αSMA+ myofibroblasts. Notably, our data suggests that fibrinogen depletion altered the ECM composition, inducing TME remodeling which led to stiffer tumors that are less metastatic. To further interrogate the role of fibrinogen in mediating metastasis, we employed an experimental metastasis model to study the colonization of tumor cells in the liver in the absence of fibrinogen. In this model, fibrinogen depletion did not impede the colonization of Pa03C cells, suggesting that fibrinogen does not mediate the colonization of tumor cells in the cascade of events for liver metastasis and is likely involved in one of the other steps of metastasis. Collectively, our data showed that pharmacologically reducing systemic fibrinogen levels impeded tumor growth and metastasis by remodeling the TME.
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    Romidepsin is a Potential Therapeutic to Treat Metastatic Osteosarcoma
    (2024-12) Seiden, Emily Elizabeth; Greenfield, Ed; Byram, Jessica; Collier, Chris; McNulty, Margaret; Pollok, Karen
    Osteosarcoma is the most common primary malignant bone tumor in children, adolescents, and dogs. Lethality is due to lung metastases. Despite standard of care, patients with lung metastases have a survival rate of only 25%. Therefore, our lab studied potential therapeutics to target lung metastases. To do this, our lab repurposed already FDA-approved oncology drugs, which saves on the extensive time and cost of traditional drug discovery. Our lab also used 3-D spheroids (sarcospheres) that mimic in vivo tumors. In initial drug screens, our lab identified romidepsin, a histone deacetylase inhibitor, as a potent inhibitor of viability of highly metastatic osteosarcoma sarcospheres that was non-toxic to normal cells. Romidepsin was additive-to-synergistic with standardof- care chemotherapy and inhibited viability in a subset of patient-derived sarcospheres. This thesis therefore focused on determining the mechanism of action of romidepsin in osteosarcoma sarcospheres and the efficacy of romidepsin in a mouse model of metastatic osteosarcoma. Romidepsin either blocked growth or increased cell death in a growth phenotype dependent manner. Sarcospheres that grow slowly showed an increase in cell death, while those that grow quickly showed a cell cycle arrest. This trend differs in patient-derived sarcospheres. Though they grow slowly, only the most sensitive patient sample showed an increase in cell death. Our sarcosphere assay may be able to be used clinically to identify which patients are most likely to respond to specific drugs. In a tail vein injection mouse model of metastatic osteosarcoma, romidepsin increased survival and reduced respiratory distress in immunocompetent mice, but not in immunocompromised mice. This indicated that romidepsin may act on the immune system to have an effect. Therefore, it may be essential to use a mouse model with an intact immune system for romidepsin to decrease tumor burden. Overall, romidepsin is a potential therapeutic to treat a subset of metastatic osteosarcoma patients.