Browsing by Author "Safa, Ahmad"

Now showing 1 - 10 of 18
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    A complex signature network that controls the upregulation of PRMT5 in colorectal cancer
    (Elsevier, 2022-03) Wei, Han; Hartley, Antja-Voy; Motolani, Aishat; Jiang, Guanglong; Safa, Ahmad; Prabhu, Lakshmi; Liu, Yunlong; Lu, Tao; Pharmacology and Toxicology, School of Medicine
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    Adapting AlphaLISA high throughput screen to discover a novel small-molecule inhibitor targeting protein arginine methyltransferase 5 in pancreatic and colorectal cancers
    (Impact Journals, 2017-05-23) Prabhu, Lakshmi; Wei, Han; Chen, Lan; Demir, Özlem; Sandusky, George; Sun, Emily; Wang, John; Mo, Jessica; Zeng, Lifan; Fishel, Melissa; Safa, Ahmad; Amaro, Rommie; Korc, Murray; Zhang, Zhong-Yin; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An in silico structure prediction suggested that PR5-LL-CM01 inhibits PRMT5 by binding with its active pocket. Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future.
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    Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondii
    (2017-06-28) Varberg, Joseph M.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Mosley, Amber; Safa, Ahmad; Vasko, Michael R.
    Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.
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    Characterization of a Novel Hunk Inhibitor in HER2+ Breast Cancer
    (2024-07) Dilday, Tinslee Y.; Yeh, Elizabeth; Fehrenbacher, Jill; Brustovetsy, Nickolay; Safa, Ahmad; Sankar, Uma
    Human Epidermal Growth Factor Receptor 2 (HER2)-targeted agents have proven to be effective, however, the development of resistance to these agents has become an obstacle in treating HER2+ breast cancer. Prior evidence implicates Hormonally Upregulated Neu-associated Kinase (HUNK) as an anti-cancer target for primary and resistant HER2+ breast cancers. An inhibitor Staurosporine (STU) has been identified as a HUNK inhibitor in HER2+ breast cancer. While STU was determined as a promising tool for inhibiting HUNK, it is a broad-spectrum kinase inhibitor and has not moved forward clinically. Therefore, identifying a more selective inhibitor of HUNK could be critical for targeting HUNK in HER2+ breast and understanding mechanisms by which HUNK promotes resistance to HER2-inhibitors. Specifically, HUNK has been implicated in promoting autophagy as a resistance mechanism in HER2+ breast cancer. Previously, we have identified that HUNK binds and phosphorylates an autophagy inhibitory protein, Rubicon, at Serine (S) 92 in 293T cells. This phosphorylation event causes Rubicon to switch to being an autophagy promoter. However, the role that Rubicon S92 plays in HER2+ breast cancer has yet to be examined. In this study, a novel inhibitor of HUNK is characterized alongside Rubicon S92 phosphorylation. This study establishes that HUNK-mediated phosphorylation of Rubicon at S92 promotes tumorigenesis in HER2/neu+ breast cancer. HUNK inhibition prevents S92 Rubicon phosphorylation in HER2/neu+ breast cancer models and inhibits both autophagy and tumorigenesis. This study characterizes a downstream phosphorylation event as a measure of HUNK activity and identifies a novel HUNK inhibitor that has meaningful efficacy toward HER2+ breast cancer.
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    Critical Role of Novel O-GlcNAcylation of S550 and S551 on the p65 Subunit of NF-κB in Pancreatic Cancer
    (MDPI, 2023-09-27) Motolani, Aishat; Martin, Matthew; Wang, Benlian; Jiang, Guanglong; Alipourgivi, Faranak; Huang, Xiumei; Safa, Ahmad; Liu, Yunlong; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    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 cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Collectively, we discovered novel serine sites of p65 O-GlcNAcylation that drive NF-κB activation and PDAC phenotypes, thus opening new avenues by inhibiting the NF-κB O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT), for PDAC treatment in the future.
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    Development of AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases
    (Royal Society of Chemistry, 2017-11-21) Prabhu, Lakshmi; Chen, Lan; Wei, Han; Demir, Özlem; Safa, Ahmad; Zeng, Lifan; Amaro, Rommie E.; O’Neil, Bert H.; Zhang, Zhongyin; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    The protein arginine methyltransferase (PRMT) family of enzymes comprises nine family members in mammals. They catalyze arginine methylation, either monomethylation or symmetric/asymmetric dimethylation of histone and non-histone proteins. PRMT methylation of its substrate proteins modulates cellular processes such as signal transduction, transcription, and mRNA splicing. Recent studies have linked overexpression of PRMT5, a member of the PRMT superfamily, to oncogenesis, making it a potential target for cancer therapy. In this study, we developed a highly sensitive (Z' score = 0.7) robotic high throughput screening (HTS) platform to discover small molecule inhibitors of PRMT5 by adapting the AlphaLISA™ technology. Using biotinylated histone H4 as a substrate, and S-adenosyl-l-methionine as a methyl donor, PRMT5 symmetrically dimethylated H4 at arginine (R) 3. Highly specific acceptor beads for symmetrically dimethylated H4R3 and streptavidin-coated donor beads bound the substrate, emitting a signal that is proportional to the methyltransferase activity. Using this powerful approach, we identified specific PRMT5 inhibitors P1608K04 and P1618J22, and further validated their efficacy and specificity for inhibiting PRMT5. Importantly, these two compounds exhibited much more potent efficacy than the commercial PRMT5 inhibitor EPZ015666 in both pancreatic and colorectal cancer cells. Overall, our work highlights a novel, powerful, and sensitive approach to identify specific PRMT5 inhibitors. The general principle of this HTS screening method can not only be applied to PRMT5 and the PRMT superfamily, but may also be extended to other epigenetic targets. This approach allows us to identify compounds that inhibit the activity of their respective targets, and screening hits like P1608K04 and P1618J22 may serve as the basis for novel drug development to treat cancer and/or other diseases.
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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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    FASN Negatively Regulates NF-kB/P65 Expression in Breast Cancer Cells by Disrupting Its Stability
    (2020-02) Barlow, Lincoln James; Lu, Tao; Zhang, Jian-Ting; Fehrenbacher, Jill; Herbert, Brittney-Shea; Safa, Ahmad
    The overexpression of the multi-domain enzyme fatty acid synthase (FASN) has long been associated with poor clinical prognosis and treatment outcome in various cancers. Previous research in the Zhang lab has determined a role for FASN in mediating increases in non-homologous end-joining (NHEJ) DNA double-strand break repair activity allowing for increased cancer cell survival, and this mechanism was found to involve inhibition of NF-kB/p65. The mechanism responsible for the regulation of NF-kB/p65 by FASN in cancer cells, however, remains unknown. To this end, I was able to determine that FASN negatively regulates both the expression and activity of NF-kB/p65 in breast cancer cells, and that this effect was likely mediated by the 16-carbon saturated fatty acid palmitate, the end product of FASN catalytic activity. Specifically, FASN was found to negatively regulate p65 expression by disrupting its protein stability as a result of an increase in poly-ubiquitination of p65 protein and subsequent proteasomal degradation. Further, I found that the phosphorylation site Thr254 of p65 is involved in the regulation of p65 protein stability by FASN, in that mutation of this residue resulted in a disruption in p65 stability. Finally, I was able to determine that FASN likely inhibits the ability of the peptidyl-prolyl cis/trans isomerase Pin1 to assist in maintaining p65 stability, in that both siRNA knockdown and pharmacological inhibition of Pin1 resulted in a reduction of p65 expression in FASN shRNA knockdown cells. The determination of this signaling mechanism serves to expand our understanding of the role of FASN in breast cancer cells and has the potential to assist in uncovering more effective ways to target the oncogenic FASN pathway to kill breast tumor cells and to overcome resistance to drug treatment.
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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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    Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue
    (Elsevier, 2023-01) Prabhu, Lakshmi; Martin, Matthew; Chen, Lan; Demir, Özlem; Jin, Jiamin; Huang, Xiumei; Motolani, Aishat; Sun, Mengyao; Jiang, Guanglong; Nakshatri, Harikrishna; Fishel, Melissa L.; Sun, Steven; Safa, Ahmad; Amaro, Rommie E.; Kelley, Mark R.; Liu, Yunlong; Zhang, Zhong-Yin; Lu, Tao; Radiation Oncology, School of Medicine
    Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays . Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth . Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.