Browsing by Author "Prabhu, Lakshmi"

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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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    Critical role of NF-κB in pancreatic cancer
    (Impact Journals, 2014-11-30) Prabhu, Lakshmi; Mundade, Rasika; Korc, Murray; Loehrer, Patrick J.; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, and in spite of intense efforts there are limited therapeutic options for patients with PDAC. PDACs harbor a high frequency of Kras mutations and other driver mutations that lead to altered signaling pathways and contribute to therapeutic resistance. Importantly, constitutive activation of nuclear factor κB (NF-κB) is frequently observed in PDAC. An increasing body of evidence suggests that both classical and non-classical NF-κB pathways play a crucial role in PDAC development and progression. In this review, we update the most recent advances regarding different aspects of NF-κB involvement in PDAC development and progression, emphasizing its potential as a therapeutic target and the need to discover pathway-specific cytosolic NF-κB regulators which could be used to design novel therapeutic strategies for PDAC.
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    Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF- B in colon cancer
    (Office of the Vice Chancellor for Research, 2015-04-17) Prabhu, Lakshmi; Mundade, Rasika; Wang, Benlian; Wei, Han; Hartley, Antja-Voy; McElyea, Kyle; Temm, Constance J.; Sandusky, George; Liu, Yunlong; Lu, Tao
    Y-box binding protein 1 (YBX1) is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor B (NF-B), has never been previously reported. Here, we show that overexpression of wild type YBX1 (wtYBX1) activates NF-B, suggesting that YBX1 is a potential NF-B activator. Furthermore, using mass spectrometry analysis, we identified novel phosphorylation of serine 165 (S165) on YBX1. Overexpression of the S165A-YBX1 mutant in either 293 cells or colon cancer HT29 cells showed dramatically reduced NF-B activating ability as compared to that of wtYBX1, confirming that S165 phosphorylation is critical for the activation of NF-B by YBX1. We further show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-B-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared to wtYBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-B activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.
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    Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer.
    (Impact Journals, 2015-10-06) Prabhu, Lakshmi; Mundade, Rasika; Wang, Benlian; Wei, Han; Hartley, Antja-Voy; Martin, Matthew; McElyea, Kyle; Temm, Constance J.; Sandusky, George; Liu, Yunlong; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of
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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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    Genetic pathways, prevention, and treatment of sporadic colorectal cancer
    (Impact Journals, LLC, 2014) Mundade, Rasika; Imperiale, Thomas F.; Prabhu, Lakshmi; Loehrer, Patrick J.; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Epithelial cancer of the colon and rectum, also known as colorectal cancer (CRC), results from a progressive accumulation of genetic and epigenetic alterations that lead to uncontrolled growth of colonocytes, the cells lining the colon and rectum. CRC is the second leading cause of cancer-related deaths and the third most common cancer in men and in women in the U.S. Of all the patients diagnosed with CRC every year, it is estimated that the vast majority of CRCs are non-hereditary "sporadic cancers" with no apparent evidence of an inherited component. Sporadic CRC results from the cumulative effects of multiple genetic and epigenetic alterations caused by somatic mutations, which may themselves be the indirect result of several environmental factors. This review examines our current understanding of the major genetic alterations leading to colon cancer, options for prevention and early detection of CRC, and the currently available treatment approaches that may target these different genetic alterations.
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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.
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    Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer
    (American Society for Biochemistry and Molecular Biology, 2017-02-24) Martin, Matthew; Hua, Laiqing; Wang, Benlian; Wei, Han; Prabhu, Lakshmi; Hartley, Antja-Voy; Jiang, Guanglong; Liu, Yunlong; Lu, Tao; Medical and Molecular Genetics, School of Medicine
    Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer.
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    PRMT5 is a therapeutic target in choroidal neovascularization
    (Nature, 2023) Muniyandi, Anbukkarasi; Martin, Matthew; Sishtla, Kamakshi; Motolani, Aishat; Sun, Mengyao; Jensen, Nathan R.; Qi, Xiaoping; Boulton, Michael E.; Prabhu, Lakshmi; Lu, Tao; Corson, Timothy W.; Ophthalmology, School of Medicine
    Ocular neovascular diseases including neovascular age-related macular degeneration (nvAMD) are widespread causes of blindness. Patients’ non-responsiveness to currently used biologics that target vascular endothelial growth factor (VEGF) poses an unmet need for novel therapies. Here, we identify protein arginine methyltransferase 5 (PRMT5) as a novel therapeutic target for nvAMD. PRMT5 is a well-known epigenetic enzyme. We previously showed that PRMT5 methylates and activates a proangiogenic and proinflammatory transcription factor, the nuclear factor kappa B (NF-κB), which has a master role in tumor progression, notably in pancreatic ductal adenocarcinoma and colorectal cancer. We identified a potent and specific small molecule inhibitor of PRMT5, PR5-LL-CM01, that dampens the methylation and activation of NF-κB. Here for the first time, we assessed the antiangiogenic activity of PR5-LL-CM01 in ocular cells. Immunostaining of human nvAMD sections revealed that PRMT5 is highly expressed in the retinal pigment epithelium (RPE)/choroid where neovascularization occurs, while mouse eyes with laser induced choroidal neovascularization (L-CNV) showed PRMT5 is overexpressed in the retinal ganglion cell layer and in the RPE/choroid. Importantly, inhibition of PRMT5 by PR5-LL-CM01 or shRNA knockdown of PRMT5 in human retinal endothelial cells (HRECs) and induced pluripotent stem cell (iPSC)-derived choroidal endothelial cells (iCEC2) reduced NF-κB activity and the expression of its target genes, such as tumor necrosis factor α (TNF-α) and VEGF-A. In addition to inhibiting angiogenic properties of proliferation and tube formation, PR5-LL-CM01 blocked cell cycle progression at G1/S-phase in a dose-dependent manner in these cells. Thus, we provide the first evidence that inhibition of PRMT5 impedes angiogenesis in ocular endothelial cells, suggesting PRMT5 as a potential therapeutic target to ameliorate ocular neovascularization.