Browsing by Author "Pridham, Kevin J."
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- Casein Kinase 1 Epsilon Regulates Glioblastoma Cell SurvivalVarghese, Robin T.; Young, Sarah; Pham, Lily; Liang, Yanping; Pridham, Kevin J.; Guo, Sujuan; Murphy, Susan F.; Kelly, Deborah F.; Sheng, Zhi (Nature, 2018-09-11)Glioblastoma is the most common malignant brain cancer with a dismal prognosis. The difficulty in treating glioblastoma is largely attributed to the lack of effective therapeutic targets. In our previous work, we identified casein kinase 1 ε (CK1ε, also known as CSNK1E) as a potential survival factor in glioblastoma. However, how CK1ε controls cell survival remains elusive and whether targeting CK1ε is a possible treatment for glioblastoma requires further investigation. Here we report that CK1ε was expressed at the highest level among six CK1 isoforms in glioblastoma and enriched in high-grade glioma, but not glia cells. Depletion of CK1ε remarkably inhibited the growth of glioblastoma cells and suppressed self-renewal of glioblastoma stem cells, while having limited effect on astrocytes. CK1ε deprivation activated β-catenin and induced apoptosis, which was further counteracted by knockdown of β-catenin. The CK1ε inhibitor IC261, but not PF-4800567, activated β-catenin and blocked the growth of glioblastoma cells and glioblastoma stem cells. Congruently, IC261 elicited a robust growth inhibition of human glioblastoma xenografts in mice. Together, our results demonstrate that CK1ε regulates the survival of glioblastoma cells and glioblastoma stem cells through β-catenin signaling, underscoring the importance of targeting CK1ε as an effective treatment for glioblastoma.
- Connexin 43 confers chemoresistance through activating PI3KPridham, Kevin J.; Shah, Farah; Hutchings, Kasen R.; Sheng, Kevin L.; Guo, Sujuan; Liu, Min; Kanabur, Pratik; Lamouille, Samy Y.; Lewis, Gabrielle; Morales, Marc; Jourdan, L. Jane; Grek, Christina L.; Ghatnekar, Gautam S.; Varghese, Robin T.; Kelly, Deborah F.; Gourdie, Robert G.; Sheng, Zhi (Springer Nature, 2022-01-12)Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.
- Functional Blockade of Small GTPase RAN Inhibits Glioblastoma Cell ViabilitySheng, Kevin L.; Pridham, Kevin J.; Sheng, Zhi; Lamouille, Samy Y.; Varghese, Robin T. (Frontiers, 2019-01-08)Glioblastoma, the most common malignant tumor in the brain, lacks effective treatments and is currently incurable. To identify novel drug targets for this deadly cancer, the publicly available results of RNA interference screens from the Project Achilles database were analyzed. Ten candidate genes were identified as survival genes in 15 glioblastoma cell lines. RAN, member RAS oncogene family (RAN) was expressed in glioblastoma at the highest level among all candidates based upon cDNA microarray data. However, Kaplan-Meier survival analysis did not show any correlation between RAN mRNA levels and patient survival. Because RAN is a small GTPase that regulates nuclear transport controlled by karyopherin subunit beta 1 (KPNB1), RAN was further analyzed together with KPNB1. Indeed, GBM patients with high levels of RAN also had more KPNB1 and levels of KPNB1 alone did not relate to patient prognosis. Through a Cox multivariate analysis, GBM patients with high levels of RAN and KPNB1 showed significantly shorter life expectancy when temozolomide and promotermethylation of O6-methylguanine DNA methyltransferase were used as covariates. These results indicate that RAN and KPNB1 together are associated with drug resistance and GBM poor prognosis. Furthermore, the functional blockade of RAN and KPNB1 by importazole remarkably suppressed cell viability and activated apoptosis in GBM cells expressing high levels of RAN, while having a limited effect on astrocytes and GBM cells with undetectable RAN. Together, our results demonstrate that RAN activity is important for GBM survival and the functional blockade of RAN/KPNB1 is an appealing therapeutic approach.
- Immunoregulatory and neutrophil-like monocyte subsets with distinct single-cell transcriptomic signatures emerge following brain injuryGudenschwager Basso, Erwin K.; Ju, Jing; Soliman, Eman; de Jager, Caroline; Wei, Xiaoran; Pridham, Kevin J.; Olsen, Michelle L.; Theus, Michelle H. (2024-02-03)Monocytes represent key cellular elements that contribute to the neurological sequela following brain injury. The current study reveals that trauma induces the augmented release of a transcriptionally distinct CD115+/Ly6Chi monocyte population into the circulation of mice pre-exposed to clodronate depletion conditions. This phenomenon correlates with tissue protection, blood–brain barrier stability, and cerebral blood flow improvement. Uniquely, this shifted the innate immune cell profile in the cortical milieu and reduced the expression of pro-inflammatory Il6, IL1r1, MCP-1, Cxcl1, and Ccl3 cytokines. Monocytes that emerged under these conditions displayed a morphological and gene profile consistent with a subset commonly seen during emergency monopoiesis. Single-cell RNA sequencing delineated distinct clusters of monocytes and revealed a key transcriptional signature of Ly6Chi monocytes enriched for Apoe and chitinase-like protein 3 (Chil3/Ym1), commonly expressed in pro-resolving immunoregulatory monocytes, as well as granule genes Elane, Prtn3, MPO, and Ctsg unique to neutrophil-like monocytes. The predominate shift in cell clusters included subsets with low expression of transcription factors involved in monocyte conversion, Pou2f2, Na4a1, and a robust enrichment of genes in the oxidative phosphorylation pathway which favors an anti-inflammatory phenotype. Transfer of this monocyte assemblage into brain-injured recipient mice demonstrated their direct role in neuroprotection. These findings reveal a multifaceted innate immune response to brain injury and suggest targeting surrogate monocyte subsets may foster tissue protection in the brain.
- Investigating Novel Targets to Inhibit Cancer Cell SurvivalPridham, Kevin J. (Virginia Tech, 2018-04-18)Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.
- A large-scale RNA interference screen identifies genes that regulate autophagy at different stagesGuo, Sujuan; Pridham, Kevin J.; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R.; Sheng, Zhi (Nature Publishing Group, 2018-02-12)Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.
- The leucine-NH4(+) uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeastCherkasova, Vera; Iben, James R.; Pridham, Kevin J.; Kessler, Alan C.; Maraia, Richard J. (2021-06-21)The sla1(+) gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1 Delta) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4(+) inhibits leucine uptake and growth of sla1 Delta cells. Here, transcriptome analysis reveals that genes upregulated in sla1 Delta cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4(+) media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1 Delta GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1 Delta cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4(+)-effects, we isolated sla1 Delta spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4(+)-sensitive leucine uptake linked to TOR. We show that H-3-leucine uptake by SSR-any1-F32V cells in NH4(+)-media is more robust than by sla1 Delta cells. Moreover, F32V may alter any1(+) function in sla1 Delta vs. sla1(+) cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.
- Monocyte proinflammatory phenotypic control by ephrin type A receptor 4 mediates neural tissue damageKowalski, Elizabeth A.; Soliman, Eman; Kelly, Colin; Basso, Erwin Kristobal Gudenschwager; Leonard, John; Pridham, Kevin J.; Ju, Jing; Cash, Alison; Hazy, Amanda; de Jager, Caroline; Kaloss, Alexandra M.; Ding, Hanzhang; Hernandez, Raymundo D.; Coleman, Gabe; Wang, Xia; Olsen, Michelle L.; Pickrell, Alicia M.; Theus, Michelle H. (American Society for Clinical Investigation, 2022-08-08)Circulating monocytes have emerged as key regulators of the neuroinflammatory milieu in a number of neuropathological disorders. Ephrin type A receptor 4 (Epha4) receptor tyrosine kinase, a prominent axon guidance molecule, has recently been implicated in the regulation of neuroinflammation. Using a mouse model of brain injury and a GFP BM chimeric approach, we found neuroprotection and a lack of significant motor deficits marked by reduced monocyte/macrophage cortical infiltration and an increased number of arginase-1(+) cells in the absence of BM-derived Epha4. This was accompanied by a shift in monocyte gene profile from pro- to antiinflammatory that included increased Tek (Tie2 receptor) expression. Inhibition of Tie2 attenuated enhanced expression of M2-like genes in cultured Epha4-null monocytes/macrophages. In Epha4-BM-deficient mice, cortical-isolated GFP(+) monocytes/macrophages displayed a phenotypic shift from a classical to an intermediate subtype, which displayed reduced Ly6c(hi) concomitant with increased Ly6c(lo)- and Tie2-expressing populations. Furthermore, clodronate liposome-mediated monocyte depletion mimicked these effects in WT mice but resulted in attenuation of phenotype in Epha4-BM-deficient mice. This demonstrates that monocyte polarization not overall recruitment dictates neural tissue damage. Thus, coordination of monocyte proinflammatory phenotypic state by Epha4 is a key regulatory step mediating brain injury.
- Novel Protocols for Scalable Production of High Quality Purified Small Extracellular Vesicles from Bovine MilkMarsh, Spencer R.; Pridham, Kevin J.; Jourdan, L. Jane; Gourdie, Robert G. (Ivyspring International, 2021-07)Extracellular Vesicles (EVs) are cell-secreted nanovesicles that have unique potential for encapsulating and targeting “difficult-to-drug” therapeutic cargos. Milk provides an enriched source of EVs, and of particular interest to the drug delivery field, small EVs. Small EVs are distinguished from large EVs by membrane components, biogenesis mechanism and downstream functionality - in particular, small EVs are primarily composed of exosomes, which show high stability in vivo and naturally function in the targeted delivery of biological materials to cells. Moreover, bovine milk is abundantly produced by the dairy industry, widely consumed, and generally well tolerated by humans. Importantly, there is evidence that milk exosomes and small EVs are efficiently taken up into the circulation from the gut, providing the opportunity for their use in administration of therapeutics such as microRNAs or peptides not typically available via an oral route. Unfortunately, present methods for isolation do not efficiently separate EVs from milk proteins, resulting in contamination that is not desirable in a clinical-grade therapeutic. Herein, we present novel EV purification methods focused on optimized timing and levels of temperature and divalent cation chelation. Incorporation of these solubilization steps into centrifugation- and tangential flow filtration-based methods provide large amounts of purified small EVs at ultra-dense concentrations, which are substantially free from contaminating milk proteins. Remarkably, these ultra-dense isolates equal 10 to 15% of the starting volume of milk indicating a prodigious rate of small EV production by mammary glands. Our approach enables gentle, scalable production of ultrastructurally and functionally intact small EVs from milk, providing a path to their industrial scale purification for oral delivery of therapeutic biologics and small drugs.
- Peptidic Connexin43 Therapeutics in Cardiac Reparative MedicineMarsh, Spencer R.; Williams, Zachary J.; Pridham, Kevin J.; Gourdie, Robert G. (MDPI, 2021-05-05)Connexin (Cx43)-formed channels have been linked to cardiac arrhythmias and diseases of the heart associated with myocardial tissue loss and fibrosis. These pathologies include ischemic heart disease, ischemia-reperfusion injury, heart failure, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and Duchenne muscular dystrophy. A number of Cx43 mimetic peptides have been reported as therapeutic candidates for targeting disease processes linked to Cx43, including some that have advanced to clinical testing in humans. These peptides include Cx43 sequences based on the extracellular loop domains (e.g., Gap26, Gap 27, and Peptide5), cytoplasmic-loop domain (Gap19 and L2), and cytoplasmic carboxyl-terminal domain (e.g., JM2, Cx43tat, CycliCX, and the alphaCT family of peptides) of this transmembrane protein. Additionally, RYYN peptides binding to the Cx43 carboxyl-terminus have been described. In this review, we survey preclinical and clinical data available on short mimetic peptides based on, or directly targeting, Cx43, with focus on their potential for treating heart disease. We also discuss problems that have caused reluctance within the pharmaceutical industry to translate peptidic therapeutics to the clinic, even when supporting preclinical data is strong. These issues include those associated with the administration, stability in vivo, and tissue penetration of peptide-based therapeutics. Finally, we discuss novel drug delivery technologies including nanoparticles, exosomes, and other nanovesicular carriers that could transform the clinical and commercial viability of Cx43-targeting peptides in treatment of heart disease, stroke, cancer, and other indications requiring oral or parenteral administration. Some of these newly emerging approaches to drug delivery may provide a path to overcoming pitfalls associated with the drugging of peptide therapeutics.
- The Role of Class IA Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunits in GlioblastomaPridham, Kevin J.; Varghese, Robin T.; Sheng, Zhi (Frontiers, 2017-12-15)Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) plays a critical role in the pathogenesis of cancer including glioblastoma, the most common and aggressive form of brain cancer. Targeting the PI3K pathway to treat glioblastoma has been tested in the clinic with modest effect. In light of the recent finding that PI3K catalytic subunits (PIK3CA/p110 alpha, PIK3CB/p110 beta, PIK3CD/p110 delta, and PIK3CG/p110 gamma) are not functionally redundant, it is imperative to determine whether these subunits play divergent roles in glioblastoma and whether selectively targeting PI3K catalytic subunits represents a novel and effective strategy to tackle PI3K signaling. This article summarizes recent advances in understanding the role of PI3K catalytic subunits in glioblastoma and discusses the possibility of selective blockade of one PI3K catalytic subunit as a treatment option for glioblastoma.
- Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase betaPridham, Kevin J.; Hutchings, Kasen R.; Beck, Patrick; Liu, Min; Xu, Eileen; Saechin, Erin; Bui, Vincent; Patel, Chinkal; Solis, Jamie; Huang, Leah; Tegge, Allison; Kelly, Deborah F.; Sheng, Zhi (Elsevier, 2024-06-21)Resistance to chemotherapies such as temozolomide is a major hurdle to effectively treat therapy-resistant glioblastoma. This challenge arises from the activation of phosphatidylinositol 3-kinase (PI3K), which makes it an appealing therapeutic target. However, non-selectively blocking PI3K kinases PI3K⍺/β/𝛿/𝛾 has yielded undesired clinical outcomes. It is, therefore, imperative to investigate individual kinases in glioblastoma’s chemosensitivity. Here,wereport that PI3K kinases were unequally expressed in glioblastoma, with levels of PI3Kβ being the highest. Patients deficient of O6-methylguanine-DNA-methyltransferase(MGMT) and expressing elevated levels of PI3Kβ, defined as MGMT-deficient/PI3Kβ-high, were less responsive to temozolomide and experienced poor prognosis. Consistently, MGMT-deficient/PI3Kβ-high glioblastoma cells were resistant to temozolomide. Perturbation of PI3Kβ, but not other kinases, sensitized MGMTdeficient/ PI3Kβ-high glioblastoma cells or tumors to temozolomide. Moreover, PI3Kβ-selective inhibitors and temozolomide synergistically mitigated the growth of glioblastoma stem cells. Our results have demonstrated an essential role of PI3Kβ in chemoresistance, making PI3Kβ-selective blockade an effective chemosensitizer for glioblastoma.