Kevin J. Pridham

Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditionalEphA4^f/f/Tie2-CreandEphA4^f/f/VE-Cadherin-CreERT2knockout (KO) mice promotes blood–brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12,Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.

Glioblastoma is the most common central nervous system cancer and is responsible for approximately one-half of all malignant brain tumors. With a 5-year survival rate of 6.8% and a median observed survival time of only 8 months, recurrent glioblastoma is among the world’s most lethal malignancies. Temozolomide (TMZ) has become a standard chemotherapeutic for glioblastoma due to its alkylating effects and ability to cross the blood-brain barrier. Unfortunately, many patients with glioblastoma develop resistance to TMZ, severely limiting therapeutic options. Recent studies have found a link between ectopic connexin 43 (Cx43) and TMZ-resistant glioblastoma. Further studies have shown success in overcoming this resistance by combining TMZ with alpha carboxyl terminus 1 (αCT1) peptide, a clinically tested Cx43 carboxyl terminus mimetic peptide inhibitor; however, because of the short half-life of αCT1, therapeutic potential is limited, and new approaches are needed. In this study, we hypothesized that simultaneously targeting Cx43 with αCT1 and phosphoinositide 3-kinase (PI3K) using PI3K isoform-selective inhibitors would synergistically sensitize glioblastoma to TMZ treatment because p110β, one of the four PI3K catalytic subunits, is a prime survival factor for TMZ-resistant glioblastoma. We cultured newly dissected primary glioblastoma cells, glioblastoma cell lines, and glioblastoma stem cells and dosed them with various combinations of αCT1, TMZ, and one of two clinically tested p110β inhibitors (TGX-221 or GSK2636771). Cell viability was measured using an MTS viability assay. Glioblastoma cells that expressed high levels of Cx43 and p110β experienced a significant decrease in viability when dosed with a triple combination. The additive properties of these combinations were measured using an excess over Bliss (EOB) score derived from Bliss independence modeling, which showed synergistic effects as high as 81% for triple therapy combinations in TMZ-resistant cell lines. The EOB was significantly higher in triple therapies than the double or single therapies. To support our findings, we used the same combinations in primary glioblastoma cells and measured the activity of cleaved caspase 3/7 using a luminescence assay. There was a synergistic increase in caspase 3/7 activity in the triple therapy that was not seen in any of the other combinations. Identical treatment in non-pathological human astrocytes suggests that our drug combinations do not exacerbate non-selective TMZ toxicity in the brain. Collectively, our data demonstrate that simultaneous inhibition of Cx43 and PI3K is an effective approach for overcoming TMZ resistance in glioblastoma. Citation Format: Kasen R. Hutchings, Kevin J. Pridham, Min Liu, Joseph Owens, Zhi Sheng. Synergistic effects of connexin 43 inhibitor and PI3K isoform-selective inhibitors in temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3243.

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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.

Thesla1⁺gene ofSchizosachharoymces pombeencodes La protein which promotes proper processing of precursor-tRNAs. Deletion ofsla1(sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4⁺inhibits leucine uptake and growth ofsla1Δ cells. Here, transcriptome analysis reveals that genes upregulated insla1Δ 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). Thesla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling inS.pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes insla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs inS.pombe. To study the NH4⁺-effects, we isolatedsla1Δ 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 that³H-leucine uptake by SSR-any1-F32Vcells in NH4⁺-media is more robust than bysla1Δ cells. Moreover, F32V may alterany1⁺function insla1Δ 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 theany1-F32Vrescuing mutant provides an additional specific link.

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.

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 promoter methylation 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.

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.

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/p110α, PIK3CB/p110β, PIK3CD/p110δ, and PIK3CG/p110γ) 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.