identification-ecohiv-infected-cells-microglia-manipulated-transgenic

Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are ...

University of South Carolina

HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 μM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 μM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1β. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.

Inhibition of the Dead Box RNA Helicase 3 Prevents HIV-1 Tat and Cocaine-Induced Neurotoxicity by Targeting Microglia Activation.

Chemoresistance is a major obstacle in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. Here we identify hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance by developing chemoresistant TNBC tumors in vivo and characterizing their transcriptomes by RNA-sequencing. Inhibiting LOX reduces collagen cross-linking and fibronectin assembly, increases drug penetration, and downregulates ITGA5/FN1 expression, resulting in inhibition of FAK/Src signaling, induction of apoptosis and re-sensitization to chemotherapy. Similarly, inhibiting FAK/Src results in chemosensitization. These effects are observed in 3D-cultured cell lines, tumor organoids, chemoresistant xenografts, syngeneic tumors and PDX models. Re-expressing the hypoxia-repressed miR-142-3p, which targets HIF1A, LOX and ITGA5, causes further suppression of the HIF-1α/LOX/ITGA5/FN1 axis. Notably, higher LOX, ITGA5, or FN1, or lower miR-142-3p levels are associated with shorter survival in chemotherapy-treated TNBC patients. These results provide strong pre-clinical rationale for developing and testing LOX inhibitors to overcome chemoresistance in TNBC patients.

Targeting lysyl oxidase (LOX) overcomes chemotherapy resistance in triple negative breast cancer.

Stress granules (SGs) are non-membranous cytosolic protein-RNA aggregates that process mRNAs through stalled translation initiation in response to cellular stressors and in disease. DEAD-Box RNA helicase 3 (DDX3) is an active target of drug development for the treatment of viral infections, cancers, and neurodegenerative diseases. DDX3 plays a critical role in RNA metabolism, including SGs, but the role of DDX3 enzymatic activity in SG dynamics is not well understood. Here, we address this question by determining the effects of DDX3 inhibition on the dynamics of SG assembly and disassembly. We use two small molecule inhibitors of DDX3, RK33 and 16D, with distinct inhibitory mechanisms that target DDX3's ATPase activity and RNA helicase site, respectively. We find that both DDX3 inhibitors reduce the assembly of SGs, with a more pronounced reduction from RK-33. In contrast, both compounds only marginally affect the disassembly of SGs. RNA-mediated knockdown of DDX3 caused a similar reduction in SG assembly and minimal effect on SG disassembly. Collectively, these results reveal that the enzymatic activity of DDX3 is required for the assembly of SGs and pharmacological inhibition of DDX3 could be relevant for the treatment of SG-dependent pathologies.

Pharmacological inhibition of DEAD-Box RNA Helicase 3 attenuates stress granule assembly.

The unfolded protein response (UPR) is involved in the pathogenesis of metabolic disorders, yet whether variations in the UPR among individuals influence the propensity for metabolic disease remains unexplored. Using outbred deer mice as a model, we show that the intensity of UPR in fibroblasts isolated early in life predicts the extent of body weight gain after high-fat diet (HFD) administration. Contrary to those with intense UPR, animals with moderate UPR in fibroblasts and therefore displaying compromised stress resolution did not gain body weight but developed inflammation, especially in the skin, after HFD administration. Fibroblasts emerged as potent modifiers of this differential responsiveness to HFD, as indicated by the comparison of the UPR profiles of fibroblasts responding to fatty acids in vitro, by correlation analyses between UPR and proinflammatory cytokine-associated transcriptomes, and by BiP (also known as HSPA5) immunolocalization in skin lesions from animals receiving HFD. These results suggest that the UPR operates as a modifier of an individual's propensity for body weight gain in a manner that, at least in part, involves the regulation of an inflammatory response by skin fibroblasts. This article has an associated First Person interview with the first author of the paper.

Propensity to endoplasmic reticulum stress in deer mouse fibroblasts predicts skin inflammation and body weight gain.

Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2 BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2 BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2 BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2 BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2 breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2 BrCa.

Inhibition of CDK8/19 Mediator kinase potentiates HER2-targeting drugs and bypasses resistance to these agents in vitro and in vivo.

Evaluation of gene co-regulation is a powerful approach for revealing regulatory associations between genes and predicting biological function, especially in genetically diverse samples. Here, we applied this strategy to identify transcripts that are co-regulated with unfolded protein response (UPR) genes in cultured fibroblasts from outbred deer mice. Our analyses showed that the transcriptome associated with RASSF1, a tumor suppressor involved in cell cycle regulation and not previously linked to UPR, is highly correlated with the transcriptome of several UPR-related genes, such as BiP/GRP78, DNAJB9, GRP94, ATF4, DNAJC3, and CHOP/DDIT3. Conversely, gene ontology analyses for genes co-regulated with RASSF1 predicted a previously unreported involvement in UPR-associated apoptosis. Bioinformatic analyses indicated the presence of ATF4-binding sites in the RASSF1 promoter, which were shown to be operational using chromatin immunoprecipitation. Reporter assays revealed that the RASSF1 promoter is responsive to ATF4, while ablation of RASSF1 mitigated the expression of the ATF4 effector BBC3 and abrogated tunicamycin-induced apoptosis. Collectively, these results implicate RASSF1 in the regulation of endoplasmic reticulum stress-associated apoptosis downstream of ATF4. They also illustrate the power of gene coordination analysis in predicting biological functions and revealing regulatory associations between genes.

RASSF1 is identified by transcriptome coordination analysis as a target of ATF4.

Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Clustering extra centrosomes is a major coping mechanism required for faithful mitosis of cancer cells with CA that would otherwise undergo mitotic catastrophe and cell death. However, its underlying molecular mechanisms have not been fully described. Furthermore, little is known about the processes and players triggering aggressiveness of cells with CA beyond mitosis. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) to be overexpressed in tumors with CA, and its high expression is associated with dramatically worse clinical outcome. We demonstrated, for the first time, that TACC3 forms distinct functional interactomes regulating different processes in mitosis and interphase to ensure proliferation and survival of cancer cells with CA. Mitotic TACC3 interacts with the Kinesin Family Member C1 (KIFC1) to cluster extra centrosomes for mitotic progression, and inhibition of this interaction leads to mitotic cell death via multipolar spindle formation. Interphase TACC3 interacts with the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in nucleus to inhibit the expression of key tumor suppressors (e.g., p21, p16 and APAF1) driving G1/S progression, and its inhibition blocks these interactions and causes p53-independent G1 arrest and apoptosis. Notably, inducing CA by p53 loss/mutation increases the expression of TACC3 and KIFC1 via FOXM1 and renders cancer cells highly sensitive to TACC3 inhibition. Targeting TACC3 by guide RNAs or small molecule inhibitors strongly inhibits growth of organoids and breast cancer cell line- and patient-derived xenografts with CA by induction of multipolar spindles, mitotic and G1 arrest. Altogether, our results show that TACC3 is a multifunctional driver of highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this disease.

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification.

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection, marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for effective therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of the HIV and cocaine-induced transcriptomes in primary cortical cultures revealed a significant overexpression of the macrophage-specific gene, aconitate decarboxylase 1 (Acod1), caused by the combined insults of HIV and cocaine. ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. The itaconate produced facilitates cytokine production and subsequently activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. While the role of itaconate' in limiting inflammation has been studied in peripheral macrophages, its immunometabolic function remains unexplored in HIV and cocaine-exposed microglia. We assessed in this model system the potential of 4-octyl-itaconate (4OI), a cell-penetrable esterified form of itaconate known for its potent anti-inflammatory properties and potential therapeutic applications. We administered 4OI to primary cortical cultures exposed to Tat and cocaine. 4OI treatment increased the number of microglial cells in both untreated and Tat±Cocaine-treated cultures and also reversed the morphological altercations induced by Tat and cocaine. In the presence of 4OI, microglial cells also appeared more ramified, resembling the quiescent microglia. Consistent with these results, 4OI treatment inhibited the secretion of the proinflammatory cytokines IL-1α, IL-1β, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling further determined that Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (Nqo1), Glutathione S-transferase Pi (Gstp1), and glutamate cysteine ligase catalytic (Gclc), were most significantly activated in Tat-4OI treated cultures, relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development aimed at addressing HAND coupled with CUD comorbidities.

Suppression of HIV and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer.

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of HIV and cocaine-induced transcriptomes in primary cortical cultures revealed significant overexpression of the macrophage-specific gene aconitate decarboxylase 1 (Acod1). The ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. Itaconate then facilitates cytokine production and activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. However, the immunometabolic function of itaconate was unexplored in HIV and cocaine-exposed microglia. We assessed the potential of 4-octyl-itaconate (4OI), a cell-penetrable ester form of itaconate known for its anti-inflammatory properties. When primary cortical cultures exposed to Tat and cocaine were treated with 4OI, microglial cell number increased and the morphological altercations induced by Tat and cocaine were reversed. Microglial cells also appeared more ramified, resembling the quiescent microglia. 4OI treatment inhibited secretion of the proinflammatory cytokines IL-1α, IL-1β, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling determined that Nrf2 target genes were significantly activated in Tat and 4OI treated cultures relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development to treat HAND coupled with CUD comorbidities.

Article	Year
Inhibition of the Dead Box RNA Helicase 3 Prevents HIV-1 Tat and Cocaine-Induced Neurotoxicity by Targeting Microglia Activation. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology\| PubMed ID: 31802418	2020
Targeting lysyl oxidase (LOX) overcomes chemotherapy resistance in triple negative breast cancer. Nature communications\| PubMed ID: 32415208	2020
Pharmacological inhibition of DEAD-Box RNA Helicase 3 attenuates stress granule assembly. Biochemical pharmacology\| PubMed ID: 33049245	2020
Propensity to endoplasmic reticulum stress in deer mouse fibroblasts predicts skin inflammation and body weight gain. Disease models & mechanisms\| PubMed ID: 34661243	2021
Inhibition of CDK8/19 Mediator kinase potentiates HER2-targeting drugs and bypasses resistance to these agents in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 35914167	2022
RASSF1 is identified by transcriptome coordination analysis as a target of ATF4. FEBS open bio\| PubMed ID: 36723232	2023
Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification. Cell death and differentiation\| PubMed ID: 36864125	2023
Suppression of HIV and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters. bioRxiv : the preprint server for biology\| PubMed ID: 37808776	2023
Mediator kinase inhibition reverses castration resistance of advanced prostate cancer. The Journal of clinical investigation\| PubMed ID: 38546787	2024
Suppression of HIV-TAT and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters. Journal of neurovirology\| PubMed ID: 38884890	2024

Vitali Sikirzhytski

Biography

JoVE Journal Publications

Other Publications