Activation of Stimulator of Interferon Genes (STING): Promising Strategy to Overcome Immune Resistance in Prostate Cancer.

Prostate cancer (PCa) is the most frequent and second-lethal cancer among men. Despite considerable efforts to explore treatments like autologous cellular immunotherapy and immune checkpoint inhibitors, their success remains limited. The intricate tumor microenvironment (TME) and its interaction with the immune system pose significant challenges in PCa treatment. Consequently, researchers have directed their focus on augmenting the immune system's anti-tumor response by targeting the STimulator of the Interferon Genes (STING) pathway. The STING pathway is activated when foreign DNA is detected in the cytoplasm of innate immune cells, resulting in the activation of endoplasmic reticulum (ER) STING. This, in turn, triggers an augmentation of signaling, leading to the production of type I interferon (IFN) and other pro-inflammatory cytokines. Numerous studies have demonstrated that activation of the STING pathway induces immune system rejection and targeted elimination of PCa cells. Researchers have been exploring various methods to activate the STING pathway, including the use of bacterial vectors to deliver STING agonists and the combination of radiation therapy with STING agonists. Achieving effective radiation therapy with minimal side effects and optimal anti-tumor immune responses necessitates precise adjustments to radiation dosing and fractionation schedules. This comprehensive review discusses promising findings from studies focusing on activating the STING pathway to combat PCa. The STING pathway exhibits the potential to serve as an effective treatment modality for PCa, offering new hope for improving the lives of those affected by this devastating disease.

Targeted Bioluminescent Imaging of Pancreatic Ductal Adenocarcinoma Using Nanocarrier-Complexed EGFR-Binding Affibody-Gaussia Luciferase Fusion Protein.

In vivo imaging has enabled impressive advances in biological research, both preclinical and clinical, and researchers have an arsenal of imaging methods available. Bioluminescence imaging is an advantageous method for in vivo studies that allows for the simple acquisition of images with low background signals. Researchers have increasingly been looking for ways to improve bioluminescent imaging for in vivo applications, which we sought to achieve by developing a bioluminescent probe that could specifically target cells of interest. We chose pancreatic ductal adenocarcinoma (PDAC) as the disease model because it is the most common type of pancreatic cancer and has an extremely low survival rate. We targeted the epidermal growth factor receptor (EGFR), which is frequently overexpressed in pancreatic cancer cells, using an EGFR-specific affibody to selectively identify PDAC cells and delivered a Gaussia luciferase (GLuc) bioluminescent protein for imaging by engineering a fusion protein with both the affibody and the bioluminescent protein. This fusion protein was then complexed with a G5-PAMAM dendrimer nanocarrier. The dendrimer was used to improve the protein stability in vivo and increase signal strength. Our targeted bioluminescent complex had an enhanced uptake into PDAC cells in vitro and localized to PDAC tumors in vivo in pancreatic cancer xenograft mice. The bioluminescent complexes could delineate the tumor shape, identify multiple masses, and locate metastases. Through this work, an EGFR-targeted bioluminescent-dendrimer complex enabled the straightforward identification and imaging of pancreatic cancer cells in vivo in preclinical models. This argues for the targeted nanocarrier-mediated delivery of bioluminescent proteins as a way to improve in vivo bioluminescent imaging.

The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance.

Castration-resistant prostate cancer (CRPC) is fatal and therapeutically under-served. We describe a novel CRPC-restraining role for the vasodilatory soluble guanylyl cyclase (sGC) pathway. We discovered that sGC subunits are dysregulated during CRPC progression and its catalytic product, cyclic GMP (cGMP), is lowered in CRPC patients. Abrogating sGC heterodimer formation in castration-sensitive prostate cancer (CSPC) cells inhibited androgen deprivation (AD)-induced senescence, and promoted castration-resistant tumor growth. We found sGC is oxidatively inactivated in CRPC. Paradoxically, AD restored sGC activity in CRPC cells through redox-protective responses evoked to protect against AD-induced oxidative stress. sGC stimulation via its FDA-approved agonist, riociguat, inhibited castration-resistant growth, and the anti-tumor response correlated with elevated cGMP, indicating on-target sGC activity. Consistent with known sGC function, riociguat improved tumor oxygenation, decreasing the PC stem cell marker, CD44, and enhancing radiation-induced tumor suppression. Our studies thus provide the first evidence for therapeutically targeting sGC via riociguat to treat CRPC. Statement of significance: Prostate cancer is the second highest cancer-related cause of death for American men. Once patients progress to castration-resistant prostate cancer, the incurable and fatal stage, there are few viable treatment options available. Here we identify and characterize a new and clinically actionable target, the soluble guanylyl cyclase complex, in castration-resistant prostate cancer. Notably we find that repurposing the FDA-approved and safely tolerated sGC agonist, riociguat, decreases castration-resistant tumor growth and re-sensitizes these tumors to radiation therapy. Thus our study provides both new biology regarding the origins of castration resistance as well as a new and viable treatment option.

In vivo active-targeting fluorescence molecular imaging with adaptive background fluorescence subtraction.

Using active tumor-targeting nanoparticles, fluorescence imaging can provide highly sensitive and specific tumor detection, and precisely guide radiation in translational radiotherapy study. However, the inevitable presence of non-specific nanoparticle uptake throughout the body can result in high levels of heterogeneous background fluorescence, which limits the detection sensitivity of fluorescence imaging and further complicates the early detection of small cancers. In this study, background fluorescence emanating from the baseline fluorophores was estimated from the distribution of excitation light transmitting through tissues, by using linear mean square error estimation. An adaptive masked-based background subtraction strategy was then implemented to selectively refine the background fluorescence subtraction. First, an in vivo experiment was performed on a mouse intratumorally injected with passively targeted fluorescent nanoparticles, to validate the reliability and robustness of the proposed method in a stringent situation wherein the target fluorescence was overlapped with the strong background. Then, we conducted in vivo studies on 10 mice which were inoculated with orthotopic breast tumors and intravenously injected with actively targeted fluorescent nanoparticles. Results demonstrated that active targeting combined with the proposed background subtraction method synergistically increased the accuracy of fluorescence molecular imaging, affording sensitive tumor detection.

Experimental Support for the Possibility of Retrograde Genesis of Intraductal Carcinoma of the Prostate.

Background. Historically, intraductal carcinoma of the prostate (IDC-P) was postulated to be a retrograde spread of high-grade invasive prostate cancer. There is evidence that IDC-P can primarily originate in the prostatic ducts. The retrograde genesis has never been experimentally or clinically confirmed before. Methods. Biopsy proven intermediate or high-risk prostate cancer was orthotopically grafted in the prostate of severe combined immunodeficiency gamma mice. Cancer growth was monitored by serum PSA levels. The animals were sacrificed and grafted areas were histological examined. Results. Twenty-one of 23 mice survived and demonstrated rising serum PSA. In 10 of 21 animals, human prostate cancer was identified orthotopically. Except for one case where the human biopsy showed a Grade Group 2 prostate cancer and mouse graft was Grade Group 5, other 9 specimens showed comparable grades. One of the specimens demonstrated a cribriform invasive prostate cancer and adjacent IDC-P. Conclusion. These experimental data offer some evidence that invasive prostate cancer can demonstrate a retrograde spread in the prostatic ducts as IDC-P. Its ability to primarily arise in the ducts has been demonstrated in other studies. However, the issue which remains unresolved is in its most common presentation of IDC-P intermixed with high-grade invasive cancer if it is the former or the latter which came first. Possibly resolving this dilemma will shed some light on the existing controversies if IDC-P should or should not be graded when invasive cancer is present.

Boosted Radiation Bystander Effect of PSMA-Targeted Gold Nanoparticles in Prostate Cancer Radiosensitization.

Metal nanoparticles are effective radiosensitizers that locally enhance radiation doses in targeted cancer cells. Compared with other metal nanoparticles, gold nanoparticles (GNPs) exhibit high biocompatibility, low toxicity, and they increase secondary electron scatter. Herein, we investigated the effects of active-targeting GNPs on the radiation-induced bystander effect (RIBE) in prostate cancer cells. The impact of GNPs on the RIBE presents implications for secondary cancers or spatially fractionated radiotherapy treatments. Anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated with PEGylated GNPs through EDC-NHS chemistry. The media transfer technique was performed to induce the RIBE on the non-irradiated bystander cells. This study focused on the LNCaP cell line, because it can model a wide range of stages relating to prostate cancer progression, including the transition from androgen dependence to castration resistance and bone metastasis. First, LNCaP cells were pretreated with phosphate buffered saline (PBS) or PSMA-targeted GNPs (PGNPs) for 24 h and irradiated with 160 kVp X-rays (0-8 Gy). Following that, the collected culture media were filtered (sterile 0.45 µm polyethersulfone) in order to acquire PBS- and PGNP- conditioned media (CM). Then, PBS- and PGNP-CM were transferred to the bystander cells that were loaded with/without PGNPs. MTT, γ-H2AX, clonogenic assays and reactive oxygen species assessments were performed to compare RIBE responses under different treatments. Compared with 2 Gy-PBS-CM, 8 Gy-PBS-CM demonstrated a much higher RIBE response, thus validating the dose dependence of RIBE in LNCaP cells. Compared with PBS-CM, PGNP-CM exhibited lower cell viability, higher DNA damage, and a smaller survival fraction. In the presence of PBS-CM, bystander cells loaded with PGNPs showed increased cell death compared with cells that did not have PGNPs. These results demonstrate the PGNP-boosted expression and sensitivity of RIBE in prostate cancer cells.

Quantifying Radiosensitization of PSMA-Targeted Gold Nanoparticles on Prostate Cancer Cells at Megavoltage Radiation Energies by Monte Carlo Simulation and Local Effect Model.

Active targeting gold nanoparticles (AuNPs) are a very promising avenue for cancer treatment with many publications on AuNP mediated radiosensitization at kilovoltage (kV) photon energies. However, uncertainty on the effectiveness of AuNPs under clinically relevant megavoltage (MV) radiation energies hinders the clinical translation of AuNP-assisted radiation therapy (RT) paradigm. The aim of this study was to investigate radiosensitization mediated by PSMA-targeted AuNPs irradiated by a 6 MV radiation beam at different depths to explore feasibility of AuNP-assisted prostate cancer RT under clinically relevant conditions. PSMA-targeted AuNPs (PSMA-AuNPs) were synthesized by conjugating PSMA antibodies onto PEGylated AuNPs through EDC/NHS chemistry. Confocal fluorescence microscopy was used to verify the active targeting of the developed PSMA-AuNPs. Transmission electron microscopy (TEM) was used to demonstrate the intracellular biodistribution of PSMA-AuNPs. LNCaP prostate cancer cells treated with PSMA-AuNPs were irradiated on a Varian 6 MV LINAC under varying depths (2.5 cm, 10 cm, 20 cm, 30 cm) of solid water. Clonogenic assays were carried out to determine the in vitro cell survival fractions. A Monte Carlo (MC) model developed on TOPAS platform was then employed to determine the nano-scale radial dose distribution around AuNPs, which was subsequently used to predict the radiation dose response of LNCaP cells treated with AuNPs. Two different cell models, with AuNPs located within the whole cell or only in the cytoplasm, were used to assess how the intracellular PSMA-AuNP biodistribution impacts the prostate cancer radiosensitization. Then, MC-based microdosimetry was combined with the local effect model (LEM) to calculate cell survival fraction, which was benchmarked against the in vitro clonogenic assays at different depths. In vitro clonogenic assay of LNCaP cells demonstrated the depth dependence of AuNP radiosensitization under clinical megavoltage beams, with sensitization enhancement ratio (SER) of 1.14 ± 0.03 and 1.55 ± 0.05 at 2.5 cm depth and 30 cm depth, respectively. The MC microdosimetry model showed the elevated percent of low-energy photons in the MV beams at greater depth, consequently resulting in increased dose enhancement ratio (DER) of AuNPs with depth. The AuNP-induced DER reached ~5.7 and ~8.1 at depths of 2.5 cm and 30 cm, respectively. Microdosimetry based LEM accurately predicted the cell survival under 6 MV beams at different depths, for the cell model with AuNPs placed only in the cell cytoplasm. TEM results demonstrated the distribution of PSMA-AuNPs in the cytoplasm, confirming the accuracy of MC microdosimetry based LEM with modelled AuNPs distributed within the cytoplasm. We conclude that AuNP radiosensitization can be achieved under megavoltage clinical radiotherapy energies with a dependence on tumor depth. Furthermore, the combination of Monte Carlo microdosimetry and LEM will be a valuable tool to assist with developing AuNP-aided radiotherapy paradigm and drive clinical translation.

Interactions of Urea-Based Inhibitors with Prostate-Specific Membrane Antigen for Boron Neutron Capture Therapy.

In this study, molecular interactions of prostate-specific membrane antigen (PSMA) with five chemically distinct urea-based boron-containing inhibitors have been investigated at the atomic level using molecular docking and molecular dynamics simulations. The PSMA-inhibitor complexations have been analyzed by comparing their binding modes, secondary structures, root-mean-square deviations, noncovalent interactions, principal components, and binding free energies. PSMA is a cell surface glycoprotein upregulated in cancerous cells and can be targeted by boron-labeled inhibitors for boron neutron capture therapy (BNCT). The effective BNCT requires the selective boron delivery to the tumor area and highly specific PSMA-mediated cellular uptake by tumor. Thus, a potent inhibitor must exhibit both high binding affinity and high boron density. The computational results suggest that the chemical nature of inhibitors affects the binding mode and their association with PSMA is primarily dominated by hydrogen bonding, salt bridge, electrostatic, and π-π interactions. The binding free energies (-28.0, -15.2, -43.9, -23.2, and -38.2 kcal/mol) calculated using λ-dynamics for all inhibitors (In1-5) predict preferential binding that is in accordance with experimental data. Among all inhibitors, In5 was found to be the best candidate for BNCT. The binding of this inhibitor to PSMA preserved its overall secondary structure. These results provide computational insights into the coordination flexibility of PSMA and its interaction with various inhibitors. They can be used for the design and synthesis of efficient BNCT agents with improved drug selectivity and high boron percentage.

Incorporating Differential Gene Expression Analysis with Predictive Biomarkers to Identify Novel Therapeutic Drugs for Fuchs Endothelial Corneal Dystrophy.

PURPOSE: Based on the differential gene expression analysis for predictive biomarkers with RNA-Sequencing data from Fuchs endothelial corneal dystrophy (FECD) patients, we are aiming to evaluate the efficacy of Library of Integrated Network-based Cellular Signatures (LINCS) perturbagen prediction software to identify novel pharmacotherapeutic targets that can revert the pathogenic gene expression signatures and reverse disease phenotype in FECD. METHODS: A publicly available RNA-seq dataset was used to compare corneal endothelial specimens from controls and patients with FECD. Based on the differential gene expression analysis for predictive biomarkers, we evaluated the efficacy of LINCS perturbagen prediction software to identify novel therapeutic targets that can revert the pathogenic gene expression signatures and reverse disease phenotypes in FECD. RESULTS: The RNA-seq dataset of the corneal endothelial cells from FECD patients revealed the differential gene expression signatures of FECD. Many of the differential expressed genes are related to canonical pathways of the FECD pathogenesis, such as extracellular matrix reorganization and immunological response. The expression levels of genes VSIG2, IL18, and ITGB8 were significantly increased in FECD compared with control. Meanwhile, the expression levels of CNGA3, SMOX, and CERS1 were significantly lower in the FECD than in control. We employed LINCS L1000 Characteristic Direction Signature Search Engine (L1000-CDS2) to investigate pathway-based molecular treatment. L1000-CDS2 predicted that small molecule drugs such as histone deacetylase (HDAC) inhibitors might be a potential candidate to reverse the pathological gene expression signature in FECD. CONCLUSIONS: Based on differential gene expression signatures, several candidate drugs have been identified to reverse the disease phenotypes in FECD. Gene expression signature with LINCS small molecule prediction software can discover novel preclinical drug candidates for FECD.

Prostate Cancer Targeted X-Ray Fluorescence Imaging via Gold Nanoparticles Functionalized With Prostate-Specific Membrane Antigen (PSMA).

PURPOSE: The gold nanoparticle (GNP) as a promising theranostic probe has been increasingly studied. The tumor-targeting efficiency of GNPs is crucial to increase the therapeutic ratio. In this study, we developed PSMA-targeted GNPs to enhance GNP uptake in prostate cancer and developed an x-ray fluorescence imaging system to noninvasively monitor and assess GNP delivery. METHODS AND MATERIALS: For targeted therapy of prostate cancer, anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PEGylated GNPs through 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) chemistry. In vivo imaging was implemented using an in-house-developed dual-modality computed tomography (CT) and x-ray fluorescence CT (XFCT) system on mice bearing subcutaneous LNCaP prostate tumors. After intravenous administration of GNPs (15 mg/mL, 200 µL), the x-ray fluorescence signals from the tumor were collected at various time points (5 minutes to approximately 30 hours) for GNP pharmacokinetics analysis. At 24 hours after administration, x-ray fluorescence projection (XRFproj) and XFCT imaging were conducted to evaluate the prostate tumor uptake of active- and passive-targeting GNPs. Inductively coupled plasma mass spectrometry analysis was adopted as a benchmark to verify the quantification accuracy of XRFproj/XFCT imaging. RESULTS: Fluorescence microscopic imaging confirmed the enhanced (approximately 4 times) targeting efficiency of PSMA-targeted GNPs in vitro. The pharmacokinetics analysis showed enhanced tumor uptake/retention of PSMA-targeted GNPs and revealed that the peak tumor accumulation appeared at approximately 24 hours after intravenous administration. Both XRFproj and XFCT imaging presented their accuracy in quantifying GNPs within tumors noninvasively. Moreover, XFCT imaging verified its unique capabilities to simultaneously determine the heterogeneous spatial distribution and the concentration of GNPs within tumors in vivo. CONCLUSIONS: In conjunction with PSMA-targeted GNPs, XRFproj/XFCT would be a highly sensitive tool for targeted imaging of prostate cancer, benefiting the elucidation of mechanisms of GNP-assisted prostate-cancer therapy.

Identification of Novel Transcriptome Signature as a Potential Prognostic Biomarker for Anti-Angiogenic Therapy in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common and devastating type of primary brain tumor, with a median survival time of only 15 months. Having a clinically applicable genetic biomarker would lead to a paradigm shift in precise diagnosis, personalized therapeutic decisions, and prognostic prediction for GBM. Radiogenomic profiling connecting radiological imaging features with molecular alterations will offer a noninvasive method for genomic studies of GBM. To this end, we analyzed over 3800 glioma and GBM cases across four independent datasets. The Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases were employed for RNA-Seq analysis, whereas the Ivy Glioblastoma Atlas Project (Ivy-GAP) and The Cancer Imaging Archive (TCIA) provided clinicopathological data. The Clinical Proteomic Tumor Analysis Consortium Glioblastoma Multiforme (CPTAC-GBM) was used for proteomic analysis. We identified a simple three-gene transcriptome signature-SOCS3, VEGFA, and TEK-that can connect GBM's overall prognosis with genes' expression and simultaneously correlate radiographical features of perfusion imaging with SOCS3 expression levels. More importantly, the rampant development of neovascularization in GBM offers a promising target for therapeutic intervention. However, treatment with bevacizumab failed to improve overall survival. We identified SOCS3 expression levels as a potential selection marker for patients who may benefit from early initiation of angiogenesis inhibitors.

Dynamic conversion of cell sorting patterns in aggregates of embryonic stem cells with differential adhesive affinity.

BACKGROUND: Mammalian early development comprises the proliferation, differentiation, and self-assembly of the embryonic cells. The classic experiment undertaken by Townes and Holtfreter demonstrated the ability of dissociated embryonic cells to sort and self-organize spontaneously into the original tissue patterns. Here, we further explored the principles and mechanisms underlying the phenomenon of spontaneous tissue organization by studying aggregation and sorting of mouse embryonic stem (ES) cells with differential adhesive affinity in culture. RESULTS: As observed previously, in aggregates of wild-type and E-cadherin-deficient ES cells, the cell assemblies exhibited an initial sorting pattern showing wild-type cells engulfed by less adhesive E-cadherin-deficient ES cells, which fits the pattern predicted by the differential adhesive hypothesis proposed by Malcom Steinberg. However, in further study of more mature cell aggregates, the initial sorting pattern reversed, with the highly adhesive wild-type ES cells forming an outer shell enveloping the less adhesive E-cadherin-deficient cells, contradicting Steinberg's sorting principle. The outer wild-type cells of the more mature aggregates did not differentiate into endoderm, which is known to be able to sort to the exterior from previous studies. In contrast to the naive aggregates, the mature aggregates presented polarized, highly adhesive cells at the outer layer. The surface polarity was observed as an actin cap contiguously spanning across the apical surface of multiple adjacent cells, though independent of the formation of tight junctions. CONCLUSIONS: Our experimental findings suggest that the force of differential adhesive affinity can be overcome by even subtle polarity generated from strong bilateral ligation of highly adhesive cells in determining cell sorting patterns.

Orbital Hemorrhagic Necrosis, Globe Rupture, and Death From Intraorbital Injection of 1% Sodium Deoxycholate in a Murine Model.

PURPOSE: Deoxycholic acid (DCA) 1% is an injectable detergent indicated for submental fat reduction, although clinically it is being injected off-label for orbital fat prolapse. It is known to cause severe inflammation, local nerve dysfunction, and tissue necrosis, all of which could be catastrophic in the orbit and periocular region. This study evaluated the effects of periocular DCA on orbital and ocular adnexal tissues in a murine model. METHODS: Mice were treated via split-face intraorbital injections, subcutaneous injections, and topical cornea application with DCA versus phosphate-buffered saline. Whole heads were fixed, decalcified, and sectioned for orbital histology after 1-7 days. Matched pairs of human globes and mouse globes were immersed in either phosphate-buffered saline or 1% DCA for 72 hours. RESULTS: Six of 11 mice receiving intraorbital DCA injections died within minutes. Surviving mice developed severe orbital inflammatory necrosis. All orbits injected with phosphate-buffered saline were clinically and histologically normal. Six mice were treated with lower concentrations of DCA and all developed variable amounts of orbital inflammation, hemorrhage, and globe necrosis. Mice receiving subcutaneous DCA injection to the lower eyelid showed inflammatory necrosis, edema, and lid malposition. Topical application of DCA to mouse corneas caused no external or histologic changes. Human and mouse globes immersed ex vivo in DCA developed corneal edema and cataract formation without observable scleral changes. CONCLUSION: Intraorbital and periocular injection of DCA can cause devastating complications in a murine model, and significant caution is advised for off-label use in the periocular region.

Integrating Differential Gene Expression Analysis with Perturbagen-Response Signatures May Identify Novel Therapies for Thyroid-Associated Orbitopathy.

Purpose: To evaluate the efficacy of Library of Integrated Network-based Cellular Signatures (LINCS) perturbagen prediction software to identify small molecules that revert pathologic gene signature and alter disease phenotype in orbital adipose stem cells (OASCs) derived from patients with thyroid-associated orbitopathy (TAO). Methods: Differentially expressed genes identified via RNA sequencing were inputted into LINCS L1000 Characteristic Direction Signature Search Engine (L1000CDS2) to predict candidate small molecules to reverse pathologic gene expression. TAO OASC cell lines were treated in vitro with six identified small molecules (Torin-2, PX12, withaferin A, isoliquiritigenin, mitoxantrone, and MLN8054), and expression of key adipogenic and differentially expressed genes was measured with quantitative polymerase chain reaction after 7 days of treatment. OASCs were differentiated into adipocytes, treated for 15 days, and stained with Oil Red O (OD 490 nm) to evaluate adipogenic changes. Results: The expression of key differentially expressed genes (IRX1, HOXB2, S100B, and KCNA4) and adipogenic genes (peroxisome proliferator activated receptor-γ, FABP4) was significantly decreased in TAO OASCs after treatment (P < .05). In treated TAO adipocytes (n = 3), all six tested small molecules yielded significant decrease (P < .05) in Oil Red O staining. In treated non-TAO adipocytes (n = 3), only three of the drugs yielded a significant decrease in Oil Red O staining. Conclusions: Combining disease expression signatures with LINCS small molecule prediction software can identify promising preclinical drug candidates for TAO. Translational Relevance: These findings may offer insight into future potential therapeutic options for TAO and demonstrate a streamlined model to predict drug candidates for other diseases.

Targeting Cullin-RING E3 Ligases for Radiosensitization: From NEDDylation Inhibition to PROTACs.

As a dynamic regulator for short-lived protein degradation and turnover, the ubiquitin-proteasome system (UPS) plays important roles in various biological processes, including response to cellular stress, regulation of cell cycle progression, and carcinogenesis. Over the past decade, research on targeting the cullin-RING (really interesting new gene) E3 ligases (CRLs) in the UPS has gained great momentum with the entry of late-phase clinical trials of its novel inhibitors MLN4924 (pevonedistat) and TAS4464. Several preclinical studies have demonstrated the efficacy of MLN4924 as a radiosensitizer, mainly due to its unique cytotoxic properties, including induction of DNA damage response, cell cycle checkpoints dysregulation, and inhibition of NF-κB and mTOR pathways. Recently, the PROteolysis TArgeting Chimeras (PROTACs) technology was developed to recruit the target proteins for CRL-mediated polyubiquitination, overcoming the resistance that develops inevitably with traditional targeted therapies. First-in-class cell-permeable PROTACs against critical radioresistance conferring proteins, including the epidermal growth factor receptor (EGFR), androgen receptor (AR) and estrogen receptor (ER), cyclin-dependent kinases (CDKs), MAP kinase kinase 1 (MEK1), and MEK2, have emerged in the past 5 years. In this review article, we will summarize the most important research findings of targeting CRLs for radiosensitization.

Mitochondrial targeted therapy with elamipretide (MTP-131) as an adjunct to tumor necrosis factor inhibition for traumatic optic neuropathy in the acute setting.

Traumatic optic neuropathy (TON) can occur following blunt trauma to the orbit and can lead to permanent vision loss. In this study, we investigated the effectiveness of elamipretide (MTP-131), a small mitochondrially-targeted tetrapeptide, in conjunction with etanercept, a tumor necrosis factor (TNF) inhibitor, as neuroprotective agents of retinal ganglion cells (RGCs) after optic nerve trauma with sonication-induced TON (SI-TON) in mice. Treatment with intravitreal MTP-131 and subcutaneous etanercept and MTP-131 showed a 21% increase (p < 0.01) in RGC survival rate compared to PBS-treated control eyes. Subcutaneous etanercept and MTP-131 had an 11% increase (p < 0.05) in RGC survival compared to controls. Subcutaneous etanercept only group showed 20% increase (p < 0.01) in RGC survival compared to controls, while subcutaneous MTP-131 alone showed a 17% increase (p < 0.01). Surprisingly, we did not observe a synergistic effect between the two drugs in the group receiving both etanercept and MTP-131. One possible explanation for the absence of a synergistic effect is that MTP-131 and etanercept may be acting on different portions of the same pathway.

Radiation Attenuates Prostate Tumor Antiviral Responses to Vesicular Stomatitis Virus Containing IFNß, Resulting in Pronounced Antitumor Systemic Immune Responses.

Vesicular stomatitis virus (VSV) expressing IFNß induces apoptosis in multiple tumor models while maintaining an excellent safety profile. VSV-IFNß is oncoselective due to permissive replication in cells with an altered IFN pathway. The human VSV-IFNß (hIFNß) vector is currently used in clinical trials as a standalone therapy; however, we hypothesized that oncolytic virotherapy might be more effective when used in combination with radiotherapy (RT). We investigated the synergistic effects of RT and VSV-hIFNß in the subcutaneous PC3 and orthotopic LNCaP prostate xenograft models and a syngeneic RM9 prostate tumor model. VSV-IFNß combined with RT amplified tumor killing for PC3 and LNCaP xenografts, and RM9 tumors. This was attributed to the induction of proapoptotic genes leading to increased VSV-IFNß infection and replication, VSV expression, and oncolysis. In the RM9 tumors, combination therapy resulted in a robust antitumor immune response. Treated RM9 tumor-bearing mice demonstrated an increase in CD8+ and CD4+ T-cell numbers, 100% resistance to tumor rechallenge, and reduced resistance to reimplantation challenge with CD8+ knockdown. RT enhanced the activity of VSV-mediated oncolysis via attenuation of the innate antiviral response, resulting in increased VSV replication and the generation of an adaptive immune response earmarked by an increase in CD8+ lymphocyte numbers and antitumor activity. Local tumor irradiation combined with VSV-IFNß affects tumor cell death through direct and systemic activity in conjunction with pronounced antitumor immunity. IMPLICATIONS: Radiotherapy enhances VSV-mediated oncolysis and anti-tumor immunity, indicating that the ombination has promise for very high risk prostate cancer.

Fibroblast growth factor receptor 1 (FGFR1) as a therapeutic target in adenoid cystic carcinoma of the lacrimal gland.

Identification of molecular targets is the first step in developing efficacious therapeutic strategies for tumors. A tumors' biological response to perturbagens yields important information on the molecular determinants for tumor growth. The aim of this study was to characterize the response of adenoid cystic carcinoma of the lacrimal gland (LGACC) to intra-arterial cytoreductive chemotherapy (IACC) in order to identify novel targets to enhance therapy. We performed high-throughput proteomic analysis on paired samples from pre-IACC diagnostic biopsies and post-IACC excised tumor samples from 6 LGACC patients. This proteomic analysis provides a comprehensive landscape of the cellular compartments contained within the excised tumors. Interestingly, we found a strong upregulation across the fibroblast growth factor (FGF) signaling pathway, with FGF receptor 1 (FGFR1) exhibiting a consistent and significant upregulation in all post-IACC samples. We thus evaluated the therapeutic efficacy of a novel FGFR1 selective inhibitor, AZD4547, in combination with cisplatin on LGACC cells in-vitro. The combination index (CI) value (<0.895) demonstrated synergistic effect of AZD4547 and cisplatin in inhibiting cell growth and viability (p<0.02), with a differential response seen in post-IACC cultures when compared to pre-IACC cultures. The combination approach showed synergy of the drugs in the migration assay. Western blot analysis indicated a significant upregulation of cleaved caspase-3 and downregulation the expression of FGFR1 (p<0.05) with the combination treatment as compared to either agent independently. Our findings demonstrate that FGFR1 inhibition potentiates the cytoreductive effects of cisplatin and suggest a potential therapeutic benefit of using AZD4547 in the management of LGACC.

The Effect of Prostaglandin Analogue Bimatoprost on Thyroid-Associated Orbitopathy.

Purpose: We characterize the effect of bimatoprost on orbital adipose tissue in thyroid-associated orbitopathy (TAO) with clinicopathologic correlation. Methods: Orbital adipose-derived stem cells (OASCs) from types 1 and 2 TAO and control patients with and without exposure to 1 µm bimatoprost were examined via immunohistochemistry, RT-PCR, and Western blot for cell viability, migration capacity, lipid content, adipocyte morphology, mitochondrial content, and levels of adipogenic markers. A retrospective chart review was performed for clinicopathologic correlation. In mice, optical coherence tomography and pattern electroretinography were performed at baseline and at 1 month following a retrobulbar injection of bimatoprost, followed by orbital exenteration for histopathologic examination. Results: Types 1 and 2 TAO-derived cells had a significantly higher migration capacity and lipid content than those of healthy controls. With the addition of bimatoprost, types 1 and 2 TAO and control adipocytes exhibited a significant decrease in lipid content with morphologic transformation into smaller and multilocular lipid droplets, and an increase in mitochondrial load and UCP-1 expression consistent with an increase in brown adipose tissue turnover. Retrobulbar injection of bimatoprost in mice did not alter the gross morphology, retinal thickness, or ganglion cell function in vivo. Conclusions: Bimatoprost inhibits adipogenesis in OASCs and upregulates pathways involved in the browning of adipocytes. Furthermore, retrobulbar injection of bimatoprost is tolerated without immediate adverse effects in mice. Our results suggest a potential future application of prostaglandin analogues in the treatment of TAO.