Reversible chemoresistance of pancreatic cancer grown as spheroids.

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

Insights into the structure-activity relationship of the anticancer compound ZJ-101: A role played by the amide moiety.

ZJ-101, a structurally simplified analog of marine natural product superstolide A, was previously designed and synthesized in our laboratory. In the present study four new analogs of ZJ-101 were designed and synthesized to investigate the structure-activity relationship of the acetamide moiety of the molecule. The biological evaluation showed that the amide moiety is important for the molecule's anticancer activity. Replacing the amide with other functional groups such as a sulfonamide group, a carbamate group, and a urea group resulted in the decrease in anticancer activity.

Dimethyl Fumarate Protects against Lipopolysaccharide- (LPS-) Induced Sepsis through Inhibition of NF-κB Pathway in Mice.

Sepsis is one of the most severe complications and causes of mortality in the clinic. It remains a great challenge with no effective treatment for clinicians worldwide. Inhibiting the release of proinflammatory cytokines during sepsis is considered as an important strategy for treating sepsis and improving survival. In the present study, we have observed the effect of dimethyl fumarate (DMF) on lipopolysaccharide- (LPS-) induced sepsis and investigated the possible mechanism. By screening a subset of the Johns Hopkins Drug Library, we identified DMF as a novel inhibitor of nitric oxide synthesis in LPS-stimulated RAW264.7 cells, suggesting that DMF could be a potential drug to treat sepsis. To further characterize the effect of DMF on LPS signaling, TNF-α, MCP-1, G-CMF, and IL-6 expression levels were determined by using cytokine array panels. In addition, an endotoxemia model with C57BL/6 mice was used to assess the in vivo efficacy of DMF on sepsis. The survival rate was assessed, and HE staining was performed to investigate histopathological damage to the organs. DMF was found to increase the survival of septic mice by 50% and attenuate organ damage, consistent with the reduction in IL-10, IL-6, and TNF-α (inflammatory cytokines) in serum. In vitro experiments revealed DMF's inhibitory effect on the phosphorylation of p65, IκB, and IKK, suggesting that the primary inhibitory effects of DMF can be attributed, at least in part, to the inhibition of phosphorylation of IκBα, IKK as well as nuclear factor-κB (NF-κB) upon LPS stimulation. The findings demonstrate that DMF dramatically inhibits NO and proinflammatory cytokine production in response to LPS and improves survival in septic mice, raising the possibility that DMF has the potential to be repurposed as a new treatment of sepsis.

Targeting cancer with molecular glues.

Molecular glues suppress the active form of the oncogenic protein KRAS.

Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines, anti-PD-1 and poly I:C.

Glioblastoma (GBM) is a lethal cancer with limited therapeutic options. Dendritic cell (DC)-based cancer vaccines provide a promising approach for GBM treatment. Clinical studies suggest that other immunotherapeutic agents may be combined with DC vaccines to further enhance antitumor activity. Here, we report a GBM case with combination immunotherapy consisting of DC vaccines, anti-programmed death-1 (anti-PD-1) and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy, and the patient remained disease-free for 69 months. The patient received DC vaccines loaded with multiple forms of tumor antigens, including mRNA-tumor associated antigens (TAA), mRNA-neoantigens, and hypochlorous acid (HOCl)-oxidized tumor lysates. Furthermore, mRNA-TAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histocompatibility complex (MHC) class I and II antigen presentation. The treatment consisted of 42 DC cancer vaccine infusions, 26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions. The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells. No immunotherapy-related adverse events were observed during the treatment. Robust antitumor CD4+ and CD8+ T-cell responses were detected. The patient remains free of disease progression. This is the first case report on the combination of the above three agents to treat glioblastoma patients. Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient. A large-scale trial to validate these findings is warranted.

Modulation of the Endomembrane System by the Anticancer Natural Product Superstolide/ZJ-101.

Marine natural products represent a unique source for clinically relevant drugs due to their vast molecular and mechanistic diversity. ZJ-101 is a structurally simplified analog of the marine natural product superstolide A, isolated from the New Caledonian sea sponge Neosiphonia Superstes. The mechanistic activity of the superstolides has until recently remained a mystery. Here, we have identified potent antiproliferative and antiadhesive effects of ZJ-101 on cancer cell lines. Furthermore, through dose-response transcriptomics, we found unique dysregulation of the endomembrane system by ZJ-101 including a selective inhibition of O-glycosylation via lectin and glycomics analysis. We applied this mechanism to a triple-negative breast cancer spheroid model and identified a potential for the reversal of 3D-induced chemoresistance, suggesting a potential for ZJ-101 as a synergistic therapeutic agent.

An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

Synthesis and biological evaluation of biotinylated ZJ-101.

ZJ-101 is a structurally simplified analog of marine natural product superstolide A that was previously designed and synthesized in our laboratory. Biological investigation shows that ZJ-101 maintains the potent anticancer activity of the original natural product with an undefined mechanism of action. To facilitate chemical biology study, a biotinylated ZJ-101 was synthesized and biologically evaluated.

ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain.

Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain.

Following peripheral nerve injury, extracellular ATP-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

Total Synthesis of Rameswaralide Utilizing a Pharmacophore-Directed Retrosynthetic Strategy.

A pharmacophore-directed retrosynthetic strategy was applied to the first total synthesis of the cembranoid rameswaralide in order to simultaneously achieve a total synthesis while also developing a structure-activity relationship profile throughout the synthetic effort. The synthesis utilized a Diels-Alder lactonization process, including a rare kinetic resolution to demonstrate the potential of this strategy for an enantioselective synthesis providing both the 5,5,6- and, through a ring expansion, 5,5,7-tricyclic ring systems present in several Sinularia soft coral cembranoids. A pivotal synthetic intermediate, a tricyclic epoxy α-bromo cycloheptenone, displayed high cytotoxicity with interesting selectivity toward the HCT-116 colon cancer cell line. This intermediate enabled the pursuit of three unique D-ring annulation strategies including a photocatalyzed intramolecular Giese-type radical cyclization and a diastereoselective, intramolecular enamine-mediated Michael addition, with the latter annulation constructing the final D-ring to deliver rameswaralide. The serendipitous discovery of an oxidation state transposition of the tricyclic epoxy cycloheptenone proceeding through a presumed doubly vinylogous, E1-type elimination enabled the facile introduction of the required α-methylene butyrolactone. Preliminary biological tests of rameswaralide and precursors demonstrated weak cytotoxicity; however, the comparable cytotoxicity of a simple 6,7-bicyclic ß-keto ester, corresponding to the CD-ring system of rameswaralide, to that of the natural product itself suggests that such bicyclic ß-ketoesters may constitute an interesting pharmacophore that warrants further exploration.

PAAN/MIF nuclease inhibition prevents neurodegeneration in Parkinson's disease.

Parthanatos-associated apoptosis-inducing factor (AIF) nuclease (PAAN), also known as macrophage migration inhibitor factor (MIF), is a member of the PD-D/E(X)K nucleases that acts as a final executioner in parthanatos. PAAN's role in Parkinson's disease (PD) and whether it is amenable to chemical inhibition is not known. Here, we show that neurodegeneration induced by pathologic α-synuclein (α-syn) occurs via PAAN/MIF nuclease activity. Genetic depletion of PAAN/MIF and a mutant lacking nuclease activity prevent the loss of dopaminergic neurons and behavioral deficits in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Compound screening led to the identification of PAANIB-1, a brain-penetrant PAAN/MIF nuclease inhibitor that prevents neurodegeneration induced by α-syn PFF, AAV-α-syn overexpression, or MPTP intoxication in vivo. Our findings could have broad relevance in human pathologies where parthanatos plays a role in the development of cell death inhibitors targeting the druggable PAAN/MIF nuclease.

Influence of stereochemistry on the activity of rapadocin, an isoform-specific inhibitor of the nucleoside transporter ENT1.

Rapadocin is a novel rapamycin-inspired polyketide-tetrapeptide hybrid macrocycle that possesses highly potent and isoform-specific inhibitory activity against the human equilibrative nucleoside transporter 1 (hENT1). Rapadocin contains an epimerizable chiral center in phenylglycine and an olefin group, and can thus exist as a mixture of four stereoisomers. Herein, we report the first total synthesis of the four stereoisomers of rapadocin using two different synthetic strategies and the assignment of their structures. The inhibitory activity of each of the four synthetic isomers on both hENT1 and hENT2 was determined. It was found that the stereochemistry of phenylglycine played a more dominant role than the configuration of the olefin in the activity of rapadocin. These findings will guide the future design and development of rapadocin analogs as new modulators of adenosine signaling.

A method for overcoming plasma protein inhibition of tyrosine kinase inhibitors.

FMS-like tyrosine kinase 3 (FLT3) is the most frequently-mutated gene in acute myeloid leukemia and a target for tyrosine kinase inhibitors (TKI). FLT3 TKI have yielded limited improvements to clinical outcomes. One reason for this is TKI inhibition by endogenous factors. We characterized plasma protein binding of FLT3 TKI, specifically staurosporine-derivatives (STS-TKI) by alpha-1-acid glycoprotein (AGP); simulating its effects upon drug efficacy. Human AGP inhibits the anti-proliferative activity of STS-TKI in FLT3-ITD-dependent cells, with IC50 shifts higher than clinically achievable. This is not seen with non-human plasma. Mifepristone co-treatment, with its higher AGP affinity, improves TKI activity despite AGP, yielding IC50s predicted to be clinically effective. In a mouse model of AGP drug inhibition, mifepristone restores midostaurin activity. This suggests combinatorial methods for overcoming plasma protein inhibition of existing TKIs for leukemia as well as providing a platform for investigating the drug-protein interaction space for developing more potent small-molecule agents.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.

Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.

Arsenicals, the Integrated Stress Response, and Epstein-Barr Virus Lytic Gene Expression.

Following our observation that clofoctol led to Epstein-Barr virus (EBV) lytic gene expression upon activation of the integrated stress response (ISR), we decided to investigate the impact of As2O3 on viral lytic gene expression. As2O3 has also been reported to activate the ISR pathway by its activation of the heme-regulated inhibitor (HRI). Our investigations show that As2O3 treatment leads to eIF2α phosphorylation, upregulation of ATF4 and TRB3 expression, and an increase of EBV Zta gene expression in lymphoid tumor cell lines as well as in naturally infected epithelial cancer cell lines. However, late lytic gene expression and virion production were blocked after arsenic treatment. In comparison, a small molecule HRI activator also led to increased Zta expression but did not block late lytic gene expression, suggesting that As2O3 effects on EBV gene expression are also mediated through other pathways.