NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells.

BACKGROUND: Genetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models. RESULTS: We treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells. CONCLUSIONS: NB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.

Malignant tumors in tuberous sclerosis complex: a case report and review of the literature.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare, autosomal dominant genetic disease that arises from TSC1 or TSC2 genetic mutations. These genetic mutations can induce the development of benign tumors in any organ system with significant clinical implications in morbidity and mortality. In rare instances, patients with TSC can have malignant tumors, including renal cell carcinoma (RCC) and pancreatic neuroendocrine tumor (PNET). It is considered a hereditary renal cancer syndrome despite the low incidence of RCC in TSC patients. TSC is typically diagnosed in prenatal and pediatric patients and frequently associated with neurocognitive disorders and seizures, which are often experienced early in life. However, penetrance and expressivity of TSC mutations are highly variable. Herein, we present a case report, with associated literature, to highlight that there exist undiagnosed adult patients with less penetrant features, whose clinical presentation may contain non-classical signs and symptoms, who have pathogenic TSC mutations. CASE PRESENTATION: A 31-year-old female with past medical history of leiomyomas status post myomectomy presented to the emergency department for a hemorrhagic adnexal cyst. Imaging incidentally identified a renal mass suspicious for RCC. Out of concern for hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, the mass was surgically removed and confirmed as RCC. Discussion with medical genetics ascertained a family history of kidney cancer and nephrectomy procedures and a patient history of ungual fibromas on the toes. Genetic testing for hereditary kidney cancer revealed a 5'UTR deletion in the TSC1 gene, leading to a diagnosis of TSC. Following the diagnosis, dermatology found benign skin findings consistent with TSC. About six months after the incidental finding of RCC, a PNET in the pancreatic body/tail was incidentally found on chest CT imaging, which was removed and determined to be a well-differentiated PNET. Later, a brain MRI revealed two small cortical tubers, one in each frontal lobe, that were asymptomatic; the patient's history and family history did not contain seizures or learning delays. The patient presently shows no evidence of recurrence or metastatic disease, and no additional malignant tumors have been identified. CONCLUSIONS: To our knowledge, this is the first report in the literature of a TSC patient without a history of neurocognitive disorders with RCC and PNET, both independently rare occurrences in TSC. The patient had a strong family history of renal disease, including RCC, and had several other clinical manifestations of TSC, including skin and brain findings. The incidental finding and surgical removal of RCC prompted the genetic evaluation and diagnosis of TSC, leading to a comparably late diagnosis for this patient. Reporting the broad spectrum of disease for TSC, including more malignant phenotypes such as the one seen in our patient, can help healthcare providers better identify patients who need genetic evaluation and additional medical care.

Radiation therapy for pineal parenchymal tumor of intermediate differentiation: A case series and literature review.

Pineal parenchymal tumor of intermediate differentiation (PPTID) is a rare, primary tumor of the pineal gland. Due to its rarity, there is no consensus on optimal therapeutic strategies or standard characterization of the tumor's behavior. Here, we report 2 new cases of PPTID and an extensive review of the literature involving the use and extent of radiation therapy. Patient 1 is a 54-year-old male who presented with PPTID and drop metastases in the spinal cord, received cranial spinal irradiation (CSI), and experienced recurrence 3.5 years after treatment. Stereotactic body radiation therapy (SBRT) helped the patient into remission for 9 months. Patient 2 is a 32-year-old male with a local PPTID at presentation who went on to receive surgical resection followed by focused adjuvant radiation therapy to the pineal tumor bed. He then presented 6 years after treatment with extensive disseminated recurrence and died due to leptomeningeal disease (LMD) about 4 years after recurrence. The available literature on PPTID is limited and reported cases of LMD with ongoing follow-up in PPTID are scarce. Our report adds to the current known PPTID cases, contributing to the information available regarding prognosis and treatment response. Although an optimal therapeutic strategy for PPTID still cannot be determined, data from the literature suggest that utilizing radiation therapy in patients with low-risk disease and gross total resections as well as the use of upfront CSI have the potential to improve patient progression and survival outcomes.

An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1-GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

FOXM1: A Multifunctional Oncoprotein and Emerging Therapeutic Target in Ovarian Cancer.

Forkhead box M1 (FOXM1) is a member of the conserved forkhead box (FOX) transcription factor family. Over the last two decades, FOXM1 has emerged as a multifunctional oncoprotein and a robust biomarker of poor prognosis in many human malignancies. In this review article, we address the current knowledge regarding the mechanisms of regulation and oncogenic functions of FOXM1, particularly in the context of ovarian cancer. FOXM1 and its associated oncogenic transcriptional signature are enriched in >85% of ovarian cancer cases and FOXM1 expression and activity can be enhanced by a plethora of genomic, transcriptional, post-transcriptional, and post-translational mechanisms. As a master transcriptional regulator, FOXM1 promotes critical oncogenic phenotypes in ovarian cancer, including: (1) cell proliferation, (2) invasion and metastasis, (3) chemotherapy resistance, (4) cancer stem cell (CSC) properties, (5) genomic instability, and (6) altered cellular metabolism. We additionally discuss the evidence for FOXM1 as a cancer biomarker, describe the rationale for FOXM1 as a cancer therapeutic target, and provide an overview of therapeutic strategies used to target FOXM1 for cancer treatment.

Enhanced 2'-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate.

2'-Fucosyllactose (2'-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2'-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2'-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2'-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2'-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2'-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2'-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2'-FL production. The resulting engineered yeast produced 25.5 g/L of 2'-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2'-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2'-FL was secreted when a complex medium was used, 82% of the produced 2'-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2'-FL enriched yeast extract can be produced cost-effectively using the 2'-FL-accumulating yeast cells.

SIRT1 is a positive regulator of in vivo bone mass and a therapeutic target for osteoporosis.

Overexpression or pharmacological activation of SIRT1 has been shown to extend the lifespan of mice and protect against aging-related diseases. Here we show that pharmacological activation of SIRT1 protects in two models of osteoporosis. Ovariectomized female mice and aged male mice, models for post-menopausal and aging-related osteoporosis, respectively, show significant improvements in bone mass upon treatment with SIRT1 agonist, SRT1720. Further, we find that calorie restriction (CR) results in a two-fold upregulation of sirt1 mRNA expression in bone tissue that is associated with increased bone mass in CR mice. Reciprocally, SIRT1 whole-body knockout (KO) mice, as well as osteoblast and osteoclast specific KOs, show a low bone mass phenotype; though double knockout mice (containing SIRT1 deleted in both osteoblasts and osteoclasts) do not show a more severe phenotype. Altogether, these findings provide strong evidence that SIRT1 is a positive regulator of bone mass and a promising target for the development of novel therapeutics for osteoporosis.

SIRT1 is a positive regulator of the master osteoblast transcription factor, RUNX2.

Activation of SIRT1 has previously been shown to protect mice against osteoporosis through yet ill-defined mechanisms. In this study, we outline a role for SIRT1 as a positive regulator of the master osteoblast transcription factor, RUNX2. We find that ex vivo deletion of sirt1 leads to decreased expression of runx2 downstream targets, but not runx2 itself, along with reduced osteoblast differentiation. Reciprocally, treatment with a SIRT1 agonist promotes osteoblast differentiation, as well as the expression of runx2 downstream targets, in a SIRT1-dependent manner. Biochemical and luciferase reporter assays demonstrate that SIRT1 interacts with and promotes the transactivation potential of RUNX2. Intriguingly, mice treated with the SIRT1 agonist, resveratrol, show similar increases in the expression of RUNX2 targets in their calvaria (bone tissue), validating SIRT1 as a physiologically relevant regulator of RUNX2.

Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.

Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.

A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo.

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 µg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.

Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction.

Hepatocyte growth factor (HGF) has been shown to have anti-fibrotic, pro-angiogenic, and cardioprotective effects; however, it is highly unstable and expensive to manufacture, hindering its clinical translation. Recently, a HGF fragment (HGF-f), an alternative c-MET agonist, was engineered to possess increased stability and recombinant expression yields. In this study, we assessed the potential of HGF-f, delivered in an extracellular matrix (ECM)-derived hydrogel, as a potential treatment for myocardial infarction (MI). HGF-f protected cardiomyocytes from serum-starvation and induced down-regulation of fibrotic markers in whole cardiac cell isolate compared to the untreated control. The ECM hydrogel prolonged release of HGF-f compared to collagen gels, and in vivo delivery of HGF-f from ECM hydrogels mitigated negative left ventricular (LV) remodeling, improved fractional area change (FAC), and increased arteriole density in a rat myocardial infarction model. These results indicate that HGF-f may be a viable alternative to using recombinant HGF, and that an ECM hydrogel can be employed to increase growth factor retention and efficacy.

Improving neutralization potency and breadth by combining broadly reactive HIV-1 antibodies targeting major neutralization epitopes.

UNLABELLED: The isolation of broadly neutralizing HIV-1 monoclonal antibodies (MAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of MAbs for prevention and treatment of HIV-1 infection. Since many of these MAbs have been isolated in the last few years, the potency and breadth of MAb combinations have not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double-, triple-, and quadruple-MAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite, and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All MAb combinations showed substantially improved neutralization breadth compared to the corresponding single MAbs, while the neutralization potency of individual MAbs was maintained. At a 50% inhibitory concentration (IC50) cutoff of 1 µg/ml per antibody, double-MAb combinations neutralized 89 to 98% of viruses, and triple combinations neutralized 98 to 100%. Overall, the improvement of neutralization breadth was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some MAb combinations, whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan MAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 MAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment. IMPORTANCE: Over the last 5 years, numerous broadly reactive HIV-1-neutralizing MAbs have been isolated from B cells of HIV-1-infected donors. Each of these MAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes could theoretically act together to provide greater potency and breadth of virus neutralization, we tested physical mixtures of double, triple, and quadruple combinations of neutralizing MAbs targeting four major epitopes on HIV-1 Env. When tested together, antibody combinations showed substantially improved neutralization breadth compared to single MAbs. This improvement could be explained by the complementary neutralization profiles of individual MAbs. We further demonstrated that each antibody maintained its full neutralization potency when used in combination with other MAbs. These data provide a rationale for clinical use of antibody-based combinations for HIV-1 prevention and therapy.

An engineered dimeric fragment of hepatocyte growth factor is a potent c-MET agonist.

Hepatocyte growth factor (HGF), through activation of the c-MET receptor, mediates biological processes critical for tissue regeneration; however, its clinical application is limited by protein instability and poor recombinant expression. We previously engineered an HGF fragment (eNK1) that possesses increased stability and expression yield and developed a c-MET agonist by coupling eNK1 through an introduced cysteine residue. Here, we further characterize this eNK1 dimer and show it elicits significantly greater c-MET activation, cell migration, and proliferation than the eNK1 monomer. The efficacy of the eNK1 dimer was similar to HGF, suggesting its promise as a c-MET agonist.

Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants.

Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian-human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants.

Broadly neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice.

Latent reservoirs of HIV-1-infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a "shock and kill" approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR-mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice.

Lipopolysaccharide biosynthesis without the lipids: recognition promiscuity of Escherichia coli heptosyltransferase I.

Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k(cat)/K(m) of 2.7 × 10(6) M(-1) s(-1), compared to a value of 2.2 × 10(5) M(-1) s(-1) for the Kdo(2)-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.

Using ligand-based virtual screening to allosterically stabilize the activated state of a GPCR.

G-protein coupled receptors play an essential role in many biological processes. Despite an increase in the number of solved X-ray crystal structures of G-protein coupled receptors, capturing a G-protein coupled receptor in its activated state for structural analysis has proven to be difficult. An unexplored paradigm is stabilization of one or more conformational states of a G-protein coupled receptor via binding a small molecule to the intracellular loops. A short tetrazole peptidomimetic based on the photoactivated state of rhodopsin-bound structure of Gt(alpha)(340-350) was previously designed and shown to stabilize the photoactivated state of rhodopsin, the G-protein coupled receptor involved in vision. A pharmacophore model derived from the designed tetrazole tetrapeptide was used for ligand-based virtual screening to enhance the possible discovery of novel scaffolds. Maybridge Hitfinder and National Cancer Institute diversity libraries were screened for compounds containing the pharmacophore. Forty-seven compounds resulted from virtually screening the Maybridge library, whereas no hits resulted with the National Cancer Institute library. Three of the 47 Maybridge compounds were found to stabilize the MII state. As these compounds did not inhibit binding of transducin to photoactivated state of rhodopsin, they were assumed to be allosteric ligands. These compounds are potentially useful for crystallographic studies where complexes with these compounds might capture rhodopsin in its activated conformational state.