CXCR4/CXCL12 mediate autocrine cell- cycle progression in NF1-associated malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. They occur sporadically in a subset of patients with neurofibromatosis type 1 (NF1). MPNSTs are highly aggressive, therapeutically resistant, and typically fatal. Using comparative transcriptome analysis, we identified CXCR4, a G-protein-coupled receptor, as highly expressed in mouse models of NF1-deficient MPNSTs, but not in nontransformed precursor cells. The chemokine receptor CXCR4 and its ligand, CXCL12, promote MPNST growth by stimulating cyclin D1 expression and cell-cycle progression through PI3-kinase (PI3K) and ß-catenin signaling. Suppression of CXCR4 activity either by shRNA or pharmacological inhibition decreases MPNST cell growth in culture and inhibits tumorigenesis in allografts and in spontaneous genetic mouse models of MPNST. We further demonstrate conservation of these activated molecular pathways in human MPNSTs. Our findings indicate a role for CXCR4 in NF1-associated MPNST development and identify a therapeutic target.

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy.

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.

Alveolar ridge width and height changes after orthodontic space opening in patients congenitally missing maxillary lateral incisors.

The purpose of this study was to evaluate the dimensional changes of the alveolar ridge in patients with congenitally missing maxillary lateral incisors. The width and height of the alveolar ridge were compared before and after opening space for an endosseous dental implant between the central incisor and canine. Pre- and post-treatment dental stone models of 31 patients (8 males, 23 females; mean age 15.1 ± 7.9 years pre-treatment, 17.6 ± 8 years post-treatment) with unilaterally or bilaterally, congenitally missing maxillary lateral incisors were used in this study. Pre- and post-treatment measurements included: the space between the maxillary central incisor and canine, the depth of the labial concavity, and the width and height of the lateral incisor alveolar ridge. Two different techniques were used to measure the ridge width. Student's paired samples t-test was used to test for significance. The alveolar ridge underwent statistically significant width loss (Method 1: 4-8 per cent, Method 2: 13-15 per cent) during the course of orthodontic treatment. A 6-12 per cent loss in ridge height was also noted. The depth of the labial concavity between the maxillary central incisor and canine nearly doubled. There was a significant decrease in the width and height of the alveolar ridge in patients congenitally missing a maxillary lateral incisor who received orthodontic treatment to create space for an endosseous dental implant.

Protein kinase inhibitors for the treatment of prostate cancer.

INTRODUCTION: Protein kinases have emerged as targetable pathways used in metastatic prostate cancer given their role in prostatic tumor growth, proliferation and metastases. Protein kinase inhibitors are small molecules that target varying pathways including the breakpoint cluster region (BCR)-Abelson tyrosine kinase (ABL), colony stimulating factor-1 receptor (CSF1R), vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) and phosphoinositide 3-kinase (PI3K) pathways and have been studied in prostate cancer trials with variable results. In particular, cabozantinib when used in combination trials and ipatasertib, when used with abiraterone in patients who harbor phosphatase and tensin homologue (PTEN) loss, have been promising. AREAS COVERED: This article reviews the key early and late phase clinical trials currently investigating the use of protein kinase inhibitors in prostate cancer. EXPERT OPINION: While multiple kinase inhibitors show promising results in prostate cancer, none have yet garnered Food and Drug Administration (FDA) approval. Studies are ongoing with the best candidate drugs discussed herein. However, multiple drugs have failed primary endpoints in prostate cancer. Therefore, further understanding of the potential mechanisms of resistance, combination and trial design of combination therapy may help pave the way for targeting kinase inhibition in prostate cancer.

Anaplastic Features in Advanced Prostate Cancer With and Without DNA Damage Repair Mutations.

BACKGROUND: Anaplastic prostate cancer has a poor prognosis with limited treatment options. Seven clinical features of anaplastic prostate cancer have been prospectively identified. In this phase II clinical trial, we identified mutations, including DNA damage repair (DDR) mutations, in patients with metastatic castration-resistant prostate cancer (mCRPC) who were treated with durvalumab and olaparib and determined how many of them can be described as anaplastic, and we examined the overlap between anaplastic features and DDR mutations. METHODS: Eligible patients with mCRPC received prior enzalutamide, abiraterone, or both. Patients were treated with durvalumab 1500 mg i.v. every 28 days and olaparib 300 mg p.o. every 12 hours until disease progression or unacceptable toxicity. Patients underwent mandatory baseline biopsies of metastatic lesions. RESULTS: Baseline characteristics were similar between anaplastic and nonanaplastic patients. Eleven patients (20%) displayed clear anaplastic features, and 43 (78.2%) lacked anaplastic features. In the anaplastic group, 2/11 (18.2%) had germline DRR mutations, and 4/11 (36.3%) had somatic DDR mutations. In the nonanaplastic group, 7/43 (16.3%) had germline mutations, and 13/43 (30.2%) had somatic mutations. Median progression-free survival (PFS) times in patients with anaplastic features (6.5 months) and without anaplastic features (5.1 months) were similar (hazard ratio 0.998, P = .996). CONCLUSIONS: Patients with and without anaplastic features appear to have similar total rates of DDR mutations and also similar rates of somatic and germline DDR mutations. Patients with anaplastic features have a trend toward improved PFS when treated with olaparib and durvalumab compared with nonanaplastic patients.

Pembrolizumab in Combination with Axitinib as First-Line Treatment for Patients with Renal Cell Carcinoma (RCC): Evidence to Date.

Over the last 18 months, 3 immunotherapy combination regimens (ipilimumab + nivolumab, pembrolizumab + axitinib, and axitinib + avelumab) were approved by the US Food and Drug Administration for the first-line treatment of metastatic renal cell carcinoma (mRCC), making selection of the optimal first-line treatment regimen very challenging. As of April 2020, preferred first-line treatment options for mRCC are pembrolizumab + axitinib and ipilimumab + nivolumab, based on the improvement in overall survival and progression-free survival compared to sunitinib, as observed in pivotal phase III clinical trials. Because the combination of 2 drugs is typically more toxic than a monotherapy, careful attention must be given to overlapping toxicities. The pembrolizumab + axitinib combination led to grade ≥3 adverse events in 75.8% of patients (vs 70.6% in the sunitinib group), while grade ≥3 adverse events were less frequent in the nivolumab + ipilimumab group compared to the sunitinib group. Discontinuation rates due to toxicity were 10.7% for pembrolizumab + axitinib (both drugs), 22% for ipilimumab + nivolumab and were comparable with sunitinib in both studies (13.9% and 12%, respectively). The combination of pembrolizumab + axitinib may have immune-modulating functions that may provide clinical benefit without the additional toxicity observed with ipilimumab + nivolumab. In addition, this tyrosine kinase inhibitor + immune checkpoint combination should have faster treatment response in patients with larger disease burden or in more symptomatic patients, which makes this combination an excellent choice for the first-line treatment regimen for mRCC. These combinations have proven to be tolerable, though long-term results are still lacking. As treatment options for mRCC are rapidly expanding, immunotherapy combinations could potentially change the treatment paradigm, with the ultimate goal of prolonging life and eventually curing mRCC.

Exploiting defects in homologous recombination repair for metastatic, castration-resistant prostate cancer.

The PROfound trial highlights that there is a benefit in testing genes involved in homologous recombination (HR) and forms the rationale for testing in all patients with metastatic, castration-resistant prostate cancer (mCRPC). This trial also demostrates that olaparib improves progression free survival (PFS), objective response rate (ORR), and time to pain progression in patients who harbor alterations in BRCA1, BRCA2, and ATM. These are groundbreaking findings - this is the first trial that demonstrates the efficacy of olaparib versus standard therapy in a genomically-selected patient population with metastatic prostate cancer. Although this trial does not demonstrate improvements in overall survival (OS), we believe that this may be an underestimation based on trial-design. Future studies of olaparib are likely to yield further promising results.

Impact of Primary Care Access on Mortality of Lung Cancer Patients in an Underserved Community.

BACKGROUND: Lack of access to primary care physicians (PCPs) may be an important contributor to mortality differences attributed to race/ethnicity. This study examined the effects of primary care access on mortality of lung cancer patients in an underserved community. METHODS: Medical records of all newly diagnosed patients with primary lung cancer from 2012 to 2016 at a National Cancer Institute (NCI)-designated center in Bronx, New York were reviewed. Demographic data, PCP status, and residence in primary care shortage areas (PCSAs) were collected. Survival data from time of first imaging to death or the end of follow-up on January 1, 2018 were recorded. Survival analysis was performed using Kaplan-Meier and Cox hazards modeling. RESULTS: Among 1062 patients, 874 (82%) were PCSA residents, 314 (30%) were Hispanic, and 445 (42%) were African American. PCSA residents were likely Hispanics (P<0.001), African Americans (P<0.001), of lower income (P<0.001), and had advanced disease at diagnosis (P=0.01). Patients without established PCPs had more comorbidities (P=0.04), more advanced disease (P<0.001), and less in-network cancer treatment (P<0.001). PCSA residence (P=0.03, hazard ratio [HR]=1.27) and no established PCP (P<0.001, HR=1.50) were associated with increased mortality. In multivariable modeling, lack of established PCP remained a predictor of increased mortality (P=0.02, HR=1.25). DISCUSSION: Among newly diagnosed lung cancer patients, lack of established PCP is associated with increased mortality. Hispanics and African Americans increasingly resided in PCSAs, suggesting race/ethnicity mortality differences may be mediated by primary care shortage. Patients without PCPs had worse health outcomes. Effective health policy efforts to reduce mortality in lung cancer patients must include approaches to improve primary care access.

TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum.

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

Temozolomide induces the expression of the glioma Big Potassium (gBK) ion channel, while inhibiting fascin-1 expression: possible targets for glioma therapy.

OBJECTIVE: Temozolomide (TMZ) improves Glioblastoma Multiforme (GBM) patient survival. The invasive behavior of the glioma cells is the cause of GBM relapse. The glioma BK ion channel (gBK) may provide glioma cells with a mechanism to invade surrounding tissue. gBK contains epitopes that cytolytic T lymphocytes (CTLs) can recognize and kill glioma cells. Fascin-1 is an actin crosslinking molecule that supports microvilli; these membrane protrusions provide a physical defense against CTLs. TMZ was investigated to determine its effect on gBK and fascin-1 expression. RESEARCH DESIGN AND METHODS: Human glioma cells cultured in TMZ were analyzed for their altered mRNA and gBK protein levels by using quantitative real time PCR, immunostaining and cellular functional assays. RESULTS: TMZ slowed glioma cell growth and inhibited their transmigratory properties due to loss of fascin-1. TMZ induced increased gBK and HLA expression and allowed these TMZ-treated cells to become better targets for gBK-specific CTLs. CONCLUSIONS: Besides its traditional chemotherapeutic effect, TMZ can have four other targeted pathways: 1) slowed glioma cell growth; 2) inhibited glioma cell transmigration; 3) increased HLA-A2 and gBK tumor antigen production; 4) increased CTL-mediated cytolysis of the TMZ treated glioma cells due to the loss of their defensive membrane protrusions supported by fascin-1.

Lanreotide autogel in acromegaly - a decade on.

INTRODUCTION: The novel formulation of lanreotide, lanreotide (LAN) autogel (ATG), has been available in Europe since 2001 and USA from 2006 for the treatment of acromegaly. It is one of only two clinically available somatostatin analogs available for use in acromegaly. Data relating to the use of ATG in acromegaly, specifically relating to comparison to octreotide (OCT) LAR and patient acceptability and preference, have been slow to accumulate. AREAS COVERED: We performed a comprehensive review of the original literature relating to development, pharmacokinetics, acceptability and clinical efficacy of ATG. EXPERT OPINION: LAN ATG is a novel formulation of LAN consequent on self-assembly of nanotubules in water. Diffusion between molecules within the nanotubules and surrounding tissue fluid in vivo leads to pseudo first-order pharmacokinetics. Efficacy is equivalent to the alternate long-acting somatostatin analog, OCT LAR, normalizing growth hormone and IGF-I levels in around 60 and 50% respectively. Control of tumor growth is observed in over 95% of patients, with 64% seeing a clinically significant reduction in tumor size. ATG is provided in a prefilled syringe for deep subcutaneous injection, allowing self-injection, and may be administered up to 8 weeks greatly improving convenience for the patient. The data strongly support consideration of ATG as the medical therapy of choice for patients with acromegaly.

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells.

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.

Preclinical therapeutic efficacy of a novel pharmacologic inducer of apoptosis in malignant peripheral nerve sheath tumors.

Neurofibromatosis type I (NF1) is an autosomal disorder that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. Patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (MPNST), a main cause of mortality. Currently, surgery is the primary course of treatment for MPNST, but with the limitation that these tumors are highly invasive. Radiotherapy is another treatment option, but is undesirable because it can induce additional mutations. Patients with MPNST may also receive doxorubicin as therapy, but this DNA-intercalating agent has relatively low tumor specificity and limited efficacy. In this study, we exploited a robust genetically engineered mouse model of MPNST that recapitulates human NF1-associated MPNST to identify a novel small chemical compound that inhibits tumor cell growth. Compound 21 (Cpd21) inhibits growth of all available in vitro models of MPNST and human MPNST cell lines, while remaining nontoxic to normally dividing Schwann cells or mouse embryonic fibroblasts. We show that this compound delays the cell cycle and leads to cellular apoptosis. Moreover, Cpd21 can reduce MPNST burden in a mouse allograft model, underscoring the compound's potential as a novel chemotherapeutic agent.

Simultaneous bilateral spontaneous pneumothoraces in a patient with occupational asthma.

Spontaneous pneumothoraces are relatively common; however, simultaneous bilateral spontaneous pneumothoraces (SBSP) have rarely been reported. This case report describes the presentation of SBSP in a 60-year-old man with occupational asthma. He was initially started on treatment for life-threatening asthma, but an early deterioration in symptoms prompted an urgent chest radiography that established the diagnosis of bilateral pneumothoraces. This was managed with bilateral needle thoracocentesis followed by stabilisation with intercostal chest drains. He was subsequently referred to the thoracic unit for minithoracotomy, bullectomy and talc pleurodesis. This case highlights the potential difficulties in diagnosing SBSP and advocates the necessity for prompt chest radiography when managing such presentations in the acute setting.

Aqueous Redox Chemistry and the Electronic Band Structure of Liquid Water.

The electronic states of aqueous species can mix with the extended states of the solvent if they are close in energy to the band edges of water. Using density functional theory-based molecular dynamics simulation, we show that this is the case for OH(-) and Cl(-). The effect is, however, badly exaggerated by the generalized gradient approximation leading to systematic underestimation of redox potentials and spurious nonlinearity in the solvent reorganization. Drawing a parallel to charged defects in wide gap solid oxides, we conclude that misalignment of the valence band of water is the main source of error turning the redox levels of OH(-) and Cl(-) in resonant impurity states. On the other hand, the accuracy of energies of levels corresponding to strongly negative redox potentials is acceptable. We therefore predict that mixing of the vertical attachment level of CO2 and the unoccupied states of water is a real effect.

E2-25K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system.

In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.

Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane.

Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.

Budding of equine infectious anemia virus is insensitive to proteasome inhibitors.

The only retrovirus protein required for the budding of virus-like particles is the Gag protein; however, recent studies of Rous sarcoma virus (RSV) and human immunodeficiency virus have suggested that modification of Gag with ubiquitin (Ub) is also required. As a consequence, the release of these viruses is reduced in the presence of proteasome inhibitors, which indirectly reduce the levels of free Ub within the cell. Here we show that the budding of equine infectious anemia virus (EIAV) from infected equine cells is largely unaffected by these drugs, although use of one inhibitor (MG-132) resulted in a dramatic block to proteolytic processing of Gag. This lack of sensitivity was also observed in transiently transfected avian cells under conditions that greatly reduce RSV budding. Moreover, insensitivity was observed when the EIAV Gag protein was expressed in the absence of all the other virus products, indicating that they are not required for this phenotype. An activity that enables EIAV to tolerate exposure to proteasome inhibitors was mapped to the C-terminal p9 sequence, as demonstrated by the ability of an RSV Gag-p9 chimera to bud in the presence of the drugs. Intriguingly, the p9 sequence contains a short sequence motif that is similar to a surface-exposed helix of Ub, suggesting that EIAV Gag may have captured a function that allows it to bypass the need for ubiquitination. Thus, the mechanism of EIAV budding may not be substantially different from that of other retroviruses, even though it behaves differently in the presence of proteasome inhibitors.

An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells.

Skp1-Cdc53/Cul1-F-box (SCF) complexes constitute a class of E3 ubiquitin ligases. Recently, a multiprotein complex containing pVHL, elongin C and Cul2 (VEC) was shown to structurally and functionally resemble SCF complexes. Cdc53 and the Cullins can become covalently linked to the ubiquitin-like molecule Rub1/NEDD8. Inhibition of neddylation inhibits SCF function in vitro and in yeast and plants. Here we show that ongoing neddylation is likewise required for VEC function in vitro and for the degradation of SCF and VEC targets in mammalian cells. Thus, neddylation regulates the activity of two specific subclasses of mammalian ubiquitin ligases.