Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release.

Synaptotagmin 1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer; however, biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and we used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (∼180 nm) were reduced to smaller rings (∼30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in 1) clamping spontaneous release and 2) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

Targeting renal cell carcinoma with a HIF-2 antagonist.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Cholesterol stabilizes recombinant exocytic fusion pores by altering membrane bending rigidity.

SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

Beyond Amphiphilic Balance: Changing Subunit Stereochemistry Alters the Pore-Forming Activity of Nylon-3 Polymers.

Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides, with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3 polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria, whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells. To ask whether factors beyond amphiphilic balance influence polymer activities, we synthesized and evaluated new nylon-3 polymers with two stereoisomeric subunits, each bearing an ethyl side chain and an aminomethyl side chain. Subunits that differ only in stereochemistry are predicted to contribute equally to amphiphilic balance, but we observed that the stereochemical difference correlates with significant changes in biological activity profile. Antibacterial activities were not strongly affected by subunit stereochemistry, but the ability to disrupt eukaryotic cell membranes varied considerably. Experiments with planar lipid bilayers and synthetic liposomes suggested that eukaryotic membrane disruption results from polymer-mediated formation of large pores. Collectively, our results suggest that factors other than amphiphilic balance influence the membrane activity profile of synthetic polymers. Subunits that differ in stereochemistry are likely to have distinct conformational propensities, which could potentially lead to differences in the average shapes of polymer chains, even when the subunits are heterochiral. These findings highlight a dimension of polymer design that should be considered more broadly in efforts to improve specificity and efficacy of antimicrobial polymers.

Author Correction: MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Overcoming sociodemographic factors in the care of patients with testicular cancer at a safety net hospital.

BACKGROUND: The objective of this study was to determine whether standardized treatment of germ cell tumors (GCTs) could overcome sociodemographic factors limiting patient care. METHODS: The records of all patients undergoing primary treatment for GCTs at both a public safety net hospital and an academic tertiary care center in the same metropolitan area were analyzed. Both institutions were managed by the same group of physicians in the context of multidisciplinary cancer care. Patients were grouped by care center; clinicopathologic features and outcomes were analyzed. RESULTS: Between 2006 and 2018, 106 and 95 patients underwent initial treatment for GCTs at the safety net hospital and the tertiary care center, respectively. Safety net patients were younger (29 vs 33 years; P = .005) and were more likely to be Hispanic (79% vs 11%), to be uninsured (80% vs 12%; P < .001), to present via the emergency department (76% vs 8%; P < .001), and to have metastatic (stage II/III) disease (42% vs 26%; P = .025). In a multivariable analysis, an absence of lymphovascular invasion (odds ratio [OR], 0.30; P = .008) and an embryonal carcinoma component (OR, 0.36; P = .02) were associated with decreased use of adjuvant treatment for stage I patients; hospital setting was not (OR, 0.67; P = .55). For patients with stage II/III nonseminomatous GCTs, there was no difference in the performance of postchemotherapy retroperitoneal lymph node dissection between the safety net hospital and the tertiary care center (52% vs 64%; P = .53). No difference in recurrence rates was observed between the cohorts (5% vs 6%; P = .76). CONCLUSIONS: Sociodemographic factors are often associated with adverse clinical outcomes in the treatment of GCTs; they may be overcome with integrated, standardized management of testicular cancer.

MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.

mTORC1 activates SREBP-2 by suppressing cholesterol trafficking to lysosomes in mammalian cells.

mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5-/- and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.

Natural history of 'second' biochemical failure after salvage radiation therapy for prostate cancer: a multi-institution study.

OBJECTIVES: To describe the natural history of prostate cancer in men who experience a second biochemical recurrence (BCR) after salvage radiotherapy (SRT) after prostatectomy. PATIENTS AND METHODS: After undergoing SRT at one of two institutions between 1986 and 2013, 286 patients experienced a second BCR, defined as two rises in prostate-specific antigen (PSA) of ≥0.2 ng/mL above nadir. Event rates for distant metastasis (DM) or freedom from DM (FFDM), castration-resistant prostate cancer (CRPC), prostate cancer-specific survival (PCSS), and overall survival (OS) were estimated using the Kaplan-Meier method. Cox regression was used for comparative analyses. RESULTS: At a median of 6.1 years after second BCR, DM, CRPC, PCSS and OS rates were 41%, 27%, 83% and 73%, respectively. On multivariable analysis, interval to second BCR <1 year (hazard ratio [HR] 2.66, 95% confidence interval [CI] 1.71-4.14; P < 0.001], Gleason score 8-10 (HR 1.65, 95% CI 1.07-2.54; P = 0.022), and concurrent ADT during SRT (HR 1.76, 95% CI 1.08-2.88; P = 0.024) were associated with FFDM, while PCSS was associated with interval to second BCR <1 year (HR 3.00, 95% CI 1.69-5.32; P < 0.001) and concurrent ADT during SRT (HR 2.15, CI 1.13-4.08; P = 0.019). These risk factors were used to stratify patients into three groups, with 6-year FFDM rates of 71%, 59% and 33%, and PCSS rates of 89%, 79%, and 65%, respectively. CONCLUSION: Following second BCR after SRT, clinical progression is enriched in a subgroup of patients with prostate cancer, while others remain without DM for long intervals. Stratifying patients into risk groups using prognostic factors may aid counselling and future trial design.

Identification of CDCP1 as a hypoxia-inducible factor 2α (HIF-2α) target gene that is associated with survival in clear cell renal cell carcinoma patients.

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α-, but not HIF-1α-, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.

Changing trends in utilization of neoadjuvant chemotherapy in muscle-invasive bladder cancer.

INTRODUCTION: To reassess use of perioperative chemotherapy in muscle-invasive bladder cancer (MIBC) following implementation of monthly multidisciplinary meetings to facilitate optimal oncologic treatment. We previously reported from 2003 to 2008 17% of eligible patients with bladder cancer received cisplatin-based neoadjuvant chemotherapy (NAC) at our institution. MATERIALS AND METHODS: A retrospective review of all patients who underwent radical cystectomy (RC) between 2008 and 2012 was performed. Information on clinical and pathologic stage, renal function, perioperative chemotherapy (CTX) use and oncologic outcomes was collected. Rationale for utilization decisions was obtained from physician encounter notes. Primary outcome was use of CTX among eligible patients. Secondary measures were type of CTX, pathologic and survival outcomes. RESULTS: Among 261 patients undergoing RC for bladder cancer, 162 were eligible for NAC. Overall 40.7% (n = 66) received NAC, and 86.4% were given platinum. Patients given NAC were younger and had more advanced clinical stage. The degree of chronic kidney disease (CKD) (0-3) did not impact likelihood of receiving NAC. NAC patients were more likely to be downstaged to non-muscle-invasive disease (21.2% versus 7.3% p < 0.01) or have a complete pathologic response (12.1% versus 3.1% p = 0.025). Receipt of NAC did not affect oncologic outcomes. Following RC 22.3% of high risk patients (n = 112) received adjuvant chemotherapy (AC). CONCLUSIONS: Our use of cisplatin-based NAC improved from 17% to 35% and overall utilization of NAC increased from 22% to 41%. NAC led to improved pT0 rates and increased pathologic downstaging. The degree of CKD (0-3) did not impact likelihood of receiving NAC. AC use decreased in part due to higher utilization of NAC.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration.

Synaptotagmin-1 and synaptotagmin-7 are two prominent calcium sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding calcium, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examine the biophysical properties of these two synaptotagmin isoforms and compare their interactions with phospholipid membranes. We discover that synaptotagmin-1-membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a super-penetrator. We exploit these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to calcium.

Emergence of BRCA Reversion Mutations in Patients with Metastatic Castration-resistant Prostate Cancer After Treatment with Rucaparib.

BACKGROUND: Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors are approved in the USA for the treatment of patients with BRCA1 or BRCA2 (BRCA) mutated (BRCA+) metastatic castration-resistant prostate cancer (mCRPC). BRCA reversion mutations are a known mechanism of acquired resistance to PARP inhibitors in multiple cancer types, although their impact and prevalence in mCRPC remain unknown. OBJECTIVE: To examine the prevalence of BRCA reversion mutations in the plasma of patients with BRCA+ mCRPC after progression on rucaparib. DESIGN, SETTING, AND PARTICIPANTS: Men with BRCA+ mCRPC enrolled in Trial of Rucaparib in Prostate Indications 2 (TRITON2) were treated with rucaparib after progressing on one to two lines of androgen receptor-directed and one taxane-based therapy. Cell-free DNA from the plasma of 100 patients, collected at the end of treatment after confirmed progression before May 5, 2020, was queried for BRCA reversion mutations using next-generation sequencing (NGS). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association of clinical efficacy and postprogression genomics was measured in 100 patients with BRCA+ mCRPC treated with rucaparib. RESULTS AND LIMITATIONS: No baseline BRCA reversion mutations were observed in 100 BRCA+ patients. NGS identified somatic BRCA reversion mutations in 39% (39/100) of patients after progression. Reversion rates were similar for BRCA2 and BRCA1, irrespective of germline or somatic status, but higher in samples with a high tumor DNA fraction. Most patients with reversions (74%, 29/39) had two or more reversion mutations occurring subclonally at lower allele frequencies than the original BRCA mutations. The incidence of BRCA reversion mutations increased with the duration of rucaparib treatment. The frequency of reversion mutations was higher in patients with an objective (58%) or a prostate-specific antigen (69%) response compared with those without either (39% and 29%, respectively). CONCLUSIONS: These findings suggest that BRCA reversion mutations are a significant mechanism of acquired resistance to rucaparib in patients with BRCA+ mCRPC, with evidence of subclonal convergence promoting systemic resistance. PATIENT SUMMARY: Men with BRCA mutated metastatic castration-resistant prostate cancer enrolled in TRITON2 were treated with rucaparib after progressing on one to two lines of androgen receptor-directed and one taxane-based therapy. Cell-free DNA from the plasma of 100 patients, collected after radiographic or prostate-specific antigen progression before May 5, 2020, was analyzed by next-generation sequencing and queried for BRCA reversion mutations.

Mitochondrial metabolism in primary and metastatic human kidney cancers.

Most kidney cancers display evidence of metabolic dysfunction1-4 but how this relates to cancer progression in humans is unknown. We used a multidisciplinary approach to infuse 13C-labeled nutrients during surgical tumour resection in over 70 patients with kidney cancer. Labeling from [U-13C]glucose varies across cancer subtypes, indicating that the kidney environment alone cannot account for all metabolic reprogramming in these tumours. Compared to the adjacent kidney, clear cell renal cell carcinomas (ccRCC) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in organotypic slices cultured ex vivo, indicating that suppressed labeling is tissue intrinsic. Infusions of [1,2-13C]acetate and [U-13C]glutamine in patients, coupled with respiratory flux of mitochondria isolated from kidney and tumour tissue, reveal primary defects in mitochondrial function in human ccRCC. However, ccRCC metastases unexpectedly have enhanced labeling of TCA cycle intermediates compared to primary ccRCCs, indicating a divergent metabolic program during ccRCC metastasis in patients. In mice, stimulating respiration in ccRCC cells is sufficient to promote metastatic colonization. Altogether, these findings indicate that metabolic properties evolve during human kidney cancer progression, and suggest that mitochondrial respiration may be limiting for ccRCC metastasis but not for ccRCC growth at the site of origin.

Pretest Video Education Versus Genetic Counseling for Patients With Prostate Cancer: ProGen, A Multisite Randomized Controlled Trial.

PURPOSE: Germline genetic testing (GT) is recommended for men with prostate cancer (PC), but testing through traditional models is limited. The ProGen study examined a novel model aimed at providing access to GT while promoting education and informed consent. METHODS: Men with potentially lethal PC (metastatic, localized with a Gleason score of ≥8, persistent prostate-specific antigen after local therapy), diagnosis age ≤55 years, previous malignancy, and family history suggestive of a pathogenic variant (PV) and/or at oncologist's discretion were randomly assigned 3:1 to video education (VE) or in-person genetic counseling (GC). Participants had 67 genes analyzed (Ambry), with results disclosed via telephone by a genetic counselor. Outcomes included GT consent, GT completion, PV prevalence, and survey measures of satisfaction, psychological impact, genetics knowledge, and family communication. Two-sided Fisher's exact tests were used for between-arm comparisons. RESULTS: Over a 2-year period, 662 participants at three sites were randomly assigned and pretest VE (n = 498) or GC (n = 164) was completed by 604 participants (VE, 93.1%; GC, 88.8%), of whom 596 participants (VE, 98.9%; GC, 97.9%) consented to GT and 591 participants completed GT (VE, 99.3%; GC, 98.6%). These differences were not statistically significant although subtle differences in satisfaction and psychological impact were. Notably, 84 PVs were identified in 78 participants (13.2%), with BRCA1/2 PV comprising 32% of participants with a positive result (BRCA2 n = 21, BRCA1 n = 4). CONCLUSION: Both VE and traditional GC yielded high GT uptake without significant differences in outcome measures of completion, GT uptake, genetics knowledge, and family communication. The increased demand for GT with limited genetics resources supports consideration of pretest VE for patients with PC.

The evolving paradigm of second-line hormonal therapy options for castration-resistant prostate cancer.

PURPOSE OF REVIEW: The review examines recent advances in second-line hormonal therapy for the treatment of castrate-resistant prostate cancer (CRPC). RECENT FINDINGS: Recent data highlight the continued importance of androgen signaling in CRPC. These findings have spurred the development of novel inhibitors of adrenal and intra-tumoral androgen synthesis and novel androgen signaling inhibitors with activity in CRPC. In the past year abiraterone acetate, a CYP17 (17α-hydroxylase/17, 20 lyase) inhibitor, received US FDA approval for use in the treatment of metastatic CRPC in patients previously treated with docetaxel. Additionally, the novel androgen signaling inhibitor MDV3100 has been reported to confer a survival advantage compared to placebo in the same patient population. Here we review the scientific rationale for targeting androgen signaling in CRPC and the recent pivotal trials that support the use of novel second-line hormonal therapies. Additionally, we summarize ongoing preclinical and clinical efforts to ascertain and overcome mechanisms of resistance. SUMMARY: Novel inhibitors of extra-gonadal androgen synthesis and androgen receptor function demonstrate the continued importance of androgen signaling in CRPC. These agents have improved clinical outcomes for patients with metastatic CRPC.

Phase II trial of RAD001 and bicalutamide for castration-resistant prostate cancer.

UNLABELLED: Study Type--Therapy (cohort) Level of Evidence 2a. What's known on the subject? and What does the study add? Despite expanding treatment options for castration-resistant prostate cancer (CRPC), therapies with long response duration remain intangible due to prostate cancer cells' natural ability to develop iterative resistance. Androgen receptor (AR) signaling has been shown to play a critical role in CRPC and its expression is regulated by the PI3K-Akt pathway. Thus inhibition of AR signalling and PI3K-Akt-mTOR (a downstream mediator of the PI3K-Akt pathway) pathway is a logical combination in CRPC and we report a phase II trial of RAD001 and bicalutamide. Our study is the first clinical trial report of an AR inhibitor of PI3K-Akt-mTOR. The AR pathway and the PI3K-Akt-mTOR pathway are two of the most relevant growth pathway for CRPC. Despite low efficacy results from our trial there will be significant interest in the field for these data (dose, schedule, response, toxicity, trial design) as newer generations of both AR inhibitors and PI3K-Akt-mTOR inhibitors are in development and likely will be tested in combination in CRPC. OBJECTIVES: • To determine best overall response and duration of response of RAD001, a selective inhibitor of mammalian target of rapamycin, in combination with bicalutamide in castration-resistant prostate cancer (CRPC). • To characterize the toxicity profile of RAD001 in combination with bicalutamide in patients with CRPC. PATIENTS AND METHODS: • A phase II study was conducted to explore the efficacy and tolerability of RAD001 (10 mg daily) in combination with bicalutamide (50 mg daily) in men with progressive CRPC. • The primary endpoint was a composite of prostate-specific antigen (PSA) level and measurable disease response by standard criteria. • This single-stage trial with a sample size of 38 eligible patients provided 90% power to differentiate a response rate of ≥ 40% from a response rate of ≤ 20%, as expected for bicalutamide alone (α= 0.10, power = 0.90). RESULTS: • In total, 36 men were enrolled, with a median (range) age of 68 (60-72) years and median (range) baseline PSA level of 22.2 (8.4-121.3) ng/mL, and 89% had metastatic disease. • There were 31 (86%) patients had previously used bicalutamide for a median duration of 7.4 months. • There were two patients with a confirmed PSA level decline ≥ 50%. • The median (interquartile range) time to progression was 8.7 (7.9-15.9) weeks. • The most common toxicity was grade 1/2 mucositis, which was observed in 20 (56%) patients. CONCLUSION: • The combination of RAD001 and bicalutamide in men with CRPC was well tolerated but had low activity and failed to achieve the primary endpoint of improved response compared to the results previously achieved for bicalutamide alone in this population.

Phase II Trial of Sipuleucel-T and Stereotactic Ablative Body Radiation for Patients with Metastatic Castrate-Resistant Prostate Cancer.

(1) We hypothesized that adding concurrent stereotactic ablative radiotherapy (SAbR) would increase the time to progression in patients with metastatic castrate-resistant prostate cancer (mCRPCA) treated with sipuleucel-T. (2) Patients with a history of prostate cancer (PC), radiographic evidence of metastatic disease, and rising prostate-specific antigen (PSA) > 0.2 ng/dL on castrate testosterone levels were enrolled in this single-arm phase II clinical trial and treated with sipuleucel-T and SAbR. The primary endpoint was time to progression (TTP). Cellular and humoral responses were measured using ELISpot and Luminex multiplex assays, respectively. (3) Twenty patients with mCRPC were enrolled and treated with SAbR to 1−3 sites. Treatment was well tolerated with 51, 8, and 4 treatment-related grade 1, 2, and 3 toxicities, respectively, and no grade 4 or 5 adverse events. At a median follow-up of 15.5 months, the median TTP was 11.2 weeks (95% CI; 6.8−14.0 weeks). Median OS was 76.8 weeks (95% CI; 41.6−130.8 weeks). This regimen induced both humoral and cellular immune responses. Baseline M-MDSC levels were elevated in mCRPC patients compared to healthy donors (p = 0.004) and a decline in M-MDSC was associated with biochemical response (p = 0.044). Responders had lower baseline uric acid levels (p = 0.05). No clear correlation with radiographic response was observed. (4) While the regimen was safe, the PC-antigen-specific immune response induced by SAbR did not yield a synergistic clinical benefit for patients treated with sipuleucel-T compared to the historically reported outcomes.

The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.