PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization.

The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P(2)) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels to quantitatively explore PtdIns(4,5)P(2) dynamics during endocytosis in yeast. Our results indicate that PtdIns(4,5)P(2) levels increase and decline in conjunction with coat and actin assembly and disassembly, respectively. Live-cell image analysis of endocytic protein dynamics in an sjl1Delta sjl2Delta mutant, which has elevated PtdIns(4,5)P(2) levels, revealed that the endocytic machinery is still able to assemble and disassemble dynamically, albeit nonproductively. The defects in the dynamic behavior of the various endocytic proteins in this double mutant suggest that PtdIns(4,5)P(2) turnover is required for multiple stages during endocytic vesicle formation. Furthermore, our results indicate that PtdIns(4,5)P(2) turnover may act in coordination with the Ark1/Prk1 protein kinases in stimulating disassembly of the endocytic machinery.

Atezolizumab plus Bevacizumab Versus Sunitinib for Patients with Untreated Metastatic Renal Cell Carcinoma and Sarcomatoid Features: A Prespecified Subgroup Analysis of the IMmotion151 Clinical Trial.

Patients with metastatic renal cell carcinoma with sarcomatoid features (sRCC) have a poor prognosis and have shown limited responsiveness to inhibition of the VEGF pathway. We conducted a prespecified analysis of the randomised, phase 3 IMmotion151 trial in previously untreated patients with advanced or metastatic RCC to assess the effectiveness of atezolizumab + bevacizumab versus sunitinib in a subgroup of patients with sarcomatoid features. Patients whose tumour had any component of sarcomatoid features were included and received atezolizumab + bevacizumab (n = 68) or sunitinib (n = 74). Baseline characteristics were similar between the groups. Median progression-free survival was significantly longer in the group receiving atezolizumab + bevacizumab overall (8.3 vs 5.3 mo; hazard ratio [HR] 0.52 95% confidence interval [CI] 0.34-0.79) and in the subset of patients with PD-L1-positive tumours (8.6 vs 5.6 mo; HR 0.45, 95% CI 0.26-0.77). More patients receiving atezolizumab + bevacizumab achieved an objective response (49% vs 14%), including complete responses (10% vs 3%), and reported greater symptom improvements versus sunitinib. Safety was consistent with the known profiles of each drug and with that reported in the overall safety-evaluable population of IMmotion151. This analysis supports enhanced activity of atezolizumab + bevacizumab in patients with sRCC. PATIENT SUMMARY: In this report, we looked at patients with a specific type of kidney cancer (tumours with sarcomatoid features) that has been hard to treat. A treatment with two drugs (atezolizumab and bevacizumab) appeared to help patients live longer without the disease getting worse than another drug (sunitinib) that is often used. Patients who took the two drugs also said they were better able to carry out their everyday activities than patients who took sunitinib. The combination of these two drugs may work better in patients with this type of advanced kidney cancer.

Safety and Clinical Activity of Atezolizumab in Patients with Metastatic Castration-Resistant Prostate Cancer: A Phase I Study.

PURPOSE: Atezolizumab [anti-programmed death-ligand 1 (anti-PD-L1)] is well tolerated and efficacious in multiple cancers, but has not been previously evaluated in metastatic castration-resistant prostate cancer (mCRPC). This study examined the safety, efficacy, and biomarkers of atezolizumab monotherapy for mCRPC. PATIENTS AND METHODS: This phase Ia, open-label, dose-escalation and dose-expansion study (PCD4989g) enrolled patients with mCRPC who had progressed on sipuleucel-T or enzalutamide. Atezolizumab was given intravenously every 3 weeks until confirmed disease progression or loss of clinical benefit. Prespecified endpoints included safety, efficacy, biomarker analyses, and radiographic assessments. RESULTS: All 35 evaluable patients [median age, 68 years (range, 45-83 years)] received atezolizumab after ≥1 prior line of therapy; 62.9% of patients had received ≥3 prior lines. Treatment-related adverse events occurred in 21 patients (60.0%), with no deaths. One patient had a confirmed partial response (PR) per RECIST 1.1, and 1 patient had a PR per immune-related response criteria. The confirmed 50% PSA response rate was 8.6% (3 patients). Median overall survival (OS) was 14.7 months [95% confidence interval (CI): 5.9-not evaluable], with a 1-year OS rate of 52.3% (95% CI: 34-70); 2-year OS was 35.9% (95% CI: 13-59). Median follow-up was 13.0 months (range, 1.2-28.1 months). Biomarker analyses showed that atezolizumab activated immune responses; however, a composite biomarker failed to reveal consistent correlations with efficacy. CONCLUSIONS: Atezolizumab was generally well tolerated in patients with mCRPC, with a safety profile consistent with other tumor types. In heavily pretreated patients, atezolizumab monotherapy demonstrated evidence of disease control; however, its limited efficacy suggests a combination approach may be needed.

Real-world practice patterns in veterans with metastatic castration-resistant prostate cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is incurable, with most patients surviving less than 3 years. However, many treatments that extend survival have been approved in the past decade. OBJECTIVE: To describe the patient demographics, disease characteristics, treatment patterns, and outcomes in a cohort of Veterans diagnosed with mCRPC in the Veterans Health Administration. DESIGN: We identified 3,637 Veterans diagnosed with prostate cancer between January 2006 and August 2015 with evidence of mCRPC through December 2016. We described the most commonly used systemic mCRPC treatments according to mCRPC diagnosis era: Epoch 1 (2006-2010) or Epoch 2 (2011-2016). Patient demographics, disease characteristics, and treatment patterns were examined using descriptive statistics. An unadjusted Kaplan-Meier method was used to estimate the median time to biochemical progression and overall survival (OS) with 95% confidence intervals. RESULTS: The median age at initial prostate cancer diagnosis was 68 years. Approximately 67% of patients were non-Hispanic white, 29% were black, and 4% were other/unknown. A high-risk Gleason score (8-10) was reported in 748 (67%) of patients in Epoch 1 and 1578 (63%) of patients in Epoch 2, and the median prostate-specific antigen level at initial prostate cancer diagnosis was higher in Epoch 1 patients than in Epoch 2 patients (68 vs. 35 ng/ml). Following mCRPC diagnosis, the most common first-line therapies in Epoch 1 patients were docetaxel (83%) and abiraterone (9%), whereas Epoch 2 patients mainly received abiraterone (47%), docetaxel (36%), and enzalutamide (15%). In Epoch 1 and Epoch 2 patients, the median time to biochemical progression (unadjusted) was 9 and 13 months, respectively, and the median OS (unadjusted) was 15 and 23 months, respectively. CONCLUSIONS: The introduction of new therapies has resulted in increased use of the noncytotoxic agents abiraterone and enzalutamide as first-line treatment in lieu of docetaxel. Our results suggest that more recently diagnosed patients (Epoch 2) have a delayed time to biochemical progression and longer OS (unadjusted) compared with patients diagnosed earlier (Epoch 1).

Molecular Subsets in Renal Cancer Determine Outcome to Checkpoint and Angiogenesis Blockade.

Integrated multi-omics evaluation of 823 tumors from advanced renal cell carcinoma (RCC) patients identifies molecular subsets associated with differential clinical outcomes to angiogenesis blockade alone or with a checkpoint inhibitor. Unsupervised transcriptomic analysis reveals seven molecular subsets with distinct angiogenesis, immune, cell-cycle, metabolism, and stromal programs. While sunitinib and atezolizumab + bevacizumab are effective in subsets with high angiogenesis, atezolizumab + bevacizumab improves clinical benefit in tumors with high T-effector and/or cell-cycle transcription. Somatic mutations in PBRM1 and KDM5C associate with high angiogenesis and AMPK/fatty acid oxidation gene expression, while CDKN2A/B and TP53 alterations associate with increased cell-cycle and anabolic metabolism. Sarcomatoid tumors exhibit lower prevalence of PBRM1 mutations and angiogenesis markers, frequent CDKN2A/B alterations, and increased PD-L1 expression. These findings can be applied to molecularly stratify patients, explain improved outcomes of sarcomatoid tumors to checkpoint blockade versus antiangiogenics alone, and develop personalized therapies in RCC and other indications.

Patient-Reported Outcomes from the Phase III Randomized IMmotion151 Trial: Atezolizumab + Bevacizumab versus Sunitinib in Treatment-Naïve Metastatic Renal Cell Carcinoma.

PURPOSE: Patient-reported outcomes (PRO) were evaluated in the phase III IMmotion151 trial (NCT02420821) to inform overall treatment/disease burden of atezolizumab plus bevacizumab versus sunitinib in patients with previously untreated metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Patients were randomized 1:1 to receive atezolizumab 1,200 mg intravenous (i.v.) infusions every 3 weeks (q3w) plus bevacizumab 15 mg/kg i.v. q3w or sunitinib 50 mg per day orally 4 weeks on/2 weeks off. Patients completed the MD Anderson Symptom Inventory (MDASI), National Comprehensive Cancer Network Functional Assessment of Cancer Therapy-Kidney Symptom Index (FKSI-19), and Brief Fatigue Inventory (BFI) at baseline, q3w during treatment, at end of treatment, and during survival follow-up. Longitudinal and time to deterioration (TTD) analyses for core and RCC symptoms and their interference with daily life, treatment side-effect bother, and health-related quality of life (HRQOL) were evaluated. RESULTS: The intent-to-treat population included 454 and 461 patients in the atezolizumab plus bevacizumab and sunitinib arms, respectively. Completion rates for each instrument were 83% to 86% at baseline and ≥ 70% through week 54. Milder symptoms, less symptom interference and treatment side-effect bother, and better HRQOL at most visits were reported with atezolizumab plus bevacizumab versus sunitinib. The TTD HR (95% CI) favored atezolizumab plus bevacizumab for core (HR, 0.50; 0.40-0.62) and RCC symptoms (HR, 0.45; 0.37-0.55), symptom interference (HR, 0.56; 0.46-0.68), and HRQOL (HR, 0.68; 0.58-0.81). CONCLUSIONS: PROs in IMmotion151 suggest lower overall treatment burden with atezolizumab plus bevacizumab compared with sunitinib in patients with treatment-naïve mRCC and provide further evidence for clinical benefit of this regimen.

Analysis of yeast endocytic site formation and maturation through a regulatory transition point.

The earliest stages of endocytic site formation and the regulation of endocytic site maturation are not well understood. Here we analyzed the order in which the earliest proteins are detectable at endocytic sites in budding yeast and found that an uncharacterized protein, Pal1p/Ydr348cp, is also present at the initial stages of endocytosis. Because Ede1p (homologue of Eps15) and clathrin are the early-arriving proteins most important for cargo uptake, their roles during the early stages of endocytosis were examined more comprehensively. Ede1p is necessary for efficient recruitment of most early-arriving proteins, but not for the recruitment of the adaptor protein Yap1802p, to endocytic sites. The early-arriving proteins, as well as the later-arriving proteins Sla2p and Ent1/2p (homologues of Hip1R and epsins), were found to have longer lifetimes in CLC1-knockout yeast, which indicates that clathrin light chain facilitates the transition from the intermediate to late coat stages. Cargo also arrives during the early stages of endocytosis, and therefore its effect on endocytic machinery dynamics was investigated. Our results are consistent with a role for cargo in regulating the transition of endocytic sites from the early stages of formation to the late stages during which vesicle formation occurs.

Modeling vesicle traffic reveals unexpected consequences for Cdc42p-mediated polarity establishment.

BACKGROUND: Polarization in yeast has been proposed to involve a positive feedback loop whereby the polarity regulator Cdc42p orients actin cables, which deliver vesicles carrying Cdc42p to the polarization site. Previous mathematical models treating Cdc42p traffic as a membrane-free flux suggested that directed traffic would polarize Cdc42p, but it remained unclear whether Cdc42p would become polarized without the membrane-free simplifying assumption. RESULTS: We present mathematical models that explicitly consider stochastic vesicle traffic via exocytosis and endocytosis, providing several new insights. Our findings suggest that endocytic cargo influences the timing of vesicle internalization in yeast. Moreover, our models provide quantitative support for the view that integral membrane cargo proteins would become polarized by directed vesicle traffic given the experimentally determined rates of vesicle traffic and diffusion. However, such traffic cannot effectively polarize the more rapidly diffusing Cdc42p in the model without making additional assumptions that seem implausible and lack experimental support. CONCLUSIONS: Our findings suggest that actin-directed vesicle traffic would perturb, rather than reinforce, polarization in yeast.

A yeast killer toxin screen provides insights into a/b toxin entry, trafficking, and killing mechanisms.

Like Ricin, Shiga, and Cholera toxins, yeast K28 is an A/B toxin that depends on endocytosis and retrograde trafficking for toxicity. Knowledge of the specific proteins, lipids, and mechanisms required for trafficking and killing by these toxins remains incomplete. Since K28 is a model for clinically relevant toxins, we screened over 5000 yeast mutants, identifying 365 that affect K28 sensitivity. Hypersensitive mutants revealed cytoprotective pathways, including stress-activated signaling and protein degradation. Resistant mutants clustered to endocytic, lipid organization, and cell wall biogenesis pathways. Furthermore, GPI anchors and transcriptional regulation are important for K28-cell binding. Strikingly, the AP2 complex, which in metazoans links endocytic cargo to the clathrin coat, but had no assigned function in yeast, was critical for K28 toxicity. Yeast AP2 localizes to endocytic sites and has a cargo-specific function in K28 uptake. This comprehensive genetic analysis identified conserved processes important for A/B toxin trafficking and killing.

Role of Ca2+ in responses of airway epithelia to Pseudomonas aeruginosa, flagellin, ATP, and thapsigargin.

Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration ([Ca](i)) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-kappaB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca(2+) pump, releases endoplasmic reticulum Ca(2+), and triggers Ca(2+) entry through plasma membrane channels) both increased [Ca](i) but hardly stimulated NF-kappaB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-kappaB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca](i)) or Ca(2+)-free solution reduced increases in [Ca](i) due to ATP or thapsigargin and also reduced NF-kappaB activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)beta/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-kappaB-binding site was critical for activation by all agonists and for activation by [Ca](i). Thus increased [Ca](i) was not required for P. aeruginosa- or flagellin-activated NF-kappaB and IL-8 expression and secretion, and increased [Ca](i) was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca](i) synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca](i) reduced these responses. Thus [Ca](i) plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 microM and [flagellin] >10(-8) g/ml and during steady increases rather than oscillations in [Ca](i).