The Hob proteins are novel and conserved lipid-binding proteins at ER-PM contact sites.

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

Ibrutinib-rituximab followed by R-HCVAD as frontline treatment for young patients (≤65 years) with mantle cell lymphoma (WINDOW-1): a single-arm, phase 2 trial.

BACKGROUND: Induction with ibrutinib and rituximab provides an opportunity to minimise chemotherapy exposure, because upfront use of these targeted therapies could result in remission without chemotherapy and allow for consolidation with only four cycles of chemotherapy instead of the conventional eight. We aimed to determine the activity and safety of ibrutinib-rituximab induction followed by shortened chemoimmunotherapy (four cycles) with rituximab plus hyper-fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (R-HCVAD) alternating with methotrexate-cytarabine in previously untreated patients with mantle cell lymphoma. METHODS: We did a single-centre, single-arm, phase 2 trial in previously untreated patients with mantle cell lymphoma. Eligible patients were aged 65 years or younger and had serum bilirubin of less than 1·5 mg/dL, creatinine clearance of 30 mL/min or more, Eastern Cooperative Oncology Group performance status of 2 or less, and cardiac ejection fraction 50% or more by echocardiogram. Patients received 12 cycles of ibrutinib-rituximab induction (part A; oral ibrutinib 560 mg daily and intravenous rituximab 375 mg/m2 weekly for the first 4 weeks and then on day 1 of cycles 3-12). As soon as patients had a complete response, four cycles of R-HCVAD alternating with methotrexate-cytarabine (part B) were administered. If they did not have a complete response or had a partial response, patients received two cycles of R-HCVAD alternating with methotrexate-cytarabine followed by reassessment, up to a total of eight cycles. Patients were taken off study if they had stable disease or progression during R-HCVAD. The primary outcome was the overall response rate after part A. The analyses were conducted on an intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT02427620. FINDINGS: 131 patients were enrolled between June 12, 2015, and Dec 6, 2018. The median age was 56 years (IQR 49-60). 58 (50%) of 117 patients had high Ki-67 (≥30%). 129 (98%, 95% CI 95-100) of 131 patients had an overall response in part A. The most common grade 3-4 adverse events were lymphocytopenia (19 [14%] of 131), skin rash (16 [12%]), thrombocytopenia (12 [9%]), infections (11 [8%]), and fatigue (ten [8%]) in part A and lymphocytopenia (96 [73%]), leukocytopenia (42 [32%]), thrombocytopenia (40 [30%]), and neutropenia (26 [20%]) in part B. There was one on-study death, which was not deemed to be treatment-related. INTERPRETATION: Induction with ibrutinib-rituximab in the frontline treatment of young patients with mantle cell lymphoma is active and safe. This approach allowed minimisation of the number of chemotherapy cycles, thereby reducing the adverse events associated with chemotherapy. Newer trials bringing the next-generation Bruton's tyrosine kinase inhibitors into the frontline setting might obviate the need for chemotherapy altogether in patients with mantle cell lymphoma. FUNDING: Pharmacyclics, Janssen.

Clinical outcomes and impact of therapeutic intervention in patients with acute myeloid leukemia who experience measurable residual disease (MRD) recurrence following MRD-negative remission.

Recurrence of MRD in AML is associated with imminent relapse unless intervened upon. Change in chemotherapy regimen and/or immediate transplant improve outcomes.

Pre-transplantation minimal residual disease with cytogenetic and molecular diagnostic features improves risk stratification in acute myeloid leukemia.

Our aim was to improve outcome prediction after allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia by combining cytogenetic and molecular data at diagnosis with minimal residual disease assessment by multicolor flow-cytometry at transplantation. Patients with acute myeloid leukemia in first complete remission in whom minimal residual disease was assessed at transplantation were included and categorized according to the European LeukemiaNet classification. The primary outcome was 1-year relapse incidence after transplantation. Of 152 patients eligible, 48 had minimal residual disease at the time of their transplant. Minimal residual disease-positive patients were older, required more therapy to achieve first remission, were more likely to have incomplete recovery of blood counts and had more adverse risk features by cytogenetics. Relapse incidence at 1 year was higher in patients with minimal residual disease (32.6% versus 14.4%, P=0.002). Leukemia-free survival (43.6% versus 64%, P=0.007) and overall survival (48.8% versus 66.9%, P=0.008) rates were also inferior in patients with minimal residual disease. In multivariable analysis, minimal residual disease status at transplantation independently predicted 1-year relapse incidence, identifying a subgroup of intermediate-risk patients, according to the European LeukemiaNet classification, with a particularly poor outcome. Assessment of minimal residual disease at transplantation in combination with cytogenetic and molecular findings provides powerful independent prognostic information in acute myeloid leukemia, lending support to the incorporation of minimal residual disease detection to refine risk stratification and develop a more individualized approach during hematopoietic stem cell transplantation.

Molecular and Genetic Analyses of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention.

BACKGROUND: Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases, including cerebral microbleeds, porencephaly, and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown, and there is a lack of effective treatment. METHODS AND RESULTS: Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small-vessel disease, recurrent hemorrhagic strokes, and age-related macroangiopathy. We showed that allelic heterogeneity, genetic context, and environmental factors such as intense exercise or anticoagulant medication modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes was a key triggering factor of ICH. Finally, we showed that treatment of mutant mice with a US Food and Drug Administration-approved chemical chaperone resulted in a decreased collagen intracellular accumulation and a significant reduction in ICH severity. CONCLUSIONS: Our data are the first to show therapeutic prevention in vivo of ICH resulting from Col4a1 mutation and imply that a mechanism-based therapy promoting protein folding might also prevent ICH in patients with COL4A1 and COL4A2 mutations.

COL4A2 mutations impair COL4A1 and COL4A2 secretion and cause hemorrhagic stroke.

Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

Direct-to-Biology Accelerates PROTAC Synthesis and the Evaluation of Linker Effects on Permeability and Degradation.

A platform to accelerate optimization of proteolysis targeting chimeras (PROTACs) has been developed using a direct-to-biology (D2B) approach with a focus on linker effects. A large number of linker analogs-with varying length, polarity, and rigidity-were rapidly prepared and characterized in four cell-based assays by streamlining time-consuming steps in synthesis and purification. The expansive dataset informs on linker structure-activity relationships (SAR) for in-cell E3 ligase target engagement, degradation, permeability, and cell toxicity. Unexpected aspects of linker SAR was discovered, consistent with literature reports on "linkerology", and the method dramatically speeds up empirical optimization. Physicochemical property trends emerged, and the platform has the potential to rapidly expand training sets for more complex prediction models. In-depth validation studies were carried out and confirm the D2B platform is a valuable tool to accelerate PROTAC design-make-test cycles.

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling.

Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface, where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway that is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer's disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans.

ESCRT-III and ER-PM contacts maintain lipid homeostasis.

Eukaryotic cells are compartmentalized into organelles by intracellular membranes. While the organelles are distinct, many of them make intimate contact with one another. These contacts were first observed in the 1950s, but only recently have the functions of these contact sites begun to be understood. In yeast, the endoplasmic reticulum (ER) makes extensive intermembrane contacts with the plasma membrane (PM), covering ∼40% of the PM. Many functions of ER-PM contacts have been proposed, including nonvesicular lipid trafficking, ion transfer, and as signaling hubs. Surprisingly, cells that lack ER-PM contacts grow well, indicating that alternative pathways may be compensating for the loss of ER-PM contact. To better understand the function of ER-PM contact sites we used saturating transposon mutagenesis to identify synthetic lethal mutants in a yeast strain lacking ER-PM contact sites. The strongest hits were components of the ESCRT complexes. The synthetic lethal mutants have low levels of some lipid species but accumulate free fatty acids and lipid droplets. We found that only ESCRT-III components are synthetic lethal, indicating that Vps4 and other ESCRT complexes do not function in this pathway. These data suggest that ESCRT-III proteins and ER-PM contact sites act in independent pathways to maintain lipid homeostasis.

Serial minimal residual disease (MRD) monitoring during first-line FCR treatment for CLL may direct individualized therapeutic strategies.

Achieving undetectable MRD (U-MRD) status after chemoimmunotherapy predicts longer progression-free and overall survival. The predictive factors and timing of relapse in patients with U-MRD and value of interim MRD analysis are ill-defined. This was a prospective study of 289 patients with CLL treated first-line with FCR. MRD analysis was performed after course 3 (C3) and at end of therapy (EOT) in bone marrow using 4-color flow cytometry (sensitivity 10-4). Eighteen percent of patients had U-MRD after C3 and 48% at EOT. U-MRD status at EOT was associated with longer PFS (median NR vs 38 mo, p < 0.001). MRD level (≤1% vs >1%) after C3 predicted greater likelihood of U-MRD status at EOT (64% vs 9%, p < 0.001). PFS was significantly longer for patients with MRD ≤1% vs >1% after C3 (median 73 mo vs 41 mo, p < 0.001), but similar for <0.01% vs 0.01-1%. Interim MRD status may therefore be used for risk stratification and to individualize therapy. Eighty-five patients with U-MRD status at EOT had yearly blood MRD monitoring; MRD re-emerged in 38/85, a median of 48 mo after EOT and preceded clinical progression by a median of 24 months, which may allow development of early intervention strategies.

ESCRTs function directly on the lysosome membrane to downregulate ubiquitinated lysosomal membrane proteins.

The lysosome plays an important role in maintaining cellular nutrient homeostasis. Regulation of nutrient storage can occur by the ubiquitination of certain transporters that are then sorted into the lysosome lumen for degradation. To better understand the underlying mechanism of this process, we performed genetic screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, Rapamycin-Induced Degradation (RapiDeg), to test the sorting defects caused by these mutants. These tests revealed that ubiquitinated vacuole membrane proteins recruit ESCRTs to the vacuole surface, where they mediate cargo sorting and direct cargo delivery into the vacuole lumen. Our findings demonstrate that the ESCRTs can function at both the late endosome and the vacuole membrane to mediate cargo sorting and intra-luminal vesicle formation.