Disruption of the ZFP574-THAP12 complex suppresses B cell malignancies in mice.

Despite the availability of life-extending treatments for B cell leukemias and lymphomas, many of these cancers remain incurable. Thus, the development of new molecular targets and therapeutics is needed to expand treatment options. To identify new molecular targets, we used a forward genetic screen in mice to identify genes required for development or survival of lymphocytes. Here, we describe Zfp574, an essential gene encoding a zinc finger protein necessary for normal and malignant lymphocyte survival. We show that ZFP574 interacts with zinc finger protein THAP12 and promotes the G1-to-S-phase transition during cell cycle progression. Mutation of ZFP574 impairs nuclear localization of the ZFP574-THAP12 complex. ZFP574 or THAP12 deficiency results in cell cycle arrest and impaired lymphoproliferation. Germline mutation, acute gene deletion, or targeted degradation of ZFP574 suppressed Myc-driven B cell leukemia in mice, but normal B cells were largely spared, permitting long-term survival, whereas complete lethality was observed in control animals. Our findings support the identification of drugs targeting ZFP574-THAP12 as a unique strategy to treat B cell malignancies.

Viable mutations of mouse midnolin suppress B cell malignancies.

In a genetic screen, we identified two viable missense alleles of the essential gene Midnolin (Midn) that were associated with reductions in peripheral B cells. Causation was confirmed in mice with targeted deletion of four of six MIDN protein isoforms. MIDN was expressed predominantly in lymphocytes where it augmented proteasome activity. We showed that purified MIDN directly stimulated 26S proteasome activity in vitro in a manner dependent on the ubiquitin-like domain and a C-terminal region. MIDN-deficient B cells displayed aberrant activation of the IRE-1/XBP-1 pathway of the unfolded protein response. Partial or complete MIDN deficiency strongly suppressed Eµ-Myc-driven B cell leukemia and the antiapoptotic effects of Eµ-BCL2 on B cells in vivo and induced death of Sp2/0 hybridoma cells in vitro, but only partially impaired normal lymphocyte development. Thus, MIDN is required for proteasome activity in support of normal lymphopoiesis and is essential for malignant B cell proliferation over a broad range of differentiation states.

Prognosis of glioblastoma patients improves significantly over time interrogating historical controls.

BACKGROUND: Glioblastoma (GBM) is the most common devastating primary brain cancer in adults. In our clinical practice, median overall survival (mOS) of GBM patients seems increasing over time. METHODS: To address this observation, we have retrospectively analyzed the prognosis of 722 newly diagnosed GBM patients, aged below 70, in good clinical conditions (i.e. Karnofsky Performance Status -KPS- above 70%) and treated in our department according to the standard of care (SOC) between 2005 and 2018. Patients were divided into two groups according to the year of diagnosis (group 1: from 2005 to 2012; group 2: from 2013 to 2018). RESULTS: Characteristics of patients and tumors of both groups were very similar regarding confounding factors (age, KPS, MGMT promoter methylation status and treatments). Follow-up time was fixed at 24 months to ensure comparable survival times between both groups. Group 1 patients had a mOS of 19 months ([17.3-21.3]) while mOS of group 2 patients was not reached. The recent period of diagnosis was significantly associated with a longer mOS in univariate analysis (HR=0.64, 95% CI [0.51 - 0.81]), p < 0.001). Multivariate Cox analysis showed that the period of diagnosis remained significantly prognostic after adjustment on confounding factors (adjusted Hazard Ratio (aHR) 0.49, 95% CI [0.36-0.67], p < 0.001). CONCLUSION: This increase of mOS over time in newly diagnosed GBM patients could be explained by better management of potentially associated non-neurological diseases, optimization of validated SOC, better management of treatments side effects, supportive care and participation in clinical trials.

Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3KBTBD2.

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis.

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.

Essential requirement for IER3IP1 in B cell development.

In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for aberrant immune function, we identified animals with low percentages of B220+ cells in the peripheral blood. The causative mutation was in Ier3ip1, encoding immediate early response 3 interacting protein 1 (IER3IP1), an endoplasmic reticulum membrane protein mutated in an autosomal recessive neurodevelopmental disorder termed Microcephaly with simplified gyration, Epilepsy and permanent neonatal Diabetes Syndrome (MEDS) in humans. However, no immune function for IER3IP1 had previously been reported. The viable hypomorphic Ier3ip1 allele uncovered in this study, identical to a reported IER3IP1 variant in a MEDS patient, reveals an essential hematopoietic-intrinsic role for IER3IP1 in B cell development and function. We show that IER3IP1 forms a complex with the Golgi transmembrane protein 167A and limits activation of the unfolded protein response mediated by inositol-requiring enzyme-1α and X-box binding protein 1 in B cells. Our findings suggest that B cell deficiency may be a feature of MEDS.

OVOL2 sustains postnatal thymic epithelial cell identity.

Distinct pathways and molecules may support embryonic versus postnatal thymic epithelial cell (TEC) development and maintenance. Here, we identify a mechanism by which TEC numbers and function are maintained postnatally. A viable missense allele (C120Y) of Ovol2, expressed ubiquitously or specifically in TECs, results in lymphopenia, in which T cell development is compromised by loss of medullary TECs and dysfunction of cortical TECs. We show that the epithelial identity of TECs is aberrantly subverted towards a mesenchymal state in OVOL2-deficient mice. We demonstrate that OVOL2 inhibits the epigenetic regulatory BRAF-HDAC complex, specifically disrupting RCOR1-LSD1 interaction. This causes inhibition of LSD1-mediated H3K4me2 demethylation, resulting in chromatin accessibility and transcriptional activation of epithelial genes. Thus, OVOL2 controls the epigenetic landscape of TECs to enforce TEC identity. The identification of a non-redundant postnatal mechanism for TEC maintenance offers an entry point to understanding thymic involution, which normally begins in early adulthood.

The dual lipid desaturase/hydroxylase DEGS2 controls phytoceramide levels necessary to counter intestinal inflammation.

Intestinal immunity is dependent on barrier function to maintain quiescence. The mechanisms for the maintenance of this barrier are not fully understood. Delta 4-desaturase, sphingolipid 2 (DEGS2) is a lipid desaturase and hydroxylase that catalyzes the synthesis of ceramide and phytoceramide from dihydroceramide. Using a forward genetic approach, we found and validated a mutation in Degs2 as causative of increasing susceptibility to colitis and altering the phytoceramide balance in the colon. DEGS2 is expressed in the intestinal epithelium, and the colitis phenotype is dependent on the non-hematopoietic compartment of the mouse. In the absence of DEGS2, the colon lacks phytoceramides and accumulates large amounts of the precursor lipid dihydroceramide. In response to dextran sodium sulfate (DSS)-induced colitis, colonic epithelial cells in DEGS2-deficient mice had increased cell death and decreased proliferation compared to those in wild-type mice. These findings demonstrate that DEGS2 is needed to maintain epithelial integrity, protect against DSS-induced colitis and maintain lipid balance in vivo.

RNPS1 inhibits excessive tumor necrosis factor/tumor necrosis factor receptor signaling to support hematopoiesis in mice.

Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cell­intrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)­dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.

Optic Perineuritis Associated With Cryptococcal Meningitis Presenting With a "Hot Orbit" in a Patient With Chronic Lymphocytic Leukemia.

ABSTRACT: A 75-year-old man presented with 3 days of progressive left retro-orbital pain, eyelid swelling, tearing, and pain with extraocular movement. His medical history was significant for type II diabetes mellitus and chronic lymphocytic leukemia, stable on no therapy since diagnosis 8 years prior. The initial examination was significant for diffuse restriction of left ocular motility, marked lid edema, and mild dyschromatopsia. Computed tomography demonstrated asymmetric left periorbital soft tissue swelling and intraconal fat stranding with an irregular left optic nerve sheath complex and clear paranasal sinuses. He was hospitalized for orbital cellulitis and treated empirically with broad-spectrum intravenous antibiotics, but his visual acuity declined over the ensuing 2 days. Subsequent MRI demonstrated left-greater-than-right circumferential optic nerve sheath enhancement, and leptomeningeal enhancement. An orbital biopsy demonstrated monoclonal B-cell lymphocyte aggregation, whereas a lumbar puncture was positive for Cryptococcus antigen with subsequent demonstration of abundant Cryptococcus by Papanicolaou stain. The final diagnosis was optic perineuritis secondary to cryptococcal meningitis presenting with orbital inflammation. Although his clinical course was complicated by immune reconstitution inflammatory syndrome, symptoms and signs of optic neuropathy ultimately resolved after 1 month of intensive antifungal therapy.

Factors affecting overall survival in 51 adult Filipino patients undergoing stereotactic radiosurgery for spine metastases.

Objectives: Stereotactic radiosurgery (SRS) is part of the multimodality treatment for patients with cancer. The objective of this study is to determine factors which influence overall survival (OS) of Filipino patients who underwent SRS for metastatic tumors of the spine. Methods: This is a retrospective analysis of a cohort of Filipino patients treated with spine SRS for metastatic tumors in a single institution. Putative predictors were determined by the institution's spine SRS team and described in the cohort. A Cox proportional hazards regression model was utilized to construct a model based on the predictors determined by the institution's spine SRS team. Results: A total of 51 consecutive patients with 68 spine metastases were treated with SRS at our institution. The median OS was 13.1 months (95% CI of 7.1 to 19.1). On multivariate analysis, significant predictors that are associated with OS were visceral tumor origin (adjusted HR: 3.08, 95% CI of 1.24 to 7.64, p = 0.015) and cardiovascular disease (adjusted HR: 2.50, 95% CI of 1.04 to 5.94, p = 0.039) with dose and number of fractions as co-variates [Model Wald χ2 (5, N = 51) = 11.11 (p = 0.049)]. Conclusions: The presence of visceral tumor origins and cardiovascular disease are independent factors that are associated with lower overall survival in Filipino patients with spine metastasis treated with SRS.

Elevated levels of perfluoroalkyl substances in breast cancer patients within the Greater Manila Area.

Several studies have reported exposure of humans to various endocrine disrupting chemicals (EDCs) worldwide. However, there is a lack of data regarding EDC exposures in humans living in Southeast Asian countries, such as the Philippines. Hence, this study measured levels of 41 EDCs in women residing in the Greater Manila Area, home to the second largest city in Southeast Asia. Urine samples from women with versus without breast cancer were analyzed for 11 phthalate metabolites, 8 environmental phenols, and 10 bisphenols, while serum samples were analyzed for 12 perfluoroalkyl substances (PFAS). Out of the four groups of EDCs analyzed, PFAS were significantly associated with breast cancer (adjusted OR = 13.63, 95% CI: 3.24-94.88 p-trend = 0.001 for PFDoA; adjusted OR = 9.26, 95% CI 2.54-45.10, p-trend = 0.002 for PFDA; and adjusted OR = 2.66, 95% CI: 0.95-7.66, p-trend = 0.004 for PFHxA). Long-chain PFAS levels were positively correlated with age and were significantly higher in women from Region IV-A, a heavily industrialized region, than from the National Capital Region. Overall, this study showed baseline information regarding the level of EDCs in Filipinas, providing a glimpse of EDC exposure in women living in a megalopolis city in Southeast Asia.

Epstein-Barr virus-associated intracranial myopericytoma in a child: case report and review of literature.

BACKGROUND: Myopericytomas are benign soft tissue tumors which are rarely found as intracranial masses. METHODS: A review of SCOPUS and PubMed databases for case reports and case series was done for patients with intracranial myopericytomas. Data on demographics, clinical features, imaging, surgical management employed, and outcomes were collected. RESULTS: We found a total of 9 cases in the literature and we describe an additional case from our own experience. The mean age at presentation was 50.1 years (32-64 years), with a female predominance. Most tumors were in the posterior fossa and were multifocal in AIDS patients. The most common clinical manifestations were visual disturbances, headaches, and vomiting. An association with EBV was reported in two cases and was also found in our own case. Gross total excision and even subtotal excision were definitive therapies with no recurrences reported with a mean follow-up of 22.7 months. Our case is the first EBV-associated intracranial myopericytoma in a child. CONCLUSION: Intracranial myopericytomas are rare; some have an association with EBV and immunodeficient states. Surgery is the only necessary treatment and outcomes are generally favorable for these benign neoplasms.

Sulfatides are endogenous ligands for the TLR4-MD-2 complex.

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4-MD-2 activation by endogenous ligands. Sulfatides (3-O-sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4-MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4-MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4-MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4-MD-2 and activate or inhibit this complex.

Applicability of the Paris System for veterans: high rates of undiagnosed low-grade urothelial neoplasia.

BACKGROUND: The Paris System for Reporting Urinary Cytology (TPS) is a recently developed standardized terminology system. It is well-established that urine cytology has low sensitivity for detecting low-grade urothelial neoplasia (LGUN). Though the majority of tumors are low-grade, surveillance of these lesions is important to monitor for possible progression. Herein, we compared TPS to our veteran integrated system network (VISN) to assess its applicability. We also introduced semi-quantitative scoring to further evaluate cytomorphologic features of high-grade urothelial carcinoma (HGUC). MATERIALS AND METHODS: Voided and instrumented urine cytology specimens and concurrent biopsies were reviewed from Sept 2018 - Jan 2020. Cytologic diagnoses reported using the VISN institutional system were reevaluated by staff cytopathologists and categorized according to TPS. A semi-quantitative scoring system to evaluate cytomorphologic features was devised. RESULTS: Cytology and surgical specimens from 105 patients were reviewed. The VISN and TPS reporting systems were compared and showed similar sensitivities and specificities for the detection of HGUC. Rates of biopsy-proven LGUN were high for the negative for high-grade urothelial carcinoma category (NHGUC; 27/53, 50.9%) and atypical urothelial cells (AUC; 14/30; 46.7%) compared to suspicious/positive (0/22, 0%) categories. Major and minor criteria as outlined in TPS were evaluated semi-quantitatively. CONCLUSIONS: Urine cytology has limited sensitivity for LGUN regardless of the cytologic reporting system used. There was a high rate of LGUN following NHGUC/AUC diagnoses in the Veteran population. Coarse chromatin was determined to be the least sensitive criterion for the detection of high-grade lesions and irregular chromatin rim was most specific.

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization.

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.

Lumbar facet cyst tuberculosis: a rare cause of cauda equina compression. Illustrative case.

BACKGROUND: Spinal tuberculosis may present in atypical form to involve only the posterior spinal element with relative sparing of the anterior vertebral body and intervertebral disc. Recognition of this unusual pattern is important to avoid delay in diagnosis and treatment. OBSERVATIONS: The authors report a case of a 59-year-old woman with right-sided radiculopathy and motor weakness. Her lumbosacral magnetic resonance imaging showed a large heterogeneous cyst arising from the right L4-5 facet joint. Laminectomy with excision of the cyst was performed. During surgery, the cyst contained cheese-like material that, on histopathological examination, revealed focal aggregates of tuberculous granuloma. Postoperatively, the patient recovered remarkably with no interval development of instability or any deformity. LESSONS: Facet cyst tuberculosis is rare but should be considered in the differential diagnosis in patients coming from endemic regions. Laminectomy with excision of the cyst along with concurrent antitubercular chemotherapy is a safe and durable treatment option in this case.

Virus Bioresistor (VBR) for Detection of Bladder Cancer Marker DJ-1 in Urine at 10 pM in One Minute.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

A Pilot Cancer-Phenome Biobanking System in a Low-Resource Southeast Asian Setting: The Philippine General Hospital Biobank Experience.

Biobanking has become an indispensable tool for translational research and health innovations. While the field of biobanking has progressed and evolved globally, biobanking in developing Association of Southeast Asian Nations (ASEAN) countries such as the Philippines remains underrepresented because of several challenges often encountered in these low- and middle-income countries. Recently, the Philippine government has undertaken enormous efforts to advancing research and development in the country, and one of the current research pursuits is the establishment of biobanks, with the hope of attaining more discoveries and innovations in the future. Given that cancer remains a leading cause of death in the Philippines, the Philippine government supported the establishment of a cancer biobank at the Philippine General Hospital (PGH). In this study, we present a specific use case of biobanking activity at the PGH Biobank, to build a cohort of biospecimens from Filipino patients with breast, endometrial, and ovarian cancer. This initiative is part of a biomonitoring study (1) to assess environmental exposures and possible risk factors in the Philippine population and (2) to develop a system of culturing human cells from Filipino patients for subsequent in vitro studies. We discuss issues faced and the solutions developed during the implementation of the biobank. Strong research collaboration, a funding source, basic infrastructure, and appropriate technology helped initiate this pilot biobank in the Philippines. Overall, the experiences of establishing the PGH Biobank may help other institutions in low-resource countries to set up cancer biobanks.