Citations

Abstract Abstract 576 Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character. We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures: No relevant conflicts of interest to declare.

Abstract Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.

Abstract The diagnostic utility of somatic mutations in the context of cytopenias is unclear: clonal hematopoiesis can be found in healthy individuals, patients with aplastic anemia (AA), clonal cytopenia of undetermined significance (CCUS) and myelodysplastic syndrome (MDS). We examined a cohort of 207 well‐characterized cytopenic patients with a 640‐gene next generation sequencing (NGS) panel and compared its diagnostic utility with a “virtual” 41 gene panel. The TET2 , SF3B1 , ASXL1 , and TP53 were the most commonly mutated genes (frequency > 10%). Mutations in the 640‐gene panel show high sensitivity (98.3%) but low specificity (47.6%) for diagnosis of MDS. Notably, mutations of splicing factors and genes in the RAS pathway are relatively specific to MDS. Furthermore, high variant allele frequency (VAF) predicts MDS: when the VAF is set at 20%, the positive predictive value (PPV) for MDS is 95.9%, with a specificity of 95.3%. The presence of two or more somatic mutations with ≥10% VAF showed a PPV of 95.2%. While the “virtual” 41‐gene panel showed a mild decrease in sensitivity (95.7% vs 98.3%), 100% specificity was observed when either VAF was set at ≥20% (100% vs 95.3%), or two or more somatic mutations had VAFs ≥ 10%. Our study shows targeted gene panel sequencing improves the diagnostic approach and accuracy for unexplained cytopenia, with its high sensitivity and high PPV for MDS when applying VAF cutoffs. Furthermore, a 41‐gene panel was shown to have at least comparable performance characteristics to the large 640‐gene panel.

PURPOSEDrug development is becoming increasingly expensive and time consuming. Drug repurposing is one potential solution to accelerate drug discovery. However, limited research exists on the use of electronic health record (EHR) data for drug repurposing, and most published studies have been conducted in a hypothesis-driven manner that requires a predefined hypothesis about drugs and new indications. Whether EHRs can be used to detect drug repurposing signals is not clear. We want to demonstrate the feasibility of mining large, longitudinal EHRs for drug repurposing by detecting candidate noncancer drugs that can potentially be used for the treatment of cancer.PATIENTS AND METHODSBy linking cancer registry data to EHRs, we identified 43,310 patients with cancer treated at Vanderbilt University Medical Center (VUMC) and 98,366 treated at the Mayo Clinic. We assessed the effect of 146 noncancer drugs on cancer survival using VUMC EHR data and sought to replicate significant associations (false discovery rate < .1) using the identical approach with Mayo Clinic EHR data. To evaluate replicated signals further, we reviewed the biomedical literature and clinical trials on cancers for corroborating evidence.RESULTSWe identified 22 drugs from six drug classes (statins, proton pump inhibitors, angiotensin-converting enzyme inhibitors, β-blockers, nonsteroidal anti-inflammatory drugs, and α-1 blockers) associated with improved overall cancer survival (false discovery rate < .1) from VUMC; nine of the 22 drug associations were replicated at the Mayo Clinic. Literature and cancer clinical trial evaluations also showed very strong evidence to support the repurposing signals from EHRs.CONCLUSIONMining of EHRs for drug exposure–mediated survival signals is feasible and identifies potential candidates for antineoplastic repurposing. This study sets up a new model of mining EHRs for drug repurposing signals.

Reactive airway diseases are significant sources of pulmonary morbidity in neonatal and pediatric patients. Supplemental oxygen exposure in premature infants contributes to airway diseases such as asthma and promotes development of airway remodeling, characterized by increased airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition. Decreased plasma membrane caveolin-1 (CAV1) expression has been implicated in airway disease and may contribute to airway remodeling and hyperreactivity. Here, we investigated the impact of clinically relevant moderate hyperoxia (50% O2) on airway remodeling and caveolar protein expression in a neonatal mouse model. Within 12 h of birth, litters of B6129SF2J mice were randomized to room air (RA) or 50% hyperoxia exposure for 7 days with or without caveolin-1 scaffolding domain peptide (CSD; caveolin-1 mimic; 10 µl, 0.25 mM daily via intraperitoneal injection) followed by 14 days of recovery in normoxia. Moderate hyperoxia significantly increased airway reactivity and decreased pulmonary compliance at 3 wk. Histologic assessment demonstrated airway wall thickening and increased ASM mass following hyperoxia. RNA from isolated ASM demonstrated significant decreases in CAV1 and cavin-1 in hyperoxia-exposed animals while cavin-3 was increased. Supplementation with intraperitoneal CSD mitigated both the physiologic and histologic changes observed with hyperoxia. Overall, these data show that moderate hyperoxia is detrimental to developing airway and may predispose to airway reactivity and remodeling. Loss of CAV1 is one mechanism through which hyperoxia produces these deleterious effects. Supplementation of CAV1 using CSD or similar analogs may represent a new therapeutic avenue for blunting hyperoxia-induced pulmonary damage in neonates.

The prevalence of Lynch syndrome (LS) varies significantly in different populations, suggesting that ethnic features might play an important role. We enrolled 3330 consecutive Chinese patients who had surgical resection for newly diagnosed colorectal cancer. Universal screening for LS was implemented, including immunohistochemistry for mismatch repair (MMR) proteins, BRAFV600E mutation test and germline sequencing. Among the 3250 eligible patients, MMR protein deficiency (dMMR) was detected in 330 (10.2%) patients. Ninety‐three patients (2.9%) were diagnosed with LS. Nine (9.7%) patients with LS fulfilled Amsterdam criteria II and 76 (81.7%) met the revised Bethesda guidelines. Only 15 (9.7%) patients with absence of MLH1 on IHC had BRAFV600E mutation. One third (33/99) of the MMR gene mutations have not been reported previously. The age of onset indicates risk of LS in patients with dMMR tumors. For patients older than 65 years, only 2 patients (5.7%) fulfilling revised Bethesda guidelines were diagnosed with LS. Selective sequencing of all cases with dMMR diagnosed at or below age 65 years and only of those dMMR cases older than 65 years who fulfill revised Bethesda guidelines results in 8.2% fewer cases requiring germline testing without missing any LS diagnoses. While the prevalence of LS in Chinese patients is similar to that of Western populations, the spectrum of constitutional mutations and frequency of BRAFV600E mutation is different. Patients older than 65 years who do not meet the revised Bethesda guidelines have a low risk of LS, suggesting germline sequencing might not be necessary in this population.

ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.

Introduction: In 2015, the US Alzheimer’s Disease Centers (ADC) implemented Version 3 of the Uniform Data Set (UDS). This paper describes the history of Version 3 development and the UDS data that are freely available to researchers. Methods: UDS Version 3 was developed after years of coordination between the National Institute on Aging-appointed Clinical Task Force (CTF), clinicians from ∼30 ADCs, and the National Alzheimer’s Coordinating Center (NACC). The CTF recognized the need for updates to align with the state of the science in dementia research, while being flexible to the diverse needs and diseases studied at the ADCs. Version 3 also developed a nonproprietary neuropsychological battery. Results: This paper focuses on the substantial Version 3 changes to the UDS forms related to clinical diagnosis and characterization of clinical symptoms to match updated consensus-based diagnostic criteria. Between March 2015 and March 2018, 4820 participants were enrolled using UDS Version 3. Longitudinal data were available for 25,337 of the 37,568 total participants using all UDS versions. Discussion: The results from utilization of the UDS highlight the possibility for numerous research institutions to successfully collaborate, produce, and use standardized data collection instruments for over a decade.

ABSTRACT Recent studies have demonstrated an effect of neurotrophins, particularly brain‐derived neurotrophic factor (BDNF), on airway contractility [ via increased airway smooth muscle (ASM) intracellular calcium [Ca 2+ ] i ] and remodeling (ASM proliferation and extracellular matrix formation) in the context of airway disease. In the present study, we examined the role of BDNF in allergen‐induced airway inflammation using 2 transgenic models: 1 ) tropomyosin‐related kinase B (TrkB) conditional knockin (TrkBKI) mice allowing for inducible, reversible disruption of BDNF receptor kinase activity by administration of 1NMPP1, a PP1 derivative, and 2) smooth muscle‐specific BDNF knockout (BDNF fl/fl /SMMHCll Cre/0 ) mice. Adult mice were intranasally challenged with PBS or mixed allergen ( Altemaria alternata, Aspergillus fumigatus , house dust mite, and ovalbumin) for 4 wk. Our data show that administration of 1NMPP1 in TrkBKI mice during the 4‐wk allergen challenge blunted airway hyper‐responsiveness (AHR) and reduced fibronectin mRNA expression in ASM layers but did not reduce inflammation per se . Smooth muscle‐specific deletion of BDNF reduced AHR and blunted airway fibrosis but did not significantly alter airway inflammation. Together, our novel data indicate that TrkB signaling is a key modulator of AHR and that smooth muscle‐derived BDNF mediates these effects during allergic airway inflammation.—Britt, R. D., Jr., Thompson, M. A., Wicher, S. A., Manlove, L. J., Roesler, A., Fang, Y.‐H., Roos, C., Smith, L., Miller, J. D., Pabelick, C. M., Prakash, Y. S. Smooth muscle brain‐derived neurotrophic factor contributes to airway hyperreactivity in a mouse model of allergic asthma. FASEB J. 33, 3024–3034 (2019). www.fasebj.org

TPS3619 Background: Cancer stem cells are considered to be fundamentally important for resistance to therapy, recurrence and metastasis. Napabucasin is a first-in-class cancer stemness inhibitor in development identified by its ability to inhibit STAT3-driven gene transcription and spherogenesis of cancer stem cells (Li et al, PNAS 112(6):1839, 2015). Preclinically, napabucasin sensitizes cancer cells to chemotherapeutics, including 5-FU and irinotecan. Encouraging anticancer activity in advanced CRC was observed in a phase Ib/II (Bendell et al, GI ASCO 2017) study of 63 pts with disease control rate (DCR) of 93% (28/30) and overall response rate (ORR) of 33% (10/30) in FOLFIRI-naïve pts who have had an on-study RECIST evaluation. On the basis of these data, a phase III trial is being conducted in North America, Europe, Australia, and Asia. Methods: This study (ClinicalTrials.gov NCT02753127) will assess the efficacy of napabucasin+FOLFIRI vs FOLFIRI in pts with mCRC (n = 1250). Addition of bevacizumab (bev) is permissible per investigator choice. Pts must have failed 1 prior line of therapy with oxaliplatin and a fluoropyrimidine +/- bev for metastatic disease. Pts are randomized 1:1 to receive napabucasin 240 mg PO BID plus FOLFIRI bi-weekly, or FOLFIRI bi-weekly (bev may be added to FOLFIRI by investigator choice) and stratified by geography, time to progression on 1st-line therapy, RAS mutation status, bev as part of study treatment and primary tumor location. Treatment will continue until disease progression, or another discontinuation criterion. Primary endpoint is overall survival (OS) in the general study population (ITT) (HR 0.80 for OS improvement from 12.54 to 15.68 months); secondary endpoints include OS in the biomarker positive (biomarker+) population, progression free survival (PFS) in the ITT population, PFS in biomarker+ population, ORR and DCR in the ITT and in biomarker+ populations, safety and quality of life. Also, blood and tumor archival tissue will be assessed for PK and biomarker analyses. Global enrollment is underway. Clinical trial information: NCT02753127.