Management of aortoesophageal fistula primarily using esophageal preservation.

Objective: Aortoesophageal fistula is a rare, life-threatening condition. There is no consensus regarding the surgical management of the esophagus in this condition. Methods: We retrospectively evaluated 13 patients diagnosed with aortoesophageal fistulas at a single institution from 2003 to 2021. Descriptive statistics were used to analyze patient characteristics, operative characteristics, and patient outcomes. Kaplan-Meier survival analysis was performed. Results: Patients' mean age was 63.5 years, and 6 (46.2%) were female. The most common presenting symptoms were hemoptysis/hematemesis (69.2%), chest/back pain (46.2%), and fever (38.5%). Twelve patients (92.3%) had a history of aortic procedures. The median time between the index operation and repair of the secondary aortoesophageal fistula in the 12 patients was 5 months. The index operation was a thoracic endovascular aortic repair in 10 of 12 patients (83.3%). Eleven patients (84.6%) underwent primary esophageal repair with flap coverage (omentum or muscle). One of these patients needed an esophagectomy within 1 year. The primary surgical management of the aorta was graft excision and replacement, aside from 1 patient who underwent primary repair. The 30-day survival was 69.2%, and 1-year and 5-year survivals were 31.7%. There were no recurrent infections at the esophageal fistula site. Conclusions: Aortoesophageal fistula remains a rare condition, but its case numbers have increased with thoracic endovascular aortic repair. It continues to be a difficult condition to manage and has a high fatality rate. Esophageal-preserving surgery may be a safe and less-invasive option for patients with a small defect.

Disruption of SUV39H1-Mediated H3K9 Methylation Sustains CAR T-cell Function.

Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.

Can hip function be assessed with self-report questionnaires? Feasibility study of a French self-report version of the Harris Hip and Merle d'Aubigné scores.

INTRODUCTION: The Harris Hip Score (HHS) and the Merle D'Aubigné Postel (MDP) score both provide an objective and subjective evaluation of hip function. These scores are collected during the follow-up of patients who have a hip disease. The objectives of this prospective study were (1) to analyze the differences between the two new French self-report versions of the HHS and MDP, and the traditional surgeon-assessed HHS and MDP; (2) to analyze the correlation between the self-report HHS and MDP and the surgeon-assessed HHS and MDP; (3) to analyze the floor and ceiling effects of the two self-report scores and the reliability of these self-report scores in operated and non-operated patients. HYPOTHESIS: The French self-report HHS and MDP are sufficiently reliable to accurately estimate the patient's objective and subjective outcomes compared to the clinical examination done by a surgeon. METHODS: A prospective multicenter study was done with patients who had a hip disease. Two self-report questionnaires were completed by the patient, independently of the clinical examination done by the surgeon. The questionnaires were in French and consisted solely of checkboxes, with sample photos that corresponded to the various range of motion items in the HHS and MDP. The agreement between the self-report scores and the surgeon-assessed scores were evaluated using the intraclass correlation coefficient (ICC). Differences in the mean values were evaluated with a paired t test. RESULTS: The analysis involved 89 patients. The self-report HHS was 2.7±3.7 points (/100) lower than the surgeon-assessed HHS, but this difference was not statistically significant (p=0.34). The self-report MDP was significantly less by 1.2±2.9 points (/18) than the surgeon-assessed MDP (p=0.01). The agreement between the self-report HSS and the surgeon-assessed HSS was excellent (ICC=0.86) as was the one between the self-report MDP and the surgeon-assessed MDP (ICC=0.75). There was a strong positive correlation between the surgeon-assessed and self-report HHS in operated patients (ICC= 0.84; R=0.75; p<0.001) and in non-operated patients (ICC=0.96; R=0.89; p<0.001). This positive correlation was also found between the surgeon-assessed and self-report MDP for operated patients (ICC=0.73; R=0.62; p<0.001) and non-operated patients (ICC=0.79; R=0.64; p<0.001). A ceiling effect (maximum of 100 points) was found in 22% of patients (20/89) for the self-report HHS and in 34% of patients (30/89) for the self-report MDP (maximum of 18 points). No floor effect was observed for either questionnaire. CONCLUSION: The French version of the HHS self-report questionnaire is an excellent overall estimator of the HHS score for patients with hip osteoarthritis or fracture, whether operated or not. The addition of the MDP, whose self-report version is less accurate, is also a reliable tool. These self-report questionnaires, when validated on a larger scale, will be useful for the long-term follow-up of patients undergoing hip arthroplasty. LEVEL OF EVIDENCE: III; prospective diagnostic study.

Osimertinib tolerance in a patient with Stevens Johnson syndrome during osimertinib therapy after treatment with pembrolizumab.

BACKGROUND: Osimertinib has emerged as an important tool in the treatment of non-small cell lung cancers (NSCLC) with certain activating mutations of epidermal growth factor receptor (EGFR). However, Osimertinib may cause adverse effects, including severe cutaneous adverse reactions (SCARs) such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The risk of certain adverse effects may be increased in the setting of recent use of immune checkpoint inhibitor (ICI) therapy, although it is unclear whether recent use of ICI therapy is a risk factor for Osimertinib-induced SJS specifically. CASE PRESENTATION: We present a patient with EGFR L858R mutation-positive metastatic NSCLC who developed Osimertinib-induced SJS after recent administration of eight cycles of a pembrolizumab-containing chemotherapy regimen. Osimertinib, which was the best treatment targeting his lung cancer, was avoided due to history of SJS. Four years later, because of unresponsiveness or side effects of alternative treatments, he underwent Osimertinib challenge and tolerated it. CONCLUSION: This case highlights the importance of multi-disciplinary care and supports the hypothesis that the risk of SJS to Osimertinib is significantly higher in the context of recent administration of ICI therapy and, patients may tolerate Osimertinib after certain time has elapsed after the last dose of ICI.

Cooperative CAR targeting to selectively eliminate AML and minimize escape.

Acute myeloid leukemia (AML) poses a singular challenge for chimeric antigen receptor (CAR) therapy owing to its phenotypic heterogeneity and similarity to normal hematopoietic stem/progenitor cells (HSPCs). Here we expound a CAR strategy intended to efficiently target AML while minimizing HSPC toxicity. Quantification of target expression in relapsed/refractory patient samples and normal HSPCs reveals a therapeutic window for gated co-targeting of ADGRE2 and CLEC12A: We combine an attenuated ADGRE2-CAR with a CLEC12A-chimeric costimulatory receptor (ADCLEC.syn1) to preferentially engage ADGRE2posCLEC12Apos leukemic stem cells over ADGRE2lowCLEC12Aneg normal HSPCs. ADCLEC.syn1 prevents antigen escape in AML xenograft models, outperforms the ADGRE2-CAR alone and eradicates AML despite proximate myelopoiesis in humanized mice. Off-target HSPC toxicity is similar to that of a CD19-CAR and can be mitigated by reducing CAR T cell-derived interferon-γ. Overall, we demonstrate the ability of target density-adapted cooperative CAR targeting to selectively eliminate AML and potentially obviate the need for hematopoietic rescue.

TET2 guards against unchecked BATF3-induced CAR T cell expansion.

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

How the Other Half Sees It: E-cigarette Advertising Exposure and Subsequent E-cigarette Use Among U.S. Young Adult Cigarette Smokers.

INTRODUCTION: E-cigarette advertising may benefit young adult cigarette smokers in transitioning to using e-cigarettes. We assessed whether e-cigarette advertising exposure was associated with subsequent e-cigarette use among young adult cigarette smokers. AIMS AND METHODS: Data were from Waves 4 (2016-2018) and 5 (2018-2019) of the nationally representative Population Assessment of Tobacco and Health (PATH) study adult survey. Respondents were young adult established cigarette smokers at Wave 4 (18-34 years; n = 3391) and a subsample of those who tried to quit smoking cigarettes completely in the past year at Wave 5 (n = 1235). Multivariable logistic regressions were used to examine the associations between e-cigarette advertising exposure (by channel of exposure) and subsequent past-year e-cigarette use in general and e-cigarette use to quit smoking cigarettes, controlling for covariates. RESULTS: At Wave 5, 43.4% of smokers reported past-year use of e-cigarettes; and 14.8% of smokers who tried to completely quit smoking reported past-year use of e-cigarettes to quit. E-cigarette advertising exposure was associated with subsequent past-year e-cigarette use (adjusted odds ratio [AOR] = 1.53, p < .0001, 95% confidence interval [CI] = 1.27, 1.86) and past-year use to quit smoking cigarettes (AOR = 1.65, p < .01, 95% CI = 1.19, 2.29). Advertising exposure through brick-and-mortar stores or websites/social media was similarly associated with both e-cigarette use behaviors. DISCUSSION: Exposure to e-cigarette advertising among U.S. young adult established cigarette smokers may be associated with subsequent e-cigarette use and use to quit smoking. More research is needed to understand the features of e-cigarette advertising (eg, discounts, flavors, smoker-targeted claims) that may shape perception and behavior related to e-cigarette use among young adult smokers. IMPLICATIONS: Little is known about the associations between e-cigarette advertising exposure and e-cigarette use among young adult cigarette smokers who may benefit from switching to e-cigarettes. This study found that e-cigarette advertising exposure was positively associated with (1) subsequent e-cigarette use among U.S. young adult established cigarette smokers and (2) subsequent e-cigarette use to quit smoking cigarettes among those who tried to completely quit in the past year. These observed associations were driven by smokers who did not currently use e-cigarettes at baseline. E-cigarette advertising exposure through brick-and-mortar stores or websites/social media was also positively associated with subsequent e-cigarette use behaviors.

In-depth analysis of alternative splicing landscape in multiple myeloma and potential role of dysregulated splicing factors.

Splicing changes are common in cancer and are associated with dysregulated splicing factors. Here, we analyzed RNA-seq data from 323 newly diagnosed multiple myeloma (MM) patients and described the alternative splicing (AS) landscape. We observed a large number of splicing pattern changes in MM cells compared to normal plasma cells (NPC). The most common events were alterations of mutually exclusive exons and exon skipping. Most of these events were observed in the absence of overall changes in gene expression and often impacted the coding potential of the alternatively spliced genes. To understand the molecular mechanisms driving frequent aberrant AS, we investigated 115 splicing factors (SFs) and associated them with the AS events in MM. We observed that ~40% of SFs were dysregulated in MM cells compared to NPC and found a significant enrichment of SRSF1, SRSF9, and PCB1 binding motifs around AS events. Importantly, SRSF1 overexpression was linked with shorter survival in two independent MM datasets and was correlated with the number of AS events, impacting tumor cell proliferation. Together with the observation that MM cells are vulnerable to splicing inhibition, our results may lay the foundation for developing new therapeutic strategies for MM. We have developed a web portal that allows custom alternative splicing event queries by using gene symbols and visualizes AS events in MM and subgroups. Our portals can be accessed at http://rconnect.dfci.harvard.edu/mmsplicing/ and https://rconnect.dfci.harvard.edu/mmleafcutter/ .

Sexually dimorphic estrogen sensing in skeletal stem cells controls skeletal regeneration.

Sexually dimorphic tissues are formed by cells that are regulated by sex hormones. While a number of systemic hormones and transcription factors are known to regulate proliferation and differentiation of osteoblasts and osteoclasts, the mechanisms that determine sexually dimorphic differences in bone regeneration are unclear. To explore how sex hormones regulate bone regeneration, we compared bone fracture repair between adult male and female mice. We found that skeletal stem cell (SSC) mediated regeneration in female mice is dependent on estrogen signaling but SSCs from male mice do not exhibit similar estrogen responsiveness. Mechanistically, we found that estrogen acts directly on the SSC lineage in mice and humans by up-regulating multiple skeletogenic pathways and is necessary for the stem cell's ability to self- renew and differentiate. Our results also suggest a clinically applicable strategy to accelerate bone healing using localized estrogen hormone therapy.

A FLEXIBLE SENSITIVITY ANALYSIS APPROACH FOR UNMEASURED CONFOUNDING WITH MULTIPLE TREATMENTS AND A BINARY OUTCOME WITH APPLICATION TO SEER-MEDICARE LUNG CANCER DATA.

In the absence of a randomized experiment, a key assumption for drawing causal inference about treatment effects is the ignorable treatment assignment. Violations of the ignorability assumption may lead to biased treatment effect estimates. Sensitivity analysis helps gauge how causal conclusions will be altered in response to the potential magnitude of departure from the ignorability assumption. However, sensitivity analysis approaches for unmeasured confounding in the context of multiple treatments and binary outcomes are scarce. We propose a flexible Monte Carlo sensitivity analysis approach for causal inference in such settings. We first derive the general form of the bias introduced by unmeasured confounding, with emphasis on theoretical properties uniquely relevant to multiple treatments. We then propose methods to encode the impact of unmeasured confounding on potential outcomes and adjust the estimates of causal effects in which the presumed unmeasured confounding is removed. Our proposed methods embed nested multiple imputation within the Bayesian framework, which allow for seamless integration of the uncertainty about the values of the sensitivity parameters and the sampling variability, as well as use of the Bayesian Additive Regression Trees for modeling flexibility. Expansive simulations validate our methods and gain insight into sensitivity analysis with multiple treatments. We use the SEER-Medicare data to demonstrate sensitivity analysis using three treatments for early stage non-small cell lung cancer. The methods developed in this work are readily available in the R package SAMTx.

Robotic Resection of Pulmonary Epithelial Myoepithelial Carcinoma: A Case Report.

Background Pulmonary epithelial-myoepithelial carcinoma (P-EMC) is an extremely rare, well-differentiated, and malignant neoplasm originating from submucosal bronchial glands in the lung. EMCs arise mainly in the salivary glands. Case Description This case represents an asymptomatic 78-year-old male with a remote 75-pack-year history of smoking who presents with a solitary endobronchial lesion, which is suggestive of a primary lung EMC, detected on annual screening chest computed tomography (CT) scan. Conclusion A recent review of literature reveals less than 50 documented cases of the pulmonary subtype of this tumor worldwide. We are reporting a unique case of robot-assisted pulmonary lobectomy for a P-EMC.

Pediatric Autoimmune Ocular Myasthenia Gravis: Evaluation of Presentation and Treatment Outcomes in a Large Cohort.

BACKGROUND: In autoimmune myasthenia gravis (MG), autoantibodies target the neuromuscular junction. Ocular myasthenia gravis (OMG) is localized, affecting only extraocular and/or levator palpebrae muscles. OMG presents across all ages, varying in presentation, treatment modalities, and outcomes. Recently, there have been advances in MG/OMG treatment; their utilization and effectiveness are an important part of optimal disease management. METHODS: We completed a retrospective chart review of children aged 18 years or younger with a confirmed diagnosis of OMG presenting from 2002 to 2019. RESULTS: Forty-two patients were included with mean age at presentation of 8.5 years (2 to 18 years). Twenty-one patients (50%) had positive antibodies; 90% had acetylcholine receptor antibodies. Ten patients developed generalized symptoms with mean time to generalization of 13.6 months. Multiple logistic regression showed that older age of onset was a trend predictive factor (P = 0.054; odds ratio 1.17) for generalized disease. All patients were treated with pyridostigmine. Immunomodulating agents included steroids (15), mycophenolate mofetil (four), and intravenous immunoglobulin (one). Three patients underwent thymectomy. Twenty patients reached minimal manifestation status, and 12 achieved remission. Gender, race, and positive antibody status were not statistically significant predictors for advanced immunosuppressive therapy. CONCLUSIONS: We summarize one of the largest cohorts of pediatric patients with OMG who have undergone up-to-date diagnostic and therapeutic regimens. The predictors of outcome and treatment pathway for OMG patients suggested by this report may be further elucidated by future prospective studies.

Clinical exome sequencing in the diagnosis of pediatric neuromuscular disease.

BACKGROUND: The diagnosis of uncommon pediatric neuromuscular disease (NMD) is challenging due to genetic and phenotypic heterogeneity, yet is important to guide treatment, prognosis, and recurrence risk. Patients with diagnostically challenging presentations typically undergo extensive testing with variable molecular diagnostic yield. Given the advancement in next generation sequencing (NGS), we investigated the value of clinical whole exome sequencing (ES) in uncommon pediatric NMD. METHODS: A retrospective cohort study of 106 pediatric NMD patients with a combination of ES, chromosomal microarray (CMA), and candidate gene testing was completed at a large tertiary referral center. RESULTS: A molecular diagnosis was achieved in 37/79 (46%) patients with ES, 4/44 (9%) patients with CMA, and 15/74 (20%) patients with candidate gene testing. In 2/79 (3%) patients, a dual molecular diagnosis explaining the neuromuscular disease process was identified. A total of 42 patients (53%) who received ES remained without a molecular diagnosis at the conclusion of the study. CONCLUSIONS: Due to NGS, molecular diagnostic yield of rare neurological diseases is at an all-time high. We show that ES has a higher diagnostic rate compared to other genetic tests in a complex pediatric neuromuscular disease cohort and should be considered early in the diagnostic journey for select NMD patients with challenging presentations in which a clinical diagnosis is not evident.

Hospital Readmissions for Hyperparathyroidism After Bariatric Surgery in the United States: A National Database Review.

INTRODUCTION: The incidence and significance of hyperparathyroidism in patients after bariatric surgery have been established to some degree; however, the impact it has on the national healthcare system has not. We sought to assess the risk of readmission and related comorbidities in this patient population. METHODS: The Healthcare Cost and Utilization Project Nationwide Readmission Database was queried for all patients who underwent Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Multivariate logistic regression analysis was conducted to identify factors associated with readmission for hyperparathyroidism. RESULTS: A total of 915,792 patients between 2010 and 2015 were queried; 43.2% had undergone SG and 56.8% had RYGB. A total of 589 patients were readmitted for hyperparathyroidism; 80.8% were female and 68% had a Charlson comorbidity index ≥ 2. Factors associated with readmission were as follows: age 45-64 years (odds ratio [OR] 1.42, p=0.001), Medicare (OR 3.01, p<0.001) or Medicaid (OR 2.61, p<0.001) insurance status, lower median household income, renal failure (OR 17.14, p<0.001), hypertension (OR 2.89, p<0.001), and deficiency anemia (OR 2.62, p<0.01). CONCLUSIONS: Parathyroid axis monitoring may provide benefits to predictably high-risk patients. Appropriate surveillance may decrease the impact of bariatric hyperparathyroidism readmission on the U.S. healthcare system.

Articular cartilage regeneration by activated skeletal stem cells.

Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

Impact of a Gardening and Physical Activity Intervention in Title 1 Schools: The TGEG Study.

Background: The purpose of the Texas!Grow!Eat!Go! (TGEG) study was to assess individual and combined effects of school-based gardening and physical activity (PA) interventions on children's eating and PA behaviors and obesity status. Methods: Using a 2 × 2 design, 28 low-income schools in Texas were randomized to 1 of 4 conditions: (1) School Garden intervention (Learn!Grow!Eat!Go! [LGEG]), (2) PA intervention (Walk Across Texas [WAT!]), (3) both Garden and PA intervention (Combined), or (4) neither Garden nor PA intervention (Control). Participants included 1326 third grade students and parents (42% Hispanic; 78% free/reduced lunch). Student and parent data were collected at the beginning and end of the school year. Two different sets of analyses measuring pre-post changes in outcomes within and across conditions were estimated by factorial ANOVAs using mixed models adjusted for demographics. Results: Main effect analyses indicate that relative to children at schools that did not receive LGEG, children at schools that received LGEG, either individually or in combination with WAT!, showed significant increases in Nutrition knowledge, Vegetable preference, and Vegetable tasted (p < 0.001 in all cases). Within-group analyses show that compared to Comparison, children in the WAT! group significantly increased in the amount of time parents and children were active together (p = 0.038). In addition, children in LGEG and WAT! schools significantly decreased BMI percentile (p = 0.042, p = 0.039, respectively), relative to children in Comparison schools. Conclusions: Both the garden and PA interventions independently produced significant changes related to healthy lifestyle behaviors. However, combining the two interventions did not show greater impact than the single interventions, underscoring the need for more research to determine how to better implement comprehensive interventions at schools.

Estimation of causal effects of multiple treatments in observational studies with a binary outcome.

There is a dearth of robust methods to estimate the causal effects of multiple treatments when the outcome is binary. This paper uses two unique sets of simulations to propose and evaluate the use of Bayesian additive regression trees in such settings. First, we compare Bayesian additive regression trees to several approaches that have been proposed for continuous outcomes, including inverse probability of treatment weighting, targeted maximum likelihood estimator, vector matching, and regression adjustment. Results suggest that under conditions of non-linearity and non-additivity of both the treatment assignment and outcome generating mechanisms, Bayesian additive regression trees, targeted maximum likelihood estimator, and inverse probability of treatment weighting using generalized boosted models provide better bias reduction and smaller root mean squared error. Bayesian additive regression trees and targeted maximum likelihood estimator provide more consistent 95% confidence interval coverage and better large-sample convergence property. Second, we supply Bayesian additive regression trees with a strategy to identify a common support region for retaining inferential units and for avoiding extrapolating over areas of the covariate space where common support does not exist. Bayesian additive regression trees retain more inferential units than the generalized propensity score-based strategy, and shows lower bias, compared to targeted maximum likelihood estimator or generalized boosted model, in a variety of scenarios differing by the degree of covariate overlap. A case study examining the effects of three surgical approaches for non-small cell lung cancer demonstrates the methods.

YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Dual PAK4-NAMPT Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldenström Macroglobulinemia.

PURPOSE: p21-activated kinase 4 (PAK4) plays a significant biological and functional role in a number of malignancies, including multiple myeloma (MM). On the basis of our promising findings in MM, we here characterize PAK4 expression and role in WM cells, as well effect of dual PAK4-NAMPT inhibitor (KPT-9274) against WM cell growth and viability. EXPERIMENTAL DESIGN: We have analyzed mRNA and protein expression levels of PAK4 in WM cells, and used loss-of-function approach to investigate its contribution to WM cell viability. We have further tested the in vitro and in vivo effect of KPT-9274 against WM cell growth and viability. RESULTS: We report here high-level expression and functional role of PAK4 in WM, as demonstrated by shRNA-mediated knockdown; and significant impact of KPT-9274 on WM cell growth and viability. The growth inhibitory effect of KPT-9274 was associated with decreased PAK4 expression and NAMPT activity, as well as induction of apoptosis. Interestingly, in WM cell lines treated with KPT-9274, we detected a significant impact on DNA damage and repair genes. Moreover, we observed that apart from inducing DNA damage, KPT-9274 specifically decreased RAD51 and the double-strand break repair by the homologous recombination pathway. As a result, when combined with a DNA alkylating agents bendamustine and melphalan, KPT-9274 provided a synergistic inhibition of cell viability in WM cell lines and primary patient WM cells in vitro and in vivo. CONCLUSIONS: These results support the clinical investigation of KPT-9274 in combination with DNA-damaging agent for treatment of WM.

Non-overlapping Control of Transcriptome by Promoter- and Super-Enhancer-Associated Dependencies in Multiple Myeloma.

The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy.