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The immunomodulatory drug lenalidomide has demonstrated efficacy in patients with chronic lymphocytic leukemia (CLL), despite a lack of direct cytotoxic effects in vitro. The mechanism of lenalidomide efficacy in vivo is thought to occur via a combination of enhanced immune activity and an alteration of tumor cell–microenvironment interactions. We demonstrate in whole blood from patients with CLL that lenalidomide significantly depletes malignant B cells. Lenalidomide also induced production of interleukin-21 (IL21) and its mRNA in T cells from patients with CLL. In addition, lenalidomide enhanced upregulation of functional IL21 receptor (IL21R) on the cell surface and increased receptor mRNA in vitro. The in vitro combination of IL21 and lenalidomide enhanced IL21-mediated cytotoxicity toward CLL cells through a variety of mechanisms. We show association of cell death with upregulation of Bid by IL21, enhanced upregulation of Bid by the combination therapy, and diminished Lck and downstream BCR signaling activation of Syk and PLCG2. Collectively, we demonstrated an immune cell–tumor cell interaction through lenalidomide-mediated induction of IL21 and IL21R, with enhanced IL21-mediated cytotoxicity, which provides justification for this combination in clinical trials for patients with CLL. Cancer Immunol Res; 4(8); 698–707. ©2016 AACR.

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson’s trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

Background and Objectives The purpose of this study was to determine the pattern and timing of major wound complications (MWCs) in patients at our institution who received multimodality treatment for lower extremity soft tissue sarcoma (LE-STS) and to evaluate the impact of MWCs on tumor control and patient outcomes. Methods The medical records of 102 LE-STS patients treated with limb-sparing surgery and radiation therapy were reviewed. MWCs were defined as secondary operations with anesthesia, seroma/hematoma aspiration, admission for IV antibiotics, or persistent deep packing. Results MWCs occurred in 22% of patients, with 45% of events occurring >120 days after resection. On multivariate analysis, preoperative external beam radiation therapy (EBRT) (OR 4.29, 95% CI 1.06–17.40, P = 0.042) and skin graft placement (OR 6.39, 95% CI 1.37–29.84, P = 0.018) were found to be independent predictors of MWCs. MWC occurrence did not predict for chronic toxicity and did not impact tumor control or survival. Conclusions A considerable proportion of MWCs occur >120 days from surgical resection with preoperative EBRT and skin graft placement independent predictors for MWCs. While an additional source of morbidity, MWC occurrence did not impact tumor control, nor did it predict for chronic toxicity. J. Surg. Oncol. © 2016 Wiley Periodicals, Inc.

Purpose: Identification of predictive biomarkers is critically needed to improve selection of patients who derive the most benefit from platinum-based chemotherapy. We hypothesized that decreased expression of SMARCA4/BRG1, a known regulator of transcription and DNA repair, is a novel predictive biomarker of increased sensitivity to adjuvant platinum-based therapies in non–small cell lung cancer (NSCLC). Experimental Design: The prognostic value was tested using a gene-expression microarray from the Director's Challenge Lung Study (n = 440). The predictive significance of SMARCA4 was determined using a gene-expression microarray (n = 133) from control and treatment arms of the JBR.10 trial of adjuvant cisplatin/vinorelbine. Kaplan–Meier method and log-rank tests were used to estimate and test the differences of probabilities in overall survival (OS) and disease-specific survival (DSS) between expression groups and treatment arms. Multivariate Cox regression models were used while adjusting for other clinical covariates. Results: In the Director's Challenge Study, reduced expression of SMARCA4 was associated with poor OS compared with high and intermediate expression (P < 0.001 and P = 0.009, respectively). In multivariate analysis, compared with low, high SMARCA4 expression predicted a decrease in risk of death [HR, 0.6; 95% confidence interval (CI), 0.4–0.8; P = 0.002]. In the JBR.10 trial, improved 5-year DSS was noted only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine [HR, 0.1; 95% CI, 0.0–0.5, P = 0.002 (low); HR, 1.0; 95% CI, 0.5–2.3, P = 0.92 (high)]. An interaction test was highly significant (P = 0.01). Conclusions: Low expression of SMARCA4/BRG1 is significantly associated with worse prognosis; however, it is a novel significant predictive biomarker for increased sensitivity to platinum-based chemotherapy in NSCLC. Clin Cancer Res; 22(10); 2396–404. ©2015 AACR.

Sarcomas are malignant heterogenous tumors of mesenchymal derivation. Emerging data suggest that miRNA might have a causal role in sarcomagenesis. Herein, we used a selective miRNA screening platform to study the comparative global miRNA expression signatures in a cohort of human sarcomas with the caveat that comparisons between tumor and non-tumor cells were performed from the same patients using formalin-fixed paraffin-embedded tissue. Five histologic types were examined that included: myxoid liposarcoma, well-differentiated liposarcoma, dedifferentiated liposarcoma, pleomorphic rhabdomyosarcoma, and synovial sarcoma. In addition, soft-tissue lipomas and normal fat were included as a separate set of controls for the lipogenic tumors. Clustering analysis showed a distinct global difference in expression patterns between the normal and sarcoma tissues. Expression signatures in an unsupervised hierarchical clustering analysis revealed tight clustering in synovial and myxoid liposarcomas, and the least clustering was observed in the pleomorphic rhabdomyosarcoma subtype. MiR-145 showed underexpression in pleomorphic rhabdomyosarcoma, well-differentiated liposarcoma, and synovial sarcoma. Unexpectedly, we found that a set of muscle-specific microRNAs (miRNAs; myomiRs): miR-133, miR-1, and miR-206 was significantly underexpressed in well-differentiated liposarcoma and synovial sarcoma, suggesting that they may function as tumor suppressors as described in muscle-relevant rhabdomyosarcomas. In addition, a tight linear progression of miRNA expression was identified from normal fat to dedifferentiated liposarcoma. These results suggest that miRNA expression profiles could elucidate classes of miRNAs that may elicit tumor-relevant activities in specific sarcoma subtypes.

SUMMARY After spinal cord injury (SCI), normally innocuous visceral or somatic stimuli can trigger uncontrolled reflex activation of sympathetic circuitry, causing pathological dysautonomia. We show that remarkable structural remodeling and plasticity occur within spinal autonomic circuitry, creating abnormal sympathetic reflexes that promote dysautonomia. However, when mice are treated early after SCI with human-equivalent doses of the US Food and Drug Administration (FDA)-approved drug gabapentin (GBP), it is possible to block multi-segmental excitatory synaptogenesis and abolish sprouting of autonomic neurons that innervate immune organs and sensory afferents that trigger pain and autonomic dysreflexia (AD). This “prophylactic GBP” regimen decreases the frequency and severity of AD and protects against SCI-induced immune suppression. These benefits persist even 1 month after stopping treatment. GBP could be repurposed to prevent dysautonomia in at-risk individuals with high-level SCI., In Brief Brennan et al. show that α2δ−1 calcium channel subunits drive remarkable structural reorganization of autonomic circuitry and autonomic dysfunction after spinal cord injury. Early (prophylactic) post-injury treatment with gabapentin, an FDA-approved drug, prevents α2δ−1-dependent structural changes and autonomic dysfunction. Prophylactic gabapentin could be repurposed clinically for at-risk individuals., Graphical Abstract

SCOPE Persons with metabolic syndrome (MetS) absorb less vitamin E than healthy controls. It is hypothesized that absorption of fat-soluble vitamins (FSV) A and D2 would also decrease with MetS status and that trends would be reflected in lipidomic responses between groups. METHODS AND RESULTS Following soymilk consumption (501 IU vitamin A, 119 IU vitamin D2 ), the triglyceride-rich lipoprotein fractions (TRL) from MetS and healthy subjects (n = 10 age- and gender-matched subjects/group) are assessed using LC-MS/MS. Absorption is calculated using area under the time-concentration curves (AUC) from samples collected at 0, 3, and 6 h post-ingestion. MetS subjects have ≈6.4-fold higher median vitamin A AUC (retinyl palmitate) versus healthy controls (P = 0.07). Vitamin D2 AUC is unaffected by MetS status (P = 0.48). Untargeted LC-MS lipidomics reveals six phospholipids and one cholesterol ester with concentrations correlating (r = 0.53-0.68; P

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Purpose: ARID1A (AT-rich interactive domain 1A) is a member of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, and plays important roles in the regulation of gene transcription and DNA damage responses. ARID1A mutations occur frequently in non-small cell lung cancer (NSCLC), but the functions of ARID1A loss in the initiation, progression and maintenance of NSCLC remains largely unknown. This study aimed to investigate the clinical, pathological, and functional significance of ARID1A in NSCLC. Experimental design: We first analyzed the prognostic value of ARID1A using a gene-expression microarray from the Director's Challenge Lung Study. Kaplan-Meier method and Log-rank tests were used to estimate the correlation between ARID1A expression and overall survival (OS). Univariate and multivariate Cox regression models were used to investigate the relationship between ARID1A and other clinical covariates. We further established stable ARID1A knockdown H1299 and H358 cell lines, and ARID1A exogenously expressing H460 and A549 cell lines. We performed functional assays to study the effects of ARID1A on proliferation, migration, invasion, cell cycle progression, sensitivity to chemotherapy, and in vivo tumor growth. Results: In the Director's Challenge Study, decreased expression of ARID1A was associated with lymph node dissemination, higher T-stage (T3/T4), and worse survival compared with high expression in patients in the whole group (n=440) and subgroup who received surgery alone and no adjuvant therapy (n=328). ARID1A ablation by shRNA suppressed cell proliferation, promoted cell mobility, increased G2/M phase cell cycle distribution, and protected NSCLC cells from chemotherapy; whereas overexpression of ARID1A showed the opposite biological effects. Consistent with in vitro findings, in vivo tumor xenografts demonstrated ARID1A depletion led to reductions in cell growth but resistance of NSCLC to chemotherapy. Conclusions: Our results suggest that loss of ARID1A suppresses NSCLC cell growth perhaps by inducing G2/M arrest, but promotes cell migration/invasion, and protect cells from chemotherapy. Further study of ARID1A is needed to better define its roles in the initiation and progression of NSCLC and response to chemotherapy. Citation Format: Linlin Yang, Changxian Shen, Xiaokui Mo, Terence M. Williams. ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4155.

Exogenous sequences in bacteria have been found in many tumors, but their effect on outcomes remains largely unknown. We hypothesize that intratumoral microbes elicit effects through the recruitment of immune cells, such as CD8+ T-cells that are important for immunotherapy response. Through ORIEN (Oncology Research Information Exchange Network), we obtained RNA-seq data from biopsies of 480 tumors (Melanoma=16, Bladder=104, Colorectal=20 Renal Cell Carcinoma=20, Sarcoma=118, Lung=202) from patients treated at The Ohio State University Comprehensive Cancer Center. Reads were aligned to human and exogenous genomes using TopHat2 and Kraken2/Bracken, respectively. Human gene expression was deconvolved to relative abundances of immune cells using CIBERSORT. Across cancers, an average of 99.87% of reads were aligned to the human reference genome. The immune cell types CD8+ T-cells, activated dendritic cells and M1 macrophages were significantly enriched in lung cancer (p-values Citation Format: Xiaokui Mo, Daniel Spakowicz, Rebecca Hoyd. Intra-tumoral microbes correlate with immune cell fractions in lung cancer biopsies not other cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3338.

Glioblastoma (GBM), a mostly lethal brain tumor, acquires large amounts of free fatty acids (FAs) to promote cell growth. But how the cancer avoids lipotoxicity is unknown. Here, we identify that GBM upregulates diacylglycerol-acyltransferase 1 (DGAT1) to store excess FAs into triglycerides and lipid droplets. Inhibiting DGAT1 disrupted lipid homeostasis and resulted in excessive FAs moving into mitochondria for oxidation, leading to the generation of high levels of reactive oxygen species (ROS), mitochondrial damage, cytochrome c release, and apoptosis. Adding N-acetyl-cysteine or inhibiting FA shuttling into mitochondria decreased ROS and cell death induced by DGAT1 inhibition. We show in xenograft models that targeting DGAT1 blocked lipid droplet formation, induced tumor cell apoptosis, and markedly suppressed GBM growth. Together, our study demonstrates that DGAT1 upregulation protects GBM from oxidative damage and maintains lipid homeostasis by facilitating storage of excess FAs. Targeting DGAT1 could be a promising therapeutic approach for GBM.

OBJECTIVES: Lycopene intake is associated with reduced risk of prostate cancer, especially lethal disease. Tangerine tomatoes contain highly bioavailable lycopene isomers, compared to red tomatoes, but impact of isomers on cancer preventive bioactivity are unknown. Our goals are: (1) to determine if feeding tomato containing diets with differing lycopene isomers inhibit transgenic adenocarcinoma of the mouse prostate (TRAMP) carcinogenesis, and (2) to determine key early metabolic and transcriptional pathways through which tomatoes act to prevent carcinogenesis. METHODS: We examine prostate carcinogenesis in the TRAMP model, comparing two tomatoes with distinct lycopene isomer profiles: all-trans-lycopene-rich red tomatoes and cis-lycopene-isomer-rich tangerine tomatoes. Weanling TRAMP and wild type (WT) mice were fed AIN-93 G control, 10% tangerine tomato + AIN-93 G (w/w), or 10% red tomato + AIN-93 G (w/w) diet until 10 or 18 wks. At 10 wks., prostate RNA-seq and plasma metabolomics were performed on TRAMP and WT samples. At 18 wks., plasma carotenoid analysis and histopathology was performed on TRAMP mice. RESULTS: At 18 wks., plasma lycopene concentrations were 2.84-fold greater in tangerine tomato-fed mice than red tomato-fed mice (p

4083 Background: The presence of certain bacteria among or adjacent to tumor cells may contribute to colorectal cancer (CRC) development. However, the effect of the tumor microbiome on survival in CRC patients undergoing treatment is poorly understood. We hypothesize that intra-tumoral microbes correlate with overall survival (OS) in CRC patients. Methods: We obtained RNA-seq data from CRC tumor biopsies from patients treated at The Ohio State University Comprehensive Cancer Center as part of the Oncology Research Information Exchange Network (ORIEN). Reads were aligned to human and exogenous genomes using TopHat2 and Kraken2/Bracken, respectively. Results: The analyzed cohort included 99 CRC patients with an age range from 31-83 years, 62% female, and 44% with metastatic CRC. Therapies received prior to sample collection were grouped into chemotherapy with or without radiation (37%), antiVEGF/EGFR therapies (33%), no systemic therapy (23%), immunotherapy (3%); 3% were unknown. Overall, eleven bacteria were significantly associated with shorter OS, including a species in the genus Clostridium and Vibrio. Conversely, five other bacteria including several commensal gut microbes, were associated with longer OS. In patients who received chemotherapy with or without radiation (n = 38), several microbes were significantly associated with shorter OS, including a member of the genus Streptomyces. Only three bacteria were significantly associated with longer OS. In the patients who received antiVEGF/EGFR therapies (bevacizumab, cetuximab, panitumumab) (n = 33), several bacterial taxa were associated with shorter OS. In addition, bacteria including a member of the genera Bacillus and Staphylococcus were significantly associated with metastatic CRCs. (p < 0.05 for all, Fisher’s Exact tests). Conclusions: This study suggests that demonstrating the presence or absence of certain microbes in tumor biopsies could have important therapeutic implications for CRC patients. Only bacteria (no fungi, viruses, archaea, etc.) were found to significantly associate with OS across the entire cohort and within treatment subsets. The presence of bacteria was mostly, but not always, associated with worse OS. Antibiotics targeted towards bacterial species associated with negative outcomes could have the potential to improve OS in CRC patients.

e14503 Background: NSCLC with driver mutations may be managed with SRS, WBRT, and TKIs. Prior studies have demonstrated a worse survival with a delay in brain radiotherapy. We sought to determine if there is a difference in CNS PFS, OS, and cumulative incidence of intracranial brain failure (CI IBF) with next generation TKIs. Methods: A total of 69 patients with driver mutation NSCLC patients with brain metastasis were included. CNS PFS was measured from brain metastasis diagnosis. CI ICF was measured as any new or progressive disease on MRI Brain imaging. Results: The 1 year OS was 65.7% vs 60% (p = 0.9) for the RT and TKI arms. The CNS PFS was 82% vs 72% and CI ICF 13.5% vs 22% in the RT vs TKI arms, respectively. Conclusions: There was no difference in overall survival in our cohort when giving TKI therapy upfront or delayed with radiation. There was a clinically meaningful improvement in CNS PFS at 1 year but was not significant. There findings warrant further study.

BACKGROUND: Tumor aggressiveness and hypoxia are linked to acidosis in the tumor microenvironment (TME). We hypothesized that low pre-treatment serum bicarbonate, potentially correlating with an acidic and hypoxic TME, predicts for poor outcomes after stereotactic body radiation therapy (SBRT) for non-small cell lung cancer (NSCLC). METHODS: We included patients with localized NSCLC treated to a biologically effective dose (BED) ≥ 100 Gy, with available pre-treatment bicarbonate values within 3 months of treatment. We used receiver operating characteristic analysis to determine the bicarbonate concentration optimally predicting for primary tumor recurrence, and evaluated its association with recurrence and survival. We validated our findings in an independent cohort of patients from three collaborating institutions. RESULTS: A total of 110 patients and 114 tumors were included in the training cohort, with median follow-up of 15.0 months. Bicarbonate < 26 mEq/L was associated with primary tumor recurrence on univariate (HR = 5.92; 95% CI 1.69–24.88; p = 0.005) and multivariate analysis (HR = 5.48; 95% CI 1.37–25.19; p = 0.020). The validation cohort consisted of 195 patients and 208 tumors with median follow-up of 27.5 months. In the validation cohort, bicarbonate < 26 mEq/L was again associated with primary tumor recurrence on univariate (HR = 3.38; 95% CI 1.27–9.37; p = 0.015) and multivariate analysis (HR = 3.33; 1.18–10.07; p = 0.023). CONCLUSIONS: Pre-treatment bicarbonate predicts for primary tumor control in NSCLC treated with SBRT and may be useful for risk stratification. These findings should be confirmed prospectively.

Purpose Noninvasive measurement of mechanical properties of brain tissue using magnetic resonance elastography (MRE) has been a promising method for investigating neurologic disorders such as multiple sclerosis, hydrocephalus, and Alzheimer's. However, because of the regional and directional dependency of brain stiffness, estimating anisotropic stiffness is important. This study investigates isotropic and anisotropic stiffness as a function of age as well as the correlation between isotropic and anisotropic stiffness. Methods MRE and diffusion tensor imaging (DTI) were performed on 28 healthy subjects with age ranges between 18-62 y. Isotropic and anisotropic stiffness was measured and compared with age for different regions of interest such as the thalamus, corpus callosum, gray matter, white matter, and whole brain. Results Isotropic stiffness in gray matter (rs = -0.57; P = 0.001) showed a significant decrease with age. Anisotropic stiffness in gray matter showed a significant decrease with age in C11 through C66 and in the thalamus, only in C33 . Between anisotropic and isotropic stiffness, gray matter showed a significant positive correlation in C11 through C66 , C22 and C66 showed a significant negative correlation in the thalamus and whole brain, and C44 showed a negative correlation in the corpus callosum. No significant difference between genders was observed in any measurements. Conclusion This study demonstrated a change in isotropic and anisotropic stiffness with age in different regions of the brain along with a correlation of anisotropic stiffness to isotropic stiffness.

Spinal cord injury (SCI) damages supraspinal control over spinal autonomic circuitry. As a result, normally innocuous visceral or somatic stimuli (e.g. distended bladder or bowel) trigger pathological dysautonomia caused by uncontrolled reflex activation of sympathetic circuitry. In this report we show that remarkable structural remodeling and synaptic plasticity occurs within spinal autonomic circuitry and that this increases the frequency of autonomic dysreflexia (AD), a life-threatening condition of episodic vascular hypertension that develops progressively in SCI animals and people. We also prove that maladaptive plasticity can be prevented if SCI mice are treated with human equivalent doses of the FDA-approved drug, gabapentin (GBP) before the onset of plasticity. Although GBP is used clinically to treat pain or spasticity after symptom onset, we tested the hypothesis that GBP would prevent maladaptive plasticity if it were used as a prophylactic therapy, starting before the onset of a critical period of enhanced synaptogenesis with maintenance dosing continued for up to 35d post-injury. Using this novel treatment regimen, we show that prophylactic GBP successfully blocks multi-segmental excitatory synaptogenesis within spinal autonomic lamina, abolishes sprouting of autonomic neurons that innervate immune organs and also the sensory afferents that trigger pain and AD. Functionally, this treatment protocol delays the onset and decreases the frequency of spontaneous AD, confers resistance to experimentally induced AD, and protects mice from SCI-induced immune suppression. These data show that GBP could be repurposed as a prophylactic therapy in at-risk individuals with a high-level SCI to prevent the pathological effects of autonomic dysfunction.

Nonhuman sequences have been found in many tumors, but their effect on outcomes remains poorly understood. We hypothesize that intratumoral microbes affect the recruitment of immune cells through local immunostimulatory effects including activation nucleic acid sensing pathways. We obtained RNA-seq data from 480 tumor biopsies including melanoma (16), bladder (104), colorectal (20), renal cell carcinoma (20), sarcoma (118), and lung (202) from patients treated at The Ohio State University Comprehensive Cancer Center as part of the Oncology Research Information Exchange Network (ORIEN). Reads were aligned to human and exogenous genomes using TopHat2 and Kraken2/Bracken, respectively. Human gene expression was deconvolved to absolute abundances of immune cells using CIBERSORT. An average of 99.87% of reads aligned to the human reference genome across all samples. Inferred counts of tumor-infiltrating lymphocytes (TILs), and particularly the immune cell types CD8+ T-cells and M1 macrophages, were significantly enriched in lung cancer relative to the other tested cancers (p-values Citation Format: Daniel Spakowicz, Rebecca Hoyd, YunZhou Liu, Janhavi Sahasrabudhe, Malvenderjit J. Singh, Isaac Arefi, Andrew Denney, David Carbone, Xiaokui Mo. Intratumoral microbes correlate with tumor-infiltrating lymphocytes in lung cancer RNAseq [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B30.

Neutrophil-lymphocyte ratio (NLR) has been associated with mortality in several disease sites. We hypothesized that NLR is associated with inferior outcomes in localized non-small cell lung cancer (NSCLC) treated with stereotactic body radiotherapy (SBRT).We evaluated the association of pre-treatment NLR, obtained within 6 months of starting SBRT, with overall survival, as well as primary tumor, regional, and distant recurrence. Multivariate Cox regression was then used to assess pre-treatment NLR as a predictor of mortality. We validated our findings in an independent cohort of patients treated at two other institutions. In a secondary analysis, we also evaluated the association of post-treatment NLR with mortality in the training cohort.A total of 156 patients and 166 tumors were included in the training cohort with a median follow-up of 13.4 months. After dichotomization by median, NLR 3.6 was associated with mortality on univariate (p = 0.010) and multivariate analysis (p = 0.023). In the validation cohort, NLR 3.6 was similarly associated with mortality on univariate (p = 0.031) and multivariate (p = 0.007) analysis. In a secondary analysis in the training cohort, we found post-treatment NLR was significantly increased compared to pre-treatment NLR (p 0.001) and associated with mortality on univariate analysis (p = 0.005) and multivariate analysis (p = 0.010).Pre-treatment NLR 3.6 is associated with mortality in patients treated with SBRT. This finding was validated in an independent cohort of patients treated at two other institutions. Additionally, post-treatment NLR was significantly increased from pre-treatment and associated with overall survival.

The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib has transformed chronic lymphocytic leukemia (CLL) therapy but requires continuous administration. These factors have spurred interest in combination treatments. Unlike with chemotherapy, CD20-directed antibody therapy has not improved the outcome of BTKi treatment. Whereas CD20 antigen density on CLL cells decreases during ibrutinib treatment, the B-cell activating factor (BAFF) and its receptor (BAFF-R) remain elevated. Furthermore, BAFF signaling via noncanonical NF-κB remains elevated with BTKi treatment. Blocking BAFF interaction with BAFF-R by using VAY-736, a humanized defucosylated engineered antibody directed against BAFF-R, antagonized BAFF-mediated apoptosis protection and signaling at the population and single-cell levels in CLL cells. Furthermore, VAY-736 showed superior antibody-dependent cellular cytotoxicity compared with CD20- and CD52-directed antibodies used in CLL. VAY-736 exhibited in vivo activity as a monotherapy and, when combined with ibrutinib, produced prolonged survival compared with either therapy alone. The in vivo activity of VAY-736 is dependent upon immunoreceptor tyrosine–based activation motif (ITAM)–mediated activation of effector cells as shown by using an ITAM-deficient mouse model. Collectively, our findings support targeting the BAFF signaling pathway with VAY-736 to more effectively treat CLL as a single agent and in combination with ibrutinib.

The purpose of this study is 1) to demonstrate reproducibility of spin echo-echo planar imaging (SE-EPI) magnetic resonance elastography (MRE) to estimate kidney stiffness; and 2) to compare SE-EPI MRE and gradient recalled echo (GRE) MRE-derived stiffness estimations in various anatomical regions of the kidney. Kidney MRE was performed on 33 healthy subjects (8 for SE-EPI MRE reproducibility and 25 for comparison with GRE MRE; age range: 22-66 years) in a 3 T MRI scanner. To demonstrate SE-EPI MRE reproducibility, subjects were scanned for the first scan and then asked to leave the scan room and repositioned again for the second (repeat) scan. Similar set-up was used for GRE MRE as well. The displacement data was then processed to obtain overall stiffness estimates of the kidney. Concordance correlation analyses were performed to determine SE-EPI MRE reproducibility and agreement between GRE MRE and SE-EPI MRE derived stiffness. A high concordance correlation (ρc = 0.95; p-value

Acute myeloid leukemia (AML) remains a significant health problem, with poor outcomes despite chemotherapy and bone marrow transplants. Although one form of AML, acute promyelocytic leukemia (APL), is successfully treated with all-trans retinoic acid (ATRA), this drug is seemingly ineffective against all other forms of AML. Here, we show that ATRA up-regulates CD38 expression on AML blasts to sufficient levels that promote antibody-mediated fratricide following the addition of anti-CD38 daratumumab (DARA). The combination of ATRA plus DARA induced Fc-dependent conjugate formation and cytotoxicity among AML blasts in vitro. Combination treatment also led to reduction in tumor volume and resulted in increased overall survival in murine engraftment models of AML. These results suggest that, although ATRA does not induce differentiation of non-APL, it may be effective as a therapy in conjunction with DARA.

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fcγ receptors (FcγRs), which become activated upon binding to immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of FcγRIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of FcγRIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which FcγRIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated FcγRIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in FcγRIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of FcγRIIb protein and that this involves ubiquitination.

Kigerl et al. show that spinal cord injury causes profound changes in gut microbiota and that these changes in gut ecology are associated with activation of GALT immune cells. They show that feeding mice probiotics after SCI confers neuroprotection and improves functional recovery., The trillions of microbes that exist in the gastrointestinal tract have emerged as pivotal regulators of mammalian development and physiology. Disruption of this gut microbiome, a process known as dysbiosis, causes or exacerbates various diseases, but whether gut dysbiosis affects recovery of neurological function or lesion pathology after traumatic spinal cord injury (SCI) is unknown. Data in this study show that SCI increases intestinal permeability and bacterial translocation from the gut. These changes are associated with immune cell activation in gut-associated lymphoid tissues (GALTs) and significant changes in the composition of both major and minor gut bacterial taxa. Postinjury changes in gut microbiota persist for at least one month and predict the magnitude of locomotor impairment. Experimental induction of gut dysbiosis in naive mice before SCI (e.g., via oral delivery of broad-spectrum antibiotics) exacerbates neurological impairment and spinal cord pathology after SCI. Conversely, feeding SCI mice commercial probiotics (VSL#3) enriched with lactic acid–producing bacteria triggers a protective immune response in GALTs and confers neuroprotection with improved locomotor recovery. Our data reveal a previously unknown role for the gut microbiota in influencing recovery of neurological function and neuropathology after SCI.

Purpose To assess MR elastography (MRE)-derived aortic shear stiffness (μMRE) measurements for: 1) reproducibility, 2) comparison to pulse wave velocity, 3) changes over the cardiac cycle, and 4) relationship with age. Methods Cardiac-gated aortic MRE was performed on 20 healthy volunteers (aged 20–73 years). For assessing reproducibility of stiffness measurements, scans were repeated per volunteer. MRE wave images were analyzed to obtain stiffness of the abdominal aorta across the cardiac cycle, and comparisons were made with subject age. Results Analysis of concordance correlation coefficient between scans 1 and 2 showed that rc = 0.86 (95% confidence interval, 0.77, 0.94) with P

Purpose To assess reproducibility in measuring left ventricular (LV) myocardial stiffness in volunteers throughout the cardiac cycle using MR elastography (MRE) and to determine its correlation with age. Methods Cardiac MRE (CMRE) was performed on 29 normal volunteers, with ages ranging from 21 to 73 years. For assessing reproducibility of CMRE-derived stiffness measurements, scans were repeated per volunteer. Wave images were acquired throughout the LV myocardium, and were analyzed to obtain mean stiffness during the cardiac cycle. CMRE-derived stiffness values were correlated to age. Results Concordance correlation coefficient revealed good interscan agreement with rc of 0.77, with P-value

Purpose Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. Experimental Design The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. Results Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. Conclusions Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae. Pediatr Blood Cancer 2015;62:1768–1774. © 2015 Wiley Periodicals, Inc.

Background Curative therapy for childhood sarcoma presents challenges when complete resection is not possible. Ionizing radiation (XRT) is used as a standard modality at diagnosis or recurrence for childhood sarcoma; however, local recurrence is still problematic. Most childhood sarcomas are TP53 wild type at diagnosis, although approximately 5–10% have MDM2 amplification or overexpression. Procedures The MDM2 inhibitor, RG7388, was examined alone or in combination with XRT (20Gy given in 2 Gy daily fractions) to immune-deficient mice bearing Rh18 (embryonal) or a total of 30 Gy in 2 Gy fractions to mice bearing Rh30 (alveolar) rhabdomyosarcoma xenografts. RG7388 was administered by oral gavage using two schedules (daily ×5; schedule 1 or once weekly; schedule 2). TP53-responsive gene products (p21, PUMA, DDB2, and MIC1) as well as markers of apoptosis were analyzed. Results RG7388 showed no significant single agent antitumor activity. Twenty Grays XRT induced complete regressions (CR) of Rh18 with 100 percent tumor regrowth by week 7, but no tumor regrowth at 20 weeks when combined with RG7388. RG7388 enhanced time to recurrence combined with XRT in Rh30 xenografts compared to 30 Gy XRT alone. RG7388 did not enhance XRT-induced local skin toxicity. Combination treatments induced TP53 responsive genes more rapidly and to a greater magnitude than single agent treatments. Conclusions RG7388 enhanced the activity of XRT in both rhabdomyosarcoma models without increasing local XRT-induced skin toxicity. Changes in TP53-responsive genes were consistent with the synergistic activity of RG7388 and XRT in the Rh18 model. Pediatr Blood Cancer 2015;62:1345–1352. © 2015 Wiley Periodicals, Inc.

Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.

CpG oligodeoxynucleotides (ODNs) upregulate the interleukin-21 receptor (IL21R) and enhance IL-21-mediated cytotoxicity in chronic lymphocytic leukemia (CLL) B cells. We demonstrate that treatment of CLL B cells with the ODN CpG-685 leads to increased IL21R expression, and that this increased expression enhances the effects of IL-21 treatment as evidenced by increased phosphorylation of JAK1, STAT1, and STAT3, as compared to IL-21 treatment without prior CpG stimulation. Induction of IL21R by CpG-685 also enhanced IL-21-mediated cytotoxicity. The mechanism by which CpG ODNs upregulate IL21R has not been elucidated, although IL21R regulation in T cells has been shown to be linked to T cell receptor-induced Sp1 binding to the IL21R promoter. Here, we demonstrate that luciferase reporter constructs containing the Sp1 binding site have increased basal luciferase activity compared to constructs lacking the Sp1 binding site, but fail to increase luciferase activity with CpG-685 stimulation in CLL B cells. By treating CLL cells with an NF-κB inhibitor, we inhibit the CpG ODN-mediated induction of IL21R, thus demonstrating that CpG-685 upregulates IL21R through an NF-κB mediated pathway. These findings suggest an alternative mechanism for induction of IL-21 receptor in CLL B cells and provide a basis for creation of future combination therapies.

Fcγ receptors (FcγR) are critical mediators of monoclonal antibody cancer therapies, as they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with natural killer (NK) cells, monocytes are also known to destroy antibody-coated targets via antibody-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. Here, we show that human monocytes produce the protease Granzyme B upon both FcγR and Toll-like Receptor (TLR) 8 activation. Treatment with TLR8 agonists elicited Granzyme B and also enhanced FcγR-mediated Granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required Granzyme B. Hence, we have identified Granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies.

The pervasive nature of estrogenic industrial and dietary compounds is a growing health concern linked to cancer, obesity, and neurological disorders. Prior analyses of endocrine disruptor action have focused primarily on the short-term consequences of exposure. However, these studies are unlikely to reflect the consequences of constant exposures common to industrialized countries. Here we examined the global effects of long-term endocrine disruption on gene transcription and estrogen signaling. Estrogen-dependent breast cancer cell lines were chronically treated with physiologically relevant levels of bisphenol A or genistein for more than 70 passages. Microarray analysis demonstrated global reprogramming of the transcriptome when compared with a similarly cultured control cell line. Estrogen-responsive targets showed diminished expression in both the presence and absence of estrogen. Estrogen receptor recruitment, H3K4 monomethylation, and deoxyribonuclease accessibility were reduced at nearby response elements. Based on these observations, we investigated the potential of long-term endocrine disruptor exposure to initiate persistent transcriptional reprogramming. Culture of chronically exposed cell lines in the absence of the endocrine disruptors did not reverse many of the signaling defects that accumulated during treatment. Taken together, these data demonstrate that chronic exposure to endocrine disrupting compounds can permanently alter physiological hormone signaling.

This study determines the reproducibility of magnetic resonance elastography (MRE) derived brain stiffness in normal volunteers and compares it against pseudotumor patients before and after lumbar puncture (LP). MRE was performed on 10 normal volunteers for reproducibility and 14 pseudotumor patients before and after LP. During LP, opening and closing cerebrospinal fluid (CSF) pressures were recorded before and after removal of CSF and correlated to brain stiffness. Stiffness reproducibility was observed (r > 0.78; p < 0.008). Whole brain opening LP stiffness was significantly (p = 0.04) higher than normals, but no significant difference (p = 0.11) in closing LP measurements. No significant correlation was observed between opening and closing pressure and brain stiffness.

Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

Background STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. Methods and findings An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing’s sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. Conclusions LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

BTBR T + Itpr3tf/J (BTBR) mice are an Autism Spectrum Disorder (ASD)-like model that exhibit behavioral and physiological deficits similar to those observed in patients with ASD. While behavioral therapy is a first line of treatment in ASD patients, comparable non-pharmacological treatments are less explored in murine models. Here, we administer a bio-behavioral intervention for BTBR mice by way of environmental enrichment (EE) — an experimental housing paradigm previously shown to improve systemic metabolism, learning/memory, anxious behavior, neurogenesis, locomotion, and immunocompetence in C57BL/6 mice. Juvenile BTBR mice were randomized to standard or EE housing and were subjected to metabolic and behavioral assessments up to 17 weeks. Following EE exposure, we report an EE-induced metabolic and behavioral phenotype. Male BTBR mice responded metabolically to EE, displaying reduced adiposity, increased lean mass, improved glycemic control, and decreased circulating leptin. The gene expressions of brain-derived neurotrophic factor (Bdnf) and its receptor (Ntrk2/TrkB) were upregulated in several brain areas in EE-BTBR males. EE-BTBR females showed modest reduction of adiposity and no changes in glycemic control, circulating leptin, or Bdnf/Ntrk2 gene expression. With regard to behavior, EE resulted in decreased anxiety, and increased social affiliation. Together, these results suggest that EE improves metabolic and behavioral health in BTBR mice.

The gene encoding PTPROt (truncated isoform of protein tyrosine phosphatase receptor-type O) is methylated and suppressed in chronic lymphocytic leukemia (CLL). PTPROt exhibits in vitro tumor-suppressor characteristics through the regulation of B-cell receptor (BCR) signaling. Here we generated transgenic (Tg) mice with B-cell-specific expression of PTPROt. Although lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared with the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice, suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt-mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of BCR signaling. These results establish the in vivo tumor-suppressive function of PTPROt and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

The purposes of this study were (i) to investigate the differences in effects between 160-kV low-energy and 6-MV high-energy X-rays, both by computational analysis and in vitro studies; (ii) to determine the effects of each on platinum-sensitized F98 rat glioma and murine B16 melanoma cells; and (iii) to describe the in vitro cytotoxicity and in vivo toxicity of a Pt(II) terpyridine platinum (Typ-Pt) complex. Simulations were performed using the Monte Carlo code Geant4 to determine enhancement in absorption of low- versus high-energy X-rays by Pt and to determine dose enhancement factors (DEFs) for a Pt-sensitized tumor phantom. In vitro studies were carried out using Typ-Pt and again with carboplatin due to the unexpected in vivo toxicity of Typ-Pt. Cell survival was determined using clonogenic assays. In agreement with computations and simulations, in vitro data showed up to one log unit reduction in surviving fractions (SFs) of cells treated with 1–4 µg/ml of Typ-Pt and irradiated with 160-kV versus 6-MV X-rays. DEFs showed radiosensitization in the 50–200 keV range, which fell to approximate unity at higher energies, suggesting marginal interactions at MeV energies. Cells sensitized with 1–5 or 7 µg/ml of carboplatin and then irradiated also showed a significant decrease (P < 0.05) in SFs. However, it was unlikely this was due to increased interactions. Theoretical and in vitro studies presented here demonstrated that the tumoricidal activity of low-energy X-rays was greater than that of high-energy X-rays against Pt-sensitized tumor cells. Determining whether radiosensitization is a function of increased interactions will require additional studies.

Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1(GemR) cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1(GemR) xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer.

Piperlongumine, a natural plant product, kills multiple cancer types with little effect on normal cells. Piperlongumine raises intracellular levels of reactive oxygen species (ROS), a phenomenon that may underlie the cancer-cell killing. Although these findings suggest that piperlongumine could be useful for treating cancers, the mechanism by which the drug selectively kills cancer cells remains unknown.We treated multiple high-grade glioma (HGG) sphere cultures with piperlongumine and assessed its effects on ROS and cell-growth levels as well as changes in downstream signaling. We also examined the levels of putative piperlongumine targets and their roles in HGG cell growth.Piperlongumine treatment increased ROS levels and preferentially killed HGG cells with little effect in normal brain cells. Piperlongumine reportedly increases ROS levels after interactions with several redox regulators. We found that HGG cells expressed higher levels of the putative piperlongumine targets than did normal neural stem cells (NSCs). Furthermore, piperlongumine treatment in HGG cells, but not in normal NSCs, increased oxidative inactivation of peroxiredoxin 4 (PRDX4), an ROS-reducing enzyme that is overexpressed in HGGs and facilitates proper protein folding in the endoplasmic reticulum (ER). Moreover, piperlongumine exacerbated intracellular ER stress, an effect that was mimicked by suppressing PRDX4 expression.Our results reveal that the mechanism by which piperlongumine preferentially kills HGG cells involves PRDX4 inactivation, thereby inducing ER stress. Therefore, piperlongumine treatment could be considered as a novel therapeutic option for HGG treatment.

Introduction: While treatments for CLL have improved in recent years, CLL remains incurable for most patients who often rely on long-term suppressive medications. These can present complications associated with undesirable side effects and the risk of relapse. NK cell therapy holds great promise due to NK cells' powerful innate anti-tumor effects, with the potential to induce deep remission or even cure. However, previous efforts have been constrained by low cell numbers and limited cytotoxicity against CLL cells. Stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) induces high levels of expansion and activation, with potent cytotoxicity against various tumor cells (Denman et al. PLoS ONE 2012). We have recently demonstrated that allogeneic NKs from normal donors, expanded using mbIL-21, are potently cytotoxic to CLL cells (Yano et al. iwCLL 2019).Here, we test this technique with autologous NK cells. Autologous therapy will allow the benefits of administering activated NK cells without the risks of immunosuppression required for allogeneic treatment. Methods: We isolated NK cells from CLL patient blood and expanded them for 21 days using IL-21 expressing feeder cells and IL-2 (Denman et al. PLoS ONE 2012). We then characterized the cytotoxic capacity of the CLL-derived expanded NKs (CLL-XNKs) using calcein release assays. We measured both direct cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) against OSU-CLL and Mec1 CLL cell lines, allogeneic primary CLL cells, and autologous CLL cells. We compared CLL-XNK cells to unstimulated NK cells from normal donors and to normal donor-derived expanded NKs (ND-XNKs), produced using the same protocol. Results: During mbIL-21 NK stimulation, CLL-derived NK cells underwent an average of 5,900-fold expansion and maintained exponential growth throughout the 21-day expansion. This growth is similar to normal donor-derived NK cells (doubling time 1.6 days for CLL-derived vs. 1.5 days for donor-derived, p = 0.26, n=5). Cytotoxicity data comparing CLL-XNK cells versus ND-XNKs and unstimulated donor-derived NK cells is included in Table 1. We tested a range of effector:target ratios and found a dose response pattern of increasing cytotoxicity from 0.3125:1 to 10:1 ratios. Interestingly, while ADCC with either antibody was superior to direct cytotoxicity, obinutuzumab was not superior to rituximab for stimulating CLL-XNK cytotoxicity, differing from our experiences with ND-XNKs (Yano et al. iwCLL 2019). First, we demonstrate that CLL-XNK cells show potent cytotoxicity against both OSU-CLL and Mec1 CLL cell lines, via both direct cytotoxicity and ADCC (Table 1). These results show CLL-XNK cells to be similar or greater in potency in comparison to both ND-XNKs and normal unstimulated NKs. CLL-XNK cells also show cytotoxicity against allogeneic primary CLL cells, with greater cytotoxic activity than unstimulated, normal donor-derived NK cells (Table 1). Interestingly, while ADCC with obinutuzumab was similar between CLL-XNKs and ND-XNKs (p=.44), direct cytotoxicity and rituximab-induced cytotoxicity were both higher with CLL-XNKs than ND-XNKs (p=.0001 and .041) (Table 1). These results contrast with previous reports that LAKs derived from CLL patients have decreased potency (Foa et al. and Santiago-Schwarz et al. Blood 1990). Finally, CLL-XNKs showed potent cytotoxicity against autologous CLL cells using both direct cytotoxicity and ADCC (Table 1). Conclusion: We have successfully expanded NK cells from CLL patients and demonstrated their cytotoxicity against CLL cell lines, unmatched CLL cells, and autologous CLL cells. These patient-derived cells are superior to normal unstimulated NKs and are similar or even better than expanded donor NK cells. Ongoing studies will explore in vivo function of these NK cells, combination with CLL-targeted treatments, and further functional measures. IL-21-expanded NK cells represent a promising new therapy for CLL in both allogeneic and autologous settings. (*MY and JRL contributed equally to this work. MY is a recipient of a Pelotonia Graduate Fellowship and JRL is a recipient of a Hendrix Summer Scholars Fellowship. This work was supported by NIH R35 CA197734.) Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding.

Introduction: While treatments for CLL have improved in recent years, CLL remains incurable for most patients who often rely on long-term suppressive medications. These can present complications associated with undesirable side effects and the risk of relapse. NK cell therapy holds great promise due to NK cells' powerful innate anti-tumor effects, with the potential to induce deep remission or even cure. However, previous efforts have been constrained by low cell numbers and limited cytotoxicity against CLL cells. Stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) induces high levels of expansion and activation, with potent cytotoxicity against various tumor cells (Denman et al. PLoS ONE 2012). We have recently demonstrated that allogeneic NKs from normal donors, expanded using mbIL-21, are potently cytotoxic to CLL cells (Yano et al. iwCLL 2019). Here, we test this technique with autologous NK cells. Autologous therapy will allow the benefits of administering activated NK cells without the risks of immunosuppression required for allogeneic treatment. Methods: We isolated NK cells from CLL patient blood and expanded them for 21 days using IL-21 expressing feeder cells and IL-2 (Denman et al. PLoS ONE 2012). We then characterized the cytotoxic capacity of the CLL-derived expanded NKs (CLL-XNKs) using calcein release assays. We measured both direct cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) against OSU-CLL and Mec1 CLL cell lines, allogeneic primary CLL cells, and autologous CLL cells. We compared CLL-XNK cells to unstimulated NK cells from normal donors and to normal donor-derived expanded NKs (ND-XNKs), produced using the same protocol. Results: During mbIL-21 NK stimulation, CLL-derived NK cells underwent an average of 5,900-fold expansion and maintained exponential growth throughout the 21-day expansion. This growth is similar to normal donor-derived NK cells (doubling time 1.6 days for CLL-derived vs. 1.5 days for donor-derived, p = 0.26, n=5). Cytotoxicity data comparing CLL-XNK cells versus ND-XNKs and unstimulated donor-derived NK cells is included in Table 1. We tested a range of effector:target ratios and found a dose response pattern of increasing cytotoxicity from 0.3125:1 to 10:1 ratios. Interestingly, while ADCC with either antibody was superior to direct cytotoxicity, obinutuzumab was not superior to rituximab for stimulating CLL-XNK cytotoxicity, differing from our experiences with ND-XNKs (Yano et al. iwCLL 2019). First, we demonstrate that CLL-XNK cells show potent cytotoxicity against both OSU-CLL and Mec1 CLL cell lines, via both direct cytotoxicity and ADCC (Table 1). These results show CLL-XNK cells to be similar or greater in potency in comparison to both ND-XNKs and normal unstimulated NKs. CLL-XNK cells also show cytotoxicity against allogeneic primary CLL cells, with greater cytotoxic activity than unstimulated, normal donor-derived NK cells (Table 1). Interestingly, while ADCC with obinutuzumab was similar between CLL-XNKs and ND-XNKs (p=.44), direct cytotoxicity and rituximab-induced cytotoxicity were both higher with CLL-XNKs than ND-XNKs (p=.0001 and .041) (Table 1). These results contrast with previous reports that LAKs derived from CLL patients have decreased potency (Foa et al. and Santiago-Schwarz et al. Blood 1990). Finally, CLL-XNKs showed potent cytotoxicity against autologous CLL cells using both direct cytotoxicity and ADCC (Table 1). Conclusion: We have successfully expanded NK cells from CLL patients and demonstrated their cytotoxicity against CLL cell lines, unmatched CLL cells, and autologous CLL cells. These patient-derived cells are superior to normal unstimulated NKs and are similar or even better than expanded donor NK cells. Ongoing studies will explore in vivo function of these NK cells, combination with CLL-targeted treatments, and further functional measures. IL-21-expanded NK cells represent a promising new therapy for CLL in both allogeneic and autologous settings. (*MY and JRL contributed equally to this work. MY is a recipient of a Pelotonia Graduate Fellowship and JRL is a recipient of a Hendrix Summer Scholars Fellowship. This work was supported by NIH R35 CA197734.) Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau.

Fatty acids are essential building blocks and energy substrates of lipids in cells. They constitute the major structural components of membrane lipids, i.e., glycerophospholipids and sphingolipids, and also serve as an important energy resource. Whereas excess fatty acids or dysregulation of fatty acid metabolism causes lipotoxicity, leading to severe cell damage. We previously identified that lipid metabolism is dramatically altered in glioblastoma (GBM), especially fatty acid synthesis, which is greatly elevated in various types of cancers. However, how tumor cells prevent excessive fatty acids accumulation from inducing lipotoxicity has rarely been studied. We recently identified that GBM greatly upregulates diacylglycerol-acyltransferase 1 (DGAT1) to direct excess fatty acids into triglycerides and lipid droplets to prevent oxidative stress. Inhibiting DGAT1 disrupts lipid homeostasis and shifts excessive fatty acids moving into mitochondria for oxidation, resulting in a high level of reactive oxygen species (ROS), mitochondrial damage, cytochrome c release and apoptosis. Inhibiting carnitine palmitoyltransferase to reduce fatty acids shuttling into mitochondria significantly decreases ROS and rescues cell death caused by DGAT1 inhibition. Xenograft models show that genetic or pharmacological inhibition of DGAT1 blocks lipid droplet formation, induces tumor cell apoptosis and markedly suppresses GBM tumor growth. Together, our study demonstrates that DGAT1 upregulation protects GBM from oxidative damage and maintains lipid homeostasis by facilitating excess fatty acids storage. Our data further show that targeting DGAT1 specifically induces lipotoxicity in tumor cells, while sparing normal brain, which is a very promising therapeutic strategy antagonizing GBM and has a high potential to shift current paradigm in treating GBM.

Chronic lymphocytic leukemia (CLL) is the most common lymphoid malignancy in the United States. It causes significant morbidity and mortality, largely through its suppressive effects on the immune system. Our group has previously demonstrated that CLL cells express CTLA4, an immune checkpoint molecule normally found on activated T cells. CTLA4 is brought to the leukemic cell surface after interaction with a CLL-reactive activated T cell and functions to decrease CD80 expression on neighboring cells, thereby decreasing T cell activation. Previous data indicated a decrease in CTLA4 expression on T cells in patients treated with ibrutinib or acalabrutinib, two irreversible Bruton’s Tyrosine Kinase (BTK) inhibitors. Here, we expand our understanding of CTLA4 regulation in CLL and demonstrate that ibrutinib treatment directly modulates CTLA4 expression on CLL cells while indirectly modulating T cell CTLA4 expression. In CLL cells, we demonstrate that in vitro treatment with ibrutinib suppresses CTLA4 at each stage of its expression - mRNA, total protein, and surface trafficking (mean reductions: mRNA 53%, p>.0001, n=18; total protein 36%, p=.044, n=10; surface protein 35%, p=.0006, n=10). In contrast, treatment with acalabrutinib (a BTK-specific inhibitor) decreases mRNA (mean reduction 44%, p=.006, n=14) while sparing protein expression and trafficking. This means that CTLA4 surface protein (and therefore CTLA4 function) is regulated by non-BTK “off-target” effects of ibrutinib, while transcription is at least partly BTK-dependent. Comparing patient samples obtained before treatment versus after five months on ibrutinib, we demonstrate that CTLA4 suppression does occur with in vivo treatment (mean reduction: 54% total, p=.047; 40% surface, p=.034; n=5). In contrast to its in vivo effects and direct suppression on CLL cells, in vitro ibrutinib treatment failed to suppress T cell expression of CTLA4. This indicates that the T cell CTLA4 modulation seen in treated patients is due to indirect effects and points to different regulatory mechanisms in T versus CLL cells. Previous published data indicates that IFNγ levels in CLL patients decrease with ibrutinib treatment – here, we show that IFNγ can stimulate CTLA4 expression on CLL cells (qPCR mean increase 80%, p=.012, n=10). This indicates a second mechanism of CTLA4 reduction in patients receiving this treatment, via loss of IFNγ stimulation. Overall, we demonstrate a new mechanism of immunomodulation by ibrutinib – decreased CTLA4 on CLL cells. We demonstrate a different regulatory mechanism driving CTLA4 expression in CLL cells vs. T cells, and show that the decrease in T cell CTLA4 expression during ibrutinib treatment occurs indirectly. While recent efforts have developed more selective BTK inhibitors in an effort to decrease medication side effects, these results indicate that off-target immunomodulatory effects may be an underappreciated benefit from ibrutinib treatment. Citation Format: Max Yano, Priscilla Do, Xiaokui Mo, Natarajan Muthusamy, John C. Byrd. Ibrutinib immunomodulation via CTLA4 downregulation in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3959.

Many cationic lipids have been developed for lipid-based nanoparticles (LNPs) for delivery of siRNA and microRNA (miRNA). However, less attention has been paid to “helper lipids”. Here, we investigated several “helper lipids” and examined their effects on the physicochemical properties such as particle size and zeta potential, as well as cellular uptake and transfection efficiency. We found that inclusion of oleic acid (OA), an unsaturated fatty acid; into the LNP formulation significantly enhanced the delivery efficacy for siRNA and miRNA. For proof-of-concept, miR-122, a liver-specific microRNA associated with many liver diseases, was used as a model agent to demonstrate the hepatic delivery efficacy both in tumor cells and in animals. Compared to Lipofectamine 2000, a commercial transfection agent, OA containing LNPs delivered microRNA-122 in a more efficient manner with a 1.8-fold increase in mature miR-122 expression and a 20% decrease in Bcl-w, a target of microRNA-122. In comparison with Invivofectamine, a commercial transfection agent specifically designed for hepatic delivery, OA containing LNPs showed comparable liver accumulation and in vivo delivery efficiency. These findings demonstrated the importance of “helper lipid” components of the LNP formulation on the cellular uptake and transfection activity of siRNA and miRNA. OA containing LNPs are a promising nanocarrier system for the delivery of RNA-based therapeutics in liver diseases.

Despite advances in chemo- and immunotherapeutic agents for B chronic lymphocytic leukemia (B-CLL), the undesirable adverse side effects due to non-specific cellular uptake remain to be addressed. We identified anti-CD37 monoclonal antibody immunoliposomes (ILs) as vehicles for targeted delivery to B chronic lymphocytic leukemia cells. To achieve maximal benefits for all patients, a new strategy of dual-ligand immunoliposomes (dILs) was developed. A combinatorial antibody microarray technology was adapted to quickly identify optimal antibody combinations for individual patient cells. For proof-of-concept, a B-cell specific antibody, either anti-CD19 or anti-CD20, was combined with anti-CD37 to construct dILs with enhanced selectivity and efficacy. Consistent with data from the antibody microarray, these dILs provided highly specific targeting to both leukemia cell lines and B-CLL patient cells. Compared with the single antibody ILs, the anti-CD19/CD37 dILs clearly demonstrated superior delivery efficiency and apoptosis induction to B-CLL patient cells, whereas the anti-CD20/anti-CD37 dILs were found to be the most efficient for delivery to leukemia cell lines. In addition, it was observed that anti-CD37 ILs without payload drug mediated effective CD37 cross-linking and induced potent apoptosis induction. The anti-CD19/CD20 dILs showed the improved cell apoptosis induction compared to either anti-CD19 ILs or anti-CD20 ILs. Our findings suggest that the dual-ligand ILs may provide a preferred strategy of personalized nanomedicine for the treatment of B-cell malignancies.

Background: In pediatric tumor xenograft models, tumor-derived insulin growth factor (IGF-2) results in intrinsic resistance to IGF-IR–targeted antibodies, maintaining continued tumor angiogenesis. We evaluated the antiangiogenic activity of a ligand-binding antibody (MEDI-573) alone or in combination with IGF-I receptor binding antibodies (MAB391, CP01-B02). Methods: IGF-stimulated signaling was monitored by increased Akt phosphorylation in sarcoma and human umbilical cord vascular endothelial cells (HUVEC). Angiogenesis was determined in vitro using capillary tube formation in HUVECs and in vivo using a VEGF-stimulated Matrigel assay. Tumor growth delay was examined in 4 sarcoma xenograft models. Results: The IGF ligand-binding antibody MEDI-573 suppressed Akt phosphorylation induced by exogenous IGF-I and IGF-2 in sarcoma cells. Receptor-binding antibodies suppressed IGF-I stimulation of Akt phosphorylation, but IGF-2 circumvented this effect and maintained HUVEC tube formation. MEDI-573 inhibited HUVEC proliferation and tube formation in vitro, but did not inhibit angiogenesis in vivo, probably because MEDI-573 binds murine IGF-I with low affinity. However, in vitro antiangiogenic activity of MEDI-573 was also circumvented by human recombinant IGF-I. The combination of receptor- and ligand-binding antibodies completely suppressed VEGF-stimulated proliferation of HUVECs in the presence of IGF-I and IGF-2, prevented ligand-induced phosphorylation of IGF-IR/IR receptors, and suppressed VEGF/IGF-2–driven angiogenesis in vivo. The combination of CP1-BO2 plus MEDI-573 was significantly superior to therapy with either antibody alone against IGF-I and IGF-2 secreting pediatric sarcoma xenograft models. Conclusions: These results suggest that combination of antibodies targeting IGF receptor and ligands may be an effective therapeutic strategy to block angiogenesis for IGF-driven tumors. Clin Cancer Res; 19(11); 2984–94. ©2013 AACR.