Protonation-State Dependent Modulation of Hen Egg-White Lysozyme Fibrillation under the Influence of a Short Synthetic Peptide.

Under the influence of various conditions, misfolding of soluble proteins occurs, leading to the formation of toxic insoluble amyloids. The formation and deposition of such amyloids within the body are associated with detrimental biological consequences such as the onset of several amyloid-related diseases. Previously, we established a strategy for the rational design of peptide inhibitors against amyloid formation based on the amyloidogenic-prone region of the protein. In the current study, we have designed and identified an Asp-containing rationally designed hexapeptide (SqP4) as an excellent inhibitor of hen egg-white lysozyme (HEWL) amyloid progression in vitro. First, SqP4 showed strong affinity toward the native monomeric HEWL leading to the stabilization of the native form and restriction in the unfolding process of monomeric HEWL. Second, SqP4 was found to arrest the amyloidogenic misfolded structure of HEWL in a nonfibrillar monomer-like stage. We also observed the differential effect of the protonation state of the charged amino acid (Asp) within the peptide inhibitor on the amyloid formation of HEWL and explored the reason behind the observations. The findings of this study can be implemented in future strategies for the development of potent therapeutics against other amyloid-related diseases.

A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.

Sample average treatment effect on the treated (SATT) analysis using counterfactual explanation identifies BMT and SARS-CoV-2 vaccination as protective risk factors associated with COVID-19 severity and survival in patients with multiple myeloma.

Patients with multiple myeloma (MM), an age-dependent neoplasm of antibody-producing plasma cells, have compromised immune systems and might be at increased risk for severe COVID-19 outcomes. This study characterizes risk factors associated with clinical indicators of COVID-19 severity and all-cause mortality in myeloma patients utilizing NCATS' National COVID Cohort Collaborative (N3C) database. The N3C consortium is a large, centralized data resource representing the largest multi-center cohort of COVID-19 cases and controls nationwide (>16 million total patients, and >6 million confirmed COVID-19+ cases to date). Our cohort included myeloma patients (both inpatients and outpatients) within the N3C consortium who have been diagnosed with COVID-19 based on positive PCR or antigen tests or ICD-10-CM diagnosis code. The outcomes of interest include all-cause mortality (including discharge to hospice) during the index encounter and clinical indicators of severity (i.e., hospitalization/emergency department/ED visit, use of mechanical ventilation, or extracorporeal membrane oxygenation (ECMO)). Finally, causal inference analysis was performed using the Coarsened Exact Matching (CEM) and Propensity Score Matching (PSM) methods. As of 05/16/2022, the N3C consortium included 1,061,748 cancer patients, out of which 26,064 were MM patients (8,588 were COVID-19 positive). The mean age at COVID-19 diagnosis was 65.89 years, 46.8% were females, and 20.2% were of black race. 4.47% of patients died within 30 days of COVID-19 hospitalization. Overall, the survival probability was 90.7% across the course of the study. Multivariate logistic regression analysis showed histories of pulmonary and renal disease, dexamethasone, proteasome inhibitor/PI, immunomodulatory/IMiD therapies, and severe Charlson Comorbidity Index/CCI were significantly associated with higher risks of severe COVID-19 outcomes. Protective associations were observed with blood-or-marrow transplant/BMT and COVID-19 vaccination. Further, multivariate Cox proportional hazard analysis showed that high and moderate CCI levels, International Staging System (ISS) moderate or severe stage, and PI therapy were associated with worse survival, while BMT and COVID-19 vaccination were associated with lower risk of death. Finally, matched sample average treatment effect on the treated (SATT) confirmed the causal effect of BMT and vaccination status as top protective factors associated with COVID-19 risk among US patients suffering from multiple myeloma. To the best of our knowledge, this is the largest nationwide study on myeloma patients with COVID-19.

Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial-mesenchymal Transition in Aggressive Variant Prostate Cancers.

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.

Extended Exposure Topotecan Significantly Improves Long-Term Drug Sensitivity by Decreasing Malignant Cell Heterogeneity and by Preventing Epithelial-Mesenchymal Transition.

Maximum tolerable dosing (MTD) of chemotherapeutics has long been the gold standard for aggressive malignancies. Recently, alternative dosing strategies have gained traction for their improved toxicity profiles and unique mechanisms of action, such as inhibition of angiogenesis and stimulation of immunity. In this article, we investigated whether extended exposure (EE) topotecan could improve long-term drug sensitivity by preventing drug resistance. To achieve significantly longer exposure times, we used a spheroidal model system of castration-resistant prostate cancer. We also used state-of-the-art transcriptomic analysis to further elucidate any underlying phenotypic changes that occurred in the malignant population following each treatment. We determined that EE topotecan had a much higher barrier to resistance relative to MTD topotecan and was able to maintain consistent efficacy throughout the study period (EE IC50 of 54.4 nM (Week 6) vs. MTD IC50 of 2200 nM (Week 6) vs. 83.8 nM IC50 for control (Week 6) vs. 37.8 nM IC50 for control (Week 0)). As a possible explanation for these results, we determined that MTD topotecan stimulated epithelial-mesenchymal transition (EMT), upregulated efflux pumps, and produced altered topoisomerases relative to EE topotecan. Overall, EE topotecan resulted in a more sustained treatment response and maintained a less aggressive malignant phenotype relative to MTD topotecan.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma.

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

Identification of Novel Peptides as Potential Modulators of Aß42 Amyloidogenesis: An in silico Approach.

AIMS: Alzheimer's disease is a neurodegenerative disease for which no cure is available. The presence of amyloid plaques in the extracellular space of neural cells is the key feature of this fatal disease. BACKGROUND: The proteolysis of Amyloid Precursor Protein by presenilin leads to the formation of Amyloid-beta peptides (Aß 42/40). Deposition of 42 residual Aß peptides forms fibril's structure, disrupting neuron synaptic transmission, inducing neural cell toxicity, and ultimately leading to neuron death. OBJECTIVE: Various novel peptides have been investigated via molecular docking and molecular dynamic simulation studies to investigate their effects on Aß amyloidogenesis. METHODS: The sequence-based peptides were rationally designed and investigated for their interaction with Aß42 monomer and fibril, and their influence on the structural stability of target proteins was studied. RESULTS: Analyzed docking results suggest that the peptide YRIGY (P6) has the highest binding affinity with Aß42 fibril amongst all the synthetic peptides, and the peptide DKAPFF (P12) similarly shows a better binding with the Aß42 monomer. Moreover, simulation results also suggest that the higher the binding affinity, the better the inhibitory action. CONCLUSION: These findings indicate that both the rationally designed peptides can modulate amyloidogenesis, but peptide (P6) has better potential for the disaggregation of the fibrils. In contrast, peptide P12 stabilizes the native structure of the Aß42 monomer more effectively and hence can serve as a potential amyloid inhibitor. Thus, these peptides can be explored as therapeutic agents against Alzheimer's disease. Experimental testing of these peptides for immunogenicity, stability in cellular conditions, toxic effects and membrane permeability can be the future research scope of this study.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH.

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross ß sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.

Elucidating the inhibitory effects of rationally designed novel hexapeptide against hen egg white lysozyme fibrillation at acidic and physiological pH.

Inhibition of highly ordered cross-ß-sheet-rich aggregates of misfolded amyloid proteins using rationally designed sequence-based short peptides is a promising therapeutic strategy for the treatment of neurodegenerative diseases. Here, we have explored the anti-amyloidogenic potency of a rationally designed hexapeptide (Tyr-Pro-Gln-Ile-Pro-Asn) on in vitro hen egg white lysozyme (HEWL) amyloid fibril formation at acidic pH and physiological pH using computational docking as well as various biophysical techniques such as fluorescence spectroscopy, UV-vis spectroscopy, FTIR spectroscopy, confocal microscopy and TEM. The peptide was designed based on the aggregation-prone region (APR) of HEWL and thus referred to as SqP1 (Sequence-based Peptide 1). SqP1 showed over 70% inhibition of HEWL amyloid formation at pH 2.2 and approximately 50% inhibition at pH 7.5. We propose that SqP1 binds to the APR of HEWL and interacts strongly with the Trp62/Trp63, ultimately stabilizing monomeric HEWL at both the pH conditions and preventing conformation changes in the structure of HEWL, leading to the formation of amyloidogenic fibrillar structures. A sequence-based peptide inhibitor of HEWL amyloid formation was not reported previously, making this a critical study that will further emphasize the importance of short synthetic peptides as amyloid inhibitors.

The FACT : Use of a novel intermittent pneumatic compression device to promote pre-surgery arm vein dilation in patients with chronic renal failure.

BACKGROUND: Arteriovenous fistula (AVF) creation and maturation for hemodialysis is globally a topic of importance given the poor results and high costs associated with renal care. Successful AVF (surgical or endovascular) creation requires appropriate superficial veins and quality arteries. Many procedures fail due to initial small veins with limited blood flow capacity and distensibility. Intermittent pneumatic compression has previously shown success in trials to increase superficial veins in patients with end stage renal disease post AVF. The objective of this study is to investigate the role of an intermittent pneumatic device, the Fist Assist®, to dilate cephalic arm veins in patients with advanced chronic kidney disease (CKD) prior to AVF placement. METHODS: Three centers enrolled subjects from June 2019 through July 2021. Baseline Doppler measurements of the cephalic vein in standard locations the forearm and upper arm with and without a blood pressure cuff were recorded. Patients were instructed and used Fist Assist® on their non-dominant arm for up to 4 h daily for 90 days. At approximately 3 months, Doppler measurements were repeated. The primary endpoint was cephalic vein enlargement with secondary endpoints based on percentage of veins approaching 2.5 mm in the forearm and 3.5 mm in the upper arm. RESULTS: Thirty-seven subjects with CKD (mean eGFR 13.8 mL/min) were enrolled and completed the trial. Paired-difference t-tests (one tail) for aggregate data showed significant venous dilation of the cephalic vein in both the forearm and upper arm after use with the Fist Assist® (p < 0.05). Mean differences in the forearm veins were approximately 0.6 and 1.1 mm in the upper arm cephalic vein after Fist Assist® application. There were no major complications reported by any subject during the trial. CONCLUSIONS: Fist Assist® use in patients with CKD is effective to enhance vein dilation. Forearm and upper arm cephalic veins increased on average 0.6 and 1.1 mm respectively after Fist Assist® application. This is the first trial to evaluate the effect of intermittent, focal pneumatic compression on pre-surgery vein diameter in patients with advanced CKD before AVF creation.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

Single-Cell Proteomics and Tumor RNAseq Identify Novel Pathways Associated With Clofazimine Sensitivity in PI- and IMiD- Resistant Myeloma, and Putative Stem-Like Cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy with dose-limiting toxicities and inter-individual variation in response/resistance to the standard-of-care/primary drugs, proteasome inhibitors (PIs), and immunomodulatory derivatives (IMiDs). Although newer therapeutic options are potentially highly efficacious, their costs outweigh the effectiveness. Previously, we have established that clofazimine (CLF) activates peroxisome proliferator-activated receptor-γ, synergizes with primary therapies, and targets cancer stem-like cells (CSCs) in drug-resistant chronic myeloid leukemia (CML) patients. In this study, we used a panel of human myeloma cell lines as in vitro model systems representing drug-sensitive, innate/refractory, and clonally-derived acquired/relapsed PI- and cereblon (CRBN)-negative IMiD-resistant myeloma and bone marrow-derived CD138+ primary myeloma cells obtained from patients as ex vivo models to demonstrate that CLF shows significant cytotoxicity against drug-resistant myeloma as single-agent and in combination with PIs and IMiDs. Next, using genome-wide transcriptome analysis (RNA-sequencing), single-cell proteomics (CyTOF; Cytometry by time-of-flight), and ingenuity pathway analysis (IPA), we identified novel pathways associated with CLF efficacy, including induction of ER stress, autophagy, mitochondrial dysfunction, oxidative phosphorylation, enhancement of downstream cascade of p65-NFkB-IRF4-Myc downregulation, and ROS-dependent apoptotic cell death in myeloma. Further, we also showed that CLF is effective in killing rare refractory subclones like side populations that have been referred to as myeloma stem-like cells. Since CLF is an FDA-approved drug and also on WHO's list of safe and effective essential medicines, it has strong potential to be rapidly re-purposed as a safe and cost-effective anti-myeloma drug.

Risk and Outcome of Breakthrough COVID-19 Infections in Vaccinated Patients With Cancer: Real-World Evidence From the National COVID Cohort Collaborative.

PURPOSE: To provide real-world evidence on risks and outcomes of breakthrough COVID-19 infections in vaccinated patients with cancer using the largest national cohort of COVID-19 cases and controls. METHODS: We used the National COVID Cohort Collaborative (N3C) to identify breakthrough infections between December 1, 2020, and May 31, 2021. We included patients partially or fully vaccinated with mRNA COVID-19 vaccines with no prior SARS-CoV-2 infection record. Risks for breakthrough infection and severe outcomes were analyzed using logistic regression. RESULTS: A total of 6,860 breakthrough cases were identified within the N3C-vaccinated population, among whom 1,460 (21.3%) were patients with cancer. Solid tumors and hematologic malignancies had significantly higher risks for breakthrough infection (odds ratios [ORs] = 1.12, 95% CI, 1.01 to 1.23 and 4.64, 95% CI, 3.98 to 5.38) and severe outcomes (ORs = 1.33, 95% CI, 1.09 to 1.62 and 1.45, 95% CI, 1.08 to 1.95) compared with noncancer patients, adjusting for age, sex, race/ethnicity, smoking status, vaccine type, and vaccination date. Compared with solid tumors, hematologic malignancies were at increased risk for breakthrough infections (adjusted OR ranged from 2.07 for lymphoma to 7.25 for lymphoid leukemia). Breakthrough risk was reduced after the second vaccine dose for all cancers (OR = 0.04; 95% CI, 0.04 to 0.05), and for Moderna's mRNA-1273 compared with Pfizer's BNT162b2 vaccine (OR = 0.66; 95% CI, 0.62 to 0.70), particularly in patients with multiple myeloma (OR = 0.35; 95% CI, 0.15 to 0.72). Medications with major immunosuppressive effects and bone marrow transplantation were strongly associated with breakthrough risk among the vaccinated population. CONCLUSION: Real-world evidence shows that patients with cancer, especially hematologic malignancies, are at higher risk for developing breakthrough infections and severe outcomes. Patients with vaccination were at markedly decreased risk for breakthrough infections. Further work is needed to assess boosters and new SARS-CoV-2 variants.

secDrug: a pipeline to discover novel drug combinations to kill drug-resistant multiple myeloma cells using a greedy set cover algorithm and single-cell multi-omics.

Multiple myeloma, the second-most common hematopoietic malignancy in the United States, still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs).We have created a computational pipeline that uses pharmacogenomics data-driven optimization-regularization/greedy algorithm to predict novel drugs ("secDrugs") against drug-resistant myeloma. Next, we used single-cell RNA sequencing (scRNAseq) as a screening tool to predict top combination candidates based on the enrichment of target genes. For in vitro validation of secDrugs, we used a panel of human myeloma cell lines representing drug-sensitive, innate/refractory, and acquired/relapsed PI- and IMiD resistance. Next, we performed single-cell proteomics (CyTOF or Cytometry time of flight) in patient-derived bone marrow cells (ex vivo), genome-wide transcriptome analysis (bulk RNA sequencing), and functional assays like CRISPR-based gene editing to explore molecular pathways underlying secDrug efficacy and drug synergy. Finally, we developed a universally applicable R-software package for predicting novel secondary therapies in chemotherapy-resistant cancers that outputs a list of the top drug combination candidates with rank and confidence scores.Thus, using 17AAG (HSP90 inhibitor) + FK866 (NAMPT inhibitor) as proof of principle secDrugs, we established a novel pipeline to introduce several new therapeutic options for the management of PI and IMiD-resistant myeloma.

Polygenic Ara-C Response Score Identifies Pediatric Patients With Acute Myeloid Leukemia in Need of Chemotherapy Augmentation.

PURPOSE: To establish a patient-specific polygenic score derived from cytarabine (ara-C) pathway pharmacogenomic evaluation to personalize acute myeloid leukemia (AML) treatment. MATERIALS AND METHODS: Single nucleotide polymorphisms (SNPs) in the ara-C-pathway genes were analyzed with outcome in patients from the multicenter-AML02 trial (N = 166). Multi-SNP predictor modeling was used to develop 10-SNP Ara-C_SNP score (ACS10) using top SNPs predictive of minimal residual disease and event-free survival (EFS) from the AML02-cohort and four SNPs previously associated with ara-C triphosphate levels in the AML97 trial. ACS10 was evaluated for association with outcomes in each clinical trial arms: the standard low-dose ara-C (LDAC, n = 91) and augmented high-dose ara-C (HDAC, n = 75) arms of AML02 and the standard Ara-C, daunorubicin and etoposide (ADE) (n = 465) and the augmented ADE + gemtuzumab ozogamicin (GO; n = 466) arms of AAML0531 trial. RESULTS: In the standard LDAC-arm of AML02 cohort, the low-ACS10 score group (≤ 0) had significantly worse EFS (ACS10 low v high hazard ratio [HR] = 2.81; 95% CI, 1.45 to 5.43; P = .002) and overall survival (OS; HR = 2.98; 95% CI, 1.32 to 6.75; P = .009) compared with the high-ACS10 group (score > 0). These results were validated in the standard-ADE arm of AAML0531, with poor outcome in the low-ASC10 group compared with the high-ACS10 group (EFS: HR = 1.35, 95% CI, 1.04 to 1.75, P = .026; OS: HR = 1.64, 95% CI, 1.2 to 2.22, P = .002). Within the augmented arms (AML02-HDAC and AAML0531-ADE + GO), EFS and OS did not differ between low- and high-ACS10 score groups. In both cohorts, patients with low-ACS10 consistently showed a 10-percentage point improvement in 5-year EFS with augmented therapy (AML02-HDAC or AAML0531-ADE + GO arms) than with standard therapy (AML02-LDAC or AAML0531-ADE arms). CONCLUSION: Patients with low-ACS10 score experienced significantly poor outcome when treated on standard regimen. Augmentation with either high-dose ara-C or GO addition improved outcome in low-ACS10 group. A polygenic ACS10 score can identify patients with unfavorable pharmacogenetic characteristics and offers a potential for an elective augmented therapy option.

Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) level determines steroid-resistant airway inflammation in aging.

Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly, which correlates with the changes seen in adults with asthma. However, whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here, we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism, we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR), mixed granulomatous airway inflammation comprising eosinophils and neutrophils, and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate, lung, and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes, which progressively declined with age and further by HDM-induced inflammation. Moreover, we found increased glycolytic reprogramming, maturation/activation of DCs, the proliferation of OT-II cells, and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity.

Irg1/itaconate metabolic pathway is a crucial determinant of dendritic cells immune-priming function and contributes to resolute allergen-induced airway inflammation.

Itaconate is produced from the mitochondrial TCA cycle enzyme aconitase decarboxylase (encoded by immune responsive gene1; Irg1) that exerts immunomodulatory function in myeloid cells. However, the role of the Irg1/itaconate pathway in dendritic cells (DC)-mediated airway inflammation and adaptive immunity to inhaled allergens, which are the primary antigen-presenting cells in allergic asthma, remains largely unknown. House dust mite (HDM)-challenged Irg1-/- mice displayed increases in eosinophilic airway inflammation, mucous cell metaplasia, and Th2 cytokine production with a mechanism involving impaired mite antigen presentations by DC. Adoptive transfer of HDM-pulsed DC from Irg1-deficient mice into naïve WT mice induced a similar phenotype of elevated type 2 airway inflammation and allergic sensitization. Untargeted metabolite analysis of HDM-pulsed DC revealed itaconate as one of the most abundant polar metabolites that potentially suppress mitochondrial oxidative damage. Furthermore, the immunomodulatory effect of itaconate was translated in vivo, where intranasal administration of 4-octyl itaconate 4-OI following antigen priming attenuated the manifestations of HDM-induced airway disease and Th2 immune response. Taken together, these data demonstrated for the first time a direct regulatory role of the Irg1/itaconate pathway in DC for the development of type 2 airway inflammation and suggest a possible therapeutic target in modulating allergic asthma.

The Andrographolide Analogue 3A.1 Synergizes with Taxane Derivatives in Aggressive Metastatic Prostate Cancers by Upregulation of Heat Shock Proteins and Downregulation of MAT2A-Mediated Cell Migration and Invasion.

Conventional treatment with taxanes (docetaxel-DTX or cabazitaxel-CBZ) increases the survival rates of patients with aggressive metastatic castration-resistant prostate cancer (mCRPC); however, most patients acquire resistance to taxanes. The andrographolide analog, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1), has shown anticancer activity against various cancers. In this study, we investigated the effect of 3A.1 alone and in combination with DTX/CBZ against mCRPC and their mechanism of action. Exposure to 3A.1 alone exhibited a dose- and time-dependent antitumor activity in mCRPC. Chou-Talalay's combination index (CI) values of all 3A.1 + TX combinations were less than 0.5, indicating synergism. Co-treatment of 3A.1 with TX reduced the required dose of DTX and CBZ (P < 0.05). Caspase assay (apoptosis) results concurred with in vitro cytotoxicity data. RNA sequencing (RNAseq), followed by ingenuity pathway analysis (IPA), identified that upregulation of heat-shock proteins (Hsp70, Hsp40, Hsp27, and Hsp90) and downregulation of MAT2A as the key player for 3A.1 response. Furthermore, the top treatment-induced differentially expressed genes (DEGs) belong to DNA damage, cell migration, hypoxia, autophagy (MMP1, MMP9, HIF-1α, Bag-3, H2AX, HMOX1, PSRC1), and cancer progression pathways. Most importantly, top downregulated DEG MAT2A has earlier been shown to be involved in cell migration and invasion. Furthermore, using in silico analysis on the Cancer Genome Atlas (TCGA) database, this study found that MAT2A and highly co-expressed (r > 0.7) genes, TRA2B and SF1, were associated with worse Gleason score and nodal metastasis status in prostate adenocarcinoma patients (PRAD-TCGA). Immunoblotting, comet, and migration assays corroborated these findings. These results suggest that 3A.1 may be useful in increasing the anticancer efficacy of taxanes to treat aggressive PCa. SIGNIFICANCE STATEMENT: The andrographolide analogue, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1), showed anticancer activity against metastatic castration-resistant and neuroendocrine variant prostate cancers (mCRPC/NEPC). Additionally, 3A.1 exhibited synergistic anticancer effect in combination with standard chemotherapy drugs docetaxel and cabazitaxel in mCRPC/NEPC. Post-treatment gene expression studies revealed that heat shock proteins (Hsp70, Hsp40, Hsp27, and Hsp90) and MAT2A are important in the mechanism of 3A.1 action and drug response. Furthermore, DNA damage, cell migration, hypoxia, and autophagy were crucial pathways for the anticancer activity of 3A.1.

Identification and Characterization of Key Differentially Expressed Genes Associated With Metronomic Dosing of Topotecan in Human Prostate Cancer.

Repetitive, low-dose (metronomic; METRO) drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/-), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers.

What is the optimal surgical intervention for patients with frozen shoulder and a concomitant partial-thickness rotator cuff tear?

BACKGROUND: Patients with recalcitrant frozen shoulder traditionally undergo arthroscopic capsular release. Some patients may have a concomitant partial-thickness rotator cuff tear (PTT). There is limited evidence if these PTT require repair at the same setting. We aim to compare if patients undergoing concomitant rotator cuff repair do better than patients undergoing capsular release alone. Secondarily, we aim to determine if outcomes after arthroscopic capsular release differ for patients with and without PTT. METHODS: A retrospective review of patients with frozen shoulders undergoing arthroscopic capsular release between 2012 and 2016 was performed. Patients with partial-thickness tears and patients without rotator cuff tears were included. Clinical outcomes were collected preoperatively and at 3, 6, 12 months after operation. RESULTS: There were 33 patients with PTT-15 underwent capsular release without repair (CR group), whereas 18 underwent capsular release with rotator cuff repair (RCR group). A total of 62 control patients without rotator cuff tears (No Tear) underwent arthroscopic capsular release only. For patients with PTT, there were no significant differences in preoperative demographics and function between the CR and RCR group. The CR group had significantly worse preoperative pain. At 1-year follow-up, the RCR group had significantly better internal rotation, lesser pain, and better function than the CR group. For patients undergoing capsular release only, the No Tear group had better internal rotation, lesser pain, and better function at 1 year compared with the CR group. CONCLUSION: Patients with a stiff, frozen shoulder and concomitant PTT do benefit from arthroscopic rotator cuff repair with capsular release. The benefit is evident at 1-year follow-up.