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.

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.

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.

TIVAN: tissue-specific cis-eQTL single nucleotide variant annotation and prediction.

SUMMARY: Predicting genetic regulatory variants, most of which locate in non-coding genomic regions, still remain a challenge in genetic research. Among all non-coding regulatory variants, cis-eQTL single nucleotide variants (SNVs) are of particular interest for their crucial role in regulating gene expression. Since different gene expression patterns are believed to contribute to the etiologies of different phenotypes, it is desirable to characterize the impact of cis-eQTL SNVs in a context-specific manner. Though computational methods for predicting the potential of variants being pathogenic or deleterious are well-established, methods for annotating and predicting cis-eQTL SNVs are under-developed. Here, we present TIVAN (TIssue-specific Variant ANnotation and prediction), an ensemble method of decision trees, to predict tissue-specific cis-eQTL SNVs. TIVAN is trained based on a comprehensive collection of features, including genome-wide genomic and epigenomic profiling data. As a result, TIVAN has been shown to accurately discriminate cis-eQTL SNVs from non-eQTL SNVs and perform favorably to other methods by obtaining higher five-fold cross-validation AUC values (CV-AUC) and Leave-One-Chromosome-Out predicted AUC values (LOCO-AUC) across 44 different tissues belonging to 27 different tissue classes. Finally, TIVAN consistently maintains top performance on an independent testing dataset, which includes 7 tissues in 11 studies. AVAILABILITY AND IMPLEMENTATION: TIVAN software is available at https://github.com/lichen-lab/TIVAN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Sequence and structure-based peptides as potent amyloid inhibitors: A review.

Misfolded and natively disordered globular proteins tend to aggregate together in an interwoven fashion to form fibrous, proteinaceous deposits referred to as amyloid fibrils. Formation and deposition of such insoluble fibrils are the characteristic features of a broad group of diseases, known as amyloidosis. Some of these proteins are known to cause several degenerative disorders in humans, such as Amyloid-Beta (Aß) in Alzheimer's disease (AD), human Islet Amyloid Polypeptide (hIAPP, amylin) in type 2 diabetes, α-synuclein (α-syn) in Parkinson's disease (PD) and so on. The fact that these proteins do not share any significant sequence or structural homology in their native states make therapy quite challenging. However, it is observed that aggregation-prone proteins and peptides tend to adopt a similar type of secondary structure during the formation of fibrils. Rationally designed peptides can be a potent inhibitor that has been shown to disrupt the fibril structure by binding specifically to the amyloidogenic region(s) within a protein. The following review will analyze the inhibitory potency of both sequence-based and structure-based small peptides that have been shown to inhibit amyloidogenesis of proteins such as Aß, human amylin, and α-synuclein.

Risk factors of lateral epicondylitis: A meta-analysis.

INTRODUCTION: Lateral Epicondylitis is commonly associated with numerous anatomical and mechanical risk factors. Thus far, there have been no reviews synthesising the risk factors of lateral epicondylitis. We hence aimed to perform a meta-analysis of factors associated with lateral epicondylitis. METHODOLOGY: We searched MEDLINE, Scopus and Web of Science for 1032 articles. Eventually, based on our exclusion criteria, we had 33 articles remaining for our systematic review. 15 of these articles were used for our meta-analysis. Data was analysed using Mantel-Haenszel statistics and random effect models where appropriate. RESULTS: Females had a 1.29 times higher odds of sustaining lateral epicondylitis (OR Males: Females = 0.77, 95% CI = 0.67-0.89, Z = 3.52, I2 = 33%, p < 0.001). The odds of an individual with a current or past tobacco smoking history sustaining lateral epicondylitis was 1.49 times that of an individual with no tobacco smoking history (95% CI = 1.18-1.87, Z = 3.40, I2 = 0%, p < 0.001). There was no statistical difference in sustaining lateral epicondylitis when comparing individuals with a current tobacco smoking history to individuals with a past or no tobacco smoking history (OR = 1.18, 95% CI = 0.91-1.51, Z = 1.26, I2 = 0%, p = 0.21). Neither was there a statistical difference in sustaining lateral epicondylitis when comparing individuals with a BM ≥ 25 to those with a BMI<25 (OR = 1.12, 95% CI = 0.69-1.83, Z = 0.46, I2 = 62%, p = 0.65). CONCLUSION: Female gender and a positive and past smoking history were associated with lateral epicondylitis. Further studies should focus on identifying other associations with lateral epicondylitis and the pathophysiological explanation of such associations.

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

OBJECTIVES: The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. METHODS: A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. RESULTS: Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. CONCLUSIONS: Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer.

Comprehensive genetic analysis of cytarabine sensitivity in a cell-based model identifies polymorphisms associated with outcome in AML patients.

A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10(-5)) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10(-6)) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084.

Outcomes are favorable after arthroscopic treatment of osteochondritis dissecans of the talus.

Arthroscopic treatment of osteochondritis dissecans (OCD) of the talus has resulted in outcomes as good as, or better than, those after arthrotomy. We noted a lack of prospective studies investigating the outcomes of arthroscopic treatment. As such, we conducted a prospective study investigating the functional outcomes, pain scores, patient satisfaction, and expectation scores of patients undergoing arthroscopic treatment of OCD of the talus, hypothesizing that these patients would have good outcomes and satisfaction. A total of 61 patients underwent arthroscopic chondroplasty, removal of loose bodies, and microfracture for OCD of the talus and completed ≥1 year of follow-up. We evaluated patients pre- and postoperatively at 6 and 12 months using the Ankle-Hindfoot score, visual analog scale for pain, and Medical Outcomes Study short-form 36 questionnaires. We also evaluated the patients' expectations and satisfaction. The mean Ankle-Hindfoot score improved significantly from 53.0 ± 14.3 points preoperatively to 77.8 ± 19.1 at 6 months and 83.1 ± 18.3 at 12 months after arthroscopic treatment (p < .001). The overall scores at the final follow-up visit were excellent for 30 (49%), good for 6 (10%), fair for 18 (30%), and poor for 7 (11%). The patients also experienced significant improvement in the visual analog scale score and physical component score of the short-form 36 questionnaire (p < .001). Of the 61 patients, 67% had their expectations fulfilled and 74% were satisfied with their surgery at 12 months of follow-up. Arthroscopic treatment of OCD of the talus continues to be a successful procedure to alleviate pain and loss of function. It is also associated with improvements to quality of life and good patient satisfaction.

De-epithelialized fasciocutaneous turnover flap for recurrent calcaneal wound with osteomyelitis.

Recurrent ulcerations of the foot and ankle almost always present a challenge to lower extremity surgeons. Recalcitrant heel ulcerations with osteomyelitis are especially difficult to treat because of the lack of soft tissue coverage. The turnover flap is a simple, fast, and effective treatment method for lower extremity wounds. It is a de-epithelialized fasciocutaneous flap harvested from the adjacent area of the wound. We believe it is an underused technique for advanced wound closure in the lower extremity. It offers several advantages compared with traditional, more difficult to perform, flaps. We have seen an excellent result 18 months after using the turnover flap in a patient with recurrent posterior heel ulceration with calcaneal osteomyelitis.

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.

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.

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.

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.

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.

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.

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.