Uncovering miRNA-mRNA Regulatory Networks Related to Olaparib Resistance and Resensitization of BRCA2MUT Ovarian Cancer PEO1-OR Cells with the ATR/CHK1 Pathway Inhibitors.

Resistance to olaparib is the major obstacle in targeted therapy for ovarian cancer (OC) with poly(ADP-ribose) polymerase inhibitors (PARPis), prompting studies on novel combination therapies to enhance olaparib efficacy. Despite identifying various mechanisms, understanding how OC cells acquire PARPi resistance remains incomplete. This study investigated microRNA (miRNA) expression in olaparib-sensitive (PEO1, PEO4) and previously established olaparib-resistant OC cell lines (PEO1-OR) using high-throughput RT-qPCR and bioinformatic analyses. The role of miRNAs was explored regarding acquired resistance and resensitization with the ATR/CHK1 pathway inhibitors. Differentially expressed miRNAs were used to construct miRNA-mRNA regulatory networks and perform functional enrichment analyses for target genes with miRNet 2.0. TCGA-OV dataset was analyzed to explore the prognostic value of selected miRNAs and target genes in clinical samples. We identified potential processes associated with olaparib resistance, including cell proliferation, migration, cell cycle, and growth factor signaling. Resensitized PEO1-OR cells were enriched in growth factor signaling via PDGF, EGFR, FGFR1, VEGFR2, and TGFßR, regulation of the cell cycle via the G2/M checkpoint, and caspase-mediated apoptosis. Antibody microarray analysis confirmed dysregulated growth factor expression. The addition of the ATR/CHK1 pathway inhibitors to olaparib downregulated FGF4, FGF6, NT-4, PLGF, and TGFß1 exclusively in PEO1-OR cells. Survival and differential expression analyses for serous OC patients revealed prognostic miRNAs likely associated with olaparib resistance (miR-99b-5p, miR-424-3p, and miR-505-5p) and resensitization to olaparib (miR-324-5p and miR-424-3p). Essential miRNA-mRNA interactions were reconstructed based on prognostic miRNAs and target genes. In conclusion, our data highlight distinct miRNA profiles in olaparib-sensitive and olaparib-resistant cells, offering molecular insights into overcoming resistance with the ATR/CHK1 inhibitors in OC. Moreover, some miRNAs might serve as potential predictive signature molecules of resistance and therapeutic response.

Breast cancer but not the menopausal status is associated with small changes of the gut microbiota.

Background: Possible relationships between gut dysbiosis and breast cancer (BC) development and progression have been previously reported. However, the results of these metagenomics studies are inconsistent. Our study involved 88 patients diagnosed with breast cancer and 86 cancer-free control women. Participants were divided into groups based on their menopausal status. Fecal samples were collected from 47 and 41 pre- and postmenopausal newly diagnosed breast cancer patients and 51 and 35 pre- and postmenopausal controls, respectively. In this study, we performed shotgun metagenomic analyses to compare the gut microbial community between pre- and postmenopausal BC patients and the corresponding controls. Results: Firstly, we identified 12, 64, 158, and 455 bacterial taxa on the taxonomy level of phyla, families, genera, and species, respectively. Insignificant differences of the Shannon index and ß-diversity were found at the genus and species levels between pre- and postmenopausal controls; the differences concerned only the Chao index at the species level. No differences in α-diversity indexes were found between pre- and postmenopausal BC patients, although ß-diversity differed these subgroups at the genus and species levels. Consistently, only the abundance of single taxa differed between pre- and postmenopausal controls and cases, while the abundances of 14 and 23 taxa differed or tended to differ between premenopausal cases and controls, and between postmenopausal cases and controls, respectively. There were similar differences in the distribution of enterotypes. Of 460 bacterial MetaCyc pathways discovered, no pathways differentiated pre- and postmenopausal controls or BC patients, while two and one pathways differentiated cases from controls in the pre- and postmenopausal subgroups, respectively. Conclusion: While our findings did not reveal an association of changes in the overall microbiota composition and selected taxa with the menopausal status in cases and controls, they confirmed differences of the gut microbiota between pre- and postmenopausal BC patients and the corresponding controls. However, these differences were less extensive than those described previously.

The Risk Function of Breast and Ovarian Cancers in the Avrami-Dobrzynski Cellular Phase-Transition Model.

Specifying the role of genetic mutations in cancer development is crucial for effective screening or targeted treatments for people with hereditary cancer predispositions. Our goal here is to find the relationship between a number of cancerogenic mutations and the probability of cancer induction over the lifetime of cancer patients. We believe that the Avrami-Dobrzynski biophysical model can be used to describe this mechanism. Therefore, clinical data from breast and ovarian cancer patients were used to validate this model of cancer induction, which is based on a purely physical concept of the phase-transition process with an analogy to the neoplastic transformation. The obtained values of model parameters established using clinical data confirm the hypothesis that the carcinogenic process strongly follows fractal dynamics. We found that the model's theoretical prediction and population clinical data slightly differed for patients with the age below 30 years old, and that might point to the existence of an ancillary protection mechanism against cancer development. Additionally, we reveal that the existing clinical data predict breast or ovarian cancers onset two years earlier for patients with BRCA1/2 mutations.

Iron-triggered signaling via ETS1 and the p38/JNK MAPK pathway regulates Bmp6 expression.

BMP6 is an iron-sensing cytokine whose transcription in liver sinusoidal endothelial cells (LSECs) is enhanced by high iron levels, a step that precedes the induction of the iron-regulatory hormone hepcidin. While several reports suggested a cell-autonomous induction of Bmp6 by iron-triggered signals, likely via sensing of oxidative stress by the transcription factor NRF2, other studies proposed the dominant role of a paracrine yet unidentified signal released by iron-loaded hepatocytes. To further explore the mechanisms of Bmp6 transcriptional regulation, we used female mice aged 10-11 months, which are characterized by hepatocytic but not LSEC iron accumulation, and no evidence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 mRNA levels as compared to young controls, but do not show a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 expression in response to iron exposure. By analyzing transcriptomic data of FACS-sorted LSECs from aged versus young mice, as well as early after iron citrate injections, we identified ETS1 as a candidate transcription factor involved in Bmp6 transcriptional regulation. By performing siRNA-mediated knockdown, small-molecule treatments, and chromatin immunoprecipitation in primary LSECs, we show that Bmp6 transcription is regulated by iron via ETS1 and p38/JNK MAP kinase-mediated signaling, at least in part independently of NRF2. Thereby, these findings identify the new components of LSEC iron sensing machinery broadly associated with cellular stress responses.

MCPIP1 functions as a safeguard of early embryonic development.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase that has been described as a key negative modulator of inflammation. MCPIP1 also controls numerous tumor-related processes, such as proliferation, apoptosis and differentiation. In this study, we utilized a zebrafish model to investigate the role of Mcpip1 during embryogenic development. Our results demonstrated that during embryogenesis, the expression of the zc3h12a gene encoding Mcpip1 undergoes dynamic changes. Its transcript levels gradually increase from the 2-cell stage to the spherical stage and then decrease rapidly. We further found that ectopic overexpression of wild-type Mcpip1 but not the catalytically inactive mutant form resulted in an embryonic lethal phenotype in zebrafish embryos (24 hpf). At the molecular level, transcriptomic profiling revealed extensive changes in the expression of genes encoding proteins important in the endoplasmic reticulum stress response and in protein folding as well as involved in the formation of primary germ layer, mesendoderm and endoderm development, heart morphogenesis and cell migration. Altogether, our results demonstrate that the expression of zc3h12a must be tightly controlled during the first cell divisions of zebrafish embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper embryo development.

Diarrheal-associated gut dysbiosis in cancer and inflammatory bowel disease patients is exacerbated by Clostridioides difficile infection.

Introduction: Low diversity gut dysbiosis can take different forms depending on the disease context. In this study, we used shotgun metagenomic sequencing and gas chromatography-mass spectrometry (GC-MS) to compared the metagenomic and metabolomic profiles of Clostridioides (Clostridium) difficile diarrheal cancer and inflammatory bowel disease (IBD) patients and defined the additive effect of C. difficile infection (CDI) on intestinal dysbiosis. Results: The study cohort consisted of 138 case-mix cancer patients, 43 IBD patients, and 45 healthy control individuals. Thirty-three patients were also infected with C. difficile. In the control group, three well-known enterotypes were identified, while the other groups presented with an additional Escherichia-driven enterotype. Bacterial diversity was significantly lower in all groups than in healthy controls, while the highest level of bacterial species richness was observed in cancer patients. Fifty-six bacterial species had abundance levels that differentiated diarrheal patient groups from the control group. Of these species, 52 and 4 (Bacteroides fragilis, Escherichia coli, Klebsiella pneumoniae, and Ruminococcus gnavus) were under-represented and over-represented, respectively, in all diarrheal patient groups. The relative abundances of propionate and butyrate were significantly lower in fecal samples from IBD and CDI patients than in control samples. Isobutyrate, propanate, and butyrate concentrations were lower in cancer, IBD, and CDI samples, respectively. Glycine and valine amino acids were over- represented in diarrheal patients. Conclusion: Our data indicate that different external and internal factors drive comparable profiles of low diversity dysbiosis. While diarrheal-related low diversity dysbiosis may be a consequence of systemic cancer therapy, a similar phenotype is observed in cases of moderate to severe IBD, and in both cases, dysbiosis is exacerbated by incidence of CDI.

Impaired iron recycling from erythrocytes is an early hallmark of aging.

Aging affects iron homeostasis, as evidenced by tissue iron loading and anemia in the elderly. Iron needs in mammals are met primarily by iron recycling from senescent red blood cells (RBCs), a task chiefly accomplished by splenic red pulp macrophages (RPMs) via erythrophagocytosis. Given that RPMs continuously process iron, their cellular functions might be susceptible to age-dependent decline, a possibility that has been unexplored to date. Here, we found that 10- to 11-month-old female mice exhibit iron loading in RPMs, largely attributable to a drop in iron exporter ferroportin, which diminishes their erythrophagocytosis capacity and lysosomal activity. Furthermore, we identified a loss of RPMs during aging, underlain by the combination of proteotoxic stress and iron-dependent cell death resembling ferroptosis. These impairments lead to the retention of senescent hemolytic RBCs in the spleen, and the formation of undegradable iron- and heme-rich extracellular protein aggregates, likely derived from ferroptotic RPMs. We further found that feeding mice an iron-reduced diet alleviates iron accumulation in RPMs, enhances their ability to clear erythrocytes, and reduces damage. Consequently, this diet ameliorates hemolysis of splenic RBCs and reduces the burden of protein aggregates, mildly increasing serum iron availability in aging mice. Taken together, we identified RPM collapse as an early hallmark of aging and demonstrated that dietary iron reduction improves iron turnover efficacy.

MEETING HIGHLIGHTS: THE THIRD MARIE SKLODOWSKA-CURIE SYMPOSIUM ON CANCER RESEARCH AND CARE AT ROSWELL PARK COMPREHENSIVE CANCER CENTER, BUFFALO, NY, SEPTEMBER 20-22, 2023.

Marie Sklodowska-Curie Symposia on Cancer Research and Care (MSCS-CRC) promote collaborations between cancer researchers and care providers in the United States, Canada and Central and Eastern European Countries (CEEC), to accelerate the development of new cancer therapies, advance early detection and prevention, increase cancer awareness, and improve cancer care and the quality of life of patients and their families. The third edition of MSCS-CRC, held at Roswell Park Comprehensive Cancer Center, Buffalo, NY, in September 2023, brought together 137 participants from 20 academic institutions in the US, Poland, Ukraine, Lithuania, Croatia and Hungary, together with 16 biotech and pharma entities. The key areas of collaborative opportunity identified during the meeting are a) creating of a database of available collaborative projects in the areas of early-phase clinical trials, preclinical development, and identification of early biomarkers; b) promoting awareness of cancer risks and efforts at cancer prevention; c) laboratory and clinical training; and d) sharing experience in cost-effective delivery of cancer care and improving the quality of life of cancer patients and their families. Examples of ongoing international collaborations in the above areas were discussed. Participation of the representatives of the Warsaw-based Medical Research Agency, National Cancer Institute (NCI) of the United States, National Cancer Research Institutes of Poland and Lithuania, New York State Empire State Development, Ministry of Health of Ukraine and Translational Research Cancer Center Consortium of 13 cancer centers from the US and Canada, facilitated the discussion of available governmental and non-governmental funding initiatives in the above areas.

Evidence supporting the oncogenic role of BAZ1B in colorectal cancer.

Bromodomain Adjacent to Zinc Finger Domain 1B (BAZ1B) is involved in multiple nuclear processes, and its role in tumorigenesis is emerging. However, the function of BAZ1B in colorectal cancer (CRC) remains largely unexplored. High-density tissue microarrays comprising 100 pairs of matched normal colon and treatment-naïve CRC samples were analyzed by immunohistochemistry with an anti-BAZ1B antibody. The HCT116 and SW480 CRC cell lines were used for overexpression and small hairpin RNA-mediated BAZ1B knockdown models, respectively. Both cell lines were xenografted to immunodeficient NU/J mice to assess tumor burden. The molecular consequences of alterations of BAZ1B expression were assessed by RNA-Seq of xenografts and functional analyses using the Reactome database. Immunohistochemical analysis of BAZ1B showed that BAZ1B staining intensity was higher in 93 tumor specimens and significantly correlated with tumor size (P = 0.03), but not with the presence of KRAS mutation. BAZ1B overexpression significantly increased and its knockdown inhibited the proliferation of HCT116 and SW480 cell lines, respectively. These findings were reproduced when both cell lines were grown as xenografts. RNA-Seq of HCT116 and SW480 xenografts identified 2046 and 99 differentially expressed genes (DEGs) (adjusted P ≤ 0.05), respectively. Functional annotation of DEGs identified already established as well as new molecular processes dependent on BAZ1B protein expression. In conclusion, BAZ1B is overexpressed in CRC tissue and contributes to CRC cell proliferation in vitro and in vivo. The data support the emerging oncogenic role of BAZ1B in cancerogenesis including in CRC.

Activity and rational combinations of a novel, engineered chimeric, TRAIL-based ligand in diffuse large B-cell lymphoma.

Background: TRAIL (TNF-related apoptosis inducing ligand) exhibits selective proapoptotic activity in multiple tumor types, while sparing normal cells. This selectivity makes TRAIL an attractive therapeutic candidate. However, despite encouraging activity in preclinical models, clinical trials with TRAIL mimetics/death receptor agonists demonstrated insufficient activity, largely due to emerging resistance to these agents. Herein, we investigated the cytotoxic activity of a novel, TRAIL-based chimeric protein AD-O51.4 combining TRAIL and VEGFA-derived peptide sequences, in hematological malignancies. We characterize key molecular mechanisms leading to resistance and propose rational pharmacological combinations sensitizing cells to AD-O51.4. Methods: Sensitivity of DLBCL, classical Hodgkin lymphoma, (cHL), Burkitt lymphoma (BL) and acute myeloid leukemia (AML) to AD-O51.4 was assessed in vitro with MTS assay and apoptosis tests (Annexin V/PI staining). Markers of apoptosis were assessed using immunoblotting, flow cytometry or fluorogenic caspase cleavage assays. Resistant cell lines were obtained by incubation with increasing doses of AD-O51.4. Transcriptomic analyses were performed by RNA sequencing. Sensitizing effects of selected pathway modulators (BCL2, dynamin and HDAC inhibitors) were assessed using MTS/apoptosis assays. Results: AD-O51.4 exhibited low-nanomolar cytotoxic activity in DLBCL cells, but not in other lymphoid or AML cell lines. AD-O51.4 induced death-receptor (DR) mediated, caspase-dependent apoptosis in sensitive DLBCL cells, but not in primary resistant cells. The presence of DRs and caspase 8 in cancer cells was crucial for AD-O51.4-induced apoptosis. To understand the potential mechanisms of resistance in an unbiased way, we engineered AD-O51.4-resistant cells and evaluated resistance-associated transcriptomic changes. Resistant cells exhibited changes in the expression of multiple genes and pathways associated with apoptosis, endocytosis and HDAC-dependent epigenetic reprogramming, suggesting potential therapeutic strategies of sensitization to AD-O51.4. In subsequent analyses, we demonstrated that HDAC inhibitors, BCL2 inhibitors and endocytosis/dynamin inhibitors sensitized primary resistant DLBCL cells to AD-O51.4. Conclusions: Taken together, we identified rational pharmacologic strategies sensitizing cells to AD-O51.4, including BCL2, histone deacetylase inhibitors and dynamin modulators. Since AD-O51.4 exhibits favorable pharmacokinetics and an acceptable safety profile, its further clinical development is warranted. Identification of resistance mechanisms in a clinical setting might indicate a personalized pharmacological approach to override the resistance.

The molecular network of the proteasome machinery inhibition response is orchestrated by HSP70, revealing vulnerabilities in cancer cells.

Proteasome machinery is a major proteostasis control system in human cells, actively compensated upon its inhibition. To understand this compensation, we compared global protein landscapes upon the proteasome inhibition with carfilzomib, in normal fibroblasts, cells of multiple myeloma, and cancers of lung, colon, and pancreas. Molecular chaperones, autophagy, and endocytosis-related proteins are the most prominent vulnerabilities in combination with carfilzomib, while targeting of the HSP70 family chaperones HSPA1A/B most specifically sensitizes cancer cells to the proteasome inhibition. This suggests a central role of HSP70 in the suppression of the proteasome downregulation, allowing to identify pathways impinging on HSP70 upon the proteasome inhibition. HSPA1A/B indeed controls proteasome-inhibition-induced autophagy, unfolded protein response, and endocytic flux, and directly chaperones the proteasome machinery. However, it does not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, targeting of NRF1 proves effective in decreasing the viability of cancer cells with the inhibited proteasome and HSP70.

Genetic associations with lymphomas in Polish patients: A pooled-DNA genome-wide association analysis.

Several single nucleotide polymorphisms (SNPs) associated with susceptibility to Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL) have been identified. The aim of this study was to identify susceptibility loci for HL and DLBCL in Polish patients. Altogether, DLBCL (n = 218 and HL patients (n = 224) and healthy individuals (n = 1181) were recruited. Lymphoma diagnosis was based on standard criteria. Genome-wide association study (GWAS) was performed using pooled-DNA samples on llumina Infinium Omni2.5 Exome-8 v1.3, and selected loci were replicated by TaqMan SNP genotyping of individuals. GWAS detected thirteen and seven SNPs associated with DLBCL and HL, respectively. In the replication study, six and seven SNPs reached significance after correction for multiple testing in the DLBCL and HL cohorts, respectively. One and four SNPs associated with DLBCL and HL, respectively, were localized within, and two SNPs-near the major histocompatibility complex (MHC) region. In conclusion, the majority of loci associated with HL and DLBCL aetiology in previous studies have potential roles in immune function. Our pooled-DNA GWAS enabled the identification of several susceptibility loci for DLBCL and HL in the Polish population; some of them were mapped within or adjacent to the MHC, and other associated SNPs were located outside the MHC.

Gene Expression-Based Functional Differences between the Bladder Body and Trigonal Urothelium in Adolescent Female Patients with Micturition Dysfunction.

The aim of this study is to determine the molecular differences between the urothelial transcriptomes of the bladder body and trigone. The transcriptomes of the bladder body and trigonal epithelia were analyzed by massive sequencing of total epithelial RNA. The profiles of urothelial and urinal microbiomes were assessed by amplicon sequencing of bacterial 16S rRNA genes in 17 adolescent females with pain and micturition dysfunction and control female subjects. The RNA sequencing identified 10,261 differentially expressed genes (DEGs) in the urothelia of the bladder body and trigone, with the top 1000 DEGs at these locations annotated to 36 and 77 of the Reactome-related pathways in the bladder body and trigone, respectively. These pathways represented 11 categories enriched in the bladder body urothelium, including extracellular matrix organization, the neuronal system, and 15 categories enriched in the trigonal epithelium, including RHO GTPase effectors, cornified envelope formation, and neutrophil degranulation. Five bacterial taxa in urine differed significantly in patients and healthy adolescent controls. The evaluation of their transcriptomes indicated that the bladder body and trigonal urothelia were functionally different tissues. The molecular differences between the body and trigonal urothelia responsible for clinical symptoms in adolescents with bladder pain syndrome/interstitial cystitis remain unclear.

Oncology Drug Repurposing for Sepsis Treatment.

Sepsis involves life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite three decades of efforts and multiple clinical trials, no treatment, except antibiotics and supportive care, has been approved for this devastating syndrome. Simultaneously, numerous preclinical studies have shown the effectiveness of oncology-indicated drugs in ameliorating sepsis. Here we focus on cataloging these efforts with both oncology-approved and under-development drugs that have been repositioned to treat bacterial-induced sepsis models. In this context, we also envision the exciting prospect for further standard and oncology drug combination testing that could ultimately improve clinical outcomes in sepsis.

Characteristics of the gut microbiome in esports players compared with those in physical education students and professional athletes.

Introduction: Esports is a category of competitive video games that, in many aspects, may be similar to traditional sports; however, the gut microbiota composition of players has not been yet studied. Materials and methods: Here, we investigated the composition and function of the gut microbiota, as well as short chain fatty acids (SCFAs), and amino acids, in a group of 109 well-characterized Polish male esports players. The results were compared with two reference groups: 25 endurance athletes and 36 healthy students of physical education. DNA and metabolites isolated from fecal samples were analyzed using shotgun metagenomic sequencing and mass spectrometry, respectively. Physical activity and nutritional measures were evaluated by questionnaire. Results: Although anthropometric, physical activity and nutritional measures differentiated esports players from students, there were no differences in bacterial diversity, the Bacteroidetes/Firmicutes ratio, the composition of enterotype clusters, metagenome functional content, or SCFA concentrations. However, there were significant differences between esports players and students with respect to nine bacterial species and nine amino acids. By contrast, all of the above-mentioned measures differentiated professional athletes from esports players and students, with 45 bacteria differentiating professional athletes from the former and 31 from the latter. The only species differentiating all three experimental groups was Parabacteroides distasonis, showing the lowest and highest abundance in esports players and athletes, respectively. Conclusion: Our study confirms the marked impact of intense exercise training on gut microbial structure and function. Differences in lifestyle and dietary habits between esports players and physical education students appear to not have a major effect on the gut microbiota.

Loss of epidermal MCPIP1 is associated with aggressive squamous cell carcinoma.

BACKGROUND: Squamous cell carcinoma (SCC) of the skin is a common form of nonmelanoma skin cancer. Monocyte chemotactic protein 1-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase with anti-inflammatory properties. In normal human skin, its expression is predominantly restricted to the suprabasal epidermis. The main aim of this study was to investigate whether MCPIP1 is involved in the pathogenesis of SCC. METHODS: We analyzed the distribution of MCPIP1 in skin biopsies of patients with actinic keratoses (AKs) and SCCs. To explore the mechanisms by which MCPIP1 may modulate tumorigenesis in vivo, we established a mouse model of chemically induced carcinogenesis. RESULTS: Skin expression of MCPIP1 changed during the transformation of precancerous lesions into cutaneous SCC. MCPIP1 immunoreactivity was high in the thickened area of the AK epidermis but was predominantly restricted to keratin pearls in fully developed SCC lesions. Accelerated development of chemically induced skin tumors was observed in mice with loss of epidermal MCPIP1 (Mcpip1eKO). Papillomas that developed in Mcpip1eKO mouse skin were larger and characterized by elevated expression of markers typical of keratinocyte proliferation and tumor angiogenesis. This phenotype was correlated with enhanced expression of IL-6, IL-33 and transforming growth factor-beta (TGF-ß). Moreover, our results demonstrated that in keratinocytes, the RNase MCPIP1 is essential for the negative regulation of genes encoding SCC antigens and matrix metallopeptidase 9. CONCLUSIONS: Overall, our results provide a mechanistic understanding of how MCPIP1 contributes to the development of epidermoid carcinoma.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Inhibition of PIM Kinases in DLBCL Targets MYC Transcriptional Program and Augments the Efficacy of Anti-CD20 Antibodies.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

The gastric microbiota in patients with Crohn's disease; a preliminary study.

The gastric microbiota in Crohn's disease (CD) has not been studied. The purpose of the study was to evaluate differences of stomach microbiota between CD patients and controls. DNA was extracted from gastric mucosal and fluid samples, from 24 CD patients and 19 controls. 16S rRNA gene sequencing identified 1511 operational taxonomic units (OTUs), of which 239 passed the low abundance and low variance filters. All but one CD patients were HP negative. Fifteen bacterial phyla were identified in at least one mucosal or fluid site. Of these, Bacteroidota and Firmicutes accounted for 70% of all phyla. Proteobacteria, Actinobacteriota, and Fusobacteriota combined accounted for 27%. There was significant difference in the relative abundance of Bacteroidota, Proteobacteria, Fusobacteriota, and Campilobacterota between CD patients and controls only in gastric corpus samples. In gastric liquid, there was a significant difference only in Actinobacteriota. Pairwise comparison identified 67 differentially abundant OTUs in at least one site. Of these, 13 were present in more than one comparison, and four differentiating OTUs (Neisseriaceae, Neisseria, Absconditabacteriales, and Microbacteriaceae) were identified at all tested sites. The results reveal significant changes in gastric microbial profiles (beta diversity, phylum, and individual taxa levels) between H. pylori-negative CD patients and controls.