Alterations Of Bcl-2 Research Articles

Anti-Cancer Efficacy of Acacia nilotica Nanoparticles: Apoptosis Induction and Tumor Regression in a DMBA-induced Breast Cancer Rat Model

Background Breast cancer remains a significant global health burden despite advancements in treatment. While conventional therapies often induce adverse effects, there is growing interest in exploring natural alternatives. Acacia nilotica, a traditionally used medicinal plant, has shown promise in cancer management. Purpose This study investigated the therapeutic potential of A. nilotica extract and nanoparticles against 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in albino rats. Methods The formation of A. nilotica crude solution and nanoparticles was done and characterized via dynamic light scattering (DLS) and Fourier-transform infrared spectroscopy (FTIR). A total of 60 female Wistar albino rats were divided into six groups (10 rats/group) and received care in compliance with the state authorities following the Saudi Arabian rules of animal protection. The animals in the first group were given distilled water, while those in the second group were administered DMBA (50 mg/kg). The third and fourth groups were treated with 10 mg/kg of the A. nilotica crude solution and nanoparticles, respectively. In the fifth and sixth groups, A. nilotica crude solution and nanoparticles were administered with 10 mg/kg after DMBA-induced breast cancer in rats, respectively. After sacrificing the rats, the blood and breast tissues were collected from each rat and processed for histological and apoptotic markers analyses. Results Our findings indicated that the average size of nanoparticles was 162.5 nm in diameter with 0.145 as a polydispersity index (PDI). Functional groups were confirmed via FTIR analysis for A. nilotica crude solution and nanoparticles. Histopathological analysis revealed a marked reduction in tumor size and cellular proliferation in nanoparticle-treated groups. Our data demonstrated that both formulations significantly inhibited tumor growth and induced apoptosis, as evidenced by altered Bcl-2 and BAX expression. Conclusion These findings suggest that A. nilotica nanoparticles warrant further investigation as a potential therapeutic strategy for breast cancer.

The BIM deletion polymorphism potentiates the survival of leukemia stem and progenitor cells and impairs response to targeted therapies.

One sixth of human cancers harbor pathogenic germline variants, but few studies have established their functional contribution to cancer outcomes. Here, we developed a humanized mouse model harboring a common East Asian polymorphism, the BIM deletion polymorphism (BDP), which confers resistance to oncogenic kinase inhibitors through generation of non-apoptotic splice isoforms. However, despite its clear role in mediating bulk resistance in patients, the BDP's role in cancer stem and progenitor cells, which initiate disease and possess altered BCL-2 rheostats compared to differentiated tumor cells, remains unknown. To study the role of the BDP in leukemia initiation, we crossed the BDP mouse into a chronic myeloid leukemia (CML) model. We found that the BDP greatly enhanced the fitness of CML cells with a three-fold greater competitive advantage, leading to more aggressive disease. The BDP conferred almost complete resistance to cell death induced by imatinib in CML stem and progenitor cells (LSPCs). Using BH3 profiling, we identified a novel therapeutic vulnerability of BDP LSPCs to MCL-1 antagonists, which we confirmed in primary human LSPCs, and in vivo. Our findings demonstrate the impact of human polymorphisms on the survival of LSPCs and highlight their potential as companion diagnostics for tailored therapies.

DOCK1 deficiency drives placental trophoblast cell dysfunction by influencing inflammation and oxidative stress, hallmarks of preeclampsia.

Preeclampsia (PE) is a globally prevalent obstetric disorder, pathologically characterized by abnormal placental development. Dysfunctions of angiogenesis, vasculogenesis and spiral artery remodeling are demonstrated to be involved in PE pathogenesis; however, the underlying mechanisms remain largely unknown. Here, we investigated the role of the dedicator of cytokinesis 1 (DOCK1), crucial molecule in various cellular processes, in PE progression using HTR-8 cells derived from first-trimester placental extravillous trophoblasts. Our analysis revealed an aberrant DOCK1 expression in the placental villi of PE patients and its impact on essential cellular functions for vascular network formation. A deficiency of DOCK1 in HTR-8 cells impaired the vascular network formation, exacerbated the expression of anti-angiogenic factor ENG, and reduced VEGF levels. Moreover, DOCK1 knockout amplified apoptosis, as indicated by an altered BCL2: BAX ratio and enhanced levels of cleaved PARP. DOCK1 depletion also boosted NF-κB activation and pro-inflammatory cytokine production (IL-6 and TNF-α). Furthermore, the mice treated with DOCK1 inhibitor, TBOPP, exhibited PE-like symptoms. These findings highlight the multifaceted roles of DOCK1 in the pathophysiology of PE, demonstrating that its deficiency can lead to placental dysfunction by orchestrating inflammatory responses and oxidative stress. These insights emphasize the pathogenic role of DOCK1 in PE development and suggest potential treatment strategies that require further exploration. In the graphical abstract, a split image of placental villi contrasts the effects of normal and reduced DOCK1 expression on preeclampsia. The left side illustrates adequate DOCK1 levels supporting healthy trophoblast function and effective spiral artery remodeling. The right side highlights the consequences of DOCK1 deficiency, leading to trophoblast dysfunction and impaired spiral artery remodeling, accompanied by angiogenic imbalance, increased inflammation, oxidative stress, and apoptosis, contributing to placental dysfunction and the development of preeclampsia.

Toxicity assessment of di(2-ethylhexyl) phthalate using zebrafish embryos: Cardiotoxic potential

Plasticizers are considered as newly emerged contaminants. They are added to plastics to increase their flexibility and softness. Phthalate plasticizers including the Di-2-ethylhexyl phthalates (DEHP) are toxic and induce adverse effects on the different organization levels of the environment. In the current study, we investigated the potential toxicity of DEHP using Zebrafish as a biological model. Five ascending concentrations of DEHP were tested in embryos throughout 96 hpf: 0.0086, 0.086, 0.86, 8.6, and 86 mg/L. Embryotoxicity assessments revealed limited lethal effects on DEHP-exposed embryos, yet notable anticipation of the hatching process was observed at 48 hpf. Although DEHP showed negligible influence on the length and pericardial area of exposed embryos, it led to multiple bodily deformities. Gene expression analyses of key cardiogenic and inflammatory genes evidenced alterations in tbx20, bcl2, and il1b expression in Zebrafish embryos at 96 h post-fertilization. Results from the cardiac function analysis displayed that DEHP significantly affected the arterial pulse and linear velocity within the Posterior Cardinal Vein (PCV) of exposed fish. These findings strongly advance that even at low concentrations, DEHP can be considered as potential toxic agent, capable of inducing cardiotoxic effects.

MicroRNA-150-5p-mediated Inhibition of Cell Proliferation, G1/S Transition, and Migration in Bladder Cancer through Targeting NEDD4-binding Protein 2-like 1 Gene.

MicroRNA-150-5p (miR-150-5p) has been implicated in the progression of several cancer types, yet its specific functional role and regulatory mechanisms in bladder cancer (BC) remain largely unexplored. Our study revealed significant downregulation of miR-150-5p and upregulation of NEDD4-binding protein 2-like 1 gene (N4BP2L1) in BC tissues compared to controls using quantitative real-time polymerase chain reaction and western blot analysis, respectively. Reduced miR-150-5p expression correlated with advanced tumor stage and lymph node metastasis, while increased N4BP2L1 levels were associated with larger tumor size by the Chi-square test. Functionally, miR-150-5p exerted significant inhibitory effects on BC cell proliferation, migration, inducing G0/G1 phase arrest, and apoptosis. We confirmed N4BP2L1 as a direct target of miR-150-5p in BC cells using luciferase reporter assay. Crucially, N4BP2L1 knockdown mimicked, while overexpression counteracted the inhibitory impacts of miR-150-5p on BC cell proliferation, migration, and invasion. In addition, N4BP2L1 overexpression reversed miR-150-5p-induced alterations in CDK4, Cyclin D1, Bcl-2, PCNA, Ki-67, N-cadherin, Bad, and E-cadherin levels in BC cells. Based on these results, it can be inferred that the miR-150-5p/N4BP2L1 axis might constitute a promising candidate for therapeutic targeting in the treatment of BC.

From gains to gaps? How Selective Androgen Receptor Modulator (SARM) YK11 impact hippocampal function: In silico, in vivo, and ex vivo perspectives

Selective Androgen Receptor Modulators (SARMs), particularly (17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic-acid-methyl-ester (YK11), are increasingly popular among athletes seeking enhanced performance. Serving as an Androgen Receptor (AR) agonist, YK11 stimulates muscle growth while inhibiting myostatin. Our study delved into the impact of YK11 on the rat hippocampus, analyzing potential alterations in neurochemical mechanisms and investigating its synergistic effects with exercise (EXE), based on the strong relationship between SARM users and regular exercise. Utilizing Physiologically Based Pharmacokinetic (PBPK) modeling, we demonstrated YK11 remarkable brain permeability, with molecular docking analysis revealing YK11 inhibitory effects on 5-alpha-reductase type II (5αR2), suggesting high cell bioavailability. Throughout a 5-week experiment, we divided the animals into the following groups: Control, YK11 (0.35 g/kg), EXE (swimming exercise), and EXE + YK11. Our findings showed that YK11 displayed a high binding affinity with AR in the hippocampus, influencing neurochemical function and modulating aversive memory consolidation, including the downregulation of the BDNF/TrkB/CREB signaling, irrespective of EXE combination. In the hippocampus, YK11 increased pro-inflammatory IL-1β and IL-6 cytokines, while reducing anti-inflammatory IL-10 levels. However, the EXE + YK11 group counteracted IL-6 effects and elevated IL-10. Analysis of apoptotic proteins revealed heightened p38 MAPK activity in response to YK11-induced inflammation, initiating the apoptotic cascade involving Bax/Bcl-2/cleaved caspase-3. Notably, the EXE + YK11 group mitigated alterations in Bcl-2 and cleaved caspase-3 proteins. In conclusion, our findings suggest that YK11, at anabolic doses, significantly alters hippocampal neurochemistry, leading to impairments in memory consolidation. This underscore concerns about the misuse risks of SARMs among athletes and challenges common perceptions of their minimal side effects.

A Two-Stage Transferred Cold Atmospheric Plasma as a Unique Therapeutic Strategy for Targeting Colon Cancer Stem Cells.

The study examines the induction of apoptosis in colon cancer stem cells (CCSCs) within a 3D culture setting, employing an innovative cold atmospheric plasma (CAP) transmission method known as two-stage transferred cold atmospheric plasma (TS-TCAP). TS-TCAP is a partially or fully ionized non-thermal gaseous mixture that comprises photons, charged and neutral particles, and free radicals, which has gained traction in biomedical applications such as cancer therapy. TS-TCAP impacts CCSCs via a continuous, two-step transport process, facilitating the efficient delivery of reactive oxygen and nitrogen species (RONS). The key cellular factors of CCSCs impacted by TS-TCAP treatment, encompassing the secretion and expression levels of IL-6 and IL-8, apoptotic cell count, and expression of BAX, BCL-2, and KI-67 proteins, were evaluated using qrt-ELISA, Annexin V, and qrt-PCR procedures, respectively. The outcomes of CCSCs treatment with TS-TCAP reveal a notable rise in the number of apoptotic cells (P<0.0001), diminished secretion, and gene expression of IL-6 and IL-8 (P<0.0001), accompanied by favorable alterations in BCL-2 and BAX gene expression (P<0.0001). Additionally, a notable decrease in KI-67 expression was observed, correlating with a reduction in CCSCs proliferation (P<0.0001). As well, this study underscores the anti-cancer potential of TS-TCAP, showcasing its efficacy in reducing CCSCs survival rates. However, further pre-clinical and clinical trials are necessary to evaluate CAP's efficacy, safety, and potential synergistic effects with other therapies thoroughly. Overall, TS-TCAP presents a promising alternative for CCSCs treatment, pending further investigation and refinement.

The effects of short-time air pollution, SO2, and ozone on biochemical, histo-pathological, oxidative stress, and carcinogenesis related genes expressions in the liver of the rats.

Air pollution is a universal issue and has significant deleterious effects on both human health and also environment. The important indicators of air pollution include ozone (O3), particulate matter (PM), nitrogen dioxide (NO2), and sulfur dioxide (SO2). This research aims to investigate the impacts of ambient air pollution (AAP), SO2, and O3 on oxidative stress parameters, liver tissue histopathology, and expression of some carcinogenesis-related genes in the hepatic tissue of rats. 32 Wistar rats were randomly allocated to four groups: the control group, the AAP group, the SO2 group (10 ppm), and the ozone group (0.6 ppm). Over a period of five consecutive weeks, the rats were exposed to the specified pollutants for 3 h daily; liver tissues were harvested and instantly fixed with formalin. Pathological changes were assessed in the tissue samples. Additionally, the RT-qPCR technique was utilized to investigate Expression alterations of BAX, p-53, BCL2, caspase-3, caspase-8 and caspase-9. Furthermore, 30 milligrams of hepatic tissues were extracted to assess the activities of oxidative stress enzymes. The liver catalase and MDA activity were elevated in the air pollution (p < .05). Also, liver GPx activity in air pollution and ozone groups was significant in comparison to the control group (p < .05). The SO2 group exhibited severe lesions in histopathology examinations. The findings revealed an alteration in liver histopathology, an induction of oxidative stress, and the expression of some apoptosis-related genes in hepatic tissues after exposure to AAP, SO2, and O3.

The alteration of apoptosis-related genes in female pelvic supportive tissues with regard to menopausal status

PurposeWe aimed to compare the expression levels of anti-apoptotic and proapoptotic genes in the parametrium, sacrouterine and round ligaments with respect to menopausal status in women presenting without any indication of pelvic organ prolapse (POP). We hypothesized that apoptosis related gene expressions in female pelvic tissues may be altered during menopause.MethodsThe study groups consisted of pre-menopausal (n = 10) and menopausal (n = 10) females who did not have POP symptoms. Three different types of tissue samples (Parametrium, Round Ligament and Sacrouterine Ligament) were obtained and RNA was isolated from these tissues. After purifying and quantifying RNA samples, qPCR was used to determine the expression levels of anti-apoptotic and pro-apoptotic genes.ResultsBCL-2 gene expression levels were significantly lower in all the tissues of menopausal patients compared to those of premenopausal patients. In comparison to premenopausal patients, the sacrouterine ligament tissue BAD expression level was significantly high (p = 0.035), and the BCL-2/BAD ratio was significantly lower in menopausal patients (p = 0.006).ConclusionApoptosis-related protein levels change during menopause; pro-apoptotic gene expressions decrease and anti-apoptotic gene expressions increase. The significant alteration of BCL-2 and BAD expression in sacrouterine ligament with respect to menopausal status was observed and this suggested that when compared to other pelvic tissues, the sacrouterine ligament, which plays a crucial role for genital organs in restoring normal pelvic anatomy and providing support, could be affected more by menopause.

Cd70 Deficiency Impairs CD4 + and CD8 + T-Cell Immune Surveillance in Bcl6-Driven Diffuse Large B-Cell Lymphomas

Anti-tumor immune responses to lymphoid malignancies are modulated by co-stimulatory and co-inhibitory pathways. Our group previously characterized the recurrent genetic alterations in primary diffuse large B-cell lymphomas (DLBCL) and identified frequent inactivating mutations or copy loss of the CD70 co-stimulatory ligand in these tumors. CD70 co-stimulation of CD27 + T cells induces antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Given the frequent combination of CD70 and BCL6 alterations in our human DLBCL series, we investigated the consequences of CD70deficiency ( Cd70 -/-) in a murine model of BCL6-driven lymphomagenesis ( Bcl6 tg/+). Cd70 -/-; Bcl6 tg/+ animals developed predominantly splenic DLBCLs with earlier onset and increased penetrance when compared to Bcl6 tg/+ mice (p&amp;lt;.01). Hence, to elucidate potential differences in T-cell mediated anti-tumor responses in Cd70 -/-; Bcl6 tg/+ versus Bcl6 tg/+ mice (and wild-type (WT) and Cd70 -/- animals) and to characterize the concurrent processes of B-cell transformation and immune evasion over time, we performed single-cell RNA sequencing of splenic cell suspensions from animals euthanized for symptoms and additional asymptomatic mice at pre-determined timepoints (6, 14 and 18 months). We applied unsupervised clustering and defined the major B- and T-cell types based on canonical lineage markers. Additionally, we reconstructed the splenic T-cell receptor (TCR) and B-cell receptor (BCR) sequences with the TRUST4 algorithm to assess their diversity and clonal expansion. TRUST4 analyses revealed clonally expanded B cells that were primarily found in specific clusters. These clusters also had an increased number of aberrant cells identified with an orthogonal approach that detects copy number alterations, InferCNV. Certain of these clusters were more abundant in aged asymptomatic Bcl6 tg/+ and Cd70 -/-; Bcl6 tg/+ animals than in symptomatic mice. Aberrant cells in these clusters had a memory-like IgM + phenotype ( Nt5e, Zeb2, Cd38, Cd44, Itgb1) and included two discrete populations: 1) previously described age-associated/autoimmune-like cells ( Tbx21, Cd11c, Cd11b) and 2) newly appreciated atypical memory cells . Additional clusters that were predominantly found in symptomatic animals included aberrant cells with increased expression of cycling genes and downregulation of lineage-defining markers and MHC II (p&amp;lt;.001). We interpret the aberrant memory-like B-cells in asymptomatic Cd70 -/-; Bcl6 tg/+ and Bcl6 tg/+ animals to be precursors of the ones detected in symptomatic mice. Importantly, malignant clonal expansion and splenomegaly occurred at earlier timepoints and to a greater extent in Cd70 -/-; Bcl6 tg/+ than Bcl6 tg/+ mice (p&amp;lt;.01). To further characterize potential anti-tumor immune responses, we examined splenic CD8 + and CD4 + T-cell subsets in tumor-bearing cohorts ( Bcl6 tg/+ and Cd70 -/-; Bcl6 tg/+) and control animals (WT and Cd70 -/-). In the tumor-bearing cohorts, we detected selective expansion of CD8 + cytotoxic T cells (CTLs) with a spectrum of activation and exhaustion markers. CD8 + CTL expansion was significantly greater in Bcl6 tg/+ mice than Cd70 -/-; Bcl6 tg/+ animals. We also identified selective expansion of a major CD4 + CTL compartment ( GzmB, Prf1, Eomes) in the tumor-bearing cohorts that persisted significantly longer in Bcl6 tg/+ than Cd70 -/-; Bcl6 tg/+ mice. In the tumor-bearing cohorts at 18 months, there was significantly greater clonal expansion of CD4 + CTLs, in comparison to CD8 + CTLs, highlighting the likely importance of this understudied cytotoxic CD4 + T-cell subset. In this regard, the downregulation of MHC II on the malignant B cells of symptomatic mice likely favored CD4 + CTL immune escape and accelerated the development of overt lymphoma. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective T-cell mediated immune response to Bcl6 tg/+-driven DLBCL and highlights the unanticipated role of CD4 + CTLs and MHC II down-regulation in the attempted development and subsequent loss of an anti-tumor immune response. Additionally, we identified previously unappreciated precursor populations of murine DLBCL that warrant further investigation in human tumors.

Genetic Alterations of Follicular Lymphoma Can Predict Response to Very Low Dose Radiotherapy

Introduction Follicular lymphoma (FL) is typically indolent, but relapses and transformation to higher grade disease are common. FL is often radiosensitive and can show complete response (CR) even to very low dose radiotherapy (VLDRT, 4Gy). There are no known genetic markers of FL radiosensitivity. We sought to identify molecular signatures of FL radiosensitivity to aid in patient selection for VLDRT. Methods We analyzed our institutional database and identified 110 FL patients with 113 tumors treated with radiotherapy (RT) and preexisting MSK-IMPACT, a targeted exon sequencing panel. For each patient, we collected key clinicodemographic information including pre- and post-RT treatment history, prior large cell lymphoma, and Ann Arbor stage at RT. For treated tumors, we obtained tumor site, grade, diameter, maximum SUV, BCL2 translocation (trans) status through fluorescence in situ hybridization, RT dose and fractionation, and the treatment strategy ( i.e. treating all sites of disease vs a subsite of disease). Tumor site was dichotomized by pelvic tumors vs all others, as this was the most frequently altered site. The presence of mutant (mut) genes compared to wild type (WT) were associated with RT response, measured by PET scan within 6 months of RT, using logistic regression. Log-rank testing and Cox proportional hazards models were used to analyze local progression free survival (LPFS), censored at start of unplanned therapy, with 2-year (2y) survival reported. Multivariate modeling was used to adjust gene associations with outcomes, controlling for the clinicodemographic and tumor characteristics mentioned above. Results The patient and tumor characteristics are summarized in table 1. We found CREBBP to be the most frequently altered gene (66% of tumors). We identified 5 signatures of altered genes, most of which showed altered CREBBP and relatively long LPFS (e.g. one signature showed CREBBP mut, TNFRSF14 mut and IRF8/STAT6 mut with a 2y LPFS 78%); however, one signature with concurrent KMT2D mut and altered BCL2 including trans and mut had significantly shorter LPFS than all other signatures (2y LPFS 47%, p &amp;lt; 0.01) and lower odds of CR relative to the other signatures (p= 0.03). CREBBP mut was the only alteration associated with increased odds of CR (Odds ratio: 2.39 (95% CI: 1.06-5.37, p = 0.04), and this effect remained significant after adjusting for pelvic disease site (p=0.04). Mutations of BCL2, IRF8, and KMT2D were associated with LPFS using log-rank testing (p&amp;lt;0.01, p=0.02, and p=0.04, respectively). BCL2 mut or concurrent BCL2 mut and BCL2 trans (mut/trans) had shorter LPFS for the overall cohort (p&amp;lt;0.01 for both), while for VLDRT, only BCL2 mut/trans was associated with shorter LPFS (p=0.02). In the VLDRT cohort, we found that CREBBP histone acetyltransferase (HAT) mut was associated with improved LPFS compared to WT (n=39, 2y LPFS 74% vs 52%, HR:0.41 (95% CI: 0.18-0.93, p=0.03)), but this was not observed for tumors receiving &amp;gt;4Gy (n=44, HR: 1.27 (95% CI: 0.32-5.08), p=0.74)). We observed that either BCL2 mut, BCL2 trans, or BCL2 mut/trans and CREBBP HAT WT had a 2y LPFS of 13% whereas BCL2 mut, BCL2 trans or BCL2 mut/trans and CREBBP HAT mut had a 2y LPFS of 83% (p&amp;lt;0.01), and this relationship persisted on multivariate analysis (Figure 1). Conclusions Incorporating genetic signatures associated with radiosensitivity, and specifically alterations involving BCL2 and CREBBP, may independently improve patient selection for RT. CREBBP HAT domain mutations are potentially targetable and may have important implications for augmenting radiosensitivity to VLDRT.

Alternative genetic alterations of MYC, BCL2, and/or BCL6 in high-grade B-cell lymphoma (HGBL) and diffuse large B-cell lymphoma (DLBCL): Can we identify different prognostic subgroups?

High-grade B-cell lymphoma (HGBL)/diffuse large B-cell lymphoma (DLBCL) with rearrangements (R) in MYC and BCL2 and/or BCL6 are correlated with poor prognosis. Little is known about the impact of other genetic alterations (gain (G) or amplification (A)) of these genes. The aim of the study was to investigate whether we can identify new prognostic subgroups. Fluorescence in situ hybridization (FISH) results from 169 HGBL/DLBCL were retrospectively categorized into: (1) concurrent MYC-R and BCL2-R and/or BCL6-R-samples with MYC-R and BCL2-R (+/- BCL6-R); n = 21, and HGBL/DLBCL with MYC-R and BCL6-R; n = 11; (2) concurrent R and G/A in MYC and BCL2 and/or BCL6 called "alternative HGBL/DLBCL"-samples with (n = 16) or without (n = 6) BCL2 involvement; (3) BCL2 and/or BCL6 alterations without MYC involvement (n = 35); (4) concurrent G/A in MYC and BCL2 and/or BCL6 without R (n = 25); and (5) "No alterations" (n = 55). Patients with HGBL/DLBCL-MYC/BCL2 and "alternative" HGBL/DLBCL (with BCL2 involvement) had significantly worse survival rates compared to the "no alterations" group. G/A of these genes in the absence of rearrangements did not show any prognostic significance. HGBL/DLBCL with MYC-R and BCL6-R without BCL2 involvement showed a better survival rate compared to HGBL/DLBCL-MYC/BCL2. According to immunohistochemistry, "double/triple" expression (DEL/TEL) did not show a significantly worse outcome compared to absent DEL/TEL. This study highlights the continued value of FISH assessment of MYC, BCL2, and BCL6 in the initial evaluation of HGBL/DLBCL with different survival rates between several genetic subgroups.

Mitigating the adverse effects of Aflatoxin B1 in LMH, IPEC-J2 and 3D4/21cells by a novel integrated agent.

This study was to evaluate the efficacy of TOXO-XL (XL), an integrated mycotoxin-mitigating agent, on aflatoxin B1 (AFB1)-induced damage in Leghorn male hepatoma (LMH), porcine jejunum epithelial cell line (IPEC-J2) and porcine alveolar macrophages (3D4/21) cells, and to explore its potential mechanisms. The results showed that 30% inhibition concentration (IC30) of AFB1 in LMH, IPEC-J2 and 3D4/21cells was 0.5, 15.0, and 2.5mg/L, respectively. Notably, cell viability, ROS, apoptosis and DNA lesion induced by AFB1 (IC30) could be ameliorated by the supplementation with XL at the dosage of 0.025, 0.025 and 0.005%, respectively. Additionally, the migration and phagocytosis abilities impaired by AFB1 were also restored by XL in 3D4/21. Further experiments revealed that XL supplementation markedly attenuated AFB1-induced inflammatory response by decreasing IL-1β, IL-6 and IL-10 in LMH, IL-6 in IPEC-J2 and IL-1β in 3D4/21cells. Meanwhile, XL supplementation reversed the alterations of BAX, BCL-2 and caspase-3 induced by AFB1 in the three cells, suggesting that AFB1-induced apoptosis may be suppressed via the mitochondria-dependent pathway. Furthermore, XL may have a protective effect on the intestinal barrier through the restoration of occludin protein. Conclusively, these findings indicated that XL could alleviate AFB1-induced cytotoxicity in the three cells, potentially through the regulation of cytokines, ROS, apoptotic and DNA damage signaling.

Punica granatum (Pomegranate) Peel Extract Pre-Treatment Alleviates Fenpropathrin-Induced Testicular Injury via Suppression of Oxidative Stress and Inflammation in Adult Male Rats.

Fenpropathrin (FNP) is one of the commonly used insecticides in agriculture and domestically, leading to environmental and health problems. The goal of the current investigation was to determine how well pomegranate peel extract (PGPE) could prevent the testicular toxicity and oxidative stress induced by FNP. Four groups of male Wistar rats were randomly assigned: negative control (corn oil), PGPE (500 mg/kg BW), positive control (FNP; 15 mg/kg BW, 1/15 LD50), and PGPE + FNP. For four weeks, the rats received their doses daily and orally via gavage. The major phytochemical components (total phenolic, flavonoids, and tannins contents) detected in PGPE by GC-MS included ellagic acid, hydroxymethylfurfurole, guanosine, and pyrogallol with high total phenolic, flavonoids, and tannin contents. FNP-treated rats showed a marked elevation in testicular levels of thiobarbituric acid-reactive substances, hydrogen peroxide, and protein carbonyl content, as well as the activity of aminotransferases and phosphatases. Meanwhile. a significant decline in body weight, gonadosomatic index, glutathione, protein contents, enzymatic antioxidants, and hydroxysteroid dehydrogenase (3β HSD, and 17β HSD) activity was observed. In addition, significant alterations in testicular P53, Cas-3, Bcl-2, IL-β, IL-10, testosterone, follicle-stimulating and luteinizing hormones, and sperm quality were detected. Furthermore, biochemical and molecular changes were corroborated testicular histological abnormalities. Moreover, PGPE-pretreated FNP-intoxicated rats demonstrated considerable improvement in the majority of the studied parameters, when compared to FNP-treated groups. Conclusively, PGPE provided a potent protective effect against the testicular toxicity caused by FNP, due to its antioxidant-active components.

Molecular classification and identification of an aggressive signature in low-grade B-cell lymphomas.

Non-follicular low-grade B-cell lymphomas (LGBCL) are biologically diverse entities that share clinical and histologic features that make definitive pathologic categorization challenging. While most patients with LGBCL have an indolent course, some experience aggressive disease, highlighting additional heterogeneity across these subtypes. To investigate the potential for shared biology across subtypes, we performed RNA sequencing and applied machine learning approaches that identified five clusters of patients that grouped independently of subtype. One cluster was characterized by inferior outcome, upregulation of cell cycle genes, and increased tumor immune cell content. Integration of whole exome sequencing identified novel LGBCL mutations and enrichment of TNFAIP3 and BCL2 alterations in the poor survival cluster. Building on this, we further refined a transcriptomic signature associated with early clinical failure in two independent cohorts. Taken together, this study identifies unique clusters of LGBCL defined by novel gene expression signatures and immune profiles associated with outcome across diagnostic subtypes.

The pathobiology of follicular lymphoma

Follicular lymphoma is one of the most frequent lymphomas. Histologically, it is characterized by a follicular (nodular) growth pattern of centrocytes and centroblasts; mixed with variable immune microenvironment cells. Clinically, it is characterized by diffuse lymphadenopathy, bone marrow involvement, and splenomegaly. It is biologically and clinically heterogeneous. In most patients it is indolent, but others have a more aggressive evolution with relapses; and transformation to diffuse large B-cell lymphoma. Tumorigenesis includes an asymptomatic preclinical phase in which premalignant B-lymphocytes with the t(14;18) chromosomal translocation acquire additional genetic alterations in the germinal centers, and clonal evolution occurs, although not all the cells progress to the tumor stage. This manuscript reviews the pathobiology and clinicopathological characteristics of follicular lymphoma. It includes a description of the physiology of the germinal center, the genetic alterations of BCL2 and BCL6, the mutational profile, the immune checkpoint, precision medicine, and highlights in the lymphoma classification. In addition, a comment and review on artificial intelligence and machine (deep) learning are made.

Genetic Profiling of Diffuse Large B-Cell Lymphoma: A Comparison Between Double-Expressor Lymphoma and Non-Double-Expressor Lymphoma.

Data are limited regarding the genetic profiling of diffuse large B-cell lymphoma (DLBCL) with double expression of MYC and BCL2 proteins without underlying rearrangements (double-expressor lymphoma [DEL]). This study aimed to describe the genetic profiling and determine the prognostic significance in patients with DEL and in those with non-DEL. Capture-based targeted sequencing was performed on 244 patients with de novo DLBCL, not otherwise specified. Immunohistochemistry staining was performed for evaluating the MYC and BCL2 expression. Among 244 patients, 46 patients had DEL, and 198 had non-DEL. KMT2D, CD58, EP300, PRDM1, TNFAIP3 and BCL2 gain or amplification (BCL2GA/AMP) were significantly more frequently altered in the DEL group. Alterations in the BCR/TLR (p = 0.021), B-cell development and differentiation (p = 0.004), and NF-κB (p = 0.034) pathways occurred more frequently in patients with DEL. Thirty-seven DEL patients and 132 non-DEL patients were included for survival analyses. DEL was not significantly associated with progression-free survival (PFS) (p = 0.60) and overall survival (OS) (p = 0.49). In DEL patients, after adjusting for the International Prognostic Index, BCL2 alteration (HR 2.516, 95%CI 1.027-6.161; p = 0.044) remained an independent predictor of inferior PFS. BCL2GA/AMP also predicted poor PFS, but with marginal statistical significance (HR 2.489, 95%CI 0.995-6.224; p = 0.051). There was difference in profiling of altered genes and signaling pathways between the DEL group and the non-DEL group. The presence of DEL alone should not be considered as an adverse prognostic indicator, and BCL2 alteration could define a subset of patients with poor prognosis within DEL.

Molecular Landscape of Primary Refractory DLBCL

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma and can be cured with frontline immunochemotherapy (IC, e.g., R-CHOP or similar) in approximately 60% of patients. DLBCL that is progressive during frontline treatment or fails to achieve a complete response (CR) by the end of treatment (EOT) is referred to as primary refractory DLBCL (prDLBCL) and has a poor prognosis. It is not possible to identify such patients at initial diagnosis. Molecular predictors are highly needed. Large scale genomic profiling has led to a broader understanding of the molecular drivers in newly diagnosed DLBCL (ndDLBCL) and have identified unique molecular subtypes. However, no classification clearly identifies prDLBCL, in part due to the small number of cases analyzed to date. This study examined the genomic landscape of prDLBCL and compared it to that of non-primary refractory DLBCL (non-prDLBCL). Methods 444 patients with ndDLBCL between 2002 and 2015 and enrolled in the Molecular Epidemiology Resource (MER) of the Mayo/Iowa Lymphoma SPORE were included. RNA and DNA were isolated from FFPE tumor samples at diagnosis and evaluated for whole exome sequencing (WES, n=404) and RNA sequencing (RNAseq, n=321). WES and RNAseq data were processed using the Bristol Myers Squibb (BMS) or Mayo in house standard pipelines, and copy number alterations (CNAs) were identified by OncoScan or WES using Nexus copy number software. PrDLBCL was defined as less than a CR by EOT and all patients received complete treatment with frontline IC. Available data for prDLBCL included WES (N=24), RNAseq (N=21), and CNAs (N=23). All statistical comparisons made were between prDLBCL and non-prDLBCL (WES N=380, RNAseq N=300, CNA N=364). Pathway analysis was performed using pathfindR. Genetic classification was performed using LymphGen and HMRN. The tumor microenvironment (TME) was analyzed using Lymphoma Microenvironment Classification (LME) and Lymphoma EcoTyper. Results Primary refractory DLBCL accounted for 6% (N=24) of ndDLBCL cases. Cell of origin was available on 22 cases, 10 (45%) were GCB, 9 (41%) were ABC, and 3 (14%) were unclassified. MYC and BCL2 and/or BCL6 rearrangements (DH/THL) were available on 16 cases, 2 (12.5%) were DH/THL and 14 (87.5%) of cases were negative (N=14). TP53 (38%, N=9/24), KMT2D (29%, N=7/24) and IGLL5 (25%, N=6/24) were the most frequent mutations in prDLBCL (Figure 1). TP53 mutations were more frequent in the prDLBCL cohort compared to non-prDLBCL cohort (15.5%, Odds ratio (OR) 3.25, CI 1.1-8.3, p=0.01). Deletions at 17p13.1 (TP53) were the most frequent copy number loss alteration, detected in 48% (N=11/23) of prDLBCL cases, compared to 24% (N=89/364) of ndDLBCL cases (OR 2.9, CI 1.1-7.2, P=0.01). Together, TP53 mutation and/or deletion was observed in 65% of prDLBCL cases (N=15/23). There was an increased frequency of gains/amplifications at 8q24.21 (MYC, OR 2.3, CI 0.82-5.9, P=0.09) and 9p24.1 (PDL1, OR 2.9, CI 1.0-7.6, P=0.03) in prDLBCL. Using the LymphGen classification, only 2 (8%) of prDLBCL cases were in the A53 subtype (hallmarked by TP53 alterations), despite a high frequency of TP53 alterations. The EZB subtype (hallmarked by EZH2 and BCL2 alterations) was the most frequent (N=7, 30% of prDLBCL cases). To identify transcriptomic signatures of prDLBCL, differential gene expression analysis was performed comparing prDLBCL to non-prDLBCL followed by pathway analysis. This analysis identified 396 upregulated and 252 downregulation genes (P<0.01). PathfindR analysis identified activation of metabolic, inflammatory, and MAPK pathways. Analysis of the TME using Lymphoma EcoTyper and LME found no dicernable differences between groups. Conclusion: DLBCL patients with primary refractory disease represent a high-risk group of patients with poor survival outcomes. While our sample size is small, we found a high frequency of TP53 alterations (mutation and deletion) and MYC copy number gain in prDLBCL cases. These findings suggest TP53 and MYC alterations may mediate primary treatment resistance and could be two important mechanisms contributing to the poor outcomes. Molecular profiling at the time of diagnosis is not yet able to identify patients at high risk of frontline treatment failure, however, these results contribute to our understanding of the genomic landscape of prDLBCL. Figure 1: Oncoplot of Mutations and Copy Number Alterations in prDLBCL Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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Abstract A39: Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL)

Abstract Interaction between CLL and its microenvironment is a major factor in drug resistance. Bone marrow derived stroma cells provide a protective and supportive niche to allow CLL cells to survive drug-induced apoptosis. Therefore, a current challenge is to develop new strategies to target the microenvironment. To understand the role of tumor microenvironment in CLL survival, we studied conditioned media (CM) derived from NKTert cells. Human NKTert stroma cells provide a survival advantage to primary CLL cells by protecting them from spontaneous apoptosis in ex-vivo culture. We identified three cytokines (IL6, IL8 and CCL2) from CM as candidates for mediating survival. We exposed CLL cells to neutralizing antibodies against each cytokine and found significant decrease in CLL viability in presence of anti-CCL2 suggesting its role in CLL survival. BH3 profiling revealed an increase in apoptotic priming with anti-CCL2 treatment as well. Using dynamic BH3 profiling (DBP), we found that anti-CCL2 increased dependence on BCL-2 as well as BCL-xL in primary CLL cells suggesting a potential role of CCL2 on CLL survival. To our surprise, we also identified a CLL group (CM independent, CMI) that were resilient to cell death in the absence of stroma support in ex-vivo culture. In addition to this, anti-CCL2 antibody showed no effect on their survival. Next, we exposed CMI CLL cells to venetoclax and found that these CLL cells were less sensitive to apoptosis compared to the CLL samples that are stroma dependent (CM dependent, CMD). BH3 profiling showed that CMI CLL cells are less primed for apoptosis. Consistent with our findings, DBP with anti-CCL2, venetoclax or the combination didn’t increase any drug induced apoptotic priming in CMI CLL samples. In our study, 80% of CMI harbor unmutated IgHV. To provide additional insight to the molecular mechanism, we performed western blot analyses for BCL-2 family proteins and found decreased BAX ad BCL-2 expression in CMI samples. This result suggests altered BCL-2 family proteins expression might be a mechanism for differential priming. Bulk RNA seq analysis revealed upregulation of focal adhesion genes and Ras signaling pathway in CMI samples. In summary, our study suggest that in the CMD subset of CLL, anti-CCL2 therapy enhances apoptotic priming and venetoclax efficacy. Furthermore, we have also functionally defined two biologically distinct CLL subgroups based on stroma dependence. Citation Format: Salma Parvin, Aditi Aryal, Shanye Yin, Matthew S. Davids, Geoffrey G. Fell, Catherine J. Wu, Anthony Letai. Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A39.