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Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy worldwide. Molecular classifications have tried to improve cure rates. We prospectively examined and correlated the mutational landscape with the clinical features and outcomes of 185 Mexican patients (median age 59.3 years, 50% women) with newly diagnosed DLBCL. A customized panel of 79 genes was designed, based on previous international series. Most patients had ECOG performance status (PS) < 2 (69.2%), advanced-stage disease (72.4%), germinal-center phenotype (68.1%), and double-hit lymphomas (14.1%). One hundred and ten (59.5%) patients had at least one gene with driver mutations. The most common mutated genes were as follows: TP53 , EZH2 , CREBBP , NOTCH1 , and KMT2D . The median follow-up was 42 months, and the 5-year relapse-free survival (RFS) and overall survival (OS) rates were 70% and 72%, respectively. In the multivariate analysis, both age > 50 years and ECOG PS > 2 were significantly associated with a worse OS. Our investigation did not reveal any discernible correlation between the presence of a specific mutation and survival. In conclusion, using a customized panel, we characterized the mutational landscape of a large cohort of Mexican DLBCL patients. These results need to be confirmed in further studies.

Background: Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown., Methods: We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional in vitro experiments., Results: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation., Conclusion: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors., (© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

We have previously shown that the transmembrane protein ODZ1 promotes cytoskeletal remodeling of glioblastoma (GBM) cells and invasion of the surrounding parenchyma through the activation of a RhoA-ROCK pathway. We also described that GBM cells can control the expression of ODZ1 through transcriptional mechanisms triggered by the binding of IL-6 to its receptor and a hypoxic environment. Epidermal growth factor (EGF) plays a key role in the invasive capacity of GBM. However, the molecular mechanisms that enable tumor cells to acquire the morphological changes to migrate out from the tumor core have not been fully characterized. Here, we show that EGF is able to induce the expression of ODZ1 in primary GBM cells. We analyzed the levels of the EGF receptor (EGFR) in 20 GBM primary cell lines and found expression in 19 of them by flow cytometry. We selected two cell lines that do or do not express the EGFR and found that EGFR-expressing cells responded to the EGF ligand by increasing ODZ1 at the mRNA and protein levels. Moreover, blockade of EGF-EGFR binding by Cetuximab, inhibition of the p38 MAPK pathway, or Additionally, the siRNA-mediated knockdown of MAPK11 (p38β MAPK) reduced the induction of ODZ1 in response to EGF. Overall, we show that EGF may activate an EGFR-mediated signaling pathway through p38β MAPK, to upregulate the invasion factor ODZ1, which may initiate morphological changes for tumor cells to invade the surrounding parenchyma. These data identify a new candidate of the EGF-EGFR pathway for novel therapeutic approaches.

Background: Glioblastoma (GBM) remains a major clinical challenge due to its invasive capacity, resistance to treatment, and recurrence. We have previously shown that ODZ1 contributes to glioblastoma invasion and that ODZ1 mRNA levels can be upregulated by epigenetic mechanisms in response to hypoxia. Herein, we have further studied the transcriptional regulation of ODZ1 in GBM stem cells (GSCs) under hypoxic conditions and analyzed whether HIF2α has any role in this regulation. Methods: We performed the experiments in three primary GSC cell lines established from tumor specimens. GSCs were cultured under hypoxia, treated with HIF regulators (DMOG, chetomin), or transfected with specific siRNAs, and the expression levels of ODZ1 and HIF2α were analyzed. In addition, the response of the ODZ1 promoter cloned into a luciferase reporter plasmid to the activation of HIF was also studied. Results: The upregulation of both mRNA and protein levels of HIF2α under hypoxia conditions correlated with the expression of ODZ1 mRNA. Moreover, the knockdown of HIF2α by siRNAs downregulated the expression of ODZ1. We found, in the ODZ1 promoter, a HIF consensus binding site (GCGTG) 1358 bp from the transcription start site (TSS) and a HIF-like site (CCGTG) 826 bp from the TSS. Luciferase assays revealed that the stabilization of HIF by DMOG resulted in the increased activity of the ODZ1 promoter. Conclusions: Our data indicate that the HIF2α-mediated upregulation of ODZ1 helps strengthen the transcriptional control of this migration factor under hypoxia in glioblastoma stem cells. The discovery of this novel transcriptional pathway identifies new targets to develop strategies that may avoid GBM tumor invasion and recurrence.

We have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1., (© 2021. The Author(s).)

Myelodysplastic syndrome with deletion of chromosome 5q (5q-syndrome) has a favorable prognosis and a low risk of transformation to acute myeloid leukemia, when treated with lenalidomide. Azacitidine leads to complete remission even as second-line therapy and in patients with clonal evolution. We report a 70 years old female without previous exposure to myelotoxic drugs, presenting with three weeks with fatigue and dyspnea. She had anemia with normal white blood cell and platelet count. Bone marrow biopsy showed 50% cellularity and the karyotype analysis revealed a (5) (q33q34) deletion in 22% of the metaphases. A diagnosis of 5q-syndrome with low risk calculated using the Revised International Prognostic Scoring System (IPSS-R), was made. Since lenalidomide was not affordable, thalidomide 100 mg/day was initiated, achieving transfusion independence for three years. Afterwards, she developed pancytopenia and a bone marrow biopsy showed erythroid and megakaryocyte dysplasia with a complex karyotype, which worsened prognosis (IPSS-R of five points). Therefore, azacitidine (by donation) was administered. She achieved complete remission with a normal karyotype and completed 12 cycles of treatment. Thereafter, she relapsed and received only supportive care for a year. She suffered an ischemic stroke and died two weeks later.

We have previously described that the NFκB pathway is upregulated during differentiation of glioblastoma stem-like cells (GSCs) which keeps differentiating GSCs in a proliferative astrocytic precursor state. However, extracellular signals and cellular mediators of this pathway are not clear yet. Here, we show that TLR4 is a key factor to promote NFκB activation in differentiating GSCs. TLR4 is upregulated during differentiation of GSCs and promotes transcriptional activation of NFκB as determined by luciferase-reporter assays and expression of NFκB target genes. Downregulation of TLR4 by shRNAs or blockade with anti-TLR4 specific antibodies drastically inhibited NFκB activity which promoted further differentiation and reduced proliferation of GSCs. We found that hyaluronic acid (HA), a main component of brain extracellular matrix, triggers the TLR4-NFκB pathway in differentiating GSCs. Moreover, HA is synthesized and released by GSCs undergoing differentiation and leads to transcriptional activation of NFκB, which is inhibited following downregulation of TLR4 or blockade of HA synthesis. Thus, we have demonstrated that during the process of differentiation, GSCs upregulate TLR4 and release the TLR4 ligand HA, which activates the TLR4-NFκB signaling pathway. This strategy may efficiently be used by differentiating GSCs to maintain their proliferative potential and consequently their tumorigenic capacity.

N-acetyltransferase 2 (NAT2) catalyzes the bioactivation and/or detoxification of drugs and carcinogens. The aim of this study was to establish the correlation between the NAT2 genotype and the acetylating phenotype in a Mexican population using sulfamethazine as a probe. From a total of 122 individuals, 73 (59.8%) were slow and 49 (40.2%) were fast acetylators. Eleven individuals (9%) had the wild-type genotype (NAT2*4/NAT2*4). The most frequent genotype was NAT2*4/NAT2*5B observed in 20.66% of individuals. In conclusion, our results show that an accurate prediction of the acetylation phenotype by genotyping can be achieved in around half of the population. Further studies with a larger number of individuals are required to establish correlations between phenotype and genotype in half of that patients having a genotype combined with slow/rapid alleles.

Background: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF., Principal Findings: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53., Conclusions/significance: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts.

NLRP2 has been shown to inhibit the NF-kappaB signaling pathway, and thus may contribute to modulate the inflammatory response, where NF-kappaB plays a major role. In this study, we report that expression of NLRP2 is induced upon differentiation of CD34+ hemopoietic progenitors into granulocyte or monocyte/macrophages. We also found that NLRP2 was up-regulated following differentiation of mesenchymal stem cells toward adipocytes. Notably, stimulation of HEK293T cells with TNF-alpha or overexpression of the p65 subunit of NF-kappaB resulted in up-regulation of NLRP2 and the formation of NF-kappaB-NLRP2 promoter complexes. Moreover, ectopic expression of p65 but not of other transcriptional regulators induced transactivation of the NLRP2 promoter. Thus, NLRP2 may control NF-kappaB activation through a regulatory loop. Nucleotide changes within the NACHT domain of other NLRP proteins have been associated with hereditary fever syndromes and chronic inflammatory diseases. We identified five single nucleotide polymorphisms present in the NACHT domain of NLRP2 by sequencing genomic DNA from 319 healthy controls. The frequencies of the rare alleles varied between 0.2 and 10%. Of note, one of these variants, I352S was unable to block the transcriptional activity of NF-kappaB and the formation of NF-kappaB-DNA-binding complexes following stimulation with TNF-alpha. Overall, our findings provide molecular insight into the expression of NLRP2 by NF-kappaB and suggest that a polymorphism within the NACHT domain of NLRP2 may contribute to the amplification of inflammatory responses due to a reduction of inhibitory signals on the NF-kappaB pathway.

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-kappaB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-kappaB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-kappaB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-alpha and the p65 subunit of NF-kappaB. Moreover, TNF-alpha and overexpression of p65 induced the formation of NF-kappaB-CARD8 promoter complexes. Thus, CARD8 may control NF-kappaB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-kappaB transcriptional activity, and abrogates the binding of NF-kappaB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-kappaB target genes (cIAP, A1), in response to TNF-alpha. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-kappaB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

BH3-only proteins are required for execution of apoptotic cell death. We have found that one of these proteins, Bik, is strongly induced in cancer cells treated with chemotherapeutic agents. Furthermore, we showed that chemotherapy-induced expression of bik is independent of p53. Consistent with its pro-apoptotic activity, blockade of bik expression reduces the adriamycin-mediated apoptotic cell death. We also found that the bik gene is transcriptionally activated by E2F proteins. Consistently, adriamycin induces the E2F-bik pathway. In addition, E2Fs transactivate bik by a p53-independent mechanism. Thus, our data indicate that transcriptional regulation of bik contributes to the efficient apoptotic response to chemotherapeutic agents.

Ipaf has been associated with apoptosis, cytokine processing, and nuclear factor-kappaB activation. Here, we describe that Ipaf is highly expressed in myelomonocytic cells and that the mRNA levels of Ipaf progressively increase during differentiation of CD34(+) progenitors to granulocytes and monocytes. Additionally, treatment with tumor necrosis factor-alpha and exposure to UV radiation induced the transcriptional activation of Ipaf in human leukemia HL-60 cells. Thus, Ipaf may contribute to modulate the response of myeloid cells to genotoxic and pro-inflammatory stimuli.

Background: Biochemical research on the etiopathogenesis of affective disorders has focused on transduction mechanisms beyond receptors, such as adenylate cyclase activity., Methods: Adenylate cyclase activity (AC) was measured in postmortem frontal cortex samples from 11 suicide victims with a firm antemortem diagnosis of major depressive disorder and 11 matched control cases. We analyzed the basal activity of the enzyme and that following stimulation with forskolin, guanine nucleotides, and the beta(1)-adrenoceptor agonist xamoterol., Results: A significant negative correlation between the period of tissue storage and the response of AC to the different stimuli assayed was observed. No difference was found in the levels of basal, forskolin-, and GTPgammaS-stimulated activity between control and major depressive disorder cases, both in the drug-free and the drug-treated subgroups. In contrast, we found a significant lower response to beta(1)-adrenoceptors agonist-stimulated AC activity in the major depressive disorder group (p <.01). This pattern of reduced response was also found in the subgroup of patients with negative toxicology for antidepressants., Conclusions: These results, directly obtained from the brain of depressed patients, reinforce the involvement of noradrenergic neurotransmission in depressive illness. They also support the relevance of cyclic adenosine monophosphate signaling pathways in the etiopathogenesis of affective disorders.

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.

Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-kappaB, a transcription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn's disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34(+) hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14(+) (monocytes), CD15(+) (granulocytes), and CD40(+)/CD86(+) (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNFalpha resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-kappaB binding element that was required for transcriptional activity in response to TNFalpha. Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative IkappaBalpha blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNFalpha or lipopolysaccharide, both p50 and p65 subunits of NF-kappaB were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-kappaB, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.