BACKGROUND/OBJECTIVES:Relapse remains the leading cause of treatment failure in B-cell acute lymphoblastic leukaemia (B-ALL), highlighting the need for biomarkers and therapeutic targets that limit leukaemic dissemination. We investigated whether histone deacetylase 6 (HDAC6), a regulator of cortactin-dependent actin remodelling, contributes to B-ALL progression and relapse. SUBJECTS/METHODS:HDAC6 expression was analysed in cohorts of 72 paediatric and 54 adult patients with B-ALL and validated in B-ALL cell lines. The functional role of HDAC6 was assessed using pharmacological inhibition and shRNA-mediated knockdown in assays of CXCL12-induced actin remodelling, transendothelial migration, bone marrow colonisation and leukaemic dissemination in vivo. Cortactin-depleted cells were used to determine pathway dependence. RESULTS:HDAC6 expression was elevated in B-ALL cell lines and patient samples and was significantly associated with relapse in both patient cohorts. Pharmacological inhibition or genetic depletion of HDAC6 impaired CXCL12-induced actin remodelling, transendothelial migration, bone marrow colonisation and leukaemic dissemination. HDAC6 inhibition failed to further reduce migration in cortactin-deficient cells, indicating that HDAC6 promotes leukaemic motility through cortactin. CONCLUSIONS:HDAC6 is a clinically relevant biomarker associated with B-ALL relapse and a critical regulator of cortactin-dependent leukaemic dissemination. Targeting HDAC6 represents a promising therapeutic strategy to limit tissue invasion and reduce relapse.

Neutrophils are crucial to innate immune responses to microbes. The engagement of opsonized pathogens by Fc gamma receptors (FcγRs) on neutrophil surfaces mediates multiple antimicrobial functions, including phagocytosis and the production of reactive oxygen species (ROS). FcγRIIIb (CD16b) is the most abundant FcγR on human neutrophils. This GPI-anchored receptor lacks an intracellular domain. The mechanisms by which FcγRIIIb transduce signals remain unclear. A FcγRIIIb-deficient phenotype has been reported in apparently healthy adults, which is intriguing given the abundance of this receptor on neutrophil surfaces and its crucial role in neutrophil activation by immune complexes. Here, we identified 2 healthy adult brothers lacking FcγRIIIb on neutrophils and characterized their neutrophil activation through FcγR engagement by opsonized Escherichia coli. Sequencing of the FCGR3B gene revealed mutations in exon 2 resulting in translation loss. In the absence of stimulation, FcγRIIIbnull neutrophils showed unaltered levels of FcγRIIa, TLR-2, TLR-4 and TLR-6, but significantly higher FcγRIIIa and FcγRIa compared to FcγRIIIb-expressing controls. Upon challenge with opsonized E. coli, increased surface expression of FcγRIa, TLR-4, and αM integrin (CD11b) was observed exclusively in FcγRIIIbnull neutrophils. Antibacterial functions stimulated by opsonized E. coli were significantly lower in FcγRIIIbnull neutrophils, including phagocytic capacity and ROS production compared to FcγRIIIb-expressing neutrophils. Overall, the absence of FcγRIIIb on human neutrophils correlated with impaired antimicrobial functions following stimulation through FcγRs. This study provides new insights into the functional relevance of FcγRIIIb and emphasizes the importance of this receptor in neutrophil responses to bacteria.

Background/Objectives: B-cell precursor acute lymphoblastic leukemia (B-ALL), the most common pediatric acute leukemia (AL), is frequently characterized by aberrant antigen expression, which aids diagnosis and prognosis. The myeloid antigen CD66c is notably frequent in B-ALL and has been proposed as a marker of disease aggressiveness and treatment response. Evaluating CD66c in Mexican pediatric patients may provide insights into disease biology. Methods: A cohort of 128 pediatric patients was referred to the Laboratory of Oncoimmunology and Cytomics of Childhood Cancer (OCL) at Instituto Mexicano del Seguro Social (IMSS) for immunophenotyping tests between March 2022 and November 2023. Additionally, control bone marrow (BM) samples were assessed. Aberrant antigen expression in hematopoietic populations and BM microenvironment stroma phenotyping were performed. Results: In total, 84.38% of B-ALL patients exhibited aberrant expression of ≥1 myeloid antigen. Among CD66c-positive patients, 13.79% had detectable measurable residual disease (MRD) during follow-up and 20.69% died. Mesenchymal stromal cells (MSCs) from patients with positive or low CD66c expression displayed inflammatory profiles. ProB leukemias with low CD66c expression were more likely to exhibit detectable MRD, increased mortality, and reduced survival. Conclusions: Low CD66c expression induces molecular stealth that could favor immune evasion and niche persistence, thereby increasing the risk of relapse and therapeutic failure.

The adhesive interactions of neutrophils with postcapillary venules during inflammation have been well studied. However, how neutrophils trigger molecular changes in endothelial cells (EC) during their extravasation requires further exploration. The endothelial actin-binding protein cortactin regulates endothelial contacts and neutrophil-endothelial interactions, but the associated mechanisms remain elusive. Hypothesizing that endothelial cortactin dynamics change during inflammation, using super-resolution confocal microscopy of inflamed mouse cremasteric venules and HUVEC, we report that neutrophil interaction with EC induces reduction in EC cortactin levels. This response was specifically mediated by neutrophil serine proteases, including cathepsin G, that were detected inside EC. The observed cortactin degradation was abolished after inhibition of serine proteases or blockade of neutrophil exocytosis. Finally, the endogenous serine protease inhibitor α1-antitrypsin suppressed cortactin degradation in vivo and reduced neutrophil adhesion and extravasation. Collectively, our data unveil a new mechanism by which neutrophils manipulate proteins inside EC to facilitate their extravasation.

B-cell acute lymphoblastic leukemia (B-ALL) remains the most common pediatric cancer, and relapse continues to be a major cause of treatment failure. Although the bone marrow microenvironment is known to promote leukemic persistence and dissemination, the mechanisms mediating these effects remain poorly defined. Here, we identify activin A as a microenvironment-derived driver of B-ALL progression and relapse. Activin A and its receptors were significantly overexpressed in pediatric and adolescent B-ALL at diagnosis and relapse. Functionally, activin A enhanced leukemic cell migration, adhesion, transendothelial migration, and bone marrow organoid colonization by promoting actin polymerization. We previously demonstrated that the actin-binding protein cortactin is a key regulator of B-ALL dissemination and relapse. Consistent with this role, pharmacological inhibition of activin A signaling suppressed transendothelial migration and organoid colonization in leukemic cells expressing high levels of cortactin and activin A receptors, whereas cells with low cortactin expression were largely unresponsive. Moreover, cortactin depletion abolished activin A-induced migration, transendothelial migration, and organoid colonization. Inhibition of ERK1/2, a major cortactin-activating kinase, similarly blocked activin A-dependent responses. Together, these findings identify a previously unrecognized activin A–ERK1/2–cortactin signaling axis that drives B-ALL dissemination and represents a therapeutic vulnerability for preventing microenvironment-driven relapse.

The endothelial barrier is a semipermeable cell layer covering the inside of blood vessels that regulates the flux of ions, macromolecules, and plasma from blood to tissues. Inflammation promotes an increase in vascular permeability, which can contribute to disease if not controlled properly. Thus, it is important to understand in detail the molecular mechanisms underlying inflammatory vascular hyperpermeability. While endothelial permeability can be measured in vitro, these assays do not recapitulate precisely the in vivo vasculature. Thus, in vivo assays are required to understand the full picture of vascular permeability regulation. Here, we describe an established assay that involves injection of Evans blue dye followed by intradermal injection of agents inducing vascular permeability. This assay is relatively easy to perform and provides reliable data on permeability regulation in vivo. Key features • Step-by-step protocol to study vascular permeability in the mouse skin. • Injection of Evans blue dye followed by intradermal injection of permeability-inducing agents allows reproducible analysis of regulatory mechanisms. • This protocol allows the analysis of different substances in the same animal. • Possibility of different dye administration routes that can be compared.

Kv1.3 is unique functional voltage-dependent K+ channel in normal and leukemic human lymphocytes. Here, we show that cell lines and primary cells of acute lymphoblastic leukemia (B-ALL) express Kv1.3 protein at high levels and that it is located at the plasma membrane. Surprisingly, and in contrast to healthy B cells, B-ALL cells lack Kv1.3 plasma membrane currents. However, B-ALL cell lines and primary B-ALL patient samples displayed a robust Ca2+-dependent KCa3.1 current, comparable to that in activated B cells. The importance of the present finding for further development of antileukemic chemotherapeutic strategies is that the lack of one of the two principal lymphocyte K+ currents, which are partly redundant in their function, could render B-ALL cells more vulnerable to treatments targeting the remaining KCa3.1 current.

Acute lymphoblastic leukemia (ALL) is a rare disease in adults, but is the most common pediatric malignancy and the leading cause of death among children with cancer worldwide. While initial treatment regimens induce remission in most patients, relapses still occur in many cases during or after treatment. Relapses are difficult to treat and continue to be one of the leading causes of ALL-related deaths. Thus, it is essential to understand the biological mechanisms underlying ALL relapses, and to identify reliable biomarkers for better relapse risk prediction and novel druggable targets for precision treatments tailored to risk profiles. Here we review the latest developments in ALL research with a focus on relapse mechanisms, and we discuss related hopes and challenges.

Ion channels are integral membrane proteins that facilitate rapid transport of small ions into and out of the cell and between organelles and cytosol. Cytolytic lymphocytes including natural killer (NK) cells principally kill virus-infected and cancer cells by releasing cytolytic granules within the immunological synapse, formed between target and effector cells. This process strongly depends on Ca2+ signaling, which in human NK cells is controlled by the PLCγ/IP3R/CRAC axis. It is believed that CRAC, a Ca2+-selective channel within the cell membrane, is a principal mediator of Ca+ entry in nonexcitable cells including immune cells. However, in addition to CRAC, the activity of other plasma membrane and organellar channels, which are permeable for Ca2+ and Na+, K+, or small anions, also plays important roles in regulating NK cell functions. In this review, we discuss the role of different ion channels in the NK-mediated immune response including members of 4 distinct families of K+-selective channels, transient receptor potential channels, purinergic receptors, and pentameric ligand-gated channels that are located in the plasma membrane and lysosomes of NK cells.

Ulcerative colitis (UC) is an inflammatory colon and rectum disease affecting approximately 5 million people worldwide. There is no cure for UC, and approximately 8% of patients with UC develop colorectal cancer (CRC) by gradual acquisition of mutations driving the formation of adenomas and their progression to adenocarcinomas and metastatic disease. CRC constitutes 10% of total cancer cases worldwide and 9% of cancer deaths. Both UC and CRC have an increasing incidence worldwide. Although the epithelium has been well studied in UC and CRC, the contribution of neutrophils is less clear. Neutrophils are rapidly recruited in excessive amounts from peripheral blood to the colon during UC, and their overactivation in the proinflammatory UC tissue environment contributes to tissue damage. In CRC, the role of neutrophils is controversial, but emerging evidence suggests that their role depends on the evolution and context of the disease. The role of neutrophils in the transition from UC to CRC is even less clear. However, recent studies propose neutrophils as therapeutic targets for better clinical management of both diseases. This review summarizes the current knowledge on the roles of neutrophils in UC and CRC.

Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell-cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

The contribution of Erk1/2 to endothelial barrier regulation is convoluted and differs depending on the vascular bed. We explored the effects of Erk1/2 inhibition on endothelial barrier maintenance and its relationship with cAMP-dependent barrier strengthening. Thus, myocardial endothelial cells (MyEnd) were isolated and protein expression, localization and activity of structural and signaling molecules involved in maintenance of endothelial function were investigated by Western blot, immunostainings and G-LISA, respectively. The transendothelial electrical resistance (TEER) from confluent MyEnd monolayers was measured and used as a direct indicator of barrier integrity in vitro. Miles assay was performed to evaluate vascular permeability in vivo. Erk1/2 inhibition with U0126 affected neither the structural organization of adherens or tight junctions nor the protein level of their components, However, TEER drop significantly upon U0126 application, but the effect was transitory as the barrier function recovered 30 min after treatment. Erk1/2 inhibition delayed cAMP-mediated barrier strengthening but did not prevent barrier fortification despite diminishing Rac1 activation. Moreover, Erk1/2 inhibition, induced vascular leakage that could be prevented by local cAMP elevation in vivo. Our data demonstrate that Erk1/2 is required to prevent vascular permeability but is not critical for cAMP-mediated barrier enhancement.

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

Journal Article Reinvigorating the JLB experience Get access Michael Schnoor, Michael Schnoor Department of Molecular Biomedicine, Cinvestav-IPN, Av. IPN 2508, 07360 Mexico-City, Mexico Corresponding author: Department of Molecular Biomedicine, Cinvestav-IPN, Av. IPN 2508, 07360 Mexico-City, Mexico. Email: mschnoor@cinvestav.mx https://orcid.org/0000-0002-0269-5884 Search for other works by this author on: Oxford Academic Google Scholar Melanie J Scott, Melanie J Scott Department of Surgery, University of Pittsburgh, 3459 Fifth Ave., Pittsburgh, PA 15213, USA Search for other works by this author on: Oxford Academic Google Scholar Luis J Montaner Luis J Montaner Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA Search for other works by this author on: Oxford Academic Google Scholar Journal of Leukocyte Biology, Volume 113, Issue 3, March 2023, Pages 229–230, https://doi.org/10.1093/jleuko/qiad008 Published: 20 January 2023 Article history Editorial decision: 16 January 2023 Received: 16 January 2023 Published: 20 January 2023 Corrected and typeset: 20 February 2023

ABSTRACT The quality of life in patients with inflammatory bowel diseases (IBD) is strongly impaired. Alterations of intestinal epithelial homeostasis contribute to the development and establishment of IBD. Intestinal Paneth and Paneth-like cells produce and secrete luminal proteins sustaining epithelial homeostasis. Here we show that IFN-γ stimulates Paneth and Paneth-like cells degranulation that triggers the proliferation of intestinal epithelial cells (IEC) in a Wnt/ β -catenin independent manner. Degranulation in Paneth and Paneth-like cells was mTORC1 and necroptosis dependent. Remarkably, lack of IFN-γ, inhibition of mTORC1, or impeding necroptosis reduces IEC proliferation cytokine-mediated. Our findings identify a new role for IFN-γ in stimulating IEC proliferation through inducing degranulation of Paneth and Paneth-like cells which is mTORC1 and necroptosis- dependent. In a mouse model of colitis, mTORC1 activation and necroptosis regulate Paneth and Paneth-like cell secretion. Furthermore, the colitogenic environment triggers PC metaplasia in the distal region of the large intestine to simulate cell proliferation. Highlights: IFN-γ stimulates proliferation, β -catenin independent. IFN-γ enhances mitochondrial activity and proliferation IFN-γ regulates PC biogenesis. mTORC1-dependent necroptosis mediates secretion in Paneth and Paneth-like cells.

Metallothionein-2 (MT-2) was originally discovered as a mediator of zinc homeostasis and cadmium detoxification. However, MT-2 has recently received increased attention because altered expression of MT-2 is closely related to various diseases such as asthma and cancers. Several pharmacological strategies have been developed to inhibit or modify MT-2, revealing its potential as drug target in diseases. Therefore, a better understanding of the mechanisms of MT-2 action is warranted to improve drug development for potential clinical applications. In this review, we highlight recent advances in determining the protein structure, regulation, binding partners, and new functions of MT-2 in inflammatory diseases and cancers.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers as it is highly resistant to chemotherapy. K-Ras mutations usually trigger the development and progression of PDAC. We hypothesized that compounds stabilizing the KRas4B/PDE6δ complex could serve as PDAC treatments. Using in silico approaches, we identified the small molecules C14 and P8 that reduced K-Ras activation in primary PDAC cells. Importantly, C14 and P8 significantly prevented tumor growth in patient-derived xenotransplants. Combined treatment with C14 and P8 strongly increased cytotoxicity in PDAC cell lines and primary cultures and showed strong synergistic antineoplastic effects in preclinical murine PDAC models that were superior to conventional therapeutics without causing side effects. Mechanistically, C14 and P8 reduced tumor growth by inhibiting AKT and ERK signaling downstream of K-RAS leading to apoptosis, specifically in PDAC cells. Thus, combined treatment with C14 and P8 may be a superior pharmaceutical strategy to improve the outcome of PDAC.