Objective Microglia play a pivotal role in the initiation and progression of Alzheimer's disease ( AD ). We here tested the therapeutic hypothesis that the Ca 2+ ‐activated potassium channel KC a3.1 constitutes a potential target for treating AD by reducing neuroinflammation. Methods To determine if KC a3.1 is relevant to AD , we tested if treating cultured microglia or hippocampal slices with A β oligomer (A β O) activated KC a3.1 in microglia, and if microglial KC a3.1 was upregulated in 5x FAD mice and in human AD brains. The expression/activity of KC a3.1 was examined by qPCR , Western blotting, immunohistochemistry, and whole‐cell patch‐clamp. To investigate the role of KC a3.1 in AD pathology, we resynthesized senicapoc, a clinically tested KC a3.1 blocker, and determined its pharmacokinetic properties and its effect on microglial activation, A β deposition and hippocampal long‐term potentiation ( hLTP ) in 5x FAD mice. Results We found markedly enhanced microglial KC a3.1 expression/activity in brains of both 5x FAD mice and AD patients. In hippocampal slices, microglial KC a3.1 expression/activity was increased by A β O treatment, and its inhibition diminished the proinflammatory and hLTP ‐impairing activities of A β O. Senicapoc exhibited excellent brain penetrance and oral availability, and in 5x FAD mice, reduced neuroinflammation, decreased cerebral amyloid load, and enhanced hippocampal neuronal plasticity. Interpretation Our results prompt us to propose repurposing senicapoc for AD clinical trials, as senicapoc has excellent pharmacological properties and was safe and well‐tolerated in a prior phase‐3 clinical trial for sickle cell anemia. Such repurposing has the potential to expedite the urgently needed new drug discovery for AD .

Mutant U2AF1-induced alternative splicing of H2afy (macroH2A1) regulates B-lymphopoiesis in mice Graphical abstract Highlights d Mutant U2AF1(S34F) induces alternative splicing of H2AFY d H2afy À/À mice have defective B cell development similar to U2AF1(S34F) mice d The H2afy1.1 splice isoform, reduced by U2AF1(S34F), regulates B cell development d H2AFY occupies the Ebf1 promoter, a master regulator of B cell development

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

Patients with multiple myeloma (MM) who are treated with lenalidomide rarely develop a secondary B-cell acute lymphoblastic leukemia (B-ALL). The clonal and biological relationship between these sequential malignancies is not yet clear. We identified 17 patients with MM treated with lenalidomide, who subsequently developed B-ALL. Samples were evaluated with sequencing, cytogenetics/FISH, immunohistochemical staining (IHC), and IgH clonality assessment. Samples were assessed for shared mutations and recurrently mutated genes. Through whole exome sequencing and cytogenetics/FISH analysis of 7 paired samples (MM versus matched B-ALL), no mutational overlap between samples was observed. Unique dominant IgH clonotypes between the tumors were observed in 5 paired MM / B-ALL samples. Across all 17 B-ALL samples, 14 (83%) had a TP53 variant detected. Three MM samples with sufficient sequencing depth (>500X) revealed rare cells (average of 0.6% VAF, or 1.2% of cells) with the same TP53 variant identified in the subsequent B-ALL sample. A lack of mutational overlap between MM and B-ALL samples shows that B-ALL developed as a second malignancy arising from a founding population of cells that probably represented unrelated clonal hematopoiesis caused by a TP53 mutation. The recurrent variants in TP53 in the B-ALL samples suggest a common path for malignant transformation that may be similar to that of TP53-mutant, treatment-related acute myeloid leukemia. The presence of rare cells containing TP53 variants in bone marrow at the initiation of lenalidomide treatment suggests that cellular populations containing TP53 variants expand in the presence of lenalidomide to increase the likelihood of B-ALL development.

No abstract