Functional characterization of phospholipase C-γ mutant protein causing both somatic ibrutinib resistance and a germline monogenic autoinflammatory disorder.

Depending on its occurrence in the germline or somatic context, a single point mutation, S707Y, of phospholipase C-γ (PLCγ) gives rise to two distinct human disease states: acquired resistance of chronic lymphocytic leukemia cells (CLL) to inhibitors of Brutons´s tyrosine kinase (Btk) and dominantly inherited autoinflammation and PLCγ-associated antibody deficiency and immune dysregulation, APLAID, respectively. The functional relationships of the PLCγS707Y mutation to other mutations causing (i) Btk inhibitor resistance of CLL cells and (ii) the APLAID-related human disease PLCγ-associated antibody deficiency and immune dysregulation, PLAID, revealing different clinical characteristics including cold-induced urticaria, respectively, are currently incompletely understood. Here, we show that PLCγS707 point mutants displayed much higher activities at 37° C than the CLL Btk inhibitor resistance mutants R665W and L845F and the two PLAID mutants, PLCγΔ19 and PLCγΔ20-22.

Therapeutic sensitivity to Rac GTPase inhibition requires consequential suppression of mTORC1, AKT, and MEK signaling in breast cancer.

Rac GTPases have oncogenic roles in cell growth, survival, and migration. We tested response to the Rac inhibitor EHT1864 in a panel of breast cancer cell lines. EHT1864-induced growth inhibition was associated with dual inhibition of the PI3K/AKT/mTORC1 and MEK/ERK pathways.

An Unusual Binding Model of the Methyl 9-Anilinothiazolo[5,4-f] quinazoline-2-carbimidates (EHT 1610 and EHT 5372) Confers High Selectivity for Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases.

Methyl 9-anilinothiazolo[5,4-f]quinazoline-2-carbimidates 1 (EHT 5372) and 2 (EHT 1610) are strong inhibitors of DYRK's family kinases. The crystal structures of the complex revealed a noncanonical binding mode of compounds 1 and 2 in DYRK2, explaining the remarkable selectivity and potency of these inhibitors. The structural data and comparison presented here provide therefore a template for further improvement of this inhibitor class and for the development of novel inhibitors selectively targeting DYRK kinases.

The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein.

Mutations in the gene encoding phospholipase C-γ (PLCγ) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ an ∼2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells.

Multiplex Antibody Detection for Noninvasive Genus-Level Diagnosis of Prosthetic Joint Infection.

We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcusspecies (Staphylococcus aureus,Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens),Streptococcus agalactiae(4 antigens), and Propionibacterium acnes(4 antigens) were selected by comparative immune proteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed.