Structural basis of reactivation of oncogenic p53 mutants by a small molecule: methylene quinuclidinone (MQ).

In response to genotoxic stress, the tumor suppressor p53 acts as a transcription factor by regulating the expression of genes critical for cancer prevention. Mutations in the gene encoding p53 are associated with cancer development. PRIMA-1 and eprenetapopt (APR-246/PRIMA-1MET) are small molecules that are converted into the biologically active compound, methylene quinuclidinone (MQ), shown to reactivate mutant p53 by binding covalently to cysteine residues. Here, we investigate the structural basis of mutant p53 reactivation by MQ based on a series of high-resolution crystal structures of cancer-related and wild-type p53 core domains bound to MQ in their free state and in complexes with their DNA response elements. Our data demonstrate that MQ binds to several cysteine residues located at the surface of the core domain. The structures reveal a large diversity in MQ interaction modes that stabilize p53 and its complexes with DNA, leading to a common global effect that is pertinent to the restoration of non-functional p53 proteins.

PRC2 loss of function confers a targetable vulnerability to BET proteins in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is a group of aggressive hematological cancers with dismal outcomes that are in need of new therapeutic options. Polycomb repressor complex 2 (PRC2) loss-of-function alterations were reported in pediatric T-ALL, yet their clinical relevance and functional consequences remain elusive. Here, we extensively analyzed PRC2 alterations in a large series of 218 adult T-ALL patients. We found that PRC2 genetic lesions are frequent events in T-ALL and are not restricted to early thymic precursor ALL. PRC2 loss of function associates with activating mutations of the IL7R/JAK/STAT pathway. PRC2-altered T-ALL patients respond poorly to prednisone and have low bone marrow blast clearance and persistent minimal residual disease. Furthermore, we identified that PRC2 loss of function profoundly reshapes the genetic and epigenetic landscapes, leading to the reactivation of stem cell programs that cooperate with bromodomain and extraterminal (BET) proteins to sustain T-ALL. This study identifies BET proteins as key mediators of the PRC2 loss of function-induced remodeling. Our data have uncovered a targetable vulnerability to BET inhibition that can be exploited to treat PRC2-altered T-ALL patients.

CARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3+ regulatory T cells.

FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.

Naturally Occurring Missense MRGPRX2 Variants Display Loss of Function Phenotype for Mast Cell Degranulation in Response to Substance P, Hemokinin-1, Human ß-Defensin-3, and Icatibant.

Human mast cells (MCs) express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR X2 (MRGPRX2). Activation of this receptor by a diverse group of cationic ligands such as neuropeptides, host defense peptides, and Food and Drug Administration-approved drugs contributes to chronic inflammatory diseases and pseudoallergic drug reactions. For most GPCRs, the extracellular (ECL) domains and their associated transmembrane (TM) domains display the greatest structural diversity and are responsible for binding different ligands. The goal of the current study was to determine if naturally occurring missense variants within MRGPRX2's ECL/TM domains contribute to gain or loss of function phenotype for MC degranulation in response to neuropeptides (substance P and hemokinin-1), a host defense peptide (human ß-defensin-3) and a Food and Drug Administration-approved cationic drug (bradykinin B2 receptor antagonist, icatibant). We have identified eight missense variants within MRGPRX2's ECL/TM domains from publicly available exome-sequencing databases. We investigated the ability of MRGPRX2 ligands to induce degranulation in rat basophilic leukemia-2H3 cells individually expressing these naturally occurring MRGPRX2 missense variants. Using stable and transient transfections, we found that all variants express in rat basophilic leukemia cells. However, four natural MRGPRX2 variants, G165E (rs141744602), D184H (rs372988289), W243R (rs150365137), and H259Y (rs140862085) failed to respond to any of the ligands tested. Thus, diverse MRGPRX2 ligands use common sites on the receptor to induce MC degranulation. These findings have important clinical implications for MRGPRX2 and MC-mediated pseudoallergy and chronic inflammatory diseases.