MYD88 mutations identify a molecular subgroup of diffuse large B-cell lymphoma with an unfavorable prognosis

The 2016 World Health Organization classification defines diffuse large B-cell lymphoma (DLBCL) subtypes based on Epstein-Barr virus (EBV) infection and oncogenic rearrangements of MYC/BCL2/BCL6 as drivers of lymphomagenesis. A subset of DLBCL, however, is characterized by activating mutations in MYD88/CD79B. We investigated whether MYD88/CD79B mutations could improve the classification and prognostication of DLBCL. In 250 primary DLBCL, MYD88/CD79B mutations were identified by allele-specific polymerase chain reaction or next-generationsequencing, MYC/BCL2/BCL6 rearrangements were analyzed by fluorescence in situ hybridization, and EBV was studied by EBV-encoded RNA in situ hybridization. Associations of molecular features with clinicopathologic characteristics, outcome, and prognosis according to the International Prognostic Index (IPI) were investigated. MYD88 and CD79B mutations were identified in 29.6% and 12.3%, MYC, BCL2, and BCL6 rearrangements in 10.6%, 13.6%, and 20.3%, and EBV in 11.7% of DLBCL, respectively. Prominent mutual exclusivity between EBV positivity, rearrangements, and MYD88/CD79B mutations established the value of molecular markers for the recognition of biologically distinct DLBCL subtypes. MYD88-mutated DLBCL had a significantly inferior 5-year overall survival than wild-type MYD88 DLBCL (log-rank; P=0.019). DLBCL without any of the studied aberrations had superior overall survival compared to cases carrying .1 aberrancy (log-rank; P=0.010). MYD88 mutations retained their adverse prognostic impact upon adjustment for other genetic and clinical variables by multivariable analysis and improved the prognostic performance of the IPI. This study demonstrates the clinical utility of defining MYD88-mutated DLBCL as a distinct molecular subtype with adverse prognosis. Our data call for sequence analysis of MYD88 in routine diagnostics of DLBCL to optimize classification and prognostication, and to guide the development of improved treatment strategies

Designed spiroketal protein Modulation

\u3cp\u3eSpiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure-based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co-activator recruitment. We solved the crystal structure of the spiroketal-hRXRα-TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co-crystal structure, the first of a designed spiroketal-protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes.\u3c/p\u3

Ligand-Based Design of Allosteric Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt

Ligand-based design of allosteric retinoic acid receptor-related orphan receptor γt (RORγt) inverse agonists

\u3cp\u3eRetinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The most potent compound, 25, displayed sub-micromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and co-crystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands, but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.\u3c/p\u3

Subtype-Specific Modulation of Estrogen Receptor–Coactivator Interaction by Phosphorylation

The estrogen receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semisynthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semisynthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveals the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ERα and the analogous tyrosine (Y488) in ERβ. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism

Therapeutic Opportunities Offered by the Excessive Lactate Production in Cancer

The majority of cancers of various tissue origin display wide portions suffering from insufficient respiration, due to permanent or transient hypoxia, which occurs during tumor development. This condition leads to the development of a glycolytic phenotype, where a compensatory lactate production takes place, in order to provide the cancer cells with sufficient amounts of energy and anabolites. Lactate is not just as a waste product of the glycolytic process, instead it plays a key role in the progression of cancer, since it promotes angiogenesis, cell migration, immune escape and radioresistance. Moreover, lactate can still constitute a metabolic fuel for oxidative tumor cells or vascular endothelial cells, and it can establish a symbiotic cell-cell shuttling system with stromal cells. Therefore, these peculiar roles of lactate in invasive tumors constitutes a high-priority target for future anti-cancer therapeutics [1]. Therapeutic interventions aimed at reducing lactate production in cancer tissues may consist of: a) reduction of glucose uptake (calorie-restricted ketogenic diet, physical exercise, inhibitors of glucose transporters); b) inhibition of enzymes involved in key-steps of glycolysis (inhibitors of hexokinase, phosphofructokinase, lactate dehydrogenase); c) block of the cellular trafficking of lactate (inhibitors of monocarboxylate transporters); d) enhancement of the mitochondrial oxidative metabolism (hyperbaric oxygen therapy, removal of inhibition of the Krebs cycle, for example, by using inhibitors of pyruvate dehydrogenase kinase) [2]. We have developed compounds that exert an anti-proliferative action on cancer cells by specific interventions on cancer metabolism, such as, inhibition of lactate dehydrogenase (LDH) activity [3,4], or reduction of glucose uptake through specific transmembrane transporters (GLUT) [5]. Furthermore, some of the LDH-inhibitors demonstrated a remarkable synergism with gemcitabine against pancreatic cancer cells in hypoxia [6]. and an improved activation of the redox-sensitive anti-cancer prodrug EO9 by means of an induced increase of the NADH/NAD+ cell ratio [7]. It is important to note that the development of agents that target lactate production, trafficking, and metabolism (by these or other methods) hold promise for treating nearly all invasive cancers, provided they present an appropriate therapeutic window. References 1) J. R. Doherty, J. L. Cleveland. J. Clin. Invest. 2013, 123, 3685–3692. 2) C. Granchi, F. Minutolo. ChemMedChem 2012, 7, 1318-1350. 3) C. Granchi, S. Roy, C. Giacomelli, et al. J. Med. Chem. 2011, 54, 1599–1612. 4) E. C. Calvaresi, C. Granchi, T. Tuccinardi, et al. ChemBioChem 2013, 14, 2263–2267. 5) T. Tuccinardi, C. Granchi, J. Iegre, et al. Bioorg. Med. Chem. Lett. 2013, 23, 6923–6927. 6) M. Maftouh, A. Avan, R. Sciarrillo, et al. Br. J. Cancer 2014, 110, 172-182. 7) S. J. Allison, J. R. P. Knight, C. Granchi, et al. Oncogenesis 2014, 3, e102; DOI: 10.1038/oncsis.2014.16

MYD88 mutations identify a molecular subgroup of diffuse large B-cell lymphoma with an unfavorable prognosis

The 2016 World Health Organization classification defines diffuse large B-cell lymphoma (DLBCL) subtypes based on Epstein-Barr virus (EBV) infection and oncogenic rearrangements of MYC/BCL2/BCL6 as drivers of lymphomagenesis. A subset of DLBCL, however, is characterized by activating mutations in MYD88/CD79B We investigated whether MYD88/CD79B mutations could improve the classification and prognostication of DLBCL. In 250 primary DLBCL, MYD88/CD79B mutations were identified by allele-specific polymerase chain reaction or next-generation-sequencing, MYC/BCL2/BCL6 rearrangements were analyzed by fluorescence in situ hybridization, and EBV was studied by EBV-encoded RNA in situ hybridization. Associations of molecular features with clinicopathologic characteristics, outcome, and prognosis according to the International Prognostic Index (IPI) were investigated. MYD88 and CD79B mutations were identified in 29.6% and 12.3%, MYC, BCL2, and BCL6 rearrangements in 10.6%, 13.6%, and 20.3%, and EBV in 11.7% of DLBCL, respectively. Prominent mutual exclusivity between EBV positivity, rearrangements, and MYD88/CD79B mutations established the value of molecular markers for the recognition of biologically distinct DLBCL subtypes. MYD88-mutated DLBCL had a significantly inferior 5-year overall survival than wild-type MYD88 DLBCL (log-rank; P=0.019). DLBCL without any of the studied aberrations had superior overall survival compared to cases carrying ≥1 aberrancy (log-rank; P=0.010). MYD88 mutations retained their adverse prognostic impact upon adjustment for other genetic and clinical variables by multivariable analysis and improved the prognostic performance of the IPI. This study demonstrates the clinical utility of defining MYD88-mutated DLBCL as a distinct molecular subtype with adverse prognosis. Our data call for sequence analysis of MYD88 in routine diagnostics of DLBCL to optimize classification and prognostication, and to guide the development of improved treatment strategies