Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin.

BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.

A Novel Bispecific T-Cell Engager (CD1a x CD3ε) BTCE Is Effective against Cortical-Derived T Cell Acute Lymphoblastic Leukemia (T-ALL) Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy burdened by poor prognosis. While huge progress of immunotherapy has recently improved the outcome of B-cell malignancies, the lack of tumor-restricted T-cell antigens still hampers its progress in T-ALL. Therefore, innovative immunotherapeutic agents are eagerly awaited. To this end, we generated a novel asymmetric (2 + 1) bispecific T-cell engager (BTCE) targeting CD1a and CD3ε (CD1a x CD3ε) starting from the development of a novel mAb named UMG2. UMG2 mAb reacts against CD1a, a glycoprotein highly expressed by cortical T-ALL cells. Importantly, no UMG2 binding was found on normal T-cells. CD1a x CD3ε induced high T-cell mediated cytotoxicity against CD1a+ T-ALL cells in vitro, as demonstrated by the concentration-dependent increase of T-cell proliferation, degranulation, induction of cell surface activation markers, and secretion of pro-inflammatory cytokines. Most importantly, in a PBMC-reconstituted NGS mouse model bearing human T-ALL, CD1a x CD3ε significantly inhibited the growth of human T-ALL xenografts, translating into a significant survival advantage of treated animals. In conclusion, CD1a x CD3ε is a novel BTCE highly active against CD1a-expressing cortical-derived T-ALL cells suitable for clinical development as an effective therapeutic option for this rare and aggressive disease.

The Family Keeps on Growing: Four Novel Fungal OYEs Characterized.

Aiming at expanding the portfolio of Old Yellow Enzymes (OYEs), which have been systematically studied to be employed in the chemical and pharmaceutical industries as useful biocatalysts, we decided to explore the immense reservoir of filamentous fungi. We drew from the genome of the two Ascomycetes Aspergillus niger and Botryotinia fuckeliana four new members of the OYE superfamily belonging to the classical and thermophilic-like subfamilies. The two BfOYEs show wider substrate spectra than the AnOYE homologues, which appear as more specialized biocatalysts. According to their mesophilic origins, the new enzymes neither show high thermostability nor extreme pH optimums. The crystal structures of BfOYE4 and AnOYE8 have been determined, revealing the conserved features of the thermophilic-like subclass as well as unique properties, such as a peculiar N-terminal loop involved in dimer surface interactions. For the classical representatives BfOYE1 and AnOYE2, model structures were built and analyzed, showing surprisingly wide open access to the active site cavities due to a shorter ß6-loop and a disordered capping subdomain.

A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans.

Aiming at expanding the biocatalytic toolbox of ene-reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic activities. We selected a new thermophilic ene-reductase homologue in Chloroflexus aggregans, a peculiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called CaOYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, CaOYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene-reductases); however, given the amazing metabolic ductility and versatility of Chloroflexus aggregans, further investigations could pinpoint peculiar chemical activities. X-ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic-like group) of the family of Old Yellow Enzymes: in the crystal packing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of CaOYE catalytic properties and crystal structure provides new details useful for enlarging knowledge, development and application of this class of enzymes.

Relative Echogenicity of Tendons and Ligaments of the Palmar Metacarpal Region in Foals from Birth to 4 Months of Age: A Longitudinal Study.

The objective of this study was to evaluate relative echogenicity of superficial and deep digital flexor tendons, the accessory ligament of the deep digital flexor tendon and interosseous muscle of the metacarpal region in foals ages 1 week to 4 months; and assess the association between echogenicity and sex or side/laterality. Seven Standardbred trotter foals were examined. Right and left metacarpal regions (palmar surface) were ultrasonographically investigated, and four regions of interest were assessed. A significant increase in echogenicity was seen in superficial and deep digital flexor tendons, accessory ligament of deep digital flexor tendon, and interosseous muscle during growth from 1 week to 4 months of age. Echogenicity of examined tendons and ligaments was not influenced by gender nor laterality. Reference values for tendon and ligament echogenicity could function as a tool to discriminate between physiological and abnormal conditions such as congenital contractural conditions.