Precision RNAi using synthetic shRNAmir target sites.

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.

Loss of EZH2 Reprograms BCAA Metabolism to Drive Leukemic Transformation.

Epigenetic gene regulation and metabolism are highly intertwined, yet little is known about whether altered epigenetics influence cellular metabolism during cancer progression. Here, we show that EZH2 and NRASG12D mutations cooperatively induce progression of myeloproliferative neoplasms to highly penetrant, transplantable, and lethal myeloid leukemias in mice. EZH1, an EZH2 homolog, is indispensable for EZH2-deficient leukemia-initiating cells and constitutes an epigenetic vulnerability. BCAT1, which catalyzes the reversible transamination of branched-chain amino acids (BCAA), is repressed by EZH2 in normal hematopoiesis and aberrantly activated in EZH2-deficient myeloid neoplasms in mice and humans. BCAT1 reactivation cooperates with NRASG12D to sustain intracellular BCAA pools, resulting in enhanced mTOR signaling in EZH2-deficient leukemia cells. Genetic and pharmacologic inhibition of BCAT1 selectively impairs EZH2-deficient leukemia-initiating cells and constitutes a metabolic vulnerability. Hence, epigenetic alterations rewire intracellular metabolism during leukemic transformation, causing epigenetic and metabolic vulnerabilities in cancer-initiating cells. SIGNIFICANCE: EZH2 inactivation and oncogenic NRAS cooperate to induce leukemic transformation of myeloproliferative neoplasms by activating BCAT1 to enhance BCAA metabolism and mTOR signaling. We uncover a mechanism by which epigenetic alterations rewire metabolism during cancer progression, causing epigenetic and metabolic liabilities in cancer-initiating cells that may be exploited as potential therapeutics.See related commentary by Li and Melnick, p. 1158.This article is highlighted in the In This Issue feature, p. 1143.

Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering.

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10-15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

A Gain-of-Function Mutation in EPO in Familial Erythrocytosis.

Familial erythrocytosis with elevated erythropoietin levels is frequently caused by mutations in genes that regulate oxygen-dependent transcription of the gene encoding erythropoietin ( EPO). We identified a mutation in EPO that cosegregated with disease with a logarithm of the odds (LOD) score of 3.3 in a family with autosomal dominant erythrocytosis. This mutation, a single-nucleotide deletion (c.32delG), introduces a frameshift in exon 2 that interrupts translation of the main EPO messenger RNA (mRNA) transcript but initiates excess production of erythropoietin from what is normally a noncoding EPO mRNA transcribed from an alternative promoter located in intron 1. (Funded by the Gebert Rüf Foundation and others.).

Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis.

Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2 In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2(-/-) mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2(+/-) mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F-expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN.

Bactericidal monoclonal antibodies specific to the lipopolysaccharide O antigen from multidrug-resistant Escherichia coli clone ST131-O25b:H4 elicit protection in mice.

The Escherichia coli sequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to live E. coli cells and the in vitro and in vivo efficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused by E. coli ST131-O25b:H4.

Tumor-driven Molecular Changes in Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) exert either tumor-stimulatory or tumor-inhibitory effect. The outcome of the tumor-MSC interaction is dictated by the tumor-specific activating signals. We analyzed the alterations in MSC phenotype in response to stimulation by tumor-secreted paracrine factors. Paracrine factors from human melanoma A375 and glioblastoma 8MGBA cells were used for prolonged culture of MSC to produce derived cells designated DIFF(A)-MSC or DIFF(G)-MSC, respectively. Derived cells were analyzed for the specific surface markers, the expression pattern of MSC markers and fibroblast-specific proteins. Changes in the cell phenotype were evaluated using scratch wound assay and tube formation in vitro; and xenotransplant growth in vivo. Our data show induced expression of vascular endothelial growth factor 2, CD146, fibroblast-specific protein, vimentin and endosialin in DIFF(A)-MSC cells. This indicates their differentiation towards the cells with features of tumor-associated fibroblasts upon stimulation with melanoma-secreted cytokines. Paracrine stimulation in DIFF(G)-MSC led to up-regulation of the genes involved in the MSC differentiation. MSC-specific surface marker characteristics were preserved in derived DIFF(A)-MSC and DIFF(G)-MSC cells. However, we observed increased proportion of CD146 and GD2 (neural ganglioside) positive cells and decreased expression of marker NG2 in the MSC exposed to tumor-conditioned medium. Melanoma-CM increased MSC migration, glioblastoma-CM compromised angiogenic capacity of MSC in vitro and the protumorigenic effect in vivo. Our data directly compare the pleiotropic effects mediated by the malignant cells on the MSC. Secreted paracrine factors from melanoma or glioblastoma differently changed molecular traits in MSC, which explains the dual role of MSC in tumor growth.

Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.