Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis.

Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain. Despite the large deletions, the two SVs cross-react with a substantial population of anti-Jo-l antibodies from myositis patients. Moreover, expression of at least one of the SVs is up-regulated in dermatomyositis patients, and cell-based experiments show that both SVs and HisRS can be secreted. We suggest that, in patients with inflammatory myositis, anti-Jo-1 antibodies may have extracellular activity.

Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants.

Drugging the epigenome in the age of precision medicine.

BACKGROUND: Modulating the epigenome has long been considered a potential opportunity for therapeutic intervention in numerous disease areas with several approved therapies marketed, primarily for cancer. Despite the overall promise of early approaches, however, these drugs have been plagued by poor pharmacokinetic and safety/tolerability profiles due in large part to off-target effects and a lack of specificity. RESULTS: Recently, there has been marked progress in the field on a new generation of epigenomic therapies which address these challenges directly by targeting defined loci with highly precise, durable, and tunable approaches. Here, we review the promise and pitfalls of epigenetic drug development to date and provide an outlook on recent advances and their promise for future therapeutic applications. CONCLUSIONS: Novel therapeutic modalities leveraging epigenetics and epigenomics with increased precision are well positioned to advance the field and treat patients across disease areas in the coming years.

Serum-circulating His-tRNA synthetase inhibits organ-targeted immune responses.

His-tRNA synthetase (HARS) is targeted by autoantibodies in chronic and acute inflammatory anti-Jo-1-positive antisynthetase syndrome. The extensive activation and migration of immune cells into lung and muscle are associated with interstitial lung disease, myositis, and morbidity. It is unknown whether the sequestration of HARS is an epiphenomenon or plays a causal role in the disease. Here, we show that HARS circulates in healthy individuals, but it is largely undetectable in the serum of anti-Jo-1-positive antisynthetase syndrome patients. In cultured primary human skeletal muscle myoblasts (HSkMC), HARS is released in increasing amounts during their differentiation into myotubes. We further show that HARS regulates immune cell engagement and inhibits CD4+ and CD8+ T-cell activation. In mouse and rodent models of acute inflammatory diseases, HARS administration downregulates immune activation. In contrast, neutralization of extracellular HARS by high-titer antibody responses during tissue injury increases susceptibility to immune attack, similar to what is seen in humans with anti-Jo-1-positive disease. Collectively, these data suggest that extracellular HARS is homeostatic in normal subjects, and its sequestration contributes to the morbidity of the anti-Jo-1-positive antisynthetase syndrome.

Human tRNA synthetase catalytic nulls with diverse functions.

Genetic efficiency in higher organisms depends on mechanisms to create multiple functions from single genes. To investigate this question for an enzyme family, we chose aminoacyl tRNA synthetases (AARSs). They are exceptional in their progressive and accretive proliferation of noncatalytic domains as the Tree of Life is ascended. Here we report discovery of a large number of natural catalytic nulls (CNs) for each human AARS. Splicing events retain noncatalytic domains while ablating the catalytic domain to create CNs with diverse functions. Each synthetase is converted into several new signaling proteins with biological activities "orthogonal" to that of the catalytic parent. We suggest that splice variants with nonenzymatic functions may be more general, as evidenced by recent findings of other catalytically inactive splice-variant enzymes.

Anti-tumor effect of CT-322 as an adnectin inhibitor of vascular endothelial growth factor receptor-2.

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an Adnectin™, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PE Gylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (K(D), 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15-60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.