Miller, B. C. | bioRxiv Science

bioRxiv2021

Microenvironmental correlates of immune checkpoint inhibitor response in human melanoma brain metastases revealed by T cell receptor and single-cell RNA sequencing

Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

cancer biology↗

bioRxiv2021

Protein crosslinking as a therapeutic strategy for SOD1-related ALS

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS). One effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers. Considerable research efforts have been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but these have not yet resulted in an approved drug. We demonstrate that a cyclic thiosulfinate cross-linker can stabilize prevalent disease-causing SOD1 variants. The degree of stabilization afforded by cyclic thiosulfinates (up to 24 {degrees}C) is unprecedented. We show this compound works rapidly in vivo with a half-life of ~3 days. The efficacy, low toxicity, and pharmacodynamics of cross-linker mediated stabilization make it a promising therapeutic approach for SOD1-related fALS. Significance statementCyclic thiosulfinate S-XL6 enables the kinetic stabilization of ALS-associated SOD1 variants, in vivo.

neuroscience↗

bioRxiv2021

Profiling the in vitro and in vivo activity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action

Streptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked {beta}-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940s, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50 and MIC90 for S-F and S-D were 2 and 4 {micro}M, and 0.25 and 0.5 {micro}M, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. There was >10-fold in vitro selectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operon E. coli identify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, gram-negative pathogens. IMPORTANCEStreptothricins are a historic class of antibiotics discovered by Waksman and colleagues in the 1940s. Toxicities associated with the streptothricin natural product mixture, also known as nourseothricin, discouraged further development. However, we found that a component of nourseothricin, streptothricin-F, retained potent activity against contemporary carbapenem-resistant Enterobacterales with significant selectivity in in vitro and in vivo assays. This included demonstration of rapid bactericidal activity in vitro and substantial therapeutic effect in the murine thigh model against a pandrug-resistant Klebsiella pneumoniae isolate at non-toxic concentrations. Through resistance mutation analysis, we identified helix 34 of 16S rRNA in the prokaryotic ribosome, and specifically bases C1054 and A1196, as critical for streptothricins activity. The mechanism of action is distinct from other known translation inhibitors. Based on promising and unique activity, we believe the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens.

microbiology↗