Rodriguez, Moses

Abstract Glioblastoma (GBM) is a rapidly fatal malignancy typically treated with radiation and temozolomide (TMZ), an alkylating chemotherapeutic. These cytotoxic therapies cause oxidative stress and DNA damage, yielding a senescent-like state of replicative arrest in surviving tumor cells. Unfortunately, recurrence is inevitable and may be driven by surviving tumor cells eventually escaping senescence. A growing number of so-called “senolytic” drugs have been recently identified that are defined by their ability to selectively eliminate senescent cells. A growing inventory of senolytic drugs is under consideration for several diseases associated with aging, inflammation, DNA damage, as well as cancer. Ablation of senescent tumor cells after radiation and chemotherapy could help mitigate recurrence by decreasing the burden of residual tumor cells at risk of recurrence. This strategy has not been previously explored for GBM. We evaluated a panel of 10 previously described senolytic drugs to determine whether any could exhibit selective activity against human GBM persisting after exposure to radiation or TMZ. Three of the 10 drugs have known activity against BCL-XL and preferentially induced apoptosis in radiated or TMZ-treated glioma. This senolytic activity was observed in 12 of 12 human GBM cell lines. Efficacy could not be replicated with BCL-2 inhibition or senolytic agents acting against other putative senolytic targets. Knockdown of BCL-XL decreased survival of radiated GBM cells, whereas knockdown of BCL-2 or BCL-W yielded no senolytic effect. Implications: These findings imply that molecularly heterogeneous GBM lines share selective senescence-induced BCL-XL dependency increase the significance and translational relevance of the senolytic therapy for latent glioma.

Background Recombinant human immunoglobulin M22 (rHIgM22) has promoted remyelination in animal models and was well tolerated in people with clinically stable multiple sclerosis. Objective Safety/tolerability of a single rHIgM22 dose was investigated following an acute relapse and to determine whether this enhanced CNS/CSF concentrations. Methods Adults (N = 27) with acute relapse were assigned to rHIgM22 (0.5 or 2.0 mg/kg) or placebo. Study included screening/steroid administration periods and 10 study visits over 6 months. rHIgM22 CSF concentrations were assessed on days 2 and 29. Pharmacokinetic and safety samples were taken for up to 60 days. Assessments included adverse events and other clinical measures. Brain magnetic resonance imaging was performed with/without gadolinium. Results rHIgM22 CSF levels were consistent with dose-dependent concentration on both days 2 and 29. Infusion was generally well tolerated during an acute relapse. Immunogenicity was mild. Most adverse events did not appear to be dose dependent, were mild/moderate, and were events often associated with multiple sclerosis. Conclusion Although limited by high variability and small sample size, the data suggest enhanced CNS uptake associated with a drop in CSF levels. This study demonstrated safety of an antibody directed to myelin and oligodendrocytes in the course of active demyelinating disease. Further research into rHIgM22 is warranted. ClinicalTrials.gov: NCT02398461 https://clinicaltrials.gov/ct2/show/NCT02398461

Abstract Objective To identify features of the gut microbiome associated with multiple sclerosis activity over time. Methods We used 16S ribosomal RNA sequencing from stool of 55 recently diagnosed pediatric‐onset multiple sclerosis patients. Microbiome features included the abundance of individual microbes and networks identified from weighted genetic correlation network analyses. Prentice‐Williams‐Peterson Cox proportional hazards models estimated the associations between features and three disease activity outcomes: clinical relapses and both new/enlarging T2 lesions and new gadolinium‐enhancing lesions on brain MRI. Analyses were adjusted for age, sex, and disease‐modifying therapies. Results Participants were followed, on average, 2.1 years. Five microbes were nominally associated with all three disease activity outcomes after multiple testing correction. These included butyrate producers Odoribacter (relapse hazard ratio = 0.46, 95% confidence interval: 0.24, 0.88) and Butyricicoccus (relapse hazard ratio = 0.49, 95% confidence interval: 0.28, 0.88). Two networks of co‐occurring gut microbes were significantly associated with a higher hazard of both MRI outcomes (gadolinium‐enhancing lesion hazard ratios (95% confidence intervals) for Modules 32 and 33 were 1.29 (1.08, 1.54) and 1.42 (1.18, 1.71), respectively; T2 lesion hazard ratios (95% confidence intervals) for Modules 32 and 33 were 1.34 (1.15, 1.56) and 1.41 (1.21, 1.64), respectively). Metagenomic predictions of these networks demonstrated enrichment for amino acid biosynthesis pathways. Interpretation Both individual and networks of gut microbes were associated with longitudinal multiple sclerosis activity. Known functions and metagenomic predictions of these microbes suggest the important role of butyrate and amino acid biosynthesis pathways. This provides strong support for future development of personalized microbiome interventions to modify multiple sclerosis disease activity.

AbstractTheiler's murine encephalomyelitis virus causes a chronic demyelinating disease in susceptible strains of mice that is similar to human multiple sclerosis. Several nonmajor histocompatibility complex–linked genes have been implicated as determinants of susceptibility or resistance to either demyelination or virus persistence. In this study, we used linkage analysis of major histocompatibility complex identical H-2d (DBA/2J × B10.D2) F2 intercross mice to identify loci associated with susceptibility to virus-induced demyelinating disease. In a 20-cM region on chromosome 14, we identified four markers, D14Mit54, D14Mit60, D14Mit61, and D14Mit90 that are significantly associated with demyelination. Because two peaks were identified, one near D14Mit54 and one near D14Mit90, it is possible that two loci in this region are involved in controlling demyelination.

Abstract BACKGROUND Radiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumors are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. METHODS Female athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected patient-derived xenograft (PDX) GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. RESULT Of nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196). However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM 39, 123 whose rate of growth was not impacted by the radiated brain. CONCLUSION These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

AbstractIncomplete relapse recovery contributes to disability accrual and earlier onset of secondary progressive multiple sclerosis. We sought to investigate the effect of age on relapse recovery. We identified patients with multiple sclerosis from two longitudinal prospective studies, with an Expanded Disability Status Scale (EDSS) score within 30 days after onset of an attack, and follow-up EDSS 6 months after attack. Adult patients with multiple sclerosis (n = 632) were identified from the Comprehensive Longitudinal Investigations in Multiple Sclerosis at Brigham study (CLIMB), and paediatric patients (n = 132) from the US Network of Paediatric Multiple Sclerosis Centers (NPMSC) registry. Change in EDSS was defined as the difference in EDSS between attack and follow-up. Change in EDSS at follow-up compared to baseline was significantly lower in children compared to adults (P = 0.001), as were several functional system scores. Stratification by decade at onset for change in EDSS versus age found for every 10 years of age, EDSS recovery is reduced by 0.15 points (P < 0.0001). A larger proportion of children versus adults demonstrated improvement in EDSS following an attack (P = 0.006). For every 10 years of age, odds of EDSS not improving increase by 1.33 times (P < 0.0001). Younger age is associated with improved recovery from relapses. Age-related mechanisms may provide novel therapeutic targets for disability accrual in multiple sclerosis.

Objective To assess real‐world effectiveness of initial treatment with newer compared to injectable disease‐modifying therapies (DMTs) on disease activity in pediatric multiple sclerosis (MS) and clinically isolated syndrome (CIS). Methods This is a cohort study of children with MS/CIS followed at 12 clinics in the US Network of Pediatric MS Centers, who received initial therapy with newer (fingolimod, dimethyl fumarate, teriflunomide, natalizumab, rituximab, ocrelizumab) or injectable (interferon‐β, glatiramer acetate) DMTs. Propensity scores (PSs) were computed, including preidentified confounders. Relapse rate while on initial DMT was modeled with negative binomial regression, adjusted for PS‐quintile. Time to new/enlarging T2‐hyperintense and gadolinium‐enhancing lesions on brain magnetic resonance imaging were modeled with midpoint survival analyses, adjusted for PS‐quintile. Results A total of 741 children began therapy before 18 years, 197 with newer and 544 with injectable DMTs. Those started on newer DMTs were older (15.2 vs injectable 14.4 years, p = 0.001) and less likely to have a monofocal presentation. In PS‐quintile–adjusted analysis, those on newer DMTs had a lower relapse rate than those on injectables (rate ratio = 0.45, 95% confidence interval (CI) = 0.29–0.70, p < 0.001; rate difference = 0.27, 95% CI = 0.14–0.40, p = 0.004). One would need to treat with newer rather than injectable DMTs for 3.7 person‐years to prevent 1 relapse. Those started on newer DMTs had a lower rate of new/enlarging T2 (hazard ratio [HR] = 0.51, 95% CI = 0.36–0.72, p < 0.001) and gadolinium‐enhancing lesions (HR = 0.38, 95% CI = 0.23–0.63, p < 0.001) than those on injectables. Interpretation Initial treatment of pediatric MS/CIS with newer DMTs led to better disease activity control compared to injectables, supporting greater effectiveness of newer therapies. Long‐term safety data for newer DMTs are required. ANN NEUROL 2020 ANN NEUROL 2020;88:42–55

Mice expressing the H2b haplotype are resistant to infection with Theiler's murine encephalomyelitis virus (TMEV), which causes chronic demyelination in susceptible mice. The prominent cytolytic T-lymphocyte (CTL) response to the VP2 antigen encoded by TMEV led us to the identification of a class I-binding peptide derived from the VP2 antigen. Escherichia coli transformants overexpressing a series of 11 overlapping VP2 protein fragments were subjected to lysis and alkali digestion, and the resultant peptide pools were tested for their abilities to sensitize RMA-S targets for lysis by CTLs. The source of effector CD8+ T cells for the assays was either freshly harvested central nervous system-infiltrating lymphocytes (CNS-IL) or CNS-IL-derived VP2-specific CTL clones and lines. A 10-residue peptide at VP2 positions 121 to 130 (VP2(121-130)) (FHAGSLLVFM) was identified that sensitized targets for lysis and formed stable complexes with H2Db class I molecules. The VP2(121-130) peptide sensitized target cells for lysis by freshly harvested CNS-IL CTLs at femtomolar concentrations. Despite its relative high level of biological activity, the VP2(121-130) peptide is distinguished from other Db-binding peptides by its lack of an asparagine residue at position five, which had been previously proposed to be a requirement for Db-peptide complexing.