Purpose: Malignant peripheral nerve sheath tumors (MPNSTs) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin dependent kinases (CDKs), commonly through loss of CDK inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs. Experimental Design:We examined patient-matched MPNSTs and precursor lesions by RNA-Seq and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors.Results: RABL6A was dramatically upregulated in human MPNSTs compared to precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G1 arrest that coincided with p27 upregulation, CDK downregulation and RB1 activation. The growth suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in vitro in a RABL6A-dependent manner and suppressed MPNST growth in vivo. Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced anti-tumorigenic activity associated with potential MPNST cell redifferentiation.Conclusions: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.

In this study, diethylnitrosamine-treated male mice were assigned to 3 groups: a 35% high fat ethanol liquid diet (EtOH) with casein as the protein source, the same EtOH liquid diet with soy protein isolate as the sole protein source (EtOH/SPI) and a chow group. EtOH feeding continued for 16 wks. As expected, EtOH increased the incidence and multiplicity of basophilic lesions and adenomas compared to the chow group, p<0.05. Soy protein replacement of casein in the EtOH diet significantly reduced adenoma progression when compared to the EtOH and EtOH/SPI group, p<0.05. Tumor reduction in the EtOH/SPI group corresponded to reduced liver injury associated with decreased hepatic tumor necrosis factor α (Tnfα) and Cd14 antigen (Cd14) expression and decreased nuclear accumulation of NFκB1 protein compared to the EtOH group (p<0.05). Detection of sphingolipids using high resolution MALDI-FTICR Imaging mass spectrometry revealed increased accumulation of long acyl chain ceramide species, and sphingosine-1-phosphate (S1P) in the EtOH group that were significantly reduced in the EtOH/SPI group. Chronic EtOH feeding also increased mRNA expression of β-catenin transcriptional targets, including cyclin D1 (Ccnd1), matrix metallopeptidase 7 (Mmp7) and glutamine synthetase (Glns), which were reduced in the EtOH/SPI group, p<0.05. We conclude that soy prevents tumorigenesis by reducing pro-inflammatory and oxidative environment resulting from EtOH-induced hepatic injury, and by reducing hepatocyte proliferation through inhibition of β-catenin signaling. These mechanisms may involve changes in sphingolipid signaling.

Background Chronic alcohol consumption leads to increased fracture risk and an elevated risk of osteoporosis by decreasing bone accrual through increasing osteoclast activity and decreasing osteoblast activity. We have shown that this mechanism involves the generation of reactive oxygen species (ROS) produced by NADPH oxidases (NOX). It was hypothesized that different dietary antioxidants, N-acetyl cysteine (NAC, 1.2mg/kg/d) and α-tocopherol (VitE, 60 mg/kg/d)) would be able to attenuate the NOX-mediated ROS effects on bone due to chronic alcohol intake. Methods To study the effects of these antioxidants, female mice received a Lieber DeCarli liquid diet containing ethanol (EtOH) with or without additional antioxidant for 8 weeks. Results Tibias displayed decreased cortical bone mineral density in both the EtOH and EtOH+antioxidant groups compared to pair-fed (PF) and PF+antioxidant groups (P<0.05). However, there was significant protection from trabecular bone loss in mice fed either antioxidant (P<0.05). MicroCT analysis demonstrated a significant decrease in bone volume (BV/TV) and trabecular number (Tb.N) (P<0.05), along with a significant increase in trabecular spacing (Tb.Sp) in the EtOH compared to PF (P<0.05). In contrast, the EtOH+NAC and EtOH+α-tocopherol did not statistically differ from their respective PF controls. Ex vivo histological sections of tibias were stained for nitrotyrosine, an indicator of intracellular damage by ROS, and tibias from mice fed EtOH exhibited significantly more staining than PF controls. EtOH treatment significantly increased the number of marrow adipocytes per mm as well as mRNA expression of aP2, an adipocyte marker in bone. Only NAC was able to reduce the number of marrow adipocytes to PF levels. EtOH fed mice exhibited reduced bone length (P<0.05) and had a reduced number of proliferating chondrocytes within the growth plate. NAC and Vitamin E prevented this (P<0.05). Conclusions These data show that alcohol’s pathological effects on bone extend beyond decreasing bone mass and suggest a partial protective effect of the dietary antioxidants NAC and α-tocopherol at these doses with regard to alcohol effects on bone turnover and bone morphology.

Background Ultra-high dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising anti-tumor efficacy compared to conventional dose rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes. Methods Cohorts of three-week-old male and female C57Bl/6 mice exposed to hypofractionated (2×10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months post-treatment. Animals were sacrificed 6 months post-irradiation and tissues analyzed for neurological and cerebrovascular decrements. Results The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin) and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and a preservation of cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels. Conclusions Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlight its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non–small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine– and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide–dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974).