National Cancer Institute Collaborative Workshop on Shaping the Landscape of Brain Metastases Research: challenges and recommended priorities.

Brain metastases are an increasing global public health concern, even as survival rates improve for patients with metastatic disease. Both metastases and the sequelae of their treatment are key determinants of the inter-related priorities of patient survival, function, and quality of life, mandating a multidimensional approach to clinical care and research. At a virtual National Cancer Institute Workshop in September, 2022, key stakeholders convened to define research priorities to address the crucial areas of unmet need for patients with brain metastases to achieve meaningful advances in patient outcomes. This Policy Review outlines existing knowledge gaps, collaborative opportunities, and specific recommendations regarding consensus priorities and future directions in brain metastases research. Achieving major advances in research will require enhanced coordination between the ongoing efforts of individual organisations and consortia. Importantly, the continual and active engagement of patients and patient advocates will be necessary to ensure that the directionality of all efforts reflects what is most meaningful in the context of patient care.

Differentiating pseudoprogression from true progression: analysis of radiographic, biologic, and clinical clues in GBM.

INTRODUCTION: Pseudoprogression (PsP) is a diagnostic dilemma in glioblastoma (GBM) after chemoradiotherapy (CRT). Magnetic resonance imaging (MRI) features may fail to distinguish PsP from early true progression (eTP), however clinical findings may aid in their distinction. METHODS: Sixty-seven patients received CRT for GBM between 2003 and 2016, and had pre- and post-treatment imaging suitable for retrospective evaluation using RANO criteria. Patients with signs of progression within the first 12-weeks post-radiation (P-12) were selected. Lesions that improved or stabilized were defined as PsP, and lesions that progressed were defined as eTP. RESULTS: The median follow up for all patients was 17.6 months. Signs of progression developed in 35/67 (52.2%) patients within P-12. Of these, 20/35 (57.1%) were subsequently defined as eTP and 15/35 (42.9%) as PsP. MRI demonstrated increased contrast enhancement in 84.2% of eTP and 100% of PsP, and elevated CBV in 73.7% for eTP and 93.3% for PsP. A decrease in FLAIR was not seen in eTP patients, but was seen in 26.7% PsP patients. Patients with eTP were significantly more likely to require increased steroid doses or suffer clinical decline than PsP patients (OR 4.89, 95% CI 1.003-19.27; p = 0.046). KPS declined in 25% with eTP and none of the PsP patients. CONCLUSIONS: MRI imaging did not differentiate eTP from PsP, however, KPS decline or need for increased steroids was significantly more common in eTP versus PsP. Investigation and standardization of clinical assessments in response criteria may help address the diagnostic dilemma of pseudoprogression after frontline treatment for GBM.

Radiation-Induced Alteration of the Brain Proteome: Understanding the Role of the Sirtuin 2 Deacetylase in a Murine Model.

Whole brain radiotherapy (WBRT) produces unwanted sequelae, albeit via unknown mechanisms. A deacetylase expressed in the central nervous system, Sirtuin 2 (SIRT2), has been linked to neurodegeneration. Therefore, we sought to challenge the notion that a single disease pathway is responsible for radiation-induced brain injury in Sirt2 wild-type (WT) and knockout (KO) mice at the proteomic level. We utilized isobaric tag for relative and absolute quantitation to analyze brain homogenates from Sirt2 WT and KO mice with and without WBRT. Selected proteins were independently verified, followed by ingenuity pathway analysis. Canonical pathways for Huntington's, Parkinson's, and Alzheimer's were acutely affected by radiation within 72 h of treatment. Although loss of Sirt2 preferentially affected both Huntington's and Parkinson's pathways, WBRT most significantly affected Huntington's-related proteins in the absence of Sirt2. Identical protein expression patterns were identified in Mog following WBRT in both Sirt2 WT and KO mice, revealing a proteomic radiation signature; however, long-term radiation effects were found to be associated with altered levels of a small number of key neurodegeneration-related proteins, identified as Mapt, Mog, Snap25, and Dnm1. Together, these data demonstrate the principle that the presence of Sirt2 can have significant effects on the brain proteome and its response to ionizing radiation.

The sex-dependent impact of PER2 polymorphism on sleep and activity in a novel mouse model of cranial-irradiation-induced hypersomnolence.

Background: Hypersomnolence is a common and disruptive side effect of cranial radiotherapy and is associated with fatigue and disturbances in mood and cognition in primary brain tumor (PBT) patients. The biological underpinnings of this effect are not understood. Our laboratory has previously found that the presence of a single nucleotide polymorphism (rs934945, G-E mutation) in the PERIOD2 (PER2) clock gene was associated with a decreased likelihood of fatigue in PBT patients. Here, we aim to understand the effects of PER2 polymorphism on radiation susceptibility within a murine model of cranial-irradiation-induced hypersomnolence (C-RIH). Methods: Male and female transgenic mice were generated using CRISPR-Cas9, replacing the endogenous mouse PER2:CRY1 binding domain with its human isoform with (hE1244 KI) or without the SNP rs934945 (hG1244 KI). Activity and sleep were monitored continuously 10 days before and after cranial irradiation (whole brain, 15Gy, single fraction). Behavioral assessments measuring anxiety, depression, and working memory were used to assess mood and cognitive changes 2 months postradiation. Results: During their active phase, hE1244 knock-ins (KIs) had less radiation-induced suppression of activity relative to hG1244 KIs and female hE1244 KIs saw a reduction of hypersomnolence over 10 days. hE1244 KIs displayed less anxiety behavior and were more ambulatory within all behavioral tests. Conclusions: The PER2 rs934945 polymorphism had long-lasting behavioral effects associated with radiation toxicity, particularly in sleep in females and the activity of all animals. Our findings shed light on biological mechanisms underlying C-RIH.

Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach.

We tested the hypothesis that the effects on gene expression of altered DNA methylation by 5-aza-2'-deoxycytidine (5-aza-CdR) and genetic (DNMT knockout) manipulation of DNA are similar, and distinct from Trichostatin A (TSA)-induced chromatin decondensation. Surprisingly, the effects of 5-aza-CdR were more similar to those of TSA than to DNMT1, DNMT3B, or double DNMT somatic cell knockout. Furthermore, the effects of 5-aza-CdR were similar at one and five days exposure, suggesting active demethylation or direct influence of both drugs on the stability of methylation and/or chromatin marks. Agents that induce gene activation through hypomethylation may have unintended consequences, since nearly as many genes were downregulated as upregulated after demethylation. In addition, a 75 kb cluster of metallothionein genes was coordinately regulated.

Histological analysis of sleep and circadian brain circuitry in cranial radiation-induced hypersomnolence (C-RIH) mouse model.

Disrupted sleep, including daytime hypersomnolence, is a core symptom reported by primary brain tumor patients and often manifests after radiotherapy. The biological mechanisms driving the onset of sleep disturbances after cranial radiation remains unclear but may result from treatment-induced injury to neural circuits controlling sleep behavior, both circadian and homeostatic. Here, we develop a mouse model of cranial radiation-induced hypersomnolence which recapitulates the human experience. Additionally, we used the model to explore the impact of radiation on the brain. We demonstrated that the DNA damage response following radiation varies across the brain, with homeostatic sleep and cognitive regions expressing higher levels of γH2AX, a marker of DNA damage, than the circadian suprachiasmatic nucleus (SCN). These findings were supported by in vitro studies comparing radiation effects in SCN and cortical astrocytes. Moreover, in our mouse model, MRI identified structural effects in cognitive and homeostatic sleep regions two-months post-treatment. While the findings are preliminary, they suggest that homeostatic sleep and cognitive circuits are vulnerable to radiation and these findings may be relevant to optimizing treatment plans for patients.

Impact of age on the circadian visual system and the sleep-wake cycle in mus musculus.

Age plays a critical role in disease development and tolerance to cancer treatment, often leading to an increased risk of developing negative symptoms including sleep disturbances. Circadian rhythms and sleep become disrupted as organisms age. In this study, we explored the behavioral alterations in sleep, circadian rhythms, and masking using a novel video system and interrogate the long-term impact of age-based changes in the non-image forming visual pathway on brain anatomy. We demonstrated the feasibility and utility of the novel system and establish that older mice have disruptions in sleep, circadian rhythms, and masking behaviors that were associated with major negative volume alterations in the non-imaging forming visual system, critical for the induction and rhythmic expression of sleep. These results provide important insights into a mechanism, showing brain atrophy is linked to age in distinct non-image forming visual regions, which may predispose older individuals to developing circadian and sleep dysfunction when further challenged by disease or treatment.

Clonal Evolution and Heterogeneity of Osimertinib Acquired Resistance Mechanisms in EGFR Mutant Lung Cancer.

Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies.

Sirt2 Regulates Radiation-Induced Injury.

Sirtuin 2 (SIRT2) plays a major role in aging, carcinogenesis and neurodegeneration. While it has been shown that SIRT2 is a mediator of stress-induced cell death, the mechanism remains unclear. In this study, we report the role of SIRT2 in mediating radiation-induced cell death and DNA damage using mouse embryonic fibroblasts (MEFs), progenitor cells and tissues from Sirt2 wild-type and genomic knockout mice, and human tumor and primary cell lines as models. The presence of Sirt2 in cells and tissues significantly enhanced the cell's sensitivity to radiation-induced cytotoxicity by delaying the dispersion of radiation-induced γ-H2AX and 53BP1 foci. This enhanced cellular radiosensitivity correlated with reduced expression of pro-survival and DNA repair proteins, and decreased DNA repair capacities involving both homologous repair and non-homologous end joining DNA repair mechanisms compared to those in Sirt2 knockout (KO) and knockdown (KD) phenotypes. Together, these data suggest SIRT2 plays a critical role in mediating the radiation-induced DNA damage response, thus regulating radiation-induced cell death and survival.

Thioredoxin reductase as a potential molecular target for anticancer agents that induce oxidative stress.

Redox-sensitive signaling factors regulate multiple cellular processes, including proliferation, cell cycle, and prosurvival signaling cascades, suggesting their potential as molecular targets for anticancer agents. It is logical to set constraints that a molecular target should meet at least one of the following criteria: (1) inhibition of prosurvival signaling pathways; (2) inhibition of cell cycle progression; or (3) enhancement of the cytotoxic effects of anticancer agents. Therefore, we hypothesized that thioredoxin reductase 1 (TR), a component of several redox-regulated pathways, might represent a potential molecular target candidate in response to agents that induce oxidative stress. To address this issue, permanent cell lines overexpressing either the wild-type (pCXN2-myc-TR-wt) or a Cys-Ser mutant (pCXN2-myc-mTR) TR gene were used, as were parental HeLa cells treated with 1-methyl-1-propyl-2-imidazolyl disulfide (IV-2), a pharmacologic inhibitor of TR. Cells were exposed to the oxidative stressors, H2O2 and ionizing radiation (IR), and analyzed for changes in signal transduction, cell cycle, and cytotoxicity. Analysis of HeLa cells overexpressing the pCXN2-myc-TR-wt gene showed increased basal activity of nuclear factor kappaB (NFkappaB) and activator protein (AP-1), whereas HeLa cells expressing a pCXN2-myc-mTR gene and HeLa cells treated with IV-2 were unable to induce NFkappaB or AP-1 activity following H2O2 or IR exposure. Fluorescence-activated cell sorting analysis showed a marked accumulation of pCXN2-myc-mTR cells in the late G1 phase, whereas pCXN2-myc-TR-wt cells showed a decreased G1 subpopulation. Chemical inhibition of TR with IV-2 also completely inhibited cellular proliferation at concentrations between 10 and 25 micromol/L, resulting in a G1 phase cell cycle arrest consistent with the results from cells expressing the pCXN2-myc-mTR gene. Following exposure to H2O2 and IR, pCXN2-myc-mTR- and IV-2-treated cells were significantly more sensitive to oxidative stress-induced cytotoxicity as measured by clonogenic survival assays. Finally, IV-2-treated cells showed increased tumor cell death when treated with H2O2 and IR. These results identify TR as a potential target to enhance the cytotoxic effects of agents that induce oxidative stress, including IR.

SIRT2 interacts with ß-catenin to inhibit Wnt signaling output in response to radiation-induced stress.

UNLABELLED: Wnt signaling is critical to maintaining cellular homeostasis via regulation of cell division, mitigation of cell stress, and degradation. Aberrations in Wnt signaling contribute to carcinogenesis and metastasis, whereas sirtuins have purported roles in carcinogenesis, aging, and neurodegeneration. Therefore, the hypothesis that sirtuin 2 (SIRT2) directly interacts with ß-catenin and whether this interaction alters the expression of Wnt target genes to produce an altered cellular phenotype was tested. Coimmunoprecipitation studies, using mouse embryonic fibroblasts (MEF) from Sirt2 wild-type and genomic knockout mice, demonstrate that ß-catenin directly binds SIRT2. Moreover, this interaction increases in response to oxidative stress induced by ionizing radiation. In addition, this association inhibits the expression of important Wnt target genes such as survivin (BIRC5), cyclin D1 (CCND1), and c-myc (MYC). In Sirt2 null MEFs, an upregulation of matrix metalloproteinase 9 (MMP9) and decreased E-cadherin (CDH1) expression is observed that produces increased cellular migration and invasion. Together, these data demonstrate that SIRT2, a tumor suppressor lost in multiple cancers, inhibits the Wnt signaling pathway in nonmalignant cells by binding to ß-catenin and that SIRT2 plays a critical role in the response to oxidative stress from radiation. IMPLICATIONS: Disruption of the SIRT2-ß-catenin interaction represents an endogenous therapeutic target to prevent transformation and preserve the integrity of aging cells against exogenous stressors such as reactive oxygen species.

Caveolin-1 expression is maintained in rat and human astroglioma cell lines.

Caveolin-1 is the principal structural and functional component of caveolae, a plasmalemmal compartment that has been proposed to sequester lipid and protein components that participate in transmembrane signal transduction processes. Multiple studies reveal a reduction in the expression level of caveolin-1 mRNA and protein in many carcinomas as well as transformed cells. The human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). Collectively, these data have been taken to imply that caveolin-1 may function in a tumor suppressor capacity. To determine if a reduction in the expression level of caveolin-1 mRNA and protein accompanied the transformation of astrocytes, we undertook studies of two transformed rat astroglial cell lines, C6 and DI TNC(1), as well as several cell lines derived from human glioblastoma tumors: T98G, U87MG, U118MG, U138MG, and U373MG. Ultrastructural, immunolocalization, immunoblot, and Northern blot analyses demonstrated that caveolin-1 message and protein were expressed in all rat and human glioma cells. The localization pattern, buoyant density, and detergent-insolubility property of caveolin-1 protein were indistinguishable from that determined for nontransformed type 1 astrocytes in culture. Nucleotide sequence analyses of caveolin-1 cDNAs indicate that mutations are not present in the caveolin-1 sequence in any of the glioma cell types. Taken together with previous analyses, these data indicate that, at least for astrocytes, the process of transformation in and of itself is not solely sufficient to reduce the level of caveolin-1 expression, and that caveolin-1 expression in and of itself is not solely sufficient to prevent the acquisition of a transformed phenotype.