Temozolomide (TMZ) is an important first-line treatment for glioblastoma (GBM), but there are limitations to TMZ response in terms of durability and dependence on the promoter methylation status of ...the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT-promoter-hypermethylated (MGMT-M) GBMs are more sensitive to TMZ than MGMT-promoter-hypomethylated (MGMT-UM) GBMs. Moreover, TMZ resistance is inevitable even in TMZ-sensitive MGMT-M GBMs. Hence, epigenetic reprogramming strategies are desperately needed in order to enhance TMZ response in both MGMT-M and MGMT-UM GBMs. In this study, we present novel evidence that the epigenetic reactivation of Tumor Suppressor Candidate 3 (TUSC3) can reprogram sensitivity of GBM stem cells (GSCs) to TMZ irrespective of MGMT promoter methylation status. Interrogation of TCGA patient GBM datasets confirmed TUSC3 promoter regulation of TUSC3 expression and also revealed a strong positive correlation between TUSC3 expression and GBM patient survival. Using a combination of loss-of-function, gain-of-function and rescue studies, we demonstrate that TUSC3 reactivation is associated with enhanced TMZ response in both MGMT-M and MGMT-UM GSCs. Further, we provide novel evidence that the demethylating agent 5-Azacitidine (5-Aza) reactivates TUSC3 expression in MGMT-M GSCs, whereas the combination of 5-Aza and MGMT inhibitor Lomeguatrib is necessary for TUSC3 reactivation in MGMT-UM GSCs. Lastly, we propose a pharmacological epigenetic reactivation strategy involving TUSC3 that leads to significantly prolonged survival in MGMT-M and MGMT-UM orthotopic GSCs models. Collectively, our findings provide a framework and rationale to further explore TUSC3-mediated epigenetic reprogramming strategies that could enhance TMZ sensitivity and outcomes in GBM. Mechanistic and translational evidence gained from such studies could contribute towards optimal design of impactful trials for MGMT-UM GBMs that currently do not have good treatment options.
Stemness reprogramming remains a largely unaddressed principal cause of lethality in glioblastoma (GBM). It is therefore of utmost importance to identify and target mechanisms that are essential for ...GBM stemness and self-renewal. Previously, we implicated BIRC3 as an essential mediator of therapeutic resistance and survival adaptation in GBM. In this study, we present novel evidence that BIRC3 has an essential noncanonical role in GBM self-renewal and stemness reprogramming. We demonstrate that BIRC3 drives stemness reprogramming of human GBM cell lines, mouse GBM cell lines and patient-derived GBM stem cells (GSCs) through regulation of BMP4 signaling axis. Specifically, BIRC3 induces stemness reprogramming in GBM through downstream inactivation of BMP4 signaling. RNA-Seq interrogation of the stemness reprogramming hypoxic (pseudopalisading necrosis and perinecrosis) niche in GBM patient tissues further validated the high BIRC3/low BMP4 expression correlation. BIRC3 knockout upregulated BMP4 expression and prevented stemness reprogramming of GBM models. Furthermore, siRNA silencing of BMP4 restored stemness reprogramming of BIRC3 knockout in GBM models. In vivo silencing of BIRC3 suppressed tumor initiation and progression in GBM orthotopic intracranial xenografts. The stemness reprograming of both GSCs and non-GSCs populations highlights the impact of BIRC3 on intra-tumoral cellular heterogeneity GBM. Our study has identified a novel function of BIRC3 that can be targeted to reverse stemness programming of GBM.
Summary Background Despite its common use in cancer treatment, radiotherapy has not yet entered the era of precision medicine, and there have been no approaches to adjust dose based on biological ...differences between or within tumours. We aimed to assess whether a patient-specific molecular signature of radiation sensitivity could be used to identify the optimum radiotherapy dose. Methods We used the gene-expression-based radiation-sensitivity index and the linear quadratic model to derive the genomic-adjusted radiation dose (GARD). A high GARD value predicts for high therapeutic effect for radiotherapy; which we postulate would relate to clinical outcome. Using data from the prospective, observational Total Cancer Care (TCC) protocol, we calculated GARD for primary tumours from 20 disease sites treated using standard radiotherapy doses for each disease type. We also used multivariable Cox modelling to assess whether GARD was independently associated with clinical outcome in five clinical cohorts: Erasmus Breast Cancer Cohort (n=263); Karolinska Breast Cancer Cohort (n=77); Moffitt Lung Cancer Cohort (n=60); Moffitt Pancreas Cancer Cohort (n=40); and The Cancer Genome Atlas Glioblastoma Patient Cohort (n=98). Findings We calculated GARD for 8271 tissue samples from the TCC cohort. There was a wide range of GARD values (range 1·66–172·4) across the TCC cohort despite assignment of uniform radiotherapy doses within disease types. Median GARD values were lowest for gliomas and sarcomas and highest for cervical cancer and oropharyngeal head and neck cancer. There was a wide range of GARD values within tumour type groups. GARD independently predicted clinical outcome in breast cancer, lung cancer, glioblastoma, and pancreatic cancer. In the Erasmus Breast Cancer Cohort, 5-year distant-metastasis-free survival was longer in patients with high GARD values than in those with low GARD values (hazard ratio 2·11, 95% 1·13–3·94, p=0·018). Interpretation A GARD-based clinical model could allow the individualisation of radiotherapy dose to tumour radiosensitivity and could provide a framework to design genomically-guided clinical trials in radiation oncology. Funding None.
We previously developed a multigene expression model of tumor radiation sensitivity index (RSI) with clinical validation in multiple independent cohorts (breast, rectal, esophageal, and head and neck ...patients). The purpose of this study was to assess differences between RSI scores in primary colon cancer and metastases.
Patients were identified from our institutional review board-approved prospective observational protocol. A total of 704 metastatic and 1362 primary lesions were obtained from a de-identified metadata pool. RSI was calculated using the previously published rank-based algorithm. An independent cohort of 29 lung or liver colon metastases treated with 60 Gy in 5 fractions stereotactic body radiation therapy (SBRT) was used for validation.
The most common sites of metastases included liver (n=374; 53%), lung (n=116; 17%), and lymph nodes (n=40; 6%). Sixty percent of metastatic tumors, compared with 54% of primaries, were in the RSI radiation-resistant peak, suggesting metastatic tumors may be slightly more radiation resistant than primaries (P=.01). In contrast, when we analyzed metastases based on anatomical site, we uncovered large differences in RSI. The median RSIs for metastases in descending order of radiation resistance were ovary (0.48), abdomen (0.47), liver (0.43), brain (0.42), lung (0.32), and lymph nodes (0.31) (P<.0001). These findings were confirmed when the analysis was restricted to lesions from the same patient (n=139). In our independent cohort of treated lung and liver metastases, lung metastases had an improved local control rate compared to that in patients with liver metastases (2-year local control rate of 100% vs 73.0%, respectively; P=.026).
Assessment of radiation sensitivity between primary and metastatic tissues of colon cancer histology revealed significant differences based on anatomical location of metastases. These initial results warrant validation in a larger clinical cohort.
We have interrogated a 12-chemokine gene expression signature (GES) on genomic arrays of 14,492 distinct solid tumors and show broad distribution across different histologies. We hypothesized that ...this 12-chemokine GES might accurately predict a unique intratumoral immune reaction in stage IV (non-locoregional) melanoma metastases. The 12-chemokine GES predicted the presence of unique, lymph node-like structures, containing CD20⁺ B cell follicles with prominent areas of CD3⁺ T cells (both CD4⁺ and CD8⁺ subsets). CD86⁺, but not FoxP3⁺, cells were present within these unique structures as well. The direct correlation between the 12-chemokine GES score and the presence of unique, lymph nodal structures was also associated with better overall survival of the subset of melanoma patients. The use of this novel 12-chemokine GES may reveal basic information on in situ mechanisms of the anti-tumor immune response, potentially leading to improvements in the identification and selection of melanoma patients most suitable for immunotherapy.
We assessed the radiosensitivity of lung metastases on the basis of primary histologic type by using a validated gene signature and model lung metastases for the gnomically adjusted radiation dose ...(GARD).
Tissue samples were identified from our prospective observational protocol. The radiosensitivity index (RSI) 10-gene assay was run on samples and calculated alongside the GARD by using the previously published algorithms. A cohort of 105 patients with 137 lung metastases treated with stereotactic body radiation therapy (SBRT) at our institution was used for clinical correlation.
A total of 138 unique metastatic lung lesions from our institution’s tissue biorepository were identified for inclusion. There were significant differences in the RSI of lung metastases on the basis of histology. In order of decreasing radioresistance, the median RSIs for the various histologic types of cancer were endometrial adenocarcinoma (0.49), soft-tissue sarcoma (0.47), melanoma (0.44), rectal adenocarcinoma (0.43), renal cell carcinoma (0.33), head and neck squamous cell cancer (0.33), colon adenocarcinoma (0.32), and breast adenocarcinoma (0.29) (p = 0.002). We modeled the GARD for these samples and identified the biologically effective dose necessary to optimize local control. The 12- and 24-month Kaplan-Meier rates of local control for radioresistant versus radiosensitive histologic types from our clinical correlation cohort after lung SBRT were 92%/87% and 100%, respectively (p = 0.02).
In this analysis, we have noted significant differences in radiosensitivity on the basis of primary histologic type of lung metastases and have modeled the biologically effective dose necessary to optimize local control. This study suggests that primary histologic type may be an additional factor to consider in selection of SBRT dose to the lung and that dose personalization may be feasible.
Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic ...mechanism of action, we systematically analyzed CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo antitumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of costimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development of novel optimized immune receptors.
In sex role reversed species, predominantly females evolve sexually selected traits, such as ornaments and/or weapons. Female ornaments are common and their function well documented in many species, ...whether sex role reversed or not. However, sexually selected female weapons seem totally absent except for small wing spurs in three jacana species, present in both males and females. This poor female weaponry is in sharp contrast to the situation in species with conventional sex roles： males com- monly have evolved sexually selected weapons as well as ornaments. At the same time, females in many taxa have naturally se- lected weapons, used in competition over resources or in predator defence. Why are sexually selected weapons then so rare, al- most absent, in females？ Here I briefly review weaponry in females and the function of these weapons, conclude that the near ab- sence of sexually selected weapons begs an explanation, and suggest that costs of sexually selected weapons may exceed costs of ornaments. Females are more constrained when evolving sexually selected traits compared to males, at least compared to those males that do not provide direct benefits, as trait costs reduce a female＇s fecundity. I suggest that this constraining trade-off between trait and fecundity restricts females to evolve ornaments but rarely weapons. The same may apply to paternally investing males. Whether sexually selected weapons actually are more costly than sexually selected ornaments remains to be investigated
While STING-activating agents have shown limited efficacy in early-phase clinical trials, multiple lines of evidence suggest the importance of tumor cell-intrinsic STING function in mediating ...antitumor immune responses. Although STING signaling is impaired in human melanoma, its restoration through epigenetic reprogramming can augment its antigenicity and T cell recognition. In this study, we show that reversal of methylation silencing of STING in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type-I IFN induction, which, in tumor-bearing mice, can induce tumor regression through a CD8
T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest that pharmacological restoration of STING signaling through epigenetic reprogramming might improve the therapeutic efficacy of STING agonists.
Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to ...chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.
PDF
