A NOVEL ROLE FOR SMAD6 IN THE BIOLOGY OF PANCREATIC CANCER

Pancreatic cancer is a nearly uniformly lethal disease, with the five-year survival rate remaining a dismal five percent. Recently, our laboratory has associated the loss of SMAD4, a critical mediator of TGF-β and BMP signaling, with the presence of widely disseminated pancreatic cancer. Therefore, we sought to obtain a better understanding of these pathways in the biology of pancreatic cancer through analysis of SMAD4 loss and SMAD6 elevation. To gain a better understanding of the role of SMAD4 loss in pancreatic cancer, we established isogenic cell lines from pancreatic cancer cells that were SMAD4-deficient, such that these cell lines were SMAD4-complemented. We were able to show that restoration of SMAD4 into these cell lines hindered their ability to proliferate, migrate, and invade through matrigel in vitro. Furthermore, we sought to gain an understanding of the utility of SMAD4 as a marker for the efficacy of chemotherapy. We have shown that SMAD4 status is predictive of a small, but statistically significant, in vitro response to cisplatin and irinotecan and resistance to gemcitabine. Second, we identified SMAD6, previously described as an inhibitor of both TGF-β and BMP, as being differentially expressed in pancreatic cancer cell lines and tissues. SMAD6 expression was found not to be regulated by genetic mutation or degree of RNA transcription, but through aberrant proteolytic degradation. It is through loss of this regulatory mechanism that SMAD6 expression becomes elevated, leading to increased levels of proliferation, migration, and invasion through matrigel, in vitro, and increased metastatic burden in patients. Despite its previously described role as an inhibitor of TGF-β and BMP signaling, we have determined that the effects of SMAD6 on pancreatic cancer cells are independent of this function. Rather, we have identified a nuclear role for SMAD6: SMAD6 is able to bind to DNA in pancreatic cancer, and is constitutively located in the nucleus of pancreatic cancer cells. Therefore, both SMAD4 loss and SMAD6 overexpression negatively impact the biology of pancreatic cancer, albeit through different mechanisms. Further study will continue to elucidate the role of these proteins in the biology of pancreatic cancer

ATRX loss in pediatric glioma results in epigenetic dysregulation of G2/M checkpoint maintenance and sensitivity to ATM inhibition

ATRX is a histone chaperone protein recurrently mutated in pediatric glioma. The mechanism which mediates the proliferative advantage of ATRX loss in pediatric glioma remains unexplained. Recent data revealed a distinct pattern of DNA binding sites of the ATRX protein using ChIP-seq in mouse neuronal precursor cells (mNPCs). Using the ATRX peaks identified in p53-/- mNPCs, we confirmed that ATRX binding sites were significantly enriched in gene promoters (p \u3c 0.0001) and CpG islands (p \u3c 0.0001) compared with random regions. Gene set enrichment (GSE) analysis identified that cell cycle and regulation of cell cycle were among the most significantly enriched gene sets (p=2.52e-16 and 1.61e-9, respectively). We found that ATRX loss resulted in dysfunction of G2/M checkpoint maintenance: (1) ATRX-deficient pediatric glioblastoma (GBM) cells exhibited a seven-fold increase in mitotic index at 16 hours after sub-lethal radiation, and (2) murine GBM cells with ATRX knockdown demonstrated impaired pChk1 signaling on western blot at multiple time points after radiation compared to controls (p=0.0187). Notably, the ATM signaling (pChk2) remained intact in those cells, suggesting a potential therapeutic target. ATRX-deficient mouse cells were uniquely sensitive to ATM inhibitors at 1 uM alongside 8 Gy radiation compared to controls with intact ATRX (AZD0156: p=0.0027 and AZD01390: p=0.0436). Mice intra-cranially implanted with ATRX-deficient GBM cells showed improved survival (n=10, p=0.0018) when treated with AZD0156 combined with radiation. Our findings suggest that ATRX loss in glioma results in unique sensitivity to ATM inhibition via epigenetic dysregulation of G2/M checkpoint maintenance

Alternative lengthening of telomeres, ATRX loss and H3â K27M mutations in histologically defined pilocytic astrocytoma with anaplasia

Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fiftyâ seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3â 75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomereâ specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3â K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRXâ (20/24, 83%) or ALTâ /ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3â K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PAâ A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3â K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147190/1/bpa12646_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147190/2/bpa12646.pd

ATRX-Deficient High-Grade Glioma Cells Exhibit Increased Sensitivity to RTK and PDGFR Inhibitors

High-grade glioma, including anaplastic astrocytoma and glioblastoma (GBM) patients, have a poor prognosis due to the lack of effective treatments. Therefore, the development of new therapeutic strategies to treat these gliomas is urgently required. Given that high-grade gliomas frequently harbor mutations in the SNF2 family chromatin remodeler ATRX, we performed a screen to identify FDA-approved drugs that are toxic to ATRX-deficient cells. Our findings reveal that multi-targeted receptor tyrosine kinase (RTK) and platelet-derived growth factor receptor (PDGFR) inhibitors cause higher cellular toxicity in high-grade glioma ATRX-deficient cells. Furthermore, we demonstrate that a combinatorial treatment of RTKi with temozolomide (TMZ)–the current stand-ard of care treatment for GBM patients–causes pronounced toxicity in ATRX-deficient high-grade glioma cells. Our findings suggest that combinatorial treatments with TMZ and RTKi may increase the therapeutic window of opportunity in patients who suffer high-grade gliomas with ATRX mu-tations. Thus, we recommend incorporating the ATRX status into the analyses of clinical trials with RTKi and PDGFRi.This work was supported by grants from Danish National Research Foundation (DNRF115), Danish Cancer Society (KBVU-2017_R167-A11063), European Research Council (ERC-2015-STG- 679068), Nordea-fonden (02-2017-1749) and the Spanish Ministry of Science and Innovation (PID2020- 119329RB-I00). David Pladevall-Morera was supported with a PhD scholarship from the Lundbeck Foundation (R218-2016-415) and funding from Dansk Kræftforskningsfond. María Castejón-Griñán holds an Incorporación fellowship from the Junta de Andalucía. Paula Aguilera was supported with a Juan de la Cierva formación fellowship from the MICINN and an Incorporación fellowship from the Junta de Andalucía. Toyota Fonden and Læge Sofus Carl Emil Friis og hustru Olga Doris Fonden funded the acquisition of the high-content microscope used in this study

Subependymal giant cell astrocytoma-like astrocytoma: a neoplasm with a distinct phenotype and frequent neurofibromatosis type-1-association

Neurofibromatosis type-1 is a familial genetic syndrome associated with a predisposition to develop peripheral and central nervous system neoplasms. We have previously reported on a subset of gliomas developing in these patients with morphologic features resembling subependymal giant cell astrocytoma, but the molecular features of these tumors remain undefined. A total of 14 tumors were studied and all available slides were reviewed. Immunohistochemical stains and telomere-specific FISH were performed on all cases. In addition, next-generation sequencing was performed on 11 cases using a platform targeting 644 cancer-related genes. The average age at diagnosis was 28 years (range: 4–60, 9F/5M). All tumors involved the supratentorial compartment. Tumors were predominantly low grade (n = 12), with two high-grade tumors, and displayed consistent expression of glial markers. Next-generation sequencing demonstrated inactivating NF1 mutations in 10 (of 11) cases. Concurrent TSC2 and RPTOR mutations were present in two cases (1 sporadic and 1 neurofibromatosis type-1-associated). Interestingly, alternative lengthening of telomeres was present in 4 (of 14) (29%) cases. However, an ATRX mutation associated with aberrant nuclear ATRX expression was identified in only one (of four) cases with alternative lenghtening of telomeres. Gene variants in the DNA helicase RECQL4 (n = 2) and components of the Fanconi anemia complementation group (FANCD2, FANCF, FANCG) (n = 1) were identified in two alternative lenghtening of telomere-positive/ATRX-intact cases. Other variants involved genes related to NOTCH signaling, DNA maintenance/repair pathways, and epigenetic modulators. There were no mutations identified in DAXX, PTEN, PIK3C genes, TP53, H3F3A, HIST1H3B, or in canonical hotspots of IDH1, IDH2, or BRAF. A subset of subependymal giant cell astrocytoma-like astrocytomas are alternative lenghtening of telomere-positive and occur in the absence of ATRX alterations, thereby suggesting mutations in other DNA repair/maintenance genes may also facilitate alternative lenghtening of telomeres. These findings suggest that subependymal giant cell astrocytoma-like astrocytoma represents a biologically distinct group that merits further investigation

Telomere alterations in neurofibromatosis type 1-associated solid tumors

The presence of Alternative lengthening of telomeres (ALT) and/or ATRX loss, as well as the role of other telomere abnormalities, have not been formally studied across the spectrum of NF1-associated solid tumors. Utilizing a telomere-specific FISH assay, we classified tumors as either ALT-positive or having long (without ALT), short, or normal telomere lengths. A total of 426 tumors from 256 NF1 patients were evaluated, as well as 99 MPNST tumor samples that were sporadic or of unknown NF1 status. In the NF1-glioma dataset, ALT was present in the majority of high-grade gliomas: 14 (of 23; 60%) in contrast to only 9 (of 47; 19%) low-grade gliomas (p\u2009=\u20090.0009). In the subset of ALT-negative glioma cases, telomere lengths were estimated and we observed 17 (57%) cases with normal, 12 (40%) cases with abnormally long, and only 1 (3%) case with short telomeres. In the NF1-associated malignant nerve sheath tumor (NF1-MPNST) set (n\u2009=\u200975), ALT was present in 9 (12%). In the subset of ALT-negative NF1-MPNST cases, telomeres were short in 9 (38%), normal in 14 (58%) and long in 1 (3%). In the glioma set, overall survival was significantly decreased for patients with ALT-positive tumors (p\u2009<\u20090.0001). In the NF1-MPNST group, overall survival was superior for patients with tumors with short telomeres (p\u2009=\u20090.003). ALT occurs in a subset of NF1-associated solid tumors and is usually restricted to malignant subsets. In contrast, alterations in telomere lengths are more prevalent than ALT

Correlation of immune phenotypes derived from H&E-stained whole slide images with prognosis and response to checkpoint inhibitors in NSCLC.

International audience8539 Background: The classification of tumors as inflamed, excluded or desert based on spatial patterns of tumor infiltrating lymphocytes (TILs) is a potential biomarker of patients likely to respond to checkpoint inhibitors (CPI). However, the subjectivity of manual methods to assess these immune phenotypes (IPs) and poor standardization in the methods and thresholds to define IPs have hampered their clinical adoption. Here, we describe a data-driven approach to inform IP threshold selection on hematoxylin and eosin (H&E)-stained whole slide images (WSI) by maximizing differences in overall survival (OS) between IPs. Methods: A model to classify the IPs of NSCLC samples from H&E images was developed using PathExplore models applied to a TCGA non-small cell lung cancer (NSCLC) cohort of LUAD (N=459) and LUSC (N=424). TIL densities were extracted within cancer and stroma for 0.01 mm2 patches tiled across each WSI. Cut-offs to define cancer and stroma patches as hot or cold were defined based on the 75th and 50th percentiles, respectively, of cancer TIL (cTIL) densities (386 cTIL/mm2) and stroma TIL (sTIL) densities (423 sTIL/mm2) in a TCGA cohort of 4,082 H&E-stained WSI from 10 epithelial tumor types. Hierarchical fitting yielded optimal thresholds minimizing the p-values of OS differences between IPs, leading to classifications of inflamed (iIP, >40% cancer hot patches), excluded (eIP, ≤40% cancer hot patches; >45% stromal hot patches) and desert (dIP, ≤40% cancer hot patches; ≤45% stromal hot patches). The model was then deployed in a clinical cohort of PD-(L)1 inhibitor-treated NSCLC patients (N=95) enrolled in the BIP precision medicine study (NCT02534649; Institut Bergonié, Bordeaux, France). Model-predicted IPs were compared to progression-free survival (PFS) and OS. FDR correction was done with Benjamini-Hochberg. Results: In the TCGA NSCLC cohort, model-predicted iIP (N=196) and eIP (N=607) patients had significantly better OS compared to dIP (N=80; HR=0.53, p=0.003 and HR=0.59, p=0.003, respectively). In the clinical cohort, cTIL density and fraction of hot epithelial patches were significantly associated with PFS (HR=0.64, q=0.04 and HR=0.69, q=0.04, respectively). PFS was significantly shorter in model-predicted eIP patients (N=46) compared to iIP (N=39; HR=0.54, p=0.045). Notably, in PD-L1(-) patients (N=43, tumor proportion score ≤1%), iIP patients had longer PFS than eIP and dIP patients (HR=0.35, p=0.02). No difference in PFS was observed for PD-L1(+) patients. Conclusions: We developed a data-driven approach for predicting IPs using patch-level TIL features. Model-predicted IPs were prognostic in a TCGA NSCLC dataset and predictive of PFS in a CPI- treated clinical NSCLC cohort. Association of IP and PFS was independent of PD-L1 status, potentially allowing the identification of PD-L1(-) patients who may derive greater benefit from CPI