Background Glioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)–wildtype GBM long-term survivors (LTS) to date. Methods Retrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression. Results Of the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks. Conclusions While our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.
O 6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes alkyl groups at the O 6-position of guanine in DNA. MGMT expression is reduced or absent in many tumor types derived from a diverse range of tissues, most notably in glioma. Low MGMT expression confers significant sensitivity to DNA alkylating agents such as temozolomide (TMZ), providing a natural therapeutic index over normal tissue. In this study, we sought to identify novel approaches which could maximally exploit the therapeutic index between tumor cells and normal tissues based on MGMT expression, as a means to enhance selective tumor cell killing. TMZ, unlike other alkylators, activated the Ataxia Telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) axis in a manner that was highly dependent on MGMT status. TMZ induced growth delay, DNA double-strand breaks, and G2/M cell cycle arrest, which led to ATR-dependent phosphorylation of Chk1; this effect was dependent on reduced MGMT expression. Treatment of MGMT-deficient cells with TMZ increased sensitivity to ATR inhibitors both in vitro and in vivo across numerous tumor cell types. Taken together, this study reveals a novel approach for selectively targeting MGMT-deficient cells with ATR inhibitors and TMZ. As ATR inhibitors are currently being tested in clinical trials, and TMZ is a commonly used chemotherapeutic, this approach is clinically actionable. Furthermore, this interaction potently exploits a DNA-repair defect found in many cancers.
Background Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. Methods PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH and phosphoproteomics. Results Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Conclusions Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.
Cancer cells have several hallmarks that define their neoplastic behavior. One is their unabated replicative potential that allows cells to continually proliferate, and thereby contribute to increasing tumor burden. The progression of a cell through the cell cycle is regulated by a series of checkpoints that ensures successful transmission of genetic information, as well as various cellular components, including organelles and protein complexes to the two resulting daughter cells. The mitochondrial reticulum undergoes coordinated changes in shape to correspond with specific stages of the cell cycle, the most dramatic being complete mitochondrial fragmentation prior to cytokinesis. To determine whether mitochondrial fission is a required step to ensure proper mitochondrial segregation into two daughter cells, we investigated the importance of mitochondrial dynamics to cell cycle progression. We found that mitochondrial hyperfusion promotes a defect in cell cycle progression characterized by an inability for cells to exit G2/M. Additionally, extended periods of persistent mitochondrial fusion led to robust caspase-dependent cell death. The cell death signals were coordinated through activation and cleavage of caspase-8, promoting a potent death response. These results demonstrate the importance of mitochondrial dynamics in cell cycle progression, and that inhibiting mitochondrial fission regulators may provide a therapeutic strategy to target the replicative potential of cancer cells.
Human tissue samples commonly preserved as formalin-fixed paraffin-embedded (FFPE) tissues after diagnostic or surgical procedures in the clinic represent an invaluable source of clinical specimens for indepth characterization of signaling networks to assess therapeutic options. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. Additionally, pTyr analysis that may otherwise inform therapeutic interventions for patients has been limited by the requirement for large amounts of frozen tissue. Here we describe a method for highly sensitive, quantitative analysis of pTyr signaling networks, with hundreds of sites quantified from 1-2 10-µm sections of FFPE tissue specimens. A combination of optimized magnetic bead-based sample processing, optimized pTyr enrichment strategies, and TMT multiplexing enabled in depth coverage of pTyr signaling networks from small amounts of input material. Phosphotyrosine profiles of flash frozen and FFPE tissues derived from the same tumors suggested that FFPE tissues preserve pTyr signaling characteristics in patient-derived xenografts and archived clinical specimens. pTyr analysis of FFPE tissue sections from breast cancer tumors as well as lung cancer tumors highlighted patient-specific oncogenic driving kinases, indicating potential targeted therapies for each patient. These data suggest the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens.
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