Browsing by Subject "Telomere"

Telomeres are the ends of chromosomes which are composed of repetitive DNA sequence and telomere associated proteins. In C. elegans, the protein F39H2.5 was found to associate with the telomere, regulating both telomere length and genomic integrity. F39H2.5 is a member of the β-CASP family of proteins that are known to possess nuclease activity on DNA substrates. I thus sought to address whether any of the human β-CASP family proteins associated with telomeres. Here I show that hSnm1B localized to the telomere indirectly, via interaction with the double-stranded telomere binding protein TRF2. The terminal 37 amino acids of hSnm1B are necessary and sufficient for binding TRF2, and moreover that binding to TRF2 stabilized hSnm1B protein by preventing ubiquitination. In the absence of exogenous TRF2 this domain acted as a degron, promoting protein instability. I thus termed the domain the Protection And INstability (PAIN) domain. I hypothesize that TRF2 binding ensures that hSnm1B will only accumulate at telomeres by preventing the degradation of hSnm1B. However, hSnm1B stability appears to be further regulated, as telomere specific DNA damage stabilized hSnm1B independent of the PAIN domain. Thus, it appears that the telomere associated protein, hSnm1B, is regulated by protein stability in a manner that is both dependent and independent of the PAIN domain.

Malignant gliomas account for more than 80% of all primary brain malignancies and 14,000 deaths in the U.S. annually. Despite aggressive treatment, malignant gliomas are largely fatal, as their invasive nature renders them prone to rapid recurrence. Gliomas are classified according to histopathologic criteria which are critical to treatment planning, as certain subtypes show increased sensitivity to particular therapeutic agents. However, gliomas often exhibit extensive tumor heterogeneity and ambiguity among histologic features, leading to subjectivity in diagnosis and low concordance rates among neuropathologists.

Recently, a number of large-scale genomic studies identified mutations in the TERT promoter and IDH1/2 in ~80% of all gliomas. Based on the occurrence of these mutations, gliomas can be classified into objective molecular subtypes that stratify patients into clear prognostic subgroups more effectively than by histology alone. However, current sequencing-based methods to identify these alterations are limited by low sensitivity (40% tumor cellularity), a major constraint on their clinical utility in the context of diffusely infiltrative gliomas. Importantly, this work also revealed that 20% of glioblastomas lack these alterations, delineating a subset of tumors known as the TERT promoter wildtype-IDH wildtype (TERTpWT-IDHWT) glioblastomas.

Preliminary studies indicate that TERT promoter and IDH mutations can effectively stratify the majority (80%) of patients into clinically-relevant genetic subtypes, however current mutation detection methods lack sensitivity (Sanger sequencing) or are overly time-consuming (next-generation sequencing). Here, we report the development of a qPCR-based approach which can provide more sensitive and rapid detection of these mutations and practical utility in glioma diagnosis by detecting low-abundance mutations (e.g., poorly sampled tumors). Finally, we report the genetic landscape of TERTpWT-IDHWT glioblastomas using whole exome and whole genome sequencing, revealing that these tumors harbor a unique set of genetic alterations and exhibit distinct genetic mechanisms of telomere maintenance from other known subgroups of GBM, including recurrent SMARCAL1 mutations and rearrangements upstream of TERT. Using cell-based assays and markers of alternative lengthening of telomeres (ALT), we provide evidence showing that SMARCAL1 acts as a tumor and ALT suppressor and that loss of function cancer-associated mutations are involved in ALT mechanism of telomere maintenance.

These studies have identified the key underlying genetic alterations that characterize TERTpWT-IDHWT glioblastomas, and can serve as biomarkers for more accurate diagnosis and treatment of this glioma subgroup. By developing a sensitive diagnostic for the critical TERTp and IDH alterations, we facilitate accurate diagnosis and prognostication of glioma patients.