Maya Ramy

Introduction. Glioblastoma multiforme (GBM) is a grade IV glioma that accounts for 52% of primary brain tumors with a median survival of 14-15 months after diagnosis with current treatment measures ^1-3. The majority of GBM arise via reactivation of telomerase which grants cells their immortality. However, in the last decade a mechanism termed Alternative Lengthening of Telomeres (ALT) has been identified and implicated as the sole mechanism of telomere elongation in 30% of GBM^4,5. Studies have found that nearly all ALT+ tumors contained inactivating mutations in the ATRX gene. ATRX gene products are essential histone proteins which normally function in conjunction with death domain-associated protein (DAXX) and histone H3.3 as a heterodimer that maintains the heterochromatin state of DNA following mitosis and prevents telomere elongation by silencing the transcription of telomeric repeat containing RNAs (TERRA). Mutations in ATRX lead to dysfunctional gene products that cannot associate with DAXX or H3.3, leaving the DNA in a euchromatin state, and allowing TERRA to elongate telomeres via ALT⁶. Recently, a mouse model was created to analyze ATRX-deficient GBM. Methods. The Sleeping Beauty transposase system was used to block the ALT pathway using shATRX micro RNAs and was inserted into host genomic DNA along with shp53 and NRAS (protooncogene). Control groups received only shp53 and NRAS. In vivo luminescence was used to ensure complete plasmid uptake. H&E staining was used to assess GBM growth in ALT+ and ALT- mice. Fluorescence in situ hybridization (FISH) was used to visualize telomere track expansions via ALT in control and shATRX cells. Calculated total cell fluorescence (CTCF) used to quantify cell differences. Single- and double-stranded DNA damaging agents and radiation were used to assess the effect of ATRX silencing on ALT activation in GBM. Results. ATRX loss significantly accelerated GBM growth and decreased survival in mice. ATRX deficient tumors contained a distinct set of cells with increased CTCF, indicative of ALT physiology, that was not present in control GBM. Single stranded DNA break chemotherapeutic agents showed no slowing of progression of GBM while double stranded agents and radiation showed significant slowing in shATRX GBM but not in control GBM. Conclusions. ALT+, shATRX tumors respond very well to dsDNA break chemotherapeutic agents and radiation and show a higher rate of survival in patients currently treated with these methods⁷. This calls for deeper exploration into these possible agents among ALT+ shATRX GBM populations in human clinical trials.

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