DNA Topoisomerases Participate in Fragility of the Oncogene RET

Abstract

Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APH-induced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication.

Emergence of RET rearrangement co-existing with activated EGFR mutation in EGFR -mutated NSCLC patients who had progressed on first- or second-generation EGFR TKI

“…Alternatively the RET rearrangement may exist as a minor clone in the our three EGFR-mutated NSCLC samples and could have expanded clonally while the activated EGFR mutated tumor clone was inhibited by EGFR TKI, a clinical scenario analogous to the appearance of the gatekeeper T790M mutation during EGFR TKI [10] However, we did not detect the existence of RET rearrangement in the pre-EGFR TKI tumor sample at >500x coverage. Intron 11 of RET is a known site of fragile DNA secondary structures and is thought to be more susceptible to topoisomerase I and topoisomerase II mediated DNA breakage and increased recombination in response to radiation-induced DNA damage [11][12]. Recent RET breakpoint analysis suggests small, 2.0 kilobase, nonspecific regions spanning RET exon 11 to intron 11 are involved in nearly all RET-rearranged NSCLC [13].…”

RET Gene Fusions in Malignancies of the Thyroid and Other Tissues

“…Moreover, breakpoints in RET intron 11 map close to DNA topoisomerase 1/2 predicted cleavage sites, suggesting that DNA topoisomerases may play a role in RET fusion events [102]. Accordingly, during replication and transcription, DNA is unwound by DNA helicase, resulting in torsion that can be removed by topoisomerases through transient introduction of DNA breakage, thus potentially favoring gene rearrangements in cancer cells [102]. Finally, tissue-specific organization of chromatin territories may affect the frequency as well as the specificity of RET fusion with specific gene partners.…”

“…Chromosomal fragile sites are DSB-prone genome regions, in particular, under conditions that reduce DNA replication or upon the exposure to several chemical agents. It is worth noting that RET and NCOA4 are located within the fragile site FRA10G, and CCDC6 is located within the fragile site FRA10C, a fact that may facilitate their rearrangement [100][101][102][103]. Moreover, breakpoints in RET intron 11 map close to DNA topoisomerase 1/2 predicted cleavage sites, suggesting that DNA topoisomerases may play a role in RET fusion events [102].…”

“…It is worth noting that RET and NCOA4 are located within the fragile site FRA10G, and CCDC6 is located within the fragile site FRA10C, a fact that may facilitate their rearrangement [100][101][102][103]. Moreover, breakpoints in RET intron 11 map close to DNA topoisomerase 1/2 predicted cleavage sites, suggesting that DNA topoisomerases may play a role in RET fusion events [102]. Accordingly, during replication and transcription, DNA is unwound by DNA helicase, resulting in torsion that can be removed by topoisomerases through transient introduction of DNA breakage, thus potentially favoring gene rearrangements in cancer cells [102].…”

Genomic instability in fragile sites—still adding the pieces

“…Another critical role of Topo I involves the recognition and preferential cleavage of ssDNA within the duplex stem of DNA secondary structures. It was shown that the location of aphidicolin‐induced breakpoints in the RET oncogene harbored within FRA10G overlap with predicted secondary structure features, which are also presumed Topo I cleavage sites . After treatment with both aphidicolin and a Topo I inhibitor, a significant decrease in the level of DNA breaks within the RET region was observed, confirming the involvement of Topo I in the processing of structural impediments emerging within CFSs under replication stress conditions .…”