Most cancers rely on telomerase to extend and maintain telomeres, but 4-11% of cancers grow via a telomerase-independent pathway called alternative lengthening of telomere (ALT). ALT cancer uses homologous recombination (HR)-dependent telomere elongation pathway, and often has mutations in ATP-dependent helicase ATRX or histone H3.3 chaperone DAXX. Menin is a scaffold protein characterized by interactions with a wide range of proteins and acts as a promoter or suppressor in tumor progression. However, the role of menin in telomere maintenance or any specific implications for ALT pathway is not known. Therefore, I investigated the role of menin in ALT cancer and the molecular basis underlying telomere maintenance. I used U2OS and SJSA-1 osteosarcoma cell lines as in vitro models of TERT-/ALT+ and TERT+/ALT- cancer cells. U2OS cells lack ATRX expression due to genetic mutation. I found that menin inhibitor, MI-503 resulted in growth defects in both cell types. To determine the role of menin in response to global replication stress, cells were treated with hydroxyurea. RNAi-mediated menin knockdown or MI-503 treatment compromised the induction of DNA damage markers such as γH2AX and phosphorylated RPA2 in U2OS, while the induction of γH2AX but not of RPA2 phosphorylation was abolished in SJSA-1. The effects of menin in ALT phenotype were also analyzed. Biomarkers such as APBs (ALT associated PML body) and TIFs (telomere dysfunction induced foci) were affected by menin inhibitor. In addition, inhibition of menin increased the colocalization of DAXX-PML in SJSA-1 cells, but had no effect in U2OS cells. Also, TCGA analysis showed that menin expression was higher in GBM (Glioblastoma) and LGG (Low grade glioma) patients with longer telomere length in ATRX wild type context. But when ATRX was mutated, such relationships between menin abundance and telomere length were not observed. Thus, I concluded that menin is involved in the DAXX-ATRX-H3.3 axis, DNA repair, and telomere maintenance in an ATRX-dependent and independent manner. In ATRX-defective ALT cancers, menin is supposed to be mainly involved in telomere maintenance via homologous recombination, and the inhibition of menin has the potential to decrease the viability of ALT+ cancer cells. My results revealed an oncogenic ALT cancer-specific role of menin.
대안적 텔로미어 유지기작(ALT)은 텔로머레이즈 비의존적으로 텔로미어를 신장시키는 텔로미어 유지기작이다. ALT 암은 히스톤 샤페론 ATRX와 DAXX가 돌연변이 되어있기 때문에 불안정한 텔로미어를 가지게 된다. 이로 인해 상동재조합이 활성화되며 이에 의존하여 텔로미어를 신장시킨다. Menin은 DNA 수선을 조절한다. 또한 히스톤 샤페론 DAXX의 H3.3 결합영역과 menin 결합영역이 일치하는 것으로 알려져 있다. 그러나 ALT 암에서 menin의 효과나 기전이 제대로 알려진바는 없다. 따라서 나는 ALT 암에서 menin의 역할과 그 기전에 대해 연구를 진행했다. ALT 암에서 menin을 억제했을 시, 세포 생존률과 ALT 표현형이 감소하였다. Menin이 억제된 경우 DNA 수선 신호인 γH2AX와 RPA 인산화가 감소하였으나, ALT 암의 DAXX-PML 공동발현은 변화하지 않았다. 또한 ALT 암에서 ATRX가 야생형으로 존재할 경우, menin의 발현이 높을수록 따른 텔로미어 길이가 길었으나, ATRX가 돌연변이 되어있을 경우 menin 발현에 따른 텔로미어 길이의 차이는 없었다. 따라서 menin은 ATRX 의존적으로 텔로미어 길이, DAXX-PML 공동발현, 그리고 DNA 수선에 관여할 가능성이 있음을 발견했다. 또한 ALT 암은 ATRX가 돌연변이 되어 있기 때문에 다른 암과 달리 menin이 상동재조합에 주로 관여하며, menin을 억제할 시 상동재조합 억제를 통해 ALT 암을 감소시킬 가능성을 보았다. 나는 ALT암에 특이적인 메닌의 발암성 역할을 밝혀냈다.