Abstract
Abstract 75
Activating NOTCH1 mutations are found in 40–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, most leukemia-associated NOTCH1 mutations fail to induce leukemia. This observation suggests that cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. In murine retroviral/transposon insertional mutagenesis screens, induction of the Zmiz1 gene was frequently associated with activation of the Notch1 receptor during leukemogenesis (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT-like family that has been implicated for prostate cancer survival. It directly interacts with the androgen receptor to enhance its transcriptional activity. To investigate the role of ZMIZ1 during leukemogenesis, activating NOTCH1 mutations and ZMIZ1 were transduced into hematopoietic progenitor cells. These cells were then used to reconstitute lethally irradiated mice. ZMIZ1 or NOTCH1 alone failed to induce T-ALL after 1 year of observation. In contrast, ZMIZ1 and NOTCH1 in combination induced T-ALL with ∼50% penetrance by 100 days after transplantation. These data show that ZMIZ1 can promote leukemogenesis in cooperation with NOTCH1. To determine the relevance of ZMIZ1 to human leukemia, we screened 15 primary human adult T-ALL samples for ZMIZ1 mRNA and protein. 20% expressed ZMIZ1. In publically available data sets, ZMIZ1 gene expression was significantly enriched by ∼2-fold in early thymocyte precursor ALL (ETP-ALL) samples. The ETP-ALL subgroup comprises about 13% of all T-ALL and may have a highly unfavorable prognosis. We discovered expression of ZMIZ1 in two ETP-like cell lines. To investigate whether ZMIZ1 is a potential therapeutic target, we transduced these cell lines with shRNA directed against ZMIZ1. ZMIZ1 inhibition reduced cell size, increased apoptosis by ∼2-fold, and reduced growth by 75–94%. Furthermore, ZMIZ1 knockdown overcame resistance to NOTCH signaling blockade with g-secretase inhibitors. Since the glucocorticoid receptor is highly homologous to the androgen receptor, we considered the possibility that ZMIZ1 inhibition may promote glucocorticoid resistance. However, we treated ZMIZ1-inhibited T-ALL cell lines with increasing doses of dexamethasone. ZMIZ1-inhibited T-ALL cell lines were twice as sensitive to dexamethasone than uninhibited cells. These data suggest that ZMIZ1 is required for leukemia growth and survival. Inhibition of ZMIZ1 may potentially enhance targeting of T-ALL with NOTCH pathway inhibitors and glucocorticoids. To determine the mechanism underlying ZMIZ1 function, we performed gene expression profiling. We identified C-MYC as a potential downstream target of ZMIZ1. C-MYC is also a direct target of NOTCH1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous C-MYC expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by ∼7000 fold, induced C-MYC target gene expression, and rescued the C-MYC dependent cell line. ZMIZ1 inhibition lowered C-MYC levels by ∼93%. The interaction between ZMIZ1 and NOTCH appeared to be specific for C-MYC, as modulation of ZMIZ1 levels did not affect the NOTCH1 target genes Hes1, Dtx1, and Cd25. Downregulation of C-MYC partly phenocopied the effects of ZMIZ1 downregulation. However, ectopic expression of C-MYC failed to rescue the growth of ZMIZ1-inhibited cells. These data suggest that C-MYC is an essential but insufficient downstream effector of ZMIZ1 function. In conclusion, ZMIZ1 is new potential therapeutic target in a subset of T-ALL. It functionally interacts with NOTCH1 to promote C-MYC expression and activity.
Disclosures:
No relevant conflicts of interest to declare.
Androgen receptor as potential therapeutic target in metastatic endometrial cancer
