To this end, we first confirmed that MLN4924 treatment indeed caused NEDD8 removal from cullin-1 (Fig. develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination. == Introduction == SAG, also known as RBX2, ROC2 (Regulator of Cullins) or RNF7 (RING finger protein-7), was originally cloned in our laboratory as a redox-inducible antioxidant protein[1], and later characterized as the second RING family member of the SCF E3 ubiquitin ligase (for review, see[2]). We and others have previously shown that in cell culture systems, SAG overexpression inhibited apoptosis induced by various agents, including redox[1],[3], nitric oxide[4], ischemia/hypoxia[5], heat shock[6], neurotoxins and 1-methyl-4-phenylpyridinium[7], and UV-irradiation[8]. SAG over-expression also promoted the S-phase entry and cell growth under serum starved conditions[9]. Furthermore, SAG transgenic expression in mouse skin inhibited tumor formation at the early stage, but enhances tumor growth at the later stage in a DMBA-TPA carcinogenesis model[10]. On the other hand, SAG knockdown by anti-sense or siRNA oligoes inhibited tumor cell growth[11], and enhanced apoptosis induced by etoposide and TRAIL[12]. TheSagknockout in mouse caused embryonic lethality, which is associated with growth retardation and other developmental defects[13], whereasSagknockout in mES cells induced radiosensitization[14], and blocked their endothelial differentiation[13]. These cellular functions were mediated via its antioxidant activity by scavenging ROS[1],[4],[6], and via its E3 ubiquitin ligase activity by promoting the degradation of p27, c-Jun, pro-caspase-3, IB, HIF-1, and NOXA in a cell context dependent manner[9],[10],[12],[15],[16],[17]. Importantly, SAG was overexpressed in carcinomas of lung, colon, stomach and liver, which was associated with poor prognosis in lung cancer patients[11],[17],[18]. The findings suggest that SAG may play a role in human tumorigenesis and could serve as an anticancer target. Retinoic acids (RAs) are natural and synthetic derivatives of vitamin A[19]. The binding of RAs to their nuclear receptors, including RAR or RXR, PPAR in the homo- and hetero-dimer formats[20], caused transactivation of more than 500 genes[21]that regulates many signaling pathways[22], and cellular processes including embryonic development, organogenesis, cell homeostasis, cell growth, differentiation and apoptosis[23],[24],[25],[26]. Given its activity in inducing differentiation, apoptosis, and growth arrest, RA has been long used for the treatment and prevention of various types of human cancers[19],[27],[28],[29],[30], particularly as a differentiation therapy agent for the treatment of AML (Acute Myeloid Leukemia)[31],[32]. Significantly, RA is very effective for the treatment of one type AML, acute promyelocytic leukemia (APL) with a cure rate in more than 75% of patients[26]. However, the complete success of this therapy was hindered by the development of RA resistance with subsequent disease relapses[30], demanding combinational therapies, most effectively with arsenic trioxide[33], and other chemotherapeutic drugs, as well as targeted therapies, including TRAIL[26],[27],[30]. Here we report that cellular sensitivity to RA is significantly increased upon inactivation of SAG E3 ubiquitin ligase via genetic deletion or pharmacological inhibition. Specifically, instead of inducing Nandrolone propionate differentiation in wild type mES cells, RA induced apoptosis inSag/mES cells. In two SAG high-expressing AML lines, HL60 and KG-1, MLN4924, a small molecule inhibitor of SAG E3, effectively sensitized otherwise resistant cells to Nandrolone propionate RA via induction of apoptosis which is associated with accumulation of pro-apoptotic proteins, c-JUN and NOXA. Thus, our study reveals that SAG is a RA sensitizing target and provides the first proof-of-concept evidence that MLN4924 can Nandrolone propionate be further developed as a RA chemo-sensitizer for the treatment of AML. == Results FRPHE == == Sag deletion sensitized mouse embryonic stem cell to RA == Our recent study showed that Sag/mES cells failed to undergo endothelial differentiation to form cystic embryoid bodies upon the withdrawal of LIF (leukemia inhibitory factor) from culture media[13]. Here we determined if Sag/mES cells were also defective in RA-induced differentiation. Using two independent pairs of mES clones with Sag+/+vs. Sag/background[14],.