Ahamed Saleem

Identification of phosphorylation sites of TOPORS and a role for serine 98 in the regulation of ubiquitin but not SUMO E3 ligase activity.

TOPORS is the first example of a protein that possesses both ubiquitin and SUMO E3 ligase activity. The ubiquitination activity maps to a conserved RING domain in the N-terminal region of the protein, which is not required for sumoylation activity. Similar to other E3 ligases, it is likely that the ubiquitin and sumoylation activities of TOPORS are regulated by post-translational modifications. Therefore, we employed mass spectrometry to identify post-translational modifications of TOPORS. Several putative phosphorylated regions were identified in conserved regions of the protein. We investigated the role of phosphorylation of serine 98, which is adjacent to the RING domain, in both cells and in vitro. Mutation of serine 98 to aspartic acid resulted in an increase in the ubiquitin ligase activity of TOPORS both in cells and in vitro. In addition, this mutation increased the binding of TOPORS to the E2 enzyme UbcH5a both in vitro and in cells. Conversely, a phospho-deficient mutant (S98A) exhibited little change in ubiquitin ligase activity compared to wild-type TOPORS, both in cells and in vitro. Neither of the mutants affected the localization of TOPORS to punctate nuclear regions. In addition, neither mutant affected the SUMO ligase activity of TOPORS in cells or in vitro. Molecular modeling studies support a role for serine 98 in regulating TOPORS-E2 interactions. Our findings indicate that phosphorylation of serine 98 regulates the ubiquitin but not the SUMO ligase activity of TOPORS, consistent with a potential binary switch function for TOPORS in protein ubiquitination versus sumoylation.

Sequential topoisomerase targeting and analysis of mechanisms of resistance to topotecan in patients with acute myelogenous leukemia

Resistance to topoisomerase I (TOP1)-targeting drugs such as topotecan often involves upregulation of topoisomerase II (TOP2), with accompanying increased sensitivity to TOP2-targeting drugs such as etoposide. This trial was designed to investigate sequential topoisomerase targeting in the treatment of patients with high-risk acute myelogenous leukemia. An initial cohort of patients received topotecan and cytosine arabinoside daily for 5 days. Serial samples of circulating mononuclear cells were examined to evaluate peak elevations of TOP2-&agr; protein expression. In subsequent cohorts, etoposide was administered daily for 3 days, beginning 6 h after initiation of the topotecan infusion. The etoposide dose was escalated to determine a maximum-tolerated dose. Circulating mononuclear cells were analyzed for TOP1 mutations and ABCG2 protein expression. In addition, systemic and intracellular topotecan concentrations were measured. Thirty-one patients were enrolled. On the basis of TOP1-&agr; protein levels in three patients with peripheral blast counts greater than 50%, etoposide administration began 6 h after initiation of the topotecan/cytosine arabinoside infusion. Using this schedule of administration, the maximum-tolerated dose of etoposide was 90 mg/m2. No TOP1 mutations were identified, but increases in ABCG2 expression during the infusion were observed in mononuclear cells from two of four evaluable patients. Administration of etoposide 6 h after initiation of a topotecan/cytosine arabinoside infusion is feasible and is associated with clinical activity. Analysis of TOP2-&agr; protein levels in this small number of patients indicated that peak increases occurred earlier than expected based on earlier publications. Upregulation of ABCG2 was detected in circulating cells and may represent an inducible form of drug resistance that should be investigated further.