A Novel Nano-Immunoassay (NIA) Reveals Inhibition of PI3K and MAPK Pathways in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndrome (MDS) Treated with the Multi-Kinase Inhibitor On 01910.Na (Rigosertib),

Abstract 3808

The ability to quantify changes in protein activity in a clinical setting is important for the development of therapeutics that target cancer signaling pathways. We have developed a highly sensitive microfluidic nano-immunoassay (NIA) to quantify unphosphorylated, single- and multi- phosphorylated isoforms of proteins in patient specimens. Using as little as 2 nanoliters of cellular lysate, we can measure over 40 proteomic parameters, and assess changes in proteins that mediate signal transduction, cell cycle progression and apoptosis. The styryl sulfone mitotic inhibitor, rigosertib, inhibits multiple kinases in vitro, including the PI3 Kinase, MAP Kinase and PLK-1 signaling pathways. In order to develop potential biomarkers of response to this agent, we first used NIA to demonstrate that rigosertib decreases specific phosphorylated MEK isoforms in the MAPK pathway and AKT isoforms in the PI3K pathway in the TF1 erythroleukemia cell line in vitro. Next, to determine if these changes occur in MDS patients treated with rigosertib, we used NIA to quantify MEK and AKT isoforms in bone marrow CD34+ cells sampled before and at sequential time points after initiating rigosertib treatment in patients enrolled in our prospective single institution Phase II study “A Phase 2, Single-Arm Study To Assess The Efficacy and Safety Of 72-Hour Continuous Intravenous Dosing Of ON 01910.Na (Rigosertib) Administered Every Other Week in Myelodysplastic Syndrome Patients with Trisomy 8 or Classified as Intermediate-1, 2 or High Risk”. To date, we have analyzed 14 specimens from 5 patients. Three patients who responded to treatment (marrow complete response or stable disease) exhibited a 20% mean decrease in MEK1 phosphorylation. In contrast, two patients whose disease progressed on treatment exhibited a 15% mean increase in MEK1 phosphorylation. In the PI3 Kinase pathway, we found that AKT2 phosphorylation decreased by an average of 15% in responders, whereas the two patients whose disease progressed on treatment exhibited a 2% decrease and an 18% increase in AKT2 phosphorylation, respectively. NIA is able to measure changes in different phosphorylated isoforms of AKT2; thus, we were able to further resolve changes in AKT2 phosphorylation into 4 distinct phosphorylated isoforms. Interestingly, NIA revealed that the most abundant of these isoforms, isoform 3, was preferentially suppressed upon successful rigosertib treatment: isoform 3 decreased by a mean of 28% in the three patients who responded to the drug. In contrast, the two patients whose disease progressed on treatment exhibited a 10% decrease and a 22% increase in isoform 3, respectively. Our results suggest that a possible mechanism of action of rigosertib in MDS patients might be through the inhibition of both the PI3K and the MAPK pathways, and raise the hypothesis that the drug may preferentially target specific phosphorylated isoforms within each pathway. We have shown that NIA can be used to measure isoforms of phospho-MEK and phospho-AKT as potential biomarkers of rigosertib activity in MDS.