Abstract 4136: Single-cell network profiling (SCNP) to evaluate the proteomic profiles associated with ON 01910.Na treatment of MDS patients (Pts)
Authors: Cesano Alessandra , Soper David , Banville Steve , Tran Mai , Seetharam Mahesh , Kumar Ramesh , Wilhelm Francois , Greenberg Peter 
Published: August 2014

Background: Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on intracellular signaling cascades at the single cell level. ON 01910.Na has been reported to inhibit polo-like kinase 1, PI3-kinase and Akt pathways. In an ongoing phase II study in Int-1, 2 or high risk MDS pts refractory to hypomethylating agents, biomarker assays are being performed to assess CD34+ cell functional signaling profiles associated with biological activity of ON 01910.Na.

Objectives: The objectives were to simultaneously compare the functional effects of a panel of modulators on different signaling pathways (such as the PI3K and the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) pathway) to identify specific proteomic profiles associated with the biological activity of and response to ON 01910.Na in MDS pts.

Methods: MDS pt bone marrow samples were collected at baseline and after treatment cycles 1, 3, 5, & 7. Bone marrow mononuclear cells (BMMCs) were isolated and cryopreserved for longitudinal analysis. Activation of signaling pathways was measured with fluorochrome-conjugated antibodies that recognize p-Erk1/1 (T202/Y204), p-Akt (S473), p-S6 (S235/236), p-Stat1 (Y701), p-Stat3 (S727), and p-Stat5 (Y694). BMMCs were modulated with FMS-like tyrosine kinase 3 ligand (FLTL3), stem cell factor (SCF), granulocyte colony stimulating factor (G-CSF), or granulocyte-monocyte colony stimulating factor (GM-CSF) for 15 minutes. Cells were processed for SCNP by fixation, permeabilization, and incubation with fluorochrome-conjugated antibodies.

Results: SCNP analyses in Pt ON103 (progressed to AML after completion of trial) showed that frequency of CD34+ cells increased during the course of the clinical trial; when modulated with either FLT3L or SCF, compared to baseline findings, CD34+ cells exhibited increased p-S6 and p-Akt responsiveness with treatment; and interestingly, CD34+ cell responsiveness to G-CSF decreased (p-Stat1 and p-Stat5) while no signaling was observed in response to GM-CSF. In contrast, SCNP analyses in Pt ON104 (stable disease), showed that frequency of CD34+ cells was maintained throughout treatment; when modulated with either FLT3L or SCF, CD34+ cells exhibited decreased p-S6, p-Akt, p-Erk (slight); and while CD34+ cell responsiveness to G-CSF decreased (p-Stat1 and p-Stat5), a robust p-Stat5 response was induced by GM-CSF which increased during the course of the clinical trial.Conclusions: The data here suggest that measurement of intracellular signaling responses using SCNP is feasible using BMMCs from pts with MDS. Interrogating key signaling pathways thought to be involved in the action of ON 01910.Na provides a tool for developing functional proteomic signaling profiles associated with biological activity of this novel cell cycle active agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4136. doi:10.1158/1538-7445.AM2011-4136