A nano-immunoassay system for monitoring targeted drug impact on signaling pathways from small samples of primary hematopoietic malignancies

C22

Measuring the effect of targeted drug therapy on signal transduction pathways is important for drug development and cancer treatment. Cancer is caused by activation of oncogenes and/or inactivation of tumor suppressor genes. Regulation of many of these oncogenes and tumor suppressor genes occurs through signal transduction pathways which are often the point of intervention of targeted therapeutics. Specific oncogene inhibition can be an effective therapy for cancer, as documented in animal models and in human cancers. Using a mouse model of lymphoma we have previously shown that suppressing the activation of the MYC oncogene induces sustained tumor regression. Here we demonstrate the use of a nano-immunoassay platform (Firefly^TM system) to measure changes in the expression and activation of a variety of onco/signaling proteins. By measuring the response of AKT, ERK, MYC, MEK, STAT and JNK in malignant cells before and after treatment we hypothesize that patterns will be revealed indicative of therapeutic clinical response. The levels of the different proteins were measured with high accuracy and sensitivity in as few as 400 cells. Since this technology separates different phosphorylated forms of a protein based on their isoelectric point, a single pan-specific antibody was able to distinguish between the phosphorylated and non-phosphorylated forms of each protein. Interestingly, we were able to identify an effect of the small-molecule tyrosine kinase inhibitor, Gleevec, on specific isoforms of ERK protein in patients responding to therapy not seen in refractory patients. Hence, this novel technique can measure the levels of key signaling proteins in oncogenic pathways from very small samples. Comparison of this technique was performed with Western blot and phospho-protein FACS analyses. This approach can potentially be used for high throughput analysis of microscopic clinical specimens for biomarker discovery, molecular diagnostics, clinical screening and to monitor changes in signaling during therapeutic interventions.