Development and validation of a CyTOF assay for measuring phosphoproteins in AML blast cell populations in whole blood from AML patients

CDC7 (Cell Division Cycle 7-related protein kinase), also known as DBF4-dependent kinase (DDK), is a critical serine/threonine cell cycle protein kinase indispensable for maintaining DNA replication fork progression and stability, particularly under conditions of replication stress. CDC7 activates fork protection and restart mechanisms, including the phosphorylation and activation of MCM2 (Minichromosome Maintenance protein 2) at Ser40. Inhibition of CDC7 disrupts the capacity of cancer cells to ameliorate replication stress and repair DNA damage, leading to the accumulation of DNA damage, incomplete DNA replication, apoptosis, and cellular demise. Consequently, CDC7 represents a promising therapeutic target in cancer, especially for hematologic malignancies such as acute myeloid leukemia (AML). Given that pMCM2 (phosphorylated MCM2) serves as a biomarker of CDC7 inhibition, the development of a CyTOF assay to quantify pMCM2 and gH2AX (a downstream marker of dsDNA damage) in cells holds significant utility in clinical trials evaluating CDC7 inhibitors. Precise measurement of these phosphoproteins in whole blood AML blast cells for example is expected to provide insights into a drug's mechanism of action and its efficacy in AML patients. We present the development and validation of a cytometry by time-of-flight (CyTOF) assay designed to measure pMCM2 and gH2AX in blast cell populations prevalent in AML patients and comparing a one step fixation process and a two-step fixation process. Initial investigations demonstrated the feasibility of characterizing the phenotype of AML blast cells using cell surface markers such as CD34, HLADR, CD33, and CD123, as well as assessing the cellular state with respect to the presence or absence of pMCM2 and gH2AX, and quantifying changes in Median Channel Value (MCV) in response to proliferation stimulators or inhibitors. We further detail the development and validation of the assay, along with an assessment of the comparability between a one-step PROT-1 fixation process versus two-step SLST (Stable Lyse Stable Store) fixation process. The finalized assay utilizing the SLST fixation process could facilitate the monitoring of treatment response and the identification of potential biomarkers in clinical trials, which could have utility in investigating disruptions to the DNA damage response (DDR) in blood cancers. (AI was used to refine the text of this abstract)