The Autocrine Secreted PDZD2 Functions As a Novel Tumor Suppressor in AML, Inducing Growth Inhibition and Cell Cycle Arrest

Epigenetic deregulation is a hallmark of acute myeloid leukemia (AML) and we have previously reported that recurring epigenetic abnormalities can be found across all AML subtypes, irrespective of cytogenetic or molecular drivers. Among the genes recurrently hypermethylated in AML we identified PDZD2 (PDZ domain-containing protein 2), which was silenced through DNA hypermethylation in over 90% of AML patients. PDZD2 processing generates two functional proteins: full-length PDZD2 and secreted PDZD2 (sPDZD2), a product of PDZD2 cleavage by caspase 3 at its C-terminal. While little is known about this protein's role in AML, work in solid tumors has shown that sPDZD2 can act as an autocrine tumor suppressor leading to senescence or quiescence of malignant cells. Given the role of PDZD2 in cancer and its almost universal loss in AML, we hypothesize that PDZD2 is a novel tumor suppressor in AML and that treatment with recombinant sPDZD2 represents a novel avenue for therapeutic targeting of AMLs. To test this hypothesis, we first characterized PDZD2 expression pattern in a panel of 13 AML cell lines and normal hematopoietic CD34 + cells. Quantification of PDZD2 mRNA levels in AML cell lines confirmed its almost universal downregulation when compared to normal CD34 + cells. Subcellular localization analysis in normal hematopoietic stem and progenitor cells demonstrated the presence of full-length PDZD2 in the cytoplasmic and nuclear fractions. In addition, treatment of a subset of the cell lines with the hypomethylating agent Decitabine at 500 nM for 72 h resulted in upregulation of PDZD2 by a range of 2.5 to 100-fold change, validating the observation that PDZD2 gets silenced through DNA hypermethylation. At the protein level, while full-length PDZD2 was detectable in approximately half the AML cell lines tested, detailed analysis revealed that in these cell lines PDZD2 was misprocessed, failing to get cleaved, and resulting in accumulation of the full-length version of the protein in the membranous (ER) fraction of the cell, while this is not observed in normal CD34 + cells. We next tested the effect of re-exposing AML cell lines to sPDZD2 by treating a panel of 10 AML cell lines with increasing doses of recombinant sPDZD2. sPDZD2 was added to the culture media every 12 h and cell growth was evaluated over a period of 8 days. We analyzed cell proliferation and viability every 48 h as well as cell cycle and expression of differentiation markers (CD11b and CD15) a