PDZD2 Is Essential for Steady-State Hematopoiesis and Its 37-Kda Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP and erythroid progenitors, however, it is almost universally lost through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37kDa protein known as secreted PDZD2 (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system.

To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using an ELISA-based detection method. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected significantly lower levels in AMLs (169 ± 17.53 vs 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines secreted significantly lower levels of sPDZD2 into the culture medium than HSPCs (28.71 ± 1.76 vs 4.62 ± 1.19 ng/mL; p<0.0001). Notably, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition (CellTiter-Glo, p<0.05 for all), differentiation (confirmed by upregulation of CD15/CD11b by flow cytometry) and cell cycle arrest in G0/G1. qRT-PCR analysis of cell cycle regulators in MV4-11 cells treated with r-sPDZD2 confirmed upregulation of CDKN2A (4.01-fold) and E2F4 (4.66-fold) and marked downregulation of multiple cyclin genes, including cyclins D and E, compatible with the cell cycle arrest observed by flow cytometry. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot for validation. In vitro treatment with r-sPDZD2 led to inhibition of ERK1/2 (T202/Y204, T185/Y187) and CREB (S133) phosphorylation, as well as decreased ß-Catenin expression and increased GSK3ß (S9), RSK1/2 (S221/S227) and p53 (S46) phosphorylation.

In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a (39 vs 15%) and CD71 (58 vs 26%). To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2^Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts (9.04 ± 0.26 vs 7.58 ± 0.47 x10⁶/µL) and hemoglobin levels (14.88 ± 0.46 vs 12.30 ± 0.81 g/dL) (p<0.05 for both), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis.

In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML.

No relevant conflicts of interest to declare.