Abnormal Expression of Ndfip2 and Cbl in Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients: Role of Ubiquitin Proteasome System in Myeloid Neoplasms and Normal Hematopoiesis

Nedd4-like E3 ligases regulate a wide variety of cellular processes, including ubiquitin-mediated trafficking, lysosomal or proteasomal degradation and nuclear translocation of proteins. Ndfip1 and Ndfip2 are endosomal PY-motif containing proteins and are activators of Nedd4 family members. Ndfip proteins are also regulators of PTEN/Akt/mTOR and MAP kinase and are induced upon T-cell activation. Cbl (or c-Cbl) is a highly conserved RING finger ubiquitin ligase (E3) and regulates signaling by multiple tyrosine kinases (TKs) through interaction with their SH2 and SH3 domains. Cbl proteins negatively regulate TK receptors, mediating the ubiquitination and degradation of activated TKs. In addition to its E3-dependent negative role, Cbl mediates positive pro-oncogenic effect when E3 activity is lost. Hematopoietic stem cells (HSC) of Cbl −/− mice exhibit augmented pool size, hyperproliferation and enhanced long-term repopulation capacity. HSC of Cbl −/− mice are also hyper responsive for thrombopoietin and display elevated levels of STAT5 phosphorylation.

The aim was to determine NDFIP1, NDFIP2 and CBL expression in acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) and myelodysplastic syndrome (MDS) patients and to correlate gene expression with clinical features. Furthermore, we analyzed the expression during erythroid, granulocytic and megakaryocytic differentiation, using cell line models.

NDFIP1, NDFIP2 and CBL expression was verified, by q-PCR, in bone marrow aspirates from 15 normal donors, 38 MDS (24 low-risk and 14 high-risk MDS), 28 AML and 5 APL patients, at the time of diagnosis. NB4, a promyelocytic leukemia cell line, was treated with 10⁻⁶ M of All-trans retinoic acid (ATRA) for granulocytic differentiation. KU812 was treated with 100μM Hydroxyurea (HU) and 50μM Hemin (HE) for erythroid differentiation. K562 was stimulated with 20nM of Phorbol-13 myristate-12 acetate (PMA) for megakaryocytic differentiation.

NDFIP2 gene expression was significantly decreased in AML (0.34 [1.7–0.03] vs. 0.59 [1.98–0.01], P=0.04) and was increased in APL, compared with normal donors (3.33 [4.73–0.32] vs. 0.59 [1.98–0.01], P=0.018) and was also different between each other (P=0.002). CBL was significantly lower in MDS, AML and APL patients, compared with control group (0.36 [3.13–0.05], 0.22 [0.68–0.002], 0.10 [0.18–0.01] vs 0.55 [1.52–0], respectively, with all P<0.01). NDFIP1 expression was not different between all subgroups (P>0.05). In AML, NDFIP1 expression was negatively correlated with peripheral lymphocyte counts (r= −0.39 and P=0.044) and blast counts (r=-0.44 and P=0.02). NDFIP2 expression in AML showed a negative correlation with peripheral lymphocyte counts (r=-0.41, P=0.03) and CBL expression was positively correlated with platelet counts in the same population (r=0.54, P=0.003). In MDS patients, NDFIP1 and NDFIP2 expression was not different from the control group. However, in high-risk MDS, NDFIP1 expression was negatively correlated with bone marrow and peripheral neutrophil counts (r= −0.63, P=0.01; r=−0.60, P=0.002, respectively). During differentiation assay for granulocytic (NB4), erythroid (KU812) and megakaryocytic (K562) lineages, there was an increased expression of NDFIP1 (eight-fold, two-fold and three-fold increase, respectively), NDFIP2 (three-fold, two-fold and four-fold increase, respectively) and CBL (ten-fold, two-fold and three-fold increase, respectively).

Ubiquitin-proteasome system (UPS) plays an essential role in the homeostasis of cellular protein traffic and degradation, regulating cell fate, together with autophagy and apoptosis. Disruption of UPS is essential for leukemogenesis and has potential therapeutic implications. NDFIP2 and CBL are important elements of UPS. Our results show that CBL and NDFIP2 are less expressed in AML (except APL) and correlate with platelet, peripheral blasts and lymphocyte counts. The upregulation of these genes during normal hematopoiesis sheds light on their importance in the myeloid differentiation process. The higher expression of NDFIP2 in APL may be related to a better differentiation potential and autophagy activation. Moreover, the correlation of higher levels of NDFIP1 with lower neutrophil counts in high risk MDS suggests that this disease may still have functional response to T-cell activation.

No relevant conflicts of interest to declare.