The Giant HECT E3 Ubiquitin Ligase HERC1 Is Aberrantly Expressed in Myeloid Related Disorders and It Is a Novel BCR-ABL1 Binding Partner

Abstract

HERC E3 subfamily members are parts of the E3 ubiquitin ligases and key players for a wide range of cellular functions. Though the involvement of the Ubiquitin Proteasome System in blood disorders has been broadly studied, so far the role of large HERCs in this context remains unexplored. In the present study we examined the expression of the large HECT E3 Ubiquitin Ligase, HERC1, in blood disorders. Our findings revealed that HERC1 gene expression was severely downregulated both in acute and in chronic myelogenous leukemia at diagnosis, while it is restored after complete remission achievement. Instead, in Philadelphia the negative myeloproliferative neoplasm HERC1 level was peculiarly controlled, being very low in Primary Myelofibrosis and significantly upregulated in those Essential Thrombocytemia specimens harboring the mutation in the calreticulin gene. Remarkably, in CML cells HERC1 mRNA level was associated with the BCR-ABL1 kinase activity and the HERC1 protein physically interacted with BCR-ABL1. Furthermore, we found that HERC1 was directly tyrosine phosphorylated by the ABL kinase. Overall and for the first time, we provide original evidence on the potential tumor-suppressing or -promoting properties, depending on the context, of HERC1 in myeloid related blood disorders.

Keywords: Bcr-Abl1; E3 ubiquitin ligases; HERC1; gene expression; myeloid neoplasms; protein–protein interaction; ubiquitin proteasome system.

Figure 1

The HERC1 gene expression is differently regulated in bone marrow (BM) and peripheral blood (PB) and it is dysregulated in haematological malignancies. HERC1 transcript was assayed in healthy donors including both PB (red dots) and BM (dark red dots), and in a panel of myeloid related disorders at diagnosis, namely Chronic Myeloid Leukemia (CML) (PB = 37, BM = 35), Acute Myeloid Leukemia (AML) (PB = 17, BM = 80), MyeloProliferative Neoplasms (MPNs) (PB = 75) by RT-qPCR. The HERC1 mRNA quantity was expressed as 2^−ΔΔCt after normalization against GUSB. HERC1 gene expression was significantly downregulated in newly diagnosed CML and AML, with median values of 0.9 and 1 respectively, when compared to healthy specimens (median = 4.01). In MPNs the HERC1 expression fluctuated from 0.4 to 12.65 with a median value of 3.3 and thus not significantly different from healthy subjects. p-values were calculated on the differences in HERC1 gene expression observed between patients and the healthy control specimens. * p < 0.05, **** p < 0.0001.

Figure 2

The HERC1 transcript is severely downmodulated in acute myeloid leukemia independently from genetic alteration. (A) HERC1 level is significantly downregulated in both PB (n = 17) and BM (n = 80) specimens derived from AML at diagnosis. (B) The stratification of the AML samples according to the different cytogenetic categories and to the most common genetic variants displayed that the HERC1 transcript downregulation is a common feature shared by almost all the different groups with the exception of the IDH2 mutated cohort, in which its gene expression is comparable to that of the controls. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

HERC1 mRNA is differentially and peculiarly expressed in MPNs patients. On the whole, the expression pattern of HERC1 in MPNs appeared to be rather scattered and variable. Among MPNs, when compared to the healthy subjects, the CALR mutated Essential Thrombocythemia (ET) specimens (A) displayed a significantly higher amount of HERC1 transcript, (p ≤ 0.0001). On the contrary, the JAK2 mutated, and non-mutated, specimens showed levels comparable to the control counterpart. (B) Differently, the Primary Myelofibrosis (PMF) patients displayed a rather steep downregulation of HERC1 gene expression. However, the JAK2V617F mutated samples showed higher HERC1 levels in comparison to the mutation free cohort. (C) In Polycythemia Vera (PV) patients the HERC1 mRNA level was slightly, but significantly, lower than that of the healthy subjects (** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4

HERC1 gene expression is differently regulated in CML patients during the evolution of the disease. (A) HERC1 gene was differentially expressed during the different CML phases being severely down-regulated at diagnosis, both in BM and PB CML specimens, increased at remission and declined again at the onset of relapse. (B) Similarly to the mRNA, in primary CML cells the HERC1 protein amount was barely detectable at diagnosis and at the relapse onset. On the contrary, at remission the protein level was comparable to that of healthy control. Immunofluorescence was performed on cytospun cells by using specific rabbit polyclonal anti HERC1 antibody. DAPI staining (blue) indicated the cell’s nuclei. The green signal corresponds to the HERC1 protein. * p < 0.05, **** p < 0.0001.

Figure 5

HERC1 mRNA and protein amounts are regulated by the Bcr-Abl1 tyrosine kinase activity. (A) Primary bone marrow derived from leukemic cells of newly diagnosed CML patients (n = 5) displayed a modest increase in HERC1 mRNA quantity after in vitro Imatinib (2.5 µM) treatment. (B,C) HEK-293T transiently and exogenously expressing the constitutively active p210 BCR-ABL1 tyrosine kinase exhibited a severe down-modulation of HERC1 protein examined by immunofluorescence (B) and Western Blot (C). DAPI staining (blue) indicated the cells nuclei while the green signal was corresponding to HERC1 protein. (D) HERC1 mRNA level was significantly reduced in HEK-293T overexpressing the BCR-ABL1 (** p < 0.01).

Figure 6

HERC1 is a novel interactor and a potential substrate of Bcr-Abl1. (A) The subcellular localization of either the HERC1 or Bcr-Abl1 (p210) proteins were assessed by staining cytospun K562 cells with anti-HERC1 and anti-Bcr specific antibodies. The immunofluorescence assay revealed that the localization of the two proteins was mostly restricted to the cytoplasmic compartment and they co-localized. (B) In K562 cells BCR-ABL1 and HERC1 proteins form a complex. The immunoprecipitated BCR-ABL1 was employed to pull-down HERC1 from K562 total cell lysate. The presence of HERC1 in the immunoprecipitated sample was determined by immunoblotting the membrane with HERC1 antibody (upper panel). Subsequently probing with a tyrosine phospho-specific antibody proved the phosphorylated HERC1 status. Vinculin was used as loading control (lower panel). (C) In vitro kinase assay with purified c-ABL and HERC1 substrate, obtained after HERC1 immunoprecipitation on 293T, performed in the absence or presence of 5 µM Imatinib treatment for 30 min. (D) The effect of the Imatinib showed in (C) was measured quantitatively using the ImageJ software. The bands intensity, corresponding to HERC1-P-Tyr protein, were quantified and normalized to the amount of HERC1 protein signal for each lane (the max value was set at 1). Normalized arbitrary units of three different experiments with SD are shown in the bar graphs. * p < 0.05.