Altered Expression of Neddylation Pathway Proteins in Myelodysplastic Syndromes

Introduction:NEDDylation is a posttranslational protein modification in which the ubiquitin-like protein NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is conjugated to target proteins through a series of enzymatic steps involving E1, E2, and E3 enzymes specific for the NEDDylation pathway. NEDDylation and de-NEDDylation play essential roles in the ubiquitin-proteasome system, influencing protein turnover and cellular homeostasis through conformational and functional alteration of target proteins. NEDD8 conjugation proteins are upregulated in some cancers, which may therefore become susceptible to an antineoplastic effect of NEDDylation inhibitors. A phase I clinical trial of pevonedistat (MLN4924, TAK-924), a first-in-class NEDD8-activating enzyme inhibitor, in AML and MDS showed modest clinical activity as a single agent (Swords et al, 2015), and a subsequent phase 1b trial demonstrated the feasibility of combining azacitidine (Aza) with pevonedistat (Swords et al, 2016). A phase II trial with pevonedistat plus Aza versus single-agent Aza is ongoing in patients with higher-risk MDS, CMML, or low-blast AML. As yet, there is no data regarding the activity or dysfunction of the NEDDylation pathway in MDS, though such knowledge may substantiate the use of NEDDylation inhibitors, either alone or combined with hypomethylating agents. We examined NEDDylation pathway protein expression and its prognostic relevance in MDS.

Methods: We built a tissue microarray (TMA) with bone marrow biopsies from 119 MDS patients, 40 AML patients, and 11 normal controls. Immunohistochemistry (IHC) protocols were established in our lab for the detection of NEDD8, NAE1 (NEDD8 activating enzyme), and UBE2M (ubiquitin conjugating enzyme E2 M, also known as UBC12). Semi-quantitative analysis was done according to Remmele-Stegner immunoreactive score (IRS) with a point system from 0 to 12. Expression was categorized as absent (0-2), weak (3-4), moderate (5-8) or strong (9-12). Clinical follow-up information on 116 patients was available from the Düsseldorf MDS Registry.

Results: MDS subtypes were as follows: 3 MDS-SLD, 3 MDS-SLD-RS, 59 MDS-MLD, 5 MDS del(5q), 18 MDS-EB-1, 11 MDS-EB-II, 5 CMML-I, 3 CMML-II, 3 sAML, 6 not classified. Median age was 68 (range 21-94). Distribution among IPSSR risk groups: 13 (11.2%) very high risk, 11 (9.5%) high risk, 24 (20.7%) intermediate risk, 29 (25%) low risk, 18 (15.5%) very low risk, and 21 not classified. Of 96 evaluable patients, 46 (39.7%) showed an abnormal karyotype. Ten patients who received disease-modifying treatment prior to biopsy were excluded from survival analysis. On IHC, all 40 AMLs and 11 normal controls were clearly positive (IRS>4) for all three NEDDylation pathway proteins. MDS patients showed a different pattern. While NEDD 8 was positive in the majority (60.6%), NAE1 and UBE2M were expressed in only 30.8% and 29.4%, respectively. For each protein, expression was not correlated with MDS type (WHO 2016), IPSS-R risk group, overall survival or AML transformation. Patients with strong expression of all three proteins survived longer (51 vs. 26 months), but the difference did not reach statistical significance (p=0.10). Low expression of NAE1 and UBE2M significantly correlated with the presence of karyotype anomalies (p=0.01 and p=0.02, respectively).

Conclusion: In contrast to upregulation in some solid tumors, and also in contrast to the AML samples on our tissue microarray, expression of NEDD8 conjugation pathway proteins was low in a substantial proportion of MDS patients. It is unclear whether such patients may benefit from further inhibition of the NEDDylation pathway. The statistically significant association between low or absent expression of NAE1 and UBE2M, and the presence of karyotype anomalies, suggests that low activity of the NEDDylation pathway may contribute to chromosomal instability. A useful mechanism of NEDDylation pathway inhibition is envisaged in the context of treatment with demethylating agents: Since NEDD8 increases DNMT3b-dependent DNA methylation (Shamay et al, 2010), and DNMT3b gene amplification (linked to higher levels of the corresponding mRNA and protein) has been reported to predict resistance to DNA demethylating drugs (Simo-Riudalbas et al, 2011), NEDDylation pathway inhibition may reduce the development of resistance to hypomethylating therapy.