Frequency of CDK2 , CCNE1 Copy Number Variation in Acral Melanoma and Implications for CDK Inhibitor in Targeted Therapy

Background: Acral melanoma have a high frequency of cell cycle-related gene copy number variation. However, the status and clinical significance of CNVs of CDK 2 and CCNE1 have not been fully elucidated. Methods: A total of 490 acral melanoma samples were examined for CNVs of CDK 2 and CCNE1 using QuantiGenePlex DNA Assay. Correlations of CDK2 and CCNE1 CNVs to clinicopathologic features and prognosis of acral melanoma were evaluated.The sensitivity of cell lines and cell-derived xenograft (CDX) containing CCNE1 CNVs to CDK inhibitor AT7519,Dinaciclib and proteasome inhibitor Bortezomib were also analyzed. Results: Among the 490 samples,140 cases, 139 cases and 39 cases respectively showed CDK2 gain (28.5%), CCNE1 gain (28.3%) and CDK2 gain plus CCNE1 gain (8.0%).The median progression-free survival (PFS) time for acral patients with CCNE1 gain was significantly shorter than that for patients without CCNE1 gain (17.0 versus 27.0 months; P =0.002). Furthermore, CCNE1 gain was an independent prognostic factor for patients receiving chemotherapy. The pan-CDK inhibitor AT7519 could inhibit the cell proliferation, induce apoptosis and cause cell cycle arrest in G2 phase of acral melanoma cells and inhibit the tumor growth of CDX with CCNE1 gain. Dinaciclib and Bortezomib showed CCNE1 copy number independent inhibitory effects on the proliferation of melanoma cells. Conclusions: CDK2 and CCNE1 copy number variations were frequent in acral melanoma and CCNE1 gain may be a useful biomarker to predict the outcome of receiving chemotherapy in patients with acral melanoma. In addition, our study provides a basis for the use of CDK inhibitor in the treatment of acral melanoma.


Background
In Asians, acral melanomas accounts for 41.8-58% of melanomas and is the most common subtype [1 − 5].Acral melanomas showed a different genomic landscape from cutaneous melanoma, with a lower mutations burden ,a higher frequency genomic instability and widespread copy number changes [6,7].Cell-cycle aberrations were significantly higher in acral melanomas and were signifificantly associated with poor melanoma-specifific survival [6 − 8].
Cyclin E1 plays a role in cell cycle progression [9 − 11], inhibition of apoptosis [12], DNA transcription [13], replication [14], and DNA repair [13]. Post-translational regulation of cyclin E1 is involved in multiple proteasomal degradation pathways, including BTB-Cullin3-Rbx1 [15] and SCF Fbw7 [16] pathways. According to TCGA database, the expression of CCNE1 and CDK2 in cutaneous melanoma tumor tissue are much higher than those in normal tissues [17].The gain frequency of CCNE1 and CDK2 in cutaneous melanomas is about 16.3% and 12.5% respectively [17].However, there is still a lack of data on the frequency of CCNE1 and CDK2 copy number variation in large samples from acral melanoma. CCNE1 gain is related to poor survival in ovarian cancer, endometrioid endometrial carcinomas and breast cancer [18 − 21]. CCNE1-amplified ovarian cancer were insensitive to chemotherapy containing cisplatin [22] or CDK4/6 inhibitor PD0332991 [23].Unfortunately,there are no drugs specifically for target CCNE1. But CDK inhibitor and proteasome inhibitor bortezomib may be effective therapeutic drugs for tumors carrying CCNE1 amplification [22,24].Based on the above-mentioned factors,CCNE1 and CDK2 copy number variation may be a potential therapeutic target.While in melanoma,clinical trials and preclinical studies targeted CCNE1 or CDK2 5 gain have not yet been carried out.
Identifying predictive biomarkers and patient subsets which are most likely to benefit from certain inhibitors is important for the clinical development of new drugs.In this study,we investigated the CNV of CCNE1 and CDK2 in 490 acral melanoma patients, analyzed the correlation with clinical prognosis and evaluate the sensitivity to CDK inhibitor AT7519,Dinaciclib and proteasome inhibitor bortezomib.Our study indicates that CCNE1 gain may be a potential biomarker for predicting the treatment's effect in acral melanoma and AT7519 may selectively target acral melanoma patients with CCNE1 gain. For each test sample, normalized signal was divided by the reference DNA sample (G1521, Promega, Madison, WI) for CCNE1 and CDK2, and the values were multiplied by the known copy number of each gene in the reference genome. When the relative copy number is greater than or equal to 3.0, the copy number of CCNE1 or CDK2 is determined to be gain.

Patients and Tissue Samples
When the relative copy number is less than 2.0, the copy number of gene is determined to be lost.

Statistical Analysis
Statistical analyses were performed using SPSS 20.0 software. All statistical analyses were two-sided, and P < 0.05 was considered as statistically significant. The statistical significance between the outcomes of PFS and OS were calculated using Kaplan-Meier method.Cox proportional-hazards regression analysis was conducted to estimate the hazard ratio (HR).

Results
Aberrations of C D K 2 a n d C C N E 1 i n a c r a l m e l a n o m a Among the 490 acral melanoma patients, the overall frequency of patients containing any CDK2 or CCNE1 copy number variation (CNV) (≥ 1 CNV) was 49.0% (240/490).140 cases (28.5%) contained CDK2 gain and 139 cases (28.3%) contained CCNE1 gain.Moreover, 39 cases (8.0%) contained concurrent CDK2 gain plus CCNE1 gain.Most of the copy number of samples with CDK2 or CCNE1 gain was about 3-4 copies ( Table 1).We also analyzed the correlation between CDK2 and CCNE1 copy number variations in acral melanoma and there was not significantly different (P = 0.874, Supplementary Table S1). Since targeted therapy of melanoma has been explored, we also analyzed the mutation frequency of genes that have been confirmed as promising targets in acral melanoma samples. In the samples containing either CDK2 or CCNE1 CNV (≥ 1 CNV), 12.2%, 19.6%, 14.8% and 4.8% of them also contained mutations in BRAF,KIT , NRAS or PDGFRA respectively.CDK2 and CCNE1 CNVs and targeted gene mutations pattern were showed in In our cohort, the age (< 60 years or ≥ 60 years), gender, ulceration, thickness were all not significantly different between patients with or without CDK2 gain, CCNE1 gain or CDK2 gain plus CCNE1 gain ( Table 2). Among the patients with CDK2 gain, the proportion of patients with stage III-IV was higher than those without CDK2 gain (P = 0.010; Table 2).While the TNM stage in patients with or without CCNE1 gain or CDK2 gain plus CCNE1 gain were not significantly different ( Table 2). Impact of C D K 2 a n d C   Effect ofCDK2 a n d C  Table S2).

S e n s i t i v i t y o f M e l a n o m a C e l l s t o M u l t i p l e I n h i b i t o r s
The

Discussion
Acral melanoma arises on the hands and feet in people, which accounts for a much higher proportion of melanomas in Asians than in Caucasians. [1 − 5] Acral melanoma genomes showed greater proportions of copy number variation than in cutaneous melanomas [6].
CDK2 and CCNE1 are important molecules related to cell functions.Cyclin E1 (encoded by 14 CCNE1) -CDK2 complex controls G1 to S transition in cell-cycle [9].The complex makes the cells to initiate DNA synthesis by phosphorylation of its downstream substrates such as Rb, CDC6, NPAT and P107, so that the cells are irreversibly involved in the S phase [10,11,25]. Cyclin E1 -CDK2 complex abnormal is associated with chromosomal instability [20].
CCNE1 also an important role in inhibition of apoptosis [12], DNA transcription [13], replication [14], and DNA repair [13],mediating asymmetric cell division, specifying cell fate and in driving stem cell self-renewal [26,27] .Post-translational regulation of cyclin E1 is involved in multiple proteasomal degradation pathways, including BTB-Cullin3-Rbx1 [15] and SCF Fbw7 [16] pathways.  [23, 29 − 31]. In addition, CCNE1 gain and over expression were associated with resistance to CDK4/6 therapies in breast cancer [32].But the reasons underlying treatment failure are not entirely clear. There was a view that cells have an intact high-fifidelity system for repairing the double stranded DNA breaks which were sustained after platinum exposure [33].Enhanced self-renewal due to CCNE1 copy number variation may also be one of the reasons for the mechanism for treatment failure [29].These findings suggested that CCNE1 gain is an important prognostic factor for acral melanoma and may be a potential biomarker for new treatments.
Given that acral melanoma with CCNE1 gain has a shorter PFS after receiving chemotherapy, new therapies are needed to improve the prognosis of these  [35].Dinaciclib is a highly potent inhibitor of CDK1, CDK2, CDK5 and CDK9 that has entered Phase III clinical studies.Dinaciclib can inhibit cell cycle progression in more than 100 tumor cell lines and induce tumor regression in mouse models [36].In the study of ovarian cancer, CDK inhibitor dinaciclib combined AKT inhibitors may selectively target patients with CCNE1-amplified HGSC [37].
In an estrogen receptor-positive breast cancer study, combined targeting of both CDK4/6 and PI3K may be the treatment of patients bearing a CCNE1 gain [30]. Recent       Effect of CDK2 and CCNE1 CNVs on Response to DTIC, DDP, and Endostar combined chemotherapy. Fig.3A showed the correlation between CDK2 copy number and PFS of melanoma patients received DTIC, DDP, and Endostar combined chemotherapy. Fig.3B showed the correlation between CCNE1 copy number and PFS of melanoma patients received chemotherapy.
28 Figure 3 Effect of CDK2 and CCNE1 CNVs on Response to DTIC, DDP, and Endostar combined chemotherapy. Fig.3A showed the correlation between CDK2 copy number and PFS of melanoma patients received DTIC, DDP, and Endostar combined chemotherapy. Fig.3B showed the correlation between CCNE1 copy number and PFS of melanoma patients received chemotherapy. 29

Supplementary Files
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