MYC copy number and mRNA expression in chronic lymphocytic leukemia patients exposed to ionizing radiation due to the Chornobyl NPP accident
Summary. Some clinical and biological features indicating an unfavorable course of the disease were found in ionizing radiation (IR) — related chronic lymphocytic leukemia (CLL) patients. The MYC proto-oncogene is considered to contribute to CLL pathogenesis. Increased MYC copy number is associated with poor prognosis in CLL. Aim: To investigate the frequency of MYC gene copy number amplification in IR-exposed CLL patients and relate the findings to the MYC mRNA levels, the presence of unfavourable prognosis mutations (TP53, SF3B1, NOTCH1), and patient's outcome. Materials and Methods: The analysis of MYC copy number was carried out by real-time quantitative polymerase chain reaction (PCR) in 70 IR-exposed CLL patients. The MYC mRNA expression was measured by real-time quantitative reverse transcription PCR. Results: Increased MYC gene copy number was present in 5.7% of cases. There was a statistically significant association between increased MYC copy number and increased MYC mRNA (p < 0.014). Additionally, somatic deletion in MYC locus was found in one patient. Most of patients (80%) with detected MYC aberrations were previously untreated, suggesting that these lesions might occur early in the course of the disease. The MYC aberrations were found mutually exclusive with high risk TP53 and SF3B1 mutations, while one case was identified, where MYC amplification and NOTCH1 mutation coincided simultaneously. Regarding clinical outcome, the MYC aberrations were associated with a shorter time to first treatment (3 vs 25 months, p = 0.008) as well as reduced overall survival (60 vs 139 months). Conclusion: Our data suggest that MYC aberrations might be an early event in IR-related CLL and contribute to aggressive disease development in the absence of high risk TP53 and SF3B1 mutations.
Submitted: November 07, 2019.
Correspondence: E-mail: firstname.lastname@example.org
Abbreviations used: CLL — chronic lymphocytic leukemia; CN — copy number; IR — ionizing radiation; OS — overall survival; PCR — polymerase chain reaction; TTFT — time to first treatment.
Chronic lymphocytic leukemia (CLL) is the most common leukemia among the adults, which is characterized by a high clinical and biological variability . Multiple genomic alterations and gene mutations have been identified as drivers of the disease, which might affect significantly clinical outcome. These alterations are associated with several key cellular signal pathways and processes: DNA damage response (ATM, CHEK2, BRCC3), apoptosis and cell cycle control (TP53), NOTCH1 signalling, RNA processing and export (SF3B1, XPO4), B cell activity related pathways (TRAF2, TRAF3), MAPK signalling. Besides, dysregulation of MYC activity is considered as one of central cell pathways involved in CLL .
MYC gene, located at chromosome 8q24.21, is one of the proto-oncogenes, which is most frequently involved in human carcinogenesis . MYC encodes for a transcription factor of the helix-loop-helix/leucine zipper protein family, which in form of dimers with the protein MAX binds to specific sequences — E-boxes (canonical sequence CACGTG) in regulatory regions of target genes. MYC/MAX dimers regulate 10–15% of all human genes and thus control a variety of cellular functions, including cell cycle progression, growth, survival, differentiation and biosynthesis [4–6]. Because of its central role in cell processes, MYC is tightly regulated at both the transcriptional and translational levels . In normal cells MYC mRNA and protein have very short half-lives. Without appropriate positive regulatory signals MYC protein levels are low and insufficient to promote cellular proliferation [8–10]. In cancers the delicate balance of MYC regulation is often disturbed. Several mechanisms for MYC deregulation have been identified, including translocation, amplification, mutations, as well as mutations of cellular pathways involved in its regulation [3, 7].
In CLL, mutations in multiple genes were identified, although most of them with low frequency, which might influence MYC activity — NOTCH1, MGA, FBXW7, PTPN11, FUBP1 . MYC amplification and translocation were reported rare in general CLL cohorts (up to 4%) , however, their frequency was found increased in CLL subsets with aggressive disease, and especially in Richter transformation (up to 40%) [12, 13]. Recently Edelmann et al.  showed that MYC amplification was highly enriched in relapsed/refractory TP53-deficient CLL group (17%). Besides, both MYC amplification and high MYC expression were reported to be associated with poor prognosis in CLL patients [11, 15].
We previously found some clinical and biological features of CLL patients exposed to ionizing radiation (IR) due to Chornobyl NPP accident indicating unfavorable course of disease, such as high frequency of secondary solid tumors and Richter transformation, mainly unmutated status of heavy chain variable region (IGHV) genes with increased usage of IGHV1-69 and IGHV3-21 . Thus, we were tempted to study the frequency of MYC copy number (CN) amplification in IR-related CLL patients to estimate the influence on disease development. In this study we investigated MYC CN amplification in relation to the MYC mRNA levels, molecular markers (mutations in TP53, NOTCH1, and SF3B1 genes), and patient’s outcome.
MATERIALS AND METHODS
Patients and samples. The studied cohort included 70 IR-exposed CLL patients (61 males and 9 females), referred to the State Institution “National Research Center for Radiation Medicine of the National Academy of Medical Sciences of Ukraine”. Fifty-four of 70 CLL patients were clean-up workers, 10 — inhabitants of radionuclide contaminated areas, and 6 patients — evacuees. The study was approved by the local Ethics Review Committee, and all patients gave informed consent prior to participation in it. The diagnosis of CLL was based on clinical history, lymphocyte morphology, and immunophenotypic criteria. The clinical stage of CLL was determined based on the Binet  and Rai  staging systems, and treatment requirement was according to National Cancer Institute criteria .
Most patients (n = 61, 87.1%) were untreated at the time of sample collection, and 9 patients were treated with various therapeutic regimens using chlorambucil (5) and fludarabine (4). Previously treated patients were observed in relapse before the next course of therapy. Molecular characteristics such as mutational status of TP53, NOTCH1 genes were known for all 70 patients, SF3B1 — for 68 patients, and mutational status of IGHV genes — for 69 patients, as a result of previous studies [16, 20].
Genomic DNA and total RNA were extracted from peripheral blood mononuclear cells with the QIAamp Blood Mini Kit (Qiagen, Crawley, United Kingdom) and TRI Reagent® (Molecular Research Center Inc., Cincinnati, OH) respectively, and were stored in deep freeze until further use.
Quantitative real-time polymerase chain reaction (PCR). For MYC CN analysis quantitative real-time PCR (qPCR) was performed with the Real-time IQ Detector System (Bio-Rad) using SYBR Green chemistry. Amplification was carried out with 50 ng DNA in a 25 μl reaction mixture containing 80 nМ of each primer and Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The BRG1 gene served as an internal control. The following primers were used: for MYC — 5′-TGGATCACCTTCTGCTGGA-3′ and 5′-TCTGACACTGTCCAACTTGACC-3′ (reported by Chen H et al. ), for BRG1 — 5′-AAGAAGACTGAGCCCCGACATTC-3′ and 5′-CCGTTACTGCTAAGGCCTATGC-3′ (reported by Buscarlet et al. ). qPCR cycling conditions were as follows: an initial denaturation step of 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. All reactions were done in triplicate, and all qPCR runs included 2 calibrator samples (DNA of healthy donors). The relative CN of MYC gene was determined using the comparative ∆∆Ct method and was calculated as CN = 2-∆∆Ct (2 x 2-∆∆Ct — per diploid genome) . To estimate boundary values for 2 copies of MYC gene, qPCR amplification was performed with DNA samples of 8 healthy donors following Kindich et al. . In this study, the MYC gene CN was considered increased in sample when CN was > 2.4.
For MYC expression analysis, cDNA was synthesized using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the supplier’s instructions. qPCR was performed with the Real-time IQ Detector System (Bio-Rad) using Absolute Blue qPCR SYBR Green Master Mix (Thermo Scientific) as reported previously . The expression levels of MYC were normalized to the expression of the housekeeping gene ABL1. The following primers were used: for MYC — 5′-TCGGATTCTCTGCTCTCCTC-3′ and 5′- GAGCCTGCCTCTTTTCCAC-3′ (reported by Caraballo et al. ); for ABL1 — 5′-TGGAGATAACACTCTAAGCATAACTAAAGGT-3′ and 5′-GATGTAGTTGCTTGGGACCCA-3′ (reported by Beillard et al. ). The relative expression levels were calculated using the ∆∆Ct method. The MYC expression of peripheral blood sample of healthy donor was used as a calibrator for relative quantification. Additionally, qPCR products we analysed by electrophoresis in 1.5% agarose gel and visualization under UV light after ethidium bromide staining.
Statistical analysis. Associations with categorical clinico-biological variables were assessed with the chi-square test or the Fisher exact test, where appropriate. The Mann-Whitney U test was used to compare median levels of MYC between groups. Kaplan — Meier method was used to analyse overall survival (OS) and time to first treatment (TTFT), the log-rank statistic was used to determine significant associations between individual markers and OS or TTFT. P values <0.05 were considered statistically significant. All analyses were performed with the SPSS software package, version 13.0 (SPSS).
CLL group characteristics. The studied CLL group consisted of 61 men and 9 women, with a median age of 57.7 years (range: 39–76 years) at the time of diagnosis. Clinical and biological characteristics of patients are provided in Table 1. Sixty-one (87.1%) of patients were untreated at the time of sample collection, while 9 (12.9%) were previously treated. In the subsequent period, 48 (68.6%) of patients received therapy. Overall, 57 (81.4%) patients were treated, with the median time from diagnosis to treatment of 33 months (1–156 months).
Table 1. Baseline characteristics of observed CLL patients
Note: *IGHV mutation status data were available for 69 patients. **SF3B1 status data were available for 68 patients.
Forty-eight (69.6%) patients had unmutated IGHV genes, whereas 21 (30.4%) patients had mutated IGHV. Mutation in at least one of the three common mutated genes (TP53, SF3B1 or NOTCH1) was detected in 18 (25.7%) cases. Eight (11.4%) of patients harbored NOTCH1 mutation, in 4 (5.7%) of cases both TP53 and SF3B1 genes were found mutated, isolated TP53 and SF3B1 mutations were found in 3 cases each (3.3%). Unmutated IGHV status and presence of TP53 or SF3B1 mutations were highly predictive factors of reduced TTFT and OS in this CLL group (Table 2).
Table 2. OS and TTFT in CLL patients according to clinical and molecular characteristics
Note: NR — not reached.
Frequency of MYC aberrations. Increased MYC gene CN (CN > 2.4) was found in 4 cases (5.7% patients). The MYC CN values ranged from 2.8 to 3.4 copies/cell. Three of these patients did not receive previous treatment and one patient was previously treated. We did not reveal a decrease in MYC CN in any case of studied CLL group.
Additionally, analysis of PCR products of с.1066-1226 MYC region amplification by electrophoresis showed shortened band in one patient (Fig. 1). This patient was previously untreated. The shortened amplicon differed by more than 10 bp compared to intact PCR product (160 bp) indicating the deletion in с.1066-1226 region of MYC (protein position: 235–287 aa). We did not find similar size deletion among COSMIC-listed MYC mutations [COSMIC database (http://cancer.sanger. ac.uk/cancergenome/projects/cosmic/]. However, that MYC region contains known phosphorylation sites, where it was demonstrated in Burkitt lymphoma, somatic mutations clustered significantly . Since phosphorylation is required for ubiquitination and subsequent degradation of the MYC protein by the proteasome, abrogation of phosphorylation could lead to a decrease of protein degradation and hence, increase the stability of the MYC protein . Thus, protein stabilization due to mutations in close to phosphorylation regions might be one of mechanisms to deregulate MYC in CLL.
Fig. 1. Results of PCR amplification of с.1066-1226 MYC region (160 bp) in CLL patients. The shortened band of about 150 bp indicates somatic deletion (marked by arrow)
MYC aberrations are almost mutual exclusive with common CLL mutations. MYC aberrations (increased MYC CN and MYC deletion) were found mutually exclusive with mutations in the TP53 gene as well as SF3B1 mutations (Table 3). At that, more than half (4 of 7; 57.1%) of TP53 mutated cases were represented simultaneously with SF3B1 mutation. This frequent representation of both TP53 and SF3B1 mutations was described by us previously as distinctive feature of IR-related CLL group . Concerning NOTCH1 gene, one case was identified, where MYC amplification and NOTCH1 mutation coincided simultaneously.
Table 3. MYC aberrations and MYC expression in relation to clinical and molecular characteristics
MYC aberrations are associated with unfavorable clinical outcome. Follow-up information was available for all patients, 32 (45.7%) of whom died during the period of observation due to CLL-related causes. The estimated median OS was 117 months (95% CI = 77.5–156.5 months). Log-rank tests for the Kaplan — Meier curves demonstrated significantly reduced TTFT in CLL patients with MYC aberrations in comparison with the rest of cohort — 3 vs 25 months, p = 0.008. These patients experienced also shorter overall survival — 60 vs 139 months, p = 0.001 (Fig. 2).
Fig. 2. Survival analysis. TTFT (a) and OS (b) in CLL patients regarding presence of MYC aberrations
MYC expression. Overall MYC expression was low in most CLL cases studied. The average relative MYC expression level was 5.7 (range, 0–48.5). The cases with revealed MYC CN amplification (n = 4) showed increased MYC expression (median = 8.5, range 6.2–19.5) in comparison with the rest of the group (median = 2.7, range 0–48.5), p < 0.014 (Fig. 3). In the case with MYC deletion, low MYC level was detected.
Fig. 3. Expression levels of MYC in relation to MYC copy number. Boxplots show relative expression of MYC in CLL patients with increased MYC copy number and not-increased MYC copy number. Median, 25 and 75 percentile values, non-outlier ranges, outliers and extremes are indicated. The p value was calculated using the Mann — Whitney U test
For the MYC expression stratification, the overall mean MYC value (5.7) was used as the cut-off to designate increased vs low expression, following Stasik et al. . Thus, in the study 19 (27.9%) of cases were classified as cases with increased MYC, while 49 (72.1%) — as low MYC expressed cases. Analysis of associations of MYC expression with clinical and biological variables, and clinical outcome was done using this stratification (see Table 3).
We failed to find a significant correlation between MYC values and clinical variables. We did not detect also any correlation between MYC expression and any of the molecular markers analysed (such as unmutated IGHV, mutated TP53, SF3B1 and NOTCH1 genes). Besides, there was no significant difference in TTFT and OS between groups with increased MYC and low MYC expression. That is in line with report by Caraballo et al. , where also no associations were found between MYC expression and several clinical and biological variables, including NOTCH1 and TP53 mutations. Besides, authors reported absence of close correlation between MYC mRNA and protein level. Contradictory results were also reported, where high expression MYC levels have been described [15, 30]. Further study is necessary to clarify this issue taking in view that MYC is tightly regulated at both the transcriptional and translational levels .
The MYC gene amplification and mutations are, in addition to mutations in MYC-related pathways, mechanisms of MYC protein deregulation in CLL. MYC aberrations were detected in 5 (7.1%) cases in our study: CN amplification was present in 4 (5.7%) cases, and in one case somatic deletion was detected in MYC locus. MYC amplification was found to be associated with increased MYC mRNA expression that agrees with previous reports [11, 14]. Most of patients with MYC aberrations (80%) were previously untreated indicating that these lesions might occur early in the course of the disease. Whole-genome sequencing studies analysing clonal heterogeneity also suggested gains at 8q24 (where MYC maps) as early events during CLL development .
Overall, the frequency of MYC amplification in studied group is slightly higher in comparison with CLL cohorts reported (up to 4%) [11, 14]. At that, MYC amplification might be underestimated in our studied group since we have assessed MYC locus only. Other MYC-related CN aberrations were identified in CLL using high resolution array-based technologies recently. In addition to MYC locus amplification, focal amplifications in 8q24.21 affecting a super-enhancer region approximately 360 kb centromeric to MYC locus were detected [11, 14]. The 8q24.21 “gene desert” regulatory region contains multiple single nucleotide polymorphisms that have been associated with susceptibility to various cancers including CLL [31, 32]. According to Edelman et al. , minimally gained region in 8q24.21 in CLL encompassed three long non-coding RNAs — CASC19, CCAT1, and CASC21. CCAT1 (colon cancer associated 1) was associated with adverse risk in several solid cancers, its transcript was shown to stabilize a chromosome loop between MYC and the enhancer region . Besides, the 8q24.21 super-enhancer region was shown to contain binding sites for the NICD1 (NOTCH1 intracellular domain) transcription factor . Edelman et al.  reported focal amplifications in 8q24.21 in 1.3% of cases and MYC locus amplification — in 2.2% of cases in standard risk CLL subset.
In one case in our cohort, somatic deletion was detected in MYC region с.1066-1226 (protein position: 235-287 aa), which included partially the second and the third exons. We did not find similar deletion among COSMIC-listed MYC mutations. This MYC region contains known phosphorylation sites, in proximity to which somatic mutations clustered significantly in Burkitt lymphoma . Phosphorylation is required for ubiquitination and degradation of the MYC protein by the proteasome, and abolition of phosphorylation could lead to a decrease of protein degradation, increase the stability of the MYC protein, and thus MYC activity . Broun et al.  reported one CLL case with missense substitution Thr58Ala related to regulatory phosphorylation site in the first exon of MYC. That substitution was previously identified in Burkitt lymphoma and was shown to impair FBXW7-mediated proteasomal degradation of MYC that resulted in its activation . Thus, protein stabilization due to mutations close to phosphorylation regions might be one of mechanisms, although infrequent, to deregulate MYC in CLL.
MYC aberrations were found to be represented almost mutually exclusive with TP53, SF3B1 and NOTCH1 mutations in studied cohort. Regarding NOTCH1 mutations, this agrees with the finding that MYC is one of main target gene of NOTCH1. Tight relation was demonstrated between aberrantly strong NOTCH1 signaling, caused particularly by activating mutations in NOTCH1 gene, and increased MYC activity in CLL . SF3B1 mutations are supposed to constitute another frequent mechanism to strengthen the NOTCH1-MYC signaling axis . It was shown that the strength of NOTCH1 signaling depends on DVL2 (multi-domain protein Dishevelled 2), which inhibits transcriptional activation by NOTCH1 . Mutations in SF3B1 lead to alternative splicing of DVL2 and the resulting splice variant was shown to lack its ability to modulate NOTCH1 signaling . In line with these, NOTCH1 and SF3B1 mutations are often mutually exclusive in CLL .
Possible involvement of SF3B1 mutations in NOTCH1-MYC signaling axis might explain our results indicating mutual exclusivity of MYC aberrations and TP53 mutations in studied cohort. This contradicts the study, where the frequency of MYC amplification was found significantly increased in TP53-deficient CLL subset . However, in more than half (57.1%) of TP53 mutated cases in our cohort SF3B1 mutation was presented simultaneously. The frequent coincidence of TP53 and SF3B1 mutations was described by us previously as distinctive feature of IR-related CLL subset .
MYC aberrations were associated with reduced TTFT and OS in studied CLL cohort. The association with unfavorable outcome was observed in the absence of high risk TP53 and SF3B1 mutations. Broun et al.  previously reported that association of MYC amplification with short TTFT was independent of co-occurrence of high risk deletions 11q and 17p.
In conclusion, the results of our study suggest that MYC aberrations might be early events in IR-related CLL and contribute to aggressive disease development in the absence of high risk TP53 and SF3B1 mutations. Further studies are needed to precise the incidence and value of MYC lesions in IR-related CLL, including analysis of the 8q24 risk region near MYC for focal amplifications and somatic mutations of the entire MYC locus.
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