Epigenetic silencing of HOXA4 gene is associated with imatinib resistance, disease progression, BCR-ABL1 transcript type and smoking status among Chronic Myeloid Leukemia patients

doi:10.21203/rs.3.rs-2268696/v1

Background

We aimed to investigate the role of HOXA4 gene methylation concerning response to imatinib, and chronic myeloid leukemia (CML) progression, also investigated the impact of the type of BCR-ABL1 transcript and smoking on methylation level.

Methods

45 CML patients at different clinical stages of the disease (including 20 good responses and 25 non-mutated imatinib-resistant patients) and 15 health controls were recruited. Methylation level of HOXA4 gene promoter was evaluated by Methylation Sensitive High-Resolution Melt (MS-HRM) analysis.

Results

There was a significant difference in the mean of HOXA4 methylation percentage between two response groups (mean = 63.8; SD = 17.79 vs mean = 47.75; SD = 14.18, P = 0. 002). HOXA4 promoter hypermethylation (51–100% methylation level) indicated a higher risk for progression to advance phase (OR = 5.75; 95% CI: 1.40-23.49; P = 0.01) and imatinib resistance (OR = 8.5; 95% CI: 1.96–36.79; P = 0.004). Importantly, the patients with b2a2 transcript and smokers were associated with a higher percentage of HOXA4 methylation (OR = 7.08; 95% CI, 1.80-27.79; P = 0.005, OR = 11.8; 95% CI, 2.67–52.67; P = 0.001 respectively).

Conclusion

Our study suggests the association of the HOXA4 gene hypermethylation with imatinib resistance and CML progression. Moreover, the BCR-ABL1 transcript type and smoking have an impact on methylation levels.

Chronic myeloid leukemia

HOXA4

Hypermethylation

Imatinib mesylate

Resistance

Smoke

Chronic myeloid leukemia (CML) is a myeloproliferative cancer and its pathogenesis is associated to the BCR-ABL1 fusion oncogene. BCR-ABL1 gene is the result of reciprocal translocation of chromosomes 9 and 22, and codes for bcr-abl1 fusion protein, which has constitutive tyrosin kinase activity [1, 2]. Imatinib mesylate (IM) is first line and gold standard drug for treatment of CML patients; it targets the tyrosine kinase domain (TKD) of bcr-abl1 protein and inhibits kinase activation and signal transduction [3]. The treatment with IM has significantly enhanced patients^ۥ survival in two thirds of them [3, 4]. Despite the success of treatment with imatinib, IM resistance is a significant clinical problem and nearly 40% of patients will finally fail IM treatment [3].The evolution of resistance to IM is a multifactorial event in CML patients and different mechanisms of resistance to IM have been explained including BCR-ABL1 dependent and BCR-ABL1 independent pathways [5, 6]. The point mutations in kinase domain as BCR-ABL1 dependent mechanism are generally the cause of resistance to IM [7]. Previously, the contribution of mutations in BCR-ABL1 TKD in mediating resistance to IM among Iranian CML patients has been reported [8], and showed that mutations are not the only cause for resistance development. There are epigenetic mechanisms that independent of BCR-ABL1 gene contribute to IM resistance and may be potential factors in resistant patients who do not have mutations [9]. There are potent symptoms that drug response and clinical outcomes of CML disease are not alone determined by BCR-ABL1 gene, but are involved a number of other genetic and epigenetic aberrations that are substantial in regulating the high rate of proliferation and the prevention of apoptosis [10]. The significance of epigenetics in the development of various human cancers, especially leukaemias, has obtain much recognition in the last decade [10, 11]. Epigenetic silencing via DNA methylation in promoter CpG regions of different genes inhibits gene transcription, which leads to decreased protein expression [9]. Hypermethylaion of several genes including tumor suppressor genes have been detected to play a role in carcinogenesis through affecting normal cell growth. In addition, abnormal DNA methylation has been correlated with the formation of leukemic clones resistant to tyrosine kinase inhibitors and considered as the cause of CML progression [12]. Some studies [13–15] have indicated that hypermethylation of several genes might play a role in disease progression and development of IM resistance in CML. The homeobox (HOX) genes, comprising transcription factors are a family of four separate clusters (HOXA, HOXB, HOXC and HOXD). These genes are major regulators of embryonic development [16], hematopoitic differentiation, leukemogenesis and development of hematologic cancers [17, 18] and treatment response [19]. DNA methylation has been attributed to have an important role in abnormal control of HOX gene expression and improper expression of HOX gene is involved in development of hematopoietic malignancies [20]. Strathdee et al.[20] indicated a novel role for HOX genes especially HOXA4 and HOXA5 in development of CML as tumor suppressor genes, in spite of oncogenic role for more 5^' HOXA cluster genes. In several studies found that hypermethylation of HOXA4 and HOXA5 genes in CML and other lymphoids cancers are related to poor response to drug and progression to blast crisis (9,16,20). Therefore ,in the current study we aimed to evaluate the status and the role of aberrant promoter methylation of HOXA4 gene in relation to imatinib response and disease progression. Moreover, we have also evaluated the association of BCR-ABL1 transcript type and smoking status with methylation level of HOXA4 gene in CML patients for the first time.

Patients and healthy control specimens

A total of 45 BCR-ABL1 positive CML patients (including both good response (n = 20) and 25 imatinib resistant patients who had not mutation on BCR-ABL) and 15 healthy controls who had no history of the cancer or any other disease were recruited for evaluating of promoter methylation percentage of HOXA4 gene by methylation sensitive high resolution melt (MS-HRM) analysis. Thirty two CML patients were in chronic phase (CP) and 13 were in accelerated and blast crisis phases (AP and BC respectively), treated for at least 18 months with IM (400, 600 and 800 mg/ day) as a first line treatment. The patients were followed up for 48 months after imatinib treatment start. CML patients were classified into IM resistant and IM good responders based on their cytogenetic or molecular response according to European LeukemiaNet (ELN) 2013 criteria [21]. Based on these evaluation criteria, IM resistant patient was determined as absence of major molecular response (MMR) by 18 months and/or complete cytogenetic response (CCyR) by 12 months after start of the treatment [21]. In this study, the resistant patients selected had no mutation in kinase domain of ABL1 gene as were analyzed previously (data not shown).

DNA isolation and bisulfite treatment

The isolation of genomic DNA was performed from peripheral blood (4mL) of CML patients and normal controls using salting out method as before described [22]. The quality of extracted DNA was checked using agarose gel electrophoresis and quantified by NanoDrop (Epoch, BioTek, USA). 500 ng/µl of extracted DNA as the proper concentration was modified with sodium bisulfite using EZ DNA Methylation Gold kit (Zymo research, USA) according to company recommendation. Fully methylated (100%) and fully unmethylated (0%) DNA controls were universal and bisulfite converted (EpiTect control DNA methylated and unmethylated, Qiagen, Hilden,Germany). A serial of methylation dilution were prepared in proportions (10%, 25%, 50%, and 75%) and used as DNA standards of different methylation levels. These DNA standards were included in each experimental run to establish gradient standard curves. These curves were used to evaluate the methylation percentages of the patients and normal controls.

MS-HRM analysis

MS-HRM primers were designed using MethPrimer V2.0 online program (www.urogene.org, chinese academyof Medical Science). Primer designed based on protocol standard proposed by Wojdacz [23] with some corrections. Therefore, the primers sequences designed for HOXA4 promoter in our study were 5^´-TGTTAGTTTAGAATTTTTGAAGGATA-3^´ (forward primer) and 5^´- AAACCCTATCAAACTATAAATCCTC-3^´ (reverse primer). The primers were blasted before synthesized, using the Bisearch tool (http://bisearch.enzim.hu).

MS-HRM analysis and amplification of converted DNA was done by a step one plus real time PCR system (Applied Biosystem, USA). The HRM data was analyzed by high resolution melt analysis software. The whole reaction volume was 10 µl, which consisted of, master mix 5X HOT FIREPOL EvaGreen HRM( noROX) (Solis Biodyne Co, Estonia), 200 nM of each primers, 20 ng of bisulfited DNA and molecular grade water (DNase and RNase free water, Sinaclon Bioscience Co, Iran) was used to bring the final reaction volume to 10 µl. PCR condition was set as follow: The initial denaturation at 95^°C for 12 minutes, 45 cycles including denaturation at 95^°C for 15 seconds, annealing at 62.5^°C for 1 minute and extension at 72^°C for 30 seconds. The PCR amplification was followed by heteroduplex formation at 95^°C for 10 seconds and then at 60^°C for 1 minute. After this step, HRM analysis was carried out by 0.3^°C increments, and the temperature was retained for 15 seconds of each increment. The increments were done from 60^°C to 95^°C. To confirm accuracy and repeatability, each reaction was conducted in duplicate. In each experimental run, a negative control (no template) and set of serial percentage control (0%, 10%, 25%, 50%, 75%, 100%) were included. MS- HRM data were used to create difference plot. Annealing temperature was adjusted to obtain 50% concentration peak in order to prove the absence of bias towards the methylated or non-methylated pattern. Aligned melt curve was created by considering 0% as the reference relative fluorescent unit, using step one plus HRM software. Standard curves were drawn for all of the methylation control levels (10%, 25%, 50%, 75%, and 100%). MS-HRM analysis was done based on the comparison of melt profiles of samples to melt profiles from DNA with known methylation percentages for methylation controls.

Statistical analysis

The distributions of baseline features were compared for qualitative variables using χ 2 and Fisher exact test and for age as a quantitative variable using t- test. Logistic regression test was performed to evaluate the relationship between HOXA4 promoter methylation levels and IM resistance risk in CML patients by computation of Odds ratios (ORs) and 95% confidence intervals (CIs). Statistical analysis was performed using the SPSS 22.0 software (IBM Corporation). P values of < 0.05 were considered statistically significant.

Clinical profile

In this study, 45 CML patients including 25 BCR-ABL non- mutated IM resistant patients, 20 good response patients, and also 15 health controls were analyzed for evaluation of promoter HOXA4 methylation percentage. Clinical features including sex and age of patients, BCR-ABL type, follow up time, disease phase, smoking status and methylation level were compared between IM resistant and good response patients group. There was no significant difference in the mean of age, sex, and follow up time and BCR-ABL type between response groups (P > 0.05). However, disease phase and the smoking status difference were significant between IM resistant and good response patients (P= 0.0006, P= 0.01 respectively), as the number of smokers and also the patients with accelerated phase in IM resistant group were higher than good response group. The baseline features of the patients are shown in Table 1.

Table 1. Clinical features of the studied patients

P-value

IM good responder

IM resistance

Clinical features

0.5

20

10/10 (50%/50%)

25

15/10 (60%/ 40%)

Individuals, n (%)

Gender

Male/ Female

0.41

47.4±16.41

43.14±16.43

Age (y)

(Mean± SD)

0.34

10(50%)

16(64%)

9(36%)

BCR-ABL type, n (%)

b3a2

b2a2

0.0006**

0.07

20 (100%)

0

12(52%)

10(36%)

3(12%)

Disease phase , n (%)

Chronic

Accelerate

Blastic crisis

0.01*

6(30%)

14(70%)

17(68%)

8(32%)

Smoking status, n (%)

Smoker

Non smoker

0.49

45±12.3

48± 16.24

Follow up duration

(Mean±SD)

0.002**

47.75±14.18

63.8± 17.79

Promoter methylation level of HOXA4 gene

(Mean±SD)

Abbreviation: IM,imatinib mesylate; SD, standard deviation

^*P< 0.05 (bolded) ) was considered significant

Frequency of aberrant HOXA4 methylation

In this study, the MS-HRM analysis was done with receivable sensitivity and without any bias towards methylated or non-methylated patterns in concentration 50% methylation control as shown in Figure 1a. Aligned melt curve (Figure1b) and the difference plot (Figure 1c) show distinctive and separate peaks between all percentages of the methylation control including 0%, 10%, 25%, 50%,75% and 100%.

HRM analysis of resistant patients indicated that the methylation percentage of HOXA4 gene was distributed in the range of 25% to 100%, that most of them showed more than 60% of methylation, for good response patients in the range of 25% to 75% and for health controls 10% to 50%. There was a significant difference in the mean of promoter methylation level between IM resistant and good response patients (P=0.002) (Table 1), as the mean of methylation level was higher in IM resistant patients (mean=63.8; SD=17.79) compared with IM good response patients (mean=47.75; SD=14.18). Table 2 shows the methylation percentage frequencies of HOXA4 gene promoter in CML patients and health controls, and also in response groups, in which the methylation percentages were classified into four categories. The percentage of methylation level in high level category (75-100%) was significantly higher in CML patients than health controls and also in IM resistant group than good response group (p= 0.001, P= 0.002 respectively) (Table 2).

Table 2. Methylation percentage frequencies of HOXA4 gene in CML patients and health controls, IM resistant and good response patients

HOXA4 methylation (%)	Health control(n=15)	CML patients(n=45)	P –value	IM resistance(n=25)	Good response(n=20)	P- value
0-10	4	0	0.002**	0	0	1
25-49	5	9	0.29	3	7	0.08
50-74	6	17	0.87	7	10	0.13
75-100	0	19	0.001**	15	3	0.002**

Abbreviation: IM, imatinib mesylate .*P value < 0.05 (bolded) is significant

Association between methylation level and IM response

In this study, CML patients showed a range of 25-100% methylation level at the promoter region of HOXA4 gene and 85% of good response patients had methylation level in a range 25-50 %, because of this, for evaluation of methylation level difference between IM resistant and good response patients, the methylation percentages were classified into two categories (50% cutoff point) and cases with level of 51-100% only were considered as HOXA4 hypermethylated cases. Table 3 shows that there is a significant difference in methylation percentage 51-100% between IM resistant patients and good response cases, in other words, HOXA4 promoter hypermethylation in 51-100% level indicated a higher risk for IM resistance (OR= 8.5; 95% CI: 1.96-36.79; P= 0.004) (Table 3).

Table 3. Risk association between HOXA4 methylation level and IM response amidst CML patients

HOXA4 methylation (%)

IM resistance

(n=25)

Good response (n=20)

P value

OR

95% CI

25-50

10 (40%)

17 (85%)

-

reference

51-100

15(60%)

3(15%)

0.004^**

8.5

1.96-36.79

Abbreviation: IM, imatinib mesylate; OR, Odds ratio ; CI, Confidence interval;^*P< 0.05 (bolded) ) was considered significant

Correlation between HOXA4 hypermethylation and disease progression

In this study, hypermethylation of HOXA4 promoter was significantly more frequent in patients with advanced phases than chronic phase (69% vs 28% P= 0.01), for evaluation of methylation level differences between chronic and accelerated/ blast crisis phases patients, the methylation percentages were classified into two categories and cases with level of 51-100% only were considered as HOXA4 hypermethylated cases. Table 4 shows that there is a significant difference in methylation percentage 51-100% between chronic phase patients and advanced phase cases, in other words, HOXA4 promoter hypermethylation in 51-100% level indicated a higher risk for progression to accelerated and blast phases (OR= 5.75; 95% CI: 1.40-23.49; P= 0.01) (Table 4).

Table 4. Risk association between HOXA4 methylation level and disease progression among CML patients

HOXA4 methylation (%)

Accelerated+Blast phase

(n=13)

Chronic phase (n=32)

P value

OR

95% CI

25-50

4(30.77%)

23 (71.87%)

-

reference

51-100

9(69.23%)

9(28.13%)

0. 01^*

5.75

1.40-23.49

Abbreviation: OR,Odds ratio ; CI, Confidence interval;^*P< 0.05 (bolded) ) was considered significant

Correlation between HOXA4 methylation level and BCR-ABL1 type

Based on the patients' clinical file, 26 out of 45 patients (57.8%) had b3a2 transcript and 42.2% patients (19/45) b2a2 transcript. A significant correlation was found between HOXA4 methylation level and BCR-ABL1 type among CML patients, a risk of hypermethylation in patients with b2a2 transcript type was 7 fold higher than b3a2 type (OR= 7.08; 95% CI, 1.80-27.79; P= 0.005) (Table 5).

Table 5. Risk association of HOXA4 methylation levels with BCR-ABL1 transcript type among CML patients

Methylation level (%)	BCR-ABL type		OR, 95% CI	P value
Methylation level (%)	b3a2(n,%)	b2a2(n,%)	OR, 95% CI	P value
50-100 %	9(34.6%)	15(78.94%)	7.08 , (1.80-27.79)	0.005^*
25-49%	17 (65.4%)	4(21.06%)

Abbreviation: OR,Odds ratio ; CI, Confidence interval;^*P< 0.05 (bolded) ) was considered significant

Association between HOXA4 methylation level and smoking in CML patients

In this study, the smoking status difference was significant in IM resistant group than good responders (P= 0.01). The numbers of smokers among IM resistant patients were higher than good responders (Table 1). To investigate the relationship between smoking and methylation level, we subdivided the methylation percentages into two categories as shown in Table 6. There was significant association between smoking and hypermethylation of promoter HOXA4 gene. Risk of hypermethylation among smokers were significantly higher in comparison to non-smokers (OR= 11.8; 95% CI, 2.67-52.67; P=0.001) (Table 6).

Table 6. Risk association between HOXA4 methylation status and smoking in CML patients

Methylation level (%)

Smoker (N=23)

Non – Smoker N=22)

OR, 95% CI

P value

51-100 %

15 (65.3%)

3 (13.6%)

11.8,
(2.67-52.67)

0.001**

25-50%

8 (34.7%)

19 (86.4%)

Abbreviation: OR, Odds ratio ; CI,Confidence interval;^*P< 0.05 (bolded) ) was considered significant

However, BCR-ABL1 fusion gene is genetic hallmark, a primarily character and inducer of genomic instability associated with CML start, many molecular mechanisms contributing to drug resistance and CML progression remain to be clarified. CML is genetically homogenous at the initial phase, but the mechanism of transformation from CP to BC and response to drug is heterogeneous [10].This heterogeneity can be attributed to the molecular events consist of genetic and epigenetic such as mutations, additional chromosomal aberrations (ACAs), CpG methylation patterns and histone modification that adjust to BCR-ABL1 fusion [20,24]. There is numerous and strong evidence that epigenetic alterations such as DNA methylation, as one of the important mechanisms in control of gene expression, are implicated in drug resistance in CML that leads to leukemic clone release and disease progression [12]. In the current study, to find association of methylation status of HOXA4 gene with CML disease progression and drug response, and also relationship of smoking and BCR-ABL type with methylation level, we analyzed the methylation percentage difference among CML patients in different clinical stages and healthy controls and also among response groups. Our data show that resistant CML patients have a significant hypermethylation in studied gene for 50% cutoff point, in comparison with good response patients. In addition, the proportion of patients with hypermethylation of promoter HOXA4 gene (51%-100% methylation level) were significantly more in advanced stages (AP and BC) than CP stage. In other words, risk of disease progression in patients with hypermethylation 51-100%, 5.7 fold higher than low level category (25-50%). These findings of our study support and extend the results of previous studies [10,12,13,20].Consistent with our study, in two related study [9,16] focusing on CML and HOXA4 gene, recently illustrated that hypermethylation of HOXA4 promoter is correlated with imatinib resistance and this finding provide an alternative to BCR-ABL1 mutations in estimating IM treatment response. In other study by Amabile et al. [12] demonstrated that a single oncogenic damage causes DNA methylation alterations that act as an accelerating event in leukemia progression. This finding provides a strong view for the synergism role of both genetic and epigenetic changes in leukemia development [12].

It seems that mechanism of IM activity and its relation with HOXA4 gene was not reported before Elias'study [9]. Expression of HOXA4 gene is inactivated by hypermethylation [9], given that HOXA4 protein is a transcription factor and regulate gene expression and differentiation, so, this suggestion is reasonable that hypermethylation of HOXA4 induces gene silencing as a mechanism of increase IM resistance in CML patients [9]. In this study, we evaluated association of HOXA4 hypermethylation with BCR-ABL1 transcript type for the first time among CML patients. To the best of our knowledge, there are no prior reports in this case, except in one study determined the methylation status of DAPK1 gene among patients that no found association between BCR-ABL1 type and the methylation level of DAPK1 gene [25]. In our study, HOXA4 hypermethylation level in 50%-100% category was significantly higher among b2a2 transcript patients compared to those with b3a2 type. We indicated that hypermethylation risk of HOXA4 was seven fold higher in b2a2 transcript than b3a2 type. The clinical importance of this finding is undefined yet; however, it may be related to a mechanism underlying imatinib resistance or disease progression. It has been elucidated that secondary structure is different in b3a2 because of the existence of extra 25 amino acids that not seen in b2a2 transcript that leads to changes in structure of DNA binding domain of BCR-ABL1 kinase and then decrease tyrosine kinase activity. So b3a2 and b2a2 transcripts may have different roles in mediating signal transduction pathways in CML [26].The other studies, reported that patients with b3a2 type achieved faster and deeper responses to drug than b2a2 type [26,27]. According to these findings, structural difference probably causes the difference of methylation level between patients with b2a2 type and those with b3a2 type.

In the present study, smoking status was investigated between resistant patients and good response patients and we observed significant difference between two groups. We evaluated the relationship between smoking and methylation level for the first time, there is no previous report. There was significant association between smoking and hypermethylation of promoter HOXA4 gene. Risk of hypermethylation among smokers was significantly higher in comparison to non-smokers. It has been described that environmental factors can have different effects on DNA methylation depending on when the exposure happens, such as within prenatal life or during adulthood. Especially, cigarettes smoking as an environmental factor and serious warning for the health in worldwide, in the fields of both present smoking and prenatal exposure is a potent adjuster of DNA methylation. So, DNA methylation is a potential mechanistic linkage between present smoking and cancer, also, prenatal smoke exposure and the development of chronic diseases in adulthood [28]. Epigenetic alterations generated by smoke are mainly caused by the activation of enzymes that regulate these epigenetic modifications and often mediates the expression of several inflammatory genes, indeed, smoke- induced inflammatory reactions may be cause alteration of DNA methylation patterns. Aforementioned enzymes are responsible for life-long preservation DNA methylation during cell division; including DNA methyltransferase 1(DNMT1) that is exhibiting in proliferative cells [29].In conclusion, our findings indicated that hypermethylation of HOXA4 gene is associated with IM resistance, CML progression. In addition, we concluded that hypermethylation of HOXA4 gene is higher in the patients with b2a2 type transcript and also in the smokers. A further investigation on a more number is needed to confirm of these results that could be as potential biomarkers of disease progression and resistance to imatinib.

CML, chronic myeloid leukemia; IM, imatinib mesylate; CP, chronic phase; AP, accelerated phase; BP, blastic phase; CCyR, complete cytogenetic response; MMR, major molecular response; ELN, european leukemia net; OR, odds ratio; CI, confidence intervalse; TKD, Tyrosine kinase domain; SD, standard deviation; MS- HRM, Methylation Sensitive High Resolution Melt

We are appreciative for the help and collaboration of the patients, as part of their participation in the study

Funding: This work was supported by the Pasteur Institute of Iran [grant number 1033 ]

Conflict of interest The authors declare that they have no conflict of interest

Author contributions

DR : Accomplished the experiments and collected clinical data, MH: designed the study, analyzed the data and wrote manuscript . MK and MJ: assisted in final revision of manuscript, and all of authors reviewed and gave the final approval for the paper.

Ethics approval

This research was approved by the Ethics committee of Pasture institute of Iran, and specimens were used according to ethical standards (ethical approval no. IR.PII.REC.1397.56). Informed consent from patients, as well as from controls was obtained.

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Epigenetic silencing of HOXA4 gene is associated with imatinib resistance, disease progression, BCR-ABL1 transcript type and smoking status among Chronic Myeloid Leukemia patients

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Patients and healthy control specimens

DNA isolation and bisulfite treatment

MS-HRM analysis

Statistical analysis

Results

Clinical profile

Frequency of aberrant HOXA4 methylation

Association between methylation level and IM response

Correlation between HOXA4 hypermethylation and disease progression

Correlation between HOXA4 methylation level and BCR-ABL1 type

Association between HOXA4 methylation level and smoking in CML patients

Discussion

Abbreviations

Declarations

References

Status:

Version 1