Downregulation of the histone methyltransferase SETD2 promotes imatinib resistance in chronic myeloid leukaemia cells

Abstract Objectives Epigenetic modifiers were important players in the development of haematological malignancies and sensitivity to therapy. Mutations of SET domain‐containing 2 (SETD2), a methyltransferase that catalyses the trimethylation of histone 3 on lysine 36 (H3K36me3), were found in various myeloid malignancies. However, the detailed mechanisms through which SETD2 confers chronic myeloid leukaemia progression and resistance to therapy targeting on BCR‐ABL remain unclear. Materials and methods The level of SETD2 in imatinib‐sensitive and imatinib‐resistant chronic myeloid leukaemia (CML) cells was examined by immunoblotting and quantitative real‐time PCR. We analysed CD34+CD38− leukaemic stem cells by flow cytometry and colony formation assays upon SETD2 knockdown or overexpression. The impact of SETD2 expression alterations or small‐molecule inhibitor JIB‐04 targeting H3K36me3 loss on imatinib sensitivity was assessed by IC50, cell apoptosis and proliferation assays. Finally, RNA sequencing and ChIP‐quantitative PCR were performed to verify putative downstream targets. Results SETD2 was found to act as a tumour suppressor in CML. The novel oncogenic targets MYCN and ERG were shown to be the direct downstream targets of SETD2, where their overexpression induced by SETD2 knockdown caused imatinib insensitivity and leukaemic stem cell enrichment in CML cell lines. Treatment with JIB‐04, an inhibitor that restores H3K36me3 levels through blockade of its demethylation, successfully improved the cell imatinib sensitivity and enhanced the chemotherapeutic effect. Conclusions Our study not only emphasizes the regulatory mechanism of SETD2 in CML, but also provides promising therapeutic strategies for overcoming the imatinib resistance in patients with CML.


| INTRODUC TI ON
Chronic myeloid leukaemia (CML) is a myeloproliferative disorder caused by the malignant transformation of hematopoietic stem cells (HSCs) through BCR-ABL oncogene initiation. 1 Resulting from a t(9,22) (q34;q11) chromosome translocation, the oncogene encodes a chimeric oncoprotein with constitutive tyrosine kinase activity. [2][3][4] Imatinib, a classical tyrosine kinase inhibitor (TKI) that specifically targets the BCR-ABL oncogene, has been a front-line drug for the clinical treatment of CML, leading to cytogenetic and molecular remission of the disease. [5][6][7][8][9] However, approximately 90% of treated patients ultimately develop imatinib resistance, resulting in disease relapse and poor outcomes. [10][11][12] Approximately 50% of the CML cases with imatinib resistance have been proven to be caused by BCR-ABL kinase domain mutations (including T315I, Q252H, G250E, E255K/V and Y253H) as well as locus amplification, 10,13,14 which can be relatively well cured by second-generation (Dasatinib, Nilotinib, and Bosutinib) and third-generation (Ponatinib) TKIs. [15][16][17] Additionally, the primary resistance driven by leukaemic stem cells (LSCs) has turned out to be a troublesome challenge, demanding prompt solutions. [18][19][20][21] With their traits of self-renewal, quiescence and reduced differentiation, 19,20 the LSCs derived from the BCR-ABL-initiated malignant transformation of HSCs show BCR-ABL -independent behaviour, 10,22 a fact that is exemplified by the failure of single TKI treatments to eliminate these cells. 23 Therefore, the exploration of potential targets of LSCs and the generation of novel therapeutic approaches for their specific eradication would significantly benefit the outcomes of patients with CML.
Epigenetic modifiers are involved in various myeloid malignancies and in normal hematopoiesis. For example, DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B play key roles in uniquely regulating the differentiation of hematopoietic stem cells and progenitor cells. [24][25][26][27][28] Meanwhile, genetic alterations through DNA methylation (DNMT3A, TET2 and IDH1/2) and histone modifications (EZH2, ASXL1, KMT2A, CREBBP and HDAC2/3) are found in all types of myeloid haematological disorders. 29,30 Histone deacetylations have been recently supposed to exert a pivotal role in leukemogenesis, as exemplified by the emergence of histone deacetylase inhibitors as therapeutic measures for targeting LSCs. 20,31 SET domain-containing 2 (SETD2) is the major mammalian methyltransferase responsible for catalysing the trimethylation of histone 3 on lysine 36 (H3K36me3). 32 Mutations of SETD2 have been found in various types of tumours, such as clear cell renal cell carcinoma, 33,34 breast cancer, 35,36 glioma, 37 acute leukaemia and chronic lymphocytic leukaemia. 38,39 In the recent decades, research studies on the loss-of-function mutations of SETD2 have been carried out to investigate the initiation and propagation of acute leukaemia by equipping LSCs with increased self-renewal potential. 38,40 Specifically, the downregulation of SETD2 was shown to contribute to chemotherapeutic resistance in MLL-AF9 fusion protein-associated leukaemia. 41 In mouse models with SETD2 specifically depleted, the loss of the methyltransferase disrupted normal hematopoiesis through the impairment of hematopoietic stem cell differentiation, thereby further facilitating their malignant transformation. 42,43 Herein, we demonstrate that the downregulation of SETD2 facilitates imatinib resistance in CML cells, with LSC marker upregulation, which could be successfully rescued by SETD2 overexpression.
Additionally, by restoring the H3K36me3 level through treatment with JIB-04 (a small-molecule inhibitor of H3K36me3 demethylase 41 ), the sensitivity of CML cells towards imatinib was effectively increased, providing a potential therapeutic strategy to overcome imatinib-resistant CML.

| Cell culture and drug treatment
The

| Construction of the shRNA and SETD2overexpressing adenoviral vector
The short hairpin RNA (shRNA) lentiviral vectors targeting SETD2, ERG and MYCN were respectively constructed to establish stable knockdown cell lines in TF1-BA and KCL-22-S cells, with an empty vector as a control. After infected with lentiviruses, the cells were

| Cell proliferation assay
Cells were maintained in 96-well plates (1.5 × 10 4 cells/well) with different doses of the experimental drugs at 37°C for 72 hours, following which 10 µL of Cell Counting Kit-8 (CCK-8) reagent was added to each well and the plates were further incubated at 37°C in a humidified 5% CO 2 atmosphere for 2.5-3 hours. Finally, the absorbance at 450 nm was measured using a microplate reader.
Finally, the cells were analysed on a BD FACS Canto II system (BD Biosciences, Franklin Lakes, NJ) or a C6 flow cytometer (BD Biosciences).

| Quantitative RT-PCR
Total RNA was isolated from the cells using the TRIzol reagent (Invitrogen) and then reverse-transcribed to cDNA using the PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) according to the manufacturer's instructions. qRT-PCR was performed using the SYBR Premix ExTaq (Takara) and an Applied Biosystems 7500 Fast Real-Time PCR system. The relative expression of the mRNA was analysed using the 2 −ΔΔC t method and normalized to the expression of β-actin. All experiments were repeated three times. The PCR primer sequences used in this study are shown in Supporting Information

| Colony formation assay
Cells were maintained in methylcellulose medium (Cat#H4434; STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 20 ng/mL human interleukin-6 (Cat#200-06-20UG; PeproTech, Rocky Hill, NJ) and 20 ng/mL human granulocyte/colony-stimulating factor (Cat#300-23-10UG; PeproTech), in 35-mm dishes with incubation at 37°C under 5% CO 2 and 95% humidity. After 10-12 days, colonies were observed and counted with an inverted microscope. After discarding adaptor sequences with poor quality, clean reads were collected and analysed using the TopHat-Cufflinks-Cuffmerge-Cuffdiff pipeline with default parameters. The reads were mapped to the Homo sapiens GRCh38 reference genome using TopHat v2.0.13. We used DESeq2 for differential gene expression analysis, with the differentially expressed genes defining as |log 2 Foldchange| ≥ 1 and q value <0.05. Fold change is the ratio of the two groups after homogenization. The q value (padj value, the corrected P value) is calculated with R package (DESeq2) and adjusted by Benjamini-Hochberg method according to the P value. This experiment was conducted by Annoroad Gene Tech. (Beijing)

| mRNA sequencing analysis
Co., Ltd. The RNA-seq dataset was submitted into the GEO database, and the accession number is GSE124894.

| ChIP-quantitative PCR
To crosslink proteins to DNA, cells were incubated with 1% formaldehyde at room temperature for 10 minutes and then subjected to micrococcal nuclease treatment to digest the DNA followed by sonication to break the nuclear membrane. Finally, chromatin fragments in the range of 150-900 bp were obtained. After analysis of the digestion and concentration, the crosslinked chromatin (10 µg) was immunoprecipitated with anti-H3K36me3 antibodies

| Statistical analysis
Data with two groups were analysed using the unpaired Student's t test, and data with three or more groups were analysed by ANOVA. GraphPad Prism 7 software was used for all graphical and statistical analyses, with a P-value <0.05 (*), <0.01 (**) or <0.001 (***) considered statistically significant. Experimental results are expressed as the mean ± standard deviation.
These data indicated that the SETD2 deficiency was associated with imatinib resistance.

| SETD2 facilitates imatinib sensitivity in CML cell lines
To further investigate the role of SETD2 in the process of imatinib resistance acquisition by CML cells, we next used SETD2-targeted were checked by Western blot and qRT-PCR assays (Figure 2A,B).
We also overexpressed SETD2 in TF1-BAR cells (SETD2-OE-BAR) through electroporation with an SETD2-overexpressing plasmid, where the transfection efficiency and corresponding increased expression of H3K36me3 were also determined by Western blot and qRT-PCR assays ( Figure 2F,G). According to the cell viability assays, the SETD2-KD-BA cells were more resistant to imatinib treatment than the TF1-BA cells were (Figure 2C), whereas the SETD2-OE-BAR cells were more sensitive to imatinib than the TF1-BAR cells were  Figure S2).

| SETD2 deficiency upregulates a subset of genes involved in stemness regulation
To further study how SETD2 deficiency confers the upregulation of stem cells in imatinib-resistant CML cell lines, we performed RNA sequencing on the TF1-BA and TF1-BAR cells. Genes coding for proteins involved in super elongation regulation, hematopoietic key transcription factors and the KMT3 family members, which were reported to participate in the dysfunction of hematopoietic stem cells and their malignant transformation caused by SETD2 depletion, 43 were shown to be upregulated at the mRNA level in the TF1-BAR cells compared with that in the TF1-BA cells ( Figure 4A). These results were verified by qRT-PCR ( Figure 4B).

| ERG and/or MYCN knockdown successfully reverses the effects of SETD2 knockdown
To determine the functional relevance of ERG and MYCN with SETD2 knockdown, we used shRNAs to knock down ERG and MYCN individually or together in SETD2-KD-BA cells, and detected the knockdown efficiency by Western blot ( Figure 5A) and qRT-PCR assays ( Figure 5B

| Small-molecule inhibitor targeting H3K36me3 loss partially reverses the imatinib resistance
Given that the loss of SETD2 leads to the acquisition of imatinib resistance in TF1-BAR cells, we hypothesized that improving the expression level of H3K36me3 might restore the imatinib sensitivity of these cells, in view of the facts that SETD2 is the major  Figure S3). These studies illustrated that therapies that target H3K36me3 deficiency might be of great value in the treatment of imatinib resistance due to SETD2 loss.

| D ISCUSS I ON
In our study, we discovered that SETD2 deficiency significantly pro- Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.00, by ANOVA that the heterozygous depletion of SETD2 accelerated MLL-AF9 leukaemia propagation, whereas its homozygous depletion greatly dampened the latency. 41 Another interesting fact is that the homozygous depletion of SETD2 is detrimental to hematopoietic stem cells, 42,43 and thus, we speculate that the moderate expression of SETD2 is required for maintenance of the stemness in LSCs that is mediated by the upregulation of oncogenic transcription factors such as MYCN and ERG, both of which accelerate the development of acute leukaemia and lead to poor patient outcomes. [47][48][49][50][51] MYCN and MYC both play essential roles in tumorigenesis, 49,50,59,60 and it is intriguing that the upregulation of MYCN instead of MYC induced by H3K36me3 reduction conferring imatinib resistance. Consistently, MYCN upregulation displays a strong positive correlation with the poor outcomes of acute leukaemia patients. 49 20,31 In the present study, we found that the small molecule F I G U R E 6 JIB-04, a H3K36me3 demethylase, restores the H3K36me3 level and promotes imatinib (IM) sensitivity in TF1-BAR cells. A, Western blot assay of H3K36me3 and H3K9me3 expression changes in TF1-BAR cells after treatment with different doses of JIB-04 for 72 h. B, C, CCK-8 assay and IC 50 curves of the effects of JIB-04 alone or in combination with IM on TF1-BAR cells. D, E, Flow cytometric assay of the apoptosis of TF1-BAR cells after JIB-04 (1 μmol/L) and/or IM treatment for 72 h. F, G, Flow cytometric assay of the percentage of CD34 + CD38 − stem cells of TF1-BAR cells after JIB-04 (1 μmol/L) and/or IM treatment for 72 h. H, Colony formation assay of the effects of JIB-04 (1 μmol/L) and/or IM treatment on the quantity of stem cells in TF1-BAR cells. Bars in the graphs represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA JIB-04, an inhibitor of several demethylases, could efficiently eliminate LSCs through the apoptosis triggered by correction of the imbalanced epigenetics to restore the level of H3K36me3. JIB-04 therefore has potential for broader clinical applications, especially in imatinib resistance treatment.
Taken together, the results of our study have built an oncogenic role for SETD2 downregulation in CML with imatinib resistance.
Nonetheless, because we only measured these biological effects using in vitro experiments, further in vivo verification of these effects is needed as well as validation in clinical samples. Importantly, however, this is the first report to suggest that targeting H3K36me3 deficiency through the use of JIB-04 could be a novel strategy to improve clinical treatments for imatinib resistance. Our study paves the way for utilizing SETD2 to identify potential diagnostic strategies or drug targets to eventually cure CML, especially in patients with imatinib resistance.

ACK N OWLED G EM ENTS
The Shanghai Youth Talent

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest exist.

AUTH O R CO NTR I B UTI O N S
HHZ designed the research plan and interpreted the data; YS performed experiments and analysed data; W-QG and VM-S assisted in paper writing and editing; TV established the cell models; ZJ, WX and HZ helped to conduct the plasmid construction; ZJ and JW assisted with RNA sequencing analysis; HHZ and YS wrote the manuscript; CC, XW, YH and KL assisted in experiments.