1.JQ1 changed the viability of myeloid leukemia cell lines
Our study found that JQ1 has different changes in morphology and physiological functions of different myeloid leukemia cell lines (Fig. 1). Myeloid leukemia cells K-562 and MV-4-11 were treated with different concentrations of JQ1 (experimental group) and DMSO (control group) for ten days, respectively. The concentrations of JQ1 were 10nM, 100nM and 500nM. In K-562 cells, after JQ1 treatment, the cell proliferation ability slightly decreased. Although the number of dead cells did not change too much, the cell morphology changed significantly, which showed that there were differences in cell size, even some cells were several times larger than normal cells, and some cells had vacuoles in cells, and these effects were more obvious with the increase of JQ1 drug concentration. Mv-4-11 showed that the number of cells decreased significantly, the overall state of cells was poor, and the number of dead cells increased significantly, which may induce apoptosis or necrosis. These effects were obvious with the increase of JQ1 concentration.
2.JQ1 arrested cell cycle of MV-4-11 cell lines at G0/G1 phase
To further determine whether JQ1 attenuated cell viability by affecting the cell cycle distribution or not. Flow cytometry was performed to analyze the cell cycle distribution. In MV-4-11 cells, compared with the DMSO control group and blank control group, the percentage of cells in the G1/G0 phase increased while their S phase and G2 phase decreased after JQ1 treatment(Fig. 2a).These effects also proved by the researches of Jennifer A. Mertz. However, the treatment of JQ1 cannot arrest the cell cycle of K562 cell line at any phase (Fig. 2b). To test the possibility that whether BET inhibitor can explicitly retract cell cycle transcription, we utilized global transcriptional profiling and unbiased gene set enrichment analysis (GSEA) to deeply analysis the result of RNA-seq. We evaluated a canonical transcriptional signature of cell cycle gene sets obtained from Molecular Signatures Database (MSigDB)[20,21] and found the signature strongly correlated with downregulated of expression by JQ1 (Fig. 2c and 2e). Next, we have performed quantitative real-time PCR analysis to validate the hypothesis that JQ1 reduces the proliferation of MV-4-11 by arresting cell cycle at the G0/G1 phase. Cyclin D1 is an essential regulator of the G1–S transition in response to growth factor stimulation in cells, CDK4 and CDK6 are also critical factors for G1-S transition. We found that the relative mRNA expression of CDK6 and CYCLIN D1 dramatically decreased with the treatment of JQ1 (Fig. 2d and 2f). Besides, BRD4 is also an essential mediator of transcriptional elongation, interacting with pTEFb (positive transcription elongation factor b). CDK9(cyclin-dependent kinase-9), a core component of pTEFb, can be recruited to mitotic chromosomes contributing to increased expression of growth-promoting genes, its relative mRNA expression also decreased. The above results suggest that JQ1 may block cell proliferation from G1/G0 into the S phase; these finding are complementary with the research of Lee, D. H. and Wang, T.[25,26]. However, K-562 cells have little change in the cell cycle, which can be considered that JQ1 does not limit the viability of K-562 cells by affecting cell growth ability.
3.The effects of JQ1 on MYC of myeloid leukemia
c-Myc is a master regulatory factor of cell proliferation. In cancer, pathologic activation of c-Myc plays an essential role in disease pathogenesis by the coordinated upregulation of a transcriptional program influencing cell division, metabolic adaptation, and survival[27,28]. Therefore, we next investigated whether JQ1 has different effects on MV-4-11 and K562, respectively, which resulting in different changes in cell cycle and morphology. After a series of different time treatment of both MV-4-11 and K562 cell lines with 500nM JQ1, we performed qPCR to detect the relative mRNA levels and found that JQ1 induced and sustained decreases in MYC transcriptional levels (Fig. 3a). This was accompanied by a loss of detectable c-Myc protein in whole-cell lysates at both 48 and 72hours after treatment (Fig. 3e). We evaluated two canonical transcriptional signatures of MYC pathway's gene sets obtained from Molecular Signatures Database (MSigDB) and found two signatures strongly correlated with the downregulated expression by JQ1 (Fig. 3d). To define whether the changes of MYC transcription level with JQ1 treatment have further influence c-Myc target genes, we utilized the data from RNA-seq and selected 32 known c-Myc target genes. After 72 hours of treatment of MV-4-11 and K562 with DMSO and 500nM JQ1, collecting total RNA and performing pre-processing and quality inspection, followed by two-ended (PE) sequencing in illumine NovaSeq 6000 sequencer. We found that the MYC gene expression of MV-4-11 with the treatment of 500nM JQ1 decreased significantly as well as the c-Myc target genes also had the same significant downward trend (Fig. 3b). This effect of JQ1 is similar to the research of Ott, C. J. and Delmore, J. E. [29,30]. However, although the K562 cell line under JQ1 treatment had decreased expression of MYC gene, c-Myc target Genes did not show a significant downward trend while the expression of c-Myc target genes was similar to the DMSO control group (Fig. 3c). Therefore, the above results indicate thatMV-4-11 may affect the cell cycle by reducing the expression of MYC gene, thereby affecting cell proliferation. However, JQ1 did not significantly affect the expression of c-Myc downstream genes of K562 cells though the expression of MYC had reduced. There may exist compensation pathways for the expression of MYC gene in K562 cells to escape the effects of JQ1, which verified that JQ1 did not significantly change cellular proliferation and cell cycle of K562 cell line.
4.Effects of JQ1 are associated with Bcl2 family
After 72 hours of treatment of MV-4-11 and K562 with DMSO and 500nM JQ1, collecting total RNA and performing pre-processing and quality inspection, followed by two-ended (PE) sequencing in illumine NovaSeq 6000 sequencer. We analyzed the RNA-seq data and did KEGG analysis of the genes that differential significantly, and got top30 pathways, including apoptosis pathway (Fig. 4a). Besides, we selected 80 cancer-related genes and tested their relative mRNA expression. Excellent concordance was observed between JQ1 treatment group and the control group. There existed the difference in expression of the BCL-2 gene after JQ1 and DMSO treatment, respectively. Because BCL-2 gene is the core regulatory gene of apoptosis, which explaining that the effect of JQ1 on MV-4-11 is related to apoptosis, and the results are consistent with KEGG's analysis (Fig. 4b). However, the analysis of the cancer-related genes of K562 found no significant difference in the expression of bcl2 in the JQ1 treatment group and the control group (Fig. 4c).
In general, the balance between pro-apoptotic and anti-apoptotic protein regulators is the crucial point to determine whether apoptosis occurs. Therefore, we selected the core genes associated with anti-apoptosis and pro-apoptosis[31,32]. In the apoptosis-related genes of the MV-4-11 cell line, all anti-apoptosis genes were down-expressed after JQ1 treatment, including BCL2, BCL2A1, BCL2L1, BCL2L2, except for the MCL1 gene. While most of the significant pro-apoptosis related gene expressions are uplifted, including APAF1, BAK, BCL2L11, BCL2L12, BCL2L13, BMF, PMAIP1, therefore, the effect of JQ1 on MV-4-11 cell line is most likely achieved by overexpression of pro-apoptotic proteins and downregulation of anti-apoptotic proteins to promote apoptotic proteins (Fig. 5a). After K562 cell line treated by JQ1, the expression of other anti-apoptosis genes increased, although the expression of the core gene BCL2 decreased. Furthermore, the trend of pro-apoptosis gene changes is difficult to determine whether to promote or prevent apoptosis (Fig. 5b). To prove that the effect of JQ1 is related to apoptosis, we used qPCR to detect the mRNA expression of the BCL2 gene after JQ1 treatment at 24h, 48h, 72h, 96h and found that the expression of BCL2 gene in both cell lines significantly decreased (Fig. 5c). Besides, we used immune blot to detect caspase-3, BCL2, P53 protein expression of MV-4-11 and K562 after JQ1 treatment for 24 hours, 48 hours, found that the MV-4-11 cell line after JQ1 treatment. The expression of the executive protein caspase-3, which must be performed during the apoptosis process, increased significantly. However, the amount of BCL-2 protein increased slightly (Fig. 5d), suggesting that there may be a possible way for cells to compensate for the reduction of bcl2 protein reduction caused by apoptosis. K562 is entirely different. The cell line failed to detect the rise of the caspase-3 protein, while BCL2 protein expression also increased to resist apoptosis (Fig 5e).
5.Effects of JQ1 on apoptotic program
To further determine whether the effects of JQ1 on MV-4-11 cell line and K562 cell line are associated with apoptosis. MV-4-11 and K562 cells were exposed to a specific concentration of JQ1(500uM) or DMSO for 48h. DMSO-treated and JQ1-treated cells were collected and incubated with Annexin V-FITC and PI staining, then the apoptosis changes were detected by flow cytometry. It can be seen that there are no significant changes in K-562 cells (Fig. 5g). However, the JQ1 treatment group of MV-4-11 is significantly different from the DMSO control group, showing an increase in apoptosis rate. Besides (Fig. 5f), fetal bovine serum deprivation medium will increase the inactivation of cells in the control group. Therefore, this may be considered as the reason for PI-positive Annexin V positive in the control group.
We next evaluated a canonical transcriptional signature of the apoptosis pathway's gene sets obtained from Molecular Signatures Database (MSigDB). For MV-4-11 cell line, Gene set defined by the apoptotic related pathway is significantly enriched in JQ1-upregulated genes. However, JQ1 treatment did not exert significant changes for K562 cell lines (Fig. 6a). To further compare the differences between the performance of the two cell lines after JQ1 treatment, we further analyzed core genes obtained by GSEA analysis, which made a significant contribution to gene sets enrichment score. It was found that after JQ1 was treated in the MV-4-11 cell line, CFLAR (CASP8 and FADD like apoptosis regulator), TNF (tumor necrosis factor), CASP3, CASP6, CASP9 is upregulated significantly (Fig. 6b). It is proved that the death of MV-4-11 cells was subjected to the apoptosis pathway under the role of JQ1.
On the contrary, after JQ1 operated the K562 cell line, there was no change in the core genes in the apoptosis pathway (Fig. 6c). Besides, through the heatmap and scatter plots of differentiation genes of K562 cell line between JQ1-treatment and DMSO-control group, there is still a considerable amount of changes in K562 cell line after JQ1 treatment (Fig. 6d and 6e).The above results indicate that the effects of JQ1 on K562 cell line maybe not through the apoptosis pathway. This hypothesis is complementary with researches of Ott, C. J., and Zuber, J.[35,29].