Venetoclax enhances DNA damage induced by XPO1 inhibitors: A novel mechanism underlying the synergistic antileukaemic effect in acute myeloid leukaemia

Abstract Acute myeloid leukaemia (AML) is a highly heterogeneous haematologic malignancy with poor prognosis. We previously showed synergistic antileukaemic interaction between exportin 1 (XPO1) inhibitor KPT‐330 (Selinexor) and Bcl‐2 inhibitor venetoclax (ABT‐199) in preclinical models of AML, which was partially meditated by Mcl‐1, although the full mechanism of action remains unknown. In this study, using real‐time RT‐PCR and Western blot analysis, we show that inhibition of XPO1 via KPT‐330 or KPT‐8602 (Eltanexor) decreases the mRNA and protein levels of c‐Myc, CHK1, WEE1, RAD51 and RRM2. KPT‐330 and KPT‐8602 induce DNA damage, as determined by alkaline comet assay. In addition, we demonstrate that venetoclax enhances KPT‐330‐ and KPT‐8602‐induced DNA damage, likely through inhibition of DNA damage repair. This study provides new insight into the molecular mechanism underlying the synergistic antileukaemic activity between venetoclax and XPO1 inhibitors against AML. Our data support the clinical evaluation of this promising combination therapy for the treatment of AML.

and RNAs to across the nuclear envelope to the cytoplasm. 4,5 Dysregulation of XPO1-mediated nuclear export is evident in several haematologic malignancies and solid tumours, leading to enhanced transport of TSPs out of the nucleus. 6,7   is one of the first-generation orally bioavailable selective inhibitors of XPO1, 8,9 and its combination with dexamethasone was granted accelerated FDA approval for adult patients with relapsed or refractory multiple myeloma in July 2019. 10 KPT-330 is also undergoing clinical development in a wide range of haematologic malignancies and solid tumours. 11,12 The second-generation XPO1 inhibitor KPT-8602 (Eltanexor) has similar mechanism of action and potency as KPT-330, 13 but has greater tolerability than KPT-330 due to its lower central nervous system penetration 14 and exhibits promising antileukaemic activity against AML. 15 Our previous study demonstrated that KPT 16 Therefore, additional factors underlying the molecular mechanism remain to be elucidated.
c-Myc is an important transcription factor, regulating about 15% of genes in the whole genome. 17 c-Myc also plays a critical role in the survival of AML cells. 18,19 Several studies have demonstrated that KPT-330 can downregulate c-Myc. [20][21][22] In addition,  has been shown to induce DNA damage in cancer cells. 23 CHK1, WEE1 and RAD51 are important components of the DNA damage response (DDR) network. 24 In addition, ribonucleotide reductase (RR) catalyses the reduction in ribonucleotides into corresponding deoxyribonucleotides which are critical in DNA replication and DNA damage repair, thus plays a crucial role in maintaining genome stability. 25 Previous studies have shown that c-Myc has potential regulatory effects on CHK1, WEE1, RAD51 and RR. 21,26 Thus, it is conceivable that inhibition of XPO1 induces DNA damage in AML cells through downregulation of c-Myc, CHK1, WEE1, RAD51 and RR.
In addition, we have previously reported that venetoclax can enhance the antileukaemic activity of DNA damaging drugs via enhancing DNA damage induced by these agents, leading to synergistic antileukaemic activity against AML. 27 Based on this, we hypothesized that venetoclax would enhance XPO1 inhibitor-induced DNA damage resulting in synergistic antileukaemic activity against AML cells.

| Alkaline comet assay
AML cells were subjected to alkaline comet assay, as previously described. 28

| Nucleus and cytoplasm fractionation
Nucleus and cytoplasm fractionation was carried out as described by Buisson and colleagues. 32 3 × 10 6 cells were washed with PBS and resuspended in solution A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 1 mM DTT, 10 mM NaF, 1 mM Na 2 VO 3 and protease inhibitors purchased from Roche Diagnostics). Triton X-100 was added (final concentration of 0.1%), and then, the cells were incubated on ice for 4~5 min. Nuclei were separated from cytoplasmic proteins by centrifugation at 1400× g for 4 min and then washed with solution A three times. Nuclei were resuspended and sonicated, and then subjected to Western blot analysis. Experiments were repeated three times independently.
Representative blots are shown. The fold changes in protein densitometry measurements were compared with histone H4 in nuclear fraction or MEK in cytoplasmic fraction and then normalized to the vehicle control. Data are presented as mean ± SEM. The significance level was set at p < 0.05.

| The effect of XPO1 inhibition on c-Myc and DDR proteins in AML cell lines
KPT-330 has been reported to downregulate DNA repair proteins leading to DNA damage in tumour cells, including AML cells. 23 Based on reports that KPT-330 can downregulate c-Myc 20-22 and c-Myc has potential regulatory effects on CHK1, WEE1, RAD51 and RR, 21,26 we suspected that these may be downregulated by XPO1 inhibition. RR is composed of a regulatory subunit RRM1 and a catalytic subunit RRM2. 33   transcripts, although proteasome degradation may also play a role.

| Inhibition of XPO1 induces DNA damage
Given the reported role in DNA repair, 24 Figure S1B and C). Importantly, increased DNA damage was detected prior to cell apoptosis (Figure 2A and Figure S1A). These results demonstrate that inhibition of XPO1 induces DNA damage in AML cells.

| Venetoclax enhances DNA damage induced by inhibition of XPO1
We previously demonstrated that venetoclax impairs the repair of DNA damage induced by DNA damaging agents. 27

| DISCUSS ION
We previously reported that Mcl-1 plays an important role in the antileukaemic activity of combined inhibition of XPO1 and Bcl-2. 16 In this study, we add further insight into the mechanism of action, the cooperative induction of DNA damage. Consistent with Ranganathan et al., 21 we found that KPT-8602 downregulates c-Myc, CHK1 and RAD51. They also found that KPT-330 treatment decreases c-Myc protein level and reduces c-Myc binding to the promoters of CHK1 and RAD51 in AML cells. 21 Furthermore, they reported that KPT-330 induces DNA damage in cancer cells. 21,23 In contrast, we found that downregulation of c-Myc and increased DNA strand breaks occurred prior to downregulation of CHK1 and RAD51. Additionally, we found that XPO1 inhibition also downregulated WEE1 and RRM2, which also occurred after detection of increased DNA damage. Interestingly, we found that c-Myc inhibition significantly induced DNA damage ( Figure 3D although the exact mechanism remains unknown and beyond the scope of this study. Furthermore, WEE1 is a reported cargo protein of XPO1, 42,43 although we did not detect an increase in nuclear localized WEE1 following XPO1 inhibition. However, substantial downregulation of WEE1 protein following XPO1 inhibition may interfere with detecting a potential increase in the protein in the nucleus; thus, further studies are warranted. Our previous study showed that MV4-11 cells are more sensitive to XPO1 inhibitors than THP-1 cells. 16 In this study, we found that treatment of THP-1 cells with higher concentrations of XPO1 inhibitors induced much lesser DNA damage at a later time compared with that in MV4-11 cells, indicating that the level and time of DNA damage induction by XPO1 inhibitors are potential determinants of their antileukaemic activity against AML. In this study, we also found that XPO1 inhibition resulted in an increase in Bcl-2 in the nuclear fraction ( Figure 6D,E and Figure   S3). Venetoclax is designed to target the hydrophobic cleft of Bcl-2 composed of BH1, BH2 and BH3 domains. 44 However, Bcl-2 affects DNA repair via its BH4 domain. 39,40 Thus, our findings suggest that Bcl-2 inhibition causes increased nuclear localization of the protein, resulting in inhibition of DNA repair. These results are consistent with our previous studies demonstrating that venetoclax impairs the repair of DNA damage induced by DNA damaging agents. 27,37 In conclusion, we demonstrate that inhibition of XPO1 causes shows promising antileukaemic activity in both MV4-11 cells and AML patient-derived xenograft mouse models. 45 Therefore, our current and past findings 16 as well as those of other investigators 45 provide preclinical support for further investigations into the clinical efficacy of this promising therapy for AML.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China (NSFC 81800154; for Guan Wang) and the School of Life Sciences of Jilin University. The funders had no role in study design, data collection, analysis and interpretation of data, decision to publish or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article and in the additional files.