Inhibition of XPO1 enhances cell death induced by ABT‐199 in acute myeloid leukaemia via Mcl‐1

Abstract The antiapoptotic Bcl‐2 family proteins play critical roles in resistance to chemotherapy in acute myeloid leukaemia (AML). The Bcl‐2‐selective inhibitor ABT‐199 (Venetoclax) shows promising antileukaemic activity against AML, though Mcl‐1 limits its antileukaemic activity. XPO1 is a nuclear exporter overexpressed in AML cells and its inhibition decreases Mcl‐1 levels in cancer cells. Thus, we hypothesized that the XPO1‐selective inhibitor KPT‐330 (Selinexor) can synergize with ABT‐199 to induce apoptosis in AML cells through down‐regulation of Mcl‐1. The combination of KPT‐330 and ABT‐199 was found to synergistically induce apoptosis in AML cell lines and primary patient samples and cooperatively inhibit colony formation capacity of primary AML cells. KPT‐330 treatment decreased Mcl‐1 protein after apoptosis initiation. However, binding of Bim to Mcl‐1 induced by ABT‐199 was abrogated by KPT‐330 at the same time as apoptosis initiation. KPT‐330 treatment increased binding of Bcl‐2 to Bim but was overcome by ABT‐199 treatment, demonstrating that KPT‐330 and ABT‐199 reciprocally overcome apoptosis resistance. Mcl‐1 knockdown and overexpression confirmed its critical role in the antileukaemic activity of the combination. In summary, KPT‐330 treatment, alone and in combination with ABT‐199, modulates Mcl‐1, which plays an important role in the antileukaemic activity of the combination.


| INTRODUCTION
Despite substantially enhanced understanding of the biology of acute myeloid leukaemia (AML), standard chemotherapy (cytarabine plus an anthracycline, e.g. daunorubicin) and bone marrow transplant have remained the main treatment option for patients with AML over the course of the last four decades. This lack of change in standard of care has led to a meagre improvement in AML survival compared to other leukaemia types. Although many fit patients respond initially to standard therapy, survival rates remain low because of disease relapse. Leukaemic stem cells (LSCs), and our inability to target them, are believed to be responsible for AML relapse. 1 Bcl-2 is overexpressed in bulk AML cells and LSCs, making it a promising therapeutic target for the treatment of AML. 2 Anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xL, and Mcl-1) sequester proapoptotic Bcl-2 family proteins, such as Bim, to prevent induction of apoptosis. 3 The Bcl-2 family has been found to be dysregulated in AML, with increases in Bcl-2, Bcl-xL, and/or Mcl-1 expression associated with chemotherapy resistance and poor prognoses. 2,[4][5][6][7] Mcl-1 was previously shown to be necessary to inhibit Bak and Bax activation. [8][9][10] Thus, targeting the anti-apoptotic Bcl-2 family proteins represent a promising strategy for the treatment of AML. Although inhibitors of this family have been promising, inhibition of Bcl-xL has been associated with platelet death and subsequent thrombocytopenia, 5 which has sparked interest in the Bcl-2-selective inhibitor . Its excellent antileukaemic activity against chronic lymphocytic leukaemia led to FDA approval in April 2016. Our group and others have reported that ABT-199 has promising activity against preclinical models of AML and other cancers. [11][12][13][14][15][16][17] Results of a phase II clinical trial have shown that ABT-199 has promising clinical activity compared to current alternatives, but relapse remains a concern. 18,19 Combinations such as ABT-199 with decitabine or azacitidine can help induce remission but lack improvement in overall survival. 20 Thus, new therapeutic combinations involving ABT-199 are urgently needed to eradicate bulk AML cells and LSCs responsible for relapse.
We have shown that the small molecule inhibitor ABT-199 can disrupt this interaction in AML. 21  We previously demonstrated that the Mcl-1 small molecule inhibitor A-1210477 was able to synergize with ABT-199 to induce apoptosis in AML cell lines and primary patient samples by disrupting the Bim-Mcl-1 interaction. 23 However, the drug combination also synergized to reduce proliferation of normal peripheral blood mononuclear cells (PMNCs), albeit at higher concentrations. Thus, indirect inhibition of Mcl-1 may prove to be useful in combination with ABT-199.
Exportin 1 (XPO1), also known as chromosome region maintenance 1, is a protein which regulates the nuclear export of client proteins and has been found to play a critical role in many cancers including prostate cancer, ovarian cancer, osteosarcoma, glioma, cervical cancer, chronic lymphoid leukaemia (CLL), non-Hodgkin's lymphoma, multiple myeloma, and AML. [24][25][26][27][28] As a nuclear exporter, XPO1 plays a critical role in the intracellular localization of many proteins, as well as some mRNA transcripts, and is required for survival of solid tumors and hematological malignancies. 26

| Clinical samples
Diagnostic AML patient samples were obtained from the First Hospital of Jilin University (Table 1). Written informed consent was provided according to the Declaration of Helsinki. This study was approved by the Human Ethics Committee of The First Hospital of Jilin University. Clinical samples were screened for gene mutations and fusion genes as described previously. 14,40

| CRISPR knockdown
The lentiCRISPRv2 plasmid was a gift from Feng Zhang at the Broad Institute of MIT and Harvard (Addgene plasmid #52961). Guide RNAs were designed using the CRISPR design tool (http://crispr. mit.edu). The NTC and Mcl-1 vectors were generated using Feng Zhang's protocol, which is available on Addgene's website (www.add gene.org). Lentivirus production and transduction were carried out as described above in "shRNA Knockdown," except that psPAX2 (gift from Didier Trono at the Swiss Institute of Technology, Addgene plasmid #12260) was used instead of delta 8.2.

| Colony forming assay
Primary AML patient samples were treated with either ABT-199 or KPT-330, alone or in combination, for 24 hours. The cells were washed three times with PBS and then plated in MethoCult (Stem Cell Technologies, Vancouver, Canada) and allowed to incubate at 37°C humidified atmosphere containing 5% CO 2 /95% air for 14-16 days. Colony forming units (CFUs) were visualized using an inverted microscope and the number of colonies containing >50 cells were counted. Technical triplicates were performed.

| RT-PCR
Total RNA was extracted using TRIzol (Thermo Fisher Scientific) and cDNAs were prepared from 2 μg total RNA using random hexamer primers and a RT-PCR Kit (Thermo Fisher Scientific), and then purified using the QIAquick PCR Purification Kit (Qiagen, Germantown, MD, USA), as described previously. 44 Mcl-1 (cat no. Sigma-Aldrich) assays were performed as previously described. 36,43,48 Human PMNC samples were treated with variable concentrations of LUEDTKE ET AL.

| 6101
ABT-199 and/or KPT-330 for 72 hours. For each PMNC sample, the MTT assay was performed once due to limited sample.

| KPT-330 down-regulates Mcl-1 and disrupts its interaction with Bim
Having observed the synergy between ABT-199 and KPT-330, we

| DISCUSSION
ABT-199 has demonstrated clinical activity, though as a monotherapy relapse occurred in a mere 2.5 months and in combination with decitabine or azacitidine there was no significant improvement in overall survival rate compared to decitabine or azacitidine alone. [18][19][20] Therefore, new combination approaches for  are necessary to improve the long-term survival of AML patients.
In this study, we show that KPT-330 is able to overcome ABT-  However, the combination does appear to synergize in normal PMNC cells ex vivo, suggesting that toxicity could be a concern.
Further in vitro mechanistic studies and in vivo testing in AML mouse models is warranted to determine efficacy against LSCs as well as tolerability.