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Effects of XPO1 and FLT3 inhibition on AML
mutations constitutively activate FLT3 and its down- stream effectors MEK/ERK, PI3K/AKT, and STAT5 by phosphorylation. The latter effectors also activate target genes such as p21, p53, and cyclin D1.2,3 Thus, aberrant FLT3-mediated activation of effector proteins leads to uncontrolled proliferation, inhibition of differentiation and reduction of apoptosis in transformed hematopoietic blasts, and is also associated with poor prognosis in AML.4
Targeted therapies against FLT3 in FLT3-mutated AML using small molecule inhibitors such as sorafenib, quizar- tinib, midostaurin, crenolenib, and gilteritinib have shown clinical activity by reducing circulating leukemic blasts, and achieving temporary remission. However, these effects are apparently ineffective against leukemic stem cells in the bone marrow microenvironment and therefore the basis for temporary remission.5-10 In fact, we have reported marked upregulation of MAPK signaling follow- ing treatment with FLT3 inhibitors in AML/stroma co-cul- ture, hypoxia, and in clinical samples ex vivo; this upregu- lation could be partially overcome by the novel dual FLT3- ITD/MAPK inhibitor E6201.11 However, recent studies indicate that mutations of FLT3 are late events in leuke- mogenesis, suggesting that they are acquired rather than founder mutations in leukemia-initiating cells.12 Targeting FLT3 alone is, therefore, unlikely to be sufficient to eradi- cate leukemia-initiating cells.
Recently, exportin 1 (XPO1), also known as the nuclear export protein (CRM1), has been identified.13 XPO1 is a nuclear receptor involved in the active transport of a large number of cargo proteins, including Foxo3A, p53, p21 and NPM1, across the nuclear membrane14 along with microRNAs.15,16 XPO1 overexpression is common in hema- tologic malignancies including AML, and it was reported by us to be associated with poor disease prognosis.17
Leukemic cells depend on the continuous nuclear export of one or more oncoproteins, and the removal of tumor suppressor proteins, which require nuclear localization for their functions.18 Targeting nuclear membrane proteins such as XPO1 could, therefore, restore tumor suppressor function in AML. The small molecule XPO1 inhibitor, selinexor (KPT-330) is a first–in-class, orally bioavailable selective inhibitor of nuclear export compound that has shown promising anti-leukemia activity in vitro and in vivo.19,20 Selinexor was effective in inducing apoptosis in cells from established AML cell lines that are in the G0/G1 phase of the cell cycle,21 and targeting it also abrogated hypoxia-induced drug resistance in multiple myeloma cells.22 These results suggest that targeting XPO1 with selinexor may have potent anti-proliferative effects against non-proliferating or slowly proliferating leukemia- initiating cells in primary AML unlike the limitation observed when using FLT3 inhibitors.19 In addition, the recent results of phase I/II trials using selinexor as monotherapy (e.g. NCT02091245 and NCT02088541) or in combinations with conventional chemotherapeutic drugs (e.g. NCT02249091), have shown promising anti- leukemia activity with a high rate of blast clearance and complete remissions.20,23-28 Initial problems with gastroin- testinal toxicities and anorexia have largely been over- come by dose reduction without loss of clinical effica- cy.23,24 However, the anti-leukemia activity of selinexor in AML patients with FLT3 mutations, including those with acquired secondary mutations found in relapsed/refracto- ry disease following FLT3-targeted therapy, has not been established.
In this study, we report that selinexor has marked pro- apoptotic effects against AML cells harboring FLT3-ITD and/or TKD mutations. However, compensatory upregu- lation of phosphorylated FLT3 and its downstream signal- ing pathways was observed in most of the FLT3-mutated cell lines tested in vitro. We, therefore, combined selinexor with sorafenib. This combinatorial drug regimen achieved markedly synergistic leukemia cell killing in cells harbor- ing ITD and/or TKD mutations, which usually show resistance to FLT3-targeted therapy.29,30 Of note, the com- binatorial regimen also achieved encouraging clinical effi- cacy including molecular complete responses in an ongo- ing phase IB/II clinical trial of selinexor plus sorafenib in patients who were refractory to FLT3 inhibitor therapy. Thus, this combinatorial approach may abrogate selinex- or-mediated FLT3 activation, resulting in abrogation of resistance to FLT3 inhibitors and induction of durable remissions in patients with additional acquired FLT3 mutations.
Methods
Reagents and antibodies
Selinexor was provided by Karyopharm Therapeutics (Newton, MA, USA). Sorafenib was purchased from Selleckchem (Houston, TX, USA). Their molecular structures are shown in Online Supplementary Figure S1. The antibodies against human phospho- rylated (p)-p44/42 MAPK (ERK1/2)(Thr202/Tyr204), phospho- AKT(Ser473), phospho-FLT3(Tyr589/591), phospho- S6K(Ser240/244), AKT, S6K, Bcl-xL, C/EBPα, PU.1, STAT3, c- Myc and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA), against Bcl-2 from Dako (Carpinteria, CA, USA), against phospho-STAT5 A/B from Upstate (Lake Placid, NY, USA), against total STAT5A/B from R&D Systems Inc. (Minneapolis, MN, USA), against ERK2, FLT3, p53, IκB alpha, phospho-Stat3, and Mcl-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), against Bim and Puma from CalBiochem (San Diego, CA, USA), against HIF1α from BD Biosciences (San Diego, CA, USA), and against phospho-IκB alpha (ser32/36) from Novus (Littleton, CO, USA). The anti- luciferase antibody was purchased from Promega (Madison, WI, USA).
Acute myeloid leukemia cell lines and patients’ samples
The Baf3/FLT3, Baf3/ITD, and Baf3/D835Y cell lines were kind- ly provided by Dr. Donald Small (Department of Pediatric Oncology, Johns Hopkins University, Baltimore, MD, USA) and Baf3/ITD+D835Y and Baf3/ITD+D835H cells by Dr. Neil Shah (Department of Medicine, The University of California at San Francisco, San Francisco, CA, USA). The FLT3-inhibitor-resistant cells Baf3/ITD+F691 and Baf3/ITD+Y842, which harbor FLT3- ITD plus F691L and Y842C mutations, respectively, were estab- lished by us as described previously.30 The human AML cell lines THP-1, Kasumi-1, and MV4-11 were obtained from the American Type Culture Collection (Manassas, VA, USA), and MOLM13 and MOLM14 from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). All cell lines were validated by short tandem repeat DNA fingerprinting using the AmpFISTR Identifiler kit according to manufacturer's instructions (Applied Biosystems cat. n. 4322288). All cells were maintained in RPMI medium supplemented with 10% fetal bovine serum, and inter- leukin-3-dependent murine Baf3/FLT3 cells were maintained in the presence of 2 ng/mL of interleukin-3. The FLT3 status of the AML cell lines used in this study is shown in Table 1.
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