Simultaneous targeting of XPO1 and BCL2 as an effective treatment strategy for double-hit lymphoma

DHL cell lines RC, SU-DHL-4 (DHL4), SU-DHL-6 (DHL6), SU-DHL-10 (DHL10), and Toledo were purchased from the ATCC. DB, SU-DHL-5 (DHL5), OCI-LY19, Will2, WSU, Val, and U2932 were gifts from Dr. Lan Pham at the University of Texas MD Anderson Cancer Center. Cells were cultured in RPMI 1640, supplemented with 20% FBS (except for RC, DHL4, and Toledo, supplemented with 10% FBS), and 1% penicillin/streptomycin in a 5% CO2 humidified incubator. ABT199 (venetoclax) was obtained from Houston Methodist Pharmacy. KPT8602 and KPT330 were purchased from Selleckchem. Carfilzomib was purchased from Cayman Chemical.

Cells were plated at 2 × 10⁴ cells in 100-μl culture medium per well in 96-well plates. Drugs were added the next day in 4 replicates. Following 5 days of drug treatment (except for DHL10, which was treated for 3 days), cell viability was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega) according to the manufacturer’s instructions. Data analyses and calculation of the median effective dose (IC50) were carried out using GraphPad Prism 8 (GraphPad).

DHL cells were lysed on ice in CelLytic™ MT Cell Lysis Reagent (Sigma) supplemented with protease and phosphatase inhibitors. Protein concentration was determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher). Protein lysates were resolved on SDS-PAGE gels and transferred to PVDF membranes. The following antibodies were used for the western blot analysis: MYC, cleaved PARP, caspase 3, BCL2, XPO1, MCL1, BIM, BCL-XL, lamin B1, and GAPDH (Cell Signaling); MYC (Abcam); β-tubulin and β-actin (Proteintech).

Cytoplasmic and nuclear RNA were isolated and purified using the RNA Subcellular Isolation Kit (Active Motif) following the manufacturer’s protocol. RNA binding and purification were performed using the RNeasy Mini Kit (Qiagen). Total RNA was extracted with the RNeasy Mini Kit (Qiagen). cDNA was synthesized using the Verso cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. qPCR analysis was performed on the 7500 Real-Time PCR System using SYBR Green (Applied Biosystems). Transcript levels were normalized to GAPDH, and relative gene expression was determined with ddCt method. Primer sequences are available upon request.

The PE Annexin-V Apoptosis Detection Kit (BD Biosciences) was used to detect apoptosis following the manufacturer’s instruction. Briefly, cells were suspended in 150 μl of binding buffer and mixed with 5 μl of FITC-conjugated annexin-V and 7-AAD, followed by incubation at room temperature for 15 min in the dark. The stained cells were analyzed using a BD FACS Fortessa flow cytometer (BD Bioscience). All flow cytometric data were analyzed with FlowJo software (Tree Star).

Mice were treated for 5 days. Subsequently, spleen tumors were collected, formalin-fixed, and paraffin-embedded. Four-micrometer-thick sections were subjected to H&E and immunohistochemistry staining. For IHC, MYC (Abcam, ab32072), Ki67 (Abcam, ab16667), and cleaved caspase 3 (CST, 9661) antibodies were used. Pictures were taken with × 40 objectives on a Leica DMi8 microscope.

Animal studies and experimental protocols were approved by the Institutional Animal Care and Use Committee at Houston Methodist Research Institute (IACUC approval number AUP-1117-0053). All experimental methods were performed in accordance with the relevant national and institutional guidelines and regulations. The DHL PDX sample (DFBL-69487-V3-mCLP) was obtained from the Public Repository of Xenografts (PRoXe), which collects PDX samples from patients with informed consent. Cells (10⁶) were engrafted via tail vein injection into 6- to 8-week-old NSG mice. The animals were monitored once a week by whole-body imaging on an IVIS Lumina III platform. Treatment started upon the appearance of measurable tumors. Tumor volumes were assessed from the start of the treatment 2 times per week using the IVIS imaging system. The following treatment schemes were used: daily oral gavage with ABT199 (50 mg/kg) and KPT8602 (7.5 mg/kg) for 5 successive days, followed by 2 days off for 3 weeks. ABT199 tablets were dissolved in water by sonication; KPT8602 was dissolved in 0.5% methylcellulose plus 1% Tween-80 as reported [13‐15]. BLI data were analyzed with the Living Image software, version 4.2 (Caliper Life Sciences). Animal studies were carried out in accordance with IACUC approval at Houston Methodist Research Institute (AUP-1117-0053).

First, we examined whether XPO1 inhibition affects MYC protein levels in DHL cell lines. Treatment with the XPO1 inhibitor, KPT8602, led to a significant decrease in MYC protein expression in the majority of DHLs in our diverse cell line panel (Fig. 1a). Three DHL cell lines (SU-DHL4, Toledo, and SU-DHL6) were selected to further examine MYC regulation by XPO1 inhibitors [16]. Treatment with two specific XPO1 inhibitors, KPT330 and KPT8602, points to a dose- and time-dependent downregulation of MYC protein expression in all three cell lines (Fig. 1b, c, and Additional file 1: Figure S1). These results establish that XPO1 inhibition effectively abrogates MYC protein expression in DHL tumor cells. Decreased MYC protein level was followed by changes in gene expression of MYC downstream targets (Fig. 1d, e). Genes known to be upregulated by MYC, including ENO1, APEX1, RPL3, RPS5, SRM, and nucleolin [17], were significantly downregulated upon XPO1 inhibition. In contrast, genes reportedly suppressed by MYC, such as HBP1, P27, and P15, were upregulated upon treatment with XPO1 inhibitors. The abrogation of MYC protein expression by XPO1 inhibition was accompanied by induction of apoptosis, as manifested by the cleavage of PARP and caspase 3 (Additional file 1: Figure S1). We concluded that XPO1 suppression abrogates the function of MYC oncogene and induces mass apoptosis in DHL tumor cells.

XPO1 has been reported to mediate the nuclear export of several mRNAs encoding oncoproteins, including MYC, BCL2, and PIM1. Therefore, XPO1 inhibition may potentially reduce MYC protein expression through the nuclear retention of MYC mRNA. To test whether XPO1 inhibition affects the nuclear export of MYC mRNA, we examined the distribution of MYC transcripts by cellular fractionation followed by quantitative real-time PCR. Compartmental separation of the nuclear versus cytoplasmic fractions was well achieved as shown by the quantification of GAPDH and laminin B1 proteins, and Neat1 transcript in each compartment [18] (Additional file 1: Figure S2C–D). Interestingly, MYC nuclear to cytoplasmic ratio decreased by 50% upon XPO1 inhibition, while its mRNA abundance in the whole-cell extract increased by twofold (Fig. 1f, g). The nuclear to cytoplasmic ratio of BCL2 but BCL6 increased modestly at 16 h of posttreatment. The level and nuclear/cytoplasmic ratio of beta-actin, used as an internal control, was not affected by XPO1 inhibition (Additional file 1: Figure S2A–D). This observation indicates that XPO1 inhibition may not result in preferential nuclear retention of MYC mRNA. Therefore, the drastic downregulation of MYC protein expression upon XPO1 inhibition cannot be explained by changes in the levels and/or transport of MYC mRNA. We further ruled out the role of protein degradation as a contributing factor, as treatment with a proteasome inhibitor, carfilzomib, did not affect MYC shutdown by XPO1 inhibition (Additional file 1: Figure S2E). Taken together, these data strongly suggest that the effective downregulation of MYC protein by XPO1 inhibition may occur at the level of protein translation.

The compelling downregulation of MYC protein expression by XPO1 inhibition suggests the possibility to target MYC and BCL2 driver oncogenes in DHL by combining XPO1 and BCL2 inhibitors. To test this notion, we first determined the IC50 values for two targeted agents, ABT199 (a BCL2 inhibitor) and KPT8602 (an XPO1 inhibitor), in a panel of DHL cell lines. Among the DHL cell lines examined, some were resistant to ABT199 treatment, while others were moderately resistant to KPT8602. However, to our surprise, none of the DHL cell lines was resistant to both, lending experimental support to the combinatorial use of XPO1 and BCL2 inhibitors to eradicate DHL tumor cells (Fig. 2a–c, Additional file 1: Figure S3A–B). Further in vitro examination of the drug combination using additional DHL cell lines demonstrated a strong synergy in cell death induction (Fig. 3a, Additional file 1: Figure S3C–D), while treatment with single agent showed moderate effects. The apoptotic event resulting from the combined treatment was accompanied by enhanced PARP and caspase 3 cleavage compared with treatment with either single agents (Fig. 3b–d). The apoptosis phenotype induced by synergistic drug combination was further confirmed by flow cytometric analysis following 7AAD/annexin-V staining (Fig. 3e). MCL1 is known to play an important role in regulating the sensitivity of tumor cells to BCL2 inhibitors as MCL1 overexpression is associated with resistance to ABT199 [19, 20]. Next, we examined whether XPO1 inhibition enhances the sensitivity of tumors cells to ABT199 by downregulating MCL1. Indeed, MCL1 downregulation was observed in 8 out of 12 DHL cell lines treated with XPO1 inhibitor KPT8602 (Additional file 1: Figure S1A). In DHL6 cells, the drug combination did not decrease MCL1 protein level but induced BIM protein expression (Additional file 1: Figure S4A–C). These observations may explain the synergistic effect between XPO1 and BCL2 inhibitors in inducing apoptosis of DHL tumor cells.

Encouraged by our in vitro results, we initiated an in vivo study to test the combination therapy. Primary DHL patient-derived xenograft (PDX) tumor cells obtained from the Public Repository of Xenografts (PRoXe) were transplanted in NOD/SCID/IL2R gamma (NSG) mice [21]. These PDX tumor cells were labeled with firefly luciferase for bioluminescence imaging (BLI). Importantly, as part of the therapeutic regimen, a low dose of KPT8602 (7.5 mg/kg, half of the dose used in other studies) was used [13‐15]. Additionally, at the start of the treatment, the tumor loads were at least 5-fold higher than recommended [21, 22]. Despite the low drug dose and higher than recommended tumor loads, we obtained a significant therapeutic response. The combined treatment completely blocked tumor progression and significantly extended survival, while single agents were only moderately effective (Fig. 4a–c). Importantly, BLI signals from the skulls were reduced by 22-fold indicating the drug treatment effectively blocks tumor metastasis to the brain (Fig. 4b, Additional file 1: Figure S5). Tumor samples were also collected 5 days after drug treatment for immunohistochemical analysis of MYC expression, tumor cell proliferation, and apoptosis (Ki67 and cleaved caspase 3). The combined drug treatment drastically abrogated MYC protein expression, decreased tumor cell proliferation, and induced apoptosis (Fig. 5). Thus, combined targeting of XPO1 and BCL2 is highly effective for the treatment of human DHL in vivo.