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Chemical Therapeutics

CRM1 and BRAF Inhibition Synergize and Induce Tumor Regression in BRAF-Mutant Melanoma

Roberto A. Salas Fragomeni, Hye Won Chung, Yosef Landesman, William Senapedis, Jean-Richard Saint-Martin, Hensin Tsao, Keith T. Flaherty, Sharon Shacham, Michael Kauffman and James C. Cusack
Roberto A. Salas Fragomeni
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Hye Won Chung
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Yosef Landesman
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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William Senapedis
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Jean-Richard Saint-Martin
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Hensin Tsao
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Keith T. Flaherty
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Sharon Shacham
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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Michael Kauffman
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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James C. Cusack
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
1Harvard Medical School; 2Division of Surgical Oncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5Karyopharm Therapeutics, Natick, Massachusetts
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DOI: 10.1158/1535-7163.MCT-12-1171 Published July 2013
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Abstract

Resistance to BRAF inhibitor therapy places priority on developing BRAF inhibitor-based combinations that will overcome de novo resistance and prevent the emergence of acquired mechanisms of resistance. The CRM1 receptor mediates the nuclear export of critical proteins required for melanoma proliferation, survival, and drug resistance. We hypothesize that by inhibiting CRM1-mediated nuclear export, we will alter the function of these proteins resulting in decreased melanoma viability and enhanced BRAF inhibitor antitumoral effects. To test our hypothesis, selective inhibitors of nuclear export (SINE) analogs KPT-185, KPT-251, KPT-276, and KPT-330 were used to induce CRM1 inhibition. Analogs PLX-4720 and PLX-4032 were used as BRAF inhibitors. Compounds were tested in xenograft and in vitro melanoma models. In vitro, we found CRM1 inhibition decreases melanoma cell proliferation independent of BRAF mutation status and synergistically enhances the effects of BRAF inhibition on BRAF-mutant melanoma by promoting cell-cycle arrest and apoptosis. In melanoma xenograft models, CRM1 inhibition reduces tumor growth independent of BRAF or NRAS status and induces complete regression of BRAF V600E tumors when combined with BRAF inhibition. Mechanistic studies show that CRM1 inhibition was associated with p53 stabilization and retinoblastoma protein (pRb) and survivin modulation. Furthermore, we found that BRAF inhibition abrogates extracellular signal–regulated kinase phosphorylation associated with CRM1 inhibition, which may contribute to the synergy of the combination. In conclusion, CRM1 inhibition impairs melanoma survival in both BRAF-mutant and wild-type melanoma. The combination of CRM1 and BRAF inhibition synergizes and induces melanoma regression in BRAF-mutant melanoma. Mol Cancer Ther; 12(7); 1171–9. ©2013 AACR.

Introduction

Most cancers acquire functional survival capabilities during their development. These capabilities include apoptosis evasion, self-sufficient growth signals, and insensitivity to antigrowth signals (1). Advanced-stage melanoma shows these capabilities. For instance, a constitutively activate BRAF kinase is present in at least half of all the patients with advanced melanoma, driving melanoma proliferation (2). BRAF-targeted therapy interrupts the growth signal and suppresses melanoma proliferation. Targeting BRAF mutation has led to outstanding clinical results with limited associated toxicity (3, 4). However, the emergent resistance to BRAF inhibition limits the therapy's response duration, challenging our limited therapeutic options for advanced melanoma.

The modulation of nucleo-cytoplasmic protein transport has been suggested as a possible therapeutic strategy in the treatment of cancer (5). Protein transport between the nucleus and the cytoplasm is critical for cell maintenance, cell proliferation, and survival. Alterations in the expression of nuclear transport–related proteins, in particular Exportin 1 (XPO1, also known as chromosome region 1, CRM1) are found in melanoma and other cancers. Overexpression of CRM1 is linked to inactivation of tumor suppressor proteins, apoptosis evasion, and resistance to chemotherapy (6). In addition, CRM1 expression is of prognostic value in several types of cancer (7–10). CRM1 mediates nuclear export using nuclear export signals (NES), which are required for nuclear cargo transport from the nucleus to the cytoplasm. Selective inhibitors of nuclear export (SINE) also known as KPT-analogs (Fig. 1; KPT-185, KPT-251, KPT-276, and KPT-330) are capable of binding to the Cys-528 residue in the cargo-binding portion of the CRM1 protein successfully preventing protein transport from the nucleus to the cytoplasm (5, 11–13). The interruption of the system results in nuclear accumulation of the cargo, thereby restoring or disrupting cargo function. Among the CRM1-affected proteins, we find p42/44 mitogen-activated protein kinase (MAPK; extracellular signal–regulated kinase (ERK)1/2], p53, c-Fos, survivin, and retinoblastoma protein (pRb; refs. 5–9). Modifying the function of these proteins alters survival and proliferation of cancer (5, 6, 14). Furthermore, the modulation of the CRM1 function using SINE has achieved promising preclinical results in cancer (11, 12, 15). Clinical grade analog, KPT-330, is currently in clinical phase I trials (NCT01607905 and NCT01607892).

Figure 1.
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Figure 1.

Chemical structures for SINE analogs KPT-185, KPT-251, KPT-276, and KPT-330.

CRM1 has been found to be overexpressed in metastatic melanoma when compared with nevi or even primary melanoma lesions (6). Moreover, CRM1 expression portrays independent of BRAF mutational status (6). This makes CRM1 a potential therapeutic target for metastatic melanoma. Furthermore, the concurrent presence of CRM1-overexpression and increased BRAF activity offers the possibility of using simultaneous CRM1 and BRAF inhibition to reduce melanoma survival in BRAF-mutant melanoma. Therefore, we hypothesize that the inhibition of CRM1 in melanoma will result in impaired melanoma viability. In addition, by inhibiting independent targets using a CRM1/BRAF combination in BRAF-mutant melanoma, we will have enhanced antitumoral effects, which could translate in improved clinical outcomes.

Materials and Methods

Cell lines and reagents

Malignant melanoma cell lines where characterized and kindly donated by the Departments of Dermatology (Tsao laboratory) and Surgical Oncology (Wargo laboratory) from the Massachusetts General Hospital (MGH; Boston, MA) between 2011 and 2012. Cells were authenticated following American Type Culture Collection (ATCC) recommendations (ATCC Tech Bulletin #8), and used within 1 week after authentication. Cells were passaged for less than 6 months after received. Cell morphology and growth analysis were conducted posterior to resuscitation. Sequencing studies for PTEN, BRAF, NRAS, and p53 among others have been conducted in 2004, 2005, 2007, and in 2011 by Dr. Hensin Tsao (MGH; Supplementary Table S1). All cell lines were maintained and all experiments were carried out in Dulbecco's Modified Eagle Medium (DMEM; Sigma; D 6429) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Life Technologies). For in vitro studies, BRAF inhibitor, PLX-4032 (Selleck), and CRM1 inhibitor, KPT-251 or KPT-185 (Karyopharm Therapeutics) were used.

Cell proliferation assays

Cellular proliferation was evaluated by MTT assay (Sigma-Aldrich) following the manufacturer's instructions. Cells were plated in 96-well plates at 1,000 to 10,000 cells per well in 100 μL of media 24 hours after treatment and MTT signal was read at 72 hours after treatment. The IC50 and combination index (CI) by Chou–Talalay (16) were determined from the regression plot logarithm of the concentration versus effect using Calcusyn Software (Biosoft) v1.1. In addition, conservative isobolograms were used to show synergism and/or antagonism.

Immunoblotting, immunofluorescence, and immunohistochemistry microscopy

Immunoblotting and immunofluorescence were conducted using Cell Signaling Technology general protocols. Immunohistochemistry was conducted on paraffin-embedded tissue following IHC Products & Protocol Guide from R&D Systems. For our list of primary antibodies see Supplementary Table S2. Photomicrography images were recorded using a Nikon Eclipse 80i microscope and QImaging Retiga Exi chamber. Images were processed with QImaging software (Version 2.1). For immunohistochemistry quantification, 3 mice per group were chosen at random 24 hours after first dose of the indicated agent. Quantification was done from 3 views chosen at random from the slides of the selected mice.

Cell-cycle analysis by flow cytometry

Cell-cycle analysis was conducted using propidium iodide (PI) following the manufacturer's protocol in package insert (BD Pharmigen). A total of 3 × 104 cells per analysis were examined by flow cytometry (FACSCalibur), and analyzed using WinMDI 2.9.

Caspase-3/7 activity assay

Caspase-3/7 activity was determined using Promega #G8091 system, following manufacturer's protocol in package insert, and read using Victor3 multi-well reader (Wallac) and 1420 Wallac software.

Xenograft model

Athymic nude mice Nu/Nu (Crl:NU-Foxn1nu), 4 weeks of age were purchased from Charles River Laboratories and Taconic Farms. All animal experiments were carried out in accordance with protocols approved by the MGH Subcommittee on Research Animal Care (SRAC#2011N000037). Cell lines A375, A2058 (BRAF V600E-mutant), and Mel-Juso [BRAF wild-type (WT) and NRAS-mutant] were injected subcutaneously at 5 × 106. MeWo (BRAF and NRAS WT) was injected subcutaneously at 1 × 107. Mice were randomized using a random number generator once an average of approximately 300 mm3 tumor volume was reached (for A375 and A2058, average time was 7–9 days; for Mel-Juso average time was 45 days, and for MeWo average time was 82 days). After randomization, treatment was started. Mice were sacrificed following the guidelines by the Institutional Animal Care and Use Committee (IACUC) for MGH. Tumor volumes were determined using [D × (d2)]/2, in which D represents the largest diameter of the tumor, and d represents the largest perpendicular volume to D. Tumor volumes were normalized individually to their initial volume (volume at treatment day 1; relative tumor volume = Vx/V0; were Vx corresponds to the volume for the specific animal at a particular day and V0 corresponds to the initial volume for the given animal. BRAF inhibitor, PLX-4720 (Selleck), was diluted in dimethyl sulfoxide (DMSO) to a 20 mg/mL stock. Stock was diluted 1:10 in DMSO for daily intraperitoneal (i.p.) injections. KPT-251, KPT-276, and KPT-330 were diluted in 0.6% (w/v) Pluronic F-68 and PVP K-29/32 solution to a 7.5 mg/mL stock suspension, which was kept at room temperature, stirred, protected from light, and used within 7 days. Statistical analysis consisted of Mann–Whitney rank sum test, two-way ANOVA, and post hoc Bonferroni t test using GraphPad Prism, version 4.3. Oral administration of KPT-251 was tested to identify a dose and schedule that would yield maximum benefit with the least toxicity; we found 50 mg/kg per os every other day as the best option. For combination studies, we used: KPT-276 or KPT-330 per os 75 or 10 mg/kg, respectively, every other day, PLX-4720 i.p. or per os 25–50 mg/kg daily or their combination for 14 days. In experiments involving KPT-251 and KPT-330, all treatment groups contained 5 mice per group. For all experiments involving KPT-276, all treatment groups contained 10 mice per group. Response Evaluation Criteria in Solid Tumors (RECIST) criteria were used to classify response, and are defined as follows: complete response (CR): disappearance of all target lesions. Partial response (PR): at least a 30% decrease in the sum of the longest diameter of target lesions, taking as reference the baseline sum longest diameter. Stable disease: neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum longest diameter since the treatment started. Progressive disease: at least a 20% increase in the sum of the longest diameter of target lesions, taking as reference the smallest sum longest diameter recorded since the treatment started or the appearance of one or more new lesions (17).

Results and Discussion

CRM1 inhibition suppresses cell proliferation across a variety of cancer cell lines

Initially, we screened the SINE compounds across a variety cancer cell lines, both solid and hematologic cancers. The interruption of nuclear export using novel SINE, KPT-185, induces inhibition of cell proliferation across a variety of cancer cell lines but not on the tested normal cell lines (Fig. 2A). Cancer sensitivity to CRM1 inhibition in contrast to normal tissue may be explained by the altered expression of CRM1 and other nuclear transport proteins in cancer (5, 9, 15). Cancer sensitivity to CRM1 inhibition has led to the study of this target in several cancers with promising preclinical results (11, 12, 15, 18, 19).

Figure 2.
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Figure 2.

Box plots showing cell proliferation IC50s after XPO1 inhibition. A, IC50 doses for cell proliferation after XPO1 inhibition with KPT-185 across a panel of normal and cancer cell lines. B, comparison of BRAF inhibitor PLX-4032 and XPO1 inhibitor KPT-185 IC50 concentrations in melanoma cell lines, sorted by BRAF status. Error bars, SD; n, number of cell lines tested. GBM, glioblastoma multiforme.

CRM1 inhibition suppresses melanoma cell proliferation and is synergistic with BRAF inhibition, in vitro

Melanoma BRAF-mutant cell lines are sensitive to BRAF inhibition by PLX-4032, which leads to inhibition of cell proliferation. However, BRAF WT melanoma cell lines are relatively resistant to BRAF inhibition (Fig. 2B). CRM1 inhibition by SINE analogs results in inhibition of cell proliferation across tested melanoma cell lines. Among tested analogs, KPT-185 was almost twice as potent as KPT-251 or KPT-276 in vitro. The results were independent of BRAF mutational status (Fig. 2B and Supplementary Fig. S1A and S1B). In other words, a BRAF mutation is required for BRAF treatment sensitivity but not for sensitivity to CRM1 inhibition. The combination of BRAF and CRM1 inhibition in BRAF-mutant melanoma synergistically decreases cell proliferation in BRAF-mutant melanoma (Fig. 3A and B and Supplementary Fig. S1C). Synergy between BRAF inhibition and CRM1 inhibition was maintained when testing different SINE analogs at different molar ratios (i.e., 1:1, 1:2, and 1:10) and different time points (24–96 hours) showing similar CIs. The greatest effect of the combination on cell proliferation was observed between 48 and 72 hours. In addition, MEK/CRM1 combination, using MEK inhibitor U0126, results in similar synergy as the BRAF/CRM1 combination in BRAF-mutant melanoma (data not shown). Furthermore, MEK/BRAF/CRM1 combination further shifts the IC50 in BRAF-mutant melanoma and results in synergism for the 3 drug combination. These findings might suggest a MAPK pathway component as a responsible for the observed synergy. For the BRAF WT cell lines, the CI for the BRAF/CRM1 combination varied according to drug dose and cell line and ranged from antagonistic to synergy (Supplementary Fig. S1C).

Figure 3.
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Figure 3.

Nuclear export inhibition decreases melanoma cell proliferation and is synergistic with BRAF inhibition in vitro. A, MTT assay showing the effect of XPO1 inhibition, BRAF inhibition, and their combination on cell proliferation with their respective CI value. B, corresponding conservative isobologram for the MTT assay shown in A. Drug combinations at 1:1 ratio. Error bars, SD.

CRM1 inhibition induces cell-cycle arrest and apoptosis, and synergistically increases cell death when combined with BRAF inhibition

XPO1/CRM1 inhibition is associated with cell-cycle arrest (6, 19–21). In melanoma, SINE progressively reduces S-phase in both BRAF-mutant and WT cell lines, inducing cell-cycle arrest 24 hours after treatment (Supplementary Fig. S2A). Both G1 and/or G2 cell-cycle arrest can be observed using the CRM1 inhibitors (Supplementary Fig. S2B). During the next 48 hours, an increase in the sub-G1 population becomes statistically significant peaking at 72 hours of CRM1 inhibition. BRAF inhibition leads to a predominant G1 cell-cycle arrest in BRAF-mutant melanoma cell lines with a dose-cell line–dependent increase in the sub-G1 population (Supplementary Figs. S2C and S2D and S3A–S3D). We observed no statistically significant effect on the cell cycle after BRAF inhibition in the BRAF WT cell lines at tested doses (Supplementary Fig. S3C and S3D). The combination of both compounds induced a statistically significant greater increase in the sub-G1 population in BRAF-mutant cell lines in comparison with either single therapy at tested doses (P < 0.05; Supplementary Figs. S2B–S2D and S3A–S3D). There was a significant (P < 0.05) increase in the sub-G1 population of the BRAF WT cell lines after drug combination at higher doses (>3 μmol/L) when compared with single therapy with KPT (Supplementary Fig. S3) but no other statistically significant changes in the cell cycle. This can be attributed to reaching doses close to BRAF WT, BRAF inhibition IC50s.

We used caspase-3/7 activity as an apoptosis surrogate. Both CRM1 and BRAF inhibition increase caspase-3 and -7 activity in the tested melanoma cell lines in a dose- and time-related manner (P < 0.05; Supplementary Fig. S4). A modest effect of BRAF inhibition on caspase activity in some of the BRAF-mutant melanoma cell lines suggests cell-cycle arrest over apoptosis as the predominant mechanism impacting cell proliferation in these cell lines, under the tested conditions. These findings correlate to the results from the cell-cycle experiments.

The combination of both compounds results in a statistically significant increase in caspase activity when compared with either single therapy in BRAF-mutant melanoma. This would correlate to strong synergy (CI < 0.3) for caspase-3/7 activity on BRAF-mutant cell lines if the combination was taken as maximal effect and Chou–Talalay is applied. Despite slight increase in caspase activity after BRAF inhibition in BRAF WT cell lines after single drug treatment, the combination resulted in no statistically significant increase on caspase activity at tested doses when compared with CRM1 inhibition alone. For either single drug or combination studies, the effect on caspase activity was time- and dose-dependent and correlated to the sub-G1 changes observed in the cell cycle.

In melanoma xenograft models, CRM1 inhibition suppresses tumor growth and induces complete regression of A375 melanoma BRAF V600E tumors when combined with BRAF inhibition

Oral administration of KPT-251 suppressed tumor growth in 3 xenograft models (Supplementary Fig. S5A). Tumor growth suppression was associated with increased cleaved caspase-3 staining and a decreased Ki67 staining (Supplementary Fig. S5B). These findings suggest increased apoptosis, in addition to decreased cell proliferation. Similar to the in vitro data, the effects of CRM1 inhibition were independent of BRAF and NRAS status.

For our combination studies, BRAF inhibitor, PLX-4720 (preclinical PLX-4032 analog), was used because of better bioavailability. CRM1 inhibitors KPT-276 and KPT-330, structural analogs of KPT-251 with better bioavailability, were used to test BRAF and CRM1 combination in our BRAF V600E melanoma xenograft model. Both KPT-276 and PLX-4720 decreased tumor growth as single therapy. The combination of both inhibitors induced complete tumor regression per RECIST criteria (Fig. 4A, red line) and the difference between both single therapy and the combination therapy was statistically significant. These findings correlated to decreased cell proliferation and increased apoptosis when compared with the control group (Fig. 4B and C). The effect on proliferation is not statistically different between treatment groups. We believe the greatest contribution of the combination is the effect on apoptosis, which is significantly increased (P < 0.05) by the drug combination. Decreased cell proliferation complemented by increased apoptosis explains the observed tumor regression. Treatment was stopped after 14 days, after which tumor growth was observed in all treatment groups. Survival for the combination group was statistically longer than for either single therapy. As single therapy and in combination, both inhibitors were well tolerated with no significant effect on animal weight. Similar results were observed using clinical grade analog KPT-330 in A2058, PTEN null/BRAF-mutant melanoma cell line using a different treatment schedule (Supplementary Fig. S6A and S6B).

Figure 4.
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Figure 4.

In BRAF-mutant xenograft models, nuclear export inhibition induces delayed tumor growth as single therapy and tumor regression when combined with BRAF inhibition. A, relative tumor volumes curves for melanoma A375 xenografts after treatment with KPT-276 (XPO1 Inhibitor) at 75 mg/kg every other day, PLX-4720 (BRAF inhibitor) at 50 mg/kg i.p. every day, or their combination for 14 days. B and C, immunohistochemistry showing Ki67 and cleaved caspase-3 staining in harvested tumor samples and the quantification of the staining. A, each treatment group consisted of 10 mice. Error bars represent SEM. B, error bars represent SD. C, bar represent 200 μm. **, P < 0.01; †, control group reached tumor volume end of study threshold.

CRM1 inhibition modulates levels of p53, pRb, survivin, and ERK phosphorylation

TP53 is a known tumor suppressor capable of inducing cell-cycle arrest and apoptosis. Mutations in p53 disable its tumor suppressor abilities. Although mutations in p53 are not common in patients with melanoma (90% of melanoma tumors are p53 WT), loss of p53 tumor suppressor function is lost in many melanoma tumors owed to either, mutations in CDKN2A and/or overexpression of HDM2 (22, 23). The alteration of these p53 regulators facilitate CRM1-mediated nuclear export of p53 and cytoplasmic degradation of p53, thereby rendering the cells functionally deficient for the tumor suppressor p53. The inhibition of nuclear export using the CRM1 inhibitor favors nuclear localization of p53 and prevents cytoplasmic p53 degradation (Fig. 5A and B). The stabilization of p53 by CRM1 is independent of DNA damage (Fig. 5C) and is associated with increased levels of p53 targets (i.e., p21 and HDM2) in p53 WT melanoma but not in p53-mutant cell lines (6). TP53 knockdown induced partial reduction of CRM1 effects on cell proliferation (Fig. 5D) but not on caspase activity as single therapy or in the combination with BRAF inhibition (Supplementary Fig. S7). We can suggest that p53 has at least a partial role in the CRM1-mediated antitumoral effects. However, we must propose the existence of p53-independent mechanism(s), which contribute to the CRM1 p53-mediated effects (6). These alternative mechanisms would account for the remaining observed antitumoral activity and the resulting synergy of the combination. In addition, p53 nuclear localization is consistent after CRM1 inhibition and may serve as a marker of successful CRM1 inhibition.

Figure 5.
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Figure 5.

Nuclear export inhibition modulates levels of TP53, pRb, p-ERK, and survivin. Immunofluorescence (A) and Western blot analysis time course (B) from whole and fragmented cell lysate showing nuclear localization and stabilization of p53 after XPO1 inhibition using KPT-251 (1 μmol/L). C, Western blot analysis from nuclear lysate showing the effect of XPO1 inhibition (KPT-251; 1 μmol/L) on DNA damage markers. Etoposide (25 μmol/L) was used as a positive control for DNA damage. D, MTT assay showing the effect of XPO1 inhibition (KPT-251) on cell proliferation after p53 knockdown in A375 melanoma cell line. *, P < 0.05. E, Western blot analysis showing the effect of XPO1 inhibition, BRAF inhibition, and their combination on PARP, survivin, pRb, and ERK in A375 melanoma. DAPI, 4′,6-diamidino-2-phenylindole.

Other proteins we found to be affected by CRM1 inhibition include tumor suppressor pRb, survivin, and ERK. pRb and its phosphorylation are both affected by CRM1 and BRAF inhibition. The combination of both agents further reduces pRb and p-pRb levels (Fig. 5E). Hypophosphorylated pRb blocks proliferation by preventing the transcription of genes essential for cell-cycle progression(24). These finding correlate to our cell-cycle analysis suggesting a role for pRb in CRM1-mediated cell-cycle arrest.

Both CRM1 and BRAF inhibition decrease survivin levels. This finding is also seen after the drug combination (Fig. 5E). Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), is known as a bifunctional protein for its role in cell division and apoptosis suppression. Survivin is constantly shuttling between the nucleus and the cytoplasm. Cytoplasmic localization of survivin is associated with antiapoptotic function, whereas its nuclear pool mediates its mitotic function. However, CRM1 inhibition disrupts the cell division and antiapoptotic functions of survivin (6, 25).

CRM1 inhibition increases ERK phosphorylation in both BRAF WT and mutant (Fig. 5E; ref. 6). ERK phosphorylation after treatment with antitumoral agents has been described as a prosurvival event. Thus, phosphorylation of ERK following CRM1 inhibition could translate into enhanced chemoresistance (26). Another tentative explanation would involve ERK nuclear localization as an antiproliferative factor and further explaining CRM1-related antiproliferative effects (6, 27). Interestingly, the combination of the CRM1 inhibitor with the BRAF inhibitor prevents ERK phosphorylation (Fig. 5E) and results in synergy between the 2 compounds. Our data suggest that the protection against CRM1-induced ERK phosphorylation by BRAF-inhibition may play a role in the synergistic response to combined therapy. Furthermore, our data support that the inhibition of ERK phosphorylation may have a role in addressing ERK-mediated chemoresistance.

In conclusion, given the discovery of diverse mechanisms of acquired resistance to BRAF inhibitor therapy, which are not readily countered with pharmacologic strategies, we believe a priority should be placed on developing BRAF inhibitor-based combinations to overcome de novo resistance and prevent the emergence of these acquired resistance mechanisms. We believe achieving this goal would translate into longer duration of response (progression-free survival) and will increase the percentage of treatment responders. On the basis of our data, CRM1/BRAF inhibitor combination can offer a benefit to the melanoma patient population. The combination of CRM1 and BRAF inhibition results in a synergistic decrease of cell proliferation, and increased apoptosis in BRAF-mutant melanoma cell lines and xenograft models. We attribute the synergy of the combination to the inhibition of independent targets altering multiple essential factors of melanoma viability. In particular, we find that the abrogation of the CRM1-associated ERK phosphorylation by BRAF inhibition to be one of the contributors. Taken together, our findings offer rationale for the combination of CRM1 and BRAF inhibition in an attempt to improve the treatment of patients with BRAF-mutant melanoma. Furthermore, the effects of CRM1 inhibition as single therapy are independent of BRAF status. This suggests a role for CRM1 inhibition in the treatment of melanoma, without mutational limitations. Studies exploring the safety and efficacy of CRM1 and CRM1/BRAF inhibition in the melanoma clinical realm are needed.

Disclosure of Potential Conflicts of Interest

H. Tsao is a consultant/advisory board member for Genentech, WorldCare Clinical, and Quest. M. Kauffman has ownership interest (including patents) in Karyopharm Therapeutics. J.C. Cusack has a commercial research grant from Karyopharm Therapeutics. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: R.A.S. Fragomeni, H.W. Chung, Y. Landesman, S. Shacham, M. Kauffman, J.C. Cusack

Development of methodology: R.A.S. Fragomeni, H. Tsao, J.C. Cusack

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.A.S. Fragomeni, H.W. Chung, W. Senapedis, J.-R. Saint-Martin, H. Tsao, J.C. Cusack

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.A.S. Fragomeni, Y. Landesman, W. Senapedis, J.-R. Saint-Martin, H. Tsao, K.T. Flaherty, S. Shacham, J.C. Cusack

Writing, review, and/or revision of the manuscript: R.A.S. Fragomeni, H. Tsao, K.T. Flaherty, S. Shacham, M. Kauffman, J.C. Cusack

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.A.S. Fragomeni, W. Senapedis, J.C. Cusack

Study supervision: R.A.S. Fragomeni, Y. Landesman, H. Tsao, J.C. Cusack

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Received December 4, 2012.
  • Revision received April 17, 2013.
  • Accepted April 17, 2013.
  • ©2013 American Association for Cancer Research.

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Molecular Cancer Therapeutics: 12 (7)
July 2013
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CRM1 and BRAF Inhibition Synergize and Induce Tumor Regression in BRAF-Mutant Melanoma
Roberto A. Salas Fragomeni, Hye Won Chung, Yosef Landesman, William Senapedis, Jean-Richard Saint-Martin, Hensin Tsao, Keith T. Flaherty, Sharon Shacham, Michael Kauffman and James C. Cusack
Mol Cancer Ther July 1 2013 (12) (7) 1171-1179; DOI: 10.1158/1535-7163.MCT-12-1171

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CRM1 and BRAF Inhibition Synergize and Induce Tumor Regression in BRAF-Mutant Melanoma
Roberto A. Salas Fragomeni, Hye Won Chung, Yosef Landesman, William Senapedis, Jean-Richard Saint-Martin, Hensin Tsao, Keith T. Flaherty, Sharon Shacham, Michael Kauffman and James C. Cusack
Mol Cancer Ther July 1 2013 (12) (7) 1171-1179; DOI: 10.1158/1535-7163.MCT-12-1171
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