CRM1 and BRAF Inhibition Synergize and Induce …...CRM1 and BRAF Inhibition Synergize and Induce Tumor Regression in BRAF-Mutant Melanoma Roberto A. Salas Fragomeni1,2, Hye Won Chung1,2,

Chemical Therapeutics

CRM1 and BRAF Inhibition Synergize and Induce TumorRegression in BRAF-Mutant Melanoma

Roberto A. Salas Fragomeni1,2, Hye Won Chung1,2, Yosef Landesman5, William Senapedis5,Jean-Richard Saint-Martin5, Hensin Tsao1,3, Keith T. Flaherty1,4, Sharon Shacham5,Michael Kauffman5, and James C. Cusack1,2

AbstractResistance 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.Wehypothesize that by inhibitingCRM1-mediatednuclear export,wewill alter the function of

these proteins resulting in decreasedmelanoma viability and enhanced BRAF inhibitor antitumoral effects. To

test our hypothesis, selective inhibitors of nuclear export (SINE) analogsKPT-185,KPT-251,KPT-276, andKPT-

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 BRAFmutation 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.

IntroductionMost cancers acquire functional survival capabilities

during their development. These capabilities includeapoptosis evasion, self-sufficient growth signals, andinsensitivity to antigrowth signals (1). Advanced-stagemelanoma shows these capabilities. For instance, a con-stitutively activate BRAF kinase is present in at leasthalf of all the patients with advanced melanoma, drivingmelanoma proliferation (2). BRAF-targeted therapyinterrupts the growth signal and suppresses melanomaproliferation. Targeting BRAF mutation has led to out-standing clinical results with limited associated toxicity

(3, 4). However, the emergent resistance to BRAF inhi-bition limits the therapy’s response duration, challengingour limited therapeutic options for advanced melanoma.

The modulation of nucleo-cytoplasmic protein trans-port has been suggested as a possible therapeutic strategyin the treatment of cancer (5). Protein transport betweenthe nucleus and the cytoplasm is critical for cell mainte-nance, cell proliferation, and survival. Alterations in theexpression of nuclear transport–related proteins, in par-ticular Exportin 1 (XPO1, also known as chromosomeregion 1, CRM1) are found in melanoma and other can-cers. Overexpression of CRM1 is linked to inactivation oftumor suppressor proteins, apoptosis evasion, and resis-tance to chemotherapy (6). In addition, CRM1 expressionis of prognostic value in several types of cancer (7–10).CRM1 mediates nuclear export using nuclear export sig-nals (NES),which are required for nuclear cargo transportfrom the nucleus to the cytoplasm. Selective inhibitors ofnuclear export (SINE) also known as KPT-analogs (Fig. 1;KPT-185, KPT-251, KPT-276, and KPT-330) are capable ofbinding to the Cys-528 residue in the cargo-binding por-tion of the CRM1 protein successfully preventing proteintransport from thenucleus to the cytoplasm (5, 11–13). Theinterruption of the system results in nuclear accumulationof the cargo, thereby restoringordisrupting cargo function.

Authors' Affiliations: 1Harvard Medical School; 2Division of SurgicalOncology, Departments of 3Dermatology and 4Medicine, Hematology/Oncology, Massachusetts General Hospital, Boston; and 5KaryopharmTherapeutics, Natick, Massachusetts

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

Corresponding Author: James C. Cusack, Division of Surgical Oncology,Massachusetts General Hospital, Yawkey 7th Floor, 55 Fruit Street, Bos-ton, MA 02114. Phone: 617-724-4093; Fax: 617-724-3895; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-12-1171

�2013 American Association for Cancer Research.

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on December 6, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 24, 2013; DOI: 10.1158/1535-7163.MCT-12-1171

http://mct.aacrjournals.org/

Among the CRM1-affected proteins, we find p42/44mitogen-activated protein kinase (MAPK; extracellularsignal–regulated kinase (ERK)1/2], p53, c-Fos, survivin,and retinoblastoma protein (pRb; refs. 5–9). Modifyingthe function of these proteins alters survival and pro-liferation of cancer (5, 6, 14). Furthermore, the modu-lation of the CRM1 function using SINE has achievedpromising preclinical results in cancer (11, 12, 15). Clin-ical grade analog, KPT-330, is currently in clinical phaseI trials (NCT01607905 and NCT01607892).

CRM1has been found to be overexpressed inmetastaticmelanoma when compared with nevi or even primarymelanoma lesions (6). Moreover, CRM1 expression por-trays independent of BRAF mutational status (6). Thismakes CRM1 a potential therapeutic target for metastaticmelanoma. Furthermore, the concurrent presence ofCRM1-overexpression and increased BRAF activity offersthe possibility of using simultaneous CRM1 and BRAFinhibition to reduce melanoma survival in BRAF-mutantmelanoma. Therefore, we hypothesize that the inhibitionof CRM1 in melanoma will result in impaired melanomaviability. In addition, by inhibiting independent targetsusing a CRM1/BRAF combination in BRAF-mutant mel-anoma, wewill have enhanced antitumoral effects, whichcould translate in improved clinical outcomes.

Materials and MethodsCell lines and reagents

Malignant melanoma cell lines where characterizedand kindly donated by the Departments of Dermatology(Tsao laboratory) and Surgical Oncology (Wargo labora-tory) from the Massachusetts General Hospital (MGH;Boston, MA) between 2011 and 2012. Cells were authen-ticated following American Type Culture Collection

(ATCC) recommendations (ATCC Tech Bulletin #8), andused within 1 week after authentication. Cells werepassaged for less than 6 months after received. Cell mor-phology and growth analysis were conducted posteriorto resuscitation. Sequencing studies for PTEN, BRAF,NRAS, and p53 among others have been conducted in2004, 2005, 2007, and in 2011 by Dr. Hensin Tsao (MGH;Supplementary Table S1). All cell lines were maintainedand all experiments were carried out in Dulbecco’s Mod-ified Eagle Medium (DMEM; Sigma; D 6429) supplemen-tedwith 10% FBS (Gibco) and 1%penicillin–streptomycin(Life Technologies). For in vitro studies, BRAF inhibitor,PLX-4032 (Selleck), andCRM1 inhibitor, KPT-251 or KPT-185 (Karyopharm Therapeutics) were used.

Cell proliferation assaysCellular proliferation was evaluated by MTT assay

(Sigma-Aldrich) following themanufacturer’s instructions.Cells were plated in 96-well plates at 1,000 to 10,000 cellsper well in 100 mL of media 24 hours after treatment andMTT signal was read at 72 hours after treatment. The IC50

and combination index (CI) by Chou–Talalay (16) weredetermined from the regression plot logarithm of theconcentration versus effect using Calcusyn Software (Bio-soft) v1.1. In addition, conservative isobolograms wereused to show synergism and/or antagonism.

Immunoblotting, immunofluorescence, andimmunohistochemistry microscopy

Immunoblotting and immunofluorescence were con-ducted using Cell Signaling Technology general proto-cols. Immunohistochemistry was conducted on paraffin-embedded tissue following IHC Products & ProtocolGuide from R&D Systems. For our list of primary anti-bodies see Supplementary Table S2. Photomicrography

F3C

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Figure 1. Chemical structures forSINE analogs KPT-185, KPT-251,KPT-276, and KPT-330.

Salas Fragomeni et al.

Mol Cancer Ther; 12(7) July 2013 Molecular Cancer Therapeutics1172




images were recorded using a Nikon Eclipse 80i micro-scope and QImaging Retiga Exi chamber. Images wereprocessed with QImaging software (Version 2.1). Forimmunohistochemistry quantification, 3 mice per groupwere chosen at random 24 hours after first dose of theindicated agent. Quantification was done from 3 viewschosen at random from the slides of the selected mice.

Cell-cycle analysis by flow cytometryCell-cycle analysis was conducted using propidium

iodide (PI) following themanufacturer’s protocol in pack-age insert (BD Pharmigen). A total of 3 � 104 cells peranalysis were examined by flow cytometry (FACSCali-bur), and analyzed using WinMDI 2.9.

Caspase-3/7 activity assayCaspase-3/7 activity was determined using Promega

#G8091 system, following manufacturer’s protocol inpackage insert, and read using Victor3 multi-well reader(Wallac) and 1420 Wallac software.

Xenograft modelAthymic nude mice Nu/Nu (Crl:NU-Foxn1nu), 4

weeks of age were purchased from Charles River Labo-ratories and Taconic Farms. All animal experiments werecarried out in accordance with protocols approved bythe MGH Subcommittee on Research Animal Care(SRAC#2011N000037). Cell lines A375, A2058 (BRAFV600E-mutant), andMel-Juso [BRAFwild-type (WT) andNRAS-mutant] were injected subcutaneously at 5 � 106.MeWo (BRAF and NRAS WT) was injected subcutane-ously at 1 � 107. Mice were randomized using a randomnumber generator once an average of approximately 300mm3 tumor volume was reached (for A375 and A2058,average timewas 7–9 days; forMel-Juso average timewas45 days, and for MeWo average time was 82 days). Afterrandomization, treatment was started. Mice were sacri-ficed following the guidelines by the Institutional AnimalCare and Use Committee (IACUC) for MGH. Tumorvolumes were determined using [D � (d2)]/2, in whichD represents the largest diameter of the tumor, and drepresents the largest perpendicular volume toD. Tumorvolumes were normalized individually to their initialvolume (volume at treatment day 1; relative tumor vol-ume ¼ Vx/V0; were Vx corresponds to the volume for thespecific animal at a particular day and V0 corresponds tothe 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 inDMSO for daily intraperitoneal (i.p.) injections. KPT-251,KPT-276, and KPT-330 were diluted in 0.6% (w/v) Pluro-nic F-68 and PVP K-29/32 solution to a 7.5 mg/mL stocksuspension, whichwas kept at room temperature, stirred,protected from light, and used within 7 days. Statisticalanalysis consisted of Mann–Whitney rank sum test, two-way ANOVA, and post hoc Bonferroni t test using Graph-Pad Prism, version 4.3. Oral administration of KPT-251was tested to identify a dose and schedule that would

yieldmaximumbenefitwith the least toxicity;we found50mg/kg per os every other day as the best option. Forcombination studies, we used: KPT-276 or KPT-330 peros 75 or 10mg/kg, respectively, every other day, PLX-4720i.p. or per os 25–50mg/kg daily or their combination for 14days. In experiments involving KPT-251 and KPT-330, alltreatment groups contained 5 mice per group. For allexperiments involvingKPT-276, all treatment groups con-tained 10mice per group. Response Evaluation Criteria inSolid Tumors (RECIST) criteria were used to classifyresponse, 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 longestdiameter of target lesions, taking as reference the baselinesum longest diameter. Stable disease: neither sufficientshrinkage to qualify for PR nor sufficient increase toqualify for progressive disease, taking as reference thesmallest sum longest diameter since the treatment started.Progressive disease: at least a 20% increase in the sum ofthe longest diameter of target lesions, taking as referencethe smallest sum longest diameter recorded since thetreatment started or the appearance of one or more newlesions (17).

Results and DiscussionCRM1 inhibition suppresses cell proliferation acrossa variety of cancer cell lines

Initially, we screened the SINE compounds across avariety cancer cell lines, both solid and hematologic can-cers. The interruption of nuclear export using novel SINE,KPT-185, induces inhibition of cell proliferation across avariety of cancer cell lines but not on the tested normal celllines (Fig. 2A). Cancer sensitivity to CRM1 inhibition incontrast to normal tissue may be explained by the alteredexpression of CRM1 and other nuclear transport proteinsin cancer (5, 9, 15). Cancer sensitivity to CRM1 inhibitionhas led to the study of this target in several cancers withpromising preclinical results (11, 12, 15, 18, 19).

CRM1 inhibition suppresses melanoma cellproliferation and is synergisticwithBRAF inhibition,in vitro

Melanoma BRAF-mutant cell lines are sensitive toBRAF inhibition by PLX-4032, which leads to inhibitionof cell proliferation. However, BRAF WT melanoma celllines are relatively resistant to BRAF inhibition (Fig. 2B).CRM1 inhibition by SINE analogs results in inhibition ofcell proliferation across tested melanoma cell lines.Among tested analogs, KPT-185 was almost twice aspotent as KPT-251 or KPT-276 in vitro. The results wereindependent of BRAF mutational status (Fig. 2B andSupplementary Fig. S1A and S1B). In otherwords, a BRAFmutation is required for BRAF treatment sensitivity butnot for sensitivity to CRM1 inhibition. The combination ofBRAF and CRM1 inhibition in BRAF-mutant melanomasynergistically decreases cell proliferation in BRAF-mutant melanoma (Fig. 3A and B and Supplementary

CRM1/BRAF Inhibition in Melanoma

www.aacrjournals.org Mol Cancer Ther; 12(7) July 2013 1173




Fig. S1C). Synergy between BRAF inhibition and CRM1inhibition was maintained when testing different SINEanalogs at different molar ratios (i.e., 1:1, 1:2, and 1:10)and different time points (24–96 hours) showing similarCIs. The greatest effect of the combination on cell pro-liferation was observed between 48 and 72 hours. Inaddition, MEK/CRM1 combination, using MEK inhib-itor U0126, results in similar synergy as the BRAF/CRM1 combination in BRAF-mutant melanoma (datanot shown). Furthermore, MEK/BRAF/CRM1 combi-nation further shifts the IC50 in BRAF-mutant melanomaand results in synergism for the 3 drug combination.These findings might suggest a MAPK pathway com-ponent as a responsible for the observed synergy. Forthe BRAF WT cell lines, the CI for the BRAF/CRM1combination varied according to drug dose and cell lineand ranged from antagonistic to synergy (Supplemen-tary Fig. S1C).

CRM1 inhibition induces cell-cycle arrest andapoptosis, and synergistically increases cell deathwhen combined with BRAF inhibition

XPO1/CRM1 inhibition is associated with cell-cyclearrest (6, 19–21). Inmelanoma, SINEprogressively reducesS-phase in both BRAF-mutant andWT cell lines, inducingcell-cycle arrest 24 hours after treatment (SupplementaryFig. S2A). Both G1 and/or G2 cell-cycle arrest can beobserved using the CRM1 inhibitors (Supplementary Fig.S2B). During the next 48 hours, an increase in the sub-G1

population becomes statistically significant peaking at 72hours of CRM1 inhibition. BRAF inhibition leads to apredominant G1 cell-cycle arrest in BRAF-mutant melano-ma cell lines with a dose-cell line–dependent increase inthe sub-G1 population (Supplementary Figs. S2C and S2Dand S3A–S3D). We observed no statistically significanteffect on the cell cycle after BRAF inhibition in the BRAFWT cell lines at tested doses (Supplementary Fig. S3C and

S3D). The combination of both compounds induced astatistically significant greater increase in the sub-G1 pop-ulation in BRAF-mutant cell lines in comparison witheither single therapy at tested doses (P < 0.05; Supplemen-tary Figs. S2B–S2D and S3A–S3D). There was a significant(P < 0.05) increase in the sub-G1 population of the BRAFWT cell lines after drug combination at higher doses (>3mmol/L) when compared with single therapy with KPT(Supplementary Fig. S3) but no other statistically signifi-cant changes in the cell cycle. This can be attributed toreaching doses close to BRAF WT, BRAF inhibition IC50s.

We used caspase-3/7 activity as an apoptosis surro-gate. Both CRM1 and BRAF inhibition increase caspase-3 and -7 activity in the tested melanoma cell lines in adose- and time-related manner (P < 0.05; Supplemen-tary Fig. S4). A modest effect of BRAF inhibition oncaspase activity in some of the BRAF-mutant melanomacell lines suggests cell-cycle arrest over apoptosis asthe predominant mechanism impacting cell prolifera-tion 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 astatistically significant increase in caspase activity whencompared with either single therapy in BRAF-mutantmelanoma. This would correlate to strong synergy (CI <0.3) for caspase-3/7 activity on BRAF-mutant cell lines ifthe combination was taken as maximal effect and Chou–Talalay is applied. Despite slight increase in caspaseactivity after BRAF inhibition in BRAF WT cell linesafter single drug treatment, the combination resulted inno statistically significant increase on caspase activity attested doses when compared with CRM1 inhibitionalone. For either single drug or combination studies,the effect on caspase activity was time- and dose-depen-dent and correlated to the sub-G1 changes observed inthe cell cycle.

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Figure 2. Box plots showing cell proliferation IC50s after XPO1 inhibition. A, IC50 doses for cell proliferation after XPO1 inhibitionwith KPT-185 across a panel ofnormal and cancer cell lines. B, comparison of BRAF inhibitor PLX-4032 and XPO1 inhibitor KPT-185 IC50 concentrations in melanoma cell lines, sorted byBRAF status. Error bars, SD; n, number of cell lines tested. GBM, glioblastoma multiforme.






In melanoma xenograft models, CRM1 inhibitionsuppresses tumor growth and induces completeregression of A375 melanoma BRAF V600E tumorswhen combined with BRAF inhibitionOral administration of KPT-251 suppressed tumor

growth in 3 xenograft models (Supplementary Fig.S5A). Tumor growth suppression was associated withincreased cleaved caspase-3 staining and a decreasedKi67 staining (Supplementary Fig. S5B). These findingssuggest increased apoptosis, in addition to decreasedcell proliferation. Similar to the in vitro data, the effectsof CRM1 inhibition were independent of BRAF andNRAS status.For our combination studies, BRAF inhibitor, PLX-4720

(preclinical PLX-4032 analog), was used because of betterbioavailability. CRM1 inhibitors KPT-276 and KPT-330,structural analogs of KPT-251 with better bioavailability,were used to test BRAF and CRM1 combination in ourBRAF V600E melanoma xenograft model. Both KPT-276and 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 thecombination therapy was statistically significant. Thesefindings correlated to decreased cell proliferation andincreased apoptosis when compared with the controlgroup (Fig. 4B and C). The effect on proliferation is notstatistically different between treatment groups. Webelieve the greatest contribution of the combination is theeffect on apoptosis, which is significantly increased (P <0.05) by the drug combination. Decreased cell prolifera-tion complemented by increased apoptosis explains theobserved tumor regression. Treatment was stopped after14 days, after which tumor growth was observed in alltreatment groups. Survival for the combination groupwasstatistically longer than for either single therapy.As singletherapy and in combination, both inhibitors were welltolerated with no significant effect on animal weight.Similar results were observed using clinical grade analogKPT-330 in A2058, PTEN null/BRAF-mutant melanomacell line using a different treatment schedule (Supplemen-tary Fig. S6A and S6B).

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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 ofXPO1 inhibition, BRAF inhibition, and their combination on cell proliferation with their respective CI value. B, corresponding conservative isobologram for theMTT assay shown in A. Drug combinations at 1:1 ratio. Error bars, SD.






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

TP53 is a known tumor suppressor capable of inducingcell-cycle arrest and apoptosis. Mutations in p53 disableits tumor suppressor abilities. Although mutations in p53are not common in patients with melanoma (90% ofmelanoma tumors are p53 WT), loss of p53 tumor sup-pressor function is lost in many melanoma tumors owedto either, mutations in CDKN2A and/or overexpressionof HDM2 (22, 23). The alteration of these p53 regulatorsfacilitate CRM1-mediated nuclear export of p53 and cyto-plasmic degradation of p53, thereby rendering the cellsfunctionally deficient for the tumor suppressor p53. Theinhibition of nuclear export using the CRM1 inhibitorfavors nuclear localization of p53 and prevents cyto-plasmic p53 degradation (Fig. 5A and B). The stabilizationof 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 partialreduction of CRM1 effects on cell proliferation (Fig. 5D)but not on caspase activity as single therapy or in thecombination with BRAF inhibition (Supplementary Fig.S7).We can suggest that p53has at least a partial role in theCRM1-mediated antitumoral effects. However, we mustpropose the existence of p53-independent mechanism(s),which contribute to the CRM1 p53-mediated effects (6).These alternative mechanisms would account for theremaining observed antitumoral activity and the resultingsynergy of the combination. In addition, p53 nuclearlocalization is consistent after CRM1 inhibition and mayserve as a marker of successful CRM1 inhibition.

Other proteins we found to be affected by CRM1 inhi-bition include tumor suppressor pRb, survivin, and ERK.

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Figure 4. In BRAF-mutant xenograft models, nuclear export inhibition induces delayed tumor growth as single therapy and tumor regression whencombined with BRAF inhibition. A, relative tumor volumes curves for melanoma A375 xenografts after treatment with KPT-276 (XPO1 Inhibitor) at 75 mg/kgevery 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 Ki67and 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 mm. ��, P < 0.01; †, control group reached tumor volume end of studythreshold.






pRb and its phosphorylation are both affected by CRM1and BRAF inhibition. The combination of both agentsfurther reduces pRb and p-pRb levels (Fig. 5E). Hypopho-sphorylated pRb blocks proliferation by preventing thetranscription of genes essential for cell-cycle progression(24). These finding correlate to our cell-cycle analysis sug-gesting 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 ofapoptosis repeat-containing 5 (BIRC5), is known as abifunctional protein for its role in cell division and apo-ptosis suppression. Survivin is constantly shuttlingbetween the nucleus and the cytoplasm. Cytoplasmiclocalization of survivin is associated with antiapoptoticfunction, whereas its nuclear pool mediates its mitotic

function. However, CRM1 inhibition disrupts the celldivision and antiapoptotic functions of survivin (6, 25).

CRM1 inhibition increases ERK phosphorylation inboth BRAF WT and mutant (Fig. 5E; ref. 6). ERK phos-phorylation after treatment with antitumoral agents hasbeen described as a prosurvival event. Thus, phosphor-ylation of ERK following CRM1 inhibition could translateinto enhanced chemoresistance (26). Another tentativeexplanation would involve ERK nuclear localization asan antiproliferative factor and further explaining CRM1-related antiproliferative effects (6, 27). Interestingly, thecombination of the CRM1 inhibitor with the BRAF inhib-itor prevents ERKphosphorylation (Fig. 5E) and results insynergy between the 2 compounds. Our data suggest thatthe protection against CRM1-induced ERK phosphoryla-tion by BRAF-inhibition may play a role in the synergistic

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Figure 5. Nuclear export inhibitionmodulates levels of TP53, pRb, p-ERK, and survivin. Immunofluorescence (A) andWestern blot analysis time course (B) fromwhole and fragmented cell lysate showing nuclear localization and stabilization of p53 after XPO1 inhibition using KPT-251 (1 mmol/L). C, Western blotanalysis from nuclear lysate showing the effect of XPO1 inhibition (KPT-251; 1 mmol/L) on DNA damage markers. Etoposide (25 mmol/L) was used as apositive control for DNA damage. D, MTT assay showing the effect of XPO1 inhibition (KPT-251) on cell proliferation after p53 knockdown in A375melanoma 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, 40,6-diamidino-2-phenylindole.






response to combined therapy. Furthermore, our datasupport that the inhibition of ERK phosphorylation mayhave a role in addressingERK-mediated chemoresistance.

In conclusion, given the discovery of diversemechanisms of acquired resistance to BRAF inhibitortherapy, which are not readily countered with phar-macologic strategies, we believe a priority should beplaced on developing BRAF inhibitor-based combina-tions to overcome de novo resistance and prevent theemergence of these acquired resistance mechanisms.We believe achieving this goal would translate intolonger duration of response (progression-free surviv-al) and will increase the percentage of treatmentresponders. On the basis of our data, CRM1/BRAFinhibitor combination can offer a benefit to the mela-noma patient population. The combination of CRM1and BRAF inhibition results in a synergistic decrease ofcell proliferation, and increased apoptosis in BRAF-mutant melanoma cell lines and xenograft models. Weattribute the synergy of the combination to the inhibi-tion of independent targets altering multiple essentialfactors of melanoma viability. In particular, we findthat the abrogation of the CRM1-associated ERK phos-phorylation by BRAF inhibition to be one of the con-tributors. Taken together, our findings offer rationalefor the combination of CRM1 and BRAF inhibition inan attempt to improve the treatment of patients withBRAF-mutant melanoma. Furthermore, the effects ofCRM1 inhibition as single therapy are independent ofBRAF status. This suggests a role for CRM1 inhibition

in the treatment of melanoma, without mutationallimitations. Studies exploring the safety and efficacyof CRM1 and CRM1/BRAF inhibition in the melanomaclinical realm are needed.

Disclosure of Potential Conflicts of InterestH. Tsao is a consultant/advisory boardmember for Genentech,World-

Care Clinical, and Quest. M. Kauffman has ownership interest (includingpatents) in Karyopharm Therapeutics. J.C. Cusack has a commercialresearch grant from Karyopharm Therapeutics. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: R.A.S. Fragomeni, H.W. Chung, Y. Landesman,S. Shacham, M. Kauffman, J.C. CusackDevelopment of methodology: R.A.S. Fragomeni, H. Tsao, J.C. CusackAcquisition 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. CusackAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): R.A.S. Fragomeni, Y. Landesman, W. Sena-pedis, J.-R. Saint-Martin, H. Tsao, K.T. Flaherty, S. Shacham, J.C. CusackWriting, review, and/or revision of the manuscript: R.A.S. Fragomeni,H. Tsao, K.T. Flaherty, S. Shacham, M. Kauffman, J.C. CusackAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.A.S. Fragomeni, W. Senapedis,J.C. CusackStudy supervision:R.A.S. Fragomeni, Y. Landesman,H. Tsao, J.C. Cusack

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 4, 2012; revised April 17, 2013; accepted April 17,2013; published OnlineFirst April 24, 2013.

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2013;12:1171-1179. Published OnlineFirst April 24, 2013.Mol Cancer Ther Roberto A. Salas Fragomeni, Hye Won Chung, Yosef Landesman, et al. Regression in BRAF-Mutant MelanomaCRM1 and BRAF Inhibition Synergize and Induce Tumor

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CRM1 and BRAF Inhibition Synergize and Induce …...CRM1 and BRAF Inhibition Synergize and Induce Tumor Regression in BRAF-Mutant Melanoma Roberto A. Salas Fragomeni1,2, Hye Won Chung1,2,