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1. Background
2. Medical need
3. Existing treatments
4. Current research goals
5. Scientific rationale
6. Competitive environment
7. Potential development issues
8. Conclusions
9. Expert opinion
Review
Emerging BRAF inhibitors formelanomaFrancesco Sabbatino, Yangyang Wang, Xinhui Wang, Soldano Ferrone† &Cristina R Ferrone†Massachusetts General Hospital, Harvard Medical School, Department of Surgery,
Boston, MA, USA
Introduction: The clinical activity of BRAF inhibitor (BRAF-I) therapy is a major
breakthrough in the treatment of metastatic melanoma carrying BRAF muta-
tions. However, the therapeutic efficacy of BRAF-I therapy is limited due to
the onset of intrinsic and acquired drug resistance.
Areas covered: The role of wild-type BRAF in melanocytes and of the mutated
BRAF in the pathogenesis of melanoma is described in this article. The results
obtained with BRAF-I in patients with mutated BRAF are reviewed. The mech-
anisms driving the intrinsic and acquired BRAF-I resistance, the development
of combinatorial strategies designed to overcome them and their potential
limitations are discussed. Lastly, the many questions that have to be addressed
to optimize therapy with BRAF-I are listed.
Expert opinion: Melanoma is an aggressive form of skin cancer characterized
by poor prognosis and high mortality. The discovery of BRAF mutations which
drive melanoma tumorigenesis and the development of agents which selec-
tively inhibit mutant-activated BRAF represent a major breakthrough in the
treatment of metastatic melanoma. However, the development of drug resis-
tance underlies the need of more effective and individualized combinatorial
treatments to counteract the multiple escape mechanisms utilized by BRAF-
mutant melanoma. Although combinatorial strategies using agents which
target different protumorigenic signaling pathway components have been
shown to increase the clinical efficacy of BRAF-I, novel strategies which utilize
different antitumor mechanisms are needed.
Keywords: BRAF inhibitor, BRAF inhibitor resistance, combinatorial strategies, melanoma
Expert Opin. Emerging Drugs (2013) 18(4):431-443
1. Background
1.1 MelanomaMelanoma is an aggressive form of skin cancer arising from malignantly trans-formed melanocytes. The incidence and mortality from melanoma are rising, plac-ing significant demands on health care provision and representing a major publichealth issue. Approximately 76,250 new cases of melanoma, resulting in9,180 deaths are estimated for the United States in 2012 [1]. The reasons for thehigher incidence of melanoma remain unclear, but increased exposures to sun orultraviolet radiation are some of the major risk factors. Family history of melanoma,genetic susceptibility, environmental factors and age-related immunosuppressionmay also influence the incidence rate [2,3].
In 4.5 -- 40% of cases melanoma begins with the transformation of a benignnevus. Benign nevus might develop into a dysplastic lesion before progressing intoa radial and vertical growth phase of the melanoma. This tumor invades into thedermis and into the regional lymph nodes before disseminating to distant organs.However, not all melanomas arise from nevus and many arise through direct trans-formation of normal melanocytes [3-5].
10.1517/14728214.2013.842975 © 2013 Informa UK, Ltd. ISSN 1472-8214, e-ISSN 1744-7623 431All rights reserved: reproduction in whole or in part not permitted
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As for other types of malignancies, the prognosis of patientsaffected by melanoma depends mainly on the stage at presen-tation of the disease. Early detection of cutaneous melanomaand its early removal are the only therapeutic strategy ableto increase the percentage of cures. Therefore, identificationand treatment of suspected skin lesions play the major rolein the treatment of melanoma.While the majority of patients present with a primary cuta-
neous melanoma and are cured by surgical resection alone,12 -- 18% of patients present with metastasis to regionallymph nodes or distant sites. These patients are associatedwith poor long-term survival and are treated with palliativesystemic therapy. Further, in patients with visceral metastaticdisease, melanoma is usually rapidly fatal, with an average sur-vival of < 1 year and is associated with high morbidity [6].Palliative systemic therapy is the basis of management for
metastatic melanoma, and until 2011 chemotherapy withdacarbazine and immunotherapy with high dose of interleukin2 (HD IL-2) were the only treatments for metastatic patients,despite both failing to demonstrate an improvement of overallsurvival (OS) [7-9]. However, significant advances have beenmade in recent years. Recognition of key molecular mutationsthat drive melanoma tumorigenesis has led to the developmentof promising agents which selectively inhibit the mutant acti-vated target and, in turn, provide improved response rateswith decreased toxicity. Specifically, the discovery that 50%of all melanomas have activating mutations in the serine/threonine kinase BRAF (v-raf murine sarcoma viral oncogenehomolog B1) [10] has led to the development and subsequentthe Food and Drug Administration (FDA) approval of vemur-afenib and dabrafenib, which are both selective BRAF inhibi-tors (BRAF-I) [11-13]. Treatment with BRAF-I improves OSand induces tumor regression in ~50% of treated patientswith metastatic melanoma carrying a mutant BRAF. However,the responses are rarely complete and the median time to dis-ease progression is < 7 months due the onset of specific drugresistance. Several mechanisms of BRAF-I resistance havebeen described and clinical trials are testing new strategies toovercome the intrinsic or acquired BRAF-I resistance of mela-noma cells harboring BRAF mutations.
1.2 Biology of mutated BRAF and its role in
melanoma development and progressionBRAF is a member of the RAF family of serine threonine kin-ases (ARAF, BRAF and CRAF), which are part of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK)signaling pathway [14]. This signaling cascade promotes prolif-eration, survival and invasion by linking cell surface growthfactor receptors to the transcription of genes involved in cellcycle progression and antiapoptotic activity [15]. Activationof MAPK signaling pathway by oncogenic mutations hasbeen found in up to 90% of melanoma cases [10]. The mostcommon oncogene to be mutated in the MAPK signalingpathway in melanoma is BRAF. Approximately 50% of
all melanoma tumors harbor activating mutations inBRAF [10,16]. Although > 30 mutations in the BRAF genehave been identified in solid tumors [17], in melanoma> 80% of them result in the substitution of valine to glutamicacid at amino acid residue 600 (BRAFV600E mutation) [10,16].This mutation, as well as alternative point mutations at thesame position (V600D, V600K, V600R), lead to constitutiveactivation of BRAF and the down-stream MAPK signalingpathway [17]. This constitutive activation results in increasedcellular proliferation and oncogenic activity (Table 1) [10,18-34].
2. Medical need
2.1 BRAF-I therapy in melanomaAs mentioned earlier, melanoma incidence is increasing. Longterm survival in patients with metastatic melanoma is < 10%and their median OS is < 1 year. These findings emphasize theneed to develop novel, effective and more individualized treat-ment regimens for metastatic melanoma patients.
BRAF is a prime therapeutic target due to its oncogenicpotential and the relatively high incidence of mutations. Sor-afenib which targets BRAF and inhibits melanoma growthin vitro was the first drug to be investigated for its therapeuticeffects. It is a small-molecule, multikinase inhibitor, nonselec-tive RAF inhibitor (CRAF more than BRAF-I) which abro-gates MAPK signaling pathway [35]. However, early clinicaltrials failed to demonstrate any activity of sorafenib whenused as monotherapy or in combination with dacarbazine inpatients with wild type and mutant BRAF metastatic mela-noma [36]. The lack of sorafenib activity reflects its inabilityto selectively target mutant BRAF; as a result, intolerableoff-target side-effects are caused. The newest generation ofBRAF-I is markedly more effective than sorafenib in inhibit-ing mutant BRAF and causes much less off-target side effects.Preclinically, AZ628, XL281, GDC-0879, SB590885, dabra-fenib (GSK2118436) and vemurafenib (PLX4032 and itsanalog PLX4720) have been evaluated. Of these, vemurafeniband dabrafenib have been clinically validated [11-13].
Vemurafenib is an orally available ATP-competitive RAFinhibitor which markedly inhibits BRAFV600E (BRAF IC50,31 nM; CRAF IC50, 48 nM; WT BRAF IC50, 100 nM).Although in vitro vemurafenib inhibits a broad range ofkinases at the pharmacologically doses used, in vivo it hasonly a minimal effect on the vast majority of kinases(IC50 values > 10 mM) [37]. Vemurafenib inhibits growth ofmelanoma cells harboring BRAFV600E mutations by blockingthe activation of the MAPK signaling pathway, arresting cellsin the G1-phase of the cell cycle and inducing apoptosis byup-regulating BIM [37-40]. Vemurafenib also markedly inhibitsthe in vitro proliferation of melanoma cell lines which expressother mutations at the V600 position, such as BRAFV600D,BRAFV600R and BRAFV600K [39,41]. These effects are associ-ated with regression of established human BRAFV600E mutantmelanoma xenografts in immunodeficient mice [39]. In aPhase I study, vemurafenib demonstrated substantial tumor
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regression in 81% of patients with metastatic melanoma whohad a BRAFV600E mutation. Responses were associated withinhibition of phospho-ERK, reduction of Ki-67 positivity,shown by immunohistochemical staining, and inhibition ofglucose uptake measured by FDG-PET [42]. One and 2-yearsurvival were 50 and 38%, respectively, with a median OSof 13.8 months. The follow up Phase II study of previouslytreated patients demonstrated a response rate (RR) of 53%with a 6.8 month median duration of response [13]. Finally,a Phase III randomized control trial of previously untreatedBRAFV600E melanoma patients compared vemurafenib todacarbazine demonstrating improvements in RR (48 vs 5%),progression free survival (PFS) (5.3 vs 1.6 months) and per-cent of patients alive at 6.0 months (84 vs 64%) with a75% reduction in risk of death [11]. Based on the Phase IIand III trial data, vemurafenib was approved in late 2011 bythe FDA for unresectable metastatic BRAFmutant melanoma.
The Phase I study of dabrafenib, another selective inhibitorof BRAFV600E, demonstrated comparable results to thoseobtained with vemurafenib [43]. The Phase II study, designedto assess the efficacy of dabrafenib in patients withBRAFV600E/V600K mutation-positive metastatic melanoma,also demonstrated an improved RR (60%) and PFS(6.7 months) as compared to dacarbazine. Finally, a Phase IIIrandomized control trial of previously untreated BRAFV600E/V600K melanoma patients compared dabrafenib to dacarbazinedemonstrating improvements in RR (50 vs 6%) and PFS(5.1 vs 2.7 months), but OS data are not yet mature [12]. Basedon these data, dabrafenib has been recently approved by theFDA for unresectable metastatic BRAF mutant melanoma.
Both BRAF-I vemurafenib and dabrafenib have shownactivity in BRAFV600K mutant melanomas; while vemurafenibis not currently approved for patients with BRAFV600K muta-tion, additional studies are examining their efficacy in patientswith mutations other than BRAFV600E/K. Both BRAF-I havebeen tested in patients with brain metastasis with apparentactivity in the brain, although the number of patients treated
with vemurafenib is too small to draw definitive conclusionsas compared to the number of patients treated withdabrafenib [44].
3. Existing treatments
3.1 ChemotherapyChemotherapy is an accepted palliative therapy for stage IVmetastatic disease. Dacarbazine has been the most widelyused single chemotherapeutic agent for the treatment of met-astatic melanoma. Further, to date, for patients not carryingmutant BRAF oncogene or patients who are not eligible forinvestigational trials, dacarbazine still remains a reasonablepalliative option [45]. Treatment with dacarbazine originallyreported RR in up to 25% of patients in Phase I -- IItrials [7,46-48], but current trials have shown RR of 5 -- 6%[11,12]. Unfortunately, most responses to dacarbazine are tran-sient; in fact, only 1% of patients achieve a durable long-term response. Temozolomide (TMZ), an oral prodrug withthe same active intermediate (3-methyl-[triazen-1-yl]imidaz-ole-4-carboxamide) as dacarbazine, has been shown to be aseffective as dacarbazine in Phase III studies [49]. Fotemustine,a chloroethyl nitrosourea, has also been shown to have an effi-cacy comparable to that of dacarbazine [50,51]. Polychemother-apy regimens such as the Dartmouth regimen, whichcombines cisplatin, vinblastine, dacarbazine and tamoxifenor administration of carboplatin and taxol have been alsotested without an increase in OS as compared with dacarba-zine [52]. It is worth noting that dacarbazine has never beenshown to increase the OS as compared with the best support-ive care for metastatic melanoma patients.
3.2 ImmunotherapyThe potential involvement of immunological events in thepathogenesis and clinical course of melanoma has stimulatedinterest in the development and application of immunothera-peutic strategies for the treatment of this disease. In the course
Table 1. Effects of MAPK activation by BRAFV600E in melanoma cells.
Alterations Molecular effect Tumor effect
"" cyclin Dl expression "" p27 degradation "" cyclin/CDK4 complex Promotion of melanoma cell progressionthrough the G1-S phase of the cell cycle
"" BIM, BMF, BAD degradation ""Bcl-2.Bcl-w, Bcl-XL and Mcl-1 expression
## apoptosis Increased melanoma cell survival
## cyclic GMP phosphodiesterasePDE5A activity"" intracellular levels of cGMP
""cytosolic calcium andphosphorylation of myosin lightchain 2 of myosin light chain 2
Increased invasive and motile behavior ofmelanoma cells:reorganization of cytoskeletonactivation of cells’ migratory machineryup-regulation of matrix metalloproteinaseexpression
## HLA Class I antigen expression## TA expression"" PD-L1 expression
## recognition of melanoma cellsby the immune cells## inhibition of cytotoxic T cellactivation
Decreased recognition of melanoma cells by thecellular arm of host’s immune system
HLA: Human leukocyte antigen; PD-L1: Programmed death ligand 1; TA: Tumor antigen.
Emerging BRAF inhibitors for melanoma
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of the years, multiple strategies have been developed with thegoal to induce or enhance a patient’s immune response againsthis own tumor or to administer to patients antibodies orT cells which could target melanoma tumors [53]. The firstimmunotherapeutic agent registered in 1998 for the treat-ment of metastatic melanoma was IL-2, a T-cell growth fac-tor, which plays a crucial role in immune regulation and inT-cell proliferation. Although effective in a small number ofpatients and achieving a long-term durable response, treat-ment with HD IL-2 has been limited by the low RR(6 -- 10%) and the associated grade 3 toxicity [8].Interferon (IFN)-a-2b is the only approved drug for the
adjuvant therapy of high-risk melanoma patients. The antitu-mor activity of IFNa-2b has been suggested to be mediatedby several mechanisms. They include antiproliferative, proa-poptotic activity and up-regulation of immunologically rele-vant molecules which mediate the interaction of melanomacells with immune cells. Administration of IFNa-2b eitheras high dose soluble IFNa-2b or as pegylated IFNa-2b hasshown clinical benefits in stage III melanoma patients as indi-cated by the increase of relapse-free survival (RFS) [54,55].However, despite its potent antitumor properties, the clinicalutility of IFNa-2b in cancer therapy has been severely limitedby the substantial toxicities associated with its systemicadministration and by the lack of significant increase in OSof treated melanoma patients, as shown by a recently pub-lished meta-analysis [56].Biochemotherapy regimens combining chemokines such as
IL-2 or IFN-a with or without chemotherapy have slightlyincreased RR. However, this change was associated with a sig-nificant increase of toxicity, without improving OS andPFS [57].Recognition of crucial immunoregulatory pathways that
regulate the immune response against tumors has led to thedevelopment of novel immunotherapeutic strategies for thetreatment of metastatic melanoma, including patients harbor-ing the BRAF and NRAS mutations. One of the recentlyproven approaches used to overcome cancer immune toler-ance has been the promotion of T-cell activation by blockinginhibitory signals arising from receptors such as the cytotoxicT lymphocyte-associated antigen-4 (CTLA-4) [58].CTLA-4 receptor represents a key molecule able to decrease
T-cell activation; its activation is physiologically required toinduce tolerance to self-antigens avoiding autoimmune reac-tions. It can be envisioned as a natural brake of the immunesystem. CTLA-4 receptor is upregulated on activated T-cells and competes with CD28 for binding to the ligand B7.The binding of CTLA-4 to B7 leads to the block of costimu-latory signals needed for T-cell activation [58]. Ipilimumab is afully human CTLA-4-specific monoclonal antibody whichblocks CTLA-4 receptor activity allowing CD28 to bind toB7. Two Phase III trials have proven ipilimumab efficacy inmetastatic melanoma patients [59,60]. Data analysis demon-strated a significant OS improvement in the ipilimumabarm as compared with the gp100 vaccine arm in the first trial
and with dacarbazine in the second trial (10 vs 6.4 months;11.2 vs 9.1 months). Based on these data, ipilimumab hasbeen recently approved by the FDA for the treatment of met-astatic melanoma patients. Although effective, treatment withipilimumab is limited by the frequent associated toxicitywhich in some patients may be severe.
4. Current research goals
4.1 Limitations of BRAF-I therapy in melanomaBRAF-I are effective in the treatment of BRAF mutant mela-noma patients. However, like many other oncogene directedtherapies, such as imatinib in myeloid cell leukemia (MCL)and gastrointestinal stromal tumors (GISTs) or erlotinib innon-small-cell lung cancer (NSCLC), treatment with BRAF-I is effective only for a limited time and complete clinicalresponses are rarely seen (only 5%) due to the onset ofresistance. Resistance may be caused by clonal selection ofpre-existing cells which carry many activating mutations inoncogenes and/or inactivating mutations in oncosuppressorgenes (tumor heterogeneity). These alterations may lead toactivation of alternative pathway(s) which may induce tumorprogression and drug resistance. Resistance may be causedeither by the presence of mutation(s) in melanoma cells beforestarting the treatment (intrinsic resistance), or by the induc-tion of novel mutation(s) in the melanoma cells exposed tothe drug (acquired resistance). Whether intrinsic or acquired,the resistance of melanoma cells to BRAF-I leads to over-growth of BRAF-I-resistant melanoma cells at expenses ofBRAF-I sensitive melanoma cells in patients with BRAF-mutant melanoma tumors, when they are treated withBRAF-I. As a result, melanoma lesions are populated withBRAF-I resistant melanoma cells.
4.2 Intrinsic resistance of BRAFV600E-mutated
melanoma cells to BRAF-I therapyIntrinsic resistance to BRAF-I has been demonstrated in pre-clinical studies. A significant proportion of BRAFV600E-mutated melanoma cell lines displays intrinsic drug resistancewith different sensitivity to the growth inhibitory effectsmediated by BRAF-I [61]. It is known that melanomas carrya mutational profile and harbor concurrent alterations inmany genes including MITF, AKT3, COT, cyclin D1, cyclindependent kinase (CDK)2, CDK4 and the retinoblastoma(Rb) protein. For many of these alterations, it is unclearhow they modulate the biological behavior of melanoma cellswith BRAF mutations and their response to BRAF-I.
One mechanism of decreased BRAF-I sensitivity is repre-sented by amplification or overexpression of cyclin D1 [62].Cyclins are positive regulators of CDK, proteins involved incell cycle progression. Negative regulators of cyclin/CDK arerepresented by serine/threonine kinases such as p21 andp16. Several cyclins exhibit distinct expression and degrada-tion patterns which contribute to the temporal coordinationof each mitotic event. Specifically, cyclin D1 is required for
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cell cycle G1/S transition. Mutations, amplifications andover-expression of cyclin D1 in combination with BRAFmutations have been described in clinical samples and in mel-anoma cell lines. In BRAF-mutant melanomas, inhibition ofMAPK signaling by BRAF-I leads to the inhibition of cyclinD1 expression and a G1-phase cell cycle arrest. However,cyclin D1 overexpression causes intrinsic BRAF-I resistanceby facilitating cell cycle entry even when BRAF is inhibited.Abnormalities in the Cyclin/CDK complex are also causedby aberrations in CDK4 or in its negative regulators such asp16 or p53 [63], as well as in Rb [64].
Another mechanism involved in intrinsic BRAF-I resistance, which has been clinically validated in > 10% ofBRAF mutant melanoma patients, is represented by altera-tions in Phosphatase and tensin homolog (PTEN) [64,65].PTEN is a tumor suppressor gene which acts through itsphosphatase protein product resulting in cell cycle regulationand survival. Specifically, PTEN negatively modulates intra-cellular levels of phosphatidylinositol-3,4,5-trisphosphateand functions as a tumor suppressor by negatively regulatingPI3K/AKT signaling pathway. The latter pathway plays amajor role in cell survival. Inactivating mutations in thePTEN gene mediate an intrinsic BRAF-I resistance by anincreased activation of PI3K/AKT pathways which leads toan increased survival of melanoma cells. The increased sur-vival is caused by the suppression of proapoptotic proteinssuch as BAD, Bax and BIM and by the nuclear export of tran-scription factors such as FOXO3a which regulate severaloncosuppressor genes [65]. A similar mechanism causingBRAF-I resistance has also been shown to be mediated bythe insulin growth factor-1 receptor (IGF-1R) activation [66].
Lastly, in BRAFV600E melanoma an intrinsic resistance toBRAF-I is mediated by alterations of their interactions withtumor microenvironment [67]. By definition, tumor microen-vironment or ‘tumor stroma’ includes all those componentsthat are not cancer cells such as endothelial cells, fibroblastsand infiltrating leukocytes, as well as extracellular matrix pro-teins in the cancer microenvironment. It has been demon-strated that BRAF-I resistance may be caused by fibroblastproduction of growth factors such as the hepatocyte growthfactor (HGF) [67]. Secretion of HGF by stromal cells activatesits receptor c-Met, and reactivates the MAPK and PI3K/AKTsignaling pathways causing intrinsic BRAF-I resistance.
4.3 Acquired resistance of melanoma cells to
BRAF-I therapyUnlike the resistance to targeted therapies described in othermalignancies [68], no acquisition of secondary mutations inthe BRAF oncogene has been identified in large-scale sequencing analyses of melanoma specimens. Most ofthe mechanisms of acquired BRAF-I resistance have been iden-tified by generating in vitro resistance by continuous exposureof BRAFV600E melanoma cell lines to BRAF-I. BRAF-Iresistance can be mediated by reactivation of MAPK pathway
(MAPK-dependent) or activation of alternative signaling path-ways such as PI3K/AKT pathway (MAPK-independent) [66].
ERK reactivation can be caused by activation of compo-nents up-stream or down-stream of BRAF in the MAPK path-way; as a result, BRAF inhibition is bypassed and the ability ofBRAF-I to inhibit BRAFV600E is lost. Thus ERK reactivationby up-stream components of BRAFV600E is mediated by acti-vation of CRAF or ARAF, which bypasses the BRAF inhibi-tion (paradoxical MAPK activation, as discussed later).ARAF to CRAF activation can be caused by up-stream activa-tion of receptor tyrosine kinase (RTK) such as IGF-1R orplatelet-derived growth factor (PDGFR)b [66,69], NRASmutation [69] or amplification of ARAF or CRAF [70]. Onthe other hand, ERK reactivation because of the inabilityof BRAF-I to inhibit BRAFV600E can be mediated by trun-cated BRAFV600E spliced isoforms [71] or BRAFV600E amplifi-cation [72]. In both cases, although BRAF maintains theV600E mutation, it loses the binding site for BRAF-I or itsbinding to BRAF-I is not sufficient to inhibit BRAF dimeriza-tion which in turn leads to ERK reactivation.
Alterations in BRAF down-stream components lead toERK reactivation by bypassing BRAF dependent MEK orby directly affecting ERK activation. They include increasedexpression of the MAP kinase family member COT [73] andMEK activating mutations [74].
Lastly, MAPK-independent BRAF-I resistance can bedriven by activation of RTK. These alterations increase thesurvival and antiapoptotic signals by activating the PI3K/AKT-mammalian target of rapamycin (mTOR) pathway [75].
Therefore, many mechanisms are driving BRAF-I resistancein melanoma. However, the resistance mechanisms describedto date account for < 50% of patients whose disease relapseswhile being treated with a BRAF-I. Further, many of thedescribed mechanisms of BRAF-I resistance need to be con-firmed in additional laboratories besides the ones which aredescribed and/or need to be validated in clinical samples.
5. Scientific rationale
5.1 Strategies to overcome BRAF-I resistance in
melanomaThe characterization of the molecular mechanisms underlyingresistance to BRAF-I of BRAF mutant melanoma has lead tothe rational design of targeted combinatorial therapies. Themajority of these combinations have been validated in vitro.However, only a minority of them has been validatedin vivo and/or in a clinical setting.
Although different mechanisms have been described forBRAF-I resistance, most of them lead to the reactivation ofthe MAPK pathway or activation of the PI3K/AKT/mTORpathway. Therefore, it is not surprising that simultaneous tar-geting of components of these two pathways can delay orovercome BRAF-I resistance.
In the context of MAPK pathway reactivation, the combina-tion of a BRAF-I and a MEK inhibitor (MEK-I) in vitro is
Emerging BRAF inhibitors for melanoma
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effective in delaying and overcoming BRAF-I resistance [37].Since MEK is a down-stream component of the MAPK path-way, its inhibition may lead to ERK inhibition in the presenceof RTK, NRAS, BRAF, CRAF, ARAF, COT and MEK alter-ation(s), which lead(s) to ERK reactivation. The combinationof BRAF-I and MEK-I (trametinib) has been shown to beeffective in vitro at abrogating the resistance mediated byMEK1 mutations, COT overexpression, BRAF truncation,and acquired RAS mutations [66,69,73,76,77]. The combinationof a BRAF-I and a MEK-I or administration of MEK-I alonein Phase I and II trials did not induce a clinical response inpatients whose disease progressed after BRAF-I treatment [78].However, a recent Phase II trial evaluating the combinationof BRAF-I and MEK-I in melanoma patients carryingBRAFV600E/V600K demonstrated a statistically (p < 0.001) sig-nificant increase in PFS for the combinatorial treatment com-pared to administration of a BRAF-I alone. The RR with theBRAF-I/MEK-I combination was 76%, as compared to 54%with BRAF-I monotherapy (p = 0.03) [79].In the case of resistance mediated by increased IGF-1R sig-
naling, it has been shown in vitro that dual MEK and PI3Kinhibition or BRAF-I and IGF-1R inhibition may be an effec-tive strategy to overcome BRAF-I resistance [66]. Interestingly,in vitro resistance mediated by increased PDGFRb signaling isnot overcome by the PDGFR inhibitors sunitinib and imati-nib, but rather by the combination of an mTOR/PI3K/AKTinhibitor and a MEK/BRAF inhibitor [75]. Clinical trials havebeen initiated to examine the PI3K/mTOR inhibitor BEZ235in combination with the MEK-I MEK162 (NCT01337765).This trial is currently enrolling patients with BRAF andNRAS mutations [80].An additional approach to overcome BRAF-I resistance is
the simultaneous administration of BRAF-I and an inhibitorof the heat shock protein 90 (HSP90) [81]. This combinationovercomes an acquired resistance to BRAF-I mediated byNRAS mutation, PDGFRb up-regulation and COT overex-pression. HSP90 is a cellular chaperone required for the refold-ing of denatured proteins, cellular survival under stressconditions and the maturation of a subset of proteins. Its inhi-bition induces instability and degradation of client proteinssuch as MEK, BRAF and AKT overcoming resistance toBRAF-I. However, this strategy is likely to suffer from theside effects generated by the lack of selectivity of theHSP90 inhibitor used. To overcome these limitations, we aretesting a BRAF-I in combination with the monoclonal anti-body W9 which recognizes an epitope of the glucose regulatedprotein 94, a member of the HSP90 family [82]. This epitope isselectively expressed on the membrane of malignant cells.An additional promising approach to overcome BRAF-I
resistance is represented by the combination of BRAF-I witha monoclonal antibody which recognizes the tumor antigen(TA) chondroitin sulphate proteoglycan (CSPG)4 [83]. Thiscombination is effective in inhibiting the proliferation ofmelanoma cells and inducing their apoptosis by blockingERK and AKT activation [84].
Lastly, it has been demonstrated in vivo that BRAF-I-resis-tant melanomas become drug dependent for their continuedproliferation, such that cessation of drug administration leadsto regression of established drug-resistant tumors. In this way,the discontinuous administration of BRAF-I overcomes anddelays the onset of drug resistance [85].
6. Competitive environment
Although the clinical activity of BRAF-I therapy represents amajor breakthrough in the treatment of metastatic melanomacarrying BRAFV600E, novel targeted agents which inhibitdownstream components of BRAF in the MAPK signalingpathway and novel immunotherapeutic strategies are emerg-ing. Established and proposed treatments are summarizedin Table 2.
6.1 MEK-IMEK is down-stream of BRAF in the MAPK signaling path-way. As described earlier, activation of the MAPK signalingpathway by oncogenic mutations has been found in up to90% of melanoma cases. The most common oncogene to bemutated in the MAPK signaling pathway in melanoma isBRAF. However, 30% of all melanoma activating MAPKpathway harbor mutations in NRAS [86]. Because mutationsin both NRAS and BRAF signal through MEK 1 and 2, inhi-bition of MEK has proven to be an attractive therapeuticstrategy for both melanoma carrying BRAF and NRAS acti-vating mutations. Trametinib is a potent, highly selectiveand reversible ATP-competitive inhibitor of MEK 1 and 2.Phase I trials have shown that trametinib is safe and tolerable,with common adverse events (AEs) including rash and diar-rhea [87,88]. Its clinical activity has been reported to be impres-sive in untreated melanoma patients harboring theBRAFV600E mutation, with an RR of 40% and a medianPFS of 5.7 months. These results have been further confirmedin a randomized Phase III study, in which metastatic mela-noma patients carrying BRAFV600E/V600K were randomizedto receive trametinib or chemotherapy. Median PFS in thetrametinib group was 4.8 months, compared to 1.5 monthsin the chemotherapy group [79]. Based on these positiveresults, trametinib has been recently approved by the FDAas first-line treatment for metastatic melanoma patients carry-ing the BRAF and NRAS mutation.
Selumetinib is another potent, highly selective and revers-ible ATP noncompetitive inhibitor of MEK 1 and 2 whichis undergoing clinical evaluation for the treatment of meta-static melanoma patients. A recent Phase II trial comparingselumetinib in combination with dacarbazine versus dacarba-zine alone in untreated metastatic melanoma patients carryingBRAF mutation did not demonstrate a significant increase inOS between the 2 groups (median 13.9 vs 10.5 months) [89].However, PFS was significantly improved in the selumetinibplus dacarbazine group versus the placebo plus dacarbazinegroup with a median of 5.6 versus 3.0 months, respectively.
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6.2 ImmunotherapyCurrently, three immunotherapeutic strategies have been usedfor the treatment of e melanoma. Taking advantage of well-characterized melanoma antigens, immunization protocolsare been tested. Several TA such as MAGE, MART-1,gp100 and CSPG4 are being used as vaccines. To enhancetheir immunogenicity, they are administered in combinationwith cytokines. A Phase III randomized control trial ofadvanced melanoma patients compared the administrationof HD IL-2 in combination with gp100 vaccine to the admin-istration of HD IL-2. This trial demonstrated an increasedRR (16 vs 6%) and PFS (17.8 vs 11.1 months) [90]. Althoughencouraging, these data need to be validated in patients carry-ing BRAF mutations.
An additional strategy relies on the administration oftumor-infiltrating lymphocytes (TILs) or T cells which canbe directed to tumor masses by transfection with a T-cellreceptor specific for a human leukocyte antigen (HLA)-mela-noma antigen-derived peptide complex [91] or with a chimericmelanoma antigen receptor [92]. Administration of adoptivecell therapy (ACT) with TIL has been shown to result in thedurable complete regression of bulky metastatic melanomain patients refractory to approved treatments. Among93 patients with refractory melanoma treated with TILselected for tumor recognition following lymphodepletion,objective responses were seen in 50 -- 70% of patients, includ-ing 15 patients who had complete responses [93]. Despite thesepromising clinical results, however, the clinical efficacy ofthese strategies needs to be validated by double blindedPhase III trials. Further, the extensive effort, cost and timerequired to generate individual TIL or specific T-cell cultureslimit their use to only a few institutions.
A recently developed strategy relies on the administrationof monoclonal antibody specific for the programmed death-1 (PD-1) receptor. PD-1 is a receptor belonging to theCD28 family. It plays a role in tumor-associated mechanisms
of immune-escape. PD-1 has two ligands: PD-L1 and PD-L2,which act as down-regulators of immune response. PD-L1 and PD-L2 are widely expressed on cell membrane of mac-rophages, B lymphocytes, T-resting lymphocytes, dendriticcells and cancer cells. Several lines of evidence demonstrate apredominant role of PD-1 in melanoma immune tolerance.In order to block the inhibitory PD-1/PD-L1 pathway, sev-eral anti-PD-1 and anti-PD-L1 monoclonal antibodies havebeen generated including nivolumab (MDX-1106), pidilizu-mab (CT-011) and lambrolizumab. Early Phase I -- II clinicaltrials with each of the antibodies have proven their safety,well-tolerated administration and limited toxicity. Clinicalbenefits have been observed in metastatic melanoma patients.Preliminary data showed antitumor activity with 37 -- 38%RR. Although the complete responses were only 6% and theoverall response was 17%, one of the most impressive resultshas been that all responses and stable disease (27%) werehighly durable [94-96]. Although remarkable, these results stillrequire a detailed characterization of the mechanism(s) under-lying the response and the lack of response described inpatients treated with this type of therapy. This informationwill not only contribute to the optimization of the therapywith anti-PD-1/PD-L1monoclonal antibodies but may alsolead to the identification of predictive biomarkers ofclinical response.
7. Potential development issues
7.1 Side effects of BRAF-I therapy in melanomaThe most frequent AEs with selective BRAF-I include arthral-gia, rash, nausea, photosensitivity, fatigue, pruritus and pal-mar--plantar dysesthesia [11]. The most important toxicityrelated to BRAF-I is accelerated growth of cutaneous squa-mous cell carcinomas (SCCs) and keratoacanthomas(KAs) [11]. These lesions, although rapidly growing, can bemanaged through surgical excision, without evidence of
Table 2. Established and proposed treatments for metastatic melanoma.
Compound Company Indication Stage of
development
Mechanism
of action
Vemurafenib Roche and Plexikon Metastatic melanomapatients harboring BRAF mutation
FDA approved BRAF inhibitor
Dabrafenib GlaxoSmithKline Metastatic melanomapatients harboring BRAF mutation
FDA approved BRAF inhibitor
Trametinib GlaxoSmithKline Metastatic melanomapatients harboring BRAF mutation
FDA approved MEK inhibitor
Selumetinib AstraZeneca Metastatic melanomapatients harboring BRAF mutation
Phase I -- II trials MEK inhibitor
Ipilimumab Bristol-Myers Squibb Metastatic melanoma FDA approved Anti-CTLA4Nivolumab Bristol-Myers Squibb Metastatic melanoma Phase I -- II trials Anti-PD-1Pidilizumab Israel-based Curetech Ltd Metastatic melanoma Phase I -- II trials Anti-PD-1Lambrolizumab Merck Metastatic melanoma Phase I -- II trials Anti-PD-1MDX-1105 Bristol-Myers Squibb Metastatic melanoma Phase I -- II trials Anti-PD-L1
CTLA4: Cytotoxic T-lymphocyte antigen 4; FDA: Food and Drug Administration; PD1: Programmed death 1; PD-L1: Programmed death ligand 1.
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recurrence. The development of skin lesions has been attrib-uted to a differential effect of BRAF-I on the MAPK signalingpathway in cells carrying BRAF wild type. This phenomenonhas been coined ‘paradoxical activation’ of MAPKsignaling [97-100]. Paradoxical activation of MAPK signalingarises after treatment with a selective BRAF-I in BRAF wild-type cell lines that either harbor an oncogenic RAS mutationor have up-stream constitutive RTK activity. BRAF-I havebeen found to stimulate MEK-ERK signaling via CRAF acti-vation in the presence of an up-stream activator (e.g., acti-vated RTK, RAS mutation) in melanoma and other celllines with only wild-type BRAF. HRAS mutations are com-monly found in SCC and in KAs. Analysis of a large cohortof KAs from melanoma patients treated with BRAF-I hasdemonstrated a significant enrichment in RAS mutations [101].The preexistence of keratinocytes harboring HRAS mutationsmight provide an optimal context for a growth stimulatoryeffect of a selective BRAF-I to produce the SCC with KA fea-tures that have been observed in clinical trials with selectiveBRAF-I. It is worth noting that in the Phase II trial testingthe combination of BRAF-I and MEK-I in metastatic mela-noma patients harboring BRAFV600E, the incidence of KAand SCC in the combination group as compared to theBRAF-I monotherapy group was significantly lower [79]. Pre-clinical findings showed that addition of a MEK-I may helpto inhibit the hyperproliferative effects of BRAF inhibitionin the skin. The addition of a MEK-I to BRAF-I blocks reac-tivation of ERK, reduces skin toxicity and decreased the inci-dence of SCCs where preexisting SCC lesions with RASmutations may signal through CRAF to promoteSCC formation.
8. Conclusions
The information we have summarized indicate that i) BRAF-Itherapy is an effective treatment in metastatic melanomapatients although its clinical activity is limited in time;ii) many molecular mechanisms underlie the resistance toBRAF-I of BRAFV600E melanoma cells; and iii) several strate-gies have been developed to overcome and/or delayBRAF-I resistance. However, many questions still remainunanswered. First, some of the described mechanisms ofBRAF-I resistance and strategies to overcome them remainto be validated. Therefore, they have to be corroborated ordisproven by additional information in a conclusive way. Sec-ond, some of the mechanisms of BRAF-I resistance stillremain to be identified. Their identification will not only con-tribute to a better characterization of the multiple pathwaysutilized by melanoma cells to develop BRAF-I resistance butmay also define molecular biomarkers to allow for betterselection of effective therapeutic strategies for melanomapatients. In this regard, suppression of TORC1 as measuredby decreased phosphorylation of ribosomal protein S6, adown-stream component of PI3K/AKT/mTOR pathway, inmelanoma cells harvested from patients treated with
BRAF-I in combination with MEK-I, has been reported tobe a useful marker to identify patients who are likely torespond to this type of combinatorial therapy [102]. Third,many of the proposed strategies need to be tested in a clinicalsetting to determine their efficacy and toxicity (i.e., mTOR-/PI3K-/AKT-inhibitor and MEK-/BRAF-I or HSP90 inhibi-tor and BRAF-I). Fourth, it remains to be determined whythe single-agent BRAF-I, vemurafenib and dabrafenib displaya lower therapeutic efficacy than the recently FDA approvedMEK-I trametinib in untreated metastatic melanoma patientscarrying the BRAFV600E mutation, although they target thesame signaling pathway. Further, whether the differentialeffect of inhibitors of different components of a signalingpathway is unique of the MAPK pathway or is common toother pathways is at present not known. Last, the develop-ment of resistance to BRAF-I therapy emphasizes the needto combine targeted therapies with strategies which rely oncompletely different mechanisms, such as immunotherapy.As shown in Table 1, oncogenic BRAF signaling contributesto escape of melanoma cells from host’s immune system; asdiscussed before, targeting BRAF mutations may increasethe immunogenicity of melanoma cells. This evidence pro-vides the rationale for combining BRAF-targeting agentswith immunotherapy for the treatment of melanoma. Thispossibility is supported by several lines of evidence. First,in vitro incubation of melanoma cell lines and fresh mela-noma digests with BRAF-I enhances their recognition by mel-anoma antigen-specific T cells [103]. This effect is likely to bemediated by the up-regulation of melanoma differentiationantigens [28]. This experimental evidence has prompted thetesting of BRAF-I in combination with the administrationof TIL or HLA class I antigen restricted, melanoma antigen-specific T cells. In addition the up-regulation of PD-1 onT cells as well as of PD-L1 on melanoma cells [104] byBRAF-I has provided a strong rationale to combine BRAF-Iwith the administration of anti-PD-1 or anti-PD-L1 antibod-ies. However, the appropriate timing, sequence, duration aswell as potential side effects of these combinatorial therapiesneed to be defined.
9. Expert opinion
For several years, treatment of metastatic melanoma has beenvery disappointing. This scenario has been dramaticallychanged by the discovery of BRAF mutation in about 50%of melanoma patients and the following development ofBRAF targeted agents. BRAF-I appears to be very effectivein the treatment of melanoma with mutated BRAF. However,like many other oncogene directed therapies, such as imatinibin MCL and GIST or erlotinib in NSCLC, treatment withBRAF-I is effective only for a limited time and complete clin-ical responses are rarely seen due to the onset of resistance.These findings emphasize the need to characterize the molec-ular mechanisms underlying the BRAF-I resistance since thisinformation will be crucial to design rational combinatorial
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strategies to counteract this drug resistance. So far, many arethe mechanisms described which drive the BRAF-Iresistance and several are the strategies proposed to overcomethe found BRAF-I resistance. Most of the combinatorial strat-egies proposed rely on the combinations of one or more tar-geted agents such as MEK-I, PI3K inhibitor, mTORinhibitor and RTK inhibitor. Some of these combinationshave been already tested in melanoma patients with positiveresults. The combination of BRAF-I and MEK-I has beenshown to be more effective than treatment with BRAF-I alone in metastatic BRAFV600E melanoma patients. How-ever as well as for BRAF-I alone, treatment with BRAF-Iand MEK-I combination did not completely eliminate mela-noma cells and disease progressed after a limited time. Severalreasons may account for this failure. First, one of the majorlimitations of the novel combinatorial strategies proposed toovercome a selective BRAF-I resistance is represented by thelack of selection of patients to be enrolled in these clinical tri-als, since predictive molecular biomarkers are not available.An informative example in this regard is represented by thecombination of BRAF-I and MEK-I. This combination isexpected to be much more effective in patients who reactivatethe MAPK pathway as a mechanism of BRAF-I resistancethan in those who activate alternative pathway(s) such as thePI3K/AKT. Therefore, the preventive selection of BRAF-mutant patients carrying alterations which drive the MAPKreactivation might increase the efficacy of this treatment [102].Second, almost all the combinatorial strategies proposed toovercome the BRAF-I resistance rely on similar mechanismsof tumor cell killing through inhibition of cell proliferationand survival as well as induction of apoptosis. Melanoma aswell as many other solid tumors carries a genetic profilewith multiple alterations in several pathways. These altera-tions not only drive proliferation/survival and inhibit apopto-sis but also augment migration, invasion, metastatic potentialand neoangiogenesis and most importantly activate mecha-nisms of immune escape of cancer cells. Therefore, targetingmelanoma cells through multiple mechanisms is expected tobe much more effective than the combination of agents which
target only one or two aberrantly activated pathways. Last,although this is not the case of BRAF-I and MEK-Icombination which decreases BRAF-I toxicity, most of theproposed combinatorial strategies such as the combinationsof BRAF-I with an AKT inhibitor or an mTOR inhibitorare expected to be considerably toxic. This side effect is dueto the lack of a selective effect on melanoma cells of theused small molecules.
Promising strategies that overcome the BRAF-I resistanceand potentially completely eliminate melanoma cells rely onthe administration of drugs with different antitumor mecha-nisms. Available evidence such as the increase in HLA class Iantigens by BRAF-I or the up-regulation of PD-L1 on mela-noma cell surface after treatment with BRAF-I provides a con-vincing rationale to combine BRAF-I with chemokines(IFNa or IL-2), with monoclonal antibodies (anti-PD-L1,anti-PD-1, anti-CSPG4) or melanoma antigen-specificT cells. Up to now, although in a limited number of patients,immune strategies augmenting the recognition of cancer cellsby immune cells represent the only approach which provides along-term response. Therefore, the combination of targetedagents such as BRAF-I and immunotherapy is expected tobe most effective since decreasing the number of cancer cellswith BRAF-I is likely to reduce the rate of tumor immuneescape. This approach as well as that of the targeted agentcombinations will also greatly benefit from the biomarkerswhich facilitate the identification of patients most likely todevelop not only BRAF-I resistance but also mechanisms oftumor immune escape. Lastly, if one believes the role of can-cer initiating cells in disease recurrence and in metastaticspread it will be crucial to prove that the combinatorial strat-egies used eradicate melanoma initiating cells.
Declaration of interest
This work was supported by PHS grants RO1CA138188,RO1CA110249 and P50 CA121973 awarded by theNational Cancer Institute. The authors state no conflictof interest.
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AffiliationFrancesco Sabbatino1,2 MD PhD,
Yangyang Wang1,2 MD,
Xinhui Wang1,2 MD PhD,
Soldano Ferrone†1,2 MD PhD &
Cristina R Ferrone1,3 MD†Author for correspondence1Massachusetts General Hospital,
Harvard Medical School,
Department of Surgery,
55 Fruit Street, Boston, MA 02114, USA
Tel: +1 617 726 6087;
E-mail: [email protected] General Hospital,
Harvard Medical School,
Division of Surgical Oncology,
55 Fruit Street, Boston, MA 02114, USA3Massachusetts General Hospital,
Harvard Medical School,
Division of General Surgery,
55 Fruit Street, Boston, MA 02114, USA
Emerging BRAF inhibitors for melanoma
Expert Opin. Emerging Drugs (2013) 18(4) 443
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