The identification of mutations in the B-Raf proto-oncogene, serine–threoninekinase (BRAF V600E or V600K) as themost common mutation in melanomahas propelled efforts to develop thera-pies to inhibit BRAF kinase activity.While currently available BRAF inhibi-tors (BRAFi) offer unprecedentedimprovement of patient survival andquality of life, those changes are gener-ally transient due to the high occurrenceof BRAFi-resistant tumorsThe quest to understand mechanismsunderlying BRAFi resistance in mela-noma has prompted a large campaigndriven by both clinical and researchcommunities, which has led to identifi-cation of a common denominator under-lying resistance, namely, reactivation ofmitogen-activated protein kinase(MAPK) signaling by diverse mecha-nisms, including mutations in NRAS orMEK, BRAF splicing anomalies, or over-expression of tyrosine kinase receptors,including PDGFR or EGFR. These find-ings have led to clinical trials employingcombinations of MEK inhibitors (MEKi)and BRAFi; these approaches representan improvement over monotherapy butdo not significantly sustain survival, asresistant tumors eventually arise (Flah-erty et al., 2012; Morris et al., 2013).Thus, identification of additional mecha-nisms underlying resistance to MAPKpathway inhibitors is important toenable creation of more effective thera-pies.A recent paper from Dr. Peeper’sgroup reports a novel approach to iden-tify mechanisms conferring resistanceto MAPK inhibitors. In this study, inser-tional mutagenesis was employed toselect for genes whose elevatedexpression conferred resistance to theERK inhibitor (ERKi; SCH772984).Among key hits identified in the screenwas the master regulator of melaninbiogenesis, microphthalmia-associatedtranscription factor (MITF). This findingis consistent with an earlier report iden-tifying MITF overexpression as a mech-anism underlying resistance to MAPKpathway inhibition (Johannessen et al.,2013).Given the link between MITF overex-pression and MAPK resistance, Peeperand his colleagues next assessed MITFfunction under acquired resistance con-ditions in cultured melanoma cells sub-jected to BRAFi. Surprisingly, greaterthan half of the established BRAFi-resis-tant melanoma cell lines had lost MITFexpression, in contrast to upregulatedMITF expression seen in response toacute treatment with MAPKi seen in theremainder. The seemingly contradictoryfinding that both high and low expressionof MITF (MITF hi or MITF lo, hereafter)are associated with resistance pheno-types was confirmed in a cohort ofBRAFi-resistant tumor samples takenfrom patients, who were evaluated priorto and following treatment. Some sam-ples exhibited increased MITF expres-sion following treatment, while othersshowed MITF decreases. Furthermore,assessment of individual clones derivedfrom one patient revealed that someclones exhibited MITF upregulation,while others showed downregulation(Muller et al., 2014).How can one explain such variation inMITF expression and how could thisimpact on resistance mechanisms?Among plausible answers is phenotypicswitching, a dynamic change in a geneexpression signature previously reportedin melanoma and dependent on MITFexpression. Interestingly, in melanomacells, ‘MITF hi’ is associated with prolif-eration, whereas ‘MITF lo’ cells showincreased invasion and chemoresistance(Hoek and Goding, 2010; Saez-Ayalaet al., 2013). Correspondingly, as shownby Dr. Peeper’s laboratory, acquiredBRAFi-resistant melanoma cells downre-gulating MITF expression exhibitincreased invasiveness with correspond-ing increases in expression of markers ofthe epithelial-to-mesenchymal transition(EMT), in agreement with increased inva-siveness observed in melanoma cellsresistant to MAPK inhibition and withchemoresistance exhibited by stem-likecells (Vultur et al., 2014). Interestingly,MITF lo cells also show resistance to abroader spectrum of MAPK inhibitors orcombinations including those drugs, asreported recently by the Garraway labo-ratory (Konieczkowski, et al., 2014).Both the Pee-per and the Garr-away studiesnote that geneswhose expres-sion increases inMITF lo cells areassociated withcell survival, sug-gesting a mecha-nism underlyingMAPKi resis-tance. Dr. Garr-away’s groupidentified NF-kB as a key player promot-ing resistance in the MITF lo cells.Intriguingly, both studies also highlightthe importance of elevated expressionof receptor tyrosine kinases (RTKs)including AXL, EGFR, and PDGFR inMITF lo, in innate and acquired BRAFi-resistant cells, and both note AXL inparticular as a key RTK that confersresistance. It is noteworthy that AXL,as well as other RTKs, was upregulatedin melanomas that exhibit either intrin-sic or acquired resistance phenotypes,noting their possible negative regulationby MITF. The role of RTKs in resistancephenotypes was confirmed by eradica-tion of cells from intrinsically resistantcultures following treatment with acombination of drugs inhibiting AXL,PDGFR, and EGFR (Muller et al., 2014).Both studies also found that in acquiredresistance, AXL inhibition combinedwith BRAFi treatment more effectivelyimpaired viability of BRAFi-resistant cul-tures than did treatment with BRAFialone. Importantly, an inverse correla-tion between MITF and AXL expressionwas also seen in NRAS mutant melano-mas (Muller et al., 2014). Correspond-ingly, as shown by the Peeperlaboratory, combination treatment withinhibitors of AXL and ERK eliminatedgrowth of intrinsically MAPKi-resistantNRAS mutant melanoma cultures.Coverage on: Muller et al., (2014) LowMITF/AXL ratio predicts early resistanceto multiple targeted drugs in melanoma,Nature Communications 5, 5712, doi:10.1038/ncomms6712.Konieczkowski, et al. (2014). A mela-noma cell state distinction influencessensitivity to MAPK pathway inhibitors.Cancer Discovery 4, 816-27.
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