NEWS AND VIEWS
Pigment Cell Melanoma Res.
The UPs and DOWNs of MITF in melanoma resistance Hyungsoo Kim and Ze’ev A. Ronai e-mail: [email protected]
The identification of mutations in the BRaf proto-oncogene, serine–threonine kinase (BRAF V600E or V600K) as the most common mutation in melanoma has propelled efforts to develop therapies to inhibit BRAF kinase activity. While currently available BRAF inhibitors (BRAFi) offer unprecedented improvement of patient survival and quality of life, those changes are generally transient due to the high occurrence of BRAFi-resistant tumors The quest to understand mechanisms underlying BRAFi resistance in melanoma has prompted a large campaign driven by both clinical and research communities, which has led to identification of a common denominator underlying resistance, namely, reactivation of mitogen-activated protein kinase (MAPK) signaling by diverse mechanisms, including mutations in NRAS or MEK, BRAF splicing anomalies, or overexpression of tyrosine kinase receptors, including PDGFR or EGFR. These findings have led to clinical trials employing combinations of MEK inhibitors (MEKi) and BRAFi; these approaches represent an improvement over monotherapy but do not significantly sustain survival, as resistant tumors eventually arise (Flaherty et al., 2012; Morris et al., 2013). Thus, identification of additional mechanisms underlying resistance to MAPK pathway inhibitors is important to enable creation of more effective therapies. A recent paper from Dr. Peeper’s group reports a novel approach to identify mechanisms conferring resistance to MAPK inhibitors. In this study, insertional mutagenesis was employed to select for genes whose elevated expression conferred resistance to the
Coverage on: Muller et al., (2014) Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma, Nature Communications 5, 5712, doi: 10.1038/ncomms6712. Konieczkowski, et al. (2014). A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discovery 4, 816-27. doi: 10.1111/pcmr.12338
ERK inhibitor (ERKi; SCH772984). Among key hits identified in the screen was the master regulator of melanin biogenesis, microphthalmia-associated transcription factor (MITF). This finding is consistent with an earlier report identifying MITF overexpression as a mechanism underlying resistance to MAPK pathway inhibition (Johannessen et al., 2013). Given the link between MITF overexpression and MAPK resistance, Peeper and his colleagues next assessed MITF function under acquired resistance conditions in cultured melanoma cells subjected to BRAFi. Surprisingly, greater than half of the established BRAFi-resistant melanoma cell lines had lost MITF expression, in contrast to upregulated MITF expression seen in response to acute treatment with MAPKi seen in the remainder. The seemingly contradictory finding that both high and low expression of MITF (MITF hi or MITF lo, hereafter) are associated with resistance phenotypes was confirmed in a cohort of BRAFi-resistant tumor samples taken from patients, who were evaluated prior to and following treatment. Some samples exhibited increased MITF expression following treatment, while others showed MITF decreases. Furthermore, assessment of individual clones derived from one patient revealed that some clones exhibited MITF upregulation, while others showed downregulation (Muller et al., 2014). How can one explain such variation in MITF expression and how could this impact on resistance mechanisms? Among plausible answers is phenotypic switching, a dynamic change in a gene expression signature previously reported in melanoma and dependent on MITF expression. Interestingly, in melanoma cells, ‘MITF hi’ is associated with proliferation, whereas ‘MITF lo’ cells show increased invasion and chemoresistance (Hoek and Goding, 2010; Saez-Ayala et al., 2013). Correspondingly, as shown by Dr. Peeper’s laboratory, acquired BRAFi-resistant melanoma cells downregulating MITF expression exhibit increased invasiveness with corresponding increases in expression of markers of the epithelial-to-mesenchymal transition (EMT), in agreement with increased inva-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
siveness observed in melanoma cells resistant to MAPK inhibition and with chemoresistance exhibited by stem-like cells (Vultur et al., 2014). Interestingly, MITF lo cells also show resistance to a broader spectrum of MAPK inhibitors or combinations including those drugs, as reported recently by the Garraway laboratory (Konieczkowski, et al., 2014). Both the Pee‘combination per and the Garraway studies treatment with inhibitors of AXL note that genes whose expresand ERK sion increases in eliminated MITF lo cells are growth of associated with intrinsically cell survival, sugMAPKi-resistant gesting a mechaNRAS mutant nism underlying melanoma MAPKi resiscultures’ tance. Dr. Garraway’s group identified NF-kB as a key player promoting resistance in the MITF lo cells. Intriguingly, both studies also highlight the importance of elevated expression of receptor tyrosine kinases (RTKs) including AXL, EGFR, and PDGFR in MITF lo, in innate and acquired BRAFiresistant cells, and both note AXL in particular as a key RTK that confers resistance. It is noteworthy that AXL, as well as other RTKs, was upregulated in melanomas that exhibit either intrinsic or acquired resistance phenotypes, noting their possible negative regulation by MITF. The role of RTKs in resistance phenotypes was confirmed by eradication of cells from intrinsically resistant cultures following treatment with a combination of drugs inhibiting AXL, PDGFR, and EGFR (Muller et al., 2014). Both studies also found that in acquired resistance, AXL inhibition combined with BRAFi treatment more effectively impaired viability of BRAFi-resistant cultures than did treatment with BRAFi alone. Importantly, an inverse correlation between MITF and AXL expression was also seen in NRAS mutant melanomas (Muller et al., 2014). Correspondingly, as shown by the Peeper laboratory, combination treatment with inhibitors of AXL and ERK eliminated growth of intrinsically MAPKi-resistant NRAS mutant melanoma cultures.
1
News and Views Of further importance is the plasticity reported in the Peeper’s study, as reflected by different expression levels of MITF in clones from a single resistant tumor from one patient. Understanding what determines MITF levels in these clones is a cardinal question requiring further analysis. A possible role for SOX10, a key regulator of MITF expression, was assessed, yet the Peeper group was unable to establish a strong correlation between SOX10 and MITF low or high status in resistant clones. Dynamic epigenetic changes through altered DNA methylation also occur in both pluripotent and somatic cells and could underlie variability in MITF expression. The dramatic appearance of resistant melanoma in patients is often seen as a mirror image of the original tumor burden, suggesting that most tumors can switch phenotypes as part of their resistance mechanisms, (i.e., switch to MITF lo to enable resistance and back to MITF hi to promote proliferation). The new studies by both Peeper and Garraway groups provide important clues to guide further analysis of mechanisms
2
underlying this plasticity and its significance for melanoma resistance. The rheostat model established by the Goding group (Hoek and Goding, 2010) posited that understanding how its levels are finely tuned is key to understanding MITF’s function in melanoma cells, and that both low and high MITF can elicit similar phenotypes through diverse mechanisms. The latter may also provide an explanation for the nature of the high and low expression of MITF for melanoma resistance mechanisms. Yet, one cannot exclude the possibility that factors in addition to MITF play a causative role in driving melanoma resistance; thus, keeping an open mind toward the idea that also MITF-independent mechanisms may underlie plasticity and resistance phenotypes is warranted. The ups and downs of MITF clearly reflect the high plasticity nature of melanoma.
References Flaherty, K.T., Infante, J.R., Daud, A. et al. (2012). Combined BRAF and MEK
inhibition in melanoma with BRAF V600 mutations. N. Engl. J. Med. 367, 1694–1703. Hoek, K.S., and Goding, C.R. (2010). Cancer stem cells versus phenotypeswitching in melanoma. Pigment Cell Melanoma Res. 23, 746–759. Johannessen, C.M., Johnson, L.A., Piccioni, F. et al. (2013). A melanocyte lineage program confers resistance to MAP kinase pathway inhibition. Nature 504, 138–142. Morris, E.J., Jha, S., Restaino, C.R. et al. (2013). Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors. Cancer Discov. 3, 742–750. Saez-Ayala, M., Montenegro, M.F., Sanchez-Del-Campo, L. et al. (2013). Directed phenotype switching as an effective antimelanoma strategy. Cancer Cell 24, 105–119. Vultur, A., Villanueva, J., Krepler, C. et al. (2014). MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines. Oncogene 33, 1850–1861.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pigment Cell Melanoma Res.
The UPs and DOWNs of MITF in melanoma resistance Hyungsoo Kim and Ze’ev A. Ronai e-mail: [email protected]
The identification of mutations in the BRaf proto-oncogene, serine–threonine kinase (BRAF V600E or V600K) as the most common mutation in melanoma has propelled efforts to develop therapies to inhibit BRAF kinase activity. While currently available BRAF inhibitors (BRAFi) offer unprecedented improvement of patient survival and quality of life, those changes are generally transient due to the high occurrence of BRAFi-resistant tumors The quest to understand mechanisms underlying BRAFi resistance in melanoma has prompted a large campaign driven by both clinical and research communities, which has led to identification of a common denominator underlying resistance, namely, reactivation of mitogen-activated protein kinase (MAPK) signaling by diverse mechanisms, including mutations in NRAS or MEK, BRAF splicing anomalies, or overexpression of tyrosine kinase receptors, including PDGFR or EGFR. These findings have led to clinical trials employing combinations of MEK inhibitors (MEKi) and BRAFi; these approaches represent an improvement over monotherapy but do not significantly sustain survival, as resistant tumors eventually arise (Flaherty et al., 2012; Morris et al., 2013). Thus, identification of additional mechanisms underlying resistance to MAPK pathway inhibitors is important to enable creation of more effective therapies. A recent paper from Dr. Peeper’s group reports a novel approach to identify mechanisms conferring resistance to MAPK inhibitors. In this study, insertional mutagenesis was employed to select for genes whose elevated expression conferred resistance to the
Coverage on: Muller et al., (2014) Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma, Nature Communications 5, 5712, doi: 10.1038/ncomms6712. Konieczkowski, et al. (2014). A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discovery 4, 816-27. doi: 10.1111/pcmr.12338
ERK inhibitor (ERKi; SCH772984). Among key hits identified in the screen was the master regulator of melanin biogenesis, microphthalmia-associated transcription factor (MITF). This finding is consistent with an earlier report identifying MITF overexpression as a mechanism underlying resistance to MAPK pathway inhibition (Johannessen et al., 2013). Given the link between MITF overexpression and MAPK resistance, Peeper and his colleagues next assessed MITF function under acquired resistance conditions in cultured melanoma cells subjected to BRAFi. Surprisingly, greater than half of the established BRAFi-resistant melanoma cell lines had lost MITF expression, in contrast to upregulated MITF expression seen in response to acute treatment with MAPKi seen in the remainder. The seemingly contradictory finding that both high and low expression of MITF (MITF hi or MITF lo, hereafter) are associated with resistance phenotypes was confirmed in a cohort of BRAFi-resistant tumor samples taken from patients, who were evaluated prior to and following treatment. Some samples exhibited increased MITF expression following treatment, while others showed MITF decreases. Furthermore, assessment of individual clones derived from one patient revealed that some clones exhibited MITF upregulation, while others showed downregulation (Muller et al., 2014). How can one explain such variation in MITF expression and how could this impact on resistance mechanisms? Among plausible answers is phenotypic switching, a dynamic change in a gene expression signature previously reported in melanoma and dependent on MITF expression. Interestingly, in melanoma cells, ‘MITF hi’ is associated with proliferation, whereas ‘MITF lo’ cells show increased invasion and chemoresistance (Hoek and Goding, 2010; Saez-Ayala et al., 2013). Correspondingly, as shown by Dr. Peeper’s laboratory, acquired BRAFi-resistant melanoma cells downregulating MITF expression exhibit increased invasiveness with corresponding increases in expression of markers of the epithelial-to-mesenchymal transition (EMT), in agreement with increased inva-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
siveness observed in melanoma cells resistant to MAPK inhibition and with chemoresistance exhibited by stem-like cells (Vultur et al., 2014). Interestingly, MITF lo cells also show resistance to a broader spectrum of MAPK inhibitors or combinations including those drugs, as reported recently by the Garraway laboratory (Konieczkowski, et al., 2014). Both the Pee‘combination per and the Garraway studies treatment with inhibitors of AXL note that genes whose expresand ERK sion increases in eliminated MITF lo cells are growth of associated with intrinsically cell survival, sugMAPKi-resistant gesting a mechaNRAS mutant nism underlying melanoma MAPKi resiscultures’ tance. Dr. Garraway’s group identified NF-kB as a key player promoting resistance in the MITF lo cells. Intriguingly, both studies also highlight the importance of elevated expression of receptor tyrosine kinases (RTKs) including AXL, EGFR, and PDGFR in MITF lo, in innate and acquired BRAFiresistant cells, and both note AXL in particular as a key RTK that confers resistance. It is noteworthy that AXL, as well as other RTKs, was upregulated in melanomas that exhibit either intrinsic or acquired resistance phenotypes, noting their possible negative regulation by MITF. The role of RTKs in resistance phenotypes was confirmed by eradication of cells from intrinsically resistant cultures following treatment with a combination of drugs inhibiting AXL, PDGFR, and EGFR (Muller et al., 2014). Both studies also found that in acquired resistance, AXL inhibition combined with BRAFi treatment more effectively impaired viability of BRAFi-resistant cultures than did treatment with BRAFi alone. Importantly, an inverse correlation between MITF and AXL expression was also seen in NRAS mutant melanomas (Muller et al., 2014). Correspondingly, as shown by the Peeper laboratory, combination treatment with inhibitors of AXL and ERK eliminated growth of intrinsically MAPKi-resistant NRAS mutant melanoma cultures.
1
News and Views Of further importance is the plasticity reported in the Peeper’s study, as reflected by different expression levels of MITF in clones from a single resistant tumor from one patient. Understanding what determines MITF levels in these clones is a cardinal question requiring further analysis. A possible role for SOX10, a key regulator of MITF expression, was assessed, yet the Peeper group was unable to establish a strong correlation between SOX10 and MITF low or high status in resistant clones. Dynamic epigenetic changes through altered DNA methylation also occur in both pluripotent and somatic cells and could underlie variability in MITF expression. The dramatic appearance of resistant melanoma in patients is often seen as a mirror image of the original tumor burden, suggesting that most tumors can switch phenotypes as part of their resistance mechanisms, (i.e., switch to MITF lo to enable resistance and back to MITF hi to promote proliferation). The new studies by both Peeper and Garraway groups provide important clues to guide further analysis of mechanisms
2
underlying this plasticity and its significance for melanoma resistance. The rheostat model established by the Goding group (Hoek and Goding, 2010) posited that understanding how its levels are finely tuned is key to understanding MITF’s function in melanoma cells, and that both low and high MITF can elicit similar phenotypes through diverse mechanisms. The latter may also provide an explanation for the nature of the high and low expression of MITF for melanoma resistance mechanisms. Yet, one cannot exclude the possibility that factors in addition to MITF play a causative role in driving melanoma resistance; thus, keeping an open mind toward the idea that also MITF-independent mechanisms may underlie plasticity and resistance phenotypes is warranted. The ups and downs of MITF clearly reflect the high plasticity nature of melanoma.
References Flaherty, K.T., Infante, J.R., Daud, A. et al. (2012). Combined BRAF and MEK
inhibition in melanoma with BRAF V600 mutations. N. Engl. J. Med. 367, 1694–1703. Hoek, K.S., and Goding, C.R. (2010). Cancer stem cells versus phenotypeswitching in melanoma. Pigment Cell Melanoma Res. 23, 746–759. Johannessen, C.M., Johnson, L.A., Piccioni, F. et al. (2013). A melanocyte lineage program confers resistance to MAP kinase pathway inhibition. Nature 504, 138–142. Morris, E.J., Jha, S., Restaino, C.R. et al. (2013). Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors. Cancer Discov. 3, 742–750. Saez-Ayala, M., Montenegro, M.F., Sanchez-Del-Campo, L. et al. (2013). Directed phenotype switching as an effective antimelanoma strategy. Cancer Cell 24, 105–119. Vultur, A., Villanueva, J., Krepler, C. et al. (2014). MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines. Oncogene 33, 1850–1861.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
