Bengt Phung

MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITFlow/c-Junhigh syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITFlow/c-Junhigh melanomas to counteract their growth-promoting and immunosuppressive functions.

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

Background: Flt3 is an important regulator of hematopoiesis and is often found mutated and constitutively active in patients with acute myeloid leukemia. Results: SOCS6 is up-regulated by Flt3 activation and binds to phosphorylated Flt3. Conclusion: SOCS6 is a negative regulator of Flt3 signaling. Significance: Our results provide a role for SOCS6 in Flt3 signaling. The absence of SOCS6 promotes transformation of cells by Flt3 ITD. The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.

C-KIT Signaling Depends on Microphthalmia-Associated Transcription Factor for Effects on Cell Proliferation.

The development of melanocytes is regulated by the tyrosine kinase receptor c-KIT and the basic-helix-loop-helix-leucine zipper transcription factor Mitf. These essential melanocyte survival regulators are also well known oncogenic factors in malignant melanoma. Despite their importance, not much is known about the regulatory mechanisms and signaling pathways involved. In this study, we therefore sought to identify the signaling pathways and mechanisms involved in c-KIT mediated regulation of Mitf. We report that c-KIT stimulation leads to the activation of Mitf specifically through the c-KIT phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (Src binding site). Our study not only confirms the involvement of Ras-Erk signaling pathway in the activation of Mitf, but also establishes that Src kinase binding to Y568 and Y570 of c-KIT is required. Using specific inhibitors we observe and verify that c-KIT induced activation of Mitf is dependent on PI3-, Akt-, Src-, p38- or Mek kinases. Moreover, the proliferative effect of c-KIT is dependent on Mitf in HEK293T cells. In contrast, c-KIT Y568F and Y721F mutants are less effective in driving cell proliferation, compared to wild type c-KIT. Our results reveal novel mechanisms by which c-KIT signaling regulates Mitf, with implications for understanding both melanocyte development and melanoma.

Genome-wide DNA methylation analysis in melanoma reveals the importance of CpG methylation in MITF regulation

The microphthalmia-associated transcription factor (MITF) is a key regulator of melanocyte development and a lineage-specific oncogene in melanoma; a highly lethal cancer known for its unpredictable clinical course. MITF is regulated by multiple intracellular signaling pathways, although the exact mechanisms that determine MITF expression and activity remain incompletely understood. In this study, we obtained genome-wide DNA methylation profiles from 50 stage IV melanomas, normal melanocytes, keratinocytes, and dermal fibroblasts and utilized The Cancer Genome Atlas data for experimental validation. By integrating DNA methylation and gene expression data, we found that hypermethylation of MITF and its co-regulated differentiation pathway genes corresponded to decreased gene expression levels. In cell lines with a hypermethylated MITF-pathway, overexpression of MITF did not alter the expression level or methylation status of the MITF pathway genes. In contrast, however, demethylation treatment of these cell lines induced MITF-pathway activity, confirming that gene regulation was controlled via methylation. The discovery that the activity of the master regulator of pigmentation, MITF, and its downstream targets may be regulated by hypermethylation has significant implications for understanding the development and evolvement of melanoma.

Differential activity of c-KIT splice forms is controlled by extracellular peptide insert length.

Understanding receptor activation is important for disease intervention. Activation of the receptor tyrosine kinase c-KIT is involved in numerous diseases including melanoma, mastocytosis, multiple myeloma and gastrointestinal stromal tumors. To better understand the regulation of activation, we studied the two c-KIT isoforms, c-KIT(-) and c-KIT(+), which differ by a tetrapeptide insert GNNK, located in the extracellular juxtamembrane domain of the c-KIT(+) isoform. This region is important for regulating receptor activation. Here we show that the consecutive elimination of one amino acid at a time from the GNNK tetrapeptide insert gradually increases receptor tyrosine phosphorylation, ubiquitination, internalization and downstream MAP kinase-ERK activation. Successively decreasing the insert length progressively improves cell survival during drug treatment. Our results indicate that the length of the tetrapeptide fine-tunes receptor activity, thus providing deeper insight into c-KIT activation.

Enhanced SOX10 and KIT expression in cutaneous melanoma.

The transcription factor SOX10 and the receptor tyrosine kinase KIT are well recognized for their importance in melanocyte development. SOX10 and KIT are also highly involved in the development of melanoma. Moreover, SOX10 has been shown to be a marker for melanocytic tumors, and its protein expression can be correlated with benign nevi progression to superficial spreading melanoma but not nodular melanoma [1]. However, at present, there are no studies investigating the mRNA level of SOX10 in melanoma progression. The cell surface receptor KIT is involved in the development of melanoma and suggested to be an important oncogene in specific subtypes of the disease. Accordingly, KIT gene amplification and mutations have been found in acral melanoma, mucosal melanoma and chronic sun damaged melanoma but more importantly very rarely in cutaneous melanoma [2, 3]. The gene expression data generated by Curtin et al. and Beadling et al. are the key predicting that oncogenic KIT activity is pivotal in certain types of melanoma. However, Beadling et al. [2] in contrast to Curtin et al. [3] were not able to correlate elevated levels of gene expression to enhanced protein staining. Nevertheless, these are important findings establishing KIT’s role in non-cutaneous melanoma. We want to further clarify the importance of KIT in cutaneous melanoma because a recent study illustrated that KIT protein increased with progression from nevi to primary cutaneous melanoma and was decreased in metastasis [4]. Data from the two largest sample data sets (accession GSE3189 and GSE8401) from the Gene Expression Omnibus (GEO) were analyzed with respect to SOX10, KIT and KIT ligand (KITL) gene expression (Fig. 1). We extracted data from these studies because they are based on clinical material and because of the large sample sizes. The first set of data (Fig. 1a–c; accession GSE3189) consists in total of 70 clinical samples and was divided into normal skin, benign nevi and primary cutaneous melanoma. We only compared the expression level between the benign nevi and primary melanoma samples due to the comparable content of melanocytes. In the second dataset (Fig. 1d–f; accession GSE8401) 83 clinical samples were analyzed and divided into primary melanoma and metastatic melanoma. However, the subtype of primary melanoma was not disclosed by the original study (accession GSE8401). SOX10 and KIT gene expression were significantly increased in the primary cutaneous melanomas when compared to benign nevi (Fig. 1a, b). In contrast, the level of KITL was not statistically different between benign nevi and primary melanoma. Further analysis of these genes in the primary and metastatic material revealed that SOX10 expression is similar in that it was increased in the metastatic melanoma and KITL was unchanged (Fig. 1d, f). However, the expression of KIT is significantly reduced in the primary melanoma group compared to the metastatic group (Fig. 1e). To summarize, our results show the following: (1) SOX10 steadily increases during melanoma development and progression, (2) KIT expression is upregulated in cutaneous melanoma compared to benign nevi, (3) KIT expression is reduced in metastatic melanoma and (4) KITL expression does not change in melanoma progression. Our gene expression analysis of KIT correlates very well with the Nazarian et al. [4] study using immunohistochemical detection of KIT protein. Collectively, these results show that KIT’s role in cutaneous melanoma should L. Ronnstrand B. Phung (&) Wallenberg Laboratory, Skane University Hospital, Lund University, Inga Marie Nilssons gata 53, 205 02 Malmo, Sweden e-mail: bengt.phung@med.lu.se

KIT D816V induces SRC-mediated tyrosine phosphorylation of MITF and altered transcription program in melanoma

The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KITD816V has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KITD816V induces tyrosine phosphorylation of MITF through a triple protein complex formation between KIT, MITF, and SRC family kinases. In turn, phosphorylated MITF activates target genes that are involved in melanoma proliferation, cell-cycle progression, suppression of senescence, survival, and invasion. By blocking the triple protein complex formation, thus preventing MITF phosphorylation, the cells became hypersensitive to SRC inhibitors. We have therefore delineated a mechanism behind the oncogenic effects of KITD816V in melanoma and provided a rationale for the heightened SRC inhibitor sensitivity in KITD816V transformed cells. Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KITD816V, can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265–74. ©2017 AACR.

Abstract 5245: C-Kit isoforms predict melanoma drug sensitivity.

The receptor tyrosine kinase c-Kit is an essential regulator of melanocyte and melanoma development. Alternative splicing of c-Kit produces one long variant, c-Kit(+), with a tetrapeptide GNNK located in the extracellular juxtamembrane domain, and one short variant, c-Kit(-), which lacks this sequence. In this study we characterize for the first time the function of the GNNK tetrapeptide sequence in melanoma. Our results show that step by step elimination of the GNNK tetrapeptide gradually increases receptor tyrosine phosphorylation, ubiquitination and downstream MAP kinase ERK activation. Successively increasing insert length, progressively leads to reduced melanoma cell survival during dacarbazine or rapamycin drug treatment. Our results indicate that the ratio between c-Kit isoforms might be useful as a prognostic marker for melanoma drug response. Citation Format: Bengt Phung, Eirikur Steingrimsson, Lars Ronnstrand. C-Kit isoforms predict melanoma drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5245. doi:10.1158/1538-7445.AM2013-5245

Abstract 3093: Elucidation of c-KIT- dependent signaling to MITF

MITF, the master regulator of melanocytes, is a basic basic- helix- loop-helix- leucine zipper transcription factor that is essential for the development of melanocytes. MITF is also a lineage- survival oncogene in melanoma and recently, it has been shown that germline mutations in MITF predispose to this malignant disease. In melanoma, MITF has been proposed to act as a rheostat where high levels of MITF contribute to a proliferative phenotype whereas low levels result in a more invasive phenotype. The biological output generated by high or low levels of MITF is well characterized. However, how the activity of MITF is regulated to mediate these effects is less well known. It has been proposed that MITF is phosphorylated upon ligand induced activation of the receptor tyrosine kinase c- KIT. C-KIT engages the MAP kinase signaling cascade where ERK2 directly phosphorylates Ser73 of MITF and P90RSK phosphorylates the Ser409 residue of MITF. As a consequence, MITF activity increases and the protein is subjected to ubiquitin dependent degradation.Here, we show for the first time that c-KIT induces phosphorylation of MITF through the survival PI3 kinase- AKT pathway, the P38 stress pathway as well as the well-known MAP kinase-ERK signaling pathway. In addition, we show that the SRC kinase upstream of ERK is essential for c-KIT induced MITF activation. Moreover, we used several c-KIT phosphorylation site mutants to map the specific sites that are involved in the mediating transcription activation potential of MITF. These mutants also show less effects on MITF-mediated proliferation.We have generated phosphorylation specific antibodies against Ser73 and Ser409 of MITF and characterized the phosphorylation pattern of MITF using these reagents. Our results suggest that there is a particular relationship between Ser73 and Ser409 with subsequent effects on function. Based on our findings, we propose that phosphorylation of MITF might act as negative feedback mechanism that keeps MITF activity in check. Our model challenges the current knowledge of MITF modulation and provides insights that may have a profound impact on our understanding of the role of MITF in melanocyte and melanoma biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3093. doi:1538-7445.AM2012-3093

Abstract 3953: C-Kit and Mitf signaling: Novel involvement of the PI3K/Akt and p38 pathways

The development of melanocyte from melanoblast is regulated by the tyrosine kinase receptor c-Kit and the basic-helix-loop-heliz-leucine zipper transcription factor Mitf. It is established that Mitf activity is controlled by c-Kit activation and subsequent phosphorylation of the Erk/Mapk pathway. Serine phosphorylation (activation) of Mitf not only leads to upregulation of Mitf target gene expression but also ubiquitin dependent proteosomal degradation of Mitf. There is little knowledge surrounding the dysregulation between c-Kit and Mitf signaling that is often found in melanocyte progression to melanoma. The aim of our study is, therefore, to identify the signal transduction pathways involved in c-Kit mediated regulation of Mitf. In our study, we found c-Kit tyrosine phosphorylation sites and novel signaling pathways involved in the regulation of Mitf. Phosphorylation of c-Kit at Tyr 721 has been shown to activate the PI3K pathway and Tyr 568 and Tyr 568/570 are responsible for Src kinase mediated activation of Erk/Mapk pathway. We demonstrate that Y721F, Y568F and Y568/570F mutation of c-Kit prevented Mitf mobility shift, indicating that these tyrosine phosphorylation sites are involved in Mitf regulation. Further, we observe that treatment of c-Kit and Mitf transfected HEK293T cells with LY294002 (PI3K inhib.), Akt IV (Akt inhib.), SU6656 (Src kinase inhib.), SB203580 (p38 inhib.) or U0126 (Mek inhib.) prevented c-Kit mediated Mitf mobility shift. Similar results were also observed in the murine melanocyte, Melan- A cell line where Mitf and c-Kit are endogenously expressed. In order to further verify c-Kit9s role as Mitf regulator, we examined tyrosinase (downstream target of Mitf) promoter activity using a luciferase reporter construct. Ligand stimulation of c-Kit increased the Mitf target promoter activity. In conclusion, for the first time, we demonstrate that the signal transduction between c-Kit and Mitf is dependent on, 1) c-Kit Tyr 721 /PI3K/Akt pathway, 2) p38 pathway and 3) c-Kit Tyr 568 or Tyr 568/570 / Src kinase mediated Erk pathway. C-Kit activation leading to downstream were also shown to promote the transcriptional activity of Mitf. Our results reveal novel mechanisms by which c-Kit signaling regulates Mitf, with implication for understanding of both melanocyte development and melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3953.