Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA
Members of the melanoma team in SBP include Hyungsoo (RNF125), Marzia (Siah and PDK1) and Yongmei (Siah and SBI-756) and at the Technion lab, Ikrame Lazar (SPANX and ATE1) and Luba Turovsky (ATF2 splice variant)
THE TRANSCRIPTION FACTOR ATF2 IN MELANOMA
Our efforts to understand the role of a transcription factor (ATF2) in the development and progression of melanoma were driven by the observations that inhibition of ATF2 would block melanoma growth and metastasis. We demonstrated how peptides (10-15 aa long) effectively inhibited melanoma development, both in culture and in vivo mouse and human melanoma models.
Using genetic mouse models, we confirmed that ATF2 mediates an oncogenic role in melanoma. The Nras/Ink4a model in which ATF2 was inactivated in melanocytes revealed that melanoma development was largely lost. We found that as a nuclear protein ATF2 serves as a transcription factor, as well as a DNA damage response protein. Yet, in response to severe stress or damage, ATF2 translocates to the mitochondrial outer membrane domain, where it supports programmed cell death. Such nuclear export is lost in advanced melanomas due to upregulated expression/activity of PKC epsilon, which phosphorylates ATF2 and confers its nuclear localization. PKCɛ and ATF2-were found to facilitate resistance by transcriptionally repressing the expression of interferon-β1 (IFNβ1) and downstream type-I IFN signaling that is otherwise induced upon exposure to chemotherapy. Notably, nuclear ATF2 and low expression of IFNβ1 in melanoma tumor samples correlates with poor patient responsiveness to biochemotherapy or neoadjuvant IFN-α2a, whereas cytosolic ATF2 and induction of IFNβ1 coincides with therapeutic responsiveness.
ATF2 also silences expression of Fucose Kinase (FUK) in advanced melanomas. Suppression of FUK expression resulted in reduced adhesion and enhanced migration and metastatic capacity. Restoring expression of FUK in metastatic melanoma achieved genetically or by supplementing L-Fucose to the drinking water of animals with xenograft melanomas was effective in attenuating melanoma metastasis and reduced tumorigenesis in vivo.
ATF2 transcripts are subject to splicing, which results in the expression of number of isoforms. We identified a variant of ATF2 that lacks DNA binding capacity (hence transcriptionally inactive) elicits a gain of function phenotype (in both human splice and mouse variants). Significantly, the expression of such mutant ATF2 in Braf mutant melanocytes suffice to induce pigmentation and later melanoma development. Such tumors were developed over a period of ~200 days. The genetic support for the role of ATF2 variant as a super oncogene is being pursued by characterization of ATF2-associated proteins that uniquely bind to the ATF2 splice variant.
THE AGC KINASE PDK1 IN MELANOMA
Phosphoinositide-dependent kinase-1 (PDK1) is a serine threonine protein kinase that phosphorylates several members of the conserved AGC kinase superfamily and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In tumors, including melanoma, PDK1 is constitutively active due to its higher expression and autoactivation, as well as because of elevated PI3K activity. We demonstrated that as part of re-wired signaling pathways in melanoma, highly active ERK (due to NRAS and BRAF mutation) effectively increases the level and activity of c-Jun. Among c-Jun transcriptional targets is PDK1. Thus, in melanoma c-Jun drives effective upregulation of PDK1 expression and activity. This resulted in delayed melanoma development and significant inhibition of metastasis commonly seen in this model. Notably, we were able to phenocopy these observations using a pharmacological inhibitor developed against PDK1—GSK2234470. While these experiments were performed in Pten mutant animals, we confirmed that PDK1 inhibition would be as effective in Pten WT tumors, given that the key downstream kinases/substrates affected upon inhibition of PDK1, S6K, SGK and FOXO3a, were equally inhibited regardless Pten status.
Stratification of melanoma tumors for ATF2 and PDK1 based therapies.
In more recent studies, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1-phase, cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment.
THE UBIQUITIN LIGASES SIAH1/2 IN MELANOMA
Control of melanoma development and metastasis by the ubiquitin ligase Siah2
The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1alpha availability. Because HIF-1alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, these findings establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms.
THE UBIQUITIN LIGASE RNF125 IN MELANOMA
Downregulation of the ubiquitin ligase RNF125 underlies resistance of melanoma cells to BRAF inhibitors via JAK1 deregulation.
Search for drivers of melanoma resistance to BRAF inhibitors led us to identify downregulation of the ubiquitin ligase RNF5 in tumors of patients that exhibited resistance to vemurafenib. Downregulated expression of RNF125 was caused by low levels of SOX10 and MITF, which were identified as upstream regulator of RNF125 transcription. We identified JAK1 as RNF125 substrates in melanoma, and demonstrated the important role JAK1 plays in the resistance of melanoma to therapy. Upregulated JAK1 in BRAFi-resistant melanoma has been associated with upregulation of receptor tyrosine kinases (EGFR, AXL), which were previously linked with the resistant phenotype. Genetic or pharmacological inhibitors of JAK effectively attenuated the resistance and restored effectiveness of BRAFi. Current assessment of specific JAK1 inhibitors is underway. These studies are currently extended by Hyungsoo Kim in melanoma using genetic models.
TARGETING TRANSLATIONAL INITIATION COMPLEX IN MELANOMA
SBI-0640756 attenuates the growth of clinically unresponsive melanomas by disrupting the eIF4F translation initiation complex.
We identified and characterized the small molecule SBI-0640756 (SBI-756), a first-in-class inhibitor that targets eIF4G1 and disrupts the eIF4F complex. SBI-756 impaired the eIF4F complex assembly independently of mTOR and attenuated growth of BRAF-resistant and BRAF-independent melanomas. In the gene expression signature patterns elicited by SBI-756, DNA damage, and cell-cycle regulatory factors were prominent, with mutations in melanoma cells affecting these pathways conferring drug resistance. SBI-756 inhibited the growth of NRAS, BRAF, and NF1-mutant melanomas in vitro and delayed the onset and reduced the incidence of Nras/Ink4a melanomas in vivo. Furthermore, combining SBI-756 and a BRAFi attenuated the formation of BRAFi-resistant human tumors. These studies are currently pursued by Ikrame Lazar at the Technion Lab.
OPEN / ONGOING PROJECTS:
Splice ATF2 variants as oncogenes in melanoma - mechanism of action.
Characterization of small molecules that promote ATF2 nuclear export in culture and in vivo models?
The effect of Siah2 ubiquitin ligase on anti-tumor immunity.
The link between PDK1 and rewired melanoma metabolism.