Triple Inhibition of BRAF/MEK, CDK4/6, and HERV-K for Melanoma Treatment
Citation: Triple Inhibition of BRAF/MEK, CDK4/6, and HERV-K for Melanoma Treatment. American Research Journal of Oncology. 2018; 1(1): 1-15.
Copyright This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Malignant melanomas are the most lethal skin malignancy, notorious for aggressive growth and resistance to therapy. While the response to selective BRAF and MEK inhibitors (BRAFi, MEKi), alone and in combination, in BRAF V600-mutant melanoma is encouraging, virtually all patients rapidly develop secondary resistance. We have shown that constitutive deregulation of both BRAF-MEK-ERK and p16INK4A-CDK4/6- RB pathways occur at high frequencies in melanomas, and that suppression of BRAF/MEK, or restoration of p16INK4A expression/inhibition of CDK4/6 can block the growth melanoma cells, and simultaneous correction of both BRAF-MEK and p16INK4A-CDK4/6 compounds this effect and also triggers significant apoptosis in melanoma cells. Our data suggests that BRAF-MEK-ERK and p16INK4A-CDK4/6-RB pathways may act additively or synergistically in the malignant growth of melanoma cells, and could be jointly targeted for treatment of melanoma. We also reported that the expression of K-type human endogenous retrovirus (HERV-K) correlates with ERK activation and p16INK4A loss in melanoma cells, and can be inhibited by MEK and CDK4/6 inhibitors, especially in combination. Given that HERV-K may destabilize the genome and act downstream of BRAF-MEK and CDK4/6, we hypothesize that cells with activated HERV-K may escape the therapeutic effects of BRAF-MEK and CDK4/6 blockers, and that triple inhibition of BRAF-MEK, CDK4/6, and HERV-K should be an effective therapy for melanomas.
Keywords: BRAF mutation; NRAS mutation; CDKN2A/p16INK4A lesion; HERV-K activation; combination therapy
Malignant melanomas are the most lethal skin malignancy, notorious for aggressive growth and resistance to therapy. While the responses to selective BRAF/MEK and immune checkpoint inhibitors have been encouraging and revolutionized the treatment of metastatic melanoma, a subset of patients do not respond to these treatment, and those patients initially responded later develop acquired resistance and disease relapse. These issues of treatment resistance demonstrate the need to further understand mechanisms underlying melanomagenesis and therapy resistance. In this review, we will examine constitutive deregulation of both BRAF-MEK-ERK and p16INK4A-CDK4/6-RB pathways in melanomas and data suggest that BRAF-MEK-ERK and p16INK4A-CDK4/6- RB pathways may act additively or synergistically in the malignant growth of melanoma cells, and could be jointly targeted for treatment of melanoma. We will also examine the expression of K-type human endogenous retrovirus (HERV-K) in melanoma and the potential regulatory relationship between HERV-K, ERK activation and p16INK4A loss in melanoma cells. Given that HERV-K may destabilize the genome and act downstream of BRAF-MEK and CDK4/6, we will examine the hypothesis that cells with activated HERV-K may escape the therapeutic effects of BRAF-MEK and CDK4/6 blockers, and that triple inhibition of BRAF-MEK, CDK4/6, and HERV-K should be an effective therapy for melanomas.
BRAF is a component of the RAS-RAF-mitogen activated protein kinase/ERK kinase (MEK)-extracellular signal regulated kinase (ERK) signaling pathway, and p16INK4A (encoded by CDKN2A) is part of the p16INK4Acyclin D:cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway. Constitutive deregulation of the BRAF-MEK-ERK and p16INK4A-CDK4/6-RB pathways occur at high frequencies in melanomas. We have shown that correction of either BRAF-MEK or p16INK4A-CDK4/6 abnormalities suppresses the in vitro and in vivo growth of melanoma cells, and that simultaneous inhibition of both BRAF-MEK and p16INK4A-CDK4/6 lesions, compounds this effect and also triggers significant apoptosis 1-3. Our data suggests that BRAF-MEK-ERK and p16INK4A-CDK4/6-RB pathways may act additively or synergisticallyin the malignant growth of melanoma cells, and could be jointly targeted for treatment of melanoma. We have shown that the expression of HERV-K correlated with ERK activation and p16INK4A loss in melanoma cells, and that HERV-K expression can be inhibited by MEK and CDK4/6 inhibitors, especially in combination 4,5. Since HERV-K can be activated and may drive malignant growth of melanoma downstream of BRAF-MEK and CDK4/6 pathways, cells with activated HERV-K may escape the therapeutic effects of MEK and CDK4/6 blockers leading to acquired treatment resistance. We propose that triple inhibition of BRAF-MEK, CDK4/6, and HERV-K could be an effective combo therapy for melanoma.
BRAF/NRAS ACTIVATING MUTATIONS IN MELANOMA
In a systematic genome-wide screening for gene mutations, Davies et al. identified BRAF mutations at high frequencies, ranging from 59 to 80% in human melanoma samples; which included tumor cell lines, short-term cultures, and tumor tissues 6 . A T1799A transversion in exon 15, resulting in a V600E missense mutation, accounts for approximately 90% of mutations detected in melanoma samples 6 . In addition to melanomas, BRAF mutations have been identified in several other tumor types including thyroid, ovarian, colorectal, and lung tissues 6 . The pathway conveys extra- and intracellular signals to nuclear transcription factors that regulate gene expression in response to such signals 7-9. BRAF is one of three members of the RAF family, which include serine/threonine kinases that transduce regulatory signals from RAS through MEK, to ERK. The ERK signaling pathway plays essential roles in cell proliferation, differentiation, and survival 10-15. BRAF oncogenic mutations lead to constitutive activation of the ERK pathway and cause cellular transformation 6,16. Constitutive activation of the ERK pathway is believed to be essential in melanoma development 13-15. Pharmacological inhibition of the ERK pathway inhibited melanoma metastases in mice 17. We and others have detected BRAF mutations in over half of benign melanocytic nevi 16,18-20. In comparison, there are a very large number of melanocytic nevi in the general population compared with the relatively low incidence of melanomas 21,22. It is known clinically that nevi very often regress over time; thus, BRAF mutations alone are insufficient to cause malignant transformation in nevus cells.
About a third of all human cancers harbor mutations in one of the K-, N-, or H-RAS genes that encode an abnormal RAS protein, locked in a constitutively activated state, driving malignant transformation and tumor growth. NRAS-activating lesions are found in melanomas, but do not generally overlap with BRAFmutations in the same lesion 6,16. NRAS codons 12, 13, and 61mutations are oncogenic lesions causing constitutive activation of MEK-ERK signaling pathway 6 .NRAS mutations have been identified as one of the mechanisms to turn on phosphoinositide 3-kinase pathway leading to enhanced survival and resistance to BRAF inhibitors in melanoma cells23.
BRAF/MEK Inhibitors in Melanoma Treatment
The high frequency of BRAF hot spot T1799A lesions provides the opportunity to examine the effects of specifically blocking this mutant allele 2,16. We used RNAi to specifically inhibit the expression of the T1799A mutant BRAF (mBRAF) and observed inhibited endogenous ERK signaling in melanoma cells that are positive for the BRAF mutation 2. Importantly, mBRAF RNAi also significantly inhibited the growth of these cells in tissue culture as measured by cell counting, and colony formation assay, and tumor growth in nude mice xenograft 2.
Interestingly, melanoma cells expressing mBRAFRNAi not only grow slower, but are also darker in color (shown in cell pellets, colonies, and xenografts), and produce more mature melanosomes 2. Since melanosome maturation and melanin production are signatures signs of melanocyte differentiation, the induced melanogenesis by BRAF inhibition may represent a reversion of melanoma cells to a more differentiated state.De-differentiation is characteristic of tumors cells 24. In many cell types, it is caused by constitutive activation of the RAS/RAF/ MEK/ERK signaling 25,26. Suppression of mutant BRAF causes inhibition of the ERK signaling, which may explain the observed differentiation phenotype27,28.
Consistent with the finding that inhibition of BRAF in melanoma cells not only induces growth inhibition, but also triggers cellular differentiation, our gene expression microarray analyses using Affymetrix human genome U133 GeneChip show that several genes involved in cell cycle control, cell growth, and differentiation are potential targets for mutant BRAF (Table 1). For the microarray expression analyses, 624Mel control and mBRAF RNAi expressing cells were cultured as previously described1-3.RNA extraction, labeling, and hybridizations were performed at the Microarray Core Facility at Mount Sinai School of Medicine in New York City according to the manufacturer‘s protocols. The microarray expression data were divided into control and mBRAFRNAi groups and the ratio of mBRAF RNAi over control were calculated (Table 1).
CCND1: cyclin D1, a major downstream target of mitogenic signals 29. CCNA1: cyclin A1,a
CDK2 interacting cyclin that promotes cell cycle progression and a proliferation marker 30.
CDK3: cyclin dependent kinase 3, a CDK regulates the G1-phase of the cell cycle 31.
KITLG: KIT ligand, known to regulate developmental and functional processes of melanocytes 32.
FGF17: Fibroblast growth factor 17, involved in cell proliferation 33.
MMP1: matrix metallopeptidase 1, the matrix metalloproteinase (MMP) family degrades the extracellular matrix. MMP1 is up-regulated by the ERK signaling pathway in melanoma cells 34.
CEBPD: CCAAT/enhancer binding protein delta, transcription factor CCAAT/enhancer binding protein delta (also known as CEBPD, CRP3, CELF, NF-IL6beta) is implicated in diverse cellular functions, such as the acute phase response, adipocyte differentiation, and chromosomal stability 35.
MEOX2: mesenchyme homeobox 2, growth arrest specific mesenchyme homeo box 2, involved in patterning and differentiation 36B. S.
C. V.</author><author>Arnheiter, H.</author><author>Pachnis, V.</author></authors></
contributors><titles><title>The concerted action of Meox homeobox genes is required upstream
of genetic pathways essential for the formation, patterning and differentiation of somites</
LHX2: LIM homeobox 2, a LIM homeodomain protein involved in development 37.
HOXD1 and HOXD3: homeoboxD1 and homeobox D3, genes involved in development and cancer 38.
A. In benign nevi, CDKN2A is wild-type and turned on by gain-of-function V600E BRAF; over-expression of wild-type p16INK4A inhibits activation of the BRAF-MEK-ERK signaling pathway; HERV-K is not induced. B. In melanomas, CDKN2A/p16INK4A is damaged by UVB or other factors leading to its loss of expression (nonsense mutation or promoter hypermethylation) or loss of activity (missense mutation). BRAF-MEK-ERK signaling is not in check by p16INK4A; HERV-K is activated to further drive malignant progression.
We have been intrigued by the findings that specific inhibition of HERV-K using RNAi can block intercellular fusion-mediated colony formation of melanoma cells, and that melanoma cell intercellular fusion can be inhibited by HERV-K ENV antibodies 4 . We believe that efficient neutralizing HERV-K ENV antibodies can block intercellular fusion to stop the subsequent genetic changes that may lead to the evolution of tumor clones and emergence of more aggressive ones leading to tumor progression, metastasis, and treatment resistance.
HERV-K research and clinical applications need accurate analysis of HERV-K DNA, RNA, and proteins; which is challenged by the repetitive and homologous sequences of HERV-K elements. With new development to handle long-range sequencing and bioinformatics tools to correctly align homologous genomic sequences, we can better understand HERV-K sequence polymorphisms and copy number various in normal and cancer samples, which should facilitate the investigation of HERV-K in human health and diseases.
COMBINED INHIBITION OF BRAF/MEK, CDK4/6, AND HERV-K IN MELANOMA TREATMENT
Although BRAF and MEK inhibitors have been shown to be amazingly effective in treating metastatic melanoma. Unfortunately, the effect was not curative, and tumor cells returned back after 1 year or so. These results show that further improvements must be made in the treatment of this disease. The best way to reach a cure, we believe, relies on rational combinations of BRAF/MEK inhibitors with other agents. Such combined treatment approaches have resulted in therapeutic „cocktails“ that are effective in human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS). We propose that triple inhibition of BRAF/ MEK, CDK4/6, and HERV-K can be an effective combined therapy for melanoma.
Creating identity crisis and cell death for melanoma treatment
We delineated independent regulation of proliferation and differentiation by BRAFand CDKN2A lesions in melanoma cells 2 . Oncogenic BRAF can upregulate cyclin D through ERK pathway resulting in the activation of CDK4/6, and p16INK4A binds to and inactivates these CDKs, and activated CDKs phosphorylate and inactivate RB proteins resulting in the liberation of E2F transcription factors and cell cycle progression 1-3, therefore, both lesions lead to uncontrolled cellular proliferation consistent with their roles in the RB pathway. Note unlike melanoma cells with mutant BRAF inhibition, CDKN2A reconstituted cells are lighter in color (Figure 2) 2 . Since melanogenesis is a marker of melanocyte differentiation, the observed suppression of melanogenesis by p16INK4A and enhanced melanogenesis by BRAF inhibition, while unexpected, would suggest that proliferation and differentiation are regulated differently by BRAF and CDKN2A lesions in melanoma cells. Therefore, proliferation and differentiation are therefore separately regulated by BRAF and CDKN2A lesions in melanoma cells. It is believed that differentiation and malignancy are inversely correlated and cancer is a disease of cell differentiation 80-83, therefore, our findings have potential clinical significance.
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