Triple Inhibition of BRAF/MEK, CDK4/6, and HERV-K for Melanoma Treatment


Jianli Dong*

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.


Abstract:

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


Description:

INTRODUCTION

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.Skuntz, S.Harrigan,I.</author><author>Grigorieva, E.</author><author>Candia, A.</author><author>Wright,

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</

title><secondary-title>Development</secondary-title></titles><pages>4655-64</pages><volume>130</

volume><dates><year>2003</year></dates><urls></urls></record></Cite></EndNote>.

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.

The discovery that melanoma cells harboring BRAF V600 mutations are addicted to mutationally deregulated BRAF led to the exciting development and approval of PLX 4032 and other BRAF inhibitors for clinical use27,28. Although inhibitors of mutant BRAF have generated great excitement because approximately 80% of BRAF mutant metastatic melanomas regressed in response to the treatment which was considered rare victory in the fight against melanomas, attention was quickly drawn to the fact that regressed tumors may resurge more aggressively within 1 year after the start of therapy, indicating that more research is needed to conquer melanoma 39-42.

CDKN2A/p16INK4A Lesions in Melanoma

Multiple genetic and environmental factors have been linked to the development of melanomas. For example, ultraviolet exposure, as well as defects in the cyclin dependent kinase inhibitor 2A (CDKN2A, encodesp16INK4Aand p14ARF) locus are predisposing factors 43,44. Mutational spectra have shown that over 60% of CDKN2A gene mutations harbor UVB signature changes (A:T>T:A transversions)2,45,46. We have shown that loss of expression of CDKN2A (by Western blotting) co-exists with the hot-spot T1799A BRAF mutation in several melanoma cell lines, and that CDKN2Aand BRAF lesions functionally interact to promote the proliferation and survival of cultured melanoma cells 3,47. BRAF mutations occur in over half of benign nevi16,18-20, where BRAF is shown acting upstream of CDKN2A, leading to p16INK4A over-expression and senescence. The proportion of CDKN2A mutations in melanoma specimens co-existing with T1799A BRAF that also harbor UVB signature is unknown. Lesions in the p16INK4A-CDK4/6-RB pathway commonly co-exist and interact functionally with abnormal MEK-ERK pathway in melanoma cells 1-3. Similarly, the majority of melanomas have a deregulated p16INK4A-CDK4/6-RB pathway in which there is a loss of normal p16INK4A, resulting in disinhibition of CDK4/6. The mechanism(s) of CDKN2A and BRAF genetic interaction in the regulation of cell proliferation and survival is largely unclear. The observed induction of apoptosis and suppression of proliferation by simultaneous expressions of mutant BRAF siRNA and wild-type CDKN2A cDNA could result from double hitting of the “linear” BRAF-MEK-ERK-cyclin D:CDK4/6-RB pathways (additive effect). Alternatively, since BRAF and p16INK4A have non-overlapping activities in melanoma cells 1-3, their interaction can be non-linear and synergistic.

Loss of CDKN2A/p16INK4A in Melanoma

Genetic and epigenetic changes of CDKN2A have been identified in 30-70% of melanomas 44,48,49. Bi-allelic deletion of CDKN2A(p16INK4A null) occurs in 10-27% of melanomas 50,51. Other possible changes include mono-allelic deletion, point mutation, or promoter hypermethylation; thus resulting in various levels of p16INK4A expression/activity 49-53. p16INK4A shares coding sequences with ARF, however, the proteins are translated in different reading frames 54. ARF up-regulates p53 by interfering with p53 negative regulators, MDM2; while p16INK4A binds to and inhibits CDK4 and CDK6 and promotes cell-cycle arrest via the RB tumor suppressor pathway 7,55. Although deletions and mutations may affect both p16INK4A and ARF, several studies have identified mutations in melanoma specimens affecting only p16INK4A and lack of ARF involvement in human melanoma specimens 56-58. Mutational inactivation of RB tumor suppressor is rare in melanomas 59,60.

 a cell that lacks or has abnormal endogenous CDKN2A. We examined CDKN2A in several melanoma cell line 2,3. 624Mel cells were found to have an 18 bp deletion (GGAGGCGGGGGCGCTGC) in exon 1α2,3. The deleted sequence encodes an evolutionarily conserved six amino acids (LEAGAL) in the p16INK4A N-terminal domain 61. The sequence shows loss of heterozygosity (LOH), suggesting that either the wild-type copy of the gene is deleted or that this is a homozygous deletion. Since matched constitutional DNA sample from the patient are not available, we cannot determine the cause of the LOH. Castellano et al. 62 reported promoter hypermethylation in melanoma cells. Such data suggests that p16INK4A function is compromised in these melanoma cells. Florenes et al. 63 reported deletion of p16INK4A in WM35 cells. Consistent with this report, we were unable to detect p16INK4A by Western blotting or CDKN2A mRNA, using reverse transcriptase PCR at high cycle numbers (>40 cycles) with appropriate positive and negative controls in WM35 cells 15,45. PCR failed to amplify CDKN2A exons 1α and 2, however exon 3 was amplified and showed a normal sequence 2,3.

It is believed that the acquisition of loss-of-function p16INK4A lesions allows melanoma cells to bypass senescence/growth arrest during melanoma development 64. To examine the effects of p16INK4A reconstitution, we subcloned the wild-type CDKN2AcDNA into a pBabe-neo retroviral vector. We transduced 624Mel and WM35 melanoma cells with either vector control or pBabe-neo-CDKN2A retroviruses. Melanoma cells expressing exogenous CDKN2A cDNA grew slower than control cells consistent with the tumor suppressor role of p16INK4A. CDK4/6 kinase activity and the phosphorylation of RB proteins are inhibited by exogenous CDKN2A expression in melanoma cells 2,3,65.

CDK4/6 inhibitors and companion diagnostic assays

Several CDK inhibitors have been developed including palbociclib (Ibrance, Pfizer), ribociclib (Kisqali, Novartis),and abemaciclib (Verzenio, Eli Lilly) 66. Recently, the U.S. Food and Drug Administration (FDA) has approved the above three CDK4/6 inhibitors for patients with metastatic breast cancer, demonstrating that these drugs can be tolerated by cancer patients. There are ongoing investigation of CDK4/6 inhibitor-based therapies in melanoma 1,66-68. Our investigations have provided critical proof-of-concept preclinical studies to combine CDK4/6 with BRAF/MEK inhibitors to improve melanoma treatment. Given the increasing understanding and implementation of genetic and genomic medicine to improve cancer treatment efficacy and specificity, and the use of gene-signature, instead/in addition to tumor-or-origin as the basis of cancer treatment, for example the recent FDA approval of cancer treatment for any solid tumor with a specific genetic feature, and the ongoing clinical investigation of NCI-MATCH trial (https://www.cancer.gov/about-cancer/treatment/clinical-trials/ncisupported/nci-match), it is important to develop molecular tests that predict the efficacy of targeted melanoma therapy including CDK4/6 inhibitors. CDKN2A/p16INK4A assays should be validated for samples processed both by formalin-fixation with paraffin-embedding (FFPE) and frozen with optimal cutting temperature (OCT) compounds. Table 2 includes a list of the status of CDKN2A and p16INK4A that can be examined using molecular assays. Basically, wild-type CDKN2A without promoter hypermethylation should correlate with normal level of p16INK4A, mono-allellic deletion, various mutations and promoter hypermethylation with decreased levels of p16INK4A, whereas bi-allelic deletion should cause loss of p16INK4A expression. 

HERV-K Activation in Melanoma

Endogenous retroviruses (ERVs) are sequences in the genome thought to be derived from ancient viral infections of germline cells in humans, mammals and other vertebrates; as such their proviruses are passed on to the next generation and now remain in the genome 69-74. Most retroviruses (such as HIV-1) infect somatic cells, but in very rare cases, it is thought that exogenous retroviruses have infected germline cells allowing integrated retroviral genetic sequences to be passed on to subsequent progeny, thereby becoming ‚endogenous‘. Endogenous retroviruses have persisted in the genome of their hosts for thousands of years. Some human ERVs have been implicated in certain autoimmune diseases and cancers, such as rheumatoid arthritis, teratocarcinoma, melanoma and breast cancer 69-72. There are many thousands of endogenous retroviruses within human DNA, with human ERVs (HERVs) comprising nearly 8% of the human genome. HERVs are typically not capable of replication and appeared to be defective in some aspect. HERVs can be classified to over 20families based on tRNA specificity of the primer binding site used to initiate reverse transcription 69-72.  

We reported that K type HERVs (HERV-K) are expressed and correlate with status of MEK-ERK and p16INK4ACDK4/6 pathways in melanoma cells 75. It is interesting that we observed HERV-K expression in polyploid melanoma cells 4 . We detected polyploid cells in melanomas but not in benign nevi and all such cells expressed HERV-K envelop protein ENV 5 . Increased DNA content can be caused by mitosis error or intercellular fusion and is associated with enhanced malignant behavior 76-78. 

Potential roles of HERV-K in melanoma pathobiology

HERV-K activation was observed to be correlated with changes in growth characteristics of melanoma cells (e.g., changes in cell shape, loss of melanin, anchorage-independent growth) 75,79. These cellular changes share features that can be blocked by suppression of BRAF-MEK-ERK, especially in the presence of CDKN2A cDNA or CDK4/6 inhibitor 1-3. Of note, RAF signaling can aid in the replication and pathogenesis of the HIV and other retroviruses. HERV-K RT can reverse transcribe HERV-K RNA back to DNA, which may transpose into host genomic DNA, causing destabilization of the genome. We proposed the below model of regulatory interactions between BRAF-MEK-ERK, p16INK4A, and HERV-K in benign nevi and melanoma cells (Figure 1).

Fig1. A proposed model of interactions between BRAF-MEK-ERK, p16INK4A, and HERV-K in nevi and melanomas


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.

Parental 624Mel cells (control) and 624Mel cells expressing CDKN2A or mutant BRAFRNAi (BRAFRNAi) were grown in regular media as described previously1-3. Shown are regular pictures of cell pellets from similar numbers of the corresponding cells. CDKN2A and mBRAF inhibition induced lighter and darker colors, respectively 2 .
It is well known that malignant cells, including those of melanomas, can maintain their normal differentiation program and retain sensitivity to differentiation modulators80-83. “Differentiation therapy” that induces malignant reversion from malignant to benign phenotypes has been most successful for acute promyelocytic leukemia (APL) 80-83. Such treatment has changed APL from a previously dismal outcome into one of the most treatable forms of leukemia 80-83. The understanding of mBRAF in the regulation of differentiation process of melanoma cells may provide not only insights into the roles of the mutation in melanoma biology, but also may provide strategies by targeting mBRAF in the “differentiation therapy” of melanomas. 
We found that combined suppression of both BRAF-MEK and p16INK4A lesions not only further retarded cell growth, but also were synergistic in inducing apoptosis 1,3. During normal development, differentiation stimuli trigger the activation of microphthalamia-associated transcription factor (MITF) to promote cell-cycle arrest and initiate the melanogenesis process 84-86. Melanogenesis is a multi-step biochemical process resulting in the formation of melanin in pigment cells. There are three melanogenic factors, tyrosinase, tyrosinase-related protein-1 (TRP1), and dopachrometautomerase (DCT)/tyrosinase-related protein-2 (TRP2) that participate in the melanogenic pathway and are important melanocyte differentiation markers 84-86. Eberle et al. 87compared the expression of tyrosinase, TRP1, and DCT/TRP2 in cultured normal human melanocytes and melanoma cell lines by Northern blotting and reverse transcriptase-PCR (RT-PCR). They found that the three genes were expressed in the normal human melanocytes, but the expression was repressed in nearly all the 14 melanoma cell lines examined and was completely absent in 4 of the 14 lines 87. Hofbauer et al. 88G. F.Kamarashev, J.Geertsen, R.Boni, R.Dummer, R.J Cutan Pathol204-9251998 also found that tyrosinase expression level correlated inversely with clinical stage. Such data suggest that the normal melanogenesis mechanism is inhibited in melanoma cells. MITF and related melanogenesis factors are potential biomarkers for melanoma prognosis and treatment. 
Inhibiting BRAF also up-regulates MITF in melanogenesis, a process somewhat inhibited by p16INK4A restoration (data not shown). During cellular growth and differentiation, cells have specific spatial and temporal windows that are susceptible to exogenous influences of differentiation. Apoptosis occurs in cells with interrupted patterning programs. We propose that the differentiation process induced by BRAF-MEK inhibition is interrupted by wild-type p16INK4A and CDK4/6 inhibition, which create cell identity crisis and apoptosis (Figure 3). 
A. Differentiation therapy. Tumor cells are induced to more mature and less malignant state. B. Differentiation confusion therapy. Cells have conflict between differentiation and de-differentiation. The confused cells undergo apoptosis in response to the identity crisis. 
Melanoma is one of the most immunogenic tumors thought to due to its high mutational burden (caused by UVdamage) and MITF-induced molecular profile that make melanomas particularly susceptible to immunotherapy 89,90.Durable remission of metastatic melanoma is now achievable in some patients who receive to immunotherapy. However, it is unclear why some patients respond better than others. There are ongoing research to identify predictive biomarkers to immunotherapy and ways to enhance immunogenicity for better clinical efficacy 89,90. Given that HERV and MITF may modulate host immune reactivity73,89,90, it will be interesting to examine whether activation of HERV-K and MITF correlates with response of melanoma cells to immunotherapy, and if so, develop corresponding predictive biomarkers for melanoma treatment.
Blocking engine (BRAF-MEK), fixing brake (p16INK4A), stabilizing genome (HERV-K) for melanoma treatment
Constitutive activation of BRAF-MEK-ERK (engine on) and loss of p16INK4A (brake off) are common paradigm in cancer biology. We have demonstrated that combinatorial inhibition of BRAF-MEK and p16INK4A lesions blocks proliferation and enhanced apoptosis in melanoma cells 4,5, the expression of HERV-K correlates with MEK-ERK activation and loss of p16INK4A in melanoma specimens and can be suppressed by MEK and CDK4/6 inhibitors 5 , and combined use of MEK, CDK4/6, and HERV-K blockers enhance the inhibitory effect in cultured melanoma cells 91. Tumor development and acquired treatment resistance involve molecular and cellular changes leading to the evolution and selection of aggressive clones; for example, changes in survived cells under BRAF/MEK inhibition leading acquired treatment resistance. HERV-K encodes unique retroviral proteins, including reverse transcriptase (RT) and ENV that may be co-opted by melanoma cells leading to tumor progression and treatment resistance. It has been shown that constitutive BRAF-MEK signaling upregulation can drive aneuploidy in melanocytic cells 92, an effect is mediated, at least in part, by HERV-K retrotransposition-mediated mutagenesis and destabilized genome. We have shown that HERV-K ENV has fusogenic activity that can mediate intercellular fusion of melanoma cells 4 ). ENV may anchor tumor cells to target sites through intercellular fusions (melanoma cell as seed, ENV as root, target site as soil). Blocking HERV-K expression may stabilize the chromosome, inhibit cell fusion, and prevent evolution and the selection of more aggressive melanoma cells. Importantly, activation of HERV-K without normal cell cycle checkpoint control (due to abnormal p16INK4A-CDK4/6) may lead to acquired treatment resistance under BRAF/MEK treatment through HERV-K mediated intercellular fusion and retrotransposition. We believe that adding CDK4/6 and HERV-K inhibitors should block alternative and feedback survival routes and enhance tumor suppression under BRAF/MEK treatment. We contemplate that simultaneous inhibition of BRAF-MEK, CDK4/6, and HERV-K can broaden the pharmacodynamic profile, achieve target inhibition at lower bio-available drug concentrations, and generate additive/synergistic and long-lasting therapeutic effects. 
Several CDK inhibitors have been developed, and Novartis CDK4/6 inhibitor LEE011 (ribociclib) has received FDA approval for treating advanced breast cancer66. A wide variety of anti-retroviral drugs are available to treat HIV retrovirus93.HERV-K elements can be inhibited by some of the anti-retroviral drugs, for example reverse transcriptase inhibitors91,94. These inhibitors may be used in conjunction with BRAF-MEK and CDK4/6 inhibitors. While BRAF/MEK, CDK4/6, and retroviral drugs have been approved for clinical use, it is important to carry out clinical trials combining these agents to achieve more efficacious and long lasting effect so metastatic melanoma patients can live longer. Research and development of predictive biomarkers that can identify tumors with BRAF/MEK, CDK4/6, and HERV-K lesions should facilitate the selection of patients more likely to benefit from the combination therapy.

CONCLUSION

Constitutive deregulation of BRAF-MEK-ERK and p16INK4A-CDK4/6-RB pathways both occur at high frequencies in melanomas. We have shown that suppression of either BRAF-MEK or CDK4 inhibits cell growth, and that simultaneous inhibition of both BRAF-MEK and CDK4 compounds this effect and also triggers significant apoptosis in melanoma cells1-3. The 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 have also reported that the expression of HERV-K correlates with ERK activation and p16INK4A loss in melanoma cells and can be inhibited by MEK and CDK4/6 inhibitors, especially when in combination4,5. Given that HERV-K may drive malignant growth downstream of BRAF-MEK and CDK4/6 and can also be activated by other factors that may be independent of BRAF-MEK and CDK4/6, we contemplated that cells with an activated HERV-K may escape the therapeutic effects of MEK and CDK4/6 inhibitors, and that triple inhibition of BRAF-MEK, CDK4/6, and HERV-K, would be more effective melanoma treatment.
Author Contribution
J.D. conceived and designed the experiments described in the review; G.G. and L.L.T. participated in the preparation of the review


References

1. Li, J., Xu, M., Yang, Z., Li, A. & Dong, J. Simultaneous inhibition of MEK and CDK4 leads to potent apoptosis in human melanoma cells. Cancer Invest 28, 350-356 (2010). 2. Rotolo, S. et al. Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild-type INK4A in melanoma cells. Int J Cancer 115, 164-169 (2005).

3. Zhao, Y., Zhang, Y., Yang, Z., Li, A. & Dong, J. Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis. Biochem Biophys Res Commun 370, 509- 513 (2008).

4. Huang, G., Li, Z., Wan, X., Wang, Y. & Dong, J. Human endogenous retroviral K element encodes fusogenic activity in melanoma cells. J Carcinog 12, 5, doi:10.4103/1477-3163.109032JC-12-5 [pii] (2013).

5. Li, Z. et al. Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells. Cancer Invest 28, 1031-1037 (2010).

6. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949-954 (2002).

7. Chang, F. et al. Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review). Int J Oncol 22, 469-480 (2003).

8. Kolch, W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 351 Pt 2, 289-305 (2000).

9. Pearson, G. et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22, 153-183 (2001).

10. Dent, P. & Grant, S. Pharmacologic interruption of the mitogen-activated extracellular-regulated kinase/ mitogen-activated protein kinase signal transduction pathway: potential role in promoting cytotoxic drug action. Clin Cancer Res 7, 775-783 (2001).

11. English, J. M. & Cobb, M. H. Pharmacological inhibitors of MAPK pathways. Trends Pharmacol Sci 23, 40-45 (2002).

12. Peyssonnaux, C. & Eychene, A. The Raf/MEK/ERK pathway: new concepts of activation. Biol Cell 93, 53-62 (2001).

13. Busca, R. et al. Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. Embo J 19, 2900-2910 (2000).

14. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646-674, doi:10.1016/j. cell.2011.02.013 (2011).

15. Smalley, K. S. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 104, 527-532 (2003).

16. Dong, J. et al. BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma. Cancer Res63, 3883-3885 (2003).

17. Collisson, E. A., De, A., Suzuki, H., Gambhir, S. S. & Kolodney, M. S. Treatment of metastatic melanoma with an orally available inhibitor of the Ras-Raf-MAPK cascade. Cancer Res63, 5669-5673 (2003).

18. Pollock, P. M. et al. High frequency of BRAF mutations in nevi. Nat Genet33, 19-20 (2003).

19. Tschandl, P. et al. NRAS and BRAF mutations in melanoma-associated nevi and uninvolved nevi. PLoS One8, e69639, doi:10.1371/journal.pone.0069639 (2013).

20. Wu, J., Rosenbaum, E., Begum, S. & Westra, W. H. Distribution of BRAF T1799A(V600E) mutations across various types of benign nevi: implications for melanocytic tumorigenesis. Am J Dermatopathol 29, 534-537, doi:10.1097/DAD.0b013e3181584950 (2007).

21. Clark, W. H., Jr. & Tucker, M. A. Problems with lesions related to the development of malignant melanoma: common nevi, dysplastic nevi, malignant melanoma in situ, and radial growth phase malignant melanoma. Hum Pathol 29, 8-14 (1998).

22. Elder, D. Tumor progression, early diagnosis and prognosis of melanoma. Acta Oncol 38, 535-547 (1999).

23. Arozarena, I. & Wellbrock, C. Overcoming resistance to BRAF inhibitors. Ann Transl Med 5, 387, doi:10.21037/ atm.2017.06.09 (2017).

24. Harris, H. Putting on the brakes. Nature 427, 201 (2004).

25. Shibahara, S. et al. Regulation of pigment cell-specific gene expression by MITF. Pigment Cell Res 13 Suppl 8, 98-102 (2000).

26. Sicinska, E. et al. Requirement for cyclin D3 in lymphocyte development and T cell leukemias. Cancer Cell 4, 451-461 (2003).

27. Bollag, G. et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596-599 (2010).

28. Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 363, 809- 819 (2010).

29. Ortega, S., Malumbres, M. & Barbacid, M. Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602, 73-87 (2002).

30. Yasmeen, A., Berdel, W. E., Serve, H. & Muller-Tidow, C. E- and A-type cyclins as markers for cancer diagnosis and prognosis. Expert Rev Mol Diagn3, 617-633 (2003).

31. Keezer, S. M. & Gilbert, D. M. Evidence for a pre-restriction point Cdk3 activity. J Cell Biochem 85, 545-552 (2002).

32. Welch, J. et al. Lack of genetic and epigenetic changes in CDKN2A in melanocytic nevi. J Invest Dermatol 117, 383-384 (2001).

33. Ford-Perriss, M., Abud, H. & Murphy, M. Fibroblast growth factors in the developing central nervous system. Clin Exp Pharmacol Physiol 28, 493-503 (2001).

34. Tower, G. B., Coon, C. C., Benbow, U., Vincenti, M. P. & Brinckerhoff, C. E. Erk 1/2 differentially regulates the expression from the 1G/2G single nucleotide polymorphism in the MMP-1 promoter in melanoma cells. Biochim Biophys Acta 1586, 265-274 (2002).

35. Huang, J. et al. T cells associated with tumor regression recognize frameshifted products of the CDKN2A tumor suppressor gene locus and a mutated HLA class I gene product. J Immunol 172, 6057-6064 (2004).

36. Mankoo, B. S. et al. The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation, patterning and differentiation of somites. Development 130, 4655-4664 (2003).

37. Bulchand, S., Grove, E. A., Porter, F. D. & Tole, S. LIM-homeodomain gene Lhx2 regulates the formation of the cortical hem. Mech Dev 100, 165-175 (2001).

38. Chen, H. & Sukumar, S. HOX genes: emerging stars in cancer. Cancer Biol Ther 2, 524-525 (2003).

39. Ledford, H. Rare victory in fight against melanoma. Nature 467, 140-141 (2010).

40. Livingstone, E., Zimmer, L., Piel, S. & Schadendorf, D. PLX4032: does it keep its promise for metastatic melanoma treatment? Expert Opin Investig Drugs 19, 1439-1449 (2010).

41. Vultur, A., Villanueva, J. & Herlyn, M. Targeting BRAF in advanced melanoma: a first step toward manageable disease. Clin Cancer Res 17, 1658-1663 (2011).

42. Aplin, A. E., Kaplan, F. M. & Shao, Y. Mechanisms of Resistance to RAF Inhibitors in Melanoma. J Invest Dermatol (2011).

43. Gilchrest, B. A., Eller, M. S., Geller, A. C. & Yaar, M. The pathogenesis of melanoma induced by ultraviolet radiation. New Eng J Med 340, 1341-1348 (1999).

44. Sharpless, E. & Chin, L. The INK4a/ARF locus and melanoma. Oncogene 22, 3092-3098 (2003).

45. Kumar, R., Lundh Rozell, B., Louhelainen, J. & Hemminki, K. Mutations in the CDKN2A (p16INK4a) gene in microdissected sporadic primary melanomas. Int J Cancer 75, 193-198 (1998).

46. Peris, K. et al. UV fingerprint CDKN2a but no p14ARF mutations in sporadic melanomas. J Invest Dermatol 112, 825-826, doi:10.1046/j.1523-1747.1999.00575.x (1999).

47. Li, J., Xu, M., Yang, Z., Li, A. & Dong, J. Simultaneous inhibition of MEK and CDK4 leads to potent apoptosis in human melanoma cells. Cancer Investigation in press (2009).

48. Dutton-Regester, K. & Hayward, N. K. Reviewing the somatic genetics of melanoma: from current to future analytical approaches. Pigment Cell Melanoma Res (2012).

49. Grafstrom, E., Egyhazi, S., Ringborg, U., Hansson, J. & Platz, A. Biallelic deletions in INK4 in cutaneous melanoma are common and associated with decreased survival. Clin Cancer Res 11, 2991-2997 (2005).

50. Curtin, J. A. et al. Distinct sets of genetic alterations in melanoma. N Engl J Med 353, 2135-2147 (2005)

51. Jonsson, A., Tuominen, R., Grafstrom, E., Hansson, J. & Egyhazi, S. High frequency of p16(INK4A) promoter methylation in NRAS-mutated cutaneous melanoma. J Invest Dermatol130, 2809-2817 (2010).

52. Miller, P. J. et al. Classifying variants of CDKN2A using computational and laboratory studies. Hum Mutat32, 900-911 (2011). 53. Yang, G., Rajadurai, A. & Tsao, H. Recurrent patterns of dual RB and p53 pathway inactivation in melanoma. J Invest Dermatol 125, 1242-1251 (2005). 54. Sherr, C. J. The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2, 731-737 (2001).

55. Lowe, S. W. & Sherr, C. J. Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 13, 77-83 (2003).

56. Chin, L. The genetics of malignant melanoma: lessons from mouse and man. Nat Rev Cancer 3, 559-570 (2003).

57. Fargnoli, M. C. et al. CDKN2a/p16INK4a mutations and lack of p19ARF involvement in familial melanoma kindreds. J Invest Dermatol 111, 1202-1206 (1998).

58. Piepkorn, M. Melanoma genetics: an update with focus on the CDKN2A(p16)/ARF tumor suppressors. J Am Acad Dermatol 42, 705-722 (2000). 59. Bartkova, J. et al. The p16-cyclin D/Cdk4-pRb pathway as a functional unit frequently altered in melanoma pathogenesis. Cancer Res 56, 5475-5483 (1996).

60. Maelandsmo, G. M. et al. Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas. Br J Cancer 73, 909-916 (1996).

61. Greenblatt, M. S. et al. Detailed computational study of p53 and p16: using evolutionary sequence analysis and disease-associated mutations to predict the functional consequences of allelic variants. Oncogene 22, 1150-1163 (2003).

62. Castellano, M. et al. CDKN2A/p16 is inactivated in most melanoma cell lines. Cancer Res 57, 4868- 4875 (1997).

63. Florenes, V. A. et al. TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1. Oncogene 13, 2447-2457 (1996).

64. Bennett, D. C. How to make a melanoma: what do we know of the primary clonal events? Pigment Cell Melanoma Res 21, 27-38 (2008).

65. Castellano, M., Gabrielli, B. G., Hussussian, C. J., Dracopoli, N. C. & Hayward, N. K. Restoration of CDKN2A into melanoma cells induces morphologic changes and reduction in growth rate but not anchorage-independent growth reversal. J Invest Dermatol 109, 61-68 (1997).

66. O'Leary, B., Finn, R. S. & Turner, N. C. Treating cancer with selective CDK4/6 inhibitors. Nat Rev Clin Oncol 13, 417-430, doi:10.1038/nrclinonc.2016.26 (2016).

67. Teh, J. L. et al. An In Vivo Reporter to Quantitatively and Temporally Analyze the Effects of CDK4/6 InhibitorBased Therapies in Melanoma. Cancer Res 76, 5455-5466, doi:10.1158/0008-5472.CAN-15-3384 (2016).

68. Young, R. J. et al. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines. Pigment Cell Melanoma Res 27, 590-600, doi:10.1111/pcmr.12228 (2014).

69. Fuchs, N. V. et al. Expression of the human endogenous retrovirus (HERV) group HML-2/HERV-K does not depend on canonical promoter elements but is regulated by transcription factors Sp1 and Sp3. J Virol 85, 3436-3448(2011).

70. Hohn, O., Hanke, K. & Bannert, N. HERV-K(HML-2), the Best Preserved Family of HERVs: Endogenization, Expression, and Implications in Health and Disease. Front Oncol3, 246, doi:10.3389/fonc.2013.00246 (2013).

71. Oricchio, E. et al. Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression. Oncogene 26, 4226-4233 (2007).

72. Subramanian, R. P., Wildschutte, J. H., Russo, C. & Coffin, J. M. Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses. Retrovirology 8, 90, doi:10.1186/1742-4690-8-90 (2011).

73. Kassiotis, G. & Stoye, J. P. Immune responses to endogenous retroelements: taking the bad with the good. Nat Rev Immunol 16, 207-219, doi:10.1038/nri.2016.27 (2016).

74. Singh, S., Kaye, S., Gore, M. E., McClure, M. O. & Bunker, C. B. The role of human endogenous retroviruses in melanoma. Br J Dermatol 161, 1225-1231, doi:10.1111/j.1365-2133.2009.09415.x (2009).

75. Serafino, A. et al. The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation. Exp Cell Res 315, 849-862 (2009).

76. Lu, X. & Kang, Y. Cell fusion as a hidden force in tumor progression. Cancer Res 69, 8536-8539 (2009).

77. Panigrahi, A. K. & Pati, D. Road to the crossroads of life and death: linking sister chromatid cohesion and separation to aneuploidy, apoptosis and cancer. Crit Rev Oncol Hematol 72, 181-193 (2009).

78. Rajaraman, R., Guernsey, D. L., Rajaraman, M. M. & Rajaraman, S. R. Stem cells, senescence, neosis and selfrenewal in cancer. Cancer Cell Int 6, 25 (2006).

79. Lemaitre, C., Tsang, J., Bireau, C., Heidmann, T. & Dewannieux, M. A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion. PLoS Pathog 13, e1006451, doi:10.1371/journal.ppat.1006451 (2017).

80. Fusenig, N. E., Breitkreutz, D., Boukamp, P., Tomakidi, P. & Stark, H. J. Differentiation and tumor progression. Recent Results Cancer Res 139, 1-19 (1995).

81. Waxman, S. Differentiation therapy in acute myelogenous leukemia (non-APL). Leukemia 14, 491-496 (2000).

82. Leszczyniecka, M., Roberts, T., Dent, P., Grant, S. & Fisher, P. B. Differentiation therapy of human cancer: basic science and clinical applications. Pharmacol Ther 90, 105-156 (2001).

83. Spira, A. I. & Carducci, M. A. Differentiation therapy. Curr Opin Pharmacol 3, 338-343 (2003).

84. Fang, D. & Setaluri, V. Role of microphthalmia transcription factor in regulation of melanocyte differentiation marker TRP-1. Biochem Biophys Res Commun 256, 657-663 (1999).

85. Setaluri, V. The melanosome: dark pigment granule shines bright light on vesicle biogenesis and more. J Invest Dermatol 121, 650-660 (2003).

86. Widlund, H. R. & Fisher, D. E. Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival. Oncogene 22, 3035-3041 (2003).

87. Eberle, J., Garbe, C., Wang, N. & Orfanos, C. E. Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro. Pigment Cell Res 8, 307-313 (1995).

88. Hofbauer, G. F., Kamarashev, J., Geertsen, R., Boni, R. & Dummer, R. Tyrosinase immunoreactivity in formalinfixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution. J Cutan Pathol 25, 204-209 (1998).

89. Byrne, E. H. & Fisher, D. E. Immune and molecular correlates in melanoma treated with immune checkpoint blockade. Cancer 123, 2143-2153, doi:10.1002/cncr.30444 (2017).

90. Ko, J. S. The Immunology of Melanoma. Clin Lab Med 37, 449-471, doi:10.1016/j.cll.2017.06.001 (2017).

91. Dong, J. H., G.; Imtiaz, R.; Xu, F. The Potential Importance of K Type Human Endogenous Retroviral Elements in Melanoma Biology. Intech ISBN 978-953-51-0976-1, doi:10.5772/55264 (2013).

92. Cui, Y., Borysova, M. K., Johnson, J. O. & Guadagno, T. M. Oncogenic B-Raf(V600E) induces spindle abnormalities, supernumerary centrosomes, and aneuploidy in human melanocytic cells. Cancer Res 70, 675-684 (2010).

93. Taylor, B. S., Olender, S. A., Tieu, H. V. & Wilkin, T. J. CROI 2016: Advances in Antiretroviral Therapy. Top Antivir Med 24, 59-81 (2016).

94. Contreras-Galindo, R., Dube, D., Fujinaga, K., Kaplan, M. H. & Markovitz, D. M. Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors. J Virol 91, doi:10.1128/ JVI.01309-17 (2017).

ARJ @ SOCIAL

openaccess

Testimonials

indexings
indexings
indexings
indexings

Chicago

Arkansas