40 Emerging Mechanisms of Familial Melanoma Development

Faculty Mentor: Robert Judson-Torres (Dermatology, University of Utah)

Abstract

Often, melanoma is thought of as a disease caused by ultraviolet radiation from the sun, and while sun damage does increase the risk of melanoma, melanoma can also run in families. Hereditary melanoma is caused by inherited genetic mutations that are passed from parents to children, creating a familial predisposition to cancer. One such example is the CDKN2A gene, which is observed to be mutated in over half of all families with hereditary melanoma. Though the CDKN2A gene encodes two tumor suppressor proteins, p14^ARF and p16^INK4A, mutations of this gene primarily impact the structure of p16^INK4A. Past research has outlined that p16^INK4A canonically binds to and inhibits proteins CDK4 and CDK6, thereby preventing cells from progressing through the cell cycle. However, recent research has suggested that this canonical mechanism is not the primary means by which melanoma progresses. Thus, other noncanonical binding partners of mutant p16^INK4A must result in increased melanoma proliferation and metastasis. This review will focus on the known functions and interactions of p16^INK4A and explore other possible proteins that may contribute to hereditary melanoma development. These include IMPDH2 and EZH2, two proteins that are seen to promote growth and metastasis in cancers. Ongoing research is beginning to uncover the promising mechanisms involving these three proteins. By further understanding the mechanisms by which melanoma develops, future methods of melanoma screening can be designed to detect melanoma in its early stages, when therapeutic intervention is most effective.

Introduction

Skin cancer is one of the most common forms of cancer diagnosed in the United States, making up between 40% and 50% of all cancer diagnoses (American Cancer Society, 2025). Furthermore, melanoma is among the most aggressive cancers due to its ability to rapidly proliferate and metastasize (Sundararajan et al., 2018). Once a primary tumor metastasizes to distant parts of the body, the patient’s prognosis worsens, with a lesser probability of complete recovery (Heistein et al., 2023). Conversely, patients with melanoma have a very favorable prognosis when the melanoma is detected early and resected before it spreads to nearby tissues and lymphoid organs, with a five-year survival rate of over 97.5% (Five-Year Survival Rates | SEER Training, n.d.). Understanding the mechanisms by which melanoma evolves can provide possible methods for early detection and quicker, more effective treatment.

Aside from acquired melanoma resulting from UV-damaged DNA in melanocytes (Sample & He, 2018), specific genetic mutations have been identified as hereditary, resulting in germline melanocyte mutations and familial melanoma patterns (Li et al., 2019; Soufir et al., 1998). In over half of all families with hereditary melanoma, the CDKN2A gene is observed to be mutated, mainly altering the structure and function of the encoded p16^INK4A protein (CDKN2A Gene: MedlinePlus Genetics, 2018; Soufir et al., 1998). By studying the impact of germline CDKN2A mutations on cellular functions, we can begin to discover the mechanisms by which melanoma cells rapidly proliferate and metastasize.

p16^INK4A is known to inhibit Cyclin Dependent Kinases 4 and 6 (CDK4 and CDK6), thereby regulating the cell cycle by preventing cells from transitioning from the G1 phase to the S phase (Cánepa et al., 2007). However, recent studies have shown that this canonical pathway is not the primary mechanism by which melanoma progresses (Becker et al., 2001; Zeng et al., 2018). Thus, it is possible that hitherto undiscovered noncanonical binding partners of the p16^INK4A, related proteins, and alternate pathways may also play key roles in melanoma development.

This review will examine the known cellular properties of p16^INK4A, IMPDH2, and EZH2 proteins individually, their roles in various binding arrangements, the trends observed in melanoma case studies, and explore possible directions for future research.

Genetic Mutations Driving Melanoma Progression

Melanoma is a particularly dangerous form of cancer due to its ability to rapidly proliferate, adapt, and metastasize to different parts of the body (Lelliott et al., 2021). A standard method of treatment for melanoma is to use antiproliferative drugs to halt cell cycle progression. Drugs such as Barasertib and Dabrafenib have been developed to interfere with proteins that initiate cell cycle progression with the goal of cell cycle arrest and apoptosis (Alimbetov et al., 2018; Patra et al., 2023). Specifically, Barasertib targets Aurora Kinase B to prevent cell division (Porcelli et al., 2015), while Dabrafenib inhibits the mutant B-Raf enzyme to prevent cellular growth (Bowyer et al., 2015). Although these drugs offer some hope in curing cancer, many of these drugs fail to target cancerous cells solely and have unintended consequences on normal, healthy cells as well (Alimbetov et al., 2018). Furthermore, single agent drugs, such as Dabrafenib when used for MAPK pathway mutations, could result in cancer cells acquiring drug resistance, failing to provide a durable treatment response (Chen et al., 2022). While these drugs attempt to target specific cell cycle regulators, there are many more alternative proteins and pathways that, when understood, can expand the opportunities for more treatments and early detection methods.

Researchers initially took interest in the B-Raf enzyme, which is mutated in over half of all melanomas (Bowyer et al., 2015; Ascierto et al., 2012). The B-Raf V600E mutation results in increased expression of the mutated B-Raf protein, resulting in uncontrolled cell growth and division (Ascierto et al., 2012). Researching the effects of protein mutations, such as with B-Raf V600E, and comparing them to their wild-type counterparts, allows researchers to gain a deeper understanding of the mechanisms by which frequently mutated proteins contribute to increased melanoma growth.

Similar to the gene often mutated in encoding the B-Raf protein, the CDKN2A gene that encodes the p16^INK4A protein is also commonly mutated somatically, as well as through germline alterations (Danishevish et al., 2023; Kimura et al., 2021; Ming et al., 2020; Zuo et al., 1996). However, compared to B-Raf V600E, the mechanism by which mutant p16^INK4A drives melanoma is less well understood. The prevailing theory is that p16^INK4A, a tumor suppressor protein, functions solely to inhibit CDK4 and CDK6, thereby preventing cell cycle progression (Cánepa et al., 2007; CDKN2A, 2018; Zuo et al., 1996). However, recent research using pharmacologic inhibitors of CDK4 and CDK6 has shown little to no efficacy in preventing melanoma growth (Hung et al., 2025). This indicates that the p16^INK4A interaction with CDK4 and CDK6 may not be the only, or even the primary, mechanism by which the mutated variants of p16^INK4A drive melanoma growth. This suggests that other mechanisms must be the primary driving force behind hereditary melanoma’s uncontrolled proliferation and metastasis.

Two proteins also involved in the cell cycle, IMPDH2 and EZH2, have been of unique interest in the research community. Recent research involving these proteins suggests a possible alternative mechanism to melanoma progression (Kuser et al., 2021; Judson-Torres Lab, 2026, Manuscript in preparation). By understanding the roles of these proteins in cellular function, their interactions, and their involvement in melanoma, future detection strategies for melanoma can be pioneered.

Studying melanoma is inherently challenging because, unlike the predictable mechanisms of healthy melanocytes, melanoma dysregulates these mechanisms in unexpected ways that are difficult to explain. Furthermore, these seemingly minor point mutations in the genetic code result in drastic changes that enable cancers to evade the immune system, resist programmed cell death, outcompete healthy cells, bypass cellular checkpoints, and adapt to overcome treatments (Eddy & Chen, 2020).

Significant Findings & Interacting Proteins

The primary function of p16^INK4A is to act as a tumor suppressor protein by preventing cell proliferation through binding to and inhibiting the activity of CDK4 and CDK6 (Cánepa et al., 2007; CDKN2A, 2018; Zuo et al., 1996). EZH2 is most well-known for its role in forming a polycomb repressive complex on the CDKN2A gene, thereby silencing the expression of p16^INK4A (von Schimmelmann et al., 2016). Cytoplasmic IMPDH2 plays a crucial role in guanine nucleotide biosynthesis. Nuclear IMPDH2 plays a role in regulating cell survival in response to DNA damage (Espinar et al., 2024). EZH2 and IMPDH2 can bind and translocate to the cytoplasm, where EZH2 enhances IMPDH2’s active state enzymatic activity in nucleotide biosynthesis (Kuser et al., 2021). Ongoing research is beginning to reveal potential interactions and cellular impacts between germline variants of p16^INK4A and the mechanism of melanoma progression involving IMPDH2 and EZH2 (Judson-Torres Lab, 2026, Manuscript in preparation).

Protein Interactions Overview

Cyclin Dependent Kinase Inhibitor 2A Gene & p16 (CDKN2A & p16^INK4A)

The CDKN2A Gene is located on chromosome 9, band p21.3 (CDKN2A Gene – GeneCards | CDN2A Protein | CDN2A Antibody, n.d.; Hussussian et al., 1994). This gene encodes two tumor suppressor proteins via alternative splicing, as seen in Figure 1 (Kalatskaya, 2016). p16^INK4A is primarily known for its role in regulating the cell cycle by binding to and inhibiting Cyclin Dependent Kinases 4 and 6 (CDK4 & CDK6), while p14^ARF is a regulatory protein of p54 (Cánepa et al., 2007; CDKN2A, 2018; Stott, 1998). It has been well established that germline pathogenic variants and homozygous deletions of the CDKN2A gene predispose individuals and families to various cancers, with melanoma being the most common (Hussussian et al., 1994; Jensen et al., 2022; Soufir et al., 1998). In melanoma, loss-of-function CDKN2A alterations have been seen in 50% of primary melanomas, in over 75% of metastatic melanomas, and in the germline of 40% of families with a predisposition to cutaneous melanoma (Ming et al., 2020). Notably, the majority of alterations affect either p16^INK4A or both p16^INK4A and p14^ARF (Ming et al., 2020). It is not common for a mutation to solely affect p14^ARF, making p16^INK4A a primary target for research (Ming et al., 2020). Thus, CDKN2A mutations are most commonly found in Exon 1β, affecting only p16^INK4A, or in Exon 2, affecting either p16^INK4A or both p16^INK4A and p14^ARF (Newton et al., 2000).

Canonically, mutations to p16^INK4A impair its ability to bind to and inhibit CDK4 and CDK6, leading to failure of cell-cycle arrest (Byeon et al., 1998). However, several notable mutations have been identified that retain CDK4 and CDK6 binding, yet still result in reduced cell cycle inhibition (Becker et al., 2001; Li et al., 2019). This strongly suggests other p16^INK4A binding interactions that result in failed cell cycle inhibition could lead to melanoma progression (Becker et al., 2001).

One possible alternative interaction could include p16^INK4A regulating RNA polymerase transcription (Serizawa, 1998). p16^INK4A achieves this by inhibiting the phosphorylation of the CTD domain of RNA polymerase II through the transcription factor TFIIH (Serizawa, 1998). Furthermore, in addition to inhibiting CDK4 and CDK6 phosphorylation, p16^INK4A is also seen to inhibit CDK7 and CTD Domain Kinase activity, thereby further controlling cell cycle progression at the G1/S boundary (Nishiwaki et al., 2000). Additionally, p16^INK4A can bind to NF-κB transcription factors, thereby inhibiting the expression of numerous other genes that regulate the cell cycle (Wolff & Naumann, 1999).

Inosine Monophosphate Dehydrogenase 2 (IMPDH2)

Human cells contain both IMPDH1 and IMPDH2. Though IMPDH1 is consistently expressed in all human cells, IMPDH2 is expressed at higher levels in proliferating cells (Hager et al., 1995; Keppeke et al., 2018; Messina et al., 2004), making IMPDH2 a critical protein to examine for its unique role in cellular proliferation. Furthermore, IMPDH1 was only significantly expressed in retinal cells, whereas IMPDH2 was expressed abundantly in every other measured cell type, as seen in Figure 3 (Kofuji & Sasaki, 2020). Additionally, in mouse experiments, loss of IMPDH1 was only correlated with minor vision defects, while loss of IMPDH2 was lethal, further emphasizing the significance of IMPDH2 (Aherne, 2004; Gu et al., 2022).

Figure 4 illustrates IMPDH2’s functions in both the nucleus and the cytoplasm (Kozheznikova et al., 2012). While this review will primarily highlight the cell-cycle cytosolic function of IMPDH2, it is important to note IMPDH2’s nuclear function. Within the nucleus, IMPDH2 acts as a transcription factor by binding to cytosine and thymine-rich single-stranded DNA elements (Kozheznikova et al., 2012). As a transcription factor, IMPDH2 inhibits the expression of histone genes and the E2f gene, a key regulator of cellular proliferation (Kozheznikova et al., 2012). In addition to acting as a transcription factor, IMPDH2 also controls the DNA damage response by modulating PARP1 enzymatic activity (Espinar et al., 2024). IMPDH2 modulates PARP1 by modulating nuclear NAD+ concentrations. In high NAD+ concentrations, PARP1 detects and signals the need to repair portions of damaged DNA. When IMPDH2 reduces NAD+ concentrations, PARP1 inhibits glycolysis by blocking the activity of HK proteins, leading to PARP1 cleavage and cell death (Espinar et al., 2024; Murata et al., 2019).

Similar to p16^INK4A, cytosolic IMPDH2 is another essential protein, as it is related to cell cycle progression. IMPDH2 is most well-known for its role in guanine nucleotide biosynthesis and purine metabolism (Chaiyawat et al., 2021; Kuser et al., 2021). IMPDH2 catalyzes the oxidation of IMP to XMP, regulating the rate-limiting step in guanine synthesis (Buey et al., 2022; Messina et al., 2004). GTP, a product of IMPDH2, forms a negative feedback loop by allosterically inhibiting IMPDH2 (Johnson & Kollman, 2020); however, GTP concentrations are seen to be highly elevated in melanomas (Kofuji & Sasaki, 2020), suggesting a mechanism of increased IMPDH2 activation.

The structure of IMPDH2 plays a crucial role in cellular proliferation (Keppeke et al., 2018), suggesting a mechanism by which specific proteins may enhance or reduce IMPDH2 activity. The IMPDH2 monomers form stable tetramers, which reversibly assemble into filaments containing functional octamer subunits in response to cellular demands, particularly depletion of guanine nucleotide concentrations, especially during proliferation (Johnson & Kollman, 2020; Keppeke et al., 2018). The IMPDH2 octamer binds two tetramers via the Bateman domain. The catalytic site is located inside the octamer assembly. The octamer must open or expand to allow the entry of a substrate, then close before reopening to release the product. IMPDH2 contains three allosteric sites that bind adenine and guanine nucleotides (Buey et al., 2022). Figure 5 illustrates the binding arrangements of adenine and guanine and the observed confirmation of the octamer (Johnson & Kollman, 2020). As shown in Figure 5, excess guanine nucleotides bind to and close the octamer, inhibiting IMPDH2 from executing its enzymatic activity through negative feedback regulation.

Knowing that IMPDH2’s primary function is guanine nucleotide biosynthesis, it is notable that guanine nucleotide concentrations are over 200% higher in tumor cells than in regular cells, while adenosine nucleotide concentrations are only about 20% higher (Traut, 1994). This increase is a direct result of the regulatory actions of IMPDH2 (Kofuji & Sasaki, 2020). One possible regulatory mechanism is a binding partner of IMPDH2: EZH2 (Kuser et al., 2021).

Enhancer of Zeste Homolog 2 (EZH2)

Similar to IMPDH2, EZH2 also has nuclear and cytosolic functions within the cell (Kuser et al., 2021). EZH2’s nuclear functions are well known, as a methyl transferase that contributes to the formation of the Polycomb Repressive Complex 2 (PRC2), as shown in Figure 6 (von Schimmelmann et al., 2016). PRC2 is a repressive complex known for silencing genes that promote cell death (von Schimmelmann et al., 2016). In cells that had a PRC2 deficiency, target genes of PRC2 were upregulated, including PMAIP1, BID, IGFBP3, CDKN2A, and CDKN2B (Nakagawa et al., 2014; von Schimmelmann et al., 2016).

PMAIP1 encodes for Noxa proteins that play a role in programmed cell death via p53- mediated apoptosis (Database, 2025). Similar to PMAIP1, BID is also part of the BCL-2 family of proteins, which results in cell death (Database, 2024). BID plays a role in the initiation of apoptosis. IGFBP3 encodes growth factors linked to cancer progression and metastasis. Complete loss of this gene results in increased proliferation (colony formation) and invasiveness (metastasis) (“IGFBP3 Insulin-like Growth Factor Binding Protein 3 [(Human)],” 2021).

Finally, CDKN2A and CDKN2B encode the tumor suppressor proteins p16^INK4A and p14^ARF, and p15^INK4B, respectively (Xia et al., 2021). Thus, increased cellular concentrations of EZH2, as observed in human malignancies, are correlated with decreased p16^INK4A concentrations. Consequently, p16^INK4A concentrations were rescued by EZH2 inhibition (Nakagawa et al., 2014).

EZH2 & IMPDH2 Interactions

The cytosolic roles of EZH2 are less well understood than its nuclear roles; however, a recent study by the Mark Shackleton Lab at Monash University suggests a relationship between EZH2 and IMPDH2, illustrated in Figure 7 (Kuser et al., 2021). It is believed that IMPDH2 binds to EZH2 in the nucleus and translocates into the cytoplasm. Within the cytoplasm, EZH2 enhances the activity of IMPDH2, increasing cellular concentrations of guanine nucleotides (Kuser et al., 2021). Guanine nucleotides are linked with G-protein mechanisms (Wennerberg et al., 2005) and actomyosin cytoskeletal regulation. With increased guanine nucleotides, cells became significantly more motile through increased actomyosin contractility. Additionally, the lab observed nuclear enlargement due to enhanced ribosome biogenesis (Kuser et al., 2021). When the drug, Sapponone A, was added to the cells, interference of IMPDH2 and EZH2 binding resulted, and cellular proliferation and motility were reduced, resulting from decreased ribosome biogenesis and decreased myoactin contractility, respectively (Kuser et al., 2021). This study identified a noncanonical binding mechanism of EZH2, which led to increased IMPDH2 activity and enhanced melanoma characteristics.

Conclusion

In human melanomas, EZH2, IMPDH2, and guanine nucleotides are seen at higher concentrations than in healthy melanocytes (Kuser et al., 2021; Mahmoud et al., 2016; Nakagawa et al., 2014; Traut, 1994). Alternatively, p16^INK4A is typically downregulated in melanomas (Nakagawa et al., 2014); however, p16^INK4A concentrations are not a reliable predictor of melanoma (Inoue & Fry, 2018). EZH2 silences both tumor suppressor genes and apoptotic genes (von Schimmelmann et al., 2016). A loss of EZH2 in a cell is associated with increased expression of apoptotic genes, leading to enhanced programmed cell death (von Schimmelmann et al., 2016) and decreased aggressiveness and proliferation of human malignancies (Nakagawa et al., 2014). Additionally, IMPDH2 and EZH2 can bind and translocate to the cytoplasm, where EZH2 is seen to increase the enzymatic output of IMPDH2 (Kuser et al., 2021). Increased guanine concentrations resulting from IMPDH2 activity are associated with nuclear enlargement, attributed to enhanced ribosome biogenesis, as well as increased cellular motility and invasiveness, due to increased actomyosin contractility (Bianchi-Smiraglia et al., 2021; Kuser et al., 2021; Pelletier et al., 2020). However, inhibition of EZH2 does not solely reduce melanoma proliferation and metastasis (Kuser et al., 2021). Furthermore, p16^INK4A interactions with CDK4 and CDK6 are not primarily linked to melanoma proliferation and metastasis, indicating potential noncanonical p16^INK4A binding partners as the primary driving force behind melanoma (Becker et al., 2001).

Research in Progress & Future Directions

To achieve the long-term goal of developing a method to screen for and detect melanomas and other cancers in their earliest stages when treatment is most successful, it is critical to understand the mechanisms behind how melanoma evolves, adapts, evades the immune system, proliferates, and metastasizes. This can be achieved by studying the effects of various genetic mutations and identifying the mechanisms altered in melanoma, as compared to healthy melanocytes. A thorough understanding of the complex and interconnected relationships between multiple genes, proteins, and mechanisms is crucial for the development of future treatments and diagnostic systems.

A great deal is known about the CDKN2A gene, the p16^INK4A protein, the IMPDH2 protein, and the EZH2 protein, but there is certainly more to discover. Further research is required to fully understand how p16^INK4A, IMPDH2, and EZH2 interact. By researching how various CDKN2A mutations affect p16^INK4A and how these structural mutations impact the noncanonical pathways of p16^INK4A, we can gain a deeper understanding of the mechanism driving melanoma and why germline CDKN2A mutations in hereditary familial melanoma predispose families to melanoma occurrences, giving rise to more effective medical intervention.

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