ERK Hyperactivation Serves as a Unified Mechanism of Escape in Intrinsic and Acquired CDK4/6 Inhibitor Resistance in Acral Lentiginous Melanoma

Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in > 60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression are a unified mechanism of both intrinsic and acquired CDK4i/6i resistance. MEK and/or ERK inhibition increases CDK4i/6i efficacy in a patient-derived xenograft (PDX) model of ALM and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach to improve outcomes for patients with advanced ALM.


Introduction
Acral lentiginous melanoma (ALM) constitutes a distinct disease relative to other forms of cutaneous melanoma (CM) (i.e., super cial spreading, nodular, lentigo maligna) due, in part, to a different cell of origin (volar versus non-volar skin melanocytes) (1), the de ning acral skin sites they arise on, and a complex genomic landscape (2). Although patients with metastatic ALM suffer worse outcomes relative to patients with other subtypes of CM, the underlying molecular mechanisms responsible for ALM initiation, progression, and therapy resistance remain poorly understood (3,4). Existing standard-of-care targeted therapies (i.e., BRAF inhibitors) for CM are not available to the majority of advanced ALM patients due to a lower frequency of BRAF V600E/K mutations (20% versus 50% in other forms of CM (2)).
Further, targeted therapy is not as effective in ALM patients with BRAF V600E/K mutations (5). Additionally, the e cacy of immune checkpoint blockade (ICB) is less effective and remains poorly understood in ALM (2,5,6). Therefore, new therapy strategies tailored to the ALM patient population are critically warranted.
Recent genetic characterization of ALM patient tumor tissue has identi ed cyclin-dependent kinase 4 (CDK4)-pathway (e.g., CDK4 ampli cation, CDK6 ampli cation, CCND1 ampli cation, P16 INK4A loss) alterations in 53-82% of ALM cases (2, 7), with CDK4 ampli cation and P16 INK4A loss each independently serving as predictors of shorter patient overall survival. CDKs propel cell cycle progression and their frequent dysregulation in cancer contributes to the uncontrolled cellular proliferation, which is regarded as one of the hallmarks of cancer. ALM cell lines and patient-derived xenograft (PDX) models with CDK4 pathway alterations were reported to exhibit elevated in vivo sensitivity to CDK4/6 inhibitors (CDK4i/6i) (7), which provided rationale for the rst phase II clinical trial (NCT03454919) of palbociclib in patients with advanced ALM whose tumors exhibit CDK4-pathway aberrations (8). Unfortunately, most patients did not bene t from palbociclib monotherapy, and the median progression free survival (mPFS) was only 2.2 months. The few patients who did respond did not often experience durable tumor control, suggesting that both intrinsic-and acquired-resistance mechanisms to single-agent palbociclib arise rapidly. To date, mechanisms of resistance to CDK4i/6i in ALM remain poorly understood.
Here, we identify hyperactivation of the mitogen-activated protein kinase (MAPK) pathway and elevated cyclin D1 as functional drivers of intrinsic and acquired CDK4i/6i resistance. The MAPK pathway sustains cyclin D1 levels, and we nd in the context of intrinsic and acquired resistance to CDK4i/6i, elevated MAPK activity promotes ALM addiction to cyclin D1, which can be overcome with use of the clinical MEK inhibitor trametinib or genetic silencing of cyclin D1. Altogether, these ndings represent the seminal report of a uni ed intrinsic and acquired CDK4i/6i resistance mechanism in ALM and conclude the addition of a MEK inhibitor may increase the durability of rst-and second-line CDK4i/6i therapy in patients with advanced ALM.

Results
Genetic status of CDK4-pathway nodes does not predict protein expression or CDK4i/6i durability in ALM.
The current strategy for clinical use of CDK4i/6i in patients with advanced ALM rests upon the genetic status of CDK pathway nodes, in part due to recent evidence that Cdk4 and/or P16 INK4a copy number status may be of prognostic signi cance for ALM patients (7); stemming from this study, only patients with CDK4 gain, CCND1 gain and/or CDKN2A loss were eligible for treatment with palbociclib (8). In an independent analysis of a separate ALM patient cohort, we nd no signi cant correlation between the overall survival of ALM patients based off of Cdk4 gain or P16 INK4a loss than those without Cdk4 gain or P16 INK4a , respectively (Supplemental Fig. 1A, 1B (9)). These data suggest the prognostic signi cance of CDK4 pathway nodes may differ across patient cohorts.
We next characterized the relationship between copy number variations, mRNA levels, and baseline protein expression of CDK4 pathway nodes across a genetically diverse panel of human ALM and non-ALM cell lines (Fig. 1A, 1B). In agreement with clinical observations, CDK4 pathway nodes (CDK4, CDK6, CCND1, CDKN2A, CDKN2B) were highly dysregulated across our ALM models with evidence of CCND1, a key activator of CDK4 and CDK6, also being elevated at the protein level in ALM versus non-ALM models ( Fig. 1B). Notably, there was no consistent agreement between the genomic information and protein expression of CDK4, CDK6, or cyclin D1 in our ALM panel. ALM cell lines with CDK4, CDK6, or CCND1 genomic and transcriptomic ampli cations did not robustly display elevated CDK4, CDK6, or cyclin D1 protein expression relative to ALM models with normal gene copy numbers, respectively (Supplemental Fig. 1C, 1D, 1E). Analysis of the TCGA to understand the relationship between copy number status, mRNA level, and protein expression for cyclin D1 in patients with super cial spreading melanoma (CDK4 and CDK6 protein expression unavailable) also revealed no correlation between genomic or transcriptomic status with protein expression (Supplemental Fig. 1F). This may serve as a cautionary note for the identi cation of ALM patients who may bene t from CDK4i/6i based solely on tumor sequencing, which may prevent patients with elevated CDK4/CDK6 protein expression but no clear evidence of copy number variation in CDK4/6 pathway genes from treatment.
CDK6 protein expression has been recently demonstrated to indirectly predict for sensitivity to CDK4/6 inhibition in ER + breast cancers, non-small cell lung carcinomas, colorectal carcinomas, and super cial spreading melanomas (13). In contrast, an analysis of correlations for ALM sensitivity to CDK4i/6i revealed that CDK6 expression trends (p = 0.087) directly with palbociclib sensitivity in ALM (Supplemental Fig. 1I). The expression of CDK4 (p = 0.72) and Cyclin D1 (p = 0.6) did not correlate with ALM sensitivity to CDK4i/6i (Supplemental Fig. 1J, 1K). Alongside the rst phase II clinical trial results of CDK4i/6i treatment in patients with advanced ALM, it was proposed that low MCM7 expression and SH2B3 ampli cation serve as predictive biomarkers of poor response to CDK4i/6i (8). We nd across our ALM cell line panel that sensitivity to CDK4i/6i with abemaciclib trended, albeit not signi cantly (p = 0.057), with baseline expression of MCM7 (Supplemental Fig. 2A,2B). In contrast, we did not observe signi cant relationships between palbociclib and ribociclib sensitivity with MCM7 expression, nor did we observe a correlation between any of the 3 clinically available CDK4i/6i tested with SH2B3 expression (Supplemental Fig. 2B,2C). We next put into context our CDK4i/6i data with recent reports of potential oncogenic drivers of ALM. LZTR1, an adaptor for Cullin 3 ubiquitin ligase complexes, was proposed to serve as a driver of ALM aggressiveness (9). No signi cant relationship emerged between LZTR1 expression and CDK4i/6i sensitivity (Supplemental Fig. 2D). Further, CRKL, a signaling adaptor protein in pathways including the IGF1R-PI3K axis, was also recently proposed to serve as an oncogenic driver of ALM (14). We also did not observe a signi cant correlation between CRKL expression and CDK4i/6i sensitivity (Supplemental Fig. 2E). Altogether, these data demonstrate that the protein expression of CDK4, CDK6, and cyclin D1 do not correlate with the respective gene copy number status, and the sensitivity of ALM cells to single-agent CDK4i/6i does not correlate to: a) CDK4, CDK6, or CCND1 ampli cation or protein expression, b) reported CDK4i/6i sensitivity biomarkers (MCM7, SH2B3), or c) proposed ALM oncogenic drivers (CRKL, LZTR1).
Loss of DUSP4 expression following CDK4i/6i promotes ERK activation and drives intrinsic resistance via cyclin D1. Improvement in the e cacy of CDK4i/6i with inhibitors of the MAPK pathway has been reported in prostate adenocarcinoma (15), super cial spreading melanoma (16, 17), and uveal melanoma (18); however, a mechanistic connection between CDK4/6 and MAPK pathway activity has not been previously explored. We next investigated the MAPK pathway following CDK4i/6i in our ALM system and observe that although acute palbociclib treatment led to reduced activity of downstream CDK4/6 substrates (pRb, FOXM1) and E2F effectors (PLK1, cyclin A), a robust hyperactivation of MAPK signaling (pERK) and increased downstream cyclin D1 expression were observed across our ALM panel ( Fig. 2A). Hyperactivation of pERK and increased cyclin D1 expression were also observed following pharmacological inhibition of CDK4/6 with abemaciclib, ribociclib and genetic silencing of CDK4/6 (Supplemental Fig. 3A, 3B).
We next tested whether targeting the MAPK pathway with a MEK1/2 inhibitor (MEKi, trametinib) could ablate intrinsic CDK4i/6i resistance. Combination treatment with MEKi and CDK4i/6i decreases cell cycle proteins (FOXM1, p-Rb, cyclin D1, PLK1) and induces apoptosis (cleaved PARP) to a greater extent than what was achievable by either compound as a single-agent (Fig. 2B). Targeting the MAPK pathway at the level of ERK also increased the cell cycle arrest capacity of CDK4i/6i (Supplemental Fig. 3C). Combination treatment with MEKi and CDK4i/6i increased the 3D cytotoxicity in ALM spheroids ( Fig. 2C) relative to single-agent CDK4i/6i treatment alone. Further, concurrent MEKi + CDK4i/6i conferred the greatest antitumor durability in long-term colony formation assays (Fig. 2D), and most e ciently reduced the subpopulation of EdU + ALM cells relative to single-agent treatment alone (Fig. 2E).
It was previously reported that reactivation of the MAPK pathway following BRAFi in BRAF V600E mutant super cial spreading melanomas was driven, in part, by reduced expression of proteins that negatively regulate the pathway, including members of the Sprouty (SPRY) dual speci city phosphatase (DUSP) family (19). In our ALM cell line panel, we observe that CDK4i/6i treatment decreases protein expression of DUSP4 levels, not SPRY2 or DUSP6 ( Fig. 2A, Supplemental Fig. 3D). We next tested the hypothesis that alterations in DUSP4 protein levels could be contributing to the hyperactivation of the MAPK pathway following CDK4i/6i. Overexpression of DUSP4 ablates the activation of ERK and induction of cyclin D1 expression following CDK4i/6i (Fig. 2F), and increases the antiproliferative e cacy of CDK4i/6i, as evidenced by the greatest reduction of cell cycle machinery and improved inhibition of EdU positivity relative to CDK4i/6i treatment alone (Fig. 2G).
The MAPK pathway has been experimentally shown to regulate cyclin D1 in melanocytes and BRAF V600E super cial spreading melanoma (20), however, the connection between MAPK activity and cyclin D1 expression has not yet been established in ALM. Growing ALM cells in nutrient-replete media following serum starvation induces MAPK activity (pERK, pRSK) and downstream cyclin D1 expression, which could be blocked using MEKi or ERKi (VX-11e), demonstrating the MAPK pathway, at least in part, regulates cyclin D1 expression in ALM (Supplemental Fig. 3E). We next tested the hypothesis that ERK hyperactivation following CDK4i/6i preserves cellular proliferation by promoting cyclin D1 expression. Genetic silencing of cyclin D1 increased the cell cycle arrest potential of CDK4i/6i, evidenced by further decreased protein expression of pRb, PLK1, and FOXM1 (Fig. 2H). Genetic silencing of cyclin D1 also decreased the EdU + subpopulation relative to what CDK4i/6i alone could achieve (Fig. 2I). In summary, these data demonstrate ALM cells adaptively escape single-agent CDK4i/6i by hyperactivating the MAPK pathway via reduced DUSP4 expression. The hyperactivation of ERK activity maintains the proliferative capacity of ALM cells treated with CDK4i/6i by promoting cyclin D1 expression.
Activation of ERK in the context of acquired CDK4i/6i-resistance has not been reported in melanoma, however evidence in the breast cancer literature proposes a role for de novo HER2 mutations in estrogen receptor positive (ER + ) breast cancers (21). We sequenced for HER2 mutations and copy number variations (CNVs) in our CDK-R models with acquired CDK4i/6i resistance versus their respective therapy naïve parental, and observe no new HER2 mutations or copy number gains (Supplemental Fig. 4B). In addition, evidence in hormone receptor positive (HR + ) breast cancers suggest potential roles for upstream receptor overexpression (FGFR2) and de novo mutations in RAS, AKT1, AURKA, CCNE2, and/or ERBB2 in the activation of ERK following acquired CDK4i/6i resistance (22). We sequenced for AKT1, FGFR2, FGFR3, FGFR4, and NRAS mutations and CNVs in our CDK-R models versus their respective therapy naïve parental counterparts, and observe no new mutations in these genes. We observed a copy number gain of NRAS in the WM4324 CDK-R cells versus its respective parental. No other new copy number gains were observed (Supplemental Fig. 4B).
Elevated cyclin D expression was not observed in CDK-R cells relative to their respective parental cell lines. However, in concordance with the role of cyclin D1 in driving intrinsic CDK4i/6i-resistance, genetic silencing of cyclin D1 also resensitized CDK-R cells to palbociclib as evidenced by reduced expression of pRb, FOXM1, and PLK1 (Fig. 3J) and depletion of EdU + cells (Fig. 3K). Altogether, these results indicate that hyperactivation of the MAPK pathway drives acquired CDK4/6 inhibitor resistance, in part, through sustained cyclin D1 expression.
MEKi increases the in vivo e cacy of CDK4i/6i in therapy naïve and acquired CDK4i/6i-resistant ALM PDX.
To assess the utility of targeting MEK to increase the in vivo e cacy of CDK4i/6i, we implanted the WM4223 patient-derived xenograft (PDX) model, derived from a biopsy of a metastatic ALM that originated in the left foot of a 73 year old male patient, into NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice (Fig. 4A). After 1-2 weeks, tumors were palpable and mice were treated via oral gavage with vehicle control, palbociclib (25 mg/kg), trametinib (0.3 mg/kg) or the combination of palbociclib plus trametinib. Palbociclib and trametinib each conferred signi cant anti-tumor activity as single-agent treatments; however, the greatest therapeutic bene t was observed in mice receiving combination palbociclib plus trametinib treatment (Fig. 4B, Supplemental Fig. 5A). The ALM cell line YUSEEP, derived from the left heel, was also implanted in NSG mice and treated with vehicle control, palbociclib, trametinib, of the combination of palbociclib plus trametinib once tumors were palpable. Combination palbociclib plus trametinib treatment again conferred signi cantly greater antitumor activity relative to what could be accomplished by the single-agents alone (Fig. 4C, Supplemental Fig. 5B). Concurrent treatment with palbociclib and trametinib resulted in the greatest inhibition of cell cycle machinery (i.e., pRb, FOXM1, PLK1, cyclin D1) in lysate collected from a subset of tumor-bearing mice sacri ced after 3 days of treatment (Supplemental Fig. 5C) and conferred the greatest decrease of Ki67 staining in tumor tissue (Fig. 4D). At treatment endpoint for the WM4223 in vivo study (day 50), tumor tissue was characterized by reverse-phase protein array (RPPA) to identify the mechanism(s) of action underlying the long-term therapeutic e cacy of MEKi + CDK4i/6i relative to single-agent therapy (Fig. 4E). A total of 46 proteins were signi cantly differentially expressed between vehicle control tumors and combination palbociclib plus trametinib treated tumors that were not observed in the single-agent treated tumors (Supplemental Table). Interestingly, a signature indicative of reduced DNA repair capacity (decreased CENP-A, PARP and RPA32 protein expression) correlated with increased double strand DNA breaks in tumors treated with combination palbociclib plus trametinib (Fig. 4F, Supplemental Fig. 5D). Combination palbociclib plus trametinib treatment also resulted in the greatest cell cycle arrest signature (as seen by decreased cyclin B1, PLK1 and E2F1 protein expression (Fig. 4G)), and most signi cant induction of apoptosis (increased BAK, BID, BIM, caspase 7 cleavage, and reduced BCL2A1 protein expression) (Fig. 4H).
To assess the utility of targeting MEK to overcome acquired resistance to CDK4i/6i in vivo, YUSEEP-CDK-R cells chronically treated with palbociclib in vitro were implanted in NSG mice (Fig. 4I). In line with observations of reversible (non-heritable) mechanisms of acquired resistance to palbociclib in cholangiocarcinoma cells (23), YUSEEP-CDK-R cells that expanded in vivo during a > 3 week drug holiday again exhibited sensitivity to single-agent palbociclib ( Figure J). Although vehicle treated YUSEEP-CDK-R tumors exhibited a greater growth rate relative to palbociclib treated, the vehicle treated YUSEEP-CDK-R tumors grew slower than the vehicle treated parental YUSEEP tumors (Fig. 4C). Treatment with MEKi signi cantly blunted tumor growth of CDK4i/6i-resistant ALM cells, and the combination of CDK4i/6i + MEKi resulted in the greatest antitumor activity and decrease in cell cycle proteins (pRb, cyclin D, PLK1, FOXM1) relative to what could be achieved by the single-agents alone (Fig. 4J, Supplemental Fig. 5E, 5F). These ndings indicate that continuous pressure from CDK4i/6i is required to maintain maximal vulnerability to MEKi. Altogether, these results underscore the importance of the MAPK pathway in driving intrinsic and acquired CDK4i/6i resistance in ALM and the translational potential of MEKi to increase the in vivo antitumor activity of CDK4i/6i against therapy naïve and CDK4i/6i-resistant ALMs, via increased DNA damage, cell arrest and tumor cell death (Fig. 4K).

Discussion
ALM represents a distinct disease from other forms of CM and the therapy resistance landscape that limits the curability of patients with advanced ALM is poorly understood. ALM remains the most lethal form of CM and existing therapies effective in other forms of CM (e.g., BRAFi, ICB) are not as active in ALM for reasons that are poorly understood. Genetic alterations in the CDK4-pathway occur in the majority of ALM cases, and there is preclinical evidence that ALM cells with CDK pathway alterations are highly sensitive to CDK4i/6i (7). Unfortunately, these ndings have not translated clinically, with single agent palbociclib conferring a mPFS of less than 3 months suggesting mechanisms of resistance blunt e cacy. Enthusiasm for the clinical testing of CDK4i/6i-containing therapy strategies in patients with other forms of melanoma has grown in the past decade stemming from a) observations of CDK4pathway alterations in > 90% of CM cases, b) evidence of downstream activation of CDK4/6 as a consequence of elevated MAPK pathway signaling, and c) a role for CDK4 in ICB resistance (24). Clinical trials have commenced incorporating CDK4i/6i plus MEKi in NRAS mutant CM (25, 26), CDK4i/6i plus BRAFi/MEKi in BRAF mutant CM (27), and most recently CDK4i/6i plus immune checkpoint blockade (28). Mechanisms of intrinsic resistance to CDK4i/6i have been reported in other forms of melanoma, but no studies have reported CDK4i/6i resistance mechanisms in ALM models.
Here, we report a seminal investigation into the underlying intrinsic and acquired resistance mechanisms that blunt the therapeutic e cacy of CDK4i/6i in ALM. Our study nds that despite the robust inhibition of CDK4/6 substrates and E2F1 effector proteins by single-agent CDK4i/6i, most tumor cells remain viable following treatment with non-physiologically high concentrations of CDK4i/6i that results in a temporary cytostatic response in vitro and in vivo followed by reactivation of the cell cycle. A recent study proposed a role for elevated CDK6 expression in the intrinsic resistance of NSCLC and super cial spreading melanomas to CDK4i/6i, however we nd the opposite in ALM models whereby CDK6 expression directly trends with CDK4i/6i e cacy. It has been proposed that ALM models with CDK4 pathway alterations (de ned as CDK4 gain, CCND1 gain, CDKN2A loss) exhibit elevated sensitivity to CDK4i/6i (7), however, we could not corroborate a differential sensitivity between ALM models based off the status of the CDK4 pathway. This lack of correlation was also reported during the rst phase II clinical trial of palbociclib in ALM patients, whereby the authors concluded "neither the genetic status nor the protein expression level of CDK4, CCND1, or CDKN2A was signi cantly associated with clinical response to palbociclib" (8). Further, we nd a notable discordance between the genetic status of the CDK4 pathway and the protein expression of key nodes (e.g., CDK4, CDK6), suggesting both genetic and proteomic characterization of the CDK4 pathway should be performed when stratifying patients for treatment with CDK4i/6i.
We examined the role of MAPK pathway signaling in the context of intrinsic and acquired CDK4i/6i resistance, in part, due to evidence of possible synergies of combination CDK4i/6i plus MAPK pathway inhibitor treatment in breast cancer and super cial spreading melanomas with either NRAS or BRAF mutations. Our investigation identi ed a rapid hyperactivation of the MAPK pathway occurs within three days of CDK4i/6i treatment in the context of therapy naïve ALM, which drives downstream expression of cyclin D1. The MAPK pathway hyperactivation and increase in cyclin D1 expression each functionally drove intrinsic CDK4i/6i resistance, which could be reversed by either incorporating a clinically relevant MEK inhibitor (trametinib), an ERK inhibitor, or genetic silencing of cyclin D1. Of note, evidence of MAPK pathway hyperactivation following acute (0-72 hrs) CDK4i/6i treatment has not been reported previously in ALM or other melanoma subtypes. Although MAPK hyperactivation has been reported in other cancer types including head and neck (29) and luminal A breast (30) following acute CDK4i/6i, the underlying mechanism remains poorly understood. We here demonstrate that CDK4i/6i results in decreased expression of the negative regulator of ERK activity, DUSP4. Loss of DUSP4 is functional in the CDK4i/6iinduced activation of ERK, as DUSP4 overexpression ablates CDK4i/6i-induced ERK activation and increases the anti-proliferative activity of CDK4i/6i.
In the context of acquired resistance, we also observed a robust hyperactivation of the MAPK occurs across our panel of CDK-R cells relative to their respective parental lines. Elevated MAPK activity functionally drove resistance in CDK-R cells and could be reversed with the use a MEKi or ERKi. Notably, evidence in ER + breast cancer suggests de novo HER2 mutations can hyperactivate ERK activity in the context of acquired CDK4i/6i resistance. We con rmed in our paired parental and CDK-R ALM models the lack of de novo HER2 mutations following acquired CDK4i/6i resistance. Further, it has been observed by others that overexpression of FGFR2 and/or de novo mutations in RAS, AKT1, AURKA, CCNE, and ERBB2 could possibly contribute to MAPK activation following acquired CDK4i/6i resistance. We sequenced for AKT1, FGFR2, and NRAS mutations and CNVs in our CDK-R models with acquired CDK4i/6i resistance versus their respective therapy naïve parental line, and observe no new mutations in AKT1, FGFR2, or NRAS across our CDK-R and parental cell line pairs. We observed a copy number gain of NRAS in the WM4324 CDK-R cell versus its respective parental cell line.
Although there was no evidence of elevated cyclin D1 expression in CDK-R cells, genetic silencing experiments reveal CDK-R cells rely on cyclin D1 proliferation. In summary, our ndings de ne MAPK pathway plasticity as an underlying mechanism of intrinsic and acquired CDK4i/6i therapy resistance in ALM. Further, this body of work makes numerous clinically relevant observations for the treatment of patients with advanced ALM including that a) CDK4 and P16INK4A status does not robustly predict ALM patient survival, b) CDK4 pathway alterations do not predict ALM sensitivity to CDK4i/6i, c) CDK6 protein expression does not predict ALM sensitivity to CDK4i/6i, and d) genetic status (e.g., copy number variation) does not correlate with protein expression of CDK4 pathway nodes. These ndings provide the rationale to further investigate combination treatment of CDK4i/6i plus MEKi as rst-and second-line therapy in patients with advanced ALM.

Cell Culture and Reagents
Melanoma cell lines YUSEEP, YUHIMO, YUWERA, and YUCRATE were obtained from Ruth Halaban (Yale University) in 2020. Melanoma cell lines WM4235, WM4324, WM9, 1205Lu, WM989, WM983B, WM4380 and WM4258, as well as the PDX model WM4223 were obtained from Meenhard Herlyn (Wistar Institute) in 2020. All patient samples were collected under institutional review board (IRB) approval (31). Cell lines were tested for Mycoplasma biannually and authenticated using short-tandem repeat ngerprinting. All cell lines are cultured in RPMI-1640 (Corning,10-040-CM) supplemented with 5% fetal bovine serum (FBS; Cytiva, SH30109.03) in the presence of 5% CO 2 at 37°C. Commercially purchased compounds include palbociclib (SelleckChem, S1116), ribociclib (SelleckChem, S7440), abemaciclib (Apex Biotechnology, A1794), trametinib (SelleckChem, S2673), AZD6244 (SelleckChem, S1008) and VX-11e (SelleckChem, S7709). Immunoblotting, Cell Cycle Analysis, EdU Staining, and Fluorescent Microscopy Protein lysates were immunoblotted as previously described (32) with the following antibodies CDK4, CDK6, total Rb, phospho-Rb Ser807/811, PLK1, Cyclin A, Cyclin B1, Cyclin D1, β-Actin, FOXM1, PTEN, p16, phospho-ERK1/2 Thr202/204, total ERK1/2, and cleaved Parp from Cell Signaling Technology. Densitometric analysis was performed by utilizing the Gels function in ImageJ. Individual gel lanes were identi ed and manually outlined. The band intensity of each gel lane was then plotted by the ImageJ software and the subsequent peaks created were used to quantify the relative protein quantity. The software automatically calculates this based on the area under each peak. The results were then normalized to the protein quantity of β-Actin and control lanes. Fluorescent microscopy was performed as previously described (32) using the manufacturer's instructions from the EdU kit (Click-iT™ EdU Alexa Fluor™ 647 Imaging Kit, C10340. For cell cycle analysis, cells were plated at 1x10 5 per well in a 6-well plated and treated, as indicated. Floating and adherent cells were pooled, pelleted, washed with cold PBS and xed with 70% ethanol. Fixed cells were subsequently washed, resuspended in PBS, treated with RNase A solution and stained with propidium iodide (0.5 mg/mL, BioLegend, 421301) Cell cycle analysis was performed on Cytek™ NL-3000 and data were analyzed using FlowJo Software.

Cell Viability MTT Assay and Clonogenic Assay
For MTT assays, cells were at 2,000/well in 96-well plates and treated as indicated for 72 hours before thiazolyl blue tetrazolium bromide was added to growth medium, incubated for 4 hours at 37°C, solubilized and color was quanti ed on a 96 well plate reader (Synergy H1 microplate reader; BioTek) at the absorbance 570 nm. For clonogenic assays, cells were plated at 2-5x10 3 per well in a 6-well plate and treated twice a week for up to 4 weeks as indicated before colonies were stained with crystal violet. Plates were imaged and quanti ed by the Colony Area ImageJ plug-in. Individual wells were cropped by the software and thresholds were created automatically to remove the background. Manual cropping and thresholding was performed when image artifacts compromised the software's ability to properly identify the background. The Colony Measurer function was then used to quantify the percent area covered by cell colonies in each thresholded well.
Massively Parallel Sequencing DNA from cell lines was characterized by massively parallel sequencing using a custom-designed, targeted panel as previously described (31,33).

Reverse-Phase Protein Arrays
Proteins were isolated from tumor shears and cell lines, and RPPA analysis was performed as previously described (34). Prior antibody testing con rmed the speci city of each antibody, and direct correlation between RPPA and Western blotting results (data not shown). A logarithmic value was generated, re ecting the quantitation of the relative amount of each protein in each sample. Differences in relative protein loading were determined by the median protein expression for each sample across all measured proteins using data that had been normalized to the median value of each protein. The raw data were then divided by the relative-loading factor to determine load-corrected values. Logarithmic values for each protein were mean-centered to facilitate concurrent comparisons of different proteins (34).

In Vivo Experiments
Xenograft studies were performed using 6-8 week old NSG mice (Charles River Laboratories) in an Association for the Assessment and Accreditation of Laboratory Animal Care-accredited facility. WM4223 (5 x 10 5 ) cells or YUSEEP-CDK-R (3 x 10 5 ) cells were implanted subcutaneously into NSG mice and strati ed into the indicated treatment arms when tumors were palpable (~ 150 mm 3 ) to begin treatment.
Mice were treated with either palbociclib (25 mg/kg, oral gavage), trametinib (0.3 mg/kg oral gavage) or the combination of palbociclib and trametinib. Tumor sizes were measured every 2 days using digital calipers. Tumor volumes were calculated using the following formula: volume = 0.5 x (length x width 2 ).

TCGA correlation test
In the TCGA (The Cancer Genome Atlas) database, there are 89 skin cutaneous melanoma patients with paired open source RPPA cyclin D1 data and CCND1 mRNA data in primary tumor. Pearson correlation test was performed between cyclin D1 protein level and CCND1 mRNA TPM (transcript per million) level across 89 samples. The null hypothesis of the test is that there is no correlation between the two variables. P value larger than 0.05 indicates we cannot reject null hypothesis. There are 90 skin cutaneous melanoma patients with paired RPPA cyclin D1 data and copy number variation data of the primary tumor in the TCGA. Similarly, person correlation test was performed between cyclin D1 protein level and CCND1 copy number variation across these 90 patients.

Statistical Analysis
Data show the mean of at least 3 independent experiments. GraphPad Prism 9 statistical software was used to perform Student's t test where * indicates p < 0.05. Linear mixed models were used to estimate and compare tumor growth rates (mm 3 /day) between treatment groups.

Declarations Data availability
Copy number variation data of relevant genes for baseline Acral and non-Acral cell lines in Figure 1A, WM4223 tumor median centered protein RPPA data in Figure 4F  A panel of ALM models were treated with increasing concentrations of palbociclib, (E) ribociclib, or (F) abemaciclib for 72 hrs before cell numbers were quanti ed using MTT. Bars show S.E. mean. (G) A panel of ALM cell lines were treated with palbociclib for 3-4 weeks before colonies were xed and stained with crystal violet. Photographs are representative of three independent experiments and relative clonogenic survival quantitation is shown to the right. (H) WM4324 cells were treated with palbociclib (500 nM) for the time shown before EdU incorporation and imaging to assess cell proliferation. *p<0.05 and n³3 unless otherwise stated throughout panels.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. supplementarydataSeq.xlsx