Cyclin-dependent kinases 4 and 6 (CDK4/6) are crucial in regulating cell-cycle progression and cancer development. Targeting CDK4/6 has shown considerable promise in treating various cancers, including breast cancer. Despite significant therapeutic efficacy, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial hurdle in clinical practice. Using a coculture system, cytokine array, and quantitative high-throughput combinatorial screening, we discovered a mechanism by which the Runt-related transcription factor (RUNX) 1-platelet-derived growth factor (PDGF)-BB axis regulates palbociclib resistance in breast cancer cells. Specifically, RUNX1 functioned as a transcription factor to drive expression of PDGFB, leading to resistance to palbociclib by enhancing the Akt pathway and suppressing senescence. Furthermore, in resistant cells, RUNX1 was O-GlcNAcylated at serine 252 by O-GlcNAc transferase, resulting in the stabilization of RUNX1 by preventing ubiquitin-mediated degradation. Inhibition of the RUNX1-PDGF-BB axis by specific inhibitors overcame palbociclib resistance both in vitro and in vivo. Notably, the RUNX1-PDGF-BB axis was upregulated in resistant patient-derived xenograft lines and in patients with breast cancer following treatment with CDK4/6i. These findings not only unveil O-GlcNAcylation-mediated activation of a RUNX1-PDGF-BB pathway as a driver of palbociclib resistance but also provide clinical evidence supporting the repurposing of FDA-approved PDGFR inhibitors as a therapeutic strategy to treat patients with CDK4/6i-resistant breast cancer.Significance: RUNX1-PDGF-BB signaling drives resistance to CDK4/6 inhibition in breast cancer, providing the foundation to develop approaches to target the RUNX1-PDGF-BB axis to overcome CDK4/6 inhibitor resistance in breast cancer patients.

Breast cancer is the predominant malignancy with the majority of cases are characterized as HR+/HER2- subtype. Although cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have shown remarkable efficacy in treating this subtype when combined with endocrine therapy, the development of resistance to these inhibitors remains a significant clinical obstacle. Hence, there is an urgent need to explore innovative therapies and decipher the underlying mechanisms of resistance to CDK4/6i. In this study, we employed quantitative high-throughput combination screening (qHTCS) and genomics/proteomics approaches to uncover the molecular mechanisms driving resistance to CDK4/6i (palbociclib) in breast cancer. The comprehensive analyses revealed DDR1 as a potential factor implicated in mediating resistance to CDK4/6i. Specifically, DDR1 inhibition in combination with palbociclib exhibited remarkable synergistic effects, reducing cell survival signaling and promoting apoptosis in resistant cells. In-vivo xenograft model further validated the synergistic effects, showing a significant reduction in the resistant tumor growth. Exploration into DDR1 activation uncovered TFAP2C as a key transcription factor regulating DDR1 expression in palbociclib resistant cells and inhibition of TFAP2C re-sensitized resistant cells to palbociclib. Gene set enrichment analysis (GSEA) in the NeoPalAna trial demonstrated a significant enrichment of the TFAP2C-DDR1 gene set from patitens after palbociclib treatment, suggesting the possible activation of the TFAP2C-DDR1 axis following palbociclib exposure. Overall, this study provides crucial insights into the novel molecular landscape of palbociclib resistance in breast cancer, suggesting TFAP2C-DDR1 axis inhibition as a promising strategy to overcome resistance.

Secreted protein acidic and rich in cysteine (SPARC), a conserved secreted glycoprotein, plays crucial roles in regulating various biological processes. SPARC is highly expressed and has profound implications in several cancer types, including melanoma. Understanding the mechanisms that govern SPARC expression in cancers has the potential to lead to improved cancer diagnosis, prognosis, treatment strategies, and patient outcomes. Here, we demonstrate that histone deacetylase 10 (HDAC10) is a key regulator of SPARC expression in melanoma cells. Depletion or inhibition of HDAC10 upregulates SPARC expression, whereas overexpression of HDAC10 downregulates it. Mechanistically, HDAC10 coordinates with histone acetyltransferase p300 to modulate the state of acetylation of histone H3 at lysine 27 (H3K27ac) at SPARC regulatory elements and the recruitment of bromodomain-containing protein 4 (BRD4) to these regions, thereby fine-tuning SPARC transcription. HDAC10 depletion and resultant SPARC upregulation repress melanoma cell growth primarily by activating AMPK signaling and inducing autophagy. Moreover, SPARC upregulation due to HDAC10 depletion partly accounts for the resensitization of resistant cells to a BRAF inhibitor. Our work reveals the role of HDAC10 in gene regulation through indirect histone modification and suggests a potential therapeutic strategy for melanoma or other cancers by targeting HDAC10 and SPARC.

Histone deacetylases (HDACs) are enzymes that regulate many important biological pathways. There is a need for the development of isoform-selective HDAC inhibitors for further biological applications. Here, we report the development of trapoxin A analogues as potent and selective inhibitors of HDAC11, an enzyme that can efficiently remove long-chain fatty acyl groups from proteins. In particular, we show that one of the trapoxin A analogues, TD034, has nanomolar potency in enzymatic assays. We show that in cells, TD034 is active at low micromolar concentrations and inhibits the defatty acylation of SHMT2, a known HDAC11 substrate. The high potency and selectivity of TD034 would permit further development of HDAC11 inhibitors for biological and therapeutic applications.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

Abstract Melanomas are heterogeneous and adopt multiple transcriptional states that can confer an invasive phenotype and resistance to therapy. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity and tumor progression. Here we identify stress-induced HDAC8 activity as the driver of a neural crest stem cell (NCSC)-like transcriptional state that increased the formation of melanoma brain metastases (MBM). Exposure of melanocytes and melanoma cells to multiple different stresses led to HDAC8 activation, a switch to a NCSC gene expression signature and the adoption of an amoeboid, invasive phenotype. This cell state enhanced the survival of melanoma cells under shear stress conditions and increased the formation of metastases in the brain. Single cell RNA-seq analyses showed that HDAC8 expression was correlated with the NCSC cell state in clinical MBM specimens. ATAC-Seq and ChIP-Seq analysis showed HDAC8 to alter chromatin structure by increasing H3K27ac and accessibility at c-Jun binding sites without changing global histone acetylation. The increased accessibility of Jun binding sites was paralleled by decreased H3K27ac and accessibility at MITF binding sites and loss of melanoma-lineage gene expression. Mass spectrometry-based acetylomics demonstrated that HDAC8 deacetylated the histone acetyltransferase (HAT) EP300 leading to its enzymatic inactivation. This, in turn, led to an increased binding of EP300 to Jun-transcriptional sites and decreased binding to MITF-transcriptional sites. Increased expression of EP300 decreased invasion and increased the sensitivity of melanoma cells to multiple stresses while inhibition of EP300 function increased invasion and resistance to stress. We identified HDAC8 as a novel mediator of transcriptional co-factor inactivation and chromatin accessibility that increases MBM development. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Chao Zhang, Robert Macaulay, Daphne Dupéré-Richér, Bin Fang, John Koomen, Jiannong Li, Ann Chen, Edward Seto, Jonathan Licht, Keiran Smalley. HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3606.

SUMMARYSecreted Protein Acidic and Rich in Cysteine (SPARC), a highly conserved secreted glycoprotein, is crucial for various bioprocesses. Here we demonstrate that histone deacetylase 10 (HDAC10) is a key regulator ofSPARCexpression. HDAC10 depletion or inhibition upregulates, while overexpression of HDAC10 downregulates, SPARC expression. Mechanistically, HDAC10 coordinates with histone acetyltransferase p300 to modulate the acetylation state of histone H3 lysine 27 (H3K27ac) atSPARCregulatory elements and the recruitment of bromodomain-containing protein 4 (BRD4) to these regions, thereby tuningSPARCtranscription. HDAC10 depletion and resultant SPARC upregulation repress melanoma cell growth, primarily by induction of autophagy via activation of AMPK signaling. Moreover, SPARC upregulation due to HDAC10 depletion partly accounts for the resensitivity of resistant cells to a BRAF inhibitor. Our work reveals the role of HDAC10 in gene regulation through epigenetic modification and suggests a potential therapeutic strategy for melanoma or other cancers by targeting HDAC10 and SPARC.HighlightsHDAC10 is the primary HDAC member that tightly controls SPARC expression.HDAC10 coordinates with p300 in modulating the H3K27ac state atSPARCregulatory elements and the recruitment of BRD4 to these regions.HDAC10 depletion and resultant SPARC upregulation inhibit melanoma cell growth by inducing autophagy via activation of AMPK signaling.SPARC upregulation as a result of HDAC10 depletion resensitizes resistant cells to BRAF inhibitors.

Supplemental Materials and Methods, Tables and Figures.

Supplementary Table 1: Clinical data on samples stained for HDAC8 expression. S1) HDAC expression levels in melanoma cells S2) Cell lines that are resistant to BRAF inhibitors have upregulated c-Jun protein expression S3) Inhibiting HDAC6 does not resensitize BRAF/MEK resistant cells to BRAF inhibitors S4) BRAF/MEK inhibitor withdrawal decreases HDAC8 expression over time S5) Knocking down or elevating HDAC8 levels does not change cell proliferation S6) Expression of HDAC8 increased ERK activity and resistance upon treatment with BRAF-MEK inhibitors S7) Knocking down HDAC8 in BRAFi naïve cells increased apoptosis and BIM expression while decreasing ERK activity after BRAFi activity. S8) HDAC8 expression increases baseline RTK signaling S9) Inhibiting MET or FGFR3 partially resensitizes HDAC8 expressing cells to BRAF inhibitors S10) HDAC8 expression increases baseline c-Jun, p53, AKT and HSP60 signaling S11) Inhibiting c-Jun resensitizes HDAC8 expressing cells to a BRAF inhibitor S12) Protein sequences of c-Jun acetyl mutants S13) Acetylation-deficient c-JUN at residue 273 increases melanoma cell survival following BRAF inhibition S14) Acetylation-deficient c-Jun at residue 273 increases p53, Akt 1/2/3, STAT3, WNK1 and HSP60 S15) Acetylation-deficient c-JUN at residue 273 increases baseline EGFR signaling

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase-anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via β2- and β3-adrenergic receptors (β-ARs), which are G protein-coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives β-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes.

HDAC1 is the prototypical human histone deacetylase (HDAC) enzyme responsible for catalyzing the removal of acetyl group from lysine residues on many substrate proteins. By deacetylating histones and non-histone proteins, HDAC1 has a profound effect on the regulation of gene transcription and many processes related to cell growth and cell death, including cell cycle progression, DNA repair, and apoptosis. Early studies reveal that, like most eukaryotic proteins, the functions and activities of HDAC1 are regulated by post-translational modifications. For example, serine phosphorylation of HDAC1 by protein kinase CK2 promotes HDAC1 deacetylase enzymatic activity and alters its interactions with proteins in corepressor complexes. Here, we describe an alternative signaling pathway by which HDAC1 activities are regulated. Specifically, we discover that EGFR activity promotes the tyrosine phosphorylation of HDAC1, which is necessary for its protein stability. A key EGFR phosphorylation site on HDAC1, Tyr72, mediates HDAC1's anti-apoptotic function. Given that HDAC1 overexpression and EGFR activity are strongly related with tumor progression and cancer cell survival, HDAC1 tyrosine phosphorylation may present a possible target to manipulate HDAC1 protein levels in future potential cancer treatment strategies.

Myeloid-derived suppressor cells (MDSCs) constitute a heterogeneous population of immature myeloid cells derived from bone marrow and negatively regulate both innate and adaptive immunity in the tumor microenvironment. Previously we have demonstrated that MDSCs lacking histone deacetylase 11 (HDAC11) displayed an increased suppressive activity against CD8+ T-cells. However, the mechanisms of HDAC11 that contribute to the suppressive function of MDSCs remain unclear. Here, we show that arginase activity and NO production is significantly higher in HDAC11 knockout MDSCs when compared with wild-type (WT) controls. In the absence of HDAC11, elevated arginase level and enzymatic activity were observed preferentially in the tumor-infiltrated granulocytic MDSCs, whereas iNOS expression and NO production were increased in the tumor-infiltrated monocytic MDSCs. Of note and for the first time, we demonstrated an association between the elevated expression of immunosuppressive molecules with up-regulation of the transcription factor C/EBPβ in MDSCs lacking HDAC11. Interestingly, the highest expression of C/EBPβ was observed among CD11b+ Gr-1+ MDSCs isolated from tumor-bearing mice. The additional demonstration that HDAC11 is recruited to the promoter region of C/EBPβ in WT MDSCs suggests a novel molecular mechanism by which HDAC11 influence the expression of immunosuppressive molecules in MDSCs through regulation of C/EBPβ gene expression.

Abstract Background: Melanoma cells show great plasticity and can switch from a proliferative, drug-sensitive state to an invasive, drug-resistant phenotype through mechanisms that remain poorly defined. Hypothesis: Transcriptional and chromatin regulatory factors alter the gene expression states of cell in response to targeted therapy treatment and allow melanoma cells to persist and grow in the face of initially effective therapy. These factors/pathways may represent additional targets for anti-melanoma therapy. Methods: BRAF mutant melanoma cells were exposed to the BRAF inhibitor vemurafenib to generate cells that persisted and grew in the face of continued drug treatment. RNA-Seq, CHIP seq, ATAC-Seq and mass spectroscopy analyses were performed on persistent/resistant cells in comparison to parental cells. Additionally, histone deacetylase (HDAC) 8 and EP300 expression was enforced in drug naïve melanoma cell lines and the effects determined. Results: HDAC8 (but not other HDACs) was consistently up-regulated in BRAFi resistant melanoma cells. Increased expression of HDAC8 reduced cell growth, increased p53 activation and suppressed microphthalmia-associated transcription factor MITF, suppressing the melanocyte lineage program driven, leading to increased drug resistance. Introduction of HDAC8 or its active mutant (S39A) increased the tolerance of melanoma cells to multiples stresses (UV-irradiation, hypoxia, and BRAF-MEK inhibitor therapy), reduced their proliferation and increased their invasive potential in in vitro and in vivo models. Gene expression analyses indicate genes associated with a proliferative, drug-sensitive phenotype (e.g. DCT, MITF, MLANA, SOX10) are downregulated in HDAC8 active cells while genes correlative with an invasive, drug-resistant phenotype are upregulated (e.g. AXL, EGFR, TGFB1, WNT5A). ATAC-Seq analysis showed that HDAC8 regulates chromatin structure and decreased accessibility in the DNA binding sites of MITF and Sox10 while increasing accessibility in the DNA binding sites of c-Jun and TEADs (e.g. YAP1). Additionally, ChIP-Seq showed an increase in HDAC8 activity decreased H3K27 acetylation in the promoter region of MITF and MITF regulated genes (e.g. DCT, MLANA, PMEL, TYR). Mass spectrometry-based acetylomics and co-immunoprecipitation experiments demonstrated that active HDAC8 associated with and deacetylated the histone acetyltransferase EP300, a known coactivator of MITF in melanocytes. Increased expression of EP300 increased the sensitivity of melanoma cells to UV-irradiation, hypoxia and BRAF-MEK inhibitor therapy. Conclusions: HDAC8 associated with EP300 and mediated the switch to an invasive, stress resistant state in melanoma cells by suppressing MITF and increasing c-Jun driven transcriptional programs. Targeting HDAC8 could be a strategy to limit plasticity in advanced melanoma, increasing the efficacy of current therapies. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Bin Fang, Edward Seto, john koomen, Jonathan Licht, Keiran Smalley. HDAC8 activity regulates stress induced phenotype switching in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1116.

Abstract Activation of oncogenic KRAS is the most common driving event in lung adenocarcinoma development. Despite the existing rationale for targeting activated KRAS and its downstream effectors, the failure of clinical trials to date indicates that the mechanism of KRAS-driven malignancy remains poorly understood. Here we report that histone deacetylase 10 (HDAC10) might function as a putative tumor suppressor in mice carrying a spontaneously activated oncogenic Kras allele. Hdac10 deletion accelerated KRAS-driven early-onset lung adenocarcinomas, increased macrophage infiltration in the tumor microenvironment, and shortened survival time in mice. Highly tumorigenic and stem-like lung adenocarcinoma cells were increased in Hdac10-deleted tumors compared with Hdac10 wild-type tumors. HDAC10 regulated the stem-like properties of KRAS-expressing tumor cells by targeting SOX9. Expression of SOX9 was significantly increased in Hdac10-deleted tumor cells and depletion of SOX9 in Hdac10 knockout (KO) lung adenocarcinoma cells inhibited growth of tumorspheres. The genes associated with TGFβ pathway were enriched in Hdac10 KO tumor cells, and activation of TGFβ signaling contributed to SOX9 induction in Hdac10 KO lung adenocarcinoma cells. Overall, our study evaluates the functions and mechanisms of action of HDAC10 in lung carcinogenesis that will inform the rationale for targeting its related regulatory signaling as an anticancer strategy. Significance: These findings linking HDAC10 and lung tumorigenesis identify potential novel strategies for targeting HDAC10 as a treatment for lung cancer.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI), resulting in considerable disability and prolonged treatment. It is known that host leukocyte IL-10 production is required for S. aureus biofilm persistence in PJI. An S. aureus bursa aurealis Tn library consisting of 1,952 non-essential genes was screened for mutants that failed to induce IL-10 in myeloid-derived suppressor cells (MDSCs), which identified a critical role for bacterial lactic acid biosynthesis. We generated an S. aureus ddh/ldh1/ldh2 triple Tn mutant that cannot produce d- or l-lactate. Co-culture of MDSCs or macrophages with ddh/ldh1/ldh2 mutant biofilm produced substantially less IL-10 compared with wild-type S. aureus, which was also observed in a mouse model of PJI and led to reduced biofilm burden. Using MDSCs recovered from the mouse PJI model and in vitro leukocyte–biofilm co-cultures, we show that bacterial-derived lactate inhibits histone deacetylase 11, causing unchecked HDAC6 activity and increased histone 3 acetylation at the Il-10 promoter, resulting in enhanced Il-10 transcription in MDSCs and macrophages. Finally, we show that synovial fluid of patients with PJI contains elevated amounts of d-lactate and IL-10 compared with control subjects, and bacterial lactate increases IL-10 production by human monocyte-derived macrophages. Bacteria-derived lactate mediates inhibition of HDAC11 during Staphylococcus aureus biofilm infections, resulting in epigenetic changes that reprogramme the host immune response.

Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3'-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3'-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.

Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs), where leukocyte IL-10 production is critical for biofilm persistence. We screened a S. aureus mutant library that identified the importance of lactate biosynthetic pathways to elicit IL-10 production. A S. aureus Δddh/ldh1/ldh2 mutant, defective in both D- and L-lactate production, led to significantly decreased leukocyte IL-10 expression and re-programmed biofilm-associated monocytes to a pro-inflammatory state, resulting in reduced biofilm burden. Histone acetylation plays a central role in regulating gene expression, and we found that histone deacetylase (HDAC) activity was increased in Δddh/ldh1/ldh2-infected mice compared to WT concomitant with significant decreases in global histone 3 (H3) acetylation and H3 acetylation at the IL-10 promoter, demonstrating that S. aureus-derived lactate is a HDAC inhibitor. Lactate was found to inhibit HDAC11, which resulted in unregulated HDAC6 activity and increased IL-10 production. MDSCs and macrophages produced significantly less IL-10 in the presence of the HDAC6 inhibitor tubastatin A during co-culture with WT but not Δddh/ldh1/ldh2 biofilms, while HDAC11 KO MDSCs and macrophages co-cultured with Δddh/ldh1/ldh2 biofilms produced significantly more IL-10 compared to WT cells due to unchecked HDAC6 action. D-lactate was elevated in synovial fluid of patients with PJI compared to aseptic controls and lactate also promoted IL-10 production by human monocyte-derived macrophages. Collectively, S. aureus lactate-mediated inhibition of HDAC11 plays a critical role during biofilm infection, leading to epigenetic changes that reprogram the host immune response.

Histone deacetylases (HDACs) are conserved enzymes that regulate many cellular processes by catalyzing the removal of acetyl groups from lysine residues on histones and non-histone proteins. As appropriate for proteins that occupy such an essential biological role, HDAC activities and functions are in turn highly regulated. Overwhelming evidence suggests that the dysregulation of HDACs plays a major role in many human diseases. The regulation of HDACs is achieved by multiple different mechanisms, including posttranslational modifications. One of the most common posttranslational modifications on HDACs is reversible phosphorylation. Many HDAC phosphorylations are context-dependent, occurring in specific tissues or as a consequence of certain stimuli. Additionally, whereas phosphorylation can regulate some HDACs in a non-specific manner, many HDAC phosphorylations result in specific consequences. Although some of these modifications support normal HDAC function, aberrations can contribute to disease development. Here we review and critically evaluate how reversible phosphorylation activates or deactivates HDACs and, thereby, regulates their many functions under various cellular and physiological contexts.

Abstract Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF–MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug-resistant phenotype. Mass spectrometry–based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF–MEK inhibition. Significance: This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.