SETDB1: an emerging target for anticancer drug development

Cancer continues to rank among the world’s top causes of morbidity and mortality, and it poses particular difficulties since it can become resistant to standard treatments [1]. Despite advancements in targeted therapy, many patients experience relapse as cancer cells evolve defense mechanisms that allow them to evade the effects of the drugs. This adaptability is driven by both genetic mutations and epigenetic modifications, enabling tumors to persist and thrive even in the presence of treatment. Epigenetic processes such as DNA methylation and histone modifications play a crucial role in regulating gene expression without altering the DNA sequence itself. Since these changes are reversible and can be inherited, they offer unique therapeutic opportunities to address the dynamic nature of cancer [2,3].

The histone methyltransferase SET domain bifurcated 1 (SETDB1), which targets lysine 9 on histone H3 (H3K9), is one of the key players in this epigenetic environment. Through transcriptional repression, SETDB1 silences tumor suppressor genes, which has been closely linked to medication resistance and the advancement of cancer. Targeting epigenetic regulators like SETDB1 is a viable approach to fight cancer resistance and enhance therapy effectiveness because malignancies develop via both genetic and epigenetic routes. This editorial aims to clarify the significance of SETDB1 in cancer treatment by highlighting its function in the creation of innovative therapeutic strategies meant to combat cancer resistance [4,5].

The highly conserved pre-SET, SET, and post-SET domains found in the C-terminal region of SETDB1 are crucial for its histone methylation activity, just like those found in other proteins in the SET domain family. The complex interaction between these pre- and post-SET regions is essential to the SET domain’s catalytic activity. However, as noted by Harte et al., SETDB1 is specifically differentiated by a 347-amino acid insertion inside the SET domain, known as the I-SET region. Although its exact function is yet unknown, this insertion, which is unique to SETDB1 and conserved across species, adds an interesting element of intricacy to its function [6–8]. Furthermore, SETDB1 features an N-terminal methyl-CpG binding domain (MBD) and three tandem tudor domains (TTD). Traditionally, the MBD domain recognizes and binds CpG-rich DNA regions to mediate gene silencing, but recent studies show it is crucial for SETDB1‘s interaction with C11orf46. It is noteworthy that C11orf46 has a greater affinity for citrullinated histone H3 peptides (H3R2), suggesting that it has a regulatory function in addition to methylation. Meanwhile, TTD of SETDB1 interacts with chromatin-modifying enzymes, aiding RNA processing and transcription factor regulation. Research is still ongoing to determine the wider biological consequences of SETDB1‘s connections with citrullination and H3K9 methylation. Structural predictions from AlphaFold have also revealed additional features in C11orf46, such as CCCH motifs that resemble the DNA-binding domains of THAP-family proteins, further broadening the functional scope of this protein complex [7,8]. SETDB1‘s TTD forms multiprotein complexes that control transcriptional repression through interactions with mSin3A/B and HDAC1/2. TTD identifies peptides that are methylated at H3K9 and acetylated at H3K14, but with a low affinity for H3K9 methylation alone. The significance of this dual modification, combining active and repressive marks, is unclear. Additionally, the MBD in SETDB1 suggests a link between H3K9 methylation and DNA methylation, a key mechanism for gene silencing. The crystal structure of TTD of human SETDB1 was resolved through protein crystallography and X-ray diffraction, and is available in the Protein Data Bank under the accession code 3DLM [9].

Growing evidence highlights a strong correlation between SETDB1 overexpression and abnormal activity in various malignancies, which is associated with poor prognosis in cancer patients [10]. SETDB1 amplification plays a pivotal role in tumorigenesis and progression by promoting key processes such as cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), metastasis, resistance, and immune evasion. As mentioned above, SETDB1 primarily functions in transcriptional silencing through its H3K9 methyltransferase activity. However, it also extends its influence beyond epigenetic modifications by directly regulating crucial proteins that are essential for various cellular functions. Such interactions underscore SETDB1‘s significance beyond its canonical function, positioning it as an important player in the regulation of gene expression and tumorigenesis. For instance, activated AKT kinase supports cancer cell proliferation and survival. SETDB1 can enhance AKT activation by mediating the methylation of its K64 residue, thereby promoting the development of non-small-cell lung carcinoma (NSCLC) [11].

In hepatocellular carcinoma (HCC), SETDB1 is recognized as an oncogene, enhancing cell proliferation and migration through its interaction with T-cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) [12]. Notably, SETDB1 upregulation is strongly linked to disease progression and poor prognosis in HCC patients, with its expression observed in all metastatic sites, highlighting its critical role in driving HCC aggressiveness [12,13].

Moreover, SETDB1 has been identified as a crucial component of the PML nuclear bodies (PML-NBs), which play vital roles in various cellular functions, including the maintenance of pluripotency in embryonic stem cells and the recruitment of SUMO-1 and Sp100 proteins, which have been extensively associated with gene transcription, tumor suppression, apoptosis, antiviral responses, and DNA repair mechanisms. Cho et al. demonstrated that the knockdown of SETDB1 led to the disassembly of PML-NBs, while degradation of PML via arsenic treatment resulted in the loss of SETDB1 foci. Subsequent research revealed that the SUMO-interaction motif (SIM) on SETDB1 is essential for its interaction with PML proteins. SETDB1 variants lacking a functional SIM caused significant damage to the integrity of PML-NB structures [14].

Recent insights into the interaction between SETDB1 and key cellular regulators have revealed new therapeutic opportunities. Targeting SETDB1 to disrupt oncogenic processes is becoming a promising cancer treatment strategy. By inhibiting SETDB1, these therapies aim to reshape the epigenetic landscape and reactivate silenced tumor suppressor genes. Various approaches, including small molecule inhibitors, peptides, targeted delivery systems, and combination therapies, are being explored to develop selective SETDB1 inhibitors for clinical use [15–17].

Park et al. employed in vitro experiments and in silico screening to find small molecule inhibitors of SETDB1. They found 21 potential compounds (VH01–VH21) using the Asinex and ChemDiv databases [18]. Viral tests demonstrated that VH01 and VH06 had substantial binding affinities (K_D values of 3.26 ± 1.71 μM and 0.232 ± 0.146 μM, respectively) and effectively suppressed H3K9me3 and heterochromatin condensation. Afterward, another study used the groundbreaking work of Park et al. to identify 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a possible SETDB1 inhibitor. It showed that APQ could reduce H3K9 trimethylation with an IC₅₀ value of 65 μM. The mechanism of action of APQ was clarified by its shown ability to bind efficiently to the lysine and S-adenosyl-L-methionine (SAM) binding sites of SETDB1 [19,20].

Additionally, research has shown that combining immune checkpoint modulators, such as anti-PD1 or anti-PD-L1 therapies, with SETDB1 suppression significantly amplifies their anti-tumor effects. While SETDB1 suppression or anti-PD1 therapy alone only has modest anti-tumor effects, their combination results in a marked and sustained decrease in tumor size. In contrast, the combination of immune checkpoint inhibitors (anti-PD1 and/or anti-PD-L1) with SUV39H1 is surprisingly less effective than that of SETDB1, even though they both target the same histone mark (H3K9me3). This contrast underscores the study’s findings that while SETDB1 suppression and anti-PD1 therapy together create a synergistic effect, the combination of immune checkpoint inhibitors with SUV39H1 does not achieve the same level of effectiveness. This discrepancy suggests that although both therapies target similar epigenetic changes, they may influence different pathways within the tumor microenvironment, leading to varying treatment outcomes. This study emphasizes that specific contributions of epigenetic variables in cancer do not necessarily translate into predictions about the success of treatment combinations. As a result, this discovery suggests a promising method of treating cancer: the combination of immune checkpoint modulators with SETDB1 inhibitors [20].

Determining the regulatory mechanisms of SETDB1 and its functional consequences across different cancer types has advanced significantly. In order to silence tumor suppressors like p53 and p21, which are crucial for controlling the cell cycle and apoptosis, SETDB1 promotes H3K9 methylation at certain promoter sites. In addition to histone modification, SETDB1 stabilizes and amplifies the tumor-promoting properties of non-histone proteins like p53 and Akt by methylating them directly. This complex function of SETDB1 positions it as a prospective therapeutic target by highlighting its overexpression as a distinct signature in a number of cancer types in addition to aiding in tumor growth [4–7].

In light of this, it should be mentioned that while SETDB1 and SUV39H1 are members of the SUV39 histone methyltransferases family of proteins and thus are both responsible for H3K9me3, they function in different ways and target different parts of the genome. SUV39H1 mostly preserves heterochromatin stability at centromeres and telomeres, whereas SETDB1 primarily mediates H3K9me3 in euchromatin and at certain loci implicated in silencing tumor suppressors, inhibiting retroviruses and transposable elements [21]. It is noteworthy that SETDB1 suppression is not limited to H3K9me3, it also directly regulates important enzymes implicated in cancerous pathways, including the MYC and Wnt/β-catenin pathways, setting it apart from the SUV39H family of proteins [22,23]. This implies that SETDB1 targeting may have therapeutic benefits that go beyond epigenetic changes. According to some research, SUV39H1 can partially offset SETDB1‘s inhibition in specific genomic locations, despite the fact that SETDB1 and SUV39H1 have different preferences for chromatin localization. Related enzymes like SUV39H2 may be involved in this compensatory mechanism, which helps to maintain H3K9me3 levels. Nevertheless, this effect is not always seen in all cases and cell types, but could lessen the effectiveness of therapies that target only SETDB1 [24]. Therefore, the potential advantages of dual-targeting techniques to suppress both SETDB1 and SUV39H1 are highlighted by evidence of functional redundancy between both methyltransferases, especially in cancer cells [21,24].

The issue of creating particular SETDB1 inhibitors has not yet been overcome, though. The lack of selectivity in current inhibitors raises questions regarding possible drug resistance and off-target consequences. Further understanding of the processes behind SETDB1 amplification, overexpression, and activity is necessary to address these problems [4]. The therapeutic landscape may change dramatically as our knowledge of SETDB1‘s function in cancer expands, especially its impact on tumor and immune responses. The development of more effective and selective SETDB1 inhibitors has the potential to advance epigenetic therapeutics, providing a fresh approach to treating malignancies that are resistant to treatment and enhancing patient outcomes.

Funding Statement

The authors would like to thank FAPESP (The São Paulo Research Foundation [2017/25543–8]), CNPq (Brazilian National Council for Scientific and Technological Development [314429/2023–9 and 436791/2018–8], FUSP (The University of Sao Paulo Support Foundation) and CAPES (Coordination for the Improvement of Higher Education Personnel) for financial support and fellowship. Conselho Nacional de Desenvolvimento Científico e Tecnológico [314429/20239]; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Fundação de Amparo à Pesquisa do Estado de São Paulo [2017/25543–8]; USP - The University of Sao Paulo Support Foundation.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Financial disclosure

The authors thank FUSP, CNPq, CAPES and FAPESP.

Author contributions

Gustavo Henrique Goulart Trossini – Idea conception, manuscript writing, supervisor, project PI, manuscript correction and edition.

Haifa Hassanie – manuscript writing, bibliographic search, documental analyses

André Berndt Penteado – manuscript writing and correction, bibliographic search, documental analyses.

Marissa El-Hajje – manuscript writing and correction, bibliographic search, documental analyses.