Retinoid X Receptor agonists as selective modulators of the immune system for the treatment of cancer

Abstract

Upon heterodimerizing with other nuclear receptors, retinoid X receptors (RXR) act as ligand-dependent transcription factors, regulating transcription of critical signaling pathways that impact numerous hallmarks of cancer. By controlling both inflammation and immune responses, ligands that activate RXR can modulate the tumor microenvironment. Several small molecule agonists of these essential receptors have been synthesized. Historically, RXR agonists were tested for inhibition of growth in cancer cells, but more recent drug discovery programs screen new molecules for inhibition of inflammation or activation of immune cells. Bexarotene is the first successful example of an effective therapeutic that molecularly targets RXR; this drug was approved to treat cutaneous T cell lymphoma and is still used as a standard of care treatment for this disease. No additional RXR agonists have yet achieved FDA approval, but several promising novel compounds are being developed. In this review, we provide an overview of the multiple mechanisms by which RXR signaling regulates inflammation and tumor immunity. We also discuss the potential of RXR-dependent immune cell modulation for the treatment or prevention of cancer and concomitant challenges and opportunities.

Graphical Abstract - Immunomodulatory effects of novel retinoid X receptor (RXR) agonists in cancer prevention and treatment

The immunomodulatory effects of novel RXR agonists, such as UAB30, MSU-42011, LG100268, and V-125, have been described when used for preventing or treating cancer in several preclinical models. Upon binding to an agonist, RXR receptors function as homo- or heterodimeric transcription factors that modulate gene expression, including multiple genes that regulate immune function. RXR agonists can increase the number of antigen-presenting dendritic cells (DCs) and cytotoxic CD8+ T cells and reduce the infiltration of tumor-promoting immune cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), within the tumor microenvironment.

1. Introduction

Retinoid X receptors (RXRs) are essential modulators of human physiology and attractive targets for treating cancer (Altucci et al., 2007; Schierle & Merk, 2019). By binding to other nuclear receptors (NRs), RXRs help to regulate multiple processes including development, metabolism, and homeostasis (Evans & Mangelsdorf, 2014; Sharma et al., 2022). RXRs exist in three subtypes: RXRα (also called NR2B1, nuclear receptor subfamily 2, group B, member 1), RXRβ (NR2B2) and RXRγ (NR2B3), encoded by the RXRA, RXRB, and RXRG genes (Germain et al., 2006). Each subtype is widely expressed and is thought to have analogous cellular functions. However, the relative expression patterns of the subtypes vary in different cell types (Ahuja et al., 2003; Germain et al., 2006). One classic example is RXRγ, which is highly expressed in thyrotropes and thyrotrope-derived cells. Deficiency of the RXRγ subtype is associated with higher serum thyroid-stimulating hormone (TSH) and thyroxine levels in mice (N. S. Brown et al., 2000; Haugen et al., 1997).

RXRs are the heterodimerization partner of multiple NRs, including the peroxisome proliferator-activated receptors (PPAR, NR1C1-3), liver X receptors (LXR, NR1H2,3), farnesoid X receptors (FXR, NR1H4,5), retinoic acid receptors (RAR, NR1B1-3), vitamin D receptor (VDR, NR1l1) and thyroid hormone receptors (TR, NR1A1,2) (Mangelsdorf & Evans, 1995). RXR partners can be categorized as permissive (PPARs, LXR, and FXR) or non-permissive (RAR, VDR, and TR) (Vivat-Hannah et al., 2002; Altucci et al., 2007; Evans & Mangelsdorf, 2014; Thompson et al., 2001; Brtko & Dvorak, 2020). Agonists of either partner receptor can activate the permissive heterodimers. Furthermore, agonist binding to both RXR and a partner can result in cumulative or synergistic effects. Nonpermissive partners, in contrast, are typically activated only by ligands specific to the partner receptor but not to RXR (Evans & Mangelsdorf, 2014; Forman et al., 1995). Increasing the concentration of a RXR ligand may skew activity from one dimer to another, inducing entirely different regulatory mechanisms in various tissues (Davies et al., 2001; De Luca, 1991). In addition to their primary function as heterodimer partners, RXRs can act as homodimers and even homotetramers to control gene expression (Lefebvre et al., 2010). Once a heterodimer or homodimer binds to target DNA, it activates transcription, resulting in a vast and varied range of biological effects. This versatility permits RXRs to regulate multiple biological processes relevant to cancer, including cell proliferation, differentiation, survival, and immune cell function (Dawson & Xia, 2012; Germain et al., 2006; Menéndez-Gutiérrez et al., 2023; Nagy et al., 2012; Oliveira et al., 2018; Rőszer et al., 2013; Saito-Hakoda et al., 2015; Szanto et al., 2004).

Like other nuclear hormone receptors, RXRs have a variable N-terminal domain (NTD) followed by a central highly conserved DNA binding domain (DBD), which is linked to the C-terminal ligand binding domain (LBD) via a flexible hinge region (Figure 1A) (Penvose et al., 2019). The RXR subtypes, despite being encoded by three distinct genes, have a significant degree of sequence similarity and structural homology (de Lera et al., 2007). The DNA binding domains of RXRs are crucial for the specificity of heterodimer binding to specific DNA sequences in the promoter regions of target genes (Giguère & Evans, 2022; Petkovich & Chambon, 2022; Predki et al., 1994). The ligand-binding domains of RXRs bind lipophilic signaling molecules, such as RAs, in the hydrophobic pockets (Aranda & Pascual, 2001; Evans & Mangelsdorf, 2014). Ligand binding causes conformational changes that favor the interaction between RXR and its heterodimeric partner and the binding efficiency to DNA. It also sets off a chain of events, such as co-regulator exchange or binding, that results in positive or negative gene transcription and other biological activities (Figure 1B) (Germain et al., 2002; Sever & Glass, 2013; Egea et al., 2002; Evans & Mangelsdorf, 2014). In the absence of an agonist, the activation function (AF2) domain in the LBD promotes interaction with the corepressor complex, which inhibits transcription (Figure 1C). In the presence of a ligand, conformational changes occur in the AF2 domain that weaken corepressor interactions and encourage the binding of coactivators, which activate the transcription of target genes (Dawson & Xia, 2012; Germain et al., 2006).

Figure 1. Structure and activation of the retinoid X receptor (RXR).

(A) The structure of a RXR includes an N-terminal domain, a DNA-binding domain, hinge region, and ligand-binding domain. RXRs can form homodimers or heterodimers with various other nuclear hormone receptors; heterodimers are categorized as permissive or non-permissive. (B) Permissive dimers, such as RXR with a liver X receptor (LXR), peroxisome proliferator-activated receptor (PPAR), or farnesoid X receptor (FXR), are activated by ligand binding either to RXR or to the partner receptor and subsequent binding to response elements, specific DNA sequences in the promoter region of target genes. Simultaneous activation of both RXR and its partner receptor results in additive or synergistic activation. In contrast, (C) non-permissive heterodimers, such as RXR with a retinoic acid receptor (RAR), vitamin D receptor (VDR), or thyroid hormone receptor (TR) only respond to the ligands of the partner receptor. In the absence of ligands, heterodimers interact with corepressor complexes, which block transcription. However, upon ligand binding, corepressors are released, and coactivators are recruited, leading to chromatin alterations that activate gene expression. Panels B and C were adapted from Brtko et al., 2020.

9-cis-Retinoic acid (9-cis-RA) was initially identified as an endogenous ligand for RXR (Heyman et al., 1992; Mangelsdorf et al., 1992). However, multiple groups were unable to detect endogenous 9-cis-RA in tissues under physiological conditions (Blomhoff & Blomhoff, 2006; Calléja et al., 2006; Gundersen, 2006; Gundersen et al., 2007; Kane et al., 2005, 2008; Schmidt et al., 2003; Wolf, 2006). Therefore, 9-cis-RA may be present in sufficient amounts to bind and activate RXRs only after pharmacological or dietary supplementation (Arnhold et al., 1996; Krężel et al., 2019; Ulven et al., 2001). Subsequently, several fatty acids such as docosahexaenoic acid (DHA), oleic acid, phytanic acid (de Urquiza et al., 2000; Kitareewan et al., 1996; Lemotte et al., 1996), and other ligands (Brtko & Dvorak, 2020) were identified as potential RXR ligands. However, none of these compounds have been proven to be the putative endogenous ligand of RXR (Calléja et al., 2006; Krężel et al., 2019; Wolf, 2006). Despite this challenge, a variety of potent synthetic RXR ligands (known as “rexinoids”) have been synthesized, which have shown promising activity in multiple preclinical models of disease including cancer (Martino & Welch, 2019; Moerland et al., 2020; Reich et al., 2023; Uray et al., 2016; D. Zhang et al., 2019) and even for prevention in clinical trials (Moyer & Brown, 2023). However, most of these studies reported efficacy or studied changes only in the cancer cells following treatment with a RXR agonist.

One of the most intriguing and underexplored areas of investigation in the RXR field is the effects of RXR agonists on immune cells within the tumor microenvironment (Leal et al., 2019; Leal, Moerland, et al., 2021), which have emerged as significant druggable targets in cancer (Lu et al., 2023). A remarkable shift in cancer therapy from solely targeting cancer cells to focusing on modulating the immune response with immunotherapy has profoundly improved patient survival in certain cancers (Sanmamed & Chen, 2018; Y. Zhang & Zhang, 2020). Because of the complexities of RXR receptor dimerization, signaling crosstalk, and regulation by co-repressors and co-activators in different cell types (De Bosscher et al., 2020), understanding the biology of RXR in immunology is critical for developing new and specific RXR agonists as novel anticancer therapies. In this review, we provide an overview of the immunomodulatory effects of RXR agonists in cancer and suggest promising new areas of investigation.

2. RXR regulation of inflammation and the immune response

Numerous studies have explored the importance of RXRs in coordinating gene expression in immunity and inflammation (Núñez et al., 2010; Rőszer et al., 2013; L. Zhou et al., 2010). The function of RXR in the immune system was first studied in a mouse model with a RXRα hypomorphic allele (Du et al., 2005). The RXR receptor expressed by these mice contains modified ligand binding and heterodimerization domains that do not respond to RA and vitamin D. These mice have extensive immune system defects, including an exaggerated Th1 response but a lack of a Th2 response, which are due to aberrant antigen-presenting cell and naïve CD4 T cell activity (Du et al., 2005; Spilianakis et al., 2005). By disrupting RXRα in the T cell compartment, CD4 and CD8 lymphocyte proliferation were lower, albeit the difference was more pronounced in CD8 T cells (Stephensen et al., 2007). Consistent with the results first reported by Du et al., other researchers also observed skewing of the immune response toward a Th1-type response in mice with a hypomorphic allele of RXRα (Stephensen et al., 2007). Additionally, a Th2 phenotype could be induced by treating T cells with either 9-cis-RA or AGN194204 (Stephensen et al., 2002). These findings suggest that RXRα signaling typically suppresses naive CD4 T cell differentiation into Th1 cells and that RXRα allows naive cells to differentiate into Th2 cells. Th1 cells are typically regarded as anti-tumorigenic, while Th2 cells are considered pro-tumorigenic (Al-Yassin & Bretscher, 2018). RXR signaling therefore is expected to help maintain the Th1:Th2 balance in cancer.

The innate and adaptive immune systems play major roles in the initiation and development of many cancers (Barber, 2015; Gajewski et al., 2013; Goldszmid et al., 2014; Lisovska & Shanazarov, 2019; Wculek et al., 2020). Immune cells within tumors can have either tumor-promoting or tumor-suppressive effects independent of the Th1:Th2 balance, depending on the local tumor microenvironment (TME) (Lisovska & Shanazarov, 2019; Shiao et al., 2011). RXRs regulate the differentiation and function of multiple immune cells such as T cells, B cells (Du et al., 2005; Stephensen et al., 2007), dendritic cells (DCs) (Geissmann et al., 2003; Mohty et al., 2003), and macrophages (Casanova-Acebes et al., 2020; Kiss et al., 2013; Núñez et al., 2010). Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are both associated with tumor growth and metastasis, demonstrating the significance of the myeloid compartment in cancer (Cook & Hagemann, 2013; Haas & Obenauf, 2019; Quail & Joyce, 2013). RXRα is highly expressed in macrophages, and RXR-PPARγ heterodimers control the differentiation of human monocytes into macrophages (Thomas et al., 2012). The functional significance of RXR signaling in immune cell function, cell differentiation and proliferation has made RXR and its heterodimeric partners promising pharmacological candidates for anti-cancer therapy (de la Fuente et al., 2015; K. T. Liby & Sporn, 2016; Núñez et al., 2010; Zhu et al., 2009).

3. Mechanisms of RXR in immunomodulation

3.1. Regulatory T cells

The combination of a RXR agonist such as PA024 or tributyltin increased the expression of Foxp3 in CD4+;CD25- T cells treated with all trans-retinoic acid (ATRA) or another RAR agonist. However, RXR agonists were unable to increase Foxp3 expression when used in combination with the RAR antagonist LE540, suggesting that immunosuppressive properties of T cells depend on RAR-mediated signals. While RXR and LXR agonists suppressed the expression of IL-17 in a RXR-dependent manner, RAR inhibition did not reverse this inhibition (Takeuchi et al., 2013). ATRA and the RAR agonist Am80 increased cell-surface expression of CCR9 in mouse naive CD4 T cells, but the combination of RXR agonists such as PA024 or the environmentally harmful chemicals tributyltin and triphenyltin that bind to RXR increased the expression of CCR9. The RXR agonist PA024 also increased the expression of α4β7, CCR9, and Foxp3. Binding of both NFATc2 (nuclear factor of activated T-cells c2) to NFAT-binding sites and the RAR-RXR heterodimer to a retinoic acid response element (RARE) site in the mouse Ccr9 gene were necessary for the activation of promoter activity following treatment with RA. By binding directly to RARα and RXRα, NFATc2 increased the binding of RARα to the RARE half-site (Ohoka et al., 2011; Takeuchi et al., 2010). Taken together, these data suggest that activation of RXR enhances the RAR-dependent homing capacity of T cells.

Neurologic diseases are now known to be regulated by highly complex immunologic systems (Kipnis & Filiano, 2018; Zang et al., 2022). The RXR agonist bexarotene promoted remyelination in patients with multiple sclerosis (J. W. L. Brown et al., 2022) . However, no changes in circulating regulatory T cells (Tregs) were observed, as Treg induction likely occurs within tissues. Preclinical studies have also confirmed the anti-inflammatory properties of RXR agonists, as they decreased Th17 differentiation yet increased the induction of Treg following treatment with ATRA. Bexarotene also promoted Treg induction in human cells, but in contrast to murine cells, regulation of the Treg/Th17 axis in human cells was independent of RAR activation (Gaunt et al., 2021). The RXR agonist IRX4204 also promoted differentiation of inducible regulatory T cells (iTreg) but suppressed the differentiation of pro-inflammatory Th17 cells both in vitro and in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (Chandraratna et al., 2016).

3.2. T cell trafficking and exhaustion

Enhancing T cell trafficking to tumors can improve immune responses and overall outcomes in cancer. T cell regulation by RXR has been mostly associated with RAR heterodimerization, through vitamin A activation (Bono et al., 2016). The G protein-coupled CCR9 chemokine receptor is expressed on DCs, CD4 T cells, and B cells. DCs that express CCR9 hone to the gut and thymus and thus are involved in food allergy, autoimmunity, and immune responses to cancer. RAR, RXR and other NRs play essential roles in T cell homing and activation, mostly by modulating CCR9 expression (Pathak & Lal, 2020; Wurbel et al., 2011; Xu et al., 2020). RXR agonists also elevated activation markers and decreased exhaustion markers in primary human T cells (J. Wu et al., 2023).

3.3. Myeloid cells

Monocytes, macrophages, DCs, and neutrophils are important immune cells throughout inflammation, both in autoimmunity and cancer (Gabrilovich & Nagaraj, 2009; Mantovani et al., 2008; Morell et al., 2017). RXRs are important regulators of macrophage biology; dysregulation of this pathway and pharmacological activation or inhibition are implicated in several pathologies, including atherosclerosis, insulin resistance and diabetes, neurodegeneration, and cancer (Rőszer et al., 2013). Notably, RXR is required for populating murine resident tissue macrophages. Deletion of RXRα in hematopoietic precursor cells significantly decreased the number of resident tissue macrophage populations in many tissues, such as the liver, lung, spleen, and peritoneal cavity (Philpott et al., 2022). Additionally, RXR expression increased as monocytes differentiated (Defacque et al., 1996), and monocyte differentiation was increased by treatment with a RXR agonist (Kizaki et al., 1996). Because of the varied effects on resident macrophages and differentiation pathways, RXR activation or inhibition in macrophages can have different outcomes depending on the tissue and disease being studied.

Knockout of RXRα specifically in myeloid cells lowered the susceptibility of mice to sepsis, in part by decreasing CCL6 and CCL9 chemokine expression and by reducing leukocyte recruitment to sites of inflammation. RXR regulated transcription of CCL6 and CCL9 in macrophages does not require heterodimeric partners (Núñez et al., 2010). In viral infections, RXR activation following treatment with an agonist reduced the production of interferon by macrophages and host resistance to viral infection (Ma et al., 2014). In a mouse model of systemic lupus erythematosus (autoimmune glomerulonephritis), the lack of RXR in macrophages exacerbated the disease due to the lack of the ability of macrophages to engulf and clear apoptotic cells (Roszer et al., 2011).

In cancer, the RXR role in macrophages is highly diverse, depending on the origin of the cancer and the prevalence of resident or infiltrating macrophages. In ovarian cancer, tissue resident macrophages infiltrate the tumors early and drive tumor growth by expressing RXR that regulates chromatin accessibility and lipid metabolism. Knockout of RXR in macrophages decreased the accumulation of tissue resident macrophages in ovarian tumors and reduced tumor growth (Casanova-Acebes et al., 2020). In contrast, RXR depletion in macrophages in lung cancer had no effect on the size of the primary tumors but increased metastasis by increasing expression of prometastatic genes (Kiss et al., 2017).

4. RAR and RXR agonists for the treatment of cancer

4.1. RAR agonists for leukemia

A translocation between chromosomes 15 and 17 can result in the fusion of the promyelocytic leukemia (PML) gene and the RARα gene. This fusion creates a new pathognomonic protein, PML/RARA, that represses the expression of genes required for myeloid differentiation and that causes acute promyelocytic leukemia (APL) (Noguera et al., 2016; Wang & Chen, 2008; Welch et al., 2011). ATRA is an effective FDA-approved treatment for APL, as it induces granulocytic differentiation in APL (de Thé & Chen, 2010; Wang & Chen, 2008) and non-APL leukemia cells (Breitman et al., 1980; Honma et al., 1980, 1983). Additionally, combined treatment with ATRA and 1,25(OH)2D3, an active form of vitamin D3, enhanced the differentiation of leukemia cells (G. Brown et al., 1994; Makishima et al., 1996; Taimi et al., 1991). 1,25(OH)2D3 induced monocyte and macrophage differentiation (Abe et al., 1981; Mangelsdorf et al., 1984), while ATRA induced granulocytic differentiation (Breitman et al., 1980; Honma et al., 1980). Interestingly, combined treatment with 1,25(OH)2D3 and RAR agonists was more effective in inducing differentiation into macrophage-like cells by selectively inducing M2 macrophage markers in human myeloid leukemia cells, suggesting that the VDR and RAR signaling pathways play complementary roles in the differentiation of myeloid leukemia cells (Takahashi et al., 2014). Vitamin A deficient mice exhibited an abnormal expansion of myeloid cells in the bone marrow, spleen, and peripheral blood that could be reversed following treatment with ATRA (Carman et al., 1992). Conversely, mutations in RARα that lead to dominant-negative effects, as well as the use of RARα antagonists, blocked the maturation of myeloid cells and resulted in an increased number of myeloid precursors (Cañete et al., 2017; Martelli et al., 2015; van Bennekum et al., 1991).

Although RAR agonists are potent and effective for the treatment of leukemia, they are associated with several side effects such as skin toxicity, hypertriglyceridemia, cheilitis, conjunctivitis, and teratogenicity (Costa et al., 1995; Elmazar et al., 1996; Lippman et al., 1994), which limit their clinical use. In contrast, RXR agonists have a more favorable toxicity profile than RAR agonists, with lower toxicity even at high doses (Dragnev et al., 2007; Jurutka et al., 2013; Rendi et al., 2004). This reduction in side effects with RXR agonists is likely because RXR forms heterodimers with many other NRs and has ligands other than RA. These differences result in a broader range of physiological effects independent of retinoid signaling and allow for a wider range of gene regulation and therapeutic potential. The clinical advancement of novel RXR agonists, alone or in combination with other drugs targeting nuclear receptors, is an underexplored but highly promising approach for the treatment of leukemia (Pan & Chen, 2020).

4.2. The RXR agonist bexarotene for cutaneous T cell lymphoma

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of clonal T cell cancers defined by malignant T cell infiltration in the skin (Siegel et al., 2000). These malignant T cells are immunosuppressive and alter the cutaneous microenvironment by widely impairing cellular immunity. CTCLs have become more common in the last three decades. Mycosis fungoides (MF) is the predominant type of CTCL, accounting for approximately four new cases per million individuals each year (Paulli & Berti, 2004; Vonderheid et al., 2002). The classical subtype Sézary syndrome (SS) is more aggressive but less common, as it comprises only 3% of all cases of CTCL or approximately 3 cases per 1,000,000 people in western countries (Saulite et al., 2021). MF lesions are clinically characterized by epidermotropism of helper/memory T cells expressing CD41 and CD45RO1. On the other hand, SS manifestations include lymphadenopathy, erythroderma and detectable neoplastic T cells in the skin, lymph nodes and blood. In general, both subtypes are distinguished by increased IL-4, IL-5, and IL-10 production as well as skin infiltrating and/or circulating malignant CD4 T cells with a Th2 phenotype (Saulite I et al., 2021; R. S. Siegel et al., 2000).

CTCL therapies have been tumor-suppressive rather than curative. Oral bexarotene (Boehm et al., 1994; Querfeld et al., 2006) is the first and only selective RXR agonist to receive regulatory approval for the treatment of CTCL. This drug is approved for the treatment of advanced-stage refractory CTCL in Europe and for all stages of refractory CTCL in the US. Bexarotene has also been tested in clinical trials for the treatment of leukemia and lymphoma (Table 1). Several biological actions have been proposed for the efficacy of bexarotene in CTCL. Some argue that the most relevant mechanism of action is apoptosis of neoplastic T cells, while other studies suggest that bexarotene inhibits molecules necessary for lymphocyte adhesion to the skin and blocks T cell proliferation and IL-2 secretion in CTCL. Clinically relevant concentrations of bexarotene induce apoptosis in CTCL cell lines in vitro by upregulating apoptotic molecules, e.g., caspase-3, Bax-1 and CD95. Bexarotene also affects adhesion molecules that control T cell trafficking, resulting in T cell migration (Lee et al., 1999).

Table 1:

Clinical trials for the RXR agonist bexarotene in lymphoma and leukemia

Tumor	Drug	Target	Phase	Status	ClinicalTrials.gov
Lymphoma	Bexarotene (in CTCL)	RXR	I	Recruiting	NCT05296304
	Bexarotene (in CTCL)	RXR	IV	Completed	NCT01007448
	Bexarotene with Gemcitabine (in CTCL)	RXR	II	Completed	NCT00660231 *
	Bexarotene with Photopheresis (in CTCL)	RXR	I/II	Completed	NCT00306969 *
	Bexarotene with Rosiglitazone (in CTCL)	RXR	II	Completed	NCT00178841 *
	Bexarotene with Targretin (in CTCL)	RXR	I	Terminated	NCT00127101 *
	Bexarotene with photodynamic therapy (in CTCL)	RXR	I	Unknown	NCT00030589 *
	Bexarotene (in CTCL)	RXR	II	Completed	NCT00030849
	Bexarotene and CS-7017	RXR-PPARγ	I	Terminated	NCT01504490
	Bexarotene with liposomal doxorubicin	RXR	II	Completed	NCT00255801 *
	Bexarotene	RXR	III	Terminated	NCT00056056
	Bexarotene	RXR	III	Completed	NCT01578499
	Bexarotene	RXR	-	Not yet recruiting	NCT05106192
Leukemia	Bexarotene	RXR	II	Terminated	NCT00615784
	Bexarotene	RXR	I	Completed	NCT01001143
	Bexarotene	RXR	I	Completed	NCT00316030
	Bexarotene	RXR	II	Completed	NCT00425477

CTCL, Cutaneous T-cell lymphoma

^*: Bexarotene was combined with other drugs not known to activate RXR or RAR

Richardson and colleagues tested the effects of bexarotene treatment in SS patients on CCR4 and chemotactic expression. Cell surface expression of CCR4 on CD4 malignant T cells in SS, determined by flow cytometric analysis, corresponded with the degree of cutaneous T cell involvement. The same study also reported that most SS patients have high (>50%) CCR4 expression in the peripheral blood mononuclear cells, with the bulk of these cells being malignant CD4 T cells. In contrast, CCR4 expression was detected in only 5-15% of mononuclear cells from normal individuals. Therefore, the authors hypothesized that by downregulating CCR4 expression on the surface of neoplastic cells, bexarotene prevents the migration of malignant T cells to the skin (Richardson et al., 2007). Some authors also reported that the decreased expression of CCR4 expression by bexarotene should be considered a precursor event to apoptosis. In addition, the induction of neoplastic T cell apoptosis in CTCL likely increases Treg cell populations, which is a secondary immunological effect of bexarotene. In another study, patients with SS were compared to healthy volunteers to determine whether bexarotene regulates the Th2 cytokine IL-4. Notably, IL-4 levels declined in 45% of SS patients and 67% of healthy participants treated with bexarotene, but no change was reported in patients resistant to apoptosis. These data indicate that the Th2 cytokines, which are prevalent in SS, can also be suppressed as part of the therapeutic benefits of bexarotene (Budgin et al., 2005). Bexarotene also modifies the expression of CD25 on Sezary cells. After one week of bexarotene therapy, CD25 expression was elevated in 8 of 13 patients receiving dosages of at least 150 mg/day. Six patients had circulating Sezary cells, with upregulated CD25 expression on the tumor cells (Foss et al., 2005).

The role of RXR and its pharmacological agonists on Tregs and Th17 in autoimmune and neurologic diseases is well established, as described in Section 3.1. However, the immune modulatory effects in lymphomas are not fully known. Although bexarotene was approved to treat CTCL in 1999, its ability to decrease Foxp3 Tregs was only reported recently (Stahly et al., 2020). Implications for treatment follow-up and the use of Foxp3 expression as a clinical biomarker in CTCL are still in development. Moreover, expression of CCL22, CXCL5, CXCL10, and p19 was significantly lower following treatment with bexarotene in the TME in an EL4 mouse model of T-cell lymphoma. In patients with CTCL who responded to treatment with bexarotene, serum levels of CCL22 were decreased by 80%. Further studies confirm that the main source of CCL22 in both CTCL patients and the EL4 mouse T-cell lymphoma model is macrophages (Tanita et al., 2019).

5. Modulation of the tumor microenvironment by new RXR agonists

RXR agonists modulate gene expression and regulate cellular proliferation, differentiation, and survival, which may lead to the suppression of tumor growth (Connolly et al., 2013; Mongan & Gudas, 2007; Uray et al., 2016). Bexarotene remains the only FDA-approved RXR agonist and only for the treatment of CTCL, but it also has been tested in clinical trials for breast cancer, non-small cell lung cancer, thyroid cancer, and melanoma. In all these studies, bexarotene, which can activate RXR at nanomolar concentrations (de Almeida & Conda-Sheridan, 2019; Michellys et al., 2003), was reported to have therapeutic benefits, at least in subpopulations of patients (Boehm et al., 1994; Esteva et al., 2003; Pérez et al., 2012; Tyagi, 2005). However, at high concentrations, bexarotene retains some binding to RAR (Dragnev et al., 2000; Duvic, Hymes, et al., 2001; Duvic, Martin, et al., 2001; R. S. Siegel et al., 2000; Talpur et al., 2002), which contributes to unwanted side effects (Maminakis et al., 2018; Schlotawa et al., 2023). Additional RXR agonists have been synthesized that are more selective for RXR, more potent, and with lower toxicity. Some of these new RXR agonists include LG100268, LG101506, UAB30, Net-3IB, IRX194204, MSU-42011, and V-125; therapeutic benefits have been reported for all these compounds in preclinical studies (Boehm et al., 1994; Leal, Reich, et al., 2021; K. T. Liby & Sporn, 2016; Michellys et al., 2003; Nakayama et al., 2011; Reich et al., 2023; Vedell et al., 2013).

Multiple clinical trials have investigated RAR and RXR agonists for the treatment of solid tumors (Table 2), but they largely neglected to evaluate their impact on immune cells in the TME. With the established correlation between RXRs, RARs and immunity, numerous preclinical studies have demonstrated that in addition to their anti-tumor effects, various RAR and RXR agonists regulate diverse immune cells within the TME, suggesting new avenues of investigation for both chemoprevention and chemotherapy. This section outlines the prominent regulatory effects of RAR and RXR agonists as TME immunomodulators in various types of cancers.

Table 2:

Clinical trials of RXR and RAR agonists in solid tumors

Tumor	Drug	Target	Phase	Status	ClinicalTrials.gov
Non-melanoma Skin Cancer	Vitamin D	RXR-VDR	-	Not yet recruiting	NCT05550766
	13-Cis retinoic acid	RAR	-	Completed	NCT00025012
	UAB30	RXR	I	Recruiting	NCT03327064
Melanoma	ATRA	RARα	II	Active	NCT02403778
	13-Cis retinoic acid	RAR	-	Completed	NCT00025012
Non-Small Cell Lung Cancer	IRX4204 with Erlotinib	RXRs	I	Suspended	NCT02991651 *
	NRX194204	RXRs	II	Unknown	NCT00964132
	Bexarotene	RXR	I/II	Terminated	NCT00153842
	Bexarotene	RXR	I	Completed	NCT01116622
	Bexarotene	RXR	III	Completed	NCT00050973
	Bexarotene with erlotinib	RXR	-	Completed	NCT00125372 *
	Bexarotene with erlotinib	RXR	II	Completed	NCT00411632 *
	Bexarotene with erlotinib	RXR	II	Completed	NCT00125359 *
	Bexarotene with carboplatin and paclitaxel	RXR	III	Completed	NCT00050960 *
	Bexarotene with gefitinib	RXR	I/II	Completed	NCT00238628 *
	Bexarotene with ATRA	RXR	II	Unknown	NCT00514293 *
	ATRA	RAR	III	Unknown	NCT01041833
	13-Cis retinoic acid	RAR	II	Completed	NCT00002586
	ATRA	RARα	II	Completed	NCT00617409
	ATRA	RARα	II	Completed	NCT01048645
Myeloma	Bexarotene and CS-7017	RXR-PPARγ	I	Terminated	NCT01504490
Breast Cancer	UAB30	RXR	I	Active	NCT02876640
	Bexarotene	RXR	I	Completed	NCT03323658
	Bexarotene	RXR	II	Completed	NCT00003752
	Bexarotene (in TNBC)	RXR	I	Recruiting	NCT04664829
	Bexarotene (prevention)	RXR	I	Completed	NCT00055991
	9-Cis retinoic acid	RAR	I	Completed	NCT00001504
	ATRA	RARα	II	Recruiting	NCT04113863
Neuroblastoma	Spironolactone	RXRγ	II	Recruiting	NCT05754684
	13-Cis retinoic acid	RAR	I	Completed	NCT01208454
	Retinoic Acid with Onalta	RXR	II	Withdrawn	NCT01048086 *
Prostate Cancer	NRX194204	RXRs	II	Completed	NCT01540071
Thyroid Cancer	Bexarotene	RXR	I	Completed	NCT00718770
Head and neck cancer	13-Cis retinoic acid	RAR	III	Completed	NCT03370367
	Bexarotene	RXR	I	Completed	NCT01116622
Cervical tumors	13-Cis retinoic acid	RAR	III	Completed	NCT00001073
Esophagus	Bexarotene	RXR	I	Completed	NCT01116622

ATRA, all-trans retinoic acid; TNBC, Triple-negative breast cancer

^*: The RAR or RXR agonists were tested in combination with other drugs that are not known to activate these receptors.

5.1. Breast Cancer

Despite the numerous treatments available, aggressive subtypes such as HER2-positive and triple-negative breast cancers are still challenging to treat (Bianchini et al., 2022; Swain et al., 2023). Many RXR agonists are effective for both prevention and treatment in preclinical models of breast cancer (P. H. Brown et al., 2008; Leal et al., 2019; Leal, Moerland, et al., 2021; K. Liby et al., 2006; Reich et al., 2023), and for prevention in clinical trials (Moyer & Brown, 2023). Bexarotene alone (P. H. Brown et al., 2008) or in combination with a selective COX-2 inhibitor significantly delayed the formation of HER2/neu mammary tumors and suppressed aromatase activity more effectively than either drug alone, suggesting a promising approach for breast cancer prevention (P. H. Brown et al., 2008). Topical bexarotene has also been developed as a potential drug for prevention of breast cancer in high-risk patients (Moyer & Brown, 2023).

The immunomodulatory effects of bexarotene and other RXR agonists have only been explored in the past 5 years, either alone or in combination with immunotherapy and/or vaccines. Treatment of mammary tumors in TgMMTV-neu mice with bexarotene or 9cUAB30 alone increased Th1 plasmacytoid DCs and myeloid DCs. Pretreatment of TgMMTV-neu mice before vaccination with 150 αg of a HER2-IGFBP2-IGF1R vaccine and 5 αg of a GMCSF adjuvant increased antigen-specific T cells and IFN-γ-mediated immune responses to HER2, IGFBP2, and IGF1R. The combination of paclitaxel and bexarotene reduced tumor growth by 83% (p=0.02 compared to each individual drug) and increased the number of CD8 T cells in the tumor by 58% compared to the vehicle control group (p=0.01). Treatment with bexarotene alone also activated type 1 antigen presenting cells (APC) and increased the infiltration of CD8 T cells into the tumor (Disis et al., 2013; Stanton et al., 2017, 2021). Notably, in breast cancers with mutations in estrogen receptor alpha (ER/ESR1), RXR activation reprogrammed FOXA1 but a RXR antagonist inhibited the growth of these breast cancer cells in vitro and of a PDX organoid with a known ESR1 mutation (Y. Wu et al., 2023).

In contrast to bexarotene, which did not show immunomodulatory activity in the MMTV-Neu and MMTV-PyMT models of breast cancer, the more potent RXR agonist LG100268 decreased tumor-promoting Foxp3 CD4 T cells in both models and increased the ratio of CD8/CD4, CD25 T cells when used in combination with anti-PDL1 antibodies (Leal et al., 2019). LG100268 also reduced the infiltration of MDSCs and CD206-expressing macrophages. Newer RXR agonists, MSU-42011 and V-125, increased the percentage of activated CD8 T cells and reduced populations of immunosuppressive macrophages in MMTV-Neu mice (Leal, Moerland, et al., 2021; Reich et al., 2022, 2023).

5.2. Lung Cancer

Lung cancer is one of the most frequently diagnosed cancers, but resistance to targeted therapies remains a significant challenge and leads to very high mortality rates (R. L. Siegel et al., 2023). Immune checkpoint inhibitors, including FDA-approved antibody drugs against PD1 and PD-L1, have become a standard of care for lung cancer patients without identifiable molecular targets, but the majority of patients fail to respond (Amanam et al., 2020). RXR agonists have emerged as a potential pharmacological approach to modulate the lung TME, with enhanced anti-tumor efficacy and reduction of tumor-promoting immune populations with select RXR agonists. Although bexarotene has been used in numerous clinical trials for treating patients with lung cancer (Table 2), it has not been approved for the treatment of solid epithelial tumors, at least in part because of significant adverse effects (Qu & Tang, 2010; Ramlau et al., 2008; Tyagi, 2005). Consequently, other RXR agonists have been designed, synthesized, and screened to improve pharmacokinetic properties and to reduce toxicity. For example, LG100268 reduced the percentage of TAMs and MDSCs in a lung cancer model. Other RXR agonists skewed macrophages toward an anti-tumor phenotype and inhibited critical inflammatory mediators, such as IL-6, IL-1β, CCL9, and nitric oxide synthase (iNOS) (D. Zhang et al., 2019). Preliminary screening for other new RXR agonists tested the ability of these compounds to reduce nitric oxide production in RAW264.7 macrophage-like cells (K. Liby et al., 2006; D. Zhang et al., 2019). Moerland et al. found that the new RXR agonist MSU-42011, when combined with carboplatin and paclitaxel chemotherapies, suppressed the number of alveolar macrophages, and reduced the expression of CD206, a marker of tumor-promoting macrophages, in the lungs (Moerland et al., 2020). MSU-42011 also increased the percentage of activated CD8 cytotoxic T cells. When MSU-42011 was combined with anti-PD1 antibodies, the combination decreased total tumor burden and increased anti-tumor T cells in a Kras-driven AJ model of lung cancer (Leal, Moerland, et al., 2021).

5.3. Colon Cancer

Patients with chronic inflammatory bowel disease (IBD) have an elevated risk of developing colorectal cancer (CRC) (Kraus & Arber, 2009; Ullman & Itzkowitz, 2011). Reducing inflammation in the colon may help to prevent some cases of colon cancer. Desreumaux et al. found that RXR agonists reduced intestinal inflammation as effectively as PPARγ agonists, with a potential synergistic effect when used in combination. RXR and PPARγ agonists also significantly reduced colitis when mice were challenged with 2,4,6-trinitrobenzene sulfonic acid (TNBS) (Desreumaux et al., 2001). The intestine is the largest reservoir of macrophages in humans, with macrophages accounting for 10-20% of all mononuclear cells within the lamina propria (Bain et al., 2014; Little et al., 2014). RA from local tissue modulates a specific transcriptional program of peritoneal macrophages regulated by the transcription factor GATA-6 to control the migration of macrophages into the peritoneal cavity, which can control the initiation and development of gastrointestinal tumors (Okabe & Medzhitov, 2014). The identification of differentiation characteristics in cancer-induced macrophages is useful to help understand the correlation between TAMs and colon cancer. Two well-established macrophage polarization programs are classically activated (M1) and alternatively activated (M2) macrophages. M2 macrophages are essential for tissue remodeling and suppressing inflammatory responses, which can promote tumor progression (Jetten et al., 2014; Yunna et al., 2020). Dong et al. reported that fenretinide (4-HPR), a synthetic derivative of ATRA, reduces tumorigenesis in the colon and inhibits tumor-promoting macrophage polarization by suppressing STAT6 phosphorylation (Dong et al., 2017).

5.4. Other cancers

The effects that RAR and RXR agonists have on the immune cells within the TME to prevent and treat cancers have received increasing attention. In ovarian cancer, peritoneal macrophages infiltrate the tumors and promote tumor progression. Interestingly, deleting RXR from these macrophages induced apoptosis in tumor-promoting macrophages, leading to reduced tumor growth. Thus, targeting RXR-dependent peritoneal macrophages could be a promising therapeutic approach for treating ovarian cancer (Casanova-Acebes et al., 2020). In patients with metastatic renal cell carcinoma, ATRA significantly reduced the number of immature MDSCs and improved DCs function, leading to improved antigen-specific T cell response (Mirza et al., 2006). ATRA also inhibited the tumor-promoting M2 polarization of macrophages and reduced the infiltration of M2-type macrophages in osteosarcoma, resulting in decreased pulmonary metastasis (Q. Zhou et al., 2017). In addition, several new RXR agonists decrease expression of chemokines/cytokines needed in inflammation and prevent non-melanoma skin cancers without causing toxicity (Atigadda et al., 2022). The appearance of TAMs in the tumor stroma often indicates a poor prognosis in various neoplasms associated with angiogenesis. Notably, RA directly blocked the recruitment and activation of TAMs by monocyte chemoattractant protein-1, which typically exhibits high expression in head and neck squamous cell carcinoma (HNSCC), thereby altering the ability of TAMs to facilitate the angiogenic process (Liss et al., 2002). RA treatment also decreased TGF-β1 secretion, resulting in a significant reduction in the production of vascular endothelial growth factor (VEGF) and IL-8 induced by TAMs. As a result, these effects on TAMs in RA-treated HNSCC lead to slower growth and decreased tumor aggression due to reduced vascularity (Liss et al., 2002).

6. Conclusions and future directions

The importance of the immunomodulatory properties of RXR nuclear receptors, binding partners, and pharmacological agonists has emerged, first in diseases in which immune components contribute to disease pathogenesis, such as autoimmune and neurogenerative diseases, and more recently in cancer. Most research to date highlights the many roles of RXR in immune cell differentiation, homing and function. Our studies were the first to demonstrate that the anti-tumor effects of RXR agonists in the lung are dependent on the presence of a fully functional immune system. MSU-42011, a RXR agonist with promising anti-tumor effects in a carcinogenesis-induced murine mouse model, reduced the number and grade of tumors in the lung, but the anti-tumor effects were lost when lung cancer cells were implanted in the flank of athymic nude mice (Leal, Moerland, et al., 2021). These observations are consistent with studies that reported limited growth inhibitory or pro-apoptotic effects of many RXR agonists on epithelial cancer cells in vitro, even at concentrations > 20 μM (K. T. Liby & Sporn, 2016).

RXR biology in immune cells is dependent on the origin of the immune cell (resident versus infiltrating) and the organ in which the tumor develops. It is likely that effects of RXR agonists will differ depending on the cancer type being treated. Interestingly, in models of breast and lung cancer, with different populations of macrophages, both LG100268 and MSU-42011 reduced CD206 expression in macrophages (Leal et al., 2019; Leal, Moerland, et al., 2021; Moerland et al., 2020). However, in ovarian cancer, tissue resident macrophages expressing RXR promoted tumor growth (Casanova-Acebes et al., 2020). Notably, immune modulatory effects of bexarotene in CTCL have only been explored recently. Because patient responses to bexarotene correlated with lower expression of CCL22 in macrophages and increased CD25 expression in tumor infiltrating lymphocytes, both CCL22 and CD25 have been suggested as predictive markers of patient response (Tanita et al., 2019; Yamamoto et al., 2023).

The RXR agonist bexarotene has been tested in multiple clinical trials for several cancers (Tables 1 and 2). Despite numerous studies, bexarotene is still only used clinically to treat CTCL and is restricted to patients without other treatment options. Although bexarotene is not as toxic as traditional non-targeted chemotherapies such as cyclophosphamide and cisplatin, treatment duration for this drug is often limited because of hypothyroidism and hypertriglyceridemia (Sherman, 2003; Standeven et al., 2001; Takamura et al., 2022). Hypothyroidism is likely a consequence of RXRγ-mediated TSH gene expression, as bexarotene also causes marked reductions in serum TSH and thyroxine concentrations (Makita et al., 2019; Mathews & Gigliottie, 2021; Sherman, 2003). Thus, bexarotene serves as a useful example of the challenges that must be overcome when developing new RXR agonists.

The physicochemical properties of bexarotene, including high lipophilicity, low solubility, and poor pharmacokinetics, are unfavorable for drug delivery (Howell et al., 2001). In addition to the most frequent adverse side effects of hypothyroidism and elevated triglyceride and cholesterol levels, bexarotene has been reported to cause leukopenia and increase the risk of acute pancreatitis (Duvic, Martin, et al., 2001; Schadt, 2013). Other known side effects of bexarotene such as teratogenicity, higher risk of infections, and dermatitis are likely to be found with the majority of RXR agonists but can be clinically managed.

As a RXR is a versatile heterodimeric partner for numerous nuclear receptors, the possibility of overly broad or off-target gene activation must be addressed. However, identifying RXR agonists that selectively target specific RXR homodimers or heterodimers, thereby achieving the desired gene modulation without inadvertently inducing unwanted effects, is feasible (Hanish et al., 2018; Marshall et al., 2015). Recently, the natural product valerenic acid was discovered to be a RXR agonist with a remarkable preference for RXR homodimerization. Structural modifications enhanced RXR homodimer agonism (Zaienne et al., 2023). Other RXR agonists such as UAB110 and UAB111 are more potent than older molecules such as UAB30 and bexarotene. Notably, UAB110 treatment caused fewer toxic effects and induced different gene expression profiles than bexarotene (Melo et al., 2023).

Optimizing the structures of new RXR agonists is critical for clinical advancement. New screening paradigms, such as inhibition of nitric oxide in macrophages in vitro, which correlates with anti-tumor efficacy in vivo, and using sterol regulatory element binding proteins (SREBP) elevation as a biomarker for predicting hypertriglyceridemia (Moerland et al., 2020; Wagner et al., 2009) have been used to screen new RXR agonists. Other key priorities include the need to explore and modify molecular cores, consider isosteric replacements, and investigate less-explored scaffolds. Computational studies and molecular dynamics simulations should be used to provide valuable insights. Designing selective agonists for specific RXR subtypes is challenging but feasible (Takamatsu et al., 2008). Nanotechnology offers opportunities for encapsulating RXR agonists to help address biodistribution and solubility issues. Incorporating targeting elements like peptides and aptamers into agonists could also improve disease localization, particularly in cancer (de Almeida & Conda-Sheridan, 2019; Watanabe & Kakuta, 2018).

Despite the high potential of using RXR agonists to treat cancer, additional challenges remain. As with most targeted therapies, unpredictable efficacy may be observed with RXR-based cancer therapies, with variations in clinical outcome and length of remission. These results are often due to the heterogeneity of tumors and the immune microenvironment among patients and within individual tumors. Additionally, RXR-based therapies typically show limited single-agent activity, often resulting in growth inhibition rather than regression, which complicates the design of clinical trials. In cancer, a combination of drugs with different mechanisms will most likely be more effective than single agents. Moreover, combination treatments with an RXR agonist would limit toxicity, reduce drug resistance and make cancer stem cells more responsive to conventional chemotherapeutic agents (Issac et al., 2023).

The importance of RXR-mediated effects in macrophages on both tumor progression (Casanova-Acebes et al., 2020) and metastasis (Kiss et al., 2017) and the immunomodulation by RXR agonists in multiple tumor models (Leal et al., 2019; Leal, Moerland, et al., 2021; Moerland et al., 2020) suggests that combinations of RXR agonists with immune checkpoint inhibitors should be developed. Reported increases in CD8 cytotoxic T cells with the combination of anti-PDL1 antibodies with LG100268 and MSU-42011 support the idea that RXR agonists could increase the efficacy of immunotherapy in patients with cancer. The effects on T cell homing and the enhancement in the activation of DCs imply that RXR agonists can enhance the effects of cancer vaccines or even function as vaccine-like treatments (Disis et al., 2013; Nagy et al., 2012; Shen et al., 2018). To optimize RXR-based immunotherapy, the right RXR agonist and treatment regimen must be selected for each patient or tumor. This advancement toward precision medicine requires categorizing cancer patients based on molecular subtypes and the TME, as well as identifying reliable prognostic and predictive biomarkers (Luna & Shinohara, 2023). Key remaining questions regarding RXR biology and the pharmacological use of RXR agonists in cancer are listed in Table 3.

Table 3:

Key questions for future studies

1. Can RXR agonists be developed to preferentially target immune cells?

2. Because RXR is a central nuclear receptor that heterodimerizes with other NRs, can the binding to a specific NR be skewed by pharmacological agonists to change the immune cell populations that are targeted?

3) What are the biological effects of RXR activation in T cells and dendritic cells within the tumor microenvironment compared to autoimmune and neurodegenerative diseases?

4) What are the functions of RXR and RXR activation in resident vs. infiltrating immune populations?

5) What are the consequences of RXR agonists in cancer cells since they do not appear to induce apoptosis directly, at least in vitro?

6) Can bexarotene or new RXR agonists be combined with immunotherapy to increase response in patients with cancer?

Abbreviations

9-cis-RA

9-cis retinoic acid

AF2

activation function 2

APC

antigen presenting cell

APL

acute promyelocytic leukemia

ATRA

all trans-retinoic acid

CRC

colorectal cancer

CTCL

cutaneous T-cell lymphoma

DBD

DNA binding domain

DC

dendritic cell

DHA

docosahexaenoic acid

EAE

experimental autoimmune encephalomyelitis

ER/ESR1

estrogen receptor alpha

FXR

farnesoid X receptor

HNSCC

head and neck squamous cell carcinoma

IBD

inflammatory bowel disease

iNOS

nitric oxide synthase

iTreg

inducible regulatory T cells

LBD

ligand binding domain

LXR

liver X receptor

M1

classically activated macrophage

M2

alternatively activated macrophage

MDSC

myeloid-derived suppressor cell

MF

mycosis fungoides

NR

nuclear receptor

NR2B1/2/3

nuclear receptor subfamily 2, group B, member 1/2/3

NTD

N-terminal domain

PML

promyelocytic leukemia

PPAR

peroxisome proliferator-activated receptor

RA

retinoic acid

RAR

retinoic acid receptor

RARE

retinoic acid response element

RXR

retinoid X receptor

SREBP

sterol regulatory element binding protein

SS

Sézary syndrome

TAM

tumor-associated macrophage

TME

tumor microenvironment

TNBC

triple-negative breast cancer

TR

thyroid receptor

Treg

regulatory T cell

TSH

thyroid-stimulating hormone

VDR

vitamin D receptor

VEGF

vascular endothelial growth factor