The rexinoid V-125 reduces tumor growth in preclinical models of breast and lung cancer

Rexinoids are ligands which activate retinoid X receptors (RXRs), regulating transcription of genes involved in cancer-relevant processes. Rexinoids have anti-neoplastic activity in multiple preclinical studies. Bexarotene, used to treat cutaneous T cell lymphoma, is the only FDA-approved rexinoid. Bexarotene has also been evaluated in clinical trials for lung and metastatic breast cancer, wherein subsets of patients responded despite advanced disease. By modifying structures of known rexinoids, we can improve potency and toxicity. We previously screened a series of novel rexinoids and selected V-125 as the lead based on performance in optimized in vitro assays. To validate our screening paradigm, we tested V-125 in clinically relevant mouse models of breast and lung cancer. V-125 significantly (p < 0.001) increased time to tumor development in the MMTV-Neu breast cancer model. Treatment of established mammary tumors with V-125 significantly (p < 0.05) increased overall survival. In the A/J lung cancer model, V-125 significantly (p < 0.01) decreased number, size, and burden of lung tumors. Although bexarotene elevated triglycerides and cholesterol in these models, V-125 demonstrated an improved safety profile. These studies provide evidence that our screening paradigm predicts novel rexinoid efficacy and suggest that V-125 could be developed into a new cancer therapeutic.

Tamoxifen is known to cause side effects in patients such as hot flashes and an increased risk of endometrial carcinoma 18 . SERMs are an effective treatment and prevention option for ER + breast cancer, but fewer strategies exist for hormone receptor negative breast cancers, despite high morbidity and mortality. With their enhanced efficacy coupled with a favorable toxicity profile, rexinoids may be useful not only for cancer treatment but also for prevention. In 2002, Wu et al. tested bexarotene for prevention in MMTV-erbB2 transgenic mice. Bexarotene significantly increased tumor-free survival, even at doses as low as 10 mg/kg 19 . The more potent rexinoid LG100268 and a SERM were highly effective at delaying tumor development in a similar murine model of HER2 + breast cancer, while the combination of a SERM and LG100268 completely prevented tumor development over a 50 week period of treatment 20 .
In addition to breast cancer, rexinoids are also effective in the A/J mouse model of lung cancer 21 . These mice develop Kras mutations in the lung after being challenged by carcinogens such as vinyl and ethyl carbamate, which form epoxides and DNA adducts, leading to the development of lung adenocarcinomas 22 . Activating mutations in KRAS are one of the most common oncogenic drivers in human lung adenocarcinomas 23 . Ethyl and vinyl carbamate are found in tobacco products 24 , making the A/J model an appropriate model of NSCLC in smokers. Several rexinoids inhibit growth of lung tumors in this model, including LG100268, LG101506, and IRX194204 25,26 .
Although rexinoids showed promising activity in several preclinical models of cancer 13,19,26 , clinical trials of bexarotene in both metastatic breast cancer and NSCLC did not lead to FDA approval. To improve clinical responses, new rexinoids with greater potency are needed. As rexinoids are also effective in cancer prevention studies, the toxicity profile of rexinoids must be monitored, as patients on long-term treatment with bexarotene experienced cumulative side effects, particularly elevated triglycerides and hypothyroidism 27 . To address these challenges, we synthesized novel analogues of bexarotene using molecular modeling software to guide chemical substitutions and rational drug design. These new compounds activated RXR with nanomolar potency and stimulated RXR-regulated transcription with minimal RAR activation 28,29 .
To identify rexinoids with potential in vivo efficacy, we have developed a series of in vitro screening assays 30 . Of ten new rexinoids screened in these assays, V-125 ( Fig. 1) was selected as the lead compound, as it was the most potent in the iNOS suppression assay, an assay which correlates with efficacy in the A/J model of lung cancer 30 . The EC 50 value for RXRα activation for V-125 was 14 nM vs. 55 nM for bexarotene. Activation of sterol regulatory element-binding protein (SREBP), a biomarker of triglyceride elevation was lower with V-125 than either bexarotene or LG100268 30 . To further validate our in vitro screening paradigm, we tested the efficacy and safety of V-125 in preclinical models of breast and lung cancer. Bexarotene is approved to treat cutaneous T-cell lymphoma, and V-125 was identified as a promising novel rexinoid based on its activity in published in vitro assays 30 . The iNOS suppression in RAW 264.7 cells was determined by cell treatment with 100 nM rexinoid followed by challenge with 1 ng/mL LPS over 24 h. Griess assay measured NO production normalized to LPS-stimulated control. SREBP and RXR activation were assessed as reported in prior work 30

Materials and methods
Drugs. Synthesis of V-125 was previously described 31 . Bexarotene was purchased from LC Laboratories. Rexinoids were dissolved in a vehicle (50 ml/kg diet) of 1 part ethanol:3 parts highly purified coconut oil (Neobee oil, Thermo Fisher) and then mixed into 1 kg of powdered diet (AIN-93M, BioServ for A/J mice or 5002 rodent chow, PMI Nutrition International for MMTV-neu mice) using a commercial KitchenAid mixer. The same vehicle was used in the control diets. In the MMTV-Neu model, doses were 30 mg per kg of diet (~ 7.5 mg per kg body weight) for prevention studies and 100 mg per kg of diet (~ 25 mg per kg body weight) for treatment studies, as utilized in previous publications 20,32 . In the A/J model, doses were 40 and 80 mg per kg of diet (~ 10 and 20 mg per kg body weight), doses used in previous publications 25 . Rexinoids remain stable in diet at 4 °C for up to 6 weeks, as confirmed by liquid chromatography-mass spectrometry.
In vivo experiments. All animal studies were approved by the Institutional Animal Care and Use Committee at Michigan State University (IACUC protocol number 201800050). All protocols were carried out ethically in accordance with the Regulations for the Management of Laboratory Animals at Michigan State University and in compliance with the ARRIVE guidelines. Every effort was made to minimize suffering throughout these studies. Mice were euthanized by inhalation of carbon dioxide followed by cervical dislocation.
Lung carcinogenesis studies. Female A/J (Jackson Laboratory) mice at 7 and 8 weeks of age were injected intraperitoneally with vinyl carbamate (16 mg/kg, Toronto Research Chemicals) in a vehicle of isotonic saline. One week after the last injection of vinyl carbamate, rexinoids were fed in diet as described above. After 16 weeks of treatment, mice were euthanized and tissues collected. Lungs were inflated with PBS, and tumor number on the surface of the lungs was evaluated. The left lung was then fixed in 10% neutral buffered formalin; samples were blinded and randomized. The lung was sectioned and stained with H&E for evaluation of the histopathology on multiple sections, using previously established criteria 25 .
Breast cancer studies. For prevention studies, 10-week-old female MMTV-Neu mice 33 (Jackson Laboratory) were fed control diet or 30 mg rexinoid per kg diet. Littermate-matched controls were used whenever possible. Mice were palpated twice weekly for the development of new tumors. For treatment studies, mice were fed standard diet and palpated twice weekly until tumors reached 5 mm in diameter, at which time mice were randomized and started on control diet or 100 mg V-125 per kg of diet. Mice were fed until tumors reached IACUCdefined endpoints (overall survival studies) or for 10 days (biomarker studies). At study conclusion, mice were euthanized, tissues were harvested, and tumors and livers weighed.
Triglyceride quantification. Plasma was harvested from mice at necropsy. Triglyceride and cholesterol levels were measured using the Triglyceride Quantification Assay Kit (Abcam) or the Cholesterol Quantification Kit (Sigma-Aldrich) as per the manufacturers' recommended protocols.

Statistical analysis.
Results were expressed as the means ± standard errors as indicated in figure legends.
Kaplan-Meier survival curves from in vivo experiments were analyzed using the log rank test. Data from plasma triglyceride and cholesterol experiments were analyzed using one-way ANOVA, and significant differences between groups were determined by the Tukey HSD multiple comparison method (VassarStats.com). Data from lung experiments were analyzed by one-way ANOVA. When data fit a normal distribution, the Holm-Sidak test for multiple comparison was used. www.nature.com/scientificreports/ Quantitative PCR. qPCR was performed to validate mRNA expression of IL-6, Jun, and COX2 in tumors.
mRNA was quantified using SYBR green, and values were normalized to the housekeeping gene GAPDH.

V-125 clusters favorably in computational analyses of in vitro activity.
We have found that applying principal component analysis (PCA) in our prior work 35,36 with rexinoids has helped to visualize particularly active rexinoids with improved therapeutic potential, and to that end, we have conducted a PCA with V-125 and the nine other novel rexinoids ( Fig. 1) to visualize where they group relative to each other as well as bexarotene and LG100268 (Figs. S1, S2). The PCA analysis resulted in three groups-A, B, and C-in which rexinoids clustered according to their activities in in vitro assays. In Group A, containing bexarotene, we observed the difluorobexarotene analog (1) as well as a rexinoid with a biphenyl structural motif (9). In Group B, containing LG100268, we observed a pyridine analog of bexarotene (2) as well as a pyrimidine containing rexinoid (4) and another biphenyl rexinoid (8). In group C, containing V-125, we observed the other rexinoids with structurally similar features-3, 5, 6, and 7-though, V-125 was somewhat isolated whereas the other rexinoids in this group clustered more closely together. We envision PCA to be useful to quickly assess potential of novel rexinoids by seeing where they group according to their activities in these assays.

V-125 prevents lung tumor development in A/J mice.
A/J mice were challenged with vinyl carbamate to induce Kras mutations and subsequent lung adenocarcinomas 37 and then treated with V-125. Mice were fed control diet or rexinoids in diet starting two weeks post-carcinogen for 16 weeks. At the conclusion of the study, evaluation of lung tumor number, size, and histopathology on slides (2 per mouse) was done in a randomized and blinded manner by two independent investigators. V-125 at 80 mg/kg of diet significantly (p < 0.01, Fig. 2A Although both V-125 and bexarotene were well-tolerated in this model based on animal weights (Fig. S3A), bexarotene elevates triglycerides in animal models 21 and in human patients 38 . As shown in Fig. 2D, bexarotene significantly (p < 0.01) increased plasma triglyceride levels at both the 40 and 80 mg/kg doses (2.30 ± 0.26 nmol/ μL and 2.97 ± 0.35 nmol/μL, respectively, vs. control 0.97 ± 0.09 nmol/μL) in the A/J mice. In contrast, V-125 had no effect on plasma triglycerides. Bexarotene also significantly (p < 0.001) elevated plasma cholesterol (Fig. 2E) in a dose-dependent manner (2.08 ± 0.31 µg/μL in mice fed bexarotene at 40 mg/kg of diet and 2.80 ± 0.30 µg/ μL in mice fed bexarotene at 80 mg/kg of diet, vs control 1.27 ± 0.14 µg/μL), while V-125 did not significantly increase plasma cholesterol at either dose.
Lung tumors were classified as low, medium, or high grade based on previously established criteria 25 (Table 1). Treatment with 80 mg/kg V-125 significantly (p < 0.05) increased the proportion of tumors graded low/medium (66% vs. control 43%) and reduced (p < 0.05) the proportion of tumors classified as high grade (34% vs. control 57%). In comparison, bexarotene did not significantly change the proportions of tumors of any grade at either dose.
V-125 delays the development of HER2-positive mammary tumors in MMTV-Neu mice. MMTV-Neu mice express wild-type, unactivated Neu in mammary tissue under the control of the mouse mammary tumor virus (MMTV) promoter 33 and develop focal mammary adenocarcinomas by 25-35 weeks of age 39 . To investigate the chemopreventive effects of V-125 in this preclinical model of HER2 + breast cancer 40,41 , MMTV-neu mice were fed control diet or V-125 in diet (30 mg/kg) starting at 10 weeks of age. V-125 significantly (p < 0.001) delayed initial tumor development compared to the control group (Fig. 3A), resulting in an approximately 10 week increase in mean time to tumor development (36.1 ± 7.8 weeks vs. control 26.5 ± 4.9 weeks). Treatment with V-125 was well tolerated in this study (Fig. S3B). Bexarotene also significantly (p < 0.05) increased the time to initial tumor development in MMTV-neu mice (Fig. 3B) but was not as effective as V-125, delaying tumor development by only 4 weeks (35.9 ± 4.2 weeks vs. control 31.7 ± 4.1 weeks).

Treatment with V-125 extends overall survival in MMTV-Neu mice.
To evaluate the anti-tumor efficacy of V-125, MMTV-Neu mice with established tumors measuring 5 mm in diameter were treated with control diet or V-125 at a dose of 100 mg/kg of diet. Tumors were measured twice weekly until they reached 10 mm in diameter, at which point they were euthanized per IACUC guidelines. Time from initiation of drug diet to euthanasia was compared between groups. Treatment with V-125 significantly (p < 0.0172) increased overall survival (51.6 ± 15.8 days vs. control 32.8 ± 13.6 days) (Fig. 4A). Average tumor volume over the first 14 days of treatment was calculated and normalized to the initial tumor volume when treatment diet was started. The average change in tumor volume of the V-125-treated group was significantly (p < 0.01) lower than the average tumor volume in the control group at day 4 (0.57 ± 0.10 vs. control 1.8 ± 0.22), day 11 (0.53 ± 0.14 vs. control 2.9 ± 0.33), and day 14 (0.89 ± 0.21 vs. control 3.6 ± 0.86) of treatment (Fig. 4B).

V-125 alters immune-related biomarkers in MMTV-Neu tumors. MMTV-Neu tumors were har-
vested from mammary glands after 10 days of treatment with V-125 and sectioned for immunohistochemistry (Fig. 5). In these tumors, V-125 did not change expression of proliferating cell nuclear antigen (PCNA), a marker of cell proliferation. However, there was a marked increase in cleaved caspase 3, a marker of apoptosis, in tumors

Discussion
Our results demonstrate significant efficacy of the novel rexinoid V-125 for prevention and treatment in the MMTV-Neu model of HER2 + breast cancer and prevention in the A/J model of Kras-driven NSCLC. Notably, V-125 was more efficacious than the clinically approved rexinoid bexarotene in both models. Unlike bexarotene, V-125 did not elevate plasma triglycerides, although it did elevate plasma cholesterol levels at its highest treatment dose (100 mg/kg diet) in the MMTV-Neu model. The combination of improved efficacy and reduced toxicity in two different clinically relevant animal models suggests that V-125 is a strong candidate for further development toward a new therapy for the treatment of patients with breast or lung cancer. These studies also provide proof of principle that more effective and better tolerated rexinoids can be developed through structural modifications to existing rexinoids and further validate the in vitro screening paradigm we previously optimized. These in vitro assays demonstrated that V-125 does not elevate SREBP, a transcription factor which regulates genes responsible for lipid homeostasis (Fig. 1). Overall survival is plotted for control and V-125-treated groups (*P < 0.0172 vs. control via log rank test; N = 9-10 mice per group). (B) Tumor volume over the first 14 days of treatment with V-125 was calculated and normalized to initial tumor volume (**P < 0.01 vs. control). C. Tumor weight at the end of 10 days of treatment is presented as a percent of body weight (*P < 0.05 vs. control; N = 6-8 mice per group). Plasma triglycerides (D) and cholesterol (E) levels in mice treated with control or rexinoids (100 mg/kg diet) for 10 days were quantified using commercial kits (*P < 0.01 vs. control; # P < 0.01 vs. bexarotene; n = 6-11 mice/group). All error bars represent the standard errors. www.nature.com/scientificreports/ Elevation of SREBP in vitro predicts for increased plasma cholesterol and triglycerides in vivo 21,30 . Furthermore, V-125 has anti-inflammatory activity in the iNOS suppression assay at the nM level ( Fig. 1), which correlates with efficacy for prevention in the A/J lung cancer model, and nM activity in a RXRα reporter assay 30 . As predicted by our in vitro screening assays, V-125 treatment resulted in superior anti-tumor efficacy without elevating triglycerides as observed with bexarotene. This screening paradigm was also used to select the compound MSU42011 which, as predicted based on the in vitro screening assays, reduced lung tumor burden in A/J mice 21 .
Rexinoids have been used clinically for both treatment (NCT00003752 and NCT04664829) and prevention (NCT00055991 and NCT03323658) of breast cancer. As expected, lower doses are needed for prevention studies (30 mg/kg diet in the MMTV-neu model and 40-80 mg/kg diet in the more aggressive A/J model of lung cancer) than treatment studies (100 mg/kg in the MMTV-Neu model), as early intervention is more amenable to pharmacological intervention than treating late-stage, advanced disease 42 . A compound must be very welltolerated to warrant long-term prevention studies in patients, and lower doses will help reduce toxicity. In comparison, higher doses are used for treatment, as larger tumors have acquired multiple mutations and are more resistant to drugs. Importantly, with the combination of high anti-tumor efficacy and a favorable safety profile, rexinoids may be utilized in either clinical setting 27 . While recent strategies utilizing novel aerosolized delivery systems for administration of bexarotene have been effective in rodent models of lung cancer without inducing hypertriglyceridemia or hypercholesterolemia 43,44 , it is prudent to continue to synthesize new RXR agonists with improved efficacy and toxicity profiles in addition to optimizing drug delivery strategies. A combination of these two strategies may yield the best results.
The mechanism of action of V-125 is complex. V-125 had a limited effect on proliferation of tumor cells, visualized by PCNA expression (Fig. 5). However, V-125 induced apoptosis in MMTV-Neu tumors as evidenced by the increased expression of cleaved caspase 3. Tumor size is dependent on the balance between cancer cell growth and death 45 30 , which may contribute to anti-tumor efficacy 46 . These data, combined with the iNOS screening data in RAW 264.7 macrophage-like cells 30 , suggest that the effects of V-125 on macrophages may be necessary for anti-tumor activity. Indeed, the reduction in CD206 staining evident in MMTV-Neu tumors treated with V-125 (Fig. 5) confirms that this compound reduces populations of immunosuppressive macrophages associated with tumor progression. There are several possible explanations for this reduction: V-125 treatment may result in macrophage skewing, altering the polarization of these cells away from a tumor-promoting phenotype and towards a tumor-suppressive phenotype 47 . Alternatively, this decrease in CD206 staining in mice treated with V-125 could be the result of altered immune cell infiltration into the tumor, a change in the localization of immunosuppressive macrophages, or systemic effects on monocyte differentiation or circulation. Notably, we have previously demonstrated that novel pyrimidinyl derivative of LG100268 decreased F4/80 + lung macrophage populations in A/J mice 30 . Further studies are needed to determine how V-125 exerts these immunomodulatory effects in these models and how critical these effects are for anti-tumor efficacy. The increased PD-L1 expression in tumors treated with V-125 provides an intriguing potential avenue for the combination of rexinoids with immunotherapy. The expression of PD-L1 is a positive prognostic marker in Figure 6. V-125 differentially regulates cancer-associated genes in tumors. Mice with established mammary tumors 5 mm in diameter were treated with control or V-125 (100 mg/kg in diet) for 10 days. Total RNA was extracted for PCR array. Analysis was performed using GeneGlobe software (Qiagen), revealing the upregulation of 10 genes with a log fold change > 2 (A), and the downregulation of 10 genes with a log fold change > 2 (B). Validation was performed on IL-6 (C), Jun (D), and COX2 (E) by qPCR. N = 2 mice/group. Data shown is representative of two independent experimental runs. www.nature.com/scientificreports/ breast cancer, indicating a hot immune microenvironment 48 . In basal tumors, upregulation of PD-L1 correlates with strong local cytotoxic T cell responses and positive responses to neoadjuvant chemotherapy 48 . High PD-L1 expression and increased numbers of tumor-infiltrating lymphocytes may be predictive of patient populations that would benefit from immunotherapy 49 . As V-125 increased the expression of PD-L1 in tumors, this may bolster response to immunotherapies such as atezolizumab, a monoclonal antibody which selectively targets PD-L1 50 . Combination treatment with rexinoids and immunotherapy is a promising area for further investigation. A breast cancer PCR array revealed differentially regulated genes in mammary tumors treated with V-125. Adam23, which is increased in V-125 tumors compared to controls, is a cellular adhesion molecule that enhances progression of breast cancer when downregulated 55 . Slit2 expression in breast cancer inhibits migration 56 and is inactivated in both breast and lung cancer 57 . Importantly, IL-6 expression was lower in V-125-treated tumors, suggesting that V-125 plays a role in modulating the tumor immune compartment. This finding was confirmed by qPCR and recapitulates our previous studies with other rexinoids 58 . Future studies will explore the role of these gene products in mediating the anti-tumor activity of V-125.
In conclusion, the novel rexinoid V-125 is an effective treatment in murine models of both breast cancer and lung cancer. The favorable toxicity profile of this compound allows for use in either cancer treatment or prevention.

Data availability
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.