V-125 clusters favorably in computational analyses of in vitro activity
We have found that applying principal component analysis (PCA) in our prior work35,36 with rexinoids has helped to visualize particularly active rexinoids with improved therapeutic potential group, 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 (Fig. 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 adenocarcinomas37 and then treated with V-125. Mice were fed control diet or rexinoids in diet starting two weeks post-carcinogen for a period of 16 weeks. At the conclusion of the study, evaluation of lung tumor number, size, and histopathology in slides 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-C) reduced tumor size by 45% (0.09 ± 0.01 mm3/tumor vs. 0.16 ± 0.01 mm3/tumor in the control group), tumor number by 37% (2.07 ± 0.30 vs. control 3.3 ± 0.35), and tumor burden by 65% (0.18 ± 0.04 mm3 vs. 0.53 ± 0.08 mm3 in the control group). While there was a trend toward lower tumor parameters in mice treated with bexarotene (Table 1), these changes were not statistically significant at either the 40 or 80 mg/kg doses.
Table 1
V-125 reduces lung carcinogenesis in A/J mice
| Control | Bexarotene | V-125 |
mg/kg diet | | 40 | 80 | 40 | 80 |
# of slides/group | 30 | 28 | 30 | 30 | 28 |
# of tumors/group | 99 | 104 | 81 | 76 | 58 |
Total # tumors/slide (% control) | 3.3 ± 0.35 (100%) | 3.71 ± 0.30 (112.6%) | 2.7 ± 0.26 (81.8%) | 2.53 ± 0.28 (76.8%) | 2.07 ± 0.30* (62.8%) |
Total Tumor Volume, mm3 | 15.98 | 14.17 | 11.247 | 11.16 | 5.17 |
Ave Tumor Size (mm3)/tumor (% control) | 0.16 ± 0.01 (100%) | 0.14 ± 0.01 (84.4%) | 0.14 ± 0.02 (84.4%) | 0.15 ± 0.04 (91.0%) | 0.09 ± 0.01* (55.2%) |
Ave Tumor Burden (mm3) (% control) | 0.53 ± 0.08 (100%) | 0.51 ± 0.07 (95.0%) | 0.37 ± 0.06 (70.4%) | 0.37 ± 0.10 (69.8%) | 0.18 ± 0.04* (34.7%) |
Total # L/M Grade (% total) | 43 (43%) | 50 (48%) | 43 (53%) | 38 (50%) | 38 (66%)* |
Total # HH Grade (% total) | 56 (57%) | 54 (52%) | 38 (47%) | 38 (50%) | 20 (34%)* |
Although both V-125 and bexarotene were well-tolerated in this model based on animal weights (Fig. S3A), bexarotene elevates triglycerides in animal models21 and in human patients38. 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 criteria25 (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 estrogen receptor-negative 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) promoter32 and develop focal mammary adenocarcinomas by 25–35 weeks of age39. To investigate the tumor preventive effects of V-125 in this preclinical model of HER2 + breast cancer, MMTV-neu mice were fed control diet or V-125 in diet (30 mg/kg diet) 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).
Plasma triglycerides and cholesterol levels were measured at the end of this study (Fig. 3C-D). Bexarotene significantly (p < 0.01) increased plasma triglyceride levels (11.1 ± 1.7 nmol/µL vs. control 6.5 ± 0.98 nmol/µL). In comparison, V-125 did not elevate plasma triglycerides (6.6 ± 0.73 nmol/µL). Bexarotene also significantly (p < 0.01) raised plasma cholesterol levels (Fig. 3D) (2.1 ± 0.16 µg/µL vs control 1.5 ± 0.08 µg/µL), while V-125 had no effect.
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 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).
To identify biomarkers of V-125 efficacy, MMTV-Neu mice with established tumors were treated with 100 mg V-125/kg diet for 10 days. A treatment duration of 10 days was selected based on the drastic decrease in tumor volume observed on days 10–11 of treatment (Fig. 4B). As shown in Fig. 4C, V-125 significantly (p < 0.05) decreased tumor weight, presented as a percentage of total body weight (1.46 ± 0.82% vs. control 4.95 ± 0.95%). Ten days of treatment with 100 mg/kg diet bexarotene (Fig. S4) also significantly (p < 0.05) decreased tumor weight (2.52 ± 0.41% of total body weight vs. control 3.78 ± 0.49%), but only by 1.26%, nearly three times less than the effect observed with V-125.
When triglyceride levels in MMTV-Neu mice were evaluated, treatment with 100 mg/kg diet bexarotene for 10 days significantly (p < 0.01) elevated plasma triglyceride levels (18.0 ± 1.4 nmol/µL vs. control 9.1 ± 0.61 nmol/µL). In contrast, V-125 did not change plasma triglycerides (10.4 ± 2.3 nmol/µL vs. control 9.12 ± 0.61 nmol/µL), and the triglyceride levels observed in mice treated with V-125 were significantly (p < 0.01) lower than those in bexarotene-treated mice (Fig. 4D). Both bexarotene and V-125 significantly increased plasma cholesterol at this dose (1.87 ± 0.21 µg/µL and 1.47 ± 0.18 µg/µL respectively, vs. control 0.89 ± 0.12 µg/µL; Fig. 4E).
V-125 Alters Immune-related Biomarkers In Mmtv-neu Tumors
MMTV-Neu tumors were harvested 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 treated with V-125. As previous studies have demonstrated the immunomodulatory effects of rexinoids33, we also examined the expression of CD206 and programmed death-ligand 1 (PD-L1) in tumors. Tumors of mice treated with V-125 had a striking decrease in CD206, a cell surface marker expressed by immunosuppressive macrophages. V-125 also increased expression of PD-L1, an immune checkpoint molecule.