Humanized cereblon mice revealed two distinct therapeutic pathways of immunomodulatory drugs

Abstract

Immunomodulatory drugs (IMiDs), including thalidomide derivatives such as lenalidomide and pomalidomide, offer therapeutic benefit in several hematopoietic malignancies and autoimmune/inflammatory diseases. However, it is difficult to study the IMiD mechanism of action in murine disease models because murine cereblon (CRBN), the substrate receptor for IMiD action, is resistant to some of IMiDs therapeutic effects. To overcome this difficulty, we generated humanized cereblon (CRBN^I391V) mice thereby providing an animal model to unravel complex mechanisms of action in a murine physiological setup. In our current study, we investigated the degradative effect toward IKZF1 and CK-1α, a target substrate of IMiDs. Unlike WT mice which were resistant to lenalidomide and pomalidomide, T lymphocytes from CRBN^I391V mice responded with a higher degree of IKZF1 and CK-1α protein degradation. Furthermore, IMiDs resulted in an increase in IL-2 among CRBN^I391V mice but not in the WT group. We have also tested a thalidomide derivative, FPFT-2216, which showed an inhibitory effect toward IKZF1 protein level. As opposed to pomalidomide, FPFT-2216 and lenalidomide degrades CK-1α. Additionally, we assessed the potential therapeutic effects of IMiDs in dextran sodium sulfate (DSS)-induced colitis. In both WT and humanized mice, lenalidomide showed a significant therapeutic effect in the DSS model of colitis, while the effect of pomalidomide was less pronounced. Thus, while IMiDs' degradative effect on IKZF1 and CK-1α, and up-regulation of IL-2, is dependent on CRBN, the therapeutic benefit of IMiDs in a mouse model of inflammatory bowel disease occurs through a CRBN-IMiD binding region independent pathway.

Keywords: DSS colitis; IMiDs; Ikaros 1; casein kinase-1α; humanized cereblon.

Fig. 1.

Humanized cereblon mice respond to IMiD activity. (A) Naïve CD4⁺ T cells stimulated with anti-CD3ε (5 µg/mL) and simultaneously treated with DMSO or lenalidomide (10 µM) for the indicated time points. The IL-2 level was measured by ELISA. (B) IL-2 production was measured by RT-PCR at the indicated time points. Data are shown as the mean ± SEM of three independent experiments. (n.s, nonsignificant; P > 0.05, P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test).

Fig. 2.

Cereblon modulator, FPFT-2216, results in up-regulation of IL-2 production and degradation of IKZF1 and CK-1α. (A) Naïve CD4⁺ T cells treated with lenalidomide (100 µM), pomalidomide (10 µM), FPFT-2216 (10 µM), and IL-2 production measured by ELISA. P values for WT were nonsignificant for comparison between IMiDs and DMSO (P > 0.05). For I391V, P < 0.05 for all comparisons between IMiDs and DMSO. Student’s t test was used to calculate statistical significance. Data are shown as the mean ± SEM of three independent experiments. (B) Naïve CD4⁺ T cells were treated as A and Western blot analysis was done for IKZF1 and CK-1α.

Fig. 3.

In vivo administration of immunomodulatory drugs result in degradation of cullin4A^CRBN E3 ligase substrates. (A) Mice were administered 200 µL of control, lenalidomide (100 mg/kg), pomalidomide (50 mg/kg), FPFT-2216 (30 mg/kg) solubilized in 0.5% carboxymethylcellulose/sodium and 0.25% Tween 80 for 14 h orally by gavaging. Murine CD4⁺ T cells were isolated from spleen and Western blot was done. (B) IMiDs were administered intraperitoneally and Western blot analysis was done as in A. The data are representative of three independent experiments. n ≥ 3.

Fig. 4.

Lenalidomide protects against DSS-induced colitis. (A) DSS-colitis induction. Mice were given 3% DSS for 6 d and water only for the final 1 d, they were simultaneously treated with IMiDs and killed on day 7. (B) Colon isolated from DSS colitis-induced mice. (C) Colon length. (D) Clinical score, change in body weight. P values for WT vs. I391V were nonsignificant for all treatments. P value for DMSO vs. pomalidomide was nonsignificant for WT mice. P value for DMSO vs. lenalidomide was significant for both WT and I391V. P value for DMSO vs. pomalidomide was significant for I391V. (E) Fecal score (stool consistency) and (F) hematochezia (level of blood in stool). P values for WT vs. I391V were nonsignificant for all treatments. P values for DMSO vs. all treatments were significant for both WT and I391V mice (E and F). (G) Histopathological image of colon tissue stained with hematoxylin and eosin. (Scale bar, 10 µm.) (H) Myeloperoxidase activity from colon tissue was measured using a colorimetric method. All experiments were representatives of two independent experiments. Data are expressed as the mean ± SEM (n = 6 per group). Student’s t test was used.