Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase

Abstract

Inactivation of the retinoblastoma gene (RB1) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1^+/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1^+/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1-null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

Keywords: JARID1A; KDM5A; cancer; epigenetics; genetically engineered mouse models.

Fig. 1.

Inducible deletion of Rbp2 in vivo. (A–D) qPCR-based allelic frequency assays for the Rbp2 null allele in normal pituitary (A) and thyroid tissues (C) and for the Rbp2 floxed allele in normal pituitary (B) and thyroid tissues (D) from Rbp2^lox/lox and Rbp2^lox/lox; Cre–ER mice. Mice were treated with tamoxifen or corn oil by oral gavage daily for 5 consecutive days. Pituitary and thyroid glands were harvested for gDNA extraction 10 d after the first dose. (E) RBP2 immunoblot in pituitary tissues harvested from Rbp2^lox/lox and Rbp2^lox/lox; Cre–ER mice. Mice were treated with tamoxifen or corn oil and tissues were harvested as in A–D. For A–D, n ≥ 3 and data presented are means ± SD.

Fig. 2.

Schema of experimental design for inducible deletion of Rbp2 in tumors of Rb1^+/− mice. C57BL/6 mice of the indicated genotypes began monthly MRI at 5 mo of age to detect the development of pituitary and thyroid tumors. Once tumors were detected, the mice were administered tamoxifen (Tam) by oral gavage on 5 consecutive days. MRI scans were performed 2, 4, 6, 10, and 14 wk after initiation of tamoxifen treatment to quantify tumor volume. Additional doses of tamoxifen were given once per week until mice were euthanized due to tumor burden.

Fig. 3.

Rbp2 deletion retards the growth of established Rb1-null thyroid tumors. (A and B) Representative MRIs of thyroid tumors in Rbp2^+/+; Rb1^+/−; Cre–ER (A) and Rbp2^lox/lox; Rb1^+/−; Cre–ER (B) mice before (0 wk) and 2, 4, 6, and 10 wk after initiation of tamoxifen treatment as outlined in Fig. 2. Red arrows indicate tumors. (Scale bar: 2 mm.) Imaging results from three mice in each cohort are shown and their unique study IDs are listed at Left. In A, all three mice had progressive disease. In B, mouse 1J41 had progressive tumor growth, 3A13 had stable disease, and 1E43 had a regression at week 10. (C and D) Thyroid tumor burden in Rbp2^+/+; Rb1^+/−; Cre–ER (C) and Rbp2^lox/lox; Rb1^+/−; Cre–ER (D) mice before and 2, 4, 6, 10, and 14 wk after initiation of tamoxifen treatment as quantified by MRI. Colored lines represent individual mice. Dashed line indicates 30 mm³ tumor burden and the fraction of mice surpassing and failing to exceed this threshold is listed for each cohort. Two mice in the Rbp2^+/+ cohort had both left and right thyroid tumors at the time of enrollment. In these mice, tumor burden was calculated by summing the volumes of the two tumors at each time point. (E) Median thyroid tumor burden over time in Rbp2^+/+ and Rbp2^lox/lox cohorts represented in C and D. Error bars indicate interquartile range. P value was determined by a mean-based longitudinal mixed-effects model to accommodate repeated measurements within animals.

Fig. 4.

Rbp2 deletion retards the growth of established Rb1-null pituitary tumors. (A and B) Representative MRIs of pituitary tumors in Rbp2^+/+; Rb1^+/−; Cre–ER (A) and Rbp2^lox/lox; Rb1^+/−; Cre–ER (B) mice before (0 wk) and 2, 4, 6, and 10 wk after initiation of tamoxifen treatment as in Fig. 2. Red arrows indicate tumors. (Scale bar: 2 mm.) Imaging results from three mice in each cohort are shown and their unique study IDs are listed at Left. In A, mice 2C31 and 2C33 had progressive disease and mouse 2B43 had stable disease; in B, mouse 1E30 had progressive disease and mice 2J3 and 1J30 had stable disease. (C and D) Pituitary tumor burden in Rbp2^+/+; Rb1^+/−; Cre–ER (C) and Rbp2^lox/lox; Rb1^+/−; Cre–ER (D) mice before and 2, 4, 6, 10, and 14 wk after initiation of tamoxifen treatment as quantified by MRI. Colored lines represent individual mice. Dashed line indicates 30 mm³ tumor burden and the fraction of mice surpassing and failing to exceed this threshold is listed for each cohort. (E) Median pituitary tumor burden over time in Rbp2^+/+ and Rbp2^lox/lox cohorts represented in C and D. Error bars indicate interquartile range. P value was determined by a mean-based longitudinal mixed-effects model to accommodate repeated measurements within animals.

Fig. 5.

Rbp2 deletion in established tumors significantly extends survival of Rb1^+/− mice. (A) Kaplan–Meier survival curves of Rbp2^+/+; Rb1^+/−; Cre–ER and Rbp2^lox/lox; Rb1^+/−; Cre–ER mice treated with tamoxifen as in Fig. 2, with day 0 being the first day of tamoxifen treatment. P value was determined by log-rank test. (B) Immunohistochemical analysis of pituitary and thyroid tumors obtained at necropsy in representative tamoxifen-treated Rbp2^+/+; Rb1^+/−; Cre–ER and Rbp2^lox/lox; Rb1^+/−; Cre–ER mice from the study in A. Serial tumor tissue sections from mice in the indicated cohorts were stained with hematoxylin and eosin (H&E) and RBP2 and pRB antibodies. (Scale bar: 100 μm.) (C) Immunohistochemical analysis of thyroid tumors obtained at necropsy in representative tamoxifen-treated Rbp2^+/+; Rb1^+/−; Cre–ER and Rbp2^lox/lox; Rb1^+/−; Cre–ER mice from the study in A. Responder status for mice from the Rbp2^lox/lox cohort is indicated and based on tumor growth kinetics as depicted in Fig. 3D. Serial tumor tissue sections were stained with antibodies against RBP2, SMCX, and H3K4me3 or with H&E. (Scale bar: 1 mm, for 2× magnification main images.) (Scale bar: 50 μm, for 40× magnification Insets.) (D) Quantification of H3K4me3 staining intensity for a subset of the thyroid tumors from A analyzed as in C. Data presented are means ± SEM; *P < 0.05, **P < 0.01. Two-tailed P values were determined by unpaired t test. n.s., nonsignificant.