Human Mass Balance and Metabolite Profiling of [14 C]-Pamiparib, a Poly (ADP-Ribose) Polymerase Inhibitor, in Patients With Advanced Cancer.

Pamiparib, a selective poly (ADP-ribose) polymerase 1/2 inhibitor, demonstrated tolerability and antitumor activity in patients with solid tumors at 60 mg orally twice daily. This phase 1 open-label study (NCT03991494; BGB-290-106) investigated the absorption, metabolism, and excretion (AME) of 60 mg [14 C]-pamiparib in 4 patients with solid tumors. The mass balance in excreta, blood, and plasma radioactivity and plasma pamiparib concentration were determined along with metabolite profiles in plasma, urine, and feces. Unchanged pamiparib accounted for the most plasma radioactivity (67.2% ± 10.2%). Pamiparib was rapidly absorbed with a median time to maximum plasma concentration (Cmax ) of 2.00 hours (range, 1.00-3.05 hours). After reaching Cmax , pamiparib declined in a biphasic manner, with a geometric mean terminal half-life (t1/2 ) of 28.7 hours. Mean cumulative [14 C]-pamiparib excretion was 84.7% ± 3.5%. Pamiparib was mainly cleared through metabolism, primarily via N-oxidation and oxidation of the pyrrolidine ring. A dehydrogenated oxidative product (M3) was the most abundant metabolite in biosamples. A mean of 2.11% and 1.11% of [14 C]-pamiparib was excreted as unchanged pamiparib in feces and urine, respectively, indicating near-complete absorption and low renal clearance of parent drug. Cytochrome P450 (CYP) phenotyping demonstrated CYP2C8 and CYP3A involvement in pamiparib metabolism. These findings provide an understanding of pamiparib AME mechanisms and potential drug-drug interaction liability.

The pharmacokinetics of pamiparib in the presence of a strong CYP3A inhibitor (itraconazole) and strong CYP3A inducer (rifampin) in patients with solid tumors: an open-label, parallel-group phase 1 study.

PURPOSE: Pamiparib is an investigational, selective, oral poly(ADP-ribose) polymerase 1/2 (PARP1/2) inhibitor that has demonstrated PARP-DNA complex trapping and CNS penetration in preclinical models, as well as preliminary anti-tumor activity in early-phase clinical studies. We investigated whether the single-dose pharmacokinetic (PK) profile of pamiparib is altered by coadministration of a strong CYP3A inducer (rifampin) or a strong CYP3A inhibitor (itraconazole) in patients with solid tumors. METHODS: In this open-label, phase 1 study, adults with advanced solid tumors received either oral pamiparib 60 mg (days 1 and 10) and once-daily oral rifampin 600 mg (days 3-11) or oral pamiparib 20 mg (days 1 and 7) and once-daily oral itraconazole 200 mg (days 3-8). Primary endpoints included pamiparib maximum observed concentration (Cmax), and area under the plasma concentration-time curve from zero to last quantifiable concentration (AUC0-tlast) and infinity (AUC0-inf). Secondary endpoints included safety and tolerability. RESULTS: Rifampin coadministration did not affect pamiparib Cmax (geometric least-squares [GLS] mean ratio 0.94; 90% confidence interval 0.83-1.06), but reduced its AUC0-tlast (0.62 [0.54-0.70]) and AUC0-inf (0.57 [0.48-0.69]). Itraconazole coadministration did not affect pamiparib Cmax (1.05 [0.95-1.15]), AUC0-tlast (0.99 [0.91-1.09]), or AUC0-inf (0.99 [0.90-1.09]). There were no serious treatment-related adverse events. CONCLUSIONS: Pamiparib plasma exposure was reduced 38-43% with rifampin coadministration but was unaffected by itraconazole coadministration. Pamiparib dose modifications are not considered necessary when coadministered with CYP3A inhibitors. Clinical safety and efficacy data will be used with these results to recommend dose modifications when pamiparib is coadministered with CYP3A inducers.

Down-regulation of ARID1A is sufficient to initiate neoplastic transformation along with epigenetic reprogramming in non-tumorigenic endometriotic cells.

The chromatin remodeler AT-Rich Interactive Domain 1A (ARID1A) is frequently mutated in ovarian clear cell carcinoma (OCCC) and endometriosis precursor lesions. Here, we show that knocking down ARID1A in an immortalized endometriosis cell line is sufficient to induce phenotypic changes indicative of neoplastic transformation as evidenced by higher efficiency of anchorage-independent growth, increased propensity to adhere to collagen, and greater capacity to invade basement membrane extract in vitro. ARID1A knockdown is associated with expression dysregulation of 99 target genes, and many of these expression changes are also observed in primary OCCC tissues. Further, pathway analysis indicates these genes fall within networks highly relevant to tumorigenesis including integrin and paxillin pathways. We demonstrate that the down-regulation of ARID1A does not markedly alter global chromatin accessibility or DNA methylation but unexpectedly, we find strong increases in the active H3K27ac mark in promoter regions and decreases of H3K27ac at potential enhancers. Taken together, these data provide evidence that ARID1A mutation can be an early stage event in the oncogenic transformation of endometriosis cells giving rise to OCCC.

Serum B-cell maturation antigen: a novel biomarker to predict outcomes for multiple myeloma patients.

B-cell maturation antigen is expressed on plasma cells. In this study, we have identified serum B-cell maturation antigen as a novel biomarker that can monitor and predict outcomes for multiple myeloma patients. Compared to healthy donors, patients with multiple myeloma showed elevated serum B-cell maturation antigen levels (P<0.0001). Serum B-cell maturation antigen levels correlated with the proportion of plasma cells in bone marrow biopsies (Spearman's rho = 0.710; P<0.001), clinical status (complete response vs partial response, P=0.0374; complete response vs progressive disease, P<0.0001), and tracked with changes in M-protein levels. Among patients with non-secretory disease, serum B-cell maturation antigen levels correlated with bone marrow plasma cell levels and findings from positron emission tomography scans. Kaplan-Meier analysis demonstrated that serum B-cell maturation antigen levels above the median levels were predictive of a shorter progression-free survival (P=0.0006) and overall survival (P=0.0108) among multiple myeloma patients (n=243). Specifically, patients with serum B-cell maturation antigen levels above the median level at the time of starting front-line (P=0.0043) or a new salvage therapy (P=0.0044) were found to have shorter progression-free survival. Importantly, serum B-cell maturation antigen levels did not show any dependence on renal function and maintained independent significance when tested against other known prognostic markers for multiple myeloma such as age, serum ß2 microglobulin, hemoglobin, and bone disease. These data identify serum B-cell maturation antigen as a new biomarker to manage multiple myeloma patients.

Levels of uninvolved immunoglobulins predict clinical status and progression-free survival for multiple myeloma patients.

Multiple myeloma (MM) is characterized by the enhanced production of the same monoclonal immunoglobulin (M-Ig or M protein). Techniques such as serum protein electrophoresis and nephelometry are routinely used to quantify levels of this protein in the serum of MM patients. However, these methods are not without their shortcomings and problems accurately quantifying M proteins remain. Precise quantification of the types and levels of M-Ig present is critical to monitoring patient response to therapy. In this study, we investigated the ability of the HevyLite (HLC) immunoassay to correlate with clinical status based on levels of involved and uninvolved antibodies. In our cohort of MM patients, we observed that significantly higher ratios and greater differences of involved HLC levels compared to uninvolved HLC levels correlated with a worse clinical status. Similarly, higher absolute levels of involved HLC antibodies and lower levels of uninvolved HLC antibodies also correlated with a worse clinical status and a shorter progression-free survival. These findings suggest that the HLC assay is a useful and a promising tool for determining the clinical status and survival time for patients with multiple myeloma.

A phase 3 trial of armodafinil for the treatment of cancer-related fatigue for patients with multiple myeloma.

PURPOSE: Fatigue is a common problem among multiple myeloma (MM) patients. Armodafinil is a drug known to promote wakefulness, which is related to modafinil, a compound that improves fatigue in some cancer patients treated with chemotherapeutic agents. We investigated whether armodafinil could reduce cancer-related fatigue in MM patients. METHODS: This double-blind, placebo-controlled phase 3 trial evaluated the efficacy of armodafinil in MM patients with evidence of moderate fatigue. Patients were randomized to one of two arms: treatment-only, with armodafinil given at 150 mg/daily for 56 days, or placebo-first, with placebo given on days 1-28, followed by armodafinil administered at 150 mg daily on days 29-56. Fatigue was measured on days 1 (pre-dose: baseline), 15, 28, 43, and 56 using seven separate assessments, including four patient-reported outcomes of fatigue and related quality of life measures, as well as three objective measures of cognitive function. RESULTS: Overall toxicities were similar between treatment groups. No significant differences were observed between the placebo-first and the treatment-only arms after 28 days. Treatment with armodafinil for 28 additional days did not produce responses. Both placebo-first and treatment-only patients showed similar significant improvements in three patient-reported measures and one objective task at day 28 compared to baseline. Placebo-first patients improved on eight additional measures (one patient-reported measure, six subscales, and one objective task), suggesting a strong placebo effect in this patient population. CONCLUSIONS: Evaluation and treatment of cancer-related fatigue continues to be challenging; a clear definition of this symptom and better assessment tools are needed.

The potential of panobinostat as a treatment option in patients with relapsed and refractory multiple myeloma.

Panobinostat is an investigational and potent histone deacetylase inhibitor (HDACi) that has shown promise as an antimultiple myeloma agent in the preclinical setting. In this review, we discuss the rationale for the use of panobinostat as a combination therapy for multiple myeloma and provide an overview of recent and ongoing clinical trials testing the safety and efficacy of panobinostat for the treatment of the disease.

Carfilzomib in multiple myeloma.

INTRODUCTION: Advances in drug therapy for multiple myeloma (MM) during the previous decade have improved survival outcomes; however, the disease remains incurable as patients eventually relapse or become refractory to all available therapies. Therefore, there is a clear need for more effective and well-tolerated treatments. AREAS COVERED: We review preclinical and clinical data regarding the use of carfilzomib , a proteasome inhibitor that is structurally and mechanistically distinct from bortezomib, for the treatment of MM patients. Carfilzomib pharmacokinetics, pharmacodynamics, efficacy, safety and tolerability are summarized, based on Phase I/II trial data. EXPERT OPINION: Carfilzomib represents a significant advance in the management of relapsed and/or refractory MM patients, including those intolerant or resistant to bortezomib. High response rates have been demonstrated with carfilzomib as a single agent or in combination with alkylating agents, immunomodulators and corticosteroids, even among patients who have failed multiple prior therapies. Carfilzomib also has significant potential in the frontline setting, with encouraging response and survival rates observed for combination regimens. Further evaluation of carfilzomib-containing regimens is ongoing in Phase III trials and investigator-sponsored studies, which include combinations with novel investigational agents. These findings will shape the future role of carfilzomib for MM patients across multiple settings.

A panel of three markers hyper- and hypomethylated in urine sediments accurately predicts bladder cancer recurrence.

PURPOSE: The high risk of recurrence after transurethral resection of bladder tumor of nonmuscle invasive disease requires lifelong treatment and surveillance. Changes in DNA methylation are chemically stable, occur early during tumorigenesis, and can be quantified in bladder tumors and in cells shed into the urine. Some urine markers have been used to help detect bladder tumors; however, their use in longitudinal tumor recurrence surveillance has yet to be established. EXPERIMENTAL DESIGN: We analyzed the DNA methylation levels of six markers in 368 urine sediment samples serially collected from 90 patients with noninvasive urothelial carcinoma (Tis, Ta, T1; grade low-high). The optimum marker combination was identified using logistic regression with 5-fold cross-validation, and validated in separate samples. RESULTS: A panel of three markers discriminated between patients with and without recurrence with the area under the curve of 0.90 [95% confidence interval (CI), 0.86-0.92] and 0.95 (95% CI, 0.90-1.00), sensitivity and specificity of 86%/89% (95% CI, 74%-99% and 81%-97%) and 80%/97% (95% CI, 60%-96% and 91%-100%) in the testing and validation sets, respectively. The three-marker DNA methylation test reliably predicted tumor recurrence in 80% of patients superior to cytology (35%) and cystoscopy (15%) while accurately forecasting no recurrence in 74% of patients that scored negative in the test. CONCLUSIONS: Given their superior sensitivity and specificity in urine sediments, a combination of hyper- and hypomethylated markers may help avoid unnecessary invasive exams and reveal the importance of DNA methylation in bladder tumorigenesis.

Establishment of active chromatin structure at enhancer elements by mixed-lineage leukemia 1 to initiate estrogen-dependent gene expression.

A number of genome-wide analyses have revealed that estrogen receptor α binding to and regulation of its target genes correlate with binding of FOXA1, a pioneer factor, to nearby DNA sites in MCF-7 breast cancer cells. The enhancer element-specific histone H3K4me1/2 mark is enriched at the specific FOXA1/ERα recruitment sites in chromatin, but the mechanism by which these enhancer marks are established in chromatin before hormone treatment is unclear. Here, we show that mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERα to the enhancer element. Our study defines the mechanism by which MLL1 nucleates histone H3K4 methylation marks in CpG-enriched regions to maintain permissive chromatin architecture and allow FOXA1 and estrogen receptor α binding to transcriptional regulatory sites in breast cancer cells.

Dysregulation of uterine signaling pathways in progesterone receptor-Cre knockout of dicer.

Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, -200b, -101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/ß-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function.

Gene reactivation by 5-aza-2'-deoxycytidine-induced demethylation requires SRCAP-mediated H2A.Z insertion to establish nucleosome depleted regions.

5-Aza-2'-deoxycytidine, approved by the FDA for the treatment of myelodysplastic syndrome (MDS), is incorporated into the DNA of dividing cells where it specifically inhibits DNA methylation by forming covalent complexes with the DNA methyltransferases (DNMTs). In an effort to study the correlations between DNA methylation, nucleosome remodeling, and gene reactivation, we investigate the integrated epigenetic events that worked coordinately to reprogram the methylated and closed promoters back to permissive chromatin configurations after 5-Aza-2'-deoxycytidine treatment. The ChIP results indicate that H2A.Z is deposited at promoter regions by the Snf2-related CBP activator protein (SRCAP) complex following DNA demethylation. According to our genome-wide expression and DNA methylation profiles, we find that the complete re-activation of silenced genes requires the insertion of the histone variant H2A.Z, which facilitates the acquisition of regions fully depleted of nucleosome as demonstrated by NOMe-seq (Nucleosome Occupancy Methylome-sequencing) assay. In contrast, SRCAP-mediated H2A.Z deposition is not required for maintaining the active status of constitutively expressed genes. By combining Hpa II digestion with NOMe-seq assay, we show that hemimethylated DNA, which is generated following drug incorporation, remains occupied by nucleosomes. Our data highlight H2A.Z as a novel and essential factor involved in 5-Aza-2'-deoxycytidine-induced gene reactivation. Furthermore, we elucidate that chromatin remodeling translates the demethylation ability of DNMT inhibitors to their downstream efficacies, suggesting future therapeutic implications for chromatin remodelers.

Dynamic nucleosome-depleted regions at androgen receptor enhancers in the absence of ligand in prostate cancer cells.

Nucleosome positioning at transcription start sites is known to regulate gene expression by altering DNA accessibility to transcription factors; however, its role at enhancers is poorly understood. We investigated nucleosome positioning at the androgen receptor (AR) enhancers of TMPRSS2, KLK2, and KLK3/PSA in prostate cancer cells. Surprisingly, a population of enhancer modules in androgen-deprived cultures showed nucleosome-depleted regions (NDRs) in all three loci. Under androgen-deprived conditions, NDRs at the TMPRSS2 enhancer were maintained by the pioneer AR transcriptional collaborator GATA-2. Androgen treatment resulted in AR occupancy, an increased number of enhancer modules with NDRs without changes in footprint width, increased levels of histone H3 acetylation (AcH3), and dimethylation (H3K4me2) at nucleosomes flanking the NDRs. Our data suggest that, in the absence of ligand, AR enhancers exist in an equilibrium in which a percentage of modules are occupied by nucleosomes while others display NDRs. We propose that androgen treatment leads to the disruption of the equilibrium toward a nucleosome-depleted state, rather than to enhancer de novo "remodeling." This allows the recruitment of histone modifiers, chromatin remodelers, and ultimately gene activation. The "receptive" state described here could help explain AR signaling activation under very low ligand concentrations.

MLL2 is required in oocytes for bulk histone 3 lysine 4 trimethylation and transcriptional silencing.

During gametogenesis and pre-implantation development, the mammalian epigenome is reprogrammed to establish pluripotency in the epiblast. Here we show that the histone 3 lysine 4 (H3K4) methyltransferase, MLL2, controls most of the promoter-specific chromatin modification, H3K4me3, during oogenesis and early development. Using conditional knockout mutagenesis and a hypomorph model, we show that Mll2 deficiency in oocytes results in anovulation and oocyte death, with increased transcription of p53, apoptotic factors, and Iap elements. MLL2 is required for (1) bulk H3K4me3 but not H3K4me1, indicating that MLL2 controls most promoters but monomethylation is regulated by a different H3K4 methyltransferase; (2) the global transcriptional silencing that preceeds resumption of meiosis but not for the concomitant nuclear reorganization into the surrounded nucleolus (SN) chromatin configuration; (3) oocyte survival; and (4) normal zygotic genome activation. These results reveal that MLL2 is autonomously required in oocytes for fertility and imply that MLL2 contributes to the epigenetic reprogramming that takes place before fertilization. We propose that once this task has been accomplished, MLL2 is not required until gastrulation and that other methyltransferases are responsible for bulk H3K4me3, thereby revealing an unexpected epigenetic control switch amongst the H3K4 methyltransferases during development.

Absence of inhibin alpha and retinoblastoma protein leads to early sertoli cell dysfunction.

Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin alpha knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10-14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice.

Retinoblastoma protein plays multiple essential roles in the terminal differentiation of Sertoli cells.

Retinoblastoma protein (RB) plays crucial roles in cell cycle control and cellular differentiation. Specifically, RB impairs the G(1) to S phase transition by acting as a repressor of the E2F family of transcriptional activators while also contributing towards terminal differentiation by modulating the activity of tissue-specific transcription factors. To examine the role of RB in Sertoli cells, the androgen-dependent somatic support cell of the testis, we created a Sertoli cell-specific conditional knockout of Rb. Initially, loss of RB has no gross effect on Sertoli cell function because the mice are fertile with normal testis weights at 6 wk of age. However, by 10-14 wk of age, mutant mice demonstrate severe Sertoli cell dysfunction and infertility. We show that mutant mature Sertoli cells continue cycling with defective regulation of multiple E2F1- and androgen-regulated genes and concurrent activation of apoptotic and p53-regulated genes. The most striking defects in mature Sertoli cell function are increased permeability of the blood-testis barrier, impaired tissue remodeling, and defective germ cell-Sertoli cell interactions. Our results demonstrate that RB is essential for proper terminal differentiation of Sertoli cells.

Preimplantation mouse embryos depend on inhibitory phosphorylation of separase to prevent chromosome missegregation.

Separase is a critical protease that catalyzes the cleavage of sister chromatid cohesins to allow the separation of sister chromatids in the anaphase. Its activity must be inhibited prior to the onset of the anaphase. Two inhibitory mechanisms exist in vertebrates that block the protease activity. One mechanism is through binding and inhibition by securin, and another is phosphorylation on Ser1126 (in humans [Ser1121 in mice]). These two mechanisms are largely redundant. However, phosphorylation on Ser1121 is critical for the prevention of premature sister separation in embryonic germ cells. As a result, Ser1121-to-Ala mutation leads to depletion of germ cells in development and subsequently to infertility in mice. Here, we report that the same mutation also causes embryogenesis failure between the 8- and 16-cell stages in mice. Our results indicate a critical role of separase phosphorylation in germ cell development as well as in early embryogenesis. Thus, deregulation of separase may be a significant contributor to infertility in humans.

Deletion of Dicer in somatic cells of the female reproductive tract causes sterility.

Dicer is an evolutionarily conserved ribonuclease III that is necessary for microRNA (miRNA) processing and the synthesis of small interfering RNAs from long double-stranded RNA. Although it has been shown that Dicer plays important roles in the mammalian germline and early embryogenesis, the functions of Dicer-dependent pathways in the somatic cells of the female reproductive tract are unknown. Using a transgenic line in which Cre recombinase is driven by the anti-Müllerian hormone receptor type 2 promoter, we conditionally inactivated Dicer1 in the mesenchyme of the developing Müllerian ducts and postnatally in ovarian granulosa cells and mesenchyme-derived cells of the oviducts and uterus. Deletion of Dicer in these cell types results in female sterility and multiple reproductive defects including decreased ovulation rates, compromised oocyte and embryo integrity, prominent bilateral paratubal (oviductal) cysts, and shorter uterine horns. The paratubal cysts act as a reservoir for spermatozoa and oocytes and prevent embryos from transiting the oviductal isthmus and passing the uterotubal junction to enter the uterus for implantation. Deep sequencing of small RNAs in oviduct revealed down-regulation of specific miRNAs in Dicer conditional knockout females compared with wild type. The majority of these differentially expressed miRNAs are predicted to regulate genes important for Müllerian duct differentiation and mesenchyme-derived structures, and several of these putative target genes were significantly up-regulated upon conditional deletion of Dicer1. Thus, our findings reveal diverse and critical roles for Dicer and its miRNA products in the development and function of the female reproductive tract.

Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure.

The retinoblastoma protein (RB) regulates cell proliferation and survival by binding to the E2F family of transcription factors. Recent studies suggest that RB also regulates differentiation in a variety of cell types, including myocytes, neurons, adipocytes, and chondrocytes. Rb mutations have been found in ovarian cancer; however, the role of RB in normal and abnormal ovarian function remains unclear. To test the hypothesis that loss of Rb induces ovarian tumorigenesis, we generated an ovarian granulosa cell conditional knockout of Rb (Rb cKO) using the Cre/lox recombination system. Rb cKO females showed 100% survival and no ovarian tumor formation through 9 months of age, but they developed progressive infertility. Prepubertal Rb cKO females showed increased ovulation rates compared with controls, correlating with increased follicle recruitment, higher Fshr and Kitl mRNA levels, and lower anti-Müllerian hormone levels. In contrast, the ovulation rate of 6-wk-old females was similar to that of controls. Morphometric analysis of Rb cKO ovaries from 6-wk-old and older females showed increased follicular atresia and apoptosis. Rb cKO ovaries and preantral follicles had abnormal levels of known direct and indirect target genes of RB, including Rbl2/p130, E2f1, Ccne2, Myc, Fos, and Tgfb2. In addition, preantral follicles showed increased expression of the granulosa cell differentiation marker Inha, decreased levels of Foxl2 and Cyp19a1 aromatase, and abnormal expression of the nuclear receptors Nr5a1, Nr5a2, and Nr0b1. Taken together, our results suggest that RB is required for the temporal-specific pattern of expression of key genes involved in follicular development.