Ovarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1 -amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.

MSY2, a germ-cell-specific member of the Y-box family of DNA-͞ RNA-binding proteins, is proposed to function as a coactivator of transcription in the nucleus and to stabilize and store maternal and paternal mRNAs in the cytoplasm. In mice lacking Msy2, a normal Mendelian ratio is observed after matings between heterozygotes with equal numbers of phenotypically normal but sterile male and female homozygotes (Msy2 ؊/؊ ). Spermatogenesis is disrupted in postmeiotic null germ cells with many misshapen and multinucleated spermatids, and no spermatozoa are detected in the epididymis. Apoptosis is increased in the testes of homozygotes, and real-time RT-PCR assays reveal large reductions in the mRNA levels of postmeiotic male germ cell mRNAs and smaller reductions of meiotic germ cell transcripts. In females, there is no apparent decrease in either the number of follicles or their morphology in ovaries obtained from 2-and 8-day-old Msy2 ؊/؊ mice. In contrast, follicle number and progression are reduced in 21-day-old Msy2 ؊/؊ ovaries. In adult Msy2 ؊/؊ females, oocyte loss increases, anovulation is observed, and multiple oocyte and follicle defects are seen. Thus, Msy2 represents one of a small number of germ-cell-specific genes whose deletion leads to the disruption of both spermatogenesis and oogenesis.he highly conserved family of Y-box proteins, expressed in organisms ranging from bacteria to humans, contains a cold-shock domain essential for nucleic-acid binding and variable N and C termini that confer binding specificity (1). As DNA-binding proteins, Y-box proteins serve as transcription coactivators, recognizing DNA motifs such as CTGATTGGC͞ TC͞TAA (2). As RNA-binding proteins, Y-box proteins help stabilize mRNAs and, depending on their concentration, can inhibit or stimulate mRNA translation (3, 4).Among DNA-͞RNA-binding proteins, the mouse Y-box protein MSY2 is one of the most abundant, constituting 0.7% ( Fig. 1) and 2% of total protein in male germ cells and fully grown oocytes, respectively (5, 6). MSY2 is the mouse ortholog of the Xenopus laevis FRGY2 (2) and human Contrin proteins (7), Y-box proteins proposed to be solely expressed in germ cells (8). In the testis, Msy2 is expressed in meiotic and postmeiotic germ cells, where it is believed to function in long-term mRNA storage and stabilization because cessation of transcription in postmeiotic germ cells necessitates posttranscriptional regulation for many mRNAs encoding late-stage germ cell and spermatozoan proteins (8). In addition, MSY2 marks specific mRNAs in the nucleus for storage in the cytoplasm, providing a linkage between transcription and mRNA storage for a subset of male germ cell mRNAs (9).In the female, MSY2 protein accumulates during oocyte growth, but after fertilization, it is totally degraded by the late two-cell stage (6). As in postmeiotic male germ cells, MSY2 is located in the cytoplasm in oocytes, but, in contrast to male germ cells, where MSY2 is soluble, Ϸ70% of MSY2 is retained after permeabilization procedures that release Ͼ70...

During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting Ϸ0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former population is enriched in cell growth and ubiquitously expressed mRNAs, whereas the latter population is enriched for stored or translationally delayed, male gamete-specific transcripts. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs are transcribed from genes containing the Y-box DNA-binding motif in their promoters. In transgenic mice, mRNAs encoding exogenous GFP are directed or not directed into the MSY2-bound fraction by promoters containing or lacking the Y-box motif, respectively. We propose that MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage, thereby linking transcription and mRNA storage͞translational delay in meiotic and postmeiotic male germ cells of the mouse.mRNA storage ͉ spermatogenesis ͉ transcription and translation linkage ͉ Y-box protein

MSY2 is a member of the Y-box family of proteins solely expressed in male and female germ cells. In the male, MSY2 serves as a coactivator of transcription by binding to a consensus promoter element present in many germ cell-specific genes. In the nucleus, MSY2 marks specific mRNAs for cytoplasmic storage, stabilization, and suppression of translation. The inactivation of MSY2 by gene targeting leads to spermatogenic arrest and infertility. In testes of mice lacking MSY2, incomplete nuclear condensation is prominent in later-stage spermatids at the time of massive spermatid loss. Because MSY2 interacts with DNA and mRNAs, there are several distinct sites of action, which could be disrupted in mice that lack MSY2, resulting in the arrest of spermatogenesis. To define the molecular cause(s) of the spermatogenic arrest in mice lacking MSY2, transcriptional and posttranscriptional processes were assayed. Transcription, mRNA processing, and mRNA intracellular transport appear normal in the absence of MSY2. However, a redistribution of mRNAs from ribonucleoprotein particles to polysomes and marked decreases were detected for many meiotic and postmeiotic germ cell mRNAs, including the mRNAs encoding the transition proteins and protamines. This suggests that increased mRNA instability is a likely cause of the male infertility in Msy2-null mice.

Messenger RNA is remarkably stable during oocyte growth, thus enabling mRNAs to accumulate during the growth phase and thereby provide mRNAs that support early embryonic development. MSY2, a germ cell-specific RNA-binding protein, is implicated in regulating mRNA stability. MSY2 is essential for development because female Msy2(-/-) mice are infertile. We describe here the characterization of Msy2(-/-) oocytes. Mutant oocytes grow more slowly during the first wave of folliculogenesis, and maturation to and arrest at metaphase II is severely compromised because of aberrant spindle formation and chromosome congression. Consistent with MSY2 conferring mRNA stability is that the amount of poly(A)-containing RNA is reduced by ~25% in mutant oocytes. Stability of an exogenous mRNA injected into mutant oocytes is lower than when compared to their wild-type counterparts, and moreover, expression of wild-type MSY2 in mutant oocytes increases mRNA stability, whereas injection of a mutant form of MSY2 not capable of binding RNA does not. Transcription quiescence that normally occurs during the course of oocyte growth is not observed in mutant oocytes, and the transcriptome of mutant oocytes is markedly perturbed. These results, and those of previous studies, strongly implicate a central role of MSY2 in regulating mRNA stability.

MSY2 is implicated in regulating the stability and translation of maternal mRNAs during mouse oogenesis. We report here that by driving the expression of a transgene encoding an Msy2 hairpin dsRNA in growing oocytes using the oocyte-specific Zp3 promoter, the amount of MSY2 protein was reduced by at least 60% in fully grown oocytes. The decrease appeared specific because no decrease was observed in either non-targeted mRNAs or proteins. Fertility of transgenic females was severely reduced. Although transgenic eggs could be inseminated, the eggs did not exhibit the normal series of oscillations in intracellular Ca2+, resume meiosis, undergo cortical granule exocytosis, or ZP2 cleavage to ZP2f. Transgenic oocytes also displayed a higher incidence of both the non-surrounded nucleolus chromatin morphology, and abnormal meiotic spindle formation was observed following oocyte maturation. Transgenic oocytes contained less total mRNA (approximately 75-80% that of non-transgenic oocytes) and displayed a reduced level of protein synthesis. Moreover, several of the maturation-associated changes in protein synthesis failed to occur in the transgenic oocytes. These results support a role for MSY2 in stabilizing maternal mRNAs in growing oocytes, a process essential to generate meiotically and developmentally competent oocytes.

Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We report that CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdc2a mRNA, which activates CDC2A, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDC2A following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible.

No abstract