A defect of uterine gland development in <i>Pik3ca</i>^<i>d/d</i> mice at 20 days of age.

<p>(A) The expression of PIK3CA was examined during uterine development. Immunohistochemical staining of PIK3CA in the uterus of PD 14 (a), PD 20 (b), and PD 28 (c). The uterus of GD 3.5 was used a positive control (d). (B) Gross anatomy of control (a) and <i>Pik3ca</i>^<i>d/d</i> (b) mice at PD 20. (C) The weight of uteri was not different between control and <i>Pik3ca</i>^<i>d/d</i> mice. (D) Immunohistochemical staining of FOXA2 in control (a and c) and <i>Pik3ca</i>^<i>d/d</i> (b and d) mice. (E) Quantification of FOXA2 positive endometrial glands in uteri of control and <i>Pik3ca</i>^<i>d/d</i> mice at PD 20. (F) The expression of <i>Foxa2</i> and <i>Spink3</i> in the uteri of control and <i>Pik3ca</i>^<i>d/d</i> mice. The results represent the mean ± SEM. ***, p < 0.001; **, p < 0.01.</p

<i>Pik3ca</i>^<i>d/d</i> mice have impaired implantation.

<p>(A) Gross anatomy of implantation sites in both the control (a) and <i>Pik3ca</i>^<i>d/d</i> uterus (b) at GD 7.5. (B) The number of implantation sites were recorded in the uteri. (C) The size of implantation sites were measured. (D) The proliferative cells were assessed in control and <i>Pik3ca</i>^<i>d/d</i> uterus at GD 7.5 by immunohistochemical staining using anti-Ki67 antibody. Arrow indicates the blastocyst. The data represents the mean ± SEM. ***, p < 0.001; **, p < 0.01.</p

The defect of gland formation in <i>Pik3ca</i>^<i>d/d</i> mice.

<p>(A) Gross anatomy of 8-week-old control (a) and <i>Pik3ca</i>^<i>d/d</i> (b) mice. (B) There was a significant decrease in the ratio of uterine weight in <i>Pik3ca</i>^<i>d/d</i> mice. (C) Histology of the uterus of control (a) and <i>Pik3ca</i>^<i>d/d</i> (b) mice. The expression of FOXA2 in the uterus of control (c) and <i>Pik3ca</i>^<i>d/d</i> (d) mouse. (D) Quantification of FOXA2 positive endometrial glands in mouse uteri. The results represent the mean ± SEM. *, p < 0.05; ***, p < 0.001.</p

The generation of mouse with uterine specific <i>Pik3ca</i> ablation.

<p>(A) The expression of <i>Pik3ca</i> mRNA was evaluated in uteri by real time PCR. The data represents the mean ± SEM. *, p < 0.05. PIK3CA protein expression was assessed by Western blot (B) and immunohistochemistry (C). Total RNA and protein were prepared from whole uteri. (D) The efficiency of PIK3CA loss was evaluated in uterus of <i>Pik3ca</i>^<i>d/d</i> mice at GD 2.5 (a and d), GD 3.5 (b and e), and GD 7.5 (c and f).</p

<i>Pik3ca</i>^<i>d/d</i> mice were subfertile.

<p><i>Pik3ca</i>^<i>d/d</i> mice were subfertile.</p

<i>Pik3ca</i> is required for mouse uterine gland development and pregnancy

<div><p>The PI3K/AKT signaling pathway plays a critical role in the maintenance of equilibrium between cell survival and apoptosis. The <i>Pik3ca</i> gene is mutated in a range of human cancers. It has been found to be oncogenic, and mutations lead to constitutive activation of the PI3K/AKT pathway. The expression patterns of PIK3CA proteins in the uterus of mice during early pregnancy indicate that it may play a role in the regulation of glandular epithelial cells, which is required to support uterine receptivity. To further investigate the role of <i>Pik3ca</i> in uterine function, <i>Pik3ca</i> was conditionally ablated only in the PGR-positive cells (<i>Pgr</i>^<i>cre/+</i><i>Pik3ca</i>^<i>f/f</i>; <i>Pik3ca</i>^<i>d/d</i>). A defect of uterine gland development and decidualization led to subfertility observed in <i>Pik3ca</i>^<i>d/d</i> mice. <i>Pik3ca</i>^<i>d/d</i> mice showed significantly decreased uterine weight compared to <i>Pik3ca</i>^<i>f/f</i> mice. Interestingly, a significant decrease of gland numbers were detected in <i>Pik3ca</i>^<i>d/d</i> mice compared to control mice. In addition, we found a decrease of <i>Foxa2</i> expression, which is a known uterine gland marker in <i>Pik3ca</i>^<i>d/d</i> mice. Furthermore, the excessive proliferation of endometrial epithelial cells was observed in <i>Pik3ca</i>^<i>d/d</i> mice. Our studies suggest that <i>Pik3ca</i> has a critical role in uterine gland development and female fertility.</p></div

The expression of <i>Pik3ca</i> mRNA and protein from GD 0.5 to GD 7.5.

<p>(A) The mRNA expression of <i>Pik3ca</i> was investigated in the uteri of C57BL/6 mice during early pregnancy. <i>Pik3ca</i> expression was significantly increased at GD 5.5 and GD 6.5. (A) The localization pattern of PIK3CA proteins was investigated through immunohistochemistry in uteri of C57BL/6 mice on GD 0.5 (a and b), GD 2.5 (c and d), GD 3.5 (e and f), GD 4.5 (g and h) GD 5.5 (i and j) and GD 7.5 (k and l). An arrow indicates an embryo. The results represent the mean ± SEM. **, p < 0.01; *, p < 0.05.</p

Proliferation of endometrial cells was dysregulated in the uteri of <i>Pik3ca</i>^<i>d/d</i> mice at the pre-implantation stage.

<p>(A) Immunohistochemical analysis of Ki67 (a and b), PGR (c and d), and ESR1 (e and f) proteins at GD 3.5 in endometrium of control (a, c, and e) and <i>Pik3ca</i>^<i>d/d</i> (b, d, and f) mice. (B) Quantification of Ki67 positive cells and semi-quantitative analysis of PRG and ESR1 in uterine epithelial and stromal cells of control and <i>Pik3ca</i>^<i>d/d</i> mice. The results represent the mean ± SEM. ***, p < 0.001; *, p < 0.05.</p

Discovery of a Series of 5,11-Dihydro‑6<i>H</i>‑benzo[<i>e</i>]pyrimido[5,4‑<i>b</i>][1,4]diazepin-6-ones as Selective PI3K-δ/γ Inhibitors

Dual inhibition of PI3K-δ and PI3K-γ is an established therapeutic strategy for treatment of hematological malignancies. Reported molecules targeting PI3K-δ/γ selectively are chemically similar and based upon isoquinolin-1­(2<i>H</i>)-one or quinazolin-4­(3<i>H</i>)-one scaffolds. Here we report a chemically distinct series of potent, selective PI3K-δ/γ inhibitors based on a 5,11-dihydro-6<i>H</i>-benzo­[<i>e</i>]­pyrimido­[5,4-<i>b</i>]­[1,4]­diazepin-6-one scaffold with comparable biochemical potency and cellular effects on PI3K signaling. We envisage these molecules will provide useful leads for development of next-generation PI3K-δ/γ targeting therapeutics