Biological characterization of ETP-46321 a selective and efficacious inhibitor of phosphoinositide-3-kinases.

Inhibitors of PI3K signaling are of great therapeutic interest in oncology. The phosphoinositide-3-kinase signaling pathway is activated in a variety of solid and non-solid tumors. We have identified an imidazopyrazine derivative, ETP-46321, as a potent inhibitor of PI3Kα [Formula: see text]. The compound was 6 times less potent towards PI3Kδ and more than 200 and 60 times less potent at inhibiting PI3Kß and PI3Kγ and did not significantly inhibit the related phosphoinositide-3-kinase-related protein kinase family kinases mTOR or DNA PK (IC(50)'s > 5 µM), or an additional 287 protein kinases that were screened. ETP-46321 inhibited PI3K signaling in treated tumor cell lines, induced cell cycle arrest and inhibited VEGF-dependent sprouting of HUVEC cells. The compound was anti-proliferative and synergized with both cytotoxic and targeted therapeutics. The compound induced a reduction in the phosphorylation of Akt in U87 MG xenografts after a single treatment. The growth of colon and lung cancinoma HT-29 and A549 xenografts was delayed by once a day treatment with ETP-46321. The compound synergized with Doxotaxel in a model of ovarian cancer.

Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases.

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3Kalpha. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.

Circadian regulation of cell cycle and apoptosis proteins in mouse bone marrow and tumor.

Proapoptotic drugs such as docetaxel displayed least toxicity and highest antitumor efficacy following dosing during the circadian rest phase in mice, suggesting that cell cycle and apoptotic processes could be regulated by the circadian clock. In study 1, mouse bone marrow and/or tumor were obtained every 4 h for 24 h in C3H/HeN mice with or without MA13/C mammary adenocarcinoma in order to determine the circadian patterns in cell-cycle phase distribution and BCL-2 anti-apoptotic protein expression. In study 2, mouse bone marrow from B6D2F1 mice was sampled every 3 h for 24 h in order to confirm the BCL-2 rhythm and to study its relation with 24 h changes in the expression of proapoptotic BCL-2-associated X protein (BAX) protein and clock genes mPer2, mBmal1, mClock, and mTim mRNAs. The rhythms in G1-, S- or G2/M-phase cells were shifted in tumor compared with bone marrow. In the tumor, the mean proportion of G2/M-phase cells increased by 75% from late rest to late activity span (P from cosinor = 0.001). No 24 h rhythm was found for BCL-2 in tumors. In contrast to this, in the bone marrow, mean BCL-2 expression varied 2.8-fold in B6D2F1 mice (P=0.025) and 3- or 4.5-fold in tumor-bearing and nontumor-bearing C3H/HeN mice, with a peak during the early rest span (P=0.024 and P<0.001, respectively). BAX varied fivefold during the 24 h span with a major peak occurring near mid-activity (P=0.007). The mean mRNAs of mPer2, mClock, and mBmal1 varied twofold to threefold over the 24 h, with high values during the activity span (P<0.05). In the tumor, the circadian organization in cell-cycle phase distribution was shifted and BCL2 rhythm was ablated. Conversely, a molecular circadian clock likely regulated BCL-2 and BAX expression in the bone marrow, increasing cellular protection against apoptosis during the rest span.

Persistent twenty-four hour changes in liver and bone marrow despite suprachiasmatic nuclei ablation in mice.

Rest-activity or cortisol rhythms can be altered in cancer patients, a condition that may impair the benefits from a timed delivery of anticancer treatments. In rodents, the circadian pattern in rest-activity is suppressed by the destruction of the suprachiasmatic nuclei (SCN) in the hypothalamus. We sought whether such ablation would result in a similar alteration of cellular rhythms known to be relevant for anticancer drug chronopharmacology. The SCN of 77 B6D2F(1) mice synchronized with 12 h of light and 12 h of darkness were destroyed by electrocoagulation [SCN(-)], while 34 animals were sham operated. Activity and body temperature were recorded by telemetry. Blood and organs were sampled at one of six circadian times for determinations of serum corticosterone concentration, blood leukocyte count, reduced glutathione (GSH), and dihydropyrimidine dehydrogenase (DPD) mRNA expression in liver and cell cycle phase distribution of bone marrow cells. Sham-operated mice displayed significant 24-h rhythms in rest-activity and body temperature, whereas such rhythms were found in none and in 15% of the SCN(-) mice, respectively. SCN lesions markedly altered the rhythmic patterns in serum corticosterone and liver GSH, which became nonsinusoidal. Liver DPD mRNA expression and bone marrow cell cycle phase distribution displayed similar 24-h sinusoidal patterns in sham-operated and SCN(-) mice. These results support the existence of another light-dark entrainable pacemaker that can coordinate cellular functions in peripheral organs. They suggest that the delivery of anticancer treatments at an optimal time of day may still be beneficial, despite suppressed rest-activity or cortisol rhythms.

Disruption of circadian coordination accelerates malignant growth in mice.

An animal model (mice B6D2F1) was developed to study the consequence of suprachiasmatic nuclei (SCN) destruction on tumor growth. SCN destruction abolished the rest-activity and body temperature rhythms and markedly altered the rhythms in serum corticosterone concentration and lymphocyte count. Tumor growth was faster in mice with lesioned SCN than in controls for both tumor models studied, Glasgow osteosarcoma (GOS) and pancreatic adenocarcinoma (P03). This shows that disruption of circadian coordination accelerates malignant growth in mice, suggesting that the host circadian clock controls tumor progression.

[Rhythm of expression of BCL-2 protein of MA13/C mammaryadenocarcinoma bearing-mice]. / Rythme d'expression de la protéine BCL-2 chez la souris porteuse de l'adénocarcinome mammaire MA13/C.

We previously demonstrated a circadian rhythm in response to docetaxel chemotherapy in C3H/HeN mice bearing MA13/C mammary adenocarcinoma. We investigated the relation between this rhythm and the expression of BCL-2 in bone marrow and in tumor tissues. A circadian rhythm characterized BCL-2 expression in the bone marrow, which was hardly modified in tumor-bearing animals. BCL-2 acrophase coincided with the time of highest docetaxel tolerability and efficacy in this model. This suggests that BCL-2 protects the bone marrow from the drug toxicity, especially during the light phase.

Host circadian clock as a control point in tumor progression.

BACKGROUND: The circadian timing system controlled by the suprachiasmatic nuclei (SCN) of the hypothalamus regulates daily rhythms of motor activity and adrenocortical secretion. An alteration in these rhythms is associated with poor survival of patients with metastatic colorectal or breast cancer. We developed a mouse model to investigate the consequences of severe circadian dysfunction upon tumor growth. METHODS: The SCN of mice were destroyed by bilateral electrolytic lesions, and body activity and body temperature were recorded with a radio transmitter implanted into the peritoneal cavity. Plasma corticosterone levels and circulating lymphocyte counts were measured (n = 75 with SCN lesions, n = 64 sham-operated). Complete SCN destruction was ascertained postmortem. Mice were inoculated with implants of Glasgow osteosarcoma (n = 16 with SCN lesions, n = 12 sham-operated) or pancreatic adenocarcinoma (n = 13 with SCN lesions, n = 13 sham-operated) tumors to determine the effects of altered circadian rhythms on tumor progression. Time series for body temperature and rest-activity patterns were analyzed by spectral analysis and cosinor analysis. Parametric data were compared by the use of analysis of variance (ANOVA) and survival curves with the log-rank test. All statistical tests were two-sided. RESULTS: The 24-hour rest-activity cycle was ablated and the daily rhythms of serum corticosterone level and lymphocyte count were markedly altered in 75 mice with complete SCN destruction as compared with 64 sham-operated mice (two-way ANOVA for corticosterone: sampling time effect P<.001, lesion effect P =.001, and time x lesion interaction P<.001; for lymphocytes P =.001,.002, and.002 respectively). Body temperature rhythm was suppressed in 60 of the 75 mice with SCN lesions (P<.001). Both types of tumors grew two to three times faster in mice with SCN lesions than in sham-operated mice (two-way ANOVA: P<.001 for lesion and for tumor effects; P =.21 for lesion x tumor effect interaction). Survival of mice with SCN lesions was statistically significantly shorter compared with that of sham-operated mice (log-rank P =.0062). CONCLUSIONS: Disruption of circadian rhythms in mice was associated with accelerated growth of malignant tumors of two types, suggesting that the host circadian clock may play an important role in endogenous control of tumor progression.

Tumor-based rhythms of anticancer efficacy in experimental models.

Experimental tumor models constitute a prerequisite toward chronotherapy testing in cancer patients. Studies in experimental models are required to understand the relation between tumor rhythms and antitumor treatments efficacy. In healthy tissues, cell proliferation, and differentiation processes are regulated precisely and exhibit marked circadian rhythmicity. Experimental and human tumors can retain circadian rhythms or display altered oscillations. Healthy tissues can also display rhythm modifications, possibly related to cancer stage. Cellular rhythms modulate the metabolism of cytotoxic agents and the cellular response to them; hence, they determine the chronopharmacology of anticancer drugs. Circadian rhythms in host tolerability and/or cancer chemotherapy efficacy have been demonstrated with nontoxic doses of drugs in several experimental tumor models, while in other ones a circadian-time effect was only seen within a specific dose range. The usual coupling between tolerability and efficacy rhythms of anticancer agents has resulted in significant improvement of their therapeutic index. Results of laboratory animal studies have been extrapolated to the design of clinical cancer therapy trials involving a chronobiological approach.