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PDF file, 71KB, The data are scaled by the positive control (1�M MG132) and the negative control (DMSO). The percentage of maximum activity (Amax) is represented in function of the crossing point (concentration in �M at 50% of MG132 activity). Each data point represents a cell line; the vertical and horizontal lines represent, respectively, the median of the crossing point values (1.33�M) and the median of the Amax values (-90.06%), of BKM120 across all cell lines. The populations of cell lines least responding to GDC-0941, among which some are sensitive to BKM120, are highlighted in green.

PDF file, 300KB, A. Upon infection with viral particles obtained from the pBabe-puro-myr-Akt retroviral vectors, pools were selected upon addition of puromycin and maintained in culture for several weeks. Extracts from pools were generated for the indicated time of culture and analyzed by Western-blot for the expression of the exogenously expressed HA-tagged form of the dominant active myr-Akt protein. B. 2.5x106 MCF7-BP (upper panel) or MCF7-myr-Akt (bottom panel) were seeded in 10 cm dishes and exposed for 1 h to the indicated compounds at the indicated concentrations (in nM). Cells were then lyzed and cell extracts were analyzed by Western-blot for pathway inhibition as revealed by S473P-Akt levels.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G2–M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α–dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 μmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor–bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K. Mol Cancer Ther; 11(8); 1747–57. ©2012 AACR.

PDF file, 53KB, A and B. 5x105 A2058 cells were seeded in 10 cm dishes and incubated for 24 h either with increasing amounts of BKM120 (A) or with 5 �M of either GDC-0941 (B, top panel) or BEZ235 (B, bottom panel). Cells were then fixed, prepared as described for quantification of the population in the different phases of the cell cycle by fluorescence-activated cell sorting. G1, S and G2/M distribution for control untreated cells are described in the mean text and in Figure 4A. The activities of BKM120 on the cell cycle were plotted along to the inhibitory effects on pAkt levels (A).

PDF file, 107KB, A. 2.5x106 Rat1-myr-p110a (left) or U87MG cells were seeded in 10 cm dishes and exposed for 6h either to BKM120 (5 �M) or to the DMSO control. Cells were then lyzed and cell extracts were analyzed by Western-blot for PI3K pathway inhibition and G2/M markers as revealed by S473P-Akt (upper panel) and Ser10P-Histone H3 (bottom panel) levels, respectively. B. U87MG tumor bearing mice were treated orally, once per day for 6 days with BKM120 at the indicated doses. Tumor volumes were callipered and plotted. ns: not significant.

PDF file, 113KB, A. Effects of Paclitaxel and Nocodazole on Tubulin polymerization. Tubulin was mixed with either Paclitaxel (10 �M), Nocodazole (10 �M) or the DMSO control in the presence of GTP. The polymerization of monomeric tubulin into microtubule was started by transferring the reaction tubes from 4{degree sign}C to 37{degree sign}C, and monitored by the increase in absorbance (λ=340 nM) over a period of 60 min. B. Competition experiments of NVP-BKM120 with colchicine and podophyllotoxin by NMR spectroscopy. T1ρ relaxation of BKM120 in the presence of tubulin (50-fold excess of compound) remains unchanged after adding podophyllotoxin or colchicine, as emphasized by the drawn arrows. The spectra of the three compounds are shown in three colors at the bottom. C. Structures of GDC0941, BEZ235, Nocodazole and BKM120.

PDF file, 653KB, MDA-MB231 (left) and U87MG (right) cells grown on coverslips were treated for 24 h either with BKM120 (5 μM) or Nocodazole (100 nM) and the effects of compound treatment on microtubule dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin (microtubules), gamma tubulin (centrosomes) and DAPI (DNA). Pictures were taken with a 100X objective.

PDF file, 2791KB. A2058 (upper panels), MDA-MB231 (lower panels) and U87MG cells grown on coverslips were treated for 6 h, 6 h with a subsequent 18 h washout period or 24 h with either 5 μM BKM120 or 100 nM Nocodazole and the effects of compound treatment on microtubule dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin. Pictures were taken with a 40X objective.

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PurposeTo use preclinical and clinical pharmacokinetic (PK)/pharmacodynamic (PD) modeling to predict optimal clinical regimens of everolimus, a novel oral mammalian target of rapamycin (mTOR) inhibitor, to carry forward to expanded phase I with tumor biopsy studies in cancer patients.Patients and MethodsInhibition of S6 kinase 1 (S6K1), a molecular marker of mTOR signaling, was selected for PD analysis in peripheral blood mononuclear cells (PBMCs) in a phase I clinical trial. PK and PD were measured up to 11 days after the fourth weekly dose. A PK/PD model was used to describe the relationship between everolimus concentrations and S6K1 inhibition in PBMCs of cancer patients and in PBMCs and tumors of everolimus-treated CA20948 pancreatic tumor-bearing rats.ResultsTime- and dose-dependent S6K1 inhibition was demonstrated in human PBMCs. In the rat model, a relationship was shown between S6K1 inhibition in tumors or PBMCs and antitumor effect. This allowed development of a direct-link PK/PD model that predicted PBMC S6K1 inhibition-time profiles in patients. Comparison of rat and human profiles simulated by the model suggested that a weekly 20- to 30-mg dose of everolimus would be associated with an antitumor effect in an everolimus-sensitive tumor and that daily administration would exert a greater effect than weekly administration at higher doses.ConclusionA direct-link PK/PD model predicting the time course of S6K1 inhibition during weekly and daily everolimus administration allowed extrapolation from preclinical studies and first clinical results to select optimal doses and regimens of everolimus to explore in future clinical trials.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G2–M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-a-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α–dependent model. We observed that, in vivo , daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 μmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor–bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K. Mol Cancer Ther; 11(8); 1747–57. ©2012 AACR . This article is featured in Highlights of This Issue, [p. 1619][1] [1]: /lookup/volpage/11/1619?iss=8

The postantibiotic effect (PAE) is the persistent inhibition of bacterial growth after a brief exposure to an antibiotic. Most beta-lactams do not induce a PAE for Gram-negative bacteria, but PAEs have been reported for carbapenems and penems. This study investigated the effect of sequential doses of imipenem on the PAE for Pseudomonas aeruginosa and Escherichia coli cultures in a chemostat. The PAE for the bacterial population did not change even after six successive exposures to imipenem. Nevertheless, screening of colonies isolated after repeated drug exposure identified a single P. aeruginosa mutant whose imipenem PAE was shortened, although the MIC was unchanged. The PAEs for the parent and mutant were studied in vitro in batch culture by monitoring: (i) viable counts; (ii) electrical impedance of the culture medium; (iii) incorporation of radiolabelled N-acetyl-D-glucosamine and (iv) cell volume changes. PAEs for the parent and mutant were found to be significantly different by all in-vitro methods used. Moreover, the median cell volume in antibiotic-exposed cultures remained much smaller and less heterogeneous than in the control cultures, even though both cultures were growing at the same rate. The mutant was found to have a reduced expression of a 52 kDa outer membrane protein. These observations suggest that factors in addition to suppression of bacterial growth should be considered when studying the PAE. The PAEs of imipenem for the parent and mutant were studied in a thigh infection model in leucopenic mice. Similar PAEs were observed in vivo for both parent and mutant in one experiment and no PAEs for either organism were found in a second experiment. This study showed that although the PAE is a stable in-vitro phenomenon, the lack of correlation between the in-vitro and in-vivo results warrants caution in attributing clinical significance to the PAE of imipenem.

BACKGROUND Microtubule agents appear promising for the treatment of prostate cancer. Patupilone (epothilone B), a highly potent non-taxane microtubule stabilizing agent, was evaluated in models of androgen-independent prostate cancer. METHODS Patupilone was administered to athymic mice bearing human prostate cancer xenografts (subcutaneous DU 145 and PC-3M, orthotopic PC-3M). RESULTS One 4 mg/kg patupilone administration produced transient regression of DU 145 tumors, while two weekly administrations of 2.5 mg/kg produced stable disease followed by protracted regression, however with more pronounced body weight loss. Taxol® (15 mg/kg every other day) weakly inhibited tumor growth, but with less body weight loss. Patupilone (5 mg/kg) produced protracted growth inhibition of subcutaneous PC-3M tumors, with transient body weight loss. In mice with orthotopic PC-3M tumors, 4 or 5 mg/kg/week patupilone impaired primary tumor growth, abrogated metastases and enhanced survival, with only transient body weight loss. CONCLUSIONS These data suggest that patupilone holds promise for prostate cancer treatment. © 2005 Wiley-Liss, Inc.

The microtubule-stabilizing agent patupilone (epothilone B, EPO906) and the tyrosine kinase inhibitor imatinib (STI571, Glivec) which primarily inhibits Bcr-Abl, PDGF and c-Kit tyrosine kinase receptors, were combined in vivo to determine if any interaction would occur with respect to antitumour effect and tolerability using rat C6 glioma xenografted into nude mice. Patupilone and imatinib were administered alone or in combination at suboptimal doses. Imatinib treatment (orally once daily) was initiated 4 days after s.c. injection of rat C6 glioma cells into athymic nude mice and patupilone administration (i.v. once per week) was started 3 or 4 days after imatinib treatment. As a single agent, imatinib was inactive in the regimens selected (100 mg/kg: T/C 86% and 116%; 200 mg/kg: T/C 68% and 84%; two independent experiments), but well tolerated (gain in body weight and no mortalities). Patupilone weekly monotherapy demonstrated dose-dependent antitumour effects (1 mg/kg: T/C 67% and 70%; 2 mg/kg: T/C 32% and 63%; 4 mg/kg: T/C 3% and 46%). As expected, dose-dependent body weight losses occurred (final body weight changes at 1 mg/kg were −7% and −3%; at 2 mg/kg were −23% and −13%; and at 4 mg/kg were −33% and −15%). Combining 2 mg/kg patupilone and 200 mg/kg per day imatinib in one experiment produced a non-statistically significant trend for an improved antitumour effect over patupilone alone (combination, T/C 9%), while in the second experiment, enhancement was seen with the combination and reached statistical significance versus patupilone alone (combination, T/C 22%; P=0.008). Reduction of the imatinib dose to 100 mg/kg per day resulted in no enhancement of antitumour activity in combination with 2 mg/kg patupilone. Reduction of the patupilone dose to 1 mg/kg resulted in a reduced antitumour effect, and only a trend for synergy with either imatinib dose (combination, T/C 46% and 40%). Pooling the data from the two experiments confirmed a significant synergy for the combination of 2 mg/kg patupilone and 200 mg/kg per day imatinib (P=0.032), and a trend for synergy at the 1 mg/kg patupilone dose. Reduction in the imatinib dose to 100 mg/kg per day resulted only in additivity with either dose of patupilone. Body weight losses were dominated by the effect of patupilone, since no greater body weight loss was observed in the combination groups. Combining patupilone with high-dose imatinib produced an increased antitumour effect without affecting the tolerability of treatment in a relatively chemoresistant rat C6 glioma model. Such results indicate that further evaluation is warranted, in particular to elucidate possible mechanisms of combined action.

This study describes the first known model of bone cancer pain in the rat. Sprague-Dawley rats receiving intra-tibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells developed behavioural signs indicative of pain, including: mechanical allodynia, difference of weight bearing between hind paws and mechanical hyperalgesia. The development of the bone tumour and structural damage to the bone was monitored by radiological analysis, quantitative measurement of mineral content and histology. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) syngeneic MRMT-1 cells produced a rapidly expanding tumour within the boundaries of the tibia, causing severe remodelling of the bone. Radiographs showed extensive damage to the cortical bone and the trabeculae by day 10-14 after inoculation of 3 x 10(3) MRMT-1 cells, and by day 20, the damage was threatening the integrity of the tibial bone. While both mineral content and mineral density decreased significantly in the cancerous bone, osteoclast numbers in the peritumoural compact bone remained unchanged. However, tartarate-resistant acid phosphatase staining revealed a large number of polykariotic cells, resembling those of osteoclasts within the tumour. No tumour growth was observed after the injection of heat-killed MRMT-1 cells. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) MRMT-1 cells, heat-killed cells or vehicle did not show changes in body weight and core temperature over 19-20 days. The general activity of animals after injection with live or heat-killed MRMT-1 cells was higher than that of the control group, however, the activity of the MRMT-1 treated group declined during the progress of the disease. Rats receiving intra-tibial injections of MRMT-1 cells displayed the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12-14 or 10-12 following injection of 3 x 10(3) or 3 x 10(4) cells, respectively. These symptoms were not observed in rats receiving heat-killed cells or vehicle. Behavioural data suggest a reasonable time window for evaluation of anti-nociceptive agents between day 14 and 20 after cancer cell inoculation in this model. Acute treatment with morphine (1-3mg/kg, subcutanously (s.c.)) produced a dose-dependent reduction in the response frequency of hind paw withdrawal to von Frey filament stimulation 17 or 19 days following intra-tibial injections of 3 x 10(3) MRMT-1 cells. A significant reduction in the difference in hind limb weight bearing was also observed. Acute treatment with celebrex (10-30 mg/kg, s.c.) did not affect mechanical allodynia or difference in weight bearing in rats 20 days following treatment with 3 x 10(3) MRMT-1 cells. Although the pathophysiology of cancer pain is largely unknown, significant enhancement of glial fibrillary acidic protein (GFAP) staining in the corresponding segments of the ipsilateral spinal cord highlights the possible involvement of astrocytes. In summary, the induction of bone cancer in the rat by the syngeneic MRMT-1 mammary tumour cell line provides a valid pre-clinical model for pain associated with bone metastases. Significant mechanical hyperalgesia and allodynia develops in association with the progression of the tumour in the bone marrow cavity, while the general condition of the animal remains satisfactory. While acute treatment with morphine has some analgesic effect on hind limb sparing the selective COX-2 inhibitor, celebrex, has no influence on the pain-related behavioural changes in this model.

The purpose of the present study was to evaluate the combination of azithromycin and rifampicin on experimental chronic osteomyelitis due to Staphylococcus aureus. Alterations in bone bacterial titre, activity of tumour necrosis factor (TNF), a cytokine implicated in inflammation-induced bone pathology, and histopathological changes during infection and following antibiotic treatment were evaluated. Rats were infected with S. aureus by direct tibial inoculation and then randomized 56 days after infection to receive saline treatment or a combination of azithromycin and rifampicin (50 mg/kg po and 25 mg/kg sc respectively) once daily for 21 days. The combination of azithromycin and rifampicin was successful as determined by dramatic reduction in bone bacterial counts (approximately log 4 cfu), but regrowth of the organisms occurred suggesting that the duration of treatment was insufficient. TNF alpha mRNA and TNF activity were constantly elevated by approximately 20- and >200-fold, respectively, and remained elevated irrespective of antimicrobial treatment. Bone histology revealed extensive increase in bone turnover in both the infected and antibiotic treated bones with no difference being observed between the groups. This suggests that, in infected bone, the elevated TNF levels observed may be directly related to the bone pathology and both remain largely unchanged despite potent antibiotic therapy.

NVP-BEZ235 is a dual inhibitor of class 1 phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) catalytic activity. It induces cell death in a subset of breast cancer cell lines characterized by amplification of human epidermal growth factor receptor 2 (HER2/ErbB2) and/or activating PIK3CA mutations, but not in cell lines with loss of function of the PTEN tumor suppressor protein or KRAS mutations. In order to better understand the molecular mechanisms leading to cell death, to identify potential biomarkers of activity and to reveal pathways that may even enhance NVP-BEZ235 induced lethality, a pooled short hairpin RNA (shRNA) screen using a lentiviral-based shRNA library targeting the apoptome was performed in NVP-BEZ235-sensitive MDA-MB453 and HCC1954 breast cancer cells. This strategy led to the identification of shRNAs that sensitized cells to NVP-BEZ235-induced apoptosis. Top sensitizers included pro-survival BCL2 family members, BCL2L1 and MCL1. Validation studies were carried out with single shRNAs targeting BCL2L1 and MCL1 as well as drug combination studies of NVP-BEZ235 with ABT-263 or ABT-747, both BCL2 homology domain 3 (BH3)-only protein mimetics. Cell death assays and biochemical readouts including PARP cleavage support the use of BCL2 family inhibitors to enhance NVP-BEZ235-induced cell death in HER2 amplified/PIK3CA mutated breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2011-5371