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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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Activation of p53 by inhibitors of the p53–MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53–MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo. Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2–p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure–response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53–MDM2 inhibitors.Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257–67. ©2018 AACR.

Median of the shRNAs and RSA values. File containing median counts and log fold changes of each gene from Supplementary Data File S1. According significance values of each gene as activator or sensitizer compared to DMSO control (see Methods) are included.

Supplementary material and methods include the description for the bioanalytical method for HDM201 detection in plasma and tumor, the human and mouse gene expression analysis in vitro and in vivo, the live-cell quantification of cleaved-caspase activation, the western blot analysis, the immunohistochemistry, the splinkerette PCR for the amplification of transposon integration sites and the tumor sequencing, mapping of insertion sequences to the mouse genome and identification of common integration site, additional information on the shRNA screen and the tumor models and supplementary references. Supplementary figures include: • Fig S1: the SJSA-1 inhibition growth curves when treated with HDM201 at different doses and for different times and the data for MOLM-3. • Fig S2: the cumulative percentage of cleaved-caspase-3/7 positive cells over the time, the GI50 of HDM201, CGM097 or nutlin-3a on SJSA-1 cells and the cellular apoptosis, as judged by AUC of cleaved-caspase-3/7 positive cells, induced by these compounds. • Fig S3: the PK profile in plasma and tumor of HDM201 in SJSA-1 tumors-bearing rat after p.o. and i.v. treatment, the Bcl-xl mRNA levels in tumors after HDM201 treatment, representative images of SJSA-1 tumors stained with p53 and cleaved-caspase 3 antibodies after HDM201 treatment and the individual data for the efficacy experiment in SJSA-1 tumor-bearing rats. • Fig S4: the PD of HDM201 in PB tumor bearing nude mice after single dose administration. Supplementary tables include: • Table S1: Biochemical profile of HDM201. • Table S2: List of cell lines tested for their sensitivity to HDM201 (n=291) • Table S3: Contingency table indicating association between sensitivity to HDM201 and TP53 wild-type status. • Table S4: List of cell lines tested for their sensitivity to both MDM2 knock-down by shRNA and HDM201 (n=261) • Table S5: Contingency table indicating association between sensitivity to HDM201 and sensitivity to MDM2 shRNA. • Table S6: List of significant rescuer and sensitizer genes following both HDM201 treatment types • Table S7: Pharmacokinetic parameters for HDM201 after p.o. and i.v. dosing in rat. • Table S8: Summary of primary PK parameters for HDM201 daily regimen after single dose (Day 1) in patients. • Table S9: Summary of primary PK parameters for HDM201 daily regimen on Day 14 in patients. • Table S10: Summary of primary PK parameters for HDM201 q3w regimen after single dose in patients.

The histone 3 lysine 79 (H3K79) methyltransferase (HMT) DOT1L is known to play a critical role for growth and survival of MLL-rearranged leukemia. Serendipitous observations during high-throughput drug screens indicated that the use of DOT1L inhibitors might be expandable to multiple myeloma (MM). Through pharmacologic and genetic experiments, we could validate that DOT1L is essential for growth and viability of a subset of MM cell lines, in line with a recent report from another team. In vivo activity against established MM xenografts was observed with a novel DOT1L inhibitor. In order to understand the molecular mechanism of the dependency in MM, we examined gene expression changes upon DOT1L inhibition in sensitive and insensitive cell lines and discovered that genes belonging to the endoplasmic reticulum (ER) stress pathway and protein synthesis machinery were specifically suppressed in sensitive cells. Whole-genome CRISPR screens in the presence or absence of a DOT1L inhibitor revealed that concomitant targeting of the H3K4me3 methyltransferase SETD1B increases the effect of DOT1L inhibition. Our results provide a strong basis for further investigating DOT1L and SETD1B as targets in MM.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurring suppurating lesions of the intertriginous areas, resulting in a substantial impact on patients’ QOL. HS pathogenesis remains poorly understood. An autoimmune component has been proposed, but disease-specific autoantibodies, autoantigens, or autoreactive T cells have yet to be described. In this study, we identify a high prevalence of IgM, IgG, and IgA antibodies directed against Nε-carboxyethyl lysine (CEL), a methylglyoxal-induced advanced glycation end-product, in the sera of patients with HS. Titers of anti-CEL IgG and IgA antibodies were highly elevated in HS compared with those in healthy controls and individuals with other inflammatory skin diseases. Strikingly, the majority of anti-CEL IgG was of the IgG2 subclass and correlated independently with both disease severity and duration. Both CEL and anti-CEL‒producing plasmablasts could be isolated directly from HS skin lesions, further confirming the disease relevance of this autoimmune response. Our data point to an aberration of the methylglyoxal pathway in HS and support an autoimmune axis in the pathogenesis of this debilitating disease.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity., Transcription factors are critical regulators of cell identity. Here, the authors use computational and functional genomic approaches to show an oncogenic role of PAX8 in renal cancer. Mechanistic dissection of PAX8 functions reveal its role in activating genes associated with metabolic pathways.

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R(+) and IL-2Ra(+) regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis.

Activation of p53 by inhibitors of the p53–MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53–MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo. Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2–p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure–response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53–MDM2 inhibitors. Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257–67. ©2018 AACR.

Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.Leukemia advance online publication, 3 January 2017; doi:10.1038/leu.2016.327.

p53 is a transcription factor that plays a central role in guarding genomic stability of the cell through cell cycle arrest or induction of apoptosis. It has also been reported that p53 participates in the regulation of tumor immunity and in homeostatic regulation of immune responses. However, the immunomodulatory effect of p53 in tumor and the tumor microenvironment is not well understood. From gene expression and immunohistochemical analysis of pre- and post-treatment biopsies from patients treated with the MDM2 inhibitor NVP-CGM097, we observed upregulation of immune checkpoint transcripts (PD1 and PDL1), and an increase in the number of CD8+ tumor infiltrating lymphocytes. To investigate the immunomodulatory effect of p53 in more detail, we studied murine syngeneic models treated with NVP-HDM201, a potent and selective second generation MDM2 inhibitor. We performed multi-color FACS analysis on tumors and tumor draining lymph nodes. With NVP-HDM201 treatment, we observed increase in numbers of CD103+ DC cells, capable of antigen cross-presentation. Furthermore, NVP-HDM201 increased the percentage of Tbet+EOMES-T cells in tumors as well as tumor draining lymph nodes and also resulted in increased ratios of CD8+ Tcells/Treg in tumor. In addition, levels of both PD-L1 on CD45- cells and PD-1 on CD4+ T cells were increased. Importantly, PD-1 and PD-L1 blockade enhanced HDM201 activity in p53 WT syngeneic mouse models but not in p53 mutated models. The rate of complete tumor regressions (CR) was significantly increased with combination treatment as compared to either treatment alone. The animals that achieved complete regressions also developed long lasting anti-tumor memory against the specific tumor cells as evidenced by re-challenge experiments. Taken together, these results demonstrate that MDM2 inhibition triggered adaptive immunity which was further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in cancer patients with wildtype p53. Citation Format: Hui Qin Wang, Jinsheng Liang, Iain Mulford, Fiona Sharp, Swann Gaulis, Yan Chen, Gina Trabucco, David Quinn, Joseph D. Growney, Matthew J. Meyer, Juliet Williams, Peter Hammerman, Francesco Hofmann, Glenn Dranoff, Jeffrey Engelman, Jennifer Mataraza, Ensar Halilovic. PD-1/PD-L1 blockade enhances MDM2 inhibitor activity in p53 wild-type cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5560.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53–HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53–HDM2 inhibitors, such as NVP-CGM097. DOI: http://dx.doi.org/10.7554/eLife.06498.001, eLife digest Stress from daily activities and exposure to chemicals or UV radiation can all damage cells. Damaged cells may develop into cancerous tumors if unchecked. Normally, a protein called p53 helps to repair or eliminate damaged cells and prevent tumors from forming. The p53 protein does this by switching on or off genes that control DNA repair, cell division, and cell death. But half of all cancerous tumors have mutations that prevent p53 from doing its job. Another protein called HDM2 keeps p53 in check by binding to p53 and preventing it from switching on and off genes after the stress passes. In cancers that have normal p53, sometimes HDM2 is overly active and prevents p53 from suppressing tumor formation and growth. Scientists are developing anticancer drugs that work by targeting HDM2; this frees p53 and allows it to wipe out cancerous cells. However, it is not always clear which patients with cancer are the most likely to benefit from anti-HDM2 therapy. Jeay et al. screened hundreds of cancer cells to determine which ones are sensitive to HDM2-targeting drugs. As expected, the screen revealed that cancer cells that have mutations in the gene encoding p53 are insensitive to the anti-HDM2 drug because there is no working p53 to free up. But about 60% of the cancer cells that have normal p53 proteins also did not respond to the anti-HDM2 therapy. This finding indicates that the presence of normal p53 protein is necessary but not sufficient for tumor cells to respond to anti-HDM2 therapy. Next, Jeay et al. compared the patterns of gene expression in the cancer cells that responded to an anti-HDM2 drug with those in cells that didn't respond. The analysis showed that a group of 13 genes are expressed more in the cells that responded to the drug. All 13 genes are unexpectedly direct targets of p53, suggesting that p53 remains active in these tumor cells, even if it is not working optimally. To verify these results, Jeay et al. grew human tumors in mice and found that tumors with high expression of the 13 genes are sensitive to the anti-HDM2 drug (called NVP-CGM097). The experiments strongly suggest that this 13-gene signature can be used to determine if a patient with cancer will respond to anti-HDM2 therapy. Following on from this work, researchers have already launched an early clinical trial with the anti-HDM2 drug and will test whether this gene signature is indeed useful in a real clinical setting. DOI: http://dx.doi.org/10.7554/eLife.06498.002

Mutations in Notch3 cause CADASIL (cerebral autosomal dominant adult onset arteriopathy), which leads to stroke and dementia in humans. CADASIL arteriopathy is characterized by major alterations of vascular smooth muscle cells and the presence of specific granular osmiophilic deposits. Patients carry highly stereotyped mutations that lead to an odd number of cysteine residues within EGF-like repeats of the Notch3 receptor extracellular domain. Such mutations may alter the processing or the trafficking of this receptor, or may favor its oligomerization. In this study, we examined the Notch3 expression pattern in normal tissues and investigated the consequences of mutations on Notch3 expression in transfected cells and CADASIL brains. In normal tissues, Notch3 expression is restricted to vascular smooth muscle cells. Notch3 undergoes a proteolytic cleavage leading to a 210-kDa extracellular fragment and a 97-kDa intracellular fragment. In CADASIL brains, we found evidence of a dramatic and selective accumulation of the 210-kDa Notch3 cleavage product. Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells, in close vicinity to but not within the granular osmiophilic material. These results strongly suggest that CADASIL mutations specifically impair the clearance of the Notch3 ectodomain, but not the cytosolic domain, from the cell surface.

We have previously purified and characterized a 5-methylcytosine (5-MeC)-DNA glycosylase from 12 day old chick embryos [Jost,J.P. et al. (1995) J. Biol. Chem. 270, 9734-9739]. The activity of the purified enzyme is abolished upon treatment with proteinase K and ribonuclease A. RNA copurifies with 5-MeC-DNA glycosylase activity throughout all chromatographic steps and preparative gel electrophoresis. RNA with a length of approximately 300-500 nucleotides was isolated from the gel purified enzyme. Upon extensive treatment with proteinase K, the gel eluted and labeled RNA did not show any significant change in molecular mass. The purified RNA incubated alone or in the presence of Mg2+and deoxyribonucleotide phosphates had no 5-MeC-DNA glycosylase or demethylating activities. However, activity of 5-MeC-DNA glycosylase could be restored when the purified RNA was incubated with the inactive protein, free of RNA.

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

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 functional interaction between fibroblast growth factor 23 (FGF-23) and Klotho in the control of vitamin D and phosphate homeostasis is manifested by the largely overlapping phenotypes of Fgf23- and Klotho-deficient mouse models. However, to date, targeted inactivation of FGF receptors (FGFRs) has not provided clear evidence for an analogous function of FGFRs in this process. Here, by means of pharmacologic inhibition of FGFRs, we demonstrate their involvement in renal FGF-23/Klotho signaling and elicit their role in the control of phosphate and vitamin D homeostasis. Specifically, FGFR loss of function counteracts renal FGF-23/Klotho signaling, leading to deregulation of Cyp27b1 and Cyp24a1 and the induction of hypervitaminosis D and hyperphosphatemia. In turn, this initiates a feedback response leading to high serum levels of FGF-23. Further, we show that FGFR inhibition blocks Fgf23 transcription in bone and that this is dominant over vitamin D-induced Fgf23 expression, ultimately impinging on systemic FGF-23 protein levels. Additionally, we identify Fgf23 as a specific target gene of FGF signaling in vitro. Thus, in line with Fgf23- and Klotho-deficient mouse models, our study illustrates the essential function of FGFRs in the regulation of vitamin D and phosphate levels. Further, we reveal FGFR signaling as a novel in vivo control mechanism for Fgf23 expression in bone, suggesting a dual function of FGFRs in the FGF-23/Klotho pathway leading to vitamin D and phosphate homeostasis.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Patient selection biomarkers are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study we have identified a novel patient selection strategy for NVP-CGM097, a p53-Mdm2 inhibitor currently in a Phase I clinical trial ([NCT01760525][1]). We have analyzed the sensitivity of over 500 cell lines from the “Cancer Cell Line Encyclopedia” to the p53-Mdm2 inhibitor in cell viability assays, and intersected response data with information on gene expression and genomic alterations. This analysis has led to the identification of a gene signature consisting of 13 genes, as a predictor for sensitivity to NVP-CGM097. Interestingly, these 13 genes are known p53 downstream target genes, supporting the hypothesis that the identified gene signature is reflective of the p53 pathway functionality in sensitive cell lines. We show the performance of the signature as ROC and Precision-Recall curves in cell lines as well as in a number of human primary tumor xenografts, both unselected as well as pre-selected for p53 wild-type status. Work is now ongoing to validate this gene signature using baseline tumor biopsy samples and RECIST-based efficacy readouts in the current Phase I clinical trial. Citation Format: Sebastien Jeay, Swann Gaulis, Stephane Ferretti, Genevieve Albrecht, Louise Barys, Daniel Guthy, Ensar Halilovic, Moriko Ito, Masato Murakami, Astrid Pornon, Stephan Ruetz, Kavitha Venkatesan, Jianjun Yu, Michael Jensen, Marion Wiesmann, Jens Wuerthner, Diana Graus-Porta. A gene signature composed of 13 p53 target genes predicts for response to NVP-CGM097, a novel p53-Mdm2 inhibitor, in cell lines and in human primary tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2909. doi:10.1158/1538-7445.AM2014-2909 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01760525&atom=%2Fcanres%2F74%2F19_Supplement%2F2909.atom

NVP-BKM120 is a pan class I PI3K inhibitor that has recently entered phase II clinical trials. The compound was shown to inhibit cell proliferation and survival of cancer models displaying PI3K pathway dependency, in a dose-dependent manner, and proportionally to the extent of pathway inhibition. To further characterize NVP-BKM120, we have investigated its mechanism of action across a broad range of relevant models and concentrations of the molecule and compared it to other PI3K inhibitors (e.g. GDC0941 and ZSTK474). The effects observed on phenotypical read-outs were similar for all compounds, when tested up to concentrations necessary to achieve near complete pathway inhibition (IC90 for Akt-S473P). More profound effects were however observed with NVP-BKM120, at higher concentrations (>2 micromolar), in PI3K-independent models, suggesting that at these dose levels, NVP-BKM120 might display inhibitory activities other than PI3K. In order to determine this potential off-target activity, a gene expression profiling study was performed in a PI3K insensitive model, comparing effects of GDC0941 and NVP-BKM120 at equipotent concentrations. Gene-Set Enrichment Analysis (GSEA) revealed that NVP-BKM120 at the highest dose only (3.6 micromolar, 2 fold above the S473-Akt IC90 of PI3K sensitive models), led to increased expression of genes involved in G2 and mitotic (M) phases. Subsequent FACS analysis showed that in contrast to the other pan-PI3K inhibitors, NVP-BKM120 was indeed able to induce a strong G2/M arrest in several PI3K non addicted cell lines when used at concentrations higher than 2 micromolar. DAPI and tubulin immuno-histochemistry studies showed that the NVP-BKM120 induced block was phenotypically similar to that of Nocodazole, suggesting effects on spindle dynamics in prometaphase. Indeed, in cellular or in in vitro purified systems, NVP-BKM120 greatly reduced microtubule polymerization.Based on analysis the antitumor activity observed in vivo in PI3K-dependent animal models, it appears that efficacy is solely due to pure PI3K inhibition, as these off-target activities are generally observed at concentrations (corrected for free fraction) that could not be achieved in animals. Based on modeling of human PK data, a similar conclusion can be reached for patients, as the exposure currently observed in plasma does predict sole coverage of PI3K inhibitory activities. 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 4466. doi:10.1158/1538-7445.AM2011-4466