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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.

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.

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

UNLABELLED\ud \ud The neurite outgrowth inhibitor, Nogo-A, has been shown to be overexpressed in skeletal muscle in amyotrophic lateral sclerosis (ALS); it is both a potential biomarker and therapeutic target. We performed a double-blind, two-part, dose-escalation study, in subjects with ALS, assessing safety, pharmacokinetics (PK) and functional effects of ozanezumab, a humanized monoclonal antibody against Nogo-A. In Part 1, 40 subjects were randomized (3∶1) to receive single dose intravenous ozanezumab (0.01, 0.1, 1, 5, or 15 mg/kg) or placebo. In Part 2, 36 subjects were randomized (3∶1) to receive two repeat doses of intravenous ozanezumab (0.5, 2.5, or 15 mg/kg) or placebo, approximately 4 weeks apart. The primary endpoints were safety and tolerability (adverse events [AEs], vital signs, electrocardiogram (ECG), and clinical laboratory tests). Secondary endpoints included PK, immunogenicity, functional endpoints (clinical and electrophysiological), and biomarker parameters. Overall, ozanezumab treatment (0.01-15 mg/kg) was well tolerated. The overall incidence of AEs in the repeat dose 2.5 mg/kg and 15 mg/kg ozanezumab groups was higher than in the repeat dose placebo group and repeat dose 0.5 mg/kg ozanezumab group. The majority were considered not related to study drug by the investigators. Six serious AEs were reported in three subjects receiving ozanezumab; none were considered related to study drug. No study drug-related patterns were identified for ECG, laboratory, or vital signs parameters. One subject (repeat dose 15 mg/kg ozanezumab) showed a weak, positive anti-ozanezumab-antibody result. PK results were generally consistent with monoclonal antibody treatments. No apparent treatment effects were observed for functional endpoints or muscle biomarkers. Immunohistochemical staining showed dose-dependent co-localization of ozanezumab with Nogo-A in skeletal muscle. In conclusion, single and repeat dose ozanezumab treatment was well tolerated and demonstrated co-localization at the site of action. These findings support future studies with ozanezumab in ALS.\ud \ud TRIAL REGISTRATION\ud \ud ClinicalTrials.gov NCT00875446 GSK-ClinicalStudyRegister.com GSK ID 111330.

BACKGROUND\ud \ud Amyotrophic lateral sclerosis (ALS) is a primary progressive neurodegenerative disease characterised by neuronal loss of lower motor neurons (in the spinal cord and brainstem) and/or upper motor neurons (in the motor cortex) and subsequent denervation atrophy of skeletal muscle.\ud \ud AIM\ud \ud A comprehensive examination of muscle pathology from a cohort of clinically confirmed ALS patients, including an investigation of inflammation, complement activation, and deposition of abnormal proteins in order to compare them with findings from an age-matched, control group.\ud \ud MATERIAL AND METHODS\ud \ud 31 muscle biopsies from clinically confirmed ALS patients and 20 normal controls underwent a comprehensive protocol of histochemical and immunohistochemical stains, including HLA-ABC, C5b-9, p62, and TDP-43.\ud \ud RESULTS\ud \ud Neurogenic changes were confirmed in 30/31 ALS cases. In one case, no neurogenic changes could be detected. Muscle fibre necrosis was seen in 5/31 cases and chronic mononuclear inflammatory cell infiltration in 5/31 (2 of them overlapped with those showing muscle necrosis). In four biopsies there was an increase in the proportion of cytochrome oxidase (COX) negative fibres (2-3%). p62 faintly stained cytoplasmic bodies in eight cases and none were immunoreactive to TDP-43.\ud \ud CONCLUSION\ud \ud This large series of muscle biopsies from patients with ALS demonstrates neurogenic atrophy is a nearly uniform finding and that mild mitochondrial abnormalities and low-grade inflammation can be seen and do not rule out the diagnosis of ALS. These findings could lend support to the notion that ALS is a complex and heterogeneous disorder.

Defining predictive biomarkers and deriving appropriate patient populations for treatment are key areas of interest in the clinical development of phosphatidyl-inositol-3-kinase (PI3K) inhibitors, but currently most clinical trials do not pre-select patients using defined genetic markers. Pre-clinical studies indicated previously that in vitro sensitivity to BYL719, a potent and selective small molecule inhibitor of the α-isoform of PI3K, was increased in cell lines derived from a subset of indications, such as HER2-positive or luminal breast cancer. Moreover, BYL719 sensitivity in vitro was positively correlated with PIK3CA mutation or amplification and HER2 amplification. PTEN or BRAF mutation, on the other hand, was associated with BYL719 insensitivity. Consequently, presence of an activating PIK3CA alteration in the tumor was a pre-requisite for patients to enroll into the BYL719 first-in-human (FIM) clinical trial. In this study, we applied massive parallel sequencing to tumor specimens from patients enrolled in the BYL719 FIM trial to comprehensively assess the genetic determinants of clinical sensitivity to BYL719. Paraffin-embedded formalin-fixed archival or pre-treatment tumor specimens were obtained from 44 patients with advanced solid tumors upon enrolment into the clinical trial. DNA was isolated and then assayed by massive parallel sequencing covering either a panel of 182 cancer-related genes at Foundation Medicine or the whole exome at the Broad Institute. Mutations were identified by focusing on genes with alterations in the COSMIC data base and excluding germline variants and were subsequently correlated with clinical benefit as defined by stable disease ≥ 4 months, tumor shrinkage ≥ 20% or objective tumor response to BYL719 using a two-tailed Fisher's exact test. Focusing on a panel of 51 genes with known relevance to PI3K signaling, we observed that all pre-treatment tumor samples harbored at least one genetic abnormality and that each of the genes was altered in one or several tumor samples. TP53 (59%), APC (25%), KRAS (23%) and PTEN (18%) were the most frequently altered in addition to PIK3CA. Alterations in TP53 and KRAS were identified as statistically significant negative predictors of BYL719 sensitivity (p < 0.05), while mutations in the APC and PTEN genes were associated with a trend towards lack of benefit from treatment with BYL719. Our results suggest that definition of the genetic biomarkers that govern sensitivity to treatment with BYL719 and–most likely–other PI3Kα inhibitors in the clinic is possible upon analysis of patient's tumors by massive parallel sequencing. We also demonstrate that it is feasible to routinely obtain this information in the clinical trial setting. This implies that exploration of the genetic determinants driving sensitivity or primary resistance to targeted agents in general can now be undertaken clinically. Citation Format: Jordi Rodon, Dejan Juric, Ana-Maria Gonzalez-Angulo, Johanna Bendell, Jordan Berlin, Douglas Bootle, Kathlene Gravelin, Alan Huang, Adnan Derti, Joseph Lehar, Jens Würthner, Markus Boehm, Eliezer van Allen, Nikhil Wagle, Levi A. Garraway, Roman Yelensky, Philip J. Stephens, Vincent A. Miller, Robert Schlegel, Cornelia Quadt, José Baselga. Towards defining the genetic framework for clinical response to treatment with BYL719, a PI3Kalpha-specific inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-65. doi:10.1158/1538-7445.AM2013-LB-65