Selective MDM2 Inhibitor Research Articles

Induction Of p53 Transcription and Apoptosis By XPO1 Inhibition In Mantle Cell Lymphoma

We investigated the pathophysiological significance of MDM2 and the nuclear exporter Exportin 1 (XPO1/CRM1) on p53 transcription and their therapeutic targeting in mantle cell lymphoma (MCL). MCL is currently an incurable B-cell non-Hodgkin's lymphoma. Although TP53 mutations occur rarely (i.e., in 15 to 20% of the cases) in MCL, wild-type p53 is frequently inactivated by gene amplification of BMI1, homozygous deletion of CDKN2A (INK4a/ARF, over-expression of regulatory proteins, or gene deletion. For example, tumor cells may sequester p53 in the cytoplasm via the overexpression of proteins like MDM2 and XPO1 thereby abrogating its transcriptional anti-tumor activity. The selective MDM2 inhibitor Nutlin-3a (Vassilev, 2004) has been reported to induce p53-mediated apoptosis in MCL cells, and recently it was shown that the potent, small molecule Selective Inhibitor of Nuclear Export (SINE) induces apoptosis in MCL cells (Zhang, 2013).mRNA expression levels in MCL patient samples were determined using Oncomine (Compendia Bioscience, Ann Arbor, MI). MDM2 mRNA expression levels were not significantly higher in the MCL samples (n = 8) compared to normal B-cells ( n = 5) (P = 0.27). The MDM2 levels were also not associated with overall disease survival (P = 0.12) at 5 years (n = 63). In contrast, gene expression analysis on the same dataset demonstrated increased XPO1 mRNA expression in the MCL cells versus normal B-cells (P< 0.001). Furthermore, this higher XPO1 expression was associated with poorer prognosis in MCL patients (i.e., median overall survival: 3.2 years in low expression XPO1 cases and 1.9 years in high expression CRM1 cases, P = 0.033). mRNA levels of 11 representative p53 target genes were determined in 2 MCL (i.e., Z-138 and JVM-2) cells and were compared to those of four acute leukemia (i.e., OCI-AML3, MOLM-13, REH and NALM-6) cells. Cells from all six cell lines express wild-type p53. The MDM2 inhibitor Nutlin-3a (5 µM) poorly induced p53 target genes in the Z-138 (1.9 ± 0.12-fold) and JVM-2 cells (2.1 ± 0.23-fold) compared to acute leukemia cells (13.9 ± 5.2-fold) (P< 0.001 in both MCL cells), despite their increased p53 protein levels after Nutlin-3a treatment. The data suggest that p53-mediated transcription may be impaired in MCL via a Nutlin-induced increase in cytoplasmic p53 levels. Next, we treated Z-138 with 2 or 4 mM Nutlin-3a or with 80 or 160 nM KPT-185 for 18 hours and determined mRNA changes in p53 targets. KPT-185 induced p53 target genes more potently than Nutlin-3a (i.e., 2- to 4-times). To investigate the significance of p53-mediated apoptosis, Z-138 and JVM-2 were transduced with lentivirus encoding either negative control shRNA (ShC) or p53-specific shRNA (Shp53) and stable shRNA-expressing cells were generated. The p53-specific shRNA reduced p53 basal levels by 80 to 90%, and p53 knockdown cells were ∼80% less sensitive to Nutlin-3a and ∼60% to KPT-185, indicating that MDM2 or XPO1 inhibition rely on p53 pathway to induce apoptosis. Apoptotic induction by Nutlin-3a and KPT-185 in primary MCL cells were determined in 3 samples with wild-type p53. Treatment-specific Annexin V induction percentage in 2.5 µM Nutlin-3a-treated cells was 40.7 ± 8.0%, while it was 64.5 ± 15.0% in 100 nM KPT-185 treated cells. Treatment of 1 µM KPT-185 led to more prominent apoptosis, as evidenced by 81.8 ± 7.7% annexin V staining.Our data suggest that p53 is a component of MDM2 or XPO1 inhibition-induced apoptosis, and p53 transcription is more potently activated by XPO1 inhibition rather than MDM2 inhibition. Based on these data, we propose that active modulation of p53 by SINE XPO1 antagonism and/or MDM2 inhibition transcription would be beneficial in some cancer types to enhance p53-based cancer therapy. Disclosures:Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Andreeff:Karyopharm Therapeutics Inc.: Research Funding.

Small Molecule Inhibitors of MDM2-p53 and MDMX-p53 Interactions as New Cancer Therapeutics

Inactivation of the function of tumor suppressor p53 is common in human cancers. In approximately half of human cancers, the tumor suppressor function of p53 is inactivated by deletion or mutation of TP53, the gene encoding p53 protein. In the remaining 50% of human cancers, p53 tumor suppressor function can be effectively inhibited by oncoprotein MDM2 or its homolog MDMX. Since inhibition of p53 by MDM2 or MDMX protein is mediated by their direct interaction with p53, small-molecule inhibitors designed to block the MDM2-p53 or MDMX-p53 protein-protein interaction (MDM2 or MDMX inhibitors) can activate p53 in tumor cells retaining wild-type p53. In the last few years, several classes of potent, selective, and efficacious small molecule MDM2 inhibitors have been designed and developed, and six such compounds are being evaluated in clinical trials as new anticancer drugs. Additionally, non-peptide, small-molecule MDMX inhibitors have been reported. We review herein the design and development of potent small-molecule MDM2 and MDMX inhibitors.

Structure-Based Design of Novel Inhibitors of the MDM2–p53 Interaction

Structure-based rational design led to the discovery of novel inhibitors of the MDM2-p53 protein-protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.

Blockade of Hsp90 by 17AAG antagonizes MDMX and synergizes with Nutlin to induce p53-mediated apoptosis in solid tumors.

Strategies to induce p53 activation in wtp53-retaining tumors carry high potential in cancer therapy. Nutlin, a potent highly selective MDM2 inhibitor, induces non-genotoxic p53 activation. Although Nutlin shows promise in promoting cell death in hematopoietic malignancies, a major roadblock is that most solid cancers do not undergo apoptosis but merely reversible growth arrest. p53 inhibition by unopposed MDMX is one major cause for apoptosis resistance to Nutlin. The Hsp90 chaperone is ubiquitously activated in cancer cells and supports oncogenic survival pathways, many of which antagonize p53. The Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) is known to induce p53-dependent apoptosis. We show here that in multiple difficult-to-kill solid tumor cells 17AAG modulates several critical components that synergize with Nutlin-activated p53 signaling to convert Nutlin's transient cytostatic response into a cytotoxic killing response in vitro and in xenografts. Combined with Nutlin, 17AAG destabilizes MDMX, reduces MDM2, induces PUMA and inhibits oncogenic survival pathways, such as PI3K/AKT, which counteract p53 signaling at multiple levels. Mechanistically, 17AAG interferes with the repressive MDMX–p53 axis by inducing robust MDMX degradation, thereby markedly increasing p53 transcription compared with Nutlin alone. To our knowledge Nutlin+17AAG represents the first effective pharmacologic knockdown of MDMX. Our study identifies 17AAG as a promising synthetic lethal partner for a more efficient Nutlin-based therapy.

Abstract 106: Metastatic outcome of early-stage estrogen receptor-positive (ER+) primary breast cancers predicted by differential expression of a subset of wild-type p53 target genes

Abstract The p53 tumor suppressor regulates a plethora of biological processes including cell cycle, apoptosis, DNA repair and cellular senescence. Gene expression signatures associated with loss-of-function p53 mutations have previously been linked to poor prognosis primary breast cancers, even among estrogen receptor (ER)-positive breast cancers the majority of which expresses wildtype (wt) p53. However, the proportion of DNA-bound (direct) or non-bound (indirect) p53 target genes within these gene expression signatures remains unknown. Therefore, we utilized chromatin immunopreciptiation-sequencing (ChIP-seq) (FactorPath, Genpathway) and expression microarrays (Affymetrix U133Av2) to characterize genome-wide direct and indirect p53 target genes associated with transcriptome changes induced in ER+ MCF7 cells after upregulating wt-p53 protein by the selective MDM2 inhibitor MI-63 (Ascenta Therapeutics). ChIP-seq and expression data were mapped together using gene symbols to yield 10653 unique genes for the differential binding and expression analysis. 2239 showed differential p53 binding (MACS; p&amp;lt;1e-10) mapping within +/- 10kb of their UCSC gene coordinates. Of the 1903 unique genes found differentially expressed (SAM; FDR p &amp;lt;0.005), 539 were shown by ChIP-seq to be direct p53-bound targets and 1364 were shown to be indirect p53 target genes. Gene Ontology (GO) analysis was performed to identify enriched functional categories among the direct and indirect p53 target genes, finding 62 GO biological processes significantly enriched among direct p53 target genes (FDR p&amp;lt;0.05) most of which were related to apoptosis, in contrast to the 147 GO biological processes enriched within the indirect p53 target genes including cell cycle, DNA repair, and telomere maintenance. The average expression of these direct and indirect p53 target genes were then computed in a pooled expression microarray dataset of 303 adjuvant naïve, node-negative ER+ breast cancer cases from two independent sources (GSE2034, GSE7390) annotated for distant metastasis-free survival (DMFS), Cox analysis revealed a significant association between overall p53-regulated gene expression (direct + indirect targets) and DMFS (p=0.001), but this prognostic association was significant only for the indirect p53 targets (p &amp;lt; 0.001; with p = 0.08 for direct p53 targets) These findings suggest that even among early-stage primary ER+ breast cancers in which p53 mutations are rare, differentially expressed p53 target genes are prognostic, with those not directly bound but nonetheless transcriptionally linked to critical wt-p53 functions (e.g. cell cycle control, DNA repair) most strongly associated with subsequent metastatic outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 106.

Identification and Characterization of the First Small Molecule Inhibitor of MDMX

The p53 pathway is disrupted in virtually every human tumor. In approximately 50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. To date, peptide inhibitors of MDMX have been developed, but no small molecule inhibitors have been reported. We have developed biochemical and cell-based assays for high throughput screening of chemical libraries to identify MDMX inhibitors and identified the first MDMX inhibitor SJ-172550. This compound binds reversibly to MDMX and effectively kills retinoblastoma cells in which the expression of MDMX is amplified. The effect of SJ-172550 is additive when combined with an MDM2 inhibitor. Results from a series of biochemical and structural modeling studies suggest that SJ-172550 binds the p53-binding pocket of MDMX, thereby displacing p53. This lead compound is a useful chemical scaffold for further optimization of MDMX inhibitors that may eventually be used to treat pediatric cancers and various adult tumors that overexpress MDMX or have similar genetic lesions. When combined with selective MDM2 inhibitors, SJ-172550 may also be useful for treating tumors that express wild-type p53.

The transcription-independent mitochondrial p53 program is a major contributor to Nutlin-induced apoptosis in tumor cells

Strategies to induce p53 activation in tumors that retain wild-type p53 are promising for cancer therapy. Nutlin is a potent and selective pharmacological MDM2 inhibitor that competitively binds to its p53-binding pocket, thereby leading to non-genotoxic p53 stabilization and activation of growth arrest and apoptosis pathways. Nutlin-induced apoptosis is thought to occur via p53’s transcriptional program. Here we report that the transcription-independent mitochondrial p53 program plays an important role in Nutlin-induced p53-mediated tumor cell death. Aside from nuclear stabilization, Nutlin causes cytoplasmic p53 accumulation and translocation to mitochondria. Monoubiquitinated p53, originating from a distinct cytoplasmic pool, is the preferred p53 species that translocates to mitochondria in response to stress. Nutlin does not interfere with MDM2’s ability to monoubiquitinate p53, due to the fact that MDM2-p53 complexes are only partially disrupted and that Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity. Nutlin-induced mitochondrial p53 translocation is rapid and associated with cytochrome C release that precedes induction of p53 target genes. Specific inhibition of mitochondrial p53 translocation by Pifithrin µ reduces the apoptotic Nutlin response by 2.5-fold, underlining the significance of p53’s mitochondrial program in Nutlin-induced apoptosis. Surprisingly, blocking the transcriptional arm of p53, either via α-Amanitin or the p53-specific transcriptional inhibitor Pifithrin α, not only fails to inhibit, but greatly potentiates Nutlin-induced apoptosis. In sum, the direct mitochondrial program is a major mechanism in Nutlin-induced p53-mediated apoptosis. Moreover, at least in some tumors the transcriptional p53 activities in net balance not only are dispensable for the apoptotic Nutlin response, but appear to actively block its therapeutic effect.