MDM2 Inhibitor Nutlin-3a Research Articles

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

P-TEFb promotes cell survival upon p53 activation by suppressing intrinsic apoptosis pathway.

Positive transcription elongation factor b (P-TEFb) is the crucial player in RNA polymerase II (Pol II) pause release that has emerged as a promising target in cancer. Because single-agent therapy may fail to deliver durable clinical response, targeting of P-TEFb shall benefit when deployed as a combination therapy. We screened a comprehensive oncology library and identified clinically relevant antimetabolites and Mouse double minute 2 homolog (MDM2) inhibitors as top compounds eliciting p53-dependent death of colorectal cancer cells in synergy with selective inhibitors of P-TEFb. While the targeting of P-TEFb augments apoptosis by anti-metabolite 5-fluorouracil, it switches the fate of cancer cells by the non-genotoxic MDM2 inhibitor Nutlin-3a from cell-cycle arrest to apoptosis. Mechanistically, the fate switching is enabled by the induction of p53-dependent pro-apoptotic genes and repression of P-TEFb-dependent pro-survival genes of the PI3K-AKT signaling cascade, which stimulates caspase 9 and intrinsic apoptosis pathway in BAX/BAK-dependent manner. Finally, combination treatments trigger apoptosis of cancer cell spheroids. Together, co-targeting of P-TEFb and suppressors of intrinsic apoptosis could become a viable strategy to eliminate cancer cells.

Epichaperome Inhibition Targets Kinase and TP53 Mutant Therapy Resistant AML

The BCL-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, improves outcomes for many patients with AML and is now considered standard of care for patients older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months; 2.5 months after relapse. Mutations of FLT3, PTPN11, and RAS, leading to constitutive activation of cell survival signaling, and mutations of TP53 have been identified as key determinants of low response rates, early relapse and death. Mutant TP53 cells are also characterized by activation of signaling kinases. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. Epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, epichaperome inhibition has the potential of eliminating resistant subclones before they become dominant and cause relapse. In contrast to other HSP90 purine analog inhibitors, PU-H71(zelavespib) is a competitive inhibitor specific for the conformationally changed ATP binding site of HSP90 when incorporated into epichaperomes. We here investigate the therapeutic potential of targeting epichaperomes with PU-H71 in TP53 and oncogenic kinase mutant AML. Western blot analysis showed increased HSP90 and several signaling proteins such as STAT3 and c-Myc in TP53-knockout and mutant Molm13 cells generated by CRISPR/cas-9, compared to isogeneic wild-type (WT) controls. Using fluorochrome-labelled PU-H71 and flow cytometry, we demonstrated the presence of epichaperome in AML and AML stem/progenitor cells with TP53 and/or RAS mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively killed AML cells, including those with acquired resistance to BH3 mimetics and those with various FLT3 mutants. PU-H71 targets AML cells and stem/progenitor cells with TP53 mutations, and prolonged survival of TP53-mutant AML xenograft mice with minimal effects on mouse bone marrow LSK cells and hematopoiesis. PU-H71 increased the expression of pro-apoptotic BIM and the efficacy of VEN in AML cells and AML stem/progenitor cells with TP53 and/or RAS mutations (combination index < 1). Importantly, in a 1,000: 1 mixture of TP53-WT and TP53-R248W mutant isogenic Molm13 cells, MDM2 inhibitor nutlin3a selectively killed TP53-WT but enriched TP53-mutant Molm13 cells, as expected, and VEN treatment also favored the outgrowth of TP53-mutant cells. However, PU-H71 effectively killed TP53-WT and mutant cells (Fig. 1): 10 million TP53-WT cells were reduced to 335 cells, and 10,000 TP53-mutant cells to 26. The activity of PU-H71 against both TP53-WT and mutant AML cells was further enhanced by VEN in vivo in a xenograft model of mixed (10:1) TP53-WT and mutant Molm13 cells. Our data support the concept that the epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring TP53 and/or oncogenic kinase mutations. Inhibition of epichaperomes by PU-H71 targets AML cells/stem/progenitor cells, enhances VEN activity, and prevents the outgrowth of nutlin- or VEN-resistant TP53-mutant AML cells. This concept has entered clinical evaluation in MPN-derived secondary AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance.

SummaryAcute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.

Effects of the MDM2 inhibitor Nutlin-3a on sensitivity of pancreatic cancer cells to berberine and modified berberines in the presence and absence of WT-TP53.

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). A key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, cancer progression and other growth regulatory processes. The mouse double minute 2 homolog (MDM2) gene product is a nuclear-localized E3 ubiquitin ligase and negatively regulates the TP53 protein which results in its proteasomal degradation. Various MDM2 inhibitors have been isolated and examined in clinical trials, especially in patients with hematological malignancies. Nutlin-3a is one of the first MDM2 inhibitors isolated. Berberine (BBR) is a natural product found in many fruits and berries and used in traditional medicine for centuries. It has many biological effects, and some are anti-proliferative in nature. BBR may activate the expression of TP53 and inhibit cell cycle progression as well as other events important in cell growth. To understand more about the potential of compounds like BBR and chemical modified BBRs (NAX compounds) to sensitize PDAC cells to MDM2 inhibitors, we introduced either WT-TP53 or the pLXSN empty vector control into two PDAC cell lines, one lacking expression of TP53 (PANC-28) and one with gain-of-function mutant TP53 on both alleles (MIA-PaCa-2). Our results indicate that nutlin-3a was able to increase the sensitivity to BBR and certain NAX compounds. The effects of nutlin-3a were usually more substantial in those cells containing an introduced WT TP53 gene. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function by stabilization of the TP53 protein.

Abstract 1027: ProLiFiler and Cancer Data Miner, combined platforms for preclinical investigation to scrutinize impact of inhibitors on the KRAS, PI3K and MDM2 signaling pathways

Abstract Testing novel anti-cancer agents across large panel of tumor models covering genetic diversity of cancers is increasingly considered as a cornerstone of preclinical development. For this purpose, Reaction Biology developed “ProLiFiler” a standard panel of 140 cell lines (CLs) covering most common cancer types to evaluate anti-proliferative activity of novel drugs. Partnering has been made with 4HF Biotec and their in-silico platform, named “Cancer Data Miner”, to investigate and to understand molecular basis of drug sensitivity. Here we report the use of our platforms to realize integrative pharmacogenomic studies for three recent small molecules targeting major altered pathways in cancers. It includes SOS1::KRAS interaction inhibitor BI-3406, MDM2 inhibitor Nutlin-3a, and PI3K inhibitor Taselisib. Main goal of the study is to provide meaningful information for these three drugs regarding their efficacy and potency, the validation of their mechanism of actions (MOA), the suitable clinical indications, possible drug combinations and the predictive biomarkers of sensitivity or resistance. The three compounds are tested for anti-proliferative activity in vitro in a 2D monolayer assay using the “ProLiFiler”CLs panel. For data analytics, the resulting in vitro data are loaded on the “Cancer Data Miner” platform and connected to CL annotations including whole exome mutations, chromosomal aberrations, gene expression profiles or drug sensitivity profiles. The drug response profiles will be reported for the three compounds individually and compare between them, showing respective efficacy, potency, and CL/cancer entity selectivity. Using the MOA Finder tool, we will correlate BI-3406, Nutlin-3a, and Taselisib individual IC50 profiles to those of more than 800 compounds with known MOA that are integrated on the platform. The analyses will show the drugs most closely related to the 3 compounds and that are expected to have similar MOA. With the biomarker discovery tools, we will run high throughput statistical analyses to reveal whole exome mutations, copy number variations and expression significantly associated with drug sensitivity/resistance. For interpretation, pathway and enrichment analysis will be presented. A focus will be made on key alterations like KRAS and MAPK related genes, TP53-MDM2 and PIK3CA-PTEN to evaluate their predictivity. The work will be also completed by functional analysis, for instance, by assessing the effect of BI-3406 on ERK-MEK activation, and the impact of MDM2 inhibition on apoptotic markers. The present work will show the whole panel of analyses proposed by 140 CL-ProLiFiler and Cancer Data Miner complementary platforms, allowing to acquire key information at an early stage of drug development and helping to setup next steps such as selection of models for in vivo testing. Citation Format: Vincent Vuaroqueaux, Daniel Feger, Hoor Al-Hasani, Oliver Siedentopf, Anne-Lise Peille, Sarah Ulrich, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. ProLiFiler and Cancer Data Miner, combined platforms for preclinical investigation to scrutinize impact of inhibitors on the KRAS, PI3K and MDM2 signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1027.

Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a.

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.

A novel p53 regulator, C16ORF72/TAPR1, buffers against telomerase inhibition.

Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase (TERT), contributes to age‐associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome‐wide CRISPR screen to identify gene deletions that sensitized p53‐positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1 (TAPR1), that exhibited a synthetic‐sick relationship with TERT loss. A subsequent genome‐wide CRISPR screen in TAPR1‐disrupted cells reciprocally identified TERT as a sensitizing gene deletion. Cells lacking TAPR1 or TERT possessed elevated p53 levels and transcriptional signatures consistent with p53 upregulation. The elevated p53 response in TERT‐ or TAPR1‐deficient cells was exacerbated by treatment with the MDM2 inhibitor and p53 stabilizer nutlin‐3a and coincided with a further reduction in cell fitness. Importantly, the sensitivity to treatment with nutlin‐3a in TERT‐ or TAPR1‐deficient cells was rescued by loss of p53. These data suggest that TAPR1 buffers against the deleterious consequences of telomere erosion or DNA damage by constraining p53. These findings identify C16ORF72/TAPR1 as new regulator at the nexus of telomere integrity and p53 regulation.

MiR-10a as a therapeutic target and predictive biomarker for MDM2 inhibition in acute myeloid leukemia

Pharmacological inhibition of MDM2/4, which activates the critical tumor suppressor p53, has been gaining increasing interest as a strategy for the treatment of acute myeloid leukemia (AML). While clinical trials of MDM2 inhibitors have shown promise, responses have been confined to largely molecularly undefined patients, indicating that new biomarkers and optimized treatment strategies are needed. We previously reported that the microRNA miR-10a is strongly overexpressed in some AML, and demonstrate here that it modulates several key members of the p53/Rb network, including p53 regulator MDM4, Rb regulator RB1CC1, p21 regulator TFAP2C, and p53 itself. The expression of both miR-10a and its downstream targets were strongly predictive of MDM2 inhibitor sensitivity in cell lines, primary AML specimens, and correlated to response in patients treated with both MDM2 inhibitors and cytarabine. Furthermore, miR-10a inhibition induced synergy between MDM2 inhibitor Nutlin-3a and cytarabine in both in vitro and in vivo AML models. Mechanistically this synergism primarily occurs via the p53-mediated activation of cytotoxic apoptosis at the expense of cytoprotective autophagy. Together these findings demonstrate that miR-10a may be useful as both a biomarker to identify patients most likely to respond to cytarabine+MDM2 inhibition and also a druggable target to increase their efficacy.

Abstract 2409: FLIP(L) determines colon cancer cell fate following p53 activation

Abstract Background: How p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Understanding the mechanisms governing the switch from p53-induced cell-fates is important for optimising efficacy of p53-activating therapies, such as DNA damaging chemotherapy and radiotherapy. HDAC inhibitors (HDACi) have potential to enhance p53 induced cell death through enhancing p53 activation and altering regulation of other cell death regulatory proteins. Methods: We utilised a panel of matched p53 wild-type and deficient colorectal cancer cell line models to the potential for HDACi to augment cell death induced by direct and indirect p53 activating agents. A number of molecular (Western blot, RT-PCR), phenotypic (cell death) and functional genomic (RNA-seq, CRISPR, ChIP-seq) analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results: Here we report that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a ligand-independent TRAIL-R2/caspase-8 complex, which is distinct from the ligand-dependent DISC. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Conclusion: Acute p53-mediated transcriptional upregulation of FLIP(L) plays an unexpected nodal role in determining cell fate following p53 activation. This is mediated through two previously undescribed mechanisms, preventing apoptosis by a ligand-independent TRAIL-R2 complex and by suppressing expression of pro-apoptotic PUMA. Which, importantly imposes a critical dependence on FLIP(L) which can be overcome through combinations with class-I HDAC inhibitors such as Entinostat. Citation Format: Andrea Lees, Alexander J. McIntyre J. McIntyre, Nyree T. Crawford, Fiammetta Falcone, Chris McCann, Gerard P. Quinn, Jamie Z. Roberts, Thomas Sessler, Peter F. Gallagher, Gemma M. Gregg, Katherine McAllister, Kirsty M. McLaughlin, Wendy L. Allen, Caitriona Holohan, Laurence J. Egan, Aideen E. Ryan, Melissa Labonte-Wilson, Phillip D. Dunne, Mark Wappett, Vicky M. Coye, Patrick G. Johnston, Emma M. Kerr, Daniel B. Longley, Simon S. McDade. FLIP(L) determines colon cancer cell fate following p53 activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2409.

Targeting p63 Upregulation Abrogates Resistance to MAPK Inhibitors in Melanoma.

Targeting the MAPK pathway by combined inhibition of BRAF and MEK has increased overall survival in advanced BRAF-mutant melanoma in both therapeutic and adjuvant clinical settings. However, a significant proportion of tumors develop acquired resistance, leading to treatment failure. We have previously shown p63 to be an important inhibitor of p53-induced apoptosis in melanoma following genotoxic drug exposure. Here, we investigated the role of p63 in acquired resistance to MAPK inhibition and show that p63 isoforms are upregulated in melanoma cell lines chronically exposed to BRAF and MEK inhibition, with consequent increased resistance to apoptosis. This p63 upregulation was the result of its reduced degradation by the E3 ubiquitin ligase FBXW7. FBXW7 was itself regulated by MDM2, and in therapy-resistant melanoma cell lines, nuclear accumulation of MDM2 caused downregulation of FBXW7 and consequent upregulation of p63. Consistent with this, both FBXW7-inactivating mutations and MDM2 upregulation were found in melanoma clinical samples. Treatment of MAPK inhibitor-resistant melanoma cells with MDM2 inhibitor Nutlin-3A restored FBXW7 expression and p63 degradation in a dose-dependent manner and sensitized these cells to apoptosis. Collectively, these data provide a compelling rationale for future investigation of Nutlin-3A as an approach to abrogate acquired resistance of melanoma to MAPK inhibitor targeted therapy. SIGNIFICANCE: Upregulation of p63, an unreported mechanism of MAPK inhibitor resistance in melanoma, can be abrogated by treatment with the MDM2 inhibitor Nutlin-3A, which may serve as a strategy to overcome resistance.

Acylation of the antimicrobial peptide CAMEL for cancer gene therapy

Obtaining ideal gene delivery vectors is still a major goal in cancer gene therapy. CAMEL, a short hybrid antimicrobial peptide, can kill cancer cells by membrane lysis. In this study, we constructed a series of non-viral vectors by attaching fatty acids with different chain lengths to the N-terminus of CAMEL. Our results showed that the cellular uptake and transfection efficiency of acyl-CAMEL started to significantly increase from a chain length of 12 carbons. C18-CAMEL was screened for gene delivery because it had the highest transfection efficiency. Surprisingly, C18-CAMEL/plasmid complexes displayed strong endosomal escape activity after entering cells via endocytosis. Importantly, C18-CAMEL could deliver p53 plasmids to cancer cells and significantly inhibited cell proliferation by the expression of p53. In addition, the C18-CAMEL/p53 plasmid complexes and the MDM2 inhibitor nutlin-3a showed significantly synergistic anticancer activity against MCF-7 cells expressing wild-type p53. Conclusively, our study demonstrated that conjugation of stearic acid to antimicrobial peptides is a simple and successful approach for constructing efficient and economical non-viral vectors for cancer gene therapy.

Abstract 2964: Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a

Abstract Introduction: PPM1D (wild-type p53-inducible protein phosphatase WIP1) is a member of the PP2C family serine/threonine phosphatase involved in negative regulation of cell stress response pathways, leading to suppression of p53 after stress. To restore p53 function through MDM2 inhibition (Nutlin-3a) is a promising approach in AML. Promising results of the combination of MDM2 inhibitors and WIP1 inhibitor (WIP1i), obtained in many preclinical studies of solid tumors, paved the way for their application in AML. We investigated whether the inhibition of WIP1 (GSK2830371) could sensitize AML cell lines and primary cells to Nutlin-3a (Nut-3a) in order to obtain a novel therapeutic strategy for AML patients, based on restoration of p53 activity. Methods: In vitro viability (by WST-1 reagent) and Annexin-V/PI apoptosis assay were performed on a panel of TP53-wt AML cells (MOLM-13, MV-4-11 and OCI-AML3), on TP53-mutated NOMO-1 AML cells and on primary AML samples. Gene expression profile (GEP) and Western Blot (WB) analyses were performed on MV-4-11 and NOMO-1 after 16h. Results: Combined inhibition of increasing dosage of Nut-3a (0.5 to 5 uM) and WIP1i (5 to 20 uM) synergistically reduces TP53-wt AML cells viability, while NOMO-1 resulted to be insensitive to the combination. The combination index analyses showed a synergistically effect of the combination of both compounds on TP53-wt AML cells. Annexin V/PI staining showed that WIP1i sensitizes TP53-wt AML cell lines and primary samples to Nut-3a-induced apoptosis, when compared with single treatment. No effect was seen in NOMO-1. GEP demonstrated that MV-4-11 cells exhibits a major response to drug combination with an upregulation of cell cycle control genes, a downregulation of DNA repair machinery genes, upregulation of MDM2 and of TP53-downstream genes (eg.CDKN1A), confirming the activation of p53 pathway. NOMO-1 cells showed upregulation of resiliency-mechanism and confirmed insensitivity to both treatment. WB analysis confirmed GEP data showing an increased expression of p53 and p21 in wt-TP53 cell line after 16h of combined-treatment. Conclusions: We identified a novel synergistic drug combination between Nut-3a and WIP1i that induces apoptosis in AML cell lines and primary samples. GEP and WB of TP53-wt MV-4-11 and TP53-mutated NOMO-1 cells showed mechanisms underlying drug sensitivity and resistance giving novel insights on potential markers of response and novel drugable targets. In vivo studies are needed to confirm these preclinical data. Supported by: AIRC, FP7-NGS-PTL, Fondazione del Monte, HARMONY. Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giovanni Marconi, Martina Pazzaglia, Matteo Bocconcelli, Antonella Padella, Simona Soverini, Ilaria Iacobucci, Cristina Papayannidis, Anna Ferrari, Maria Teresa Bochicchio, Enrica Imbrogno, Michele Cavo, Andrea Ghelli Luserna di Rora, Giorgia Simonetti, Giovanni Martinelli. Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2964.

Comparison of genotoxic versus nongenotoxic stabilization of p53 provides insight into parallel stress-responsive transcriptional networks

ABSTRACTThe tumor suppressor protein p53 is activated in response to diverse intrinsic and extrinsic cellular stresses and controls a broad cell-protective gene network. Whether p53:DNA binding and subsequent transcriptional activation differs downstream of these diverse intrinsic and extrinsic activators is controversial. Using primary human fibroblasts, we assessed the genome-wide profile of p53 binding, chromatin structure, and transcriptional dynamics after either genotoxic or nongenotoxic activation of p53. Activation of p53 by treatment with either etoposide or the small-molecule MDM2 inhibitor nutlin 3A yields strikingly similar genome-wide binding of p53 and concomitant changes to local chromatin modifications and structure. DNA damage, but not p53 activation per se, leads to increased expression of genes in an inflammatory cytokine pathway. The NF-κB pathway inhibitor Bay 11-7082 abrogates etoposide-mediated activation of the inflammation gene signature but does not affect expression of canonical p53 target genes. Our data demonstrate that differential activation of p53 within the same cell type leads to highly similar genome-wide binding, chromatin dynamics, and gene expression dynamics and that DNA damage-mediated signaling through NF-κB likely controls the observed pro-inflammatory cytokine gene expression pattern.

Effects of the MDM-2 inhibitor Nutlin-3a on PDAC cells containing and lacking WT-TP53 on sensitivity to chemotherapy, signal transduction inhibitors and nutraceuticals.

Mutations at the TP53 gene are readily detected (approximately 50-75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT-TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT-TP53 activity can have in PDAC cells which normally lack WT-TP53, as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents.

Long Non-Coding RNA NEAT1 Is Upregulated By Heat Shock Factor 1 (HSF1) and Associated with Multiple Myeloma Progression

Background and Aims: Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed but not translated non-coding RNAs capable of influencing diverse cellular processes, such as proliferation, apoptosis, and cellular damage response. Long non-coding RNA (lnc RNA), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNA are deregulated in diverse human cancers and associated with disease progression, however little is available in multiple myeloma (MM). We have previously shown that lnc RNA MALAT1 was a stress response gene associated with MM progression. We found that lnc RNA NEAT1 is also highly expressed in MM cells by transcriptome analysis with next generation sequencer (NGS). NEAT1 is recently revealed to play an important role on DNA damage response (DDR) as downstream of p53, and thereby involves in carcinogenesis. However its exact role in cancers is still in controversy. In this study, we tried to elucidate role and regulation mechanism of NEAT1 during MM progression.Materials and Methods: Total 119 MM, 47 MGUS patients and 15 controls and 9 MM cell lines are subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. NEAT1 and its longer isoform NEAT1_2 RNA expressions were determined by RQ-PCR. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. The expression levels were normalized with ACTB and calculated with delta Ct value. Whole transcriptome analysis was performed in part of the samples by using Illumina Next Seq 500. MM cell lines transfected with tet-on p53 overexpression vector or tet-on sh-RNA HSF1 were used. RNase H-activating LNA™ GapmeR antisense oligonucleotides were used to knockdown lnc RNA in vitro.Results: The expression level of NEAT1 was significantly higher in MM (median 0.97) than MGUS (median 0.31) (p<0.0001). NEAT1 level did not differ in between control (median 0.38) and MGUS (p=0.97). Although the median level was not statistically different (0.046 in MM; 0.031 in MGUS; 0.127 in control), substantial number of MM cases showed very high level of NEAT1_2. In MM samples, both NEAT1 and NEAT1_2 expression did not differ according to ISS (p=0.52, p=0.29) and cytogenetic risk group (p=0.49, p=0.203). NEAT1 and MALAT1 expression was positively correlated (r=0.632, p<0.0001 in all samples, r=0.62, p<0.0001 in MM only). NEAT1_2 was also positively correlated with MALAT1 expression (r=0.49, p<0.0001 in MM), and NEAT1 (r=0.35, p<0.0001 in MM). NEAT1 expression level and RNA structure were confirmed by transcriptome analysis with NGS. Since p53 promotes NEAT1/NEAT1_2 expression, we checked correlation in between these two genes expression levels. NEAT1 expression were positively correlated with both p53 and p21 (r=0.30, p<0.0001, r=0.41, p<0.0001). Positive correlations were also found in between NEAT1 and HSP90s (r=0.29, p=0.029 with HSP90AA1, r=0.29, p=0.029 with HSP90AB1, r=0.411, p=0.0018 with HSP90B1). NEAT1 was upregulated by MDM2 inhibitor nutlin3A in p53 wild type cell lines and by tet-on p53 overexpression in p53 null KMS11. Interestingly bortezomib and doxorubicin significantly increased NEAT1 and NEAT1_2 by 5-10 folds in MM cell lines even in p53 null KMS11. HSP90 inhibitors did not affect NEAT1/NEAT1_2 expression, but inhibition of HSF1, which is upstream transcription factor of HSP90, by either HSF1 inhibitor KNK437 or tet-on sh-HSF1 attenuated NEAT1/NEAT1_2 expression induced by bortezomib. NEAT1 knockdown by GapmeR did not affect cell growth. Overall survival and progression free survival of the newly diagnosed MM patients did not differ in between high and low NEAT1/NEAT1_2 expression.Conclusion: Our results revealed that NEAT1/NEAT1_2 are regulated by heat shock pathway in addition to p53 pathway. Positive correlations of NEAT1 expression level with HSP90s level and existence of heat shock element in NEAT1 promoter region support this model. Considering the role of NEAT1/NEAT1_2 in DDR, our result suggests that this lncRNA may involve MM progression via damage response. Further studies elucidating roles of NEAT1 and other lncRNAs in MM contributes to development of novel therapy as well as to understand MM pathogenesis. DisclosuresTsukamoto:Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.

TP73 Isoforms (TAp73 and ΔNp73) Are Overexpressed in Acute Myeloid Leukemias and Potential Therapeutic Targets to Enhance Anti-Leukemia Activities of Bcl-2 and MDM2 Inhibitors

BackgroundTP73 is one of the TP53 family transcription factors and generates two isoforms, the transactivation p73 (TAp73) and the N-terminally truncated ΔNp73. TAp73 shares a homologous N-terminal activation domain with p53 and has similar pro-apoptotic function to p53. ΔNp73 lacks an activation domain and has activities distinct from TAp73. ΔNp73 has a dominant negative effect on the DNA binding of TAp73 and more importantly, of p53, since the DNA binding domain is homologous with that of TAp73 and highly similar to that of p53. In acute myeloid leukemias (AML), TP73 has been reported to be expressed except in acute promyelocytic leukemias. However, the association of TP73 isoforms with clinical and genetic characteristics and the regulation of the isoforms in AML have not been explored.Results We determined copy numbers of ΔNp73 and TAp73 mRNA levels in 78 AML samples including 31 de novo AML using droplet digital PCR (ddPCR), which allows to determine the absolute quantity of the isoforms expressed, and investigated their clinical and biological relevance. ΔNp73 and TAp73 expression was detected in 93.6% and 98.7% of AML samples at variable levels (mean ± SEM, 10.6 ± 5.0, and 106.6 ± 33.7 copies/µL, for ΔNp73 and TAp73, respectively). ΔNp73 and TAp73 mRNA levels were highly correlated (R2 = 0.72, P < 0.0001). Normal peripheral blood mononuclear cells and CD34+ hematopoietic cells showed little or no ΔNp73 and TAp73 expression (0.09 ± 0.09 and 0.42 ± 0.35 copies/µL, respectively), demonstrating that expression of ΔNp73 and TAp73 is 100 - 1,000 fold higher in AML as compared to normal hematopoietic cells. These data collectively suggests that transcriptional systems of both isoforms in AML cells are abnormally activated. Disease status (de novo or relapsed/refractory) and cytogenetic abnormalities did not correlate with ΔNp73 and TAp73 levels. However, AML with IDH1/2 mutations had 8.5-fold lower ΔNp73 expression than those with wild-type IDH1/2 (1.8 ± 0.8 vs 15.4 ± 7.7 copies/µL, P = 0.0069), with a similar trend for TAp73 (49.0 ± 20.3 vs 138.6 ± 51.4 copies/µL, P = 0.056). For de novo AML samples, those with DNMT3a and NRAS mutations had significantly higher ΔNp73, but not TAp73, than those without these mutations (21.6 ± 18.2 vs 2.5 ± 1.2 copies/µL, P = 0.017 and 5.6 ± 2.5 vs 9.7 ± 8.0 copies/µL, P = 0.047, respectively). These findings suggest that ΔNp73 and TAp73 can be differentially regulated in AML based on mutation status. To further explore the regulation of TP73 isoforms, we used MDM2 inhibitor Nutlin-3a to induce p53 which is a transcriptional inducer of ΔNp73. Indeed, MDM2 inhibition increased p73 protein levels, and knockdown of both TAp73 and ΔNp73 in AML cells enhanced apoptosis induction by Nutlin-3a (specific annexin V induction by 5 uM Nutlin-3a, 21.9 ± 1.3% vs 61.3 ± 5.2%, P = 0.0084 in OCI-AML3 cells with vector control vs Shp73, respectively), possibly due to the silencing of ΔNp73. AML cells with IDH1/2 mutations are more dependent on Bcl-2 than those without (Chan, Nat Med 2015). Intriguingly, (R)-2HG, the oncometabolite of mutant IDH1/2, reduced both TAp73 and ΔNp73 in AML cells and increased susceptibility to the Bcl-2 inhibitor ABT-199. These results imply a potential mechanism that regulates p73 isoforms by histone methylation or other epigenetic modifications in AML.Conclusion Absolute quantitation of TP73 isoforms by ddPCR revealed high expression in AML cells compared to normal hematopoietic cells. The repressed expression of TP73 isoforms in AML cells with IDH1/2 mutations or by the oncometabolite (R)-2HG suggests that epigenetic modifications through (R)-2HG can regulate TP73 transcription and enhance the anti-leukemia effect by Bcl-2 inhibition. Finally, downregulation of TP73 isoforms enhances the efficacy of MDM2 inhibitor in AML, suggesting a potential therapeutic strategy to enhance MDM2 inhibitor-mediated p53 activation. DisclosuresAndreeff:Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Astra Zeneca: Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; SentiBio: Equity Ownership; Reata: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding.

GPRC5A deficiency leads to dysregulated MDM2 via activated EGFR signaling for lung tumor development.

GPRC5A, a retinoic acid induced gene, is preferentially expressed in lung tissue. Gprc5a gene deletion leads to spontaneous lung tumor development. However, the mechanism of Gprc5a-mediated lung tumor suppression is not fully understood. Here we showed that MDM2, a p53-negative regulator, was dysregulated in Gprc5a-knockout (ko) mouse tracheal epithelial cells (KO-MTEC) compared to wild type ones. Targeting MDM2 in 1601-a Gprc5a-ko mouse derived lung tumor cell line-and A549-human lung cancer cells, by MDM2 inhibitor Nutlin-3a or small hairpin RNA (sh-RNA)-restored p53 signaling pathway, reduced cancer stem cell markers, and inhibited tumorigenicity. This suggests that dysregulated MDM2 pathway is essential for the oncogenic activities of these cells. MDM2 was found to be stabilized mainly by activated EGFR signaling as targeting EGFR by Erlotinib or sh-RNA repressed MDM2 in a transcription-independent manner. Importantly, overexpression of MDM2 and reduced GPRC5A expression at both protein and mRNA levels were frequently found in clinical human lung cancer tissues. Taken together, GPRC5A deficiency contributes to dysregulated MDM2 via activated EGFR signaling, which promotes lung tumor development.

76P - Targeting p63 upregulation may prevent the development of MAPK inhibitor resistance in melanoma

Background: Melanoma progression is often characterized by mutations in the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. Understanding regulation of these pathways has led to the development of novel targeted therapies which show high response rates. However, many patients relapse with ensuing resistant disease. P63, a p53 homologue, carries a poorer prognosis when overexpressed. In keratinocytes, degradation could be regulated by two ubiquitin ligases, MDM2 and FBXW7. This project explored the expression of p63, MDM2 and FBXW7 in MAPK-inhibitor (MAPKi) sensitive and resistant melanoma cell lines. Methods: Morphology change associated with the development of resistance was assessed by actin immunofluorescence experiments. Western blot and qRT-PCR enabled analysis of protein and RNA expression of p63, FBXW7 and MDM2, complemented by immunofluorescent staining and confocal microscopy data. Finally, a novel technique of flow cytometry enabled analysis of cell death in MAPKi-resistant melanoma cells treated with Nutlin-3A. Results: MAPKi resistance in melanoma cells is associated with increased p63 expression. Resistance is furthermore associated with reduced FBXW7 expression and enrichment of nuclear MDM2, suggesting a potential nuclear interaction between MDM2 and FBXW7. The resultant inactivation of FBXW7 explains the increased p63 expression upon MAPKi- resistance. Furthermore, by treating MAPKi-resistant cells with the MDM2-inhibitor Nutlin-3A, we were able to restore FBXW7 expression and promote p63-degradation, with resulting increased melanoma cell death. Conclusions: This project identifies FBXW7 and MDM2 as regulators of p63 expression in MAPKi-resistant melanoma and proposes a possible role for Nutlin-3A in treating advanced MAPKi-resistant melanoma. Legal entity responsible for the study: Queen Mary University of London (Blizzard Institute) - Cutaneous Research. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Abstract 982: Pharmacological DNA-PK inhibition induces ATM/p53 dependent premature senescence with immunomodulatory phenotype in irradiated cancer cells

Abstract A new generation, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks one of the two major pathways for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We show that by inhibiting DNA-PK catalytic activity in the presence of DNA DSBs, M3814 simultaneously suppresses DNA repair and a negative regulatory signal to ATM, leading to enhanced activation of the ATM dependent signaling, including CHK2 and p53-dependent cell cycle arrest. Combination treatment of proliferating p53 wild-type cancer cells (A549, A375, H460) with a single dose of ionizing radiation (2-5Gy) and sustained exposure to M3814 induced a complete G1/S and G2/M cell cycle block. Within 4-7 days of treatment cells acquired a typical senescence phenotype with large/flat morphology and β-Gal staining. Live cell imaging and BrdU labeling in A549 cells demonstrated that this phenotype is not reversible following M3814 removal, in contrast to a fully reversible senescence-like phenotype caused by selective p53 activation by MDM2 inhibitor Nutlin-3a. Isogenic p53-null A549 cells lost the ability to fully arrest their cell cycle, confirming the role of p53 in senescence induction. Analysis of mRNA from IR/M3814 induced senescent A549 and A375 cells by the Nanostring PanCancer Immune profiling panel revealed activation of a large group of genes from several immune response pathways, including interferon, cytokine/chemokine, and complement. Eighteen genes were commonly upregulated &amp;gt;3-150 fold compared to controls. These substantial changes in gene expression were built gradually and correlated with the development of senescence phenotype. Several proteins from the induced subset were measured in the cell media (MesoScale Discovery) and confirmed that they are secreted by senescent cells in the absence of M3814. Culture media from M3814-induced senescent cells showed increased immunomodulatory effect on human PBMC-derived immune cells, suggesting increased probability for clearance of IR+M3814 induced senescent cancer cells by the immune system. Citation Format: Yige Guo, Qing Sun, Xiaohong Liu, Janusz Puc, Frank Czauderna, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. Pharmacological DNA-PK inhibition induces ATM/p53 dependent premature senescence with immunomodulatory phenotype in irradiated cancer cells [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 982.