TP53 Wild-type Cell Lines Research Articles

Combination of Decitabine and Etoposide Is Highly Effective in Treating p53-Mutated MDS/AML Via Activating Notch Signaling

The elderly MDS/AML patients, especially those with TP53 mutations, remain very difficult to treat and resistant to chemotherapy. A total of 105 elderly (age above 60 years) high risk MDS (n = 84, IPSS-R score > 4.5) or AML-MRC (n = 21) patients' records were collected from three medical centers across China and retrospectively analyzed. Decitabine and low dose etoposide was found to be superior to standard decitabine alone in MDS/AML patients with higher ORR (70.0% vs 52.7%) after two cycles of treatment, the longer median durations of ORR (24.0 months vs 12 months) and CR (38.0 months vs 24.0 months), and longer event-free survival (13 months vs 9 months, p = 0.023), without significant improvement in overall survival (20 months vs 15 months, p = 0.059). Other than the TP53 mutation, no other gene mutation (including NRAS, DNMT3A, TET2, EZH2, ASXL1, RUNX1) in the D+E group could reliably predict OS and EFS when compared to wild type phenotype. Patients with TP53 mutations showed higher response rate after two cycles of D+E therapy and had prolonged overall survival (Mutation vs WT: 31 months vs 9 months, p=0.012) and event-free survival (Mutation vs WT: 24 months vs 5 months, p = 0.022) compared with p53 wild type patients in D+E group from multivariate survival analyses. P53 mutated patients receiving D+E treatment also had significantly improved OS (31 months vs 12 months, p = 0.00) and EFS (24 months vs 8 months, p = 0.00) compared to patients receiving decitabine treatment. Moreover, we found decitabine and etoposide greatly reduced TP53 mutant AML tumor burden in the NSG mice xenografts (tumor burden: 92% reduction in p53-null xenograft mice, 65% reduction in p53-wild-type xenograft mice on day 20 after leukemia cells administration, p < 0.0001). It was found that TP53 mutant cells and p53 gene-editing MOLM13 cells showed terminal neutrophil differentiation in the combo of decitabine and etoposide, demonstrating the expected changes in cell-surface CD11b expression and neutrophil functional assays such as phagocytosis. Combo of decitabine and etoposide showed much higher percentage of differentiation marker expression compared to single drug decitabine or etoposide treatment. However, neither the p53 gene-edited THP-1 cells nor the TP53 wild type cell lines displayed markers of terminal differentiation, such as elevated CD11b expression or phagocytosis. Furthermore, the percentage of CD11b-positive cells increased significantly in p53-mutated primary cells while insignificantly in TP53 wild type cells. Decitabine and etoposide induced significant differentiation thus killing p53 mutated myeloid tumor cells and primary patients' cells in vitro. We also uncovered the underlying mechanism that why D+E could induce significant differentiation in TP53 Mutant while not in TP53 wild type myeloid tumors. Notch1 signaling pathway was activated after D+E treatment in TP53 Mutant myeloid tumors, which was consistently correlated with drug-induced myeloid differentiation. Notch1 knockout or Notch inhibitor incubation abrogated drug induced differentiation in TP53 Mutant myeloid tumors. The activation of the Notch1 signaling pathway has been observed to induce disease regression and decrease leukemia stem cells in AML mice as well as significant apoptosis in the AML patient sample. We also found D+E could upregulate transcription factor transcripts such as ETS1, ETS2, which could bind specifically to PSEN1 (PS1) promoter and activate its transcription directly. PS1 represent catalytic subunit of γ-secretase complexes and is responsible for Notch recognition. While in TP53 wild type AML cells, p53 might interact with p300. p300 acts as a coactivator of Ets1 and Ets2, competition for binding to p300 with p53 may confront p300 recruitment and repress ETS transcription activation directly, which might exert its effect on PS1 repression, thus inhibiting γ-secretase complex activity. In short, integrated p53 could repress PSEN1 transcription thus interfering gamma secretase complex activity, Notch recognition and cleavage as well as abrogating drug induced Notch activation and myeloid differentiation, which could be achieved in p53 mutated state. Combination of decitabine and etoposide regimen is also relatively safe and tolerable in elderly patients, which may open up a new path of therapy in the p53 mutated AML/MDS.

Arsenic Trioxide Combine with G-CSF Triggers Distinct TP53 Mutations Acute Myeloid Leukemia Cells Ferroptosis through TP53-SLC7A11-GPX4 Pathway

Abstract ： Background ： Acute myeloid leukemia (AML) is a complex disease with diverse molecular subtypes. Approximately 8% of AML patients carry the TP53 mutation, which is associated with an aggressive disease course and dismal survival rates. Arsenic trioxide (ATO) has been remarkably effective in treating acute promyelocytic leukemia (APL), but its clinical activity in other AML subtypes remains limited. Recent evidence indicates that ATO demonstrates potential in rescuing multiple TP53 mutations effectively, offering promise as an individualized therapeutic strategy targeting mutant TP53 in leukemic cells. Our previous study showed that the upregulation of AQP9 by G-CSF enhanced the effect of ATO in non-APL AML cells. This demonstrates that ATO holds therapeutic promise in AML cases harboring distinct TP53 mutations. Methods ： We conducted a comprehensive analysis of TP53 mutations in the Analysis of Whole Genomes cohort, encompassing patients from the TCGA and OHSU databases. The frequency and distribution of TP53 mutations were determined. Median overall survival (OS) was calculated using the Kaplan-Meier method. To functionally assess the effect of TP53 mutation on AML phenotype, various cell lines harboring distinct TP53 mutation types were used for this study, including KASUMI-1 TP53 R248Q THP-1 TP53frameshift_delHL-60 TP53 deletionMOLM13 TP53 wild-type. The cellular lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay and it was also observed by fluorescence microscopy. Additionally, the RNA and protein expressions of GPX4 and SLC7A11 were analyzed by real-time quantitative Polymerase Chain Reaction (QRT-PCR) and Western Blot. Results ： In the OHSU cohort and TCGALAML database, including 1005 newly diagnosed patients with 120 TP53 mutations, we found that missense mutations are the most common TP53 mutations and R248Q has the highest mutation frequency. Furthermore, TP53 mutationwas significantly associated with lower survival ( P <0.001). ATO potently induced cell death in each cell line. The KASUMI-1 R248Q cell line was the most sensitive to ATO (IC50=0.3607) and showed the most pronounced change in the IC50 of ATO with the addition of ferrostatin-1(Fer-1, ferroptosis inhibitor). ATO induced cell death was prevented by the addition of lipophilic antioxidant ferrostatin-1, supporting the hypothesis that ATO induced cell death occurred by ferroptosis. In KASUMI-1 R248Q cell line, ATO increases the lipid oxidation levels (observed by C11 BODIPY flow cytometry assay and fluorescence microscopy), MDA levels and decreases the expression of GPX4 and SLC7A11 (including RNA level and protein level). Whereas, in MOLM13 TP53 wildtype cell line, ATO increases the lipid oxidation levels but cannot prevented by Fer-1. THP-1 TP53 frameshift_del and HL-60 TP53 deletion only decrease the RNA expression of GPX4 and SLC7A11, nothing else changed in ferroptosis. These suggest that ATO has a more pronounced effect on missense mutations such as TP53 R248Q. In KASUMI-1 R248Q cell line, associated with G-CSF can further promote lipid oxidation levels (observed by C11 BODIPY flow cytometry assay and fluorescence microscopy) and further reduce the expression of GPX4 and SLC7A11. These results suggest that G-CSF promotes ATO-induced ferroptosis in KASUMI-1 R248Q cell line. Conclusion: We have demonstrated that ATO induces AML with distinct TP53 mutation cell death by ferroptosis resulting from depletion of GPX4 and SLC7A11. ATO associated with G-CSF may be able to be a new therapeutic strategy for the treatment of AML with distinct TP53 mutation.

Abstract 6020: The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment

Abstract The purpose of this study was to understand the effect of p53 mutation status in ovarian cancer cells on the expression of repetitive elements (REs) following epigenetic treatment. High-grade serous ovarian carcinoma is the most aggressive subtype of ovarian cancer and the 5 year survival rate has remained unchanged for over two decades. Over 90% of high-grade serous ovarian carcinoma (OC) patients have a mutation in the TP53 gene, which encodes the p53 protein. P53 is a transcription factor primarily involved in maintaining genome integrity, but it can also regulate the expression of REs in the genome. REs account for more than half of the genome and these repetitive genomic elements are epigenetically silenced in healthy cells to prevent genome instability. The expression of some REs, such as long interspersed nuclear elements and endogenous retroviruses, are upregulated in human OC patients and cell lines. The mechanism by which p53 regulates expression of REs is not well-understood; the literature documents some instances of p53 activating RE transcription and others of p53 repressing RE transcription, depending on the target elements. We have previously shown that RE expression can be induced by treating OC cells with low doses of DNA methyltransferase inhibitors (DNMTis). Recently, we showed that OC cell lines with mutant p53 have higher baseline expression of REs compared to wild type (WT) cell lines. Treatment of TP53 mutant OC cell lines with DNMTis had a muted effect on RE expression compared to treatment of WT OC cell lines. To explore the relationship between TP53 mutation status and RE expression after DNMTi treatment, OC cell lines with different p53 mutation statuses were treated with 500 nM of the DNMTi 5-azacytidine. We then analyzed the expression of several REs via RT-qPCR and western blot. We hypothesized that TP53 mutant cell lines would have lower DNMTi upregulation of REs than TP53 WT cell lines. While at the RNA level RE expression was variable, at the protein level, we found that DNMTi treatment increased protein levels of several REs in TP53 WT and mutant, but not null, OC cell lines. Future work will examine the feasibility of utilizing these RE proteins as immunotherapy targets in OC. Citation Format: Khadra K. Omar, Erin E. Grundy, Olivia Cox, Melissa Hadley, Katherine B. Chiappinelli. The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6020.

Selective drug combination vulnerabilities in STAT3- and TP53-mutant malignant NK cells

Mature natural killer (NK) cell neoplasms are rare but very aggressive types of cancers. With currently available treatments, they have a very poor prognosis and, as such, are an example of group of cancers in which the development of effective precision therapies is needed. Using both short- and long-term drug sensitivity testing, we explored novel ways to target NK-cell neoplasms by combining the clinically approved JAK inhibitor ruxolitinib with other targeted agents. We profiled 7 malignant NK-cell lines in drug sensitivity screens and identified that these exhibit differential drug sensitivities based on their genetic background. In short-term assays, various classes of drugs combined with ruxolitinib seemed highly potent. Strikingly, resistance to most of these combinations emerged rapidly when explored in long-term assays. However, 4 combinations were identified that selectively eradicated the cancer cells and did not allow for development of resistance: ruxolitinib combined with the mouse double-minute 2 homolog (MDM2) inhibitor idasanutlin in STAT3-mutant, TP53 wild-type cell lines; ruxolitinib combined with the farnesyltransferase inhibitor tipifarnib in TP53-mutant cell lines; and ruxolitinib combined with either the glucocorticoid dexamethasone or the myeloid cell leukemia-1 (MCL-1) inhibitor S63845 but both without a clear link to underlying genetic features. In conclusion, using a new drug sensitivity screening approach, we identified drug combinations that selectively target mature NK-cell neoplasms and do not allow for development of resistance, some of which can be applied in a genetically stratified manner.

Karonudib Has Potent Anti-Tumor Effects in Preclinical Models of B-Cell Lymphoma

Although chemo-immunotherapy has improved survival in B-cell lymphoma patients, refractory and relapsed disease still represents a major challenge, urging for development of new therapeutics. A new approach is to target nucleotide metabolism. Karonudib (TH1579), was developed to inhibit MutT-homologue-1/Nudix hydrolase 1 (MTH1/NUDT1), an enzyme that prevents oxidized nucleotides to be incorporated into DNA. Under normal conditions with low reactive oxygen species (ROS) burden, MTH1 is not essential for cell survival. This contrasts cancer cells which frequently upregulate MTH1 to compensate for increased ROS with corresponding higher oxidized nucleotide levels, and therefore become more susceptible for MTH1 inhibition. Here, our aim was to perform preclinical testing of karonudib in B-cell lymphoma. Using two different gene expression datasets, we demonstrate that NUDT1, the gene encoding MTH1, was highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma as compared to follicular lymphoma and peripheral blood B cells from healthy donors, hence demonstrating a rationale for targeting MTH1 in aggressive B-cell lymphoma. We tested the efficacy of karonudib (0.06-1 µM) in vitro in a range of B-cell lymphoma cell lines using CellTiterGlo and by flow cytometry detection of active caspase-3 and TUNEL to identify apoptotic cells. Karonudib strongly reduced viability in all B-cell lymphoma cell lines tested (n = 12) and induced apoptosis at concentrations well tolerated by peripheral blood B cells from healthy donors. Cell cycle analysis and microscopy revealed that most cells arrested in prometaphase in the presence of karonudib. Failed spindle assembly led to mitotic arrest and subsequent apoptosis. Prometaphase arrest was seen in TP53 mutant as well as in TP53 wild type cell lines, confirming that karonudib induced apoptosis independent of TP53 mutational status. To test the efficacy of karonudib in vivo, we utilized two different lymphoma xenograft models, including an ABC DLBCL patient-derived model. Mice were treated with karonudib (90 mg/kg) or vehicle b.i.d, three times a week and tumor growth was monitored by in vivo imaging system or MR. In both models, karonudib as single agent completely controlled tumor growth, and significantly prolonged survival (p<0.0001, as compared to control mice). The specificity of MTH1 inhibitors has been debated and TH588, the first generation MTH1 inhibitor, was recently shown to bind b-tubulin and induce mitotic arrest in MTH1 knock out cell lines (Patterson et al, Cell Syst 2019). To elucidate the mechanism of karonudib in B-cell lymphoma, we generated MTH1 knock out cells using CRISPR/Cas9, and compared the functional effects of karonudib in these clones with the original lymphoma cells. We demonstrated on-target effect of the inhibitor as the MTH1 knock out clones were less sensitive towards karonudib. However, MTH1 knock out clones exhibited a similar cell cycle arrest as the wild type cells after karonudib treatment. This clearly indicates that karonudib can induce cell cycle arrest independent of MTH1, and hence has a dual mechanism of action. Our preclinical data suggest that karonudib is a promising drug with potential therapeutic use in B-cell lymphoma, and may be particular effective in aggressive lymphoma types. Disclosures Warpman Berglund: Oxcia AB: Other: shareholders; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Gad:Oxcia: Other: shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Pham:Oxcia AB: Other: Shareholder. Sanjiv:Oxcia AB: Other: Shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Helleday:Oxcia AB: Other: Shareholder; non profit Thomas Helleday Foundation for Medical Research: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Novel, Selective Inhibitors of USP7 Uncover Multiple Mechanisms of Antitumor Activity In Vitro and In Vivo.

The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. Thus, USP7 inhibition may decrease oncogene function, increase tumor suppressor function, and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of TP53-mutant cell lines in vitro Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex, polycomb repressive complex 2 (PRC2), and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Furthermore, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer.

TP53 mutant cell lines selected for resistance to MDM2 inhibitors retain growth inhibition by MAPK pathway inhibitors but a reduced apoptotic response

BackgroundEmergence of resistance to molecular targeted therapy constitutes a limitation to clinical benefits in cancer treatment. Cross-resistance commonly happens with chemotherapeutic agents but might not with targeted agents.MethodsIn the current study, TP53 wild-type cell lines with druggable MAPK pathway mutations [BRAFV600E (WM35) or NRASQ61K (SJSA-1)] were compared with their TP53 mutant sublines (WM35-R, SN40R2) derived by selection for resistance to MDM2/p53 binding antagonists.ResultsThe continued presence of the druggable MAPK pathway targets in the TP53 mutant (TP53MUT) WM35-R and SN40R2 cells was confirmed. Trametinib and vemurafenib were tested on the paired WM35/WM35-R and SJSA-1/SN40R2 cells and similar growth inhibitory effects on the paired cell lines was observed. However, apoptotic responses to trametinib and vemurafenib were greater in WM35 than WM35-R, evidenced by FACS analysis and caspase 3/7 activity, indicating that these MAPK inhibitors acted on the cells partially through p53-regulated pathways. SiRNA mediated p53 knockdown in WM35 replicated the same pattern of response to trametinib and vemurafenib as seen in WM35-R, confirming that p53 plays a role in trametinib and vemurafenib induced apoptosis. In contrast, these differences in apoptotic response between WM35 and WM35-R were not seen with the SJSA-1/SN40R2 cell line pair. This is likely due to p53 suppression by overexpressed MDM2 in SJSA-1.ConclusionThe TP53MUT cells selected by resistance to MDM2 inhibitors nevertheless retained growth inhibitory but not apoptotic response to MAPK pathway inhibitors.

MDM2- and FLT3-inhibitors in the treatment of FLT3-ITD acute myeloid leukemia, specificity and efficacy of NVP-HDM201 and midostaurin.

Prognosis for FLT3-ITD positive acute myeloid leukemia with high allelic ratio (>0.5) is poor, particularly in relapse, refractory to or unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. The combined use of compounds targeting both the mutated FLT3 receptor and cellular p53 inhibitors might be a promising treatment option for this poor risk leukemia subset. We therefore assessed MDM2 and FLT3 inhibitors as well as cytotoxic compounds used for conventional induction treatment as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. Acute myeloid leukemia cells represented all major morphologic and molecular subtypes with normal karyotype, including FLT3-ITD (>0.5) and FLT3 wild type, NPM1 mutant and NPM1 wild type, as well as TP53 mutant and TP53 wild type cell lines. Acute myeloid leukemia cells with mutated or deleted TP53 were resistant to MDM2- and FLT3-inhibitors. FLT3-ITD positive TP53 wild type acute myeloid leukemia cells were significantly more susceptible to FLT3-inhibitors than FLT3-ITD negative TP53 wild type cells. The presence of a NPM1 mutation reduced the susceptibility of TP53 wild type acute myeloid leukemia cells to the MDM2 inhibitor NVP-HDM201. Moreover, the combined use of MDM2- and FLT3-inhibitors was superior to single agent treatment, and the combination of midostaurin and NVP-HDM201 was as specific and effective against FLT3-ITD positive TP53 wild type cells as the combination of midostaurin with conventional induction therapy. In summary, the combined use of the MDM2 inhibitor NVP-HDM201 and the FLT3 inhibitor midostaurin was a most effective and specific treatment to target TP53 and NPM1 wild type acute myeloid leukemia cells with high allelic FLT3-ITD ratio. These data suggest that the combined use of NVP-HDM201 and midostaurin might be a promising treatment option particularly in FLT3-ITD positive acute myeloid leukemia relapsed or refractory to conventional therapy.

TP53 Mutational Status and ROS Effect the Expression of the Survivin-Associated Radio-Adaptive Response.

A survivin-associated radio-adaptive response, characterized by increased radiation resistance or sensitization, was induced by exposure to 5 mGy of ionizing radiation and was correlated to the TP53 mutational status of exposed cells. Ten human cancer lines were investigated: colorectal carcinomas HCT116 and RKO [TP53 wild-type (WT)] and their respective TP53 null isogenic lines; breast adenocarcinomas MCF7 (TP53 WT) and MDA-MB-231 (TP53 Mut); lung carcinomas A549 (TP53 WT) and NCI-H1975 (TP53 Mut); and pancreatic carcinomas Hs766T (TP53 WT) and Panc-1 (TP53 Mut). Radiation induced (5 mGy) changes in the subsequent responses to 2 Gy in a multi-dose paradigm. Effects on radiation sensitivity were associated with changes in survivin's intracellular translocation to the cytoplasm (TP53 WT) or nucleus (TP53 Mut). Survival responses were determined using a colony forming assay. Intracellular localization of survivin was determined by ELISA and correlated with survival response. Two 2 Gy doses had minimal effects on the intracellular translocation of survivin. When preceded 15 min earlier by a 5 mGy exposure, survivin translocated to the cytoplasm in all of the TP53 WT cell lines, and to the nuclei in the TP53 null and Mut cells. All TP53 WT cells were protected (P < 0.001) by 5 mGy exposures, while Mut cells were sensitized (P < 0.001). HCT116 and RKO TP53 WT cells were admixed with their respective isogenic TP53 null counterparts in different proportions: 75% to 25%, 50% to 50% and 25% to 75%, respectively. All mixed confluent cultures expressed enhanced radio-sensitization (P ≤ 0.047) characteristic of TP53 Mut cells, which could be inhibited by their exposure to the antioxidant N-acetyl-l-cysteine (NAC) indicating a role for intercellular signaling by reactive oxygen species (ROS). ROS signaling in propagating the survivin-mediated response is involved in both intra- and intercellular communication processes.

Abstract P3-07-21: Cytotoxic potential of the RG7388 MDM2-p53 binding antagonist and the GSK2830371 WIP1 inhibitor on MX-1 and MCF-7 human breast cancer cells

Abstract Background The tumor suppressor p53 is a central hub in molecular signaling pathways that control the integrity of the human genome. The p53 protein functions as a transcription factor and increases the expression of many cellular genes which contribute to activation of cell cycle arrest, apoptosis and DNA repair. MDM2 is another important p53 target gene, and the MDM2 protein is capable of binding directly to p53 and directing it for degradation through the ubiquitin-dependent proteolytic pathway. Inhibition of MDM2 stabilizes p53 and MDM2 inhibitors are being explored clinically as therapies. Stabilization alone may not be enough to increase the activity of p53, and posttranslational modification of p53 by phosphorylation has been proposed to be an important contributory mechanism by which p53 becomes functionally active. Therefore maintaining the phosphorylated status of p53 in tumor cells may help to enhance its growth inhibitory and pro-apoptotic role. Wild type p53 – induced phosphatase (Wip1) is a serine – threonine phosphatase which dephosphorylates central players in the DNA damage response, including p53 and may be an additional target to enhance p53-dependent treatments. Therefore this work was focused on the effect of MDM2 (RG7388) and Wip1 (GSK2830371) inhibitors on MX-1 and MCF breast carcinoma cell lines. These two cell lines were recorded to have wild type TP53 status as well as high expression of Wip1. Trial design RG7388 and GSK2830371 were tested for growth inhibition on MX-1 and MCF-7 breast cancer cell lines using the sulforhodamine B (SRB) assay. The results were further confirmed and mechanism explored by western blotting using extracted protein from drug treated cell lines. Contradictory evidence regarding the TP53 mutation status of the MX-1 cell line was clarified by direct sequencing of MX-1 DNA. Results The MCF-7 cells responded to both RG7388 and GSK2830371 with GI50 value of 0.034 µM and 2.92 µM respectively. The MX-1 cells did not respond to either drug. The results of western blotting showed there was no expression of p53 in the MX-1 cell line. Failure to respond to RG7388and also no expression of p53 in western blotting made us suspicious about the TP53 status of the MX-1 cells. The direct sequencing results confirmed that there was a 5bp deletion in exon 4 of the TP53 gene of the MX-1 cells. The c.154_158delCAATG mutation creates a stop codon at the 54th aminoacid position and results in a truncated p53 protein (p.Gln52Valfs*3). Conclusion RG7388 and GSK2838371 showed cytotoxic effects on MCF-7 cells, whereas both RG7388 and GSK2838371 had no effect on the MX-1 cell line due to the truncated p53 and loss of p53 function. In conclusion, the potency of both drugs depends on the TP53 mutation status and they are likely to be mediated via p53-dependent growth inhibition and apoptosis. Further studies are needed to evaluate the combination effect of both drugs on TP53 wild type cell lines. Citation Format: Manoharan V, Lunec J, Esfandiari A, Mahdi A, Wu C-E, Zanjirband M, Karunanayake EH, Tennekoon KH, De Silva S. Cytotoxic potential of the RG7388 MDM2-p53 binding antagonist and the GSK2830371 WIP1 inhibitor on MX-1 and MCF-7 human breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-21.

The Anti-Tumor Effect of the Ubiquitin-Activating Enzyme (UAE) Inhibitor TAK-243 on Pre-Clinical Models of Multiple Myeloma

Background:The ubiquitin-proteasome system (UPS) has been validated as a target in multiple myeloma (MM) through the success of proteasome inhibitors such as bortezomib, but drug resistance is an emerging challenge. Targeting some of the upstream components of the UPS, such as the E1 ubiquitin activating enzyme (UAE), could therefore be a promising alternative. TAK-243 (MLN7243) specifically blocks the ubiquitin conjugation cascade through the formation of a TAK-243-ubiquitin adduct, thereby inhibiting the UAE. Our aim was to explore the effectiveness of TAK-243 against pre-clinical myeloma models, and to understand some its mechanisms of action.Methods:We performed pre-clinical studies in myeloma cell lines and mouse models using TAK-243. Downstream effects were evaluated using viability, apoptosis assays, western blotting, gene expression profiling (GEP), and Reverse Phase Protein Array (RPPA) techniques.Results:MM1.S and MOLP-8 TP53 wild-type cell lines were sensitive to TAK-243, with median inhibitory concentrations (IC50) of 25 nM at 24 hours based on viability assays. In otherwise isogenic cell lines in which TP53 was suppressed using genome editing techniques, the IC50 was ~40 nM, but higher TAK-243 concentrations of 100 nM overcame resistance due to TP53 inactivation. Similarly, TAK-243 was able to overcome resistance to both conventional (dexamethasone) and novel (bortezomib, lenalidomide) drugs in paired sensitive and resistant cell line models. After treatment with TAK-243, Annexin V and TO-PRO3 staining determined that viable MM1.S cells were induced into early or late apoptosis. This was accompanied by a significant increase in cleaved caspase-3, -8, and -9 as detected by flow cytometry, and in cleaved caspase-7 detected by RPPA and western blot. Exposure to TAK-243 reduced the cellular content of ubiquitin-protein conjugates, and did not enhance expression levels of a fusion protein degraded by the proteasome in a ubiquitin-independent manner, indicating the lack of direct proteasome inhibition. GEP analysis and RPPA detected enhanced expression of p53-pathway related proteins, including MDM2, TP53, and p21 in TAK-243 treated MM1.S cells. Several mRNAs and proteins in the ER stress pathway, including ATF6, ATF4, IRE1a and XBP1 were also elevated, as were many non-coding RNAs and DNA-damage related genes. Combination experiments in MM cell lines demonstrated synergy between TAK-243 and lenalidomide, pomalidomide, panobinostat, melphalan and doxorubicin. Finally, TAK-243 demonstrated in vivo antitumor activity against MM1.S and MOLP-8 xenograft models when dosed at 12.5 mg/kg IV twice-weekly for 2 weeks (tumor growth inhibition of 60% and 73%, respectively). Elevation of BiP, ATF4, XBP1s and cleaved-caspase 3 was detected within the first 24 hrs after dosing in the sensitive MM1.S xenografts. In contrast, RPMI 8226 cells, which showed a 2000 nM IC50 in cell culture, were also resistant to TAK-243 in vivo, with no tumor growth inhibition detected.Conclusions:TAK-243 is a UAE inhibitor that is active against myeloma cells in vitro and in xenograft models in vivo, overcomes conventional and novel drug resistance, and its action is associated with stimulation of the TP53 and ER stress pathways. Thus, it may deserve further evaluation as an anti-myeloma agent. DisclosuresBerger:Takeda Pharmaceuticals: Employment. Hyer:Takeda Pharmaceuticals: Employment. Chattopadhyay:Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Shi:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Shinde:Takeda Pharmaceuticals: Employment. Kreshock:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Menon:Takeda Pharmaceuticals: Employment. Orlowski:Takeda Pharmaceuticals: Research Funding.

Initial Testing (Stage 1) of MK-8242-A Novel MDM2 Inhibitor-by the Pediatric Preclinical Testing Program.

MK-8242 is an inhibitor of MDM2 that stabilizes the tumor suppressor TP53 and induces growth arrest or apoptosis downstream of TP53 induction. MK-8242 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10.0 μM and against the PPTP in vivo xenograft panels using oral gavage on Days 1-5 and Day 15-19 at a dose of 125 mg/kg (solid tumors) or 75 mg/kg (acute lymphoblastic leukemia [ALL] models). The median IC50 for MK-8242 was 0.07 μM for TP53 wild-type cell lines versus >10 μM for TP53 mutant cell lines. MK-8242 induced a twofold or greater delay in time to event in 10 of 17 (59%) of TP53 wild-type solid tumor xenografts, excluding osteosarcoma xenografts that have very low TP53 expression. Objective responses were observed in seven solid tumor xenografts representing multiple histotypes. For the systemic-disease ALL panel, among eight xenografts there were two complete responses (CRs) and six partial responses (PRs). Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously showed maintained CR and PR, respectively. The expected pharmacodynamic responses to TP53 activation were observed in TP53 wild-type models treated with MK-8242. Pharmacokinetic analysis showed that MK-8242 drug exposure in SCID mice appears to exceed that was observed in adult phase 1 trials. MK-8242-induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This activity was observed at MK-8242 drug exposures that appear to exceed those observed in human phase 1 trials.

Chemical Inhibition of Wild-Type p53-Induced Phosphatase 1 (WIP1/PPM1D) by GSK2830371 Potentiates the Sensitivity to MDM2 Inhibitors in a p53-Dependent Manner.

Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53(Ser15) phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non-growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor-induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53(Ser15), known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53(Ser15) and its potential use as a biomarker for response to this combination regimen.

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress

Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of invitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our invitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.

Expression signature based on TP53 target genes doesn't predict response to TP53-MDM2 inhibitor in wild type TP53 tumors

A number of TP53-MDM2 inhibitors are currently under investigation as therapeutic agents in a variety of clinical trials in patients with TP53 wild type tumors. Not all wild type TP53 tumors are sensitive to such inhibitors. In an attempt to improve selection of patients with TP53 wild type tumors, an mRNA expression signature based on 13 TP53 transcriptional target genes was recently developed (Jeay et al. 2015). Careful reanalysis of TP53 status in the study validation data set of cancer cell lines considered to be TP53 wild type detected TP53 inactivating alterations in 23% of cell lines. The subsequent reanalysis of the remaining TP53 wild type cell lines clearly demonstrated that unfortunately the 13-gene signature cannot predict response to TP53-MDM2 inhibitor in TP53 wild type tumors.

Chemosensitivity of prostatic tumour cell lines under conditions of G2 block abrogation.

Conventional chemotherapy has had very limited success in the control of hormone-refractory prostate cancer. Methylxanthine derivatives, such as pentoxifylline (PTX), are known to abrogate the G2 block and enhance the toxicity of ionising irradiation and chemotherapeutic agents. It is now also established that late addition of the cytotoxic drug after irradiation under conditions of G2 block abrogation sensitises human tumour cells for cytotoxins. Here we assess whether the chemosensitivity of prostate tumour cell lines can be enhanced by the application of a low dose of drug in conjunction with a G2 block abrogator. Prostate cell lines DU145, BM1604 and LNCaP were irradiated with 7 Gy 60Co gamma-irradiation. A sub-toxic (2 mM) dose of pentoxifylline and a cytotoxic drug were added at maximum expression of the G2 cell cycle block and cell survival was determined by colony assay. Cisplatin, etoposide and vinblastine were tested at a toxic dose of 10% (TD10). In the TP53 mutant cell lines, DU145 and BM1604, dose enhancement factors (EFs) were found to be in the region of 4.20 for cisplatin, 3.70 for vinblastine, and 3.20 for etoposide. In the TP53 wild-type cell line, LNCaP, the enhancement factors were low and in the region of 1.20 for cisplatin, vinblastine and etoposide. It is clear, therefore, that toxicity enhancement factors (EFs) are greater in the TP53 mutant cell lines, DU145 and BM1604, than in the TP53 wild-type cell line, LNCaP. The results indicate that a significant enhancement of drug toxicity can be obtained if the cytotoxic drug is given under conditions of G2 block abrogation. The sensitisation of prostate cancer cells to cytotoxic drugs is particularly high in radiation-resistant TP53 mutant tumour cells. Drugs which abrogate G2 block have the potential to enhance the therapeutic index and therefore reduce the toxicity of chemotherapy drugs.

Cytotoxicity of azadirachtin A in human glioblastoma cell lines

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 μM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25–69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.

Abrogation of G(2)/M-phase block enhances the cytotoxicity of daunorubicin, melphalan and cisplatin in TP53 mutant human tumor cells.

Irradiation of human melanoma (MeWo, Be11) and squamous cell carcinoma (4451, 4197) cells induces cell cycle blocks from which the cells recover to re-enter mitosis after 40-60 h. In the TP53 mutant cell lines, MeWo and 4451, irradiation induces a G(2)-phase block, where the fraction of cells in G(2) phase reaches a maximum after 18-20 h. In the TP53 wild-type cell lines, 4197 and Be11, a G(1)- and G(2)-phase block is reached 12 and 16 h postirradiation, respectively. Addition of pentoxifylline after irradiation at the time when the number of cells in G(2) phase has reached a maximum shortens the normal recovery from G(2)-phase block to approximately 7 h. Addition of daunorubicin, melphalan and cisplatin under these conditions markedly enhanced drug toxicity. In the TP53-mutated cell lines MeWo and 4451, the survival ratio at 7 Gy measured by colony formation was 2.3-2.8, 8.6-85 and 52-74 for daunorubicin, melphalan and cisplatin, respectively. In the TP53 wild-type cell lines, the corresponding survival ratios were found to be 1.3-1.4, 2.3-3.0 and 1.2-2.6, respectively. The survival ratios are for clonogenic survival after 7 Gy and 2 mM pentoxifylline and measure the influence of drug doses that ensure 95% survival in nonirradiated controls. The results indicate that the G(2)-phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity. These effects are particularly pronounced in TP53 mutant cells and are observed at drug doses well below the clinical range.