P53 Wt Cells Research Articles

Abstract 215: Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes

Abstract Background: p53 regulates a plethora of biological processes including cellular metabolism and mitochondrial function. Using the small molecule MDM2 agonist MI-63, we previously demonstrated differential p53 upregulation and induction of global metabolomics profiles in malignant MCF-7 and non-malignant MCF-10A mammary epithelial cells undergoing different cell fates (apoptosis vs. cytostasis). In particular, p53 upregulation suppressed glycolytic metabolite pools in MCF-10A, while increasing their levels in MCF-7 cells. We postulated that this opposing metabolic response may be linked to their different cell fate outcomes. In this study, we evaluated the association between p53 induced effects on live cell metabolic flux and cell fate using different p53wt cell lines subjected to comparable p53 upregulation. Methods: We employed four p53wt cell lines: MCF-7, ZR-75-1, SJSA-1 and MCF-10A. Cell fate outcomes upon p53 upregulation (24hr, 10μM MI63) were assessed by Western blotting for cytostasis (p21) and apoptosis markers (cleaved PARP, PUMA). Expression changes in enzymes involved in proline metabolism were similarly measured. Real-time measurements of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) under control and MI-63 treatments were assessed by the Seahorse Extracellular Flux Analyzer, and specific acidification contributions from respiration and glycolysis to total ECAR were computed. Results: p53 upregulation induced apoptosis in the malignant MCF-7, ZR-75-1 and SJSA-1 cells, but not in non-malignant MCF-10A cells. MI-63 treatment decreased basal OCR in MCF-7 cells (t-test p = 0.046), but not the other cell lines. No significant differences in maximum OCR were observed between control and MI-63 treatments in any of the cell lines tested. As well, p53 upregulation did not produce any significant change in total ECAR. However, in MCF-7 cells, glycolytic ECAR was significantly increased (MI-63/Control: 4.36, Tukey test p = 0.001), consistent with our earlier metabolomics conclusions. In contrast, no significant changes in glycolytic ECAR were produced in MCF-10A, ZR-75-1 or SJSA-1 cells (MI-63/Control: 1.27, 1.32 and 1.33 respectively). Interestingly, p53 upregulation induced expression of proline dehydrogenase (PRODH) in all three malignant cell lines, but not in MCF-10A cells; and coordinated changes in other proline catabolizing enzymes (P5C dehydrogenase, P5C reductase) were seen in MCF-7 cells. Conclusion: These findings indicate that p53 induced effects on oxygen consumption and glycolysis are cell context dependent but are not likely responsible for the different cell fate outcomes observed following p53 upregulation. Rather, our evidence suggests a potential mechanistic link between p53 induced effects on proline metabolism and cell fate outcomes, indicating that this stress-activated mitochondrial pathway deserves further study. Citation Format: Yau Christina, Katya Frazier, Daniel Rothschild, Shona Mookerjee, Martin Brand, Gary Scott, Christopher C. Benz. Metabolic and mitochondrial consequences of p53 upregulation and their relation to cell fate outcomes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 215.

P53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb.

The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype.

RNA Polymerase I Inhibition with CX-5461 As a Novel Strategy to Target MYC in Multiple Myeloma

Background: The role of dysregulation of the proto-oncogene MYC in both early and late myeloma progression events is well established. Among key MYC -downstream targets is upregulation of ribosomal biogenesis, resulting in increased protein translational capacity and biomass accumulation that is characteristic of neoplastic cells. Thus, given the relationship between myeloma pathobiology, MYC dysregulation, and ribosomal biogenesis, we hypothesized that selective targeting of ribosomal RNA (rRNA) transcription with the small molecule RNA polymerase (pol) I inhibitor CX-5461 (Senhwa Biosciences) may represent a novel therapeutic strategy in myeloma.Methods: Studies with CX-5461 were performed in human myeloma cell lines, isogenic p53 wild-type (wt) and knock-out (KO) p53 cells generated using sequence-specific zinc-finger nucleases, drug-resistant cell lines, primary patient samples, and myeloma murine xenograft models using NOD-SCID IL2Rgnull mice.Results: CX-5461 treatment of p53 wt (MM1.S, MOLP-8) and p53 mutant (U266, RPMI-8226) myeloma cell lines demonstrated a time- and dose-dependent decrease in cell proliferation with a median inhibitory concentration (IC50) at nM levels after 72 hours. A corresponding increase in cleaved-PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot as well as increased Annexin V staining on flow cytometry analysis, although this was more pronounced in p53 wt versus mutant cell lines. CX-5461 also retained activity in a panel of cell lines resistant to standard myeloma therapeutic agents (bortezomib, carfilzomib, lenalidomide, and doxorubicin) and in primary patient samples, including a heavily pretreated relapsed/refractory patient and a de novo plasma cell leukemia patient with del 17p. In vivo studies using a systemic isogenic MM1.S p53 wt and KO myeloma murine xenograft model demonstrated significant improvement in median overall survival in the CX-5461-treated p53 wt cohort (41 days vs. not reached, P .05), although outcomes were more modest in the p53 KO cohort with only a trend towards improved survival (P.1) in the drug-treated mice.To probe the p53-independent effects of CX-5461, gene expression profiling and gene set enrichment analysis was performed on isogenic MM1.S and MOLP-8 p53 wt and KO myeloma cell lines treated with CX-5461 or vehicle. These results suggested downregulation of MYC downstream targets as one p53-independent effect of RNA pol I inhibition. qPCR and Western blot studies revealed rapid downregulation of MYC at the transcript level within 1-hour of CX-5461 treatment followed by decreases in MYC protein levels.Previous studies have suggested ribosomal biogenesis is tightly controlled by an auto-regulatory feedback mechanism in which ribosomal proteins such as RPL5 and RPL11 can bind to the 3'UTR of MYC mRNA and facilitate its degradation through the RNA-induced silencing complex (RISC). Because RNA pol I inhibition is known to induce a nucleolar stress response and increase the availability of free ribosomal proteins, RISC-mediated degradation of MYC mRNA was explored as one possible mechanism of CX-5461-mediated MYC downregulation. Indeed, treatment with CX-5461 led to increased pull-down of RPL5 when immunoprecipitated with the RISC subunit TAR (HIV-1) RNA Binding Protein 2 (TARBP2) compared to vehicle-treated controls, and RNA immunoprecipitation assays with the catalytic RISC subunit, Argonaute 2 (AGO2), demonstrated enrichment of MYC mRNA with CX-5461 treatment. These results suggest that CX-5461 may induce degradation of MYC through the cooperative binding of ribosomal proteins, RISC subunits, and MYC mRNA.Finally, to evaluate the role of MYC expression and ribosomal biogenesis in relation to CX-5461 sensitivity, MYC was overexpressed in the H1112 myeloma cell line, which at baseline does not harbor a MYC translocation. MYC overexpression in H1112pCDH-myc cells led to increased basal pre-rRNA transcript levels compared to H1112pCDH cells, and furthermore, led to enhanced sensitivity to CX-5461.Conclusion: RNA pol I inhibition by CX-5461 is a promising target in myeloma therapy, with downregulation of MYC representing one mechanism of action. Moreover, increased MYC expression enhances sensitivity to CX-5461, providing rationale for the clinical translation of CX-5461 for the treatment of myeloma and other MYC-driven cancers. DisclosuresO'Brien:Senhwa Biosciences, Inc.: Employment. Keats:Translational Genomic Research Institute: Employment. Orlowski:Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding.

The Hypoxia-Selective Epigenetic Agent, Rrx-001, Triggers Apoptosis and Overcomes Drug-Resistance in Multiple Myeloma Cells

Introduction The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in multiple myeloma (MM) cells (Chauhan et al, Cancer Cell 2009, 16:309-323) BM hypoxia (low oxygenation) plays a role in promoting MM cell survival, drug resistance, migration, and metastasis. Novel therapies that selectively target the MM cell in its hypoxic BM milieu may therefore overcome conventional drug resistance. Recent studies led to the development of a novel aerospace industry-derived Phase 2 molecule RRx-001 with hypoxia-selective epigenetic and NO-donating properties. A Phase I clinical trial demonstrated promising evidence of anti-tumor activity in a heavily pretreated population with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578, Reid et al, Lancet Oncology, in press). RRx-001 is currently under investigation in multiple Phase II clinical trials. Here we examined both the mechanism of action and anti-MM activity of RRx-001 using in vitro and in vivo models of MM.Methods Cell viability, apoptosis, and migration assays were performed using MTT, Annexin V staining, and transwell Inserts, respectively. ROS and NO generation was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). Synergistic anti-MM activity was assessed by isobologram analysisusing "CalcuSyn" software program. In vitro angiogenesis was assessed using matrigel capillary-like tube formation assays. DNMT1 activity was measured using DNMT1 assay kit. USP7 siRNA was purchased from Dharmacon. CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Statistical significance of data was determined using a Student's t test. RRx-001 was obtained from EpicentRx, CA, USA; USP7 inhibitor P5091, bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA.Results Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 48h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells, even in the presence of BM stromal cells. Washout experiments showed that a short time (3h) exposure of MM cells to RRx-001 triggered irreversible cell death. RRx-001-triggered apoptosis is associated with: 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNMT1 activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. Deubiqyitylating enzyme USP7 stimulates DNMT1 enzymatic activity. USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. Importantly, the combination of USP7 inhibitor P5091 and RRx-001 triggered synergistic anti-MM activity associated with a robust decrease in DNMT1 activity, as well as increased degradation of USP7 substrate MDM2 and induction of downstream p21/p53 signaling axis. In vivo studies using a subcutaneous human MM xenograft model shows that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Finally, combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity in p53-WT and p53-null MM cells, and overcomes drug resistance.Conclusion Our preclinical studies demonstrate that RRx-001, a ROS-mediated epigenetic inhibitor with anti-angiogenic properties selectively targets MM cells in vivo and synergizes with existing anti-MM agents to overcome therapeutic resistance. Our data also suggest a potential mechanism of action for RRx-001-induced epigenetic changes via USP7-DNMT1 complex and downstream p53/p21 signaling cascade. Collectively, these results provide a rationale for rapid translation of RRx-001, either alone or in combination, in a clinical trial of relapsed refractory MM. DisclosuresOronsky:epicentrx: Employment. Scicinski:epicentrx: Employment. Chauhan:Stemline Therapeutics: Consultancy.

Abstract A26: Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells

Abstract A multi-targeted approach will be necessary to eradicate glioblastoma multiforme (GBM) cells due to the immense genetic heterogeneity associated with GBM. Mouse double minute-2 (MDM2) regulates multiple signaling pathways and is a promising therapeutic target in GBM. In wild type (wt) p53 cells, MDM2 binds to wtp53, ubiquitinates it, and negatively regulates p53-mediated downstream events. In wtp53 and mutant (mt) p53 cells, MDM2 binds to and sequesters p73α thereby blocking p73α-mediated signaling. Our objective in the present studies was to determine if the p73α-MDM2 axis could be exploited to increase death of mtp53 GBM cells. We utilized MDM2 antagonists nutlin3a or RG7112 to block protein-protein interactions between MDM2-p53 and MDM2-p73α. In a panel of GBM cell lines, TMZ resistance was reduced in both wt53 and mt53 cells in the presence of MDM2 antagonists. In mtp53 cells, siRNA knockdown of p73α indicated that sensitivity to treatment was dependent on p73α levels. Isobologram analysis indicated that while dose-ratios of TMZ to MDM2 antagonists were additive to synergistic in inhibiting growth of wtp53 GBM cells, this was not the case in mtp53 GBM cells (SF118, GBM43, gain-of-function-mtp53 R273H U373 and MHBT32). Analysis of intracellular targets in mtp53 GBM cells exposed to TMZ and MDM2 antagonists indicated that p73α and MDM2 expression increased by 24 hours post-treatment. In addition, AKT activity was increased or sustained in mtp53 GBM cells following treatment with TMZ in the absence or presence of MDM2 antagonists. Since increased AKT activity may render cells resistant to therapy, the AKT inhibitor GDC0068 was evaluated in combination with TMZ and RG7112. As a measure of AKT-downstream target modulation, phosphorylation status of the Forkhead box O-class (FoxO) transcription factors (TFs) was determined. In the non-phosphorylated state, FoxO TFs upregulate expression of proteins involved in cell-death pathways. While phospho-FoxO1/FoxO3a TFs were increased in TMZ/RG7112-treated mtp53 GBM cells compared to controls, it was decreased in GDC0068-, TMZ/GDC0068- and TMZ/RG7112/GDC0068-treated mtp53 GBM cells which is consistent with inactivation of AKT and activation of FoxO TFs. Isobologram analysis of mtp53 GBM cell growth indicated that combination RG7112 and GDC0068 inhibited growth in a synergistic manner even in the absence of TMZ. For in vivo studies, an intermittent dosing regimen of TMZ/RG7112/GDC0068 was developed to avoid normal tissue toxicity. GBM43 flank tumor growth was significantly inhibited in mice with tumors treated with RG7112/GDC0068 and inhibited to a larger extent by the triple combination TMZ/RG7112/GDC0068 compared to vehicle and single-agent exposure (n=9-10 mice per group; single agent vs GDC0068/RG7112 or TMZ/RG7112/GDC0068, p&amp;lt;0.05). The present data indicate that targeting the p73α-MDM2 and AKT-FoxO signaling networks inhibit mtp53 GBM cell growth and with an appropriate dosing schedule can be utilized in vivo with an acceptable toxicity profile. Citation Format: Mohammad Reza Saadatzadeh, Haiyan Wang, Jixin Ding, Barbara J. Bailey, Eva Tonsing-Carter, Shanbao Cai, Nimita Dave, Harlan E. Shannon, Aaron Cohen- Gadol, Karen E. Pollok. Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A26.

P53 Configures the G2/M Arrest Response of Nucleostemin-Deficient Cells.

Nucleostemin (NS) protects the genome from replication-induced DNA damage and has an indispensable role in maintaining the continuous proliferation of both p53-wild-type and mutant cells. Yet, some outcomes of NS-deficient cells appear to be shaped by their p53 status, which stimulates conflicting claims on the role of p53 in executing the NS function. This disparity was conveniently attributed to the usual suspect of cell-type variations. To provide a definitive resolution, we investigated the interplay between NS and p53 in two pairs of isogenic cells, that is, genetically modified mouse embryonic fibroblast (MEF) cells and HCT116 human colon cancer cells. In MEF cells, p53 deletion further compromises rather than rescues the proliferative potential of NS-depleted cells without changing their G2/M arrest fate before prophase entry. The detrimental effect of p53 loss in NS-depleted MEF cells correlates with a dramatic increase of polyploid giant cells (PGCs) (up to 24%), which indicates aberrant mitosis. To determine how p53 shapes the response of cells to NS depletion at the molecular level, we showed that p53 turns on the expression of reprimo and MDM2 in NS-deficient MEF cells. In absence of p53, NS-deficient MEF cells exhibit increased levels of phosphorylated cdc2 (Y15) protein and cyclin B1. In cancer (HCT116) cells, NS loss leads to G2/M arrest under both p53wt and p53ko conditions and increases phosphorylated cdc2 more in p53ko than in p53wt cells, as it does in MEF cells. Unlike its effect in MEF cells, NS depletion decreases tumor growth and increases the expression of reprimo and cyclin B1 in a p53-independent manner in HCT116 cells. Our data indicate that the p53 status of NS-deficient cells orchestrates how they respond to G2/M arrest in a normal versus cancer cell distinct fashion.

ATPS-25p53 STATUS DEPENDENCE OF TUMOR TREATING FIELDS (TTFIELDS) EFFICACY AGAINST GLIOMA CANCER CELLS

TTFields are an effective anticancer treatment modality delivered via continuous, noninvasive application of low intensity, intermediate frequency, alternating electric fields. This innovative treatment approach is approved for the treatment of patients with recurrent glioblastoma and a recent phase 3 study demonstrated superior progression free and overall survival in newly diagnosed glioblastoma patients when TTFields were added to standard of care. The goal of this study was to evaluate the therapeutic responsiveness of genetically defined glioma cells, specifically the status of p53 tumor suppressor gene as a determinant of tumor response to TTFields. For in vitro studies, TTFields (1.75 V/cm RMS, 200 kHz) were applied for 72 hours using the inovitro™ system (Novocure, Haifa, Israel). The surviving fractions of glioma cell lines expressing either wild-type (wt) p53 (A-172 and U-87 MG) or mutant p53 (U-118 MG and F-98 rat glioma) were determined using cell counts and clonogenic assays. Induction of apoptosis was examined using flow cytometry based on Annexin V and 7-AminoactinomycinD (7-AAD) staining. Detection of caspase activity was conducted using a poly caspase assay kit. The effect of caspase inhibition was explored using the Z-VAD-FMK pan-caspase inhibitor. Application of TTFields in-vitro led to a significant reduction in cell count and clonogenic potential as compared to untreated cells in both p53 wt and p53 mutant cell lines. Flow cytometry analysis of Annexin V and Propidium Iodide staining revealed that TTFields-induced apoptosis is independent of p53 status. Caspase activity was increased in wt p53 cells following TTFields application, however no increase was observed in mutant p53 cells. Treatment of wt p53 cells with Z-VAD-FMK partially impaired cytotoxicity induced by TTFields treatment and completely abrogated apoptosis. In summary, our results suggest that TTFields exposure induces apoptosis by both p53-dependent and p53-independent pathways.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 3089: CDKN2A and p53 status predicts response to CDK4/6 inhibition in melanoma

Abstract The p16-CyclinD1-CDK4-RB pathway regulates G1-S cell cycle transition. This pathway is deregulated in 90% of melanomas, through amplification or increased expression of CDK4 or Cyclin D1 (CCND1), or reduced expression of the CDK inhibitor p16INK4A (CDKN2A). To explore if CDK4 may be a viable target for the treatment of human melanoma we screened a panel of melanoma cell lines with the highly selective CDK4/6 inhibitor PD-0332991 (PD-991). A majority of cell lines were found to be sensitive to PD-991, although there was a distinct population of resistant cell lines. In sensitive melanoma cell lines, treatment with PD-991 resulted in loss of hyperphosphorylated RB and a concomitant G0/G1 cell cycle arrest. Treated cells displayed an enlarged flattened morphology and an increase in SA-β-galactosidase staining compared to vehicle controls, indicative of cellular senescence. RB-null cell lines were resistant to CDK4/6 inhibition, and did not display cell cycle arrest or senescence. Expression and mutational analysis was performed to identify additional genomic predictors of sensitivity and resistance. Loss of CDKN2A/p16 correlated with sensitivity to PD-991 (p&amp;lt;0.02), and this was confirmed at the level of protein expression. Mutation of p53 and the corresponding low expression of p53 targets p21 and MDM2 correlated with resistance (p&amp;lt;0.0001). There was no increase in p53 transcriptional targets or evidence of apoptosis in response to PD-991 treatment of sensitive cells, and senescence occurs in both p53 WT and mutant cells, indicating that this response does not require functional p53. However, in p53 WT cells, combination treatment with PD-991 and the p53 pathway activator Nutlin3a results in increased sensitivity to PD-991 compared to treatment with PD-991 alone. This additive effect is not observed in p53 mutant cells, suggesting there is a functional role for WT p53 in the response to PD-991. Conclusions: These preliminary data identify RB, p16 and p53 as predictors of response to PD-991, and support further evaluation of CDK4/6 inhibitors in melanoma. Citation Format: Claire A. Martin, Laura Kirby, Stephen Wong, Grant A. McArthur, Karen E. Sheppard. CDKN2A and p53 status predicts response to CDK4/6 inhibition in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2015-3089

Interference with distinct steps of sphingolipid synthesis and signaling attenuates proliferation of U87MG glioma cells

Glioblastoma is the most common malignant brain tumor, which, despite combined radio- and chemotherapy, recurs and is invariably fatal for affected patients. Members of the sphingolipid (SL) family are potent effectors of glioma cell proliferation. In particular sphingosine-1-phosphate (S1P) and the corresponding G protein-coupled S1P receptors transmit proliferative signals to glioma cells. To investigate the contribution to glioma cell proliferation we inhibited the first step of de novo SL synthesis in p53wt and p53mut glioma cells, and interfered with S1P signaling specifically in p53wt U87MG cells. Subunit silencing (RNAi) or pharmacological antagonism (using myriocin) of serine palmitoyltransferase (SPT; catalyzing the first committed step of SL biosynthesis) reduced proliferation of p53wt but not p53mut GBM cells. In U87MG cells these observations were accompanied by decreased ceramide, sphingomyelin, and S1P content. Inhibition of SPT upregulated p53 and p21 expression and induced an increase in early and late apoptotic U87MG cells. Exogenously added S1P (complexed to physiological carriers) increased U87MG proliferation. In line, silencing of individual members of the S1P receptor family decreased U87MG proliferation. Silencing and pharmacological inhibition of the ATP-dependent cassette transporter A1 (ABCA1) that facilitates S1P efflux in astrocytes attenuated U87MG growth. Glyburide-mediated inhibition of ABCA1 resulted in intracellular accumulation of S1P raising the possibility that ABCA1 promotes S1P efflux in U87MG glioma cells thereby contributing to inside-out signaling. Our findings indicate that de novo SL synthesis, S1P receptor-mediated signaling, and ABCA1-mediated S1P efflux could provide pharmacological targets to interfere with glioma cell proliferation.

Abstract P3-05-14: Modeling chemoendocrine therapy for ER+/p53wt luminal breast cancer

Abstract Background ER+ breast cancers are predominantly p53 wildtype (wt). Despite this genotype, these cancers are relatively resistant to chemotherapy-induced apoptosis in the presence of estrogen. Thus, current clinical practice is to administer hormonal therapy and chemotherapy sequentially rather than concurrently. Using the ER+/p53wt cell line MCF-7, we previously demonstrated that ER bound by either the ER agonist estradiol or partial antagonist tamoxifen inhibits the expression of a set of proapoptotic p53 target genes, whereas the full ER antagonist fulvestrant, a selective ER downregulator (SERD), removes the ER-mediated suppression of these genes, sensitizing breast cancer cells to p53-mediated cell death .We hypothesize that effective chemoendocrine therapy requires complete ER antagonism resulting in ER downregulation, increased p53 activity, and ultimately apoptosis. Methods: To model mechanisms of tumor sensitivity and resistance to therapy in vivo, therapeutic experiments were performed in immunodeficient mice bearing ER+/p53wt tumor xenografts derived from a 61-year-old African-American female with grade 2 invasive ductal carcinoma. The mice were randomly assigned to the following treatment groups: A) fulvestrant B) tamoxifen, C) doxorubicin, D) fulvestrant plus doxorubicin, E) tamoxifen plus doxorubicin, and F) control. Tumors were measured weekly for 42 days to determine treatment effects on growth. In addition, RNA was harvested from tumors at an early time point for RNA sequencing to determine genes regulated by the treatments. Results: Combination therapy comprising doxorubicin plus fulvestrant resulted in tumor regression compared with single-agent or doxorubicin plus tamoxifen treatment, which inhibited the growth of tumors but did not lead to their regression. Conclusions: Current paradigms for the treatment of ER+ breast cancer either in the adjuvant or advanced setting have involved the sequential use of endocrine therapy and chemotherapy. This protocol is partially based on evidence of potential antagonism between tamoxifen and chemotherapy as observed in early studies. Our published work in vitro and the preclinical study presented here suggest that complete ER antagonism with SERDs such as fulvestrant is required to overcome the ability of ER to block p53-mediated apoptosis. Our results suggest treatments involving fulvestrant concurrent with chemotherapies involving p53 activation should be considered for the treatment of patients with ER+/p53wt breast cancer. Citation Format: Shiliang A Cao, Elgene Lim, Myles Brown, Shannon T Bailey. Modeling chemoendocrine therapy for ER+/p53wt luminal breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-14.

Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells.

Introductionp53 plays important roles in regulating the metabolic reprogramming of cancer, such as aerobic glycolysis. Oroxylin A is a natural active flavonoid with strong anticancer effects both in vitro and in vivo.Methodswt-p53 (MCF-7 and HCT116 cells) cancer cells and p53-null H1299 cancer cells were used. The glucose uptake and lactate production were analyzed using Lactic Acid production Detection kit and the Amplex Red Glucose Assay Kit. Then, the protein levels and RNA levels of p53, mouse double minute 2 (MDM2), and p53-targeted glycolytic enzymes were quantified using Western blotting and quantitative polymerase chain reaction (PCR), respectively. Immunoprecipitation were performed to assess the binding between p53, MDM2, and sirtuin-3 (SIRT3), and the deacetylation of phosphatase and tensin homolog (PTEN). Reporter assays were performed to assess the transcriptional activity of PTEN. In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 cells.ResultsHere, we analyzed the underlying mechanisms that oroxylin A regulated p53 level and glycolytic metabolism in wt-p53 cancer cells, and found that oroxylin A inhibited glycolysis through upregulating p53 level. Oroxylin A did not directly affect the transcription of wt-p53, but suppressed the MDM2-mediated degradation of p53 via downregulating MDM2 transcription in wt-p53 cancer cells. In further studies, we found that oroxylin A induced a reduction in MDM2 transcription by promoting the lipid phosphatase activity of phosphatase and tensin homolog, which was upregulated via sirtuin3-mediated deacetylation. In vivo, oroxylin A inhibited the tumor growth of nude mice-inoculated MCF-7 or HCT116 cells. The expression of MDM2 protein in tumor tissue was downregulated by oroxylin A as well.ConclusionsThese results provide a p53-independent mechanism of MDM2 transcription and reveal the potential of oroxylin A on glycolytic regulation in both wt-p53 and mut-p53 cancer cells. The studies have important implications for the investigation on anticancer effects of oroxylin A, and provide the academic basis for the clinical trial of oroxylin A in cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0137-1) contains supplementary material, which is available to authorized users.

NKX2-1-mediated p53 expression modulates lung adenocarcinoma progression via modulating IKKβ/NF-κB activation.

NKX2-1 plays a dual role in lung adenocarcinoma progression, but the underling mechanism is not fully understood. In the present study, we provide evidence that NKX2-1 directly regulates p53 transcription, and in turn, NKX2-1 elevates the mutant p53/NF-Y complex to up-regulate IKKβ transcription in p53-mutant cells, but NKX2-1-mediated wild-type p53 down-regulates IKKβ transcription via decreased Sp1 binding to IKKβ promoter in p53-WT cells. The IKKβ-mediated p65 nuclear localization and epithelial-to-mesenchymal transition (EMT) modulated by the NKX2-1/p53 axis is responsible for soft-agar growth, invasion, and xenograft tumour formation. Among patients, high-IKKβ mRNA tumours had higher prevalence in p53-mutant or nuclear p65 tumours than their counterparts, but not related with NKX2-1 mRNA expression. However, when tumours were divided into p53-WT and p53-mutant subgroups, NKX2-1 mRNA expression was negatively correlated with IKKβ mRNA in p53-WT subgroup, but positively related with IKKβ mRNA expression in p53-mutant subgroup. Kaplan-Meier and Cox regression analysis indicated that high NKX2-1 mRNA tumours exhibited poorer overall survival and relapse free survival than low NKX2-1 mRNA tumours in p53-WT subgroup, but the opposite was observed in p53-mutant subgroup. Therefore, we suggest that NKX2-1 as a tumour suppressor or a tumour promoter in lung adenocarcinoma progression is dependent on p53 status.

Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma

Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

Identifying the determinants of response to MDM2 inhibition.

Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2-amplified subset of p53WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

Targeting MKK3 as a novel anticancer strategy: molecular mechanisms and therapeutical implications.

Mitogen-activated protein kinase kinase 3 (MAP2K3, MKK3) is a member of the dual specificity protein kinase group that belongs to the MAP kinase kinase family. This kinase is activated by mitogenic or stress-inducing stimuli and participates in the MAP kinase-mediated signaling cascade, leading to cell proliferation and survival. Several studies highlighted a critical role for MKK3 in tumor progression and invasion, and we previously identified MKK3 as transcriptional target of mutant (mut) p53 to sustain cell proliferation and survival, thus rendering MKK3 a promising target for anticancer therapies. Here, we found that targeting MKK3 with RNA interference, in both wild-type (wt) and mutp53-carrying cells, induced endoplasmic reticulum stress and autophagy that, respectively, contributed to stabilize wtp53 and degrade mutp53. MKK3 depletion reduced cancer cell proliferation and viability, whereas no significant effects were observed in normal cellular context. Noteworthy, MKK3 depletion in combination with chemotherapeutic agents increased tumor cell response to the drugs, in both wtp53 and mutp53 cancer cells, as demonstrated by enhanced poly (ADP-ribose) polymerase cleavage and reduced clonogenic ability in vitro. In addition, MKK3 depletion reduced tumor growth and improved biological response to chemotherapeutic in vivo. The overall results indicate MKK3 as a novel promising molecular target for the development of more efficient anticancer treatments in both wtp53- and mutp53-carrying tumors.

Activation of p53 By Novel MDM2 Antagonist RG7388 Overcomes AML Inherent and Acquired Resistance to Bcl-2 Inhibitor ABT-199 (GDC-0199)

Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities.Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014).Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML.Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML.Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s > 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s > 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h.Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems.Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. DisclosuresNichols:Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.

Abstract B47: Blockade of MDM2 function synergizes with front-line chemotherapy to promote cell death in p53-wildtype and p53-mutant MYCN-amplified neuroblastoma cells

Abstract Neuroblastoma (NB) arises in the sympathetic nervous system and is the most common extracranial solid tumor of childhood. While treatment outcome for NB has improved over the years, the survival of children with disseminated, high-risk NB tumors is less than 50%. To improve treatment outcome in these patients, inhibition of murine double minute-2 (MDM2) function in the context of front-line therapy used to treat NB (cisplatin and etoposide) is being explored. MDM2 is a multi-functional protein that plays a critical role in controlling cell growth, cell migration, and the DNA damage response. Moreover, MDM2 is a downstream target of MYCN which can be commonly amplified in high-risk NB. The objective of the present study was to evaluate the extent to which the MDM2 antagonist nutlin3a alone or in combination with front-line therapies, could augment cell death in NB cells with clinically relevant molecular profiles. Cisplatin and etoposide are currently used in the clinic as front-line therapy, and when administered alone produced dose-related increases in cell death in SK-N-SH (wtp53, MYCN-nonamplified), IMR32 (wtp53, MYCN-amplified), SK-N-FI (mtp53, MYCN-nonamplified) and SK-N-DZ (mtp53, MYCN-amplified) NB cell lines. Nutlin3a also produced dose-related increases in cell death irrespective of p53 status, suggesting that p53-independent mechanisms, may play a role in increasing cell death produced by blockade of MDM2-mediated signaling. However, IC50 values for nutlin3a administered alone in the absence of DNA damage were &amp;gt;10x higher in the mtp53 cell lines compared with the wtp53 cell lines, suggesting that mechanisms other than MDM2-blockade could also be involved in promoting death in mtp53 NB cells. Using isobolographic analysis and broad ranges of compound ratios, we found that nutlin3a and cisplatin were moderately synergistic in wtp53 MYCN-nonamplified SK-N-SH and markedly synergistic in wtp53 MYCN-amplified IMR32 cells. Moreover, nutlin3a and etoposide were also moderately synergistic in these wtp53 cell lines. In mtp53 NB cells, nutlin3a was also synergistic with both cisplatin and etoposide irrespective of MYCN status. Interestingly, cisplatin and etoposide, which are frequently used together clinically, were strictly additive in all four cell lines by isobolographic analysis. Pharmacodynamic analysis was conducted in IMR32 cells (wtp53, MYCN-amplified) since this molecular profile is commonly found in high-risk NB. IMR32 cells were treated with vehicle, nutlin3a, cisplatin, and the combination of cisplatin/nutlin3a, and activation of p53 and the status of signaling pathways that regulate cell growth were assessed by Western analyses. Optimal activation of the p53 pathway was observed by 24-48 hours post-treatment which correlated with a robust downregulation of MYCN and the anti-apoptotic protein survivin. These data suggest that a combination therapy that incorporates efficient modulation of the MDM2 pathway and builds upon front-line therapies enhanced cell death of neuroblastoma cells that are highly resistant to conventional therapies. This combinatorial approach that builds upon front-line therapy is now under evaluation in orthotopic NB xenograft models. Citation Format: Karen E. Pollok, Barbara J. Bailey, Kania Katarzyna, Lawrence M. Gelbert, Harlan E. Shannon. Blockade of MDM2 function synergizes with front-line chemotherapy to promote cell death in p53-wildtype and p53-mutant MYCN-amplified neuroblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B47.

Abstract 4334: Greater resistance of mutant p53 tumor cells to hypoxia or chloroquine with glucose or pyruvate supplementation is diminished under glucose starvation

Abstract BACKGROUND. Poorly vascularized solid tumors are exposed to regions of low oxygen and glucose deprivation. These restrictions may promote either defensive autophagy or cell death through excessive reactive oxygen species (ROS), mediated by activated NADPH oxidases (NOX) which transport electrons from NADPH on the cytoplasmic side of plasma membranes to reduce molecular oxygen at the extracellular space, increasing superoxide anion radical (O2•-). .OBJECTIVES. To investigate in genetically-matched human melanoma cells with wt or mutant p53, responses to: a) autophagy inhibitor chloroquine and to glucose-lowering metformin or to hypoxia in glucose-supplemented medium; b) pyruvate in glucose-free medium under normoxia or hypoxia; c) sub-physiological glucose. EXPERIMENTAL. Responses to normoxia or hypoxia were assesed by live-dead fluorometric assays, or by crystal violet staining of surviving cells. Mechanisms influencing response to glucose limitation were studied by specific immune blotting. RESULTS. Lower susceptibility to chloroquine ± metformin involving greater basal AMPK activation was seen in physiological 5 mM glucose in mutant p53 cells. These cells were also preferentially protected from prolonged hypoxia by glucose supplementation or by pyruvate supplementation of glucose-free medium under normoxia, but not under hypoxia. Sub-physiological glucose without pyruvate induced apoptosis-associated PARP cleavage irrespective of p53 status. However, treatment with impermeable exogenous superoxide dismutase (SOD) to remove extracellular superoxide signalling across cell membranes, preferentially counteracted PARP cleavage only in wt p53 cells with lower LDHA expression after 48 hr in 2.5 mM glucose. CONCLUSIONS. Glucose availability increases resistance to hypoxia or to chloroquine ± metformin in mutant p53 cells. However, glucose starvation promotes toxicity linked to extracellular (O2•-), preferentially in mutant p53 cells. Citation Format: Manuel Rieber. Greater resistance of mutant p53 tumor cells to hypoxia or chloroquine with glucose or pyruvate supplementation is diminished under glucose starvation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2014-4334

Abstract 4556: A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells

Abstract Wee1 is an essential component of the G2-M cell cycle checkpoint machinery, providing a target for Wee1 inhibitors (e.g., MK1775) designed to potentiate the anti-tumor activity of genotoxic agents via mitotic lethality. In this setting, Wee1 inhibitor actions are primarily p53-dependent. However, recent studies have identified novel S-phase functions of Wee1 that operate independently of p53 status. Such findings present a rationale for combining Wee1 inhibitors with other targeted agents such as HDAC inhibitors (HDACIs) which interrupt DNA replication and repair. To test this hypothesis, interactions between MK1775 and the HDACIs SBHA or vorinostat were investigated in AML cells. Significantly, MK1775 interacted highly synergistically with either HDACI in both p53 wild type (wt) or mutant AML cells, manifested by pronounced apoptosis induction and attenuation of clonogenic survival. Consistent with these findings, shRNA knock-down of Wee1 significantly sensitized cells to HDACIs. Immunobloting revealed that HDACIs markedly enhanced Wee1 inhibition by MK1775, reflected by diminished Wee1 S642 phosphorylation and protein levels, as well as cdc2/Cdk1 Y15 phosphorylation, a direct downstream Wee1 kinase target. In p53-null (U937) or -mutant (MV-4-11) cells, MK1775 triggered S-phase arrest, whereas HDACI co-administration resulted in early S-phase accumulation accompanied by a marked increase in sub-G1 cells. In sharp contrast, in p53 wt MOLM-13 cells, HDACIs clearly induced G2/M arrest, an event abrogated by MK1775, arguing for context-specific cell cycle effects of this strategy. Interestingly, while MK1775/HDACI synergism persisted, shRNA p53 knock-down in p53 wt AML-3 cells partially attenuated lethal effects of combined treatment. Nevertheless, MK1775/HDACI co-treatment strikingly increased DNA damage, manifested by dramatically increases in γH2A.X generation, in both p53 wt and mutant cells. This event was associated with profound caspase activation and PARP cleavage. Notably, the MK1775/HDACI regimen was highly active against patient-derived AML cells harboring either wt or mutant p53, as well as various other mutations in cancer hotspot genes including FLT3, NRAS, KRAS, BRAF, PDGFRA, MET, STK11, SMARCB1, and APC. Furthermore, the CD34+/CD123+/CD38- population, enriched for leukemia-initiating progenitors, but not normal CD34+ hematopoietic cells, was highly susceptible to the Wee1/HDAC inhibition strategy. Finally, co-administration of MK1775 with vorinostat substantially suppressed tumor growth and significantly prolonged animal survival in a murine AML xenograft model. Together, these findings indicate that a strategy combining Wee1 inhibition (e.g., by MK1775) with HDACIs is highly active towards AML cells, including primitive progenitors, through context-specific p53-dependent and -independent mechanisms. Citation Format: Yu Zhang, Liang Zhou, Shuang Chen, Maciej Kmieciak, Hui Lin, Yun Dai, Steven Grant. A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2014-4556