Novel Small Molecule MDM2 Inhibitor MI-63 Induces p53-Dependent Apoptosis in AML Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2596-2596
Author(s):  
Ismael Samudio ◽  
Martin Dietrich ◽  
Paul Corn ◽  
Dajun Yang ◽  
Gautam Borthakur
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Wild Type ◽  
Wild Type P53 ◽  
Induced Apoptosis ◽  
Related Proteins ◽  
Mdm2 Inhibitor

Abstract Although TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). We investigated the effects of MI-63, a small molecule that activates p53 by inhibition of MDM2-p53 interaction [ Ki value of 3 nM (J Med Chem.2006;49(12):3432–5)] in AML cell lines. Treatment with MI-63 triggered apoptosis (evidenced by loss of membrane potential and externalization of phosphatidylserine) in AML cell lines with wild-type p53 (OCI-AML-3 and MOLM13) in a time and concentration-dependent manner (IC50 at 72 hrs.= 2.5 μM for OCI-AML-3 and 1 μM for MOLM-13), while a p53-null AML cell line (HL-60) was resistant (IC50 not reached at 10 μM). Moreover, knockdown of p53 in OCI-AML3 cells rendered this cell line resistant to MI-63 induced apoptosis while control vector infected OCI-AML-3 cells remained as sensitive to MI-63 similar to the parental cells. Mechanistic studies showed that MI-63 blocks G1/S phase transition in AML cells with wild-type p53 resulting in accumulation of cells in G1 phase (percentage cells inG1 phase at 24 hrs. = 88.66% vs 43.49% in cultures with DMSO control) while MI-61, a skeletally related but inactive control compound failed to do so (41.63%). Treatment with MI-63 increased cellular levels of p53 and p53 dependent proteins in OCI-AML-3 cells that include p21 and BH3-only pro-apoptotic protein Puma and pro-apoptotic multi-domain Bcl-2 family member Bax. Additionally, MI-63 induced a profound decrease in the levels of MDM4, an MDM2 homolog that has been reported to mediate resistance to the effects of nutlin-3a, suggesting that MI-63 may offer a therapeutic advantage in cells expressing high levels of MDM4. Finally, supporting the concept that increased levels of p53 modulate the apoptotic rheostat both directly, by behaving as a BH3-only protein, and indirectly by increasing the levels of sensitizer BH3-only proteins, MI-63 potently synergized with AT-101, an orally available pan inhibitor of Bcl-2, Bcl-xL and Mcl-1 (currently being evaluated as an antitumor agent in Phase I/II trials by Ascenta Therapeutics), to induce mitochondrial dysfunction and apoptosis in OCI-AML-3 cells (average combination index = 0.055±0.019). Taken together our results support preclinical evaluation of novel small molecule MI-63 alone and in combination with Bcl-2 inhibitors for the therapy of AML. The studies in primary AML samples are ongoing. Fig.1: MI-63 Induced Apoptosis Requires Intact p53 Fig.1:. MI-63 Induced Apoptosis Requires Intact p53 Fig.2: Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included) Fig.2:. Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)

The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5120-5120
Author(s):  
Hatice Demet Kiper ◽  
Burcin Tezcanli Kaymaz ◽  
Ozlem Purclutepe ◽  
Ceyda Tunakan Dalgic ◽  
Nur Selvi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Drug Exposure ◽  
Conflicts Of Interest ◽  
Time Dependent ◽  
Blue Dye ◽  
Dependent Manner ◽  
Down Regulation ◽  
Expression Levels ◽  
Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

ARF Loss, a Negative Prognostic Factor in Philadelphia-Positive Acute Lymphoblastic Leukemia, May Be Efficiently Overcome by the Small Molecule MDM2 Antagonist RG7112

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2574-2574
Author(s):  
Ilaria Iacobucci ◽  
Federica Cattina ◽  
Silvia Pomella ◽  
Annalisa Lonetti ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Prognostic Impact ◽  
Wild Type ◽  
Mdm2 Antagonist ◽  
Wild Type P53

Abstract Abstract 2574 Recently, using genome-wide single nucleotide polymorphism arrays and gene candidate deep exon sequencing, we identified lesions in CDKN2A gene, encoding p16/INK4A and p14/ARF tumor suppressors, in 27% (32/117) adult newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients and in 47% (14/30) relapsed cases. Clinically, in our cohort CDKN2A deletions were associated by univariate analysis to a worse outcome in terms of overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) (OS: 27.7 vs 38.2 months, p = 0.0206; DFS: 10.1 vs. 56.1 months, p = 0.0010; CIR: 73.3 vs 38.1, p = 0.0014). Noteworthy, the negative prognostic impact of CDKN2A deletion on DFS was also confirmed by the multivariate analysis (p = 0.0051). These results showed that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation and tumor cell growth by the action of MDM2, a negative regulator of p53, we investigated the preclinical activity of the MDM2 antagonist RG7112 in primary B-ALL patient samples and leukemic cell line models. BV-173, SUPB-15 and K562 Ph+ cell lines were incubated with increasing concentration of RG7112 (0.5–10 μM) and its inactive enantiomer for 24, 48 and 72 hours (hrs). MDM2 inhibition by RG7112 resulted in a dose and time-dependent cytotoxicity with IC50 (24 hrs) of 2 μM for BV-173 and SUPB-15 which harbor homozygous deletion of CDKN2A but wild-type p53. No significant changes in cell viability were observed in K562 p53-null cell line after incubation with RG7112. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/Propidium Iodide analysis showed a significant increase in apoptosis after 24 hrs in BV-173, SUPB-15 and in primary leukemia blasts, whereas no apoptosis was observed in K562 cells. To examine the possible mechanisms underlying RG7112-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased upon treatment with RG7112 after 24 hrs of incubation with concentrations equal to the IC50. These data demonstrate the ability of RG7112 to activate the intrinsic apoptotic pathway by a p53-dependent mechanism. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was next performed, comparing sensitive cell lines (BV-173 and SUPB-15) after 24 hrs exposure to 2 μM RG7112 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). They include genes involved in cell cycle and apoptosis control (e.g. Histone H1, TOP2, GAS41, H2AFZ) and in the down-regulation of the Hedgehog signaling (e.g. BMI1, BMP7, CDKN1C, POU3F1, CTNNB1, PTCH2) with a strong repression of stemness genes and re-activation of INK4/ARF as illustrated in Figure 1. Actually, both GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. In our data they were strongly down-regulated (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells, suggesting that these genes have a potential as new biomarkers of activity. In conclusion, inhibition of the p53–MDM2 interaction by RG7112 can activate the p53 pathway, resulting in apoptosis and inhibition of stemness genes in B-ALL with wild-type p53. Our findings provide a strong rational for further clinical investigation of RG7112 in Ph+ ALL. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione–Università 2007–2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

scholarly journals The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness

2001 ◽  
Vol 155 (7) ◽  
pp. 1129-1136 ◽  
Author(s):  
Larissa Kotelevets ◽  
Jolanda van Hengel ◽  
Erik Bruyneel ◽  
Marc Mareel ◽  
Frans van Roy ◽  
...  
Keyword(s):  
Cell Line ◽  
Phosphatase Activity ◽  
Cell Lines ◽  
Dependent Manner ◽  
Glioblastoma Cell ◽  
Wild Type ◽  
Invasive Phenotype ◽  
Lipid Phosphatase ◽  
Glioblastoma Cell Lines ◽  
E Cadherin

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell–cell aggregation, and suppressed the invasive phenotype in an E-cadherin–dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin–neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

ONC201 Exerts p53-Independent Cytotoxicity Through TRAIL and DR5 Induction In Mantle Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3822-3822
Author(s):  
Jo Ishizawa ◽  
Kensuke Kojima ◽  
Archana Dilip ◽  
Vivian R Ruvolo ◽  
Bing Z Carter ◽  
...  
Keyword(s):  
Cell Lines ◽  
Combination Index ◽  
Synergistic Effects ◽  
Pcr Analysis ◽  
Mutant P53 ◽  
Drug Resistant ◽  
Wild Type ◽  
Cytotoxic Effects ◽  
Wild Type P53 ◽  
Induced Apoptosis

Abstract ONC201 (TIC10) is a novel small molecule that induces TRAIL-dependent apoptosis in various cancer cell types and is under development to enter a first-in-man study in advanced cancer patients .It was identified in a screen for small molecules capable of up-regulating endogenous TRAIL gene transcription in a p53-independent manner (Allen JE et al, Sci Transl Med., 2013). ONC201 triggers FOXO3a activation through dual inhibition of ERK and AKT, which transcriptionally upregulates TRAIL and TNFRSF10B (TRAIL-R2/DR5) in solid tumors. Because PI3K/AKT and MEK/ERK activation have been shown to be major contributors to drug resistance, ONC201 is potentially promising since it not only promotes TRAIL activation, but also upregulates its pro-apoptotic receptor DR5. Here we report the anti-lymphoma effects of ONC201 in MCL, a presently incurable disease. We treated three human MCL cell lines with wild-type p53 (Z-138, JVM-2, and Granta-519) and two similar lines with mutant p53 (MINO and Jeko-1) with ONC201. A 72-hour ONC201 treatment induced apoptosis in all MCL cell lines. Surprisingly, the p53 mutant MINO and Jeko-1 cells were more susceptible in apoptosis assays to ONC201 than cells with wild-type p53 (Fig.1) The effective concentrations inducing cell killing (as measured by annexin V positivity) in 50%/75% of the cells in the Z-138, JVM-2, MINO, Jeko-1, and Granta-519 cells were 9.9/>10, >10/>10, 2.6/5.2, 2.7/4.6 and >10/ >10 micromolar, respectively. We also treated five primary human MCL samples (three with wild-type p53 and two with mutant p53), and found that one of the two mutant p53 samples was highly sensitive to ONC201 as were the three samples with wild-type p53. One mutant p53 sample that was less sensitive to ONC201, was also resistant to Nutlin-3a and Ibrutinib suggesting an extremely drug-resistant phenotype. Real-time PCR analysis revealed that both DR5 and TRAIL mRNAs were transcriptionally upregulated in the primary MCL samples (a relative ratio of 7.25 compared to 3.13 in controls) after 72-hour treatment with ONC201. To determine the significance of p53 functional status in ONC201-induced apoptosis, p53 wild-type Z-138 and JVM-2 cells were stably transduced with lentivirus encoding either negative control shRNA or p53-specific shRNA and were exposed to ONC201 and results demonstrated complete p53-independence. Normal human bone marrow cells and mesenchymal stem cells were completely resistant to the cytotoxic effects of ONC201, which illustrated this agent's low toxicity against normal tissues. In order to examine the role of p53 activation in ONC201-induced apoptosis in MCL cells, we combined ONC201 with the MDM2 inhibitor Nutlin-3a. The combination cytotoxic effects of this combination were synergistic in p53 wild-type Z-138 and JVM-2 cells (combination index 0.87 and 0.63, respectively). Similar synergistic effects of ONC201 combined with the BTK inhibitor Ibrutinib were observed in Z-138 and MINO cells (combination index 0.63 and 0.61, respectively). This combination also triggered synergistic apoptotic effects in two primary MCL samples with combination indexes of 0.0011 and 0.073, respectively. Conclusion ONC201 induces p53-independent apoptosis in MCL cells, and may have significant clinical impact by targeting both p53 wild type and p53 mutant drug-resistant MCL cells. ONC201 exerts synergistic effects with MDM2 and BTK inhibitors that may be explored clinically. Disclosures: Allen: Drug Company: Employment. Andreeff:Oncoceutics: SAB Other.

Preclinical Evaluation of AT219, a Small Molecule Inhibitor of MDM2 As an Anti-Myeloma Agent.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2948-2948
Author(s):  
Vijay G. Ramakrishnan ◽  
Teresa K. Kimlinger ◽  
Timothy Halling ◽  
Jessica Haug ◽  
Utkarsh Painuly ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Primary Cells ◽  
Wild Type ◽  
Drug Induced ◽  
Molecule Inhibitor ◽  
Wild Type P53 ◽  
Stock Solutions

Abstract Abstract 2948 Background: Deletions and mutations in the tumor suppressor protein p53 are an uncommon observation in new multiple myeloma (MM) patients and are observed more commonly in patients with advanced disease. p53 deletion has been observed to correlate with poor overall and progression free survival in MM patients. Wild type p53 modulates the expression levels of a broad array of proteins involved in cell cycle progression, apoptosis ultimately leading to cell cycle arrest and apoptosis. p53 is negatively regulated by MDM2. MDM2 binds to and ubiquitinates p53 marking it for proteasomal degradation. In addition, MDM2 is a direct downstream regulator of p53. Targeting the p53-MDM2 interaction by developing agents that bind to the p53 binding motif of MDM2 and reactivating p53 has therefore been an active area of research. Here, we present results from our pre-clinical studies using AT219, a small molecule inhibitor that binds to MDM2 preventing its interaction with p53. Methods: AT219 was obtained from Ascenta Therapeutics. Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Akt1/2 kinase inhibitor (Akti) was purchased from Sigma. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Mitocapture and cytochrome-c assays were also performed to confirm the induction of apoptosis in MM cell lines. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various time points. Results: AT219 induced potent cytotoxicity in MM cell lines MM1S, MM1R and H929, all three expressing wild type p53 with IC50 values of 2.5–5μM. Similar effects were observed when the above mentioned cell lines were treated with AT219 and the inhibitory effect of proliferation of these cells were examined. When MM1S or H929 cells were cultured with bone marrow stromal cells (BMSCs) derived from MM patients or with one of the three tumor promoting cytokines implicated in MM (IL6, IGF or VEGF) and treated with AT219, the drug was able to inhibit the proliferation of both cell lines to similar extents as observed when cultured independently without BMSCs or the cytokines. The increase in cytotoxicity was found to be due to cells undergoing apoptosis as observed when MM1S or H929 cells were cultured with AT219 and % apoptotic cells were measured as measured by annexin/PI, mitocapture and cytochrome c assays. AT219 was also observed to induce more potent apoptosis in primary cells obtained from new MM patients with wild type p53 than in cells obtained from relapsed MM patients with wild type p53. AT219 clearly upregulated p53 as observed by performing immunoblots after treatment with the drug in MM1S and H929 cells. In addition, MDM2 and p21 were also found to be significantly upregulated and Bax was slightly upregulated post drug treatment. Bcl2, Mcl1 and Xiap levels were down regulated. In MM1S cells AT219 treatment resulted in a slight down regulation of pAkt (Ser 473). However, in H929 cells we observed a transient upregulation of pAkt following AT219 treatment. This prompted us to test AT219 in combination with Akti on MM cell lines. Our results on both MM1S and H929 cells using AT219 in combination with Akti demonstrated synergy. We are currently testing this combination in primary cells drawn from MM patients with both wild type p53 and those with p53 deletions and mutations. Conclusions: Our studies validate the anti-MM activity of AT219 in MM patients with wild type p53. In addition to using AT219 in combination with Akti, we are testing AT219 in combination with existing anti- MM chemotherapeutic agents. Interesting results from our studies will form the basis for clinical evaluation of AT219 as a single agent or in combination with an Akt inhibitor or other agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

scholarly journals Prognostic Impact and Targeting of CRM1 in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 870-870
Author(s):  
Kensuke Kojima ◽  
Steven M. Kornblau ◽  
Vivian Ruvolo ◽  
Seshagiri Duvvuri ◽  
Richard E Davis ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Annexin V ◽  
Dependent Manner ◽  
Apoptosis Induction ◽  
Wild Type ◽  
Ic50 Values ◽  
P53 Status ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Mdm2 Inhibitor

Abstract Abstract 870 p53 is a transcription factor that prevents abnormal cell growth. Cellular levels of p53 are critically regulated by MDM2, which is frequently over-expressed in AML. Nutlin-3a disrupts MDM2-p53 interaction, increases cellular levels of p53 in both nucleus and cytoplasm, and activates p53 signaling in cells. p53 status is the major determinant of response to MDM2 inhibitors. p53 is shuttled between the nucleus and the cytoplasm, and CRM1 mediates its nuclear export. Karyopharm Therapeutics has developed novel, potent and irreversible small molecule selective inhibitors of CRM1. We hypothesized that CRM1 inhibition would enhance the nuclear activity of p53, thereby enhancing p53-mediated transcription-dependent apoptotic signaling in AML. We measured CRM1 expression in primary AML samples and investigated if blockade of nuclear export of p53 by CRM1 inhibition would enhance MDM2 inhibitor-induced apoptosis in AML. CRM1 expression was investigated in 511 patient AML samples using a validated robust reverse-phase protein array. Higher levels of CRM1 were associated with higher marrow and peripheral blast percentages (P < 0.00001). Expression was lower in those with favorable cytogenetics compared to those with intermediate or unfavorable cytogenetics (P = 0.029). CRM1 levels were higher in patients with FLT3 mutations (P = 0.003). In 3-way correlation (using distance weighted least squares), there was a clear interaction with p53 levels being highest when CRM1 was high and MDM2 levels were low. Overall survival progressively worsened as CRM1 levels increased, with median survival of 66 weeks for those with CRM1 expression in the lowest third, 47 weeks for middle third and 37 weeks in the highest third (P = 0.007). CRM1 levels did not affect remission duration (P = 0.33). The CRM1 inhibitor KPT-185 exhibited dose-dependent anti-proliferative and cytotoxic activity in AML cell lines, as evidenced by low IC50 values and high Annexin V positivity (= low ED50 values). IC50 values for wild-type p53 cells ranged from 27 to 38 nM, and for mutant p53 cells from 48 to 112 nM, suggesting that KPT-185 potently inhibits AML cell growth largely independent of p53. In contrast, apoptosis induction by KPT-185 was much more prominent in p53 wild-type than in p53-defective cells: ED50 values for Annexin V induction were 150, 90 and 85 nM in p53 wild-type and > 1000 nM in 5 of 6 p53 mutant cell lines. Stable p53 knockdown (> 90% efficiency) rendered AML cells resistant to KPT-induced apoptosis. KPT-185 induced p53 target genes TP53I3, GDF15, MDM2 and ZMAT3 partially in a p53-dependent manner. Hence, p53 was identified as major determinant of CRM1 inhibition-induced apoptosis in AML. MDM2-inhibitor Nutlin-3a induced p53 in both nucleus and cytoplasm, while CRM1 inhibition accumulated p53 in the nucleus. Treatment with KPT-185 or Nutlin-3a caused time-dependent increase in cellular p53 levels. The KPT-185/Nutlin-3a combination induced p53 more efficiently than the individual agents by accumulating p53 exclusively in the nucleus, and synergistically induced apoptosis and cell death. p53 knockdown abrogated these synergistic effects. In primary AML cells, both KPT-185 (24.7 – 36.7% Annexin V) and Nutlin-3a (13.6 – 59.8%) induced apoptosis in a dose-dependent manner. Importantly, both KPT-185 and Nutlin-3a induced apoptosis in CD34+CD38- progenitor cell populations as effectively as they did in bulk AML cells, suggesting high sensitivity of CD34+CD38- cells to CRM1 inhibition and MDM2 inhibition. KPT-185 and Nutlin-3a synergized in the induction of apoptosis in both bulk and CD34+CD38- AML progenitor cells: combination index (CI) values were 0.26 (bulk) and 0.30 (CD34+CD38-) for ED50 and 0.93 (bulk) and 0.46 (CD34+CD38-) for ED75, indicating highly synergistic (CI < 1) efficacy in apoptosis induction. The relation between p53 status and sensitivity to Nutlin-induced apoptosis has been well established. Nutlin-resistant samples were much less sensitive to KPT-185 than Nutlin-sensitive cases (12.2 ± 0.06 % versus 30.9 ± 0.04 % Annexin V, P < 0.05). Synergistic induction of apoptosis was not observed in normal cord blood CD34+CD38- cells. Collectively, CRM1 inhibition offers a novel therapeutic strategy for AML that mostly retains wild-type p53. We propose to develop novel combinatorial approaches for the therapy of AML, aimed at maximal activation of p53 and apoptosis signaling by concomitant MDM2 and CRM1 inhibition. Disclosures: Shacham: Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics: Employment. Andreeff:Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other.

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