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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.

Abstract 1490 Background: The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML). In AML, p53 mutations have been associated with poor risk cytogenetics (i.e. complex karyotype, −5/−7). However, the function of p53 can also be compromised by protein stabilization and/or expression. The implications of p53 protein expression have not been studied in AML. Methodology: We assessed p53 expression by high-throughput reverse phase protein array (RPPA) technology in 511 pts (719 samples). Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1: 2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status was determined by Sanger sequencing of exons 5 through 9 of the p53 gene. Results: Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). Cases of AML-M3 and –M6 exhibited higher expression of p53 than other FAB subtypes. p53 expression directly correlated with age (p=0.01) and CD34 (p=0.001) and inversely correlated with WBC (p=0.007), BM (p=0.0001) and PB (p=0.0001) blasts, platelets (p=0.007), HLA-DR (p=0.01), CD19 (p=0.02), and survival (p=0.01). High p53 (p53high) expression level was more associated with unfavorable cytogenetics than with favorable or intermediate cytogenetics (p=0.00001). When all cytogenetic abnormalities were considered, pts with −5 had the highest levels of p53 (p=0.00001). Pts with RAS mutations, but not those with FLT3-ITD, NPM1, or IDH1/2, had lower levels of p53 protein. When pts were divided according to the level of p53 protein expression p53high was associated with lower complete remission (CR) rates (51% vs 56%; p=??) and higher relapsed rates (82% vs 62%; p=??). The median overall survival (OS) of pts with p53high and p53low were 29.8 vs. 51 wks (p=0.009). Most cases with p53high had unfavorable cytogenetics and the effect on OS was predominantly seen in that subpopulation with p53high and p53low pts living a medina of 23.4 vs. 36 wks (p=0.07), respectively. In order to determine whether the poor outcomes associated with p53high were due to the presence of a higher rate of p53 mutations among pts with p53high, we determined the p53 mutational status of 55 pts. p53high was highly correlated with the presence of p53 mutations as the latter were detected in 17/40 pts with p53high but in only 1/16 pts with p53low. Importantly, the presence of p53high, both in the presence (29 wks) or in the absence (24 wks) of p53 mutations, was associated with significantly worse overall survival compared with pts with p53low (56 wks; p=0.05, Figure 1). Multivariate analysis indicated that p53 is a significant independent risk factor for survival in AML. The final model included: age (p=0.000001), favorable cytogenetics (0.01), unfavorable cytogenetics (p=0.00001), WBC (p=0.0005), albumin (p=0.0003), FLT3-ITD (P=0.04), and P53 (P=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), c-MET (p=0.01), FoxO3a (p=0.004), KIT (p=0.001), p38p180p182 (p0.02), BAD (p=0.0001), cleaved PARP (p=0.002), cleaved PARP (p=0.01), TCF4 (p=0.02), fibronectin (p=0.02), and hsp70 (p=0.003), and negatively with AKTp473 (p=0.01), ERK (p=0.002), mTOR (p=0.005), PI3Kp85 (p=0.002), PKCδ (p=0.00002), GAB2 (p=0.00005), beclin (p=0.007), JMJD6 (p=0.001), Gata3 (p=0.02), p21 (p=0.01), and Mdm2 (p=0.001). Conclusions: Our results suggest that high levels of p53 protein constitute a powerful marker of short survival in AML. This effect is independent of p53 mutational status. The poor outcome of pts with high level of expression of p53 in the absence of p53 mutations suggests that the p53 pathway may be functionally perturbed in a much higher proportion of pts with AML than previously recognized. These data support the use of p53 protein expression levels in prognostication and in the development of targeted therapeutics. Disclosures: No relevant conflicts of interest to declare.