ALBERT

Description: Abstract 3415 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor (TKI) has revolutionized the treatment of patients with CML. Development of resistance and limited activity in blast crisis (BC) CML are evolving problems facing this therapy. We found that XIAP, a potent caspase inhibitor, is highly expressed in CML cells, in both, cell lines and patient samples. Treatment with imatinib deceased XIAP levels in imatinib-sensitive KBM5 but much less so in imatinib-resistant KBM5STI571 cells (harboring T315I mutation) suggesting that XIAP expression in CML is regulated at least in part via Bcr-Abl and that targeting XIAP may promote cell death in CML cells by circumventing imatinib resistance. To test this, we treated BC CML cells with XIAP antisense oligonucleotide (ASO) and with SMAC mimetic ABT-10 and found that inhibition of XIAP induced apoptotic cell death with similar efficacy in KBM5 cells and KBM5STI517 cells (EC50=6.3±0.3 μM and 8.4±0.4 μM at 48 hours, respectively for ABT-10). However, we noted that inhibition of XIAP by ASO induced the expression, in both KBM5 and KBM5STI571 cells, of apoptosis repressor with caspase recruitment domain (ARC) in both mRNA and protein levels but not the expression of Bcl-2 protein. ARC is a unique antiapoptotic protein. It acts through inhibiting caspases and antagonizing the activity and function of p53 and Bax. Therefore, its induction may antagonize the effect of XIAP downregulation. Indeed, inhibition of both XIAP and ARC by ASO induced significantly more cell death than inhibiting either protein alone in both KBM5 and KBM5STI cells. Furthermore, we demonstrated that XIAP inhibition induced-apoptosis was enhanced by imatinib in KBM5, but not in KBM5STI cells. Interestingly, inhibition of Bcr-Abl tyrosine kinase by imatinib not only decreased XIAP, but also suppressed ARC levels in KBM5 but had minimal effects on the levels of these proteins in KBM5STI571 cells and enforced expression of the Bcr-Abl p185 fusion protein (in HL-60 cells) greatly increased both XIAP and ARC levels. This induction was inhibited by imatinib suggesting that ARC is also a downstream target of Bcr-Abl tyrosine kinase. Therefore, imatinib enhancing XIAP inhibition induced-apoptosis in KBM5, not KBM5STI cells can be explained at least in part by its ability to decrease XIAP and ARC levels. In conclusion, XIAP is highly expressed in CML cells and upregulated by Bcr-Abl. Targeting XIAP promotes death of BC and TKI resistant CML cells. Results suggest that XIAP is a potential target in BC and TKI resistant CML cells and that XIAP inhibition-induced apoptosis is enhanced by imatinib in TKI sensitive cells and by ARC inhibition independent of cellular responses to TKIs. Inhibition of XIAP and ARC as a novel therapeutic strategy in CML warrants further investigation. Disclosures: Koller: Isis Pharmaceuticals: Employment.

Description: p53, a key regulator of apoptosis, functions primarily upstream in the apoptotic cascade by directly and indirectly modulating Bcl-2 family of proteins. XIAP, a potent antiapoptotic protein, functions primarily downstream by suppressing caspases. Activation of p53 by MDM2 antagonist nutlin3a or inhibition of XIAP by small molecule inhibitors such as phenylurea 1396-11 was found to induce apoptosis in AML cells. Since the functions of XIAP and p53 are mediated and their activities controlled by a network of numerous components, some of which cross-regulate each other, we hypothesized that simultaneous activation of p53 and inhibition of XIAP would be a more effective at activating apoptotic signaling in AML cells. To test this idea, we treated AML cells with nutlin3a and 1396-11 and found that the combination synergistically induced cell death at 24 hours in OCIAML3 cells (combination index CI=0.200±0.047) and Molm13 cells (CI=0.565±0.082), two cell lines harboring wild type p53. Knocking down p53 expression by shRNA blunted the synergistic effect and downregulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin3a-induced apoptosis in OCI-AML3 cells, suggesting that the synergy was mediated by both p53 activation and XIAP inhibition. The specificity was further supported by data showing that inhibition of MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Although nutlin3a alone induced apoptosis in OCI-AML3 cells, the cell death was not robust and caspase-3 activation was minimal by itself even at 48 hours with 10 μM of nutlin3a. Immunoblot analysis showed increased expression of p53 and its downstream target p21. Of note, because p21 not only induces G1 cell cycle block, it additionally exhibits antiapoptotic activity that diminishes the effects of p53 activation, we also studied effects of these agents on p21 levels. When nutlin3a and 1396–11 were combined, caspase-3 activation was greatly increased and nutlin3a-induced p21 expression was significantly diminished. Moreover, in these experiments, caspase inhibition restored p21 levels and diminished apoptosis enhanced by 1396-11, suggesting that XIAP inhibition-mediated caspase activation eliminates p21, enhancing nutlin3a-induced apoptosis. Furthermore, activation of p53 by nutlin3a increased caspase-6 protein levels and induced mitochondrial release of SMAC, an antagonist of XIAP, suggesting that p53 activation shifts the balance toward apoptosis, promoting the effect of XIAP inhibition. Most importantly, p53 activation and XIAP inhibition greatly enhanced apoptosis in primary blasts from AML patients. Five out of six samples treated showed synergistic killing at 24 hours (CI=0.73±0.13), even when the cells were protected from drug-induced and spontaneous apoptosis by MS-5 stroma cells (CI=0.45±0.06). In conclusion, results demonstrate that simultaneous activation of p53 by antagonizing MDM2 and inhibition of XIAP synergistically activate apoptotic signaling pathways and promote death of AML cells, in part by modulating p21, caspases, and cytosolic SMAC levels. Since both, XIAP and p53, are presently being targeted by ongoing clinical trials in leukemia patients, the combination strategy holds promise for expedited translation into the clinic.

Description: Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34+38− AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk blasts and total CD34+ AML cells (P 〈 .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Description: Imatinib, a Bcr-Abl tyrosine kinase inhibitor has revolutionized the treatment of patients with CML. However, resistance develops due to Bcr-Abl gene mutations and various other mechanisms. Although second generation Bcr-Abl inhibitors can overcome most of the mutation driven resistance, they cannot overcome other resistance mechanisms. Furthermore, Imatinib has limited effectiveness in patients with blast crisis (BC) CML. We have previously shown that triptolide, an anti-cancer agent isolated from a Chinese herb, potently induces apoptosis in AML cells in part by decreasing the levels of XIAP and Mcl-1, two potent antiapoptotic proteins. Here we investigated its effect on Philadelphia chromosome positive (Ph+) cells and found that at low nM concentrations, triptolide induced significant cell death in K562 (IC50=113.4±3.9 nM) and KBM5 (IC50=30.0±2.1 nM) cells, two cell lines derived from BC CML patients, as well as in ALL-1 cells (IC50=113.8±1.4 nM), a cell line derived from Ph+ ALL. Interestingly, KBM5-STI571 cells, an Imatinib resistant KBM5 subline bearing the T315I mutation which is resistant to most available Bcr-Abl tyrosine kinase inhibitors, were as sensitive as KBM5 cells to triptolide. Likewise, triptolide killed Ba/F3 cells harboring BCR-ABL mutants (E255K and T315I) with similar efficacy as Ba/F3 cells carrying the wild type BCR-ABL gene. We then treated 8 samples from 7 CML patients with blasts ranging from 10–91% with triptolide (up to 100 nM) in vitro. Triptolide induced cell death in all samples tested. Importantly, 6/7 samples were from patients resistant/relapsed after Imatinib. Three were also nonresponsive to Nilotinib and one to neither Nilotinib nor Dasatinib. Next we tried to elucidate the possible apoptosis regulators involved in triptolide-induced cell death. Triptolide decreased antiapoptotic XIAP, Mcl-1 and Bcr-Abl protein levels in K562 cells and in blast cells from CML patients. Based on this observation, we treated CML cells with both triptolide and Imatinib. The combination synergistically induced cell death in K562 cells (CI=0.50±0.14). In KBM5 cells, Imatinib antagonized rather than enhanced triptolide when administrated simultaneously: Triptolide alone induced cell death with IC50=24.3±2.8 nM at 48 hours, while in combination with 1 μM Imatinib, the IC50 increased to 82.9±4.1 nM. This is probably due to the fact that Imatinib primarily blocks KBM5 cells in G0/G1 and that resting cells were less sensitive to triptolide. We therefore pretreated KBM5 cells with triptolide for 24 hrs followed by 1 μM Imatinib for 24 hrs. This sequential treatment was more effective to induce cell death in KBM5 cells (IC50=15.4±0.6 nM). Triptolide did not sensitize Imatinib resistant KBM5-STI571 cells. Conclusion: Results suggest that triptolide potently induces cell death in BC CML cells and that the cell death induced by triptolide is independent of response to Imatinib or other second generation Bcr-Abl kinase inhibitors. Triptolide could be of potential benefit to CML patients in blast crisis and CML patients failing Bcr-Abl tyrosine kinase inhibitors.

Description: Abstract 2626 Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012). ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC's roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P

Description: Acute myeloid leukemia (AML) is organized in a hierarchy with a rare population known as leukemia stem cells (LSC) capable of self-renewal and propagation of the disease. Characterization of the unique phenotypes and complex signaling pathways in LSCs that survive induction chemotherapy is essential for understanding of the mechanisms of chemoresistance and designing the strategies to eliminate residual leukemia clones. In this study, we compared signaling profiles of distinct phenotypic AML subsets in paired bone marrow (BM) samples collected at diagnosis and after achieving the complete remission (CR). Cell surface characteristics and signaling pathways activated within sub-populations of AML samples were defined using the novel technology of time-of-flight mass cytometry (CyTOF) that has the ability to perform up to 100 mutiparameter assays in single cells (Bendall et al, Science 2011). First, we validated CyTOF measurements by performing cross-comparisons of surface markers and intracellular proteins measured in AML cells with traditional multi-parametric flow cytometry (FCM). Frequencies of CD123+CD99+ population within CD34+CD38- cells were 73.7%±1.8% and 78.5%±3.7% by CyTOF and FCM. Patterns of specific activation of the intracellular proteins pSTAT5, pERK1/2 and pAKT by GM-CSF, PMA and SCF, and inhibition by selective kinase inhibitors showed excellent cross-platform consistency between CyTOF, FCM and immunoblotting. Next, mononuclear cells of 5 paired AML (at diagnosis and in CR) and of 3 normal BM (NBM) were stained with 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). A SPADE (spanning-tree progression analysis of density-normalized events) (Qiu et al, Nat Biotechnol. 2011) tree plot was generated, representing clustered expression of the cell-surface antigens. Boundaries and annotations of the AML cells were manually defined to represent distinct cell subsets (Figure 1). We used the pooled data from NBM samples, which showed identical patterns, as a reference. SPADE analysis revealed several subsets unique to the diagnostic AML samples, which were eliminated by chemotherapy; and phenotypically distinct subsets in diagnostic samples that persisted in CR. Notably, a subset defined by the “traditional LSC” markers (CD45dimCD34+CD38lowCD90-CD33-CD117+; annotation #2) was readily identified in diagnostic samples and was significantly reduced by induction chemotherapy in 2 of the 5 AML samples. In one of these samples we identified a distinct subset co-expressing LSC markers CD45dimCD34+CD38lowCD33-CD117-CD99lowCD133low (annotation #3) that was present in both diagnostic (1.1%) and CR (1.7%) BM; this subset may have contributed to the MRD detected by standard leukemia-associated immunophenotypes.Figure 1The tree plot was generated using 11 cell surface proteins in AML and NBM, and colored by the median intensity of individual markers (CD34 is shown). Phenotypes of each annotation are indicated.Figure 1. The tree plot was generated using 11 cell surface proteins in AML and NBM, and colored by the median intensity of individual markers (CD34 is shown). Phenotypes of each annotation are indicated. We next investigated intracellular signaling pathways in antigen-defined AML subpopulations using CyTOF. Activation of p-AKT and pS6 showed similar pattern in subsets defined by annotations 1, 9 and 10 at diagnosis (Figure 2A), and was largely reduced in the CR BM. In turn, activation of p-4EBP1 and p-mTOR were observed in multiple subsets (#1-5 and 9-11) in all diagnostic AML samples, especially in a subset 1 characterized by the “Progenitor” phenotype, and remained heightened in the CR samples (Figure 2B).Figure 2The heat map of the average expression of intracellular proteins in selected populations from individual samples. (A) Each column represents individual sample, and each row reflects expression of a certain protein for each annotation. (B) Signaling pathways in annotation #1 in individual samples.Figure 2. The heat map of the average expression of intracellular proteins in selected populations from individual samples. (A) Each column represents individual sample, and each row reflects expression of a certain protein for each annotation. (B) Signaling pathways in annotation #1 in individual samples. In summary, using CyTOF and SPADE, we characterized phenotype-specific intracellular signaling pathways in AML samples at diagnosis and in CR. Persistent activation of p-mTOR and p-4EBP1 are identified in the subpopulations of AML progenitors in CR, and may present the potentially targetable pathways in AML. The study is ongoing with prospective CyTOF analysis of a larger set of paired AML samples at diagnosis, CR and relapse coupled with the molecular analysis of the distinct subpopulations. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2177 The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL. Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy. Disclosures: No relevant conflicts of interest to declare.