Bulk Blasts Research Articles

Abstract SY05-01: Genetic and non-genetic mechanisms contribute to long-term clonal growth dynamics and therapy resistance.

Abstract SY05-01: Genetic and non-genetic mechanisms contribute to long-term clonal growth dynamics and therapy resistance.

CD99 Identifies Disease Stem Cells in Acute Myeloid Leukemia and the Myelodysplastic Syndromes

AbstractAbstract 210The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. Our data indicate that HSCs (Lin-CD34+CD38-CD90+CD45RA-) from MDS patients exhibit widespread gene dysregulation (3,258 differentially expressed genes, FDR<0.1), including 25 cell surface protein transcripts. As these proteins can be easily measured by flow cytometry, we hypothesized that they may represent promising biomarkers.We used flow cytometry to assess the expression of 25 candidate disease markers on HSCs in bone marrow (BM) samples from patients with therapy related MDS (n=26). Nine surface markers were upregulated on MDS HSCs as compared with cord blood HSCs in at least 50% of samples. The most frequently upregulated surface marker was CD99 (85%). CD99 was also upregulated on HSCs in 27/37 (73%) de novo MDS cases. To determine whether CD99 upregulation was specific to MDS, we analyzed 78 paired diagnosis/relapse AML specimens encompassing all non-M3 subtypes of AML and a variety of cytogenetic subgroups. CD99 was found to be frequently overexpressed both at diagnosis (81%) and at relapse (83%). Notably, the mean intensity of CD99 expression was significantly higher at relapse (p=0.007). This suggests that CD99 is selected for during relapse and may represent a high risk AML marker. To investigate whether CD99 is upregulated in other myeloid disorders, we measured CD99 in CML samples (n=12). CD99 was decreased in chronic phase CML HSCs (7/10) but increased in blast crisis CML HSCs (2/2), suggesting that it may be a marker of leukemic transformation. Nevertheless, its overexpression in low-risk MDS suggests a distinct role in MDS biology.In CD34+ AMLs, CD99 expression was significantly higher in the stem cell enriched CD34+CD38- fraction compared with bulk blasts (p=0.003). In most samples (91%), CD99 high cells within the CD34+CD38- fraction exhibited a CD90-CD45RA+ “LMPP-like” immunophenotype, previously shown to be enriched for leukemia-initiating cell activity, whereas CD99 low cells exhibited a CD45RA- phenotype resembling normal HSCs and MPPs. Thus, CD99 may be preferentially expressed at higher levels on leukemic stem cells (LSC). We therefore used CD99 expression levels to prospectively purify potential LSCs (high CD99) from residual normal HSCs (low CD99) by sorting CD34+CD38- cells from three primary AML specimens into complete methylcellulose. CD99 low cells generated more myeloid colonies than CD99 high cells (mean 33 and 12, respectively, p=0.02). PCR-based assays revealed that molecular abnormalities associated with the prospectively sorted leukemias were present in CD99 high derived colonies, but absent in CD99 low derived colonies (two Flt3-ITD+ and one AML1-ETO+ samples tested). Transplantation of CD34+CD38-CD99 low cells from a Flt3-ITD+ AML sample into sublethally irradiated NOD/SCID/IL2-R null mice (NSG) (n=2) led to lympho-myeloid engraftment at 12 weeks (2/2 transplanted mice), with Flt3-ITD absent in the engrafted cells. Together, these data indicate that CD99 expression can distinguish LSCs from residual normal HSCs.To study the function of CD99 in AML, we measured CD99 expression on 14 AML cell lines, identifying 12 with high levels, including HL60 and MOLM13. Using a lentiviral vector to introduce a CD99 shRNA into HL60 cells, we achieved an 8.2-fold reduction in CD99 surface expression. Cells expressing the CD99 shRNA exhibited decreased growth in vitro compared with vector control (p=0.05). When these cells were transplanted into NSG mice, animals transplanted with CD99 shRNA expressing cells demonstrated a significant improvement in survival compared with vector controls (median OS 26d vs. 22d, p=0.04, log-rank). Conversely, NSG mice transplanted with MOLM13 cells overexpressing CD99 (4.7-fold increased surface expression) demonstrated a significantly impaired survival as compared with vector controls (median OS 11d vs. 28d, p=0.05, log-rank).In summary, our results establish CD99 as a cell surface protein that is highly expressed in MDS HSCs, AML blasts, and LSCs. Using CD99 expression to prospectively isolate residual normal HSCs from LSCs may provide a useful tool for the performance of purged autologous stem cell transplants. Furthermore, CD99 expression appears to promote leukemic cell growth, particularly in vivo. Further studies are needed to fully establish the role of CD99 in the biology of MDS and AML. Disclosures:No relevant conflicts of interest to declare.

Survivin is highly expressed in CD34+38− leukemic stem/progenitor cells and predicts poor clinical outcomes in AML

AbstractSurvivin, 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.

High-Dimensional Analysis of Intracellular Signaling and Dasatinib Inhibition In High-Risk Pediatric Leukemia by 31-Parameter Mass Cytometry

AbstractAbstract 2761 Background:High-risk pediatric B precursor lymphoblastic leukemia (preB ALL) remains a serious cause of morbidity and mortality despite therapeutic advances with consequent improvements in survival over the past 30 years. Intracellular protein networks are the functional mediators of differentiation decisions. Perturbing these networks with chemical and biological agents to unveil their signaling potential has shed insight into key networks in other hematologic malignancies such as B cell lymphoma, AML, JMML, and myelofibrosis. The current study was designed to profile evoked signaling responses in NCI high-risk pediatric preB ALL using a next-generation ‘mass cytometry’ platform (CyTOF.., DVS Sciences). This new technology platform utilizes antibodies conjugated to chelated metal ion tags, and thereby circumvents the limitations of overlapping spectral emission associated with more conventional fluorochrome-conjugated antibodies. Thus, in this preliminary study we measured 13 surface markers and 18 functional intracellular proteins simultaneously at the single-cell level in primary patient samples, with the goal of correlating distinct signaling responses with surface marker expression and response to the Src family tyrosine kinase inhibitor, dasatinib. Methods:All procedures were approved by the Stanford University Human Subject Institutional Review Board. Signaling profiles were measured for cryopreserved diagnostic bone marrow samples from pediatric patients with NCI high-risk pediatric leukemia in the basal state and in response to a 15 minute treatment to 18 different extracellular modulators, with or without prior exposure to dasatinib for 30 minutes. An example is shown in Figure 1. Cell populations were gated based on surface marker expression. The activation states of 18 functional intracellular markers were measured within the leukemic blasts (Figure 1). Results:Within the bulk blast population, distinct signaling responses could be discerned correlating with surface marker heterogeneity. Blasts were manually gated into 5 distinct populations based on surface expression of CD45 and CD10. These populations demonstrated further heterogeneous expression of surface markers CD34, CD38 and CD19 as well as heterogeneous activation states of intracellular proteins such as pERK, NFkB, and pStat5. Robust activation of phospho-proteins proximal to the preB cell receptor was observed following treatment with preBCR cross-linking antibodies or with tyrosine phosphatase inhibitors. This robust signaling was abrogated by pre-treatment of leukemic blasts with dasatinib. Unexpectedly, concurrent treatment with IL-7 or preB cell receptor crosslinking antibodies and dasatinib sensitized cells to dasatinib inhibition, resulting in a synergistic suppression of phosphorylation below baseline values (Figure 2). Discussion:Together, these results suggest that intracellular signaling and response to therapeutic agents such as kinase inhibitors may differ based on the complex interaction of the leukemic cell and its microenvironment. This preliminary study shows the feasibility of using a novel mass cytometry platform to simultaneously monitor 18 intracellular signaling mediators on a per cell basis in primary samples from pediatric high risk ALL patients to a level of detail not previously possible. This approach allowed the identification of cells with a robust signaling phenotype, based on the activation of pERK, pStat5 and NFkB, members of pathways known to be involved proliferation and survival. These data identify potential targets for therapeutic agents such as the tyrosine kinase inhibitor dasatinib. inhibited preBCR signal transduction, and synergy with other biological agents. Further studies are ongoing to measure multidimensional signaling with CyTOF technology in additional high risk pediatric ALL samples with the goal of identifying and guiding the most beneficial therapeutic choices.EFS and KLD contributed equally to this work. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Inhibition of Bcl-2/Bcl-XL Promotes Apoptosis in Blast Crisis CML Including Quiescent Primitive Progenitor Cells Regardless of Cellular Responses to Tyrosine Kinase Inhibitors.

Abstract 646The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor revolutionized the treatment for patients with CML. Development of resistance, limited activity in blast crisis CML, and more importantly, insensitivity of quiescent primitive CD34+ CML progenitor cells are evolving problems facing this therapy. Antiapoptotic Bcl-2 proteins were known to be highly expressed in Bcr-Abl expressing cells and inhibition of Bcl-2/Bcl-XL by the selective inhibitor ABT-737 was reported to augment the killing of tyrosine kinase inhibitors in CML cells. However, its effect on quiescent primitive CD34+ CML progenitor cells is unknown. To investigate the effect of activating the apoptotic machinery in quiescent primitive CD34+CML progenitor cells, which are resistant to current therapies, we first compared the expression of antiapoptotic proteins in proliferating and quiescent primitive CD34+CML progenitor cells. Cells obtained from patients with blast crisis CML were stained with the fluorescent 5-(and 6-) carboxy-fluorescein diacetate succinimidyl ester, a cell proliferation tracking dye, and cultured in vitro for 4-6 days. Cells were then stained with CD34 antibody and FACS sorted into proliferating and quiescent CD34+/PI- CML progenitor cells. RNA levels of antiapoptotic proteins in these two cell populations (n=8) were determined by real-time RT-PCR: quiescent and proliferating primitive CD34+ CML progenitor cells expressed similar levels of Bcl-2, Bcl-XL, Mcl-1, and XIAP implying that like total blast cells, quiescent primitive CD34+CML progenitor cells may also be sensitive to agents targeting these proteins. We next treated 5 samples obtained from patients with blast crisis CML with ABT-737 and measured apoptosis in total CD34+ cells, proliferating CD34+ cells, and quiescent CD34+ cells. All 5 patients were resistant to or relapsed from imatinib and nilotinib and/or dasatinib treatments and they were insensitive to imatinib in vitro as expected. However, cells from 4 patients were sensitive to ABT-737, in bulk blasts and in both proliferating and quiescent CD34+ CML cell compartments: % specific apoptosis with 100 nM of ABT-737=40.8±7.7, 38.4±8.5, 40.0±5.1, respectively at 24 hours. Interestingly, when ABT-737 was combined with imatinib, cell death was greatly enhanced in cells from all 5 patients in all cell compartments (combination index=0.059±0.032, 0.041±0.025, 0.111±0.042, respectively). Furthermore, we showed previously, that triptolide, an antitumor agent from a Chinese herb, induces apoptosis in both proliferating and quiescent primitive CD34+CML progenitor cells by decreasing Mcl-1 which is a resistant factor for ABT-737, XIAP, and Bcr-Abl protein levels (Mak D. et al., MCT in press). When ABT-737 was combined with triptolide, a significant increase of cell death was found in total CD34+ and proliferating as well as quiescent primitive CD34+CML cells with combination index at EC50=0.57, 0.55, and 0.56, respectively in cells from the 5 patients suggesting a high degree of synergism. In summary, Bcl-2, Bcl-XL, Mcl-1, and XIAP are equally expressed in proliferating and quiescent primitive CML cells and targeting Bcl-2/Bcl-XL promotes death of blast crisis CML cells, tyrosine kinase inhibitor resistant CML cells, and quiescent primitive CD34+ CML progenitor cells. Researches suggest that the combination of apoptosis inducing agents and tyrosine kinase inhibitor is a novel strategy to overcome tyrosine kinase resistance, eradicate quiescent primitive CML progenitor cells, and improve current therapy for patients with CML. Disclosures:No relevant conflicts of interest to declare.

Seismic vibrations in bulk blasting with high-precise electronic and nonelectric blasting systems at quarries

The authors report measurement data on seismic waves in bulk blasting at quarries by using new high-precise electronic and pyrotechnic blasting systems. It is proved that both systems are efficient, intensity of seismic waves is much lower in large-scale bulk blasting. The authors implemented numerical modeling of seismic wave propagation under a short-delay bulk blast at a quarry. Influence of the blast delay parameters and their precision on the maximum level of seismic vibrations has been studied, and their optimal ranges have been established.

AML Progenitor/Stem Cells Overexpress XIAP and Are Sensitive to the Small Molecular XIAP Inhibitor 1396-11.

Chemotherapy is the primary treatment modality for AML patients. Although most patients show initial responses to chemotherapeutic agents, the majority of patients relapse, suggesting that more primitive cells in AML are resistant to chemotherapy-induced apoptosis. XIAP, a potent cellular caspase inhibitor, is highly expressed in various tumor cells and leukemic cells and contributes to chemoresistance. However, the expression levels of XIAP in early progenitor/stem cells of AML patient are unknown. To address this question, we collected blasts from bone marrow or peripheral blood of AML patients (n=11) and separated them into CD34+/CD38+ and CD34+/CD38− populations by fluorescent-activated cell sorter after immunostaining. RNA was isolated and XIAP mRNA levels were determined by Taq-Man quantitative RT-PCR in these two cell populations and compared with levels from normal CD34+/CD38+ and CD34+/CD38− cells (n=13). Our results demonstrated that XIAP is highly expressed in both progenitor (CD34+/CD38+) and stem cell (CD34+/CD38−) compartments in primary AML blasts, with XIAP mRNA levels higher in these two cell populations in AML than in the corresponding normal bone marrow progenitor/stem cells (p=0.05 and 0.08 for CD34+/CD38+ and CD34+/CD38− compartments, respectively). Our previous studies showed that inhibition of XIAP expression by antisense oligonucleotide and its suppression by small molecule chemicals induce apoptosis of AML cells. 1396–11 and 1396–34, polyphenylurea-based small molecule XIAP inhibitors bind to and inhibit the BIR2 domain of XIAP. 1396–34 was previously tested in AML (Carter, et al. Blood 105: 4043, 2005). Previous studies of 1396–11 demonstrated anti-tumor activity against human solid tumor xenografts in mice with no apparent hematopoietic or liver toxicities (Schimmer, et al. Cancer Cell 5: 25, 2004; Berezovskaya, et al. Cancer Research 65: 2378, 2005). We treated OCI-AML3, HL-60, Jurkat, and U937 leukemia cells with 1396–11 and found it to be more potent than 1396–34 in all leukemia cell lines tested. In addition, we treated AML blasts from 5 patients with 1396–11 and its inactive control 1396–28. Apoptosis was determined in total blasts and in both the CD34+/CD38+ and CD34+/CD38− compartments. Our results showed that 1396–11 induced cell death equally in bulk blasts, CD34+/CD38+ progenitor, and CD34+/CD38− stem cells. Four out of 5 AML samples showed significant apoptosis with ≤ 10 μM 1396–11. The control compound 1396–28 had minimal effect on cell survival in all 5 patient samples tested. Our studies illustrated that XIAP is overexpressed in AML progenitor/stem cells and that these cells are sensitive to a small molecule XIAP inhibitor, suggesting XIAP is a target for AML therapy, with the potential of eradicating primitive leukemic progenitor/stem cells.

Acoustic Waves in Bulk Blasts in Opencasts

This paper reports results from experimental studies of acoustic waves with amplitudes of 1–100 Pa from bulk blasts in opencasts. The wave shape, amplitude, duration, and pulse, and their dependences on the mass of the explosive at epicentral distances of 1–10 km are obtained. It is shown that the vibration amplitudes excited by acoustic waves acting on the frontage of multi‐storey buildings are several times larger than those produced by seismic waves.