Bone Marrow Of AML Patients Research Articles

Overexpression of miR-210 is Associated with Poor Prognosis of Acute Myeloid Leukemia.

BackgroundMicroRNAs play important roles in regulation of the initiation and progression of AML. MiR-210 is closely related with cancer development; however, whether miR-210 expression level correlates with clinical correlation in AML is unknown. Thus, the aim of this study was to investigate the potential relationship between miR-210 expression and AML prognosis.Material/MethodsReal-time quantitative PCR was carried out to examine the expression level of miR-210 in bone marrow and serum obtained from AML patients and healthy controls. Then the correlation between miR-210 expression and a variety of important clinical parameters (such as overall survival, relapse-free survival, and prognostic value) were further studied.ResultsThe expression level of miR-210 was significantly higher in the bone marrow and serum of AML patients than that of healthy controls (p<0.001). Moreover, miR-210 expression was associated with various AML clinicopathological parameters, including FAB classification and cytogenetics. The serum miR-210 expression level was reduced significantly when the patients achieved complete remission (p=0.02). The high miR-210 expression group had both poorer relapse-free survival (p=0.015) and worse overall survival (p=0.008). In the multivariate analysis model, miR-210 was identified as an independent prognostic marker.ConclusionsMiR-210 up-regulation was associated with poor prognosis in AML and it might be useful as a marker for predicting the clinical outcome of AML patients.

Synergism of Curcumin and Cytarabine in the Down Regulation of Multi-Drug Resistance Genes in Acute Myeloid Leukemia.

The aim of the study was to find a role of Curcumin from natural source to overcome drug resistance as well as to reduce cytotoxicity profile of the drug in Acute Myeloid Leukemia patients. Primary leukemic cells were obtained from AML patient's bone marrow. These cells were then exposed to different concentration of cytarabine and curcumin to find out IC50 values and also its effect on MDR genes like MDR1, BCRP, LRP and FLT3 by RT-PCR method. Our results suggested that curcumin down regulates MDR genes. Gene expression was decreased by 35.75, 31.30, 27.97 % for MDR1, LRP, BCRP respectively. In FLT3, it was 65.86 % for wild type and 31.79 % for FLT3-ITD. In addition to this, curcumin has also shown anti-proliferative effect as well as synergistic effect in combination with Cytarabine on primary leukemic cells. Thus, we can conclude that curcumin can be used as MDR modulator as well as chemosensitizer in combination with cytarabine, standard chemotherapeutic drug, to reduce the cytotoxicity profile as IC50 value decreases when treated in combination.

Therapeutic polo-like kinase 1 inhibition results in mitotic arrest and subsequent cell death of blasts in the bone marrow of AML patients and has similar effects in non-neoplastic cell lines

Polo-like kinase 1 (PLK1) is an important regulator of the cell cycle and is overexpressed in various solid and hematological malignancies. Small molecule inhibitors targeting PLK1, such as BI2536 or BI6727 (Volasertib) are a promising therapeutic approach in such malignancies. Here, we show a loss of specifically localized PLK1 in AML blasts in vivo, accompanied by mitotic arrest with transition into apoptosis, in bone marrow biopsies of AML patients after treatment with BI2536. We verify these results in live cell imaging experiments with the AML cell line HL-60, and demonstrate that non-neoplastic, immortalized lymphoblastoid cells are also sensitive to PLK1 inhibition. It is demonstrated that normal granulopoietic precursors have similar PLK1 expression levels as leukemic blasts. These results are in line with the adverse effects of PLK1 inhibition and underline the great potential of PLK1 inhibitors in the treatment of AML.

Correlation between ALIP-like cluster and fibrous proliferation in bone marrow of patients with acute myeloid leukemia in CR phase

This study was purposed to elucidate the prognostic values of ALIP (abnormal location of immature precursors)-like clusters and fibrous proliferation in bone marrow of AML patients in CR phase and the correlation between them. The bone marrow biopsy sections from 47 AML patients during admitting to relapse or till lost follow-up were examined retrospectively. The 47 patients were divided into pre-relapsed group and non-relapsed group according to relapse or not at end of follow-up. The concentration of ALIP-like cluster and reticulin fiber density (RFD) in sections were compared between the two groups respectively, the prognostic value of these two factors and the underlying relationship between them were estimated by statistical analysis. The results showed that ALIP-like cluster was (3.46 ± 2.71)/mm(2) during CR and RFD was (2.76% ± 1.50%) in pre-relapsed cases, which both were higher than those in non-relapsed cases (P < 0.05). Cases with ALIP-like cluster over 4/mm(2) or with RFD>1.68% showed high relapse rates of 89.5% or 95.2% respectively. RFD were (2.47% ± 2.48%) and (2.44% ± 2.23%) in cases with >4/mm(2) or ≤ 4/mm(2) respectively, there was no statistical significance (P > 0.05) . Meanwhile, the amount of ALIP-like cluster in CR not related with the paired RFD (r = 0.057, P > 0.05). It is concluded that both ALIP-like cluster in CR and RFD are poor prognostic factors for heralding early relapse. However, ALIP-like cluster and RFD show no correlation, and suggest that forming of ALIP not depends on fibrogenesis.

Mir-128 and DNA Damage In Acute Myeloid Leukemia

BackgroundmicroRNAs (miRs) are small non-coding RNAs that post-transcriptionally regulate gene expression by binding target mRNAs and hampering their translation by translation inhibition or mRNA degradation. miRs have been shown to play an important role in cancer development by controlling several pivotal cellular processes [Lee and Dutta (2009), Annu Rev Pathol 4:199-227]. In particular,miR-128 has also been associated with cancer, namely leukemia and has been shown, , with other miRs, to allow the discrimination between AML and ALL [Mi et al. (2007), PNAS 104(50):19971-6]. Moreover, it is included in a miR signature that associates a subgroup of patients with high-risk molecular features of AML with worse clinical outcome [Marcucci et al. (2008), NEJM 358(18):1919-28]. Nevertheless, all the data associating miR-128 with leukemia derives from expression array analysis and no functional studies have been performed. Therefore, the aim of this study was to understand the role of miR-128 in AML cells and in their response to some chemotherapeutic agents. MethodsHL-60 cells were transfected with miR-128 mimic (or control miR mimic) and further treated with etoposide, doxorubicin or their vehicle as control. miR expression was evaluated by RT-qPCR. The effect of miR-128 overexpression in sensitization of HL-60 cells to the effects of doxorubicin or etoposide was analysed by Trypan blue exclusion assay. Cellular proliferation (BrdU assay), cell cycle (flow cytometry following PI labeling), programmed cell death (TUNEL assay) and apoptosis (Annexin V/ PI staining) were analysed. The expression levels of proteins involved in apoptosis (caspase-3, PARP), autophagy (Beclin-1, Vps34 and LC3) and DNA damage (γ-H2AX, 53BP1) were studied (Western Blot). DNA damage was analysed with the Comet assay and by foci formation of γ-H2AX and 53BP1 proteins, visualized by immunofluorescence microscopy. miR-128 expression was analysed in samples from peripheral blood mononuclear cells (PBMCs) of 13 healthy donors and from bone marrow of 11 AML patients by RT-qPCR. ResultsmiR-128 expression was increased upon miR mimic transfection. miR-128 overexpression decreased HL-60 viable cell number to 84.3% and 81.0%, at 24 h and 48 h after transfection respectively, and sensitized HL-60 cells to both doxorubicin and etoposide. Nevertheless, miR-128 overexpression did not affect cell cycle profile, cellular proliferation, apoptosis, or the expression of apoptosis-related or autophagy-related proteins. Interestingly, miR-128 overexpression increased DNA damage analysed by Comet assay (from 3.6% in miR-control transfected cells to 8.1% in miR-128 transfected cells). This increase in DNA damage of miR-128 overexpressing cells was confirmed by verifying an increase in DNA repair foci of γ–H2AX and 53BP1 together with an increase in expression of both those proteins γ–H2AX and 53BP1. Analysis of miR-128 expression in samples from PBMCs of healthy donors and from bone marrow of AML patients showed no statistically significant differences, although the expression levels of miR-128 in the AML samples were higher than in healthy donors. ConclusionmiR-128 overexpression per sedecreased HL-60 viable cell number and sensitized cells to doxorubicin and etoposide. miR-128 increased DNA damage, which might justify the increased sensitivity that these cells presented to doxorubicin and etoposide. Concerning patient samples, a slight increase in the expression of miR-128 was found in AML bone marrow samples, when compared to PBMCs from healthy donors, suggesting that these patients maybe more susceptible to DNA damaging agents. AcknowledgmentsFundação Calouste Gulbenkian for financial support. FCT for the grants to H. Seca (SFRH/BD/47428/2008) and R. T. Lima (SFRH/BPD/68787/2010). G. M. Almeida was supported by FCT and the European Social Fund. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT. Disclosures:No relevant conflicts of interest to declare.

Creation Of Patient Derived AML Xenografts Displaying Distinct Phenotypes and Geneotypes

We have recently successfully engrafted leukemic cells from bone marrow of several AML patients into immunocompromised NOD/SCID mice. One of them, AM7577, was reported in the last year ASH1. This model displayed typical aggressive AML disease of M5 subtype, which starts at bone marrow and gradually expand to peripheral (spleen, lymphonode and peripheral blood…). AM7577 also harbors interesting genotypes of mutations for IDH2-R140Q, FLT3-ITD, DNMT3A R882H and NPM1.Our second AML PDX model, AM8096, was established similarly using bone marrow from a recurrent patient with AML-M2 disease. AML8096 displayed similarly aggressive disease and the same 100% mortality, including typical symptoms (BW loss, hunched, inactivity, labored breathing, ruffled coat and eventual mortality) and with abundant leukemic cells in bone, etc. The leukemic cells can serially be passed in mice with 100% take-rate and cause consistent disease (even with < 1e4 cells). This also creates a renewable and potentially unlimited source of leukemia cells. The leukemic cells in mice are identical to those of the original patient leukemic cells (CD45+, CD33+, CD13+, CD123+, and CD19-. However, some aspects of disease presentations are vastly different from that of AM7577. First, the peripheral symptom is significant less characterized by lower leukemic counts in peripheral blood and only slightly enlarged spleen and smaller lymphonode. Second, the leukemic cell morphology is also rather different with AM8096 demonstrating less differentiated phenotype. Third, the genotype, in contrast to AM7577, is wild-types for all the above oncogenes. While we have not identified the likely leukemogenesis driver gene for this model, we are currently performing RNAseq of AM8096, along with AM7577, in order to explore the underlying molecular mechanisms that drive both diseases. In addition, we are also investigating the drug response to standard of care (SOC), as compared to those AM7577.In summary, the two AML models could serve as useful experimental models to investigate the diverse leukemogenesis, including molecular mechanisms by genetic profiling. Ultimately, they can be used to help to identify new treatment strategies for AML.1. Liu, e.a. A unique leukemia mouse model established from AML patient with IDH2 R140Q and FLT3-ITD mutations among other common AML mutations. ASH-2012 Annual Meeting (2012). Disclosures:Li:Crown Bioscience: Employment. Liu:Crown Bioscience: Employment. An:Crown Bioscience: Employment. Wu:Crown Bioscience: Employment. Cai:Crown Bioscience: Employment. Wery:Crown Bioscience: Employment.

A Recombinant Antibody to EphA3 for the Treatment of Hematologic Malignancies: Research Update and Interim Phase 1 Study Results

Abstract 4893EphA3 is a receptor tyrosine kinase important in fetal development but apparently not in healthy adults. It is expressed in hematologic malignancies including AML, CML, MDS, MPN and Multiple Myeloma and in a range of solid tumors in particular in the stromal and vascular tissue. KB004 is a high-affinity non-fucosylated, recombinant antibody to EphA3 that triggers apoptosis and has potent antibody-dependent cellular cytotoxicity (ADCC) activity against EphA3+ cells. Direct apoptosis of EphA3+ leukemic cells ex vivo was demonstrated at antibody concentrations of 10μg/mL or greater. Expression on a CD34+ CD38− CD123+ cell population in primary AML patient bone marrow, and inhibition of long-term culture initiating cells by KB004, indicates that EphA3 may be present on leukemic stem cells (Palath et al 2010).EphA3 expression in bone marrow biopsies from AML patients was characterized by immunohistochemistry (IHC). EphA3 was detected on tumor cells by IHC in 4/10 samples analyzed. EphA3 was also present on the vasculature in AML bone marrow (6/10 samples) but not in similar tissues in bone marrow biopsies (n=2) from non-leukemic individuals. This suggests tumor vasculature as a potential further therapeutic target for KB004 in AML in addition to targeting leukemia cells directly. We are further characterizing the expression level of EphA3 in different hematologic malignancies, tumor microenvironment, and at various stages of disease progression from primary human samples.The Cynomolgus macaque was selected as a relevant species for toxicity testing since KB004 binds with equivalent affinity to human and Cynomolgus EphA3 and CD16. IHC studies demonstrated the absence of cellular expression of EphA3 in normal human or Cynomolgus monkey tissues. KB004 administered to Cynomolgus monkeys (n=44) twice weekly for 13 weeks at doses up to 100 mg/kg was well tolerated and there were no clinical or pathology adverse findings. Cardiovascular, respiratory and central nervous system functions were measured in primates after KB004 dosing 5 times over 15 days to reach steady-state exposure at doses of 10mg/kg (n=4) or 100 mg/kg (n=4). There were no KB004-related effects on body weight, food consumption, mean arterial pressure, heart rate, body temperature, neurological parameters, respiration rate, oxygen saturation, or blood gas parameters, or changes in serum concentrations of Troponin I. Qualitative evaluation of the electrocardiogram (ECG) did not reveal any electrocardiographic abnormalities and there were no effects on measured ECG intervals (i.e., duration of the QT and RR intervals, derived QTc values). The effect of KB004 on wound healing was tested in a primate incisional wound healing model in the Cynomolgus monkey. No statistically significant differences in healing were detected between control and KB004 (2×10 or 2×100mg/kg) treated animals (n=24).A Phase I clinical study has been initiated in subjects with hematologic malignancies including AML, CML, ALL, MDS and MPN. Subjects are being assessed for EphA3 protein expression at study entry and at various time points throughout the treatment period. Study objectives are to determine a maximum tolerated dose, examine the safety and tolerability profile of KB004, obtain pharmacokinetic data, describe the immunogenicity profile, and explore cell subpopulations and pharmacodynamic effects of treatments with KB004. This is an open-label, repeat administration study of weekly IV dosing of up to 17 cycles (3 doses per 21-day cycle). Dosing levels are scheduled for 20 mg (∼0.3 mg/kg), 70 mg (∼1 mg/kg), 200 mg (∼3 mg/kg) and 700 mg (∼10 mg/kg). Therapeutic antibody levels (target level 10μg/mL) are predicted to be achieved from the first dose cohort for a portion of the dosing interval. The higher doses should provide blood levels in excess of 10 μg/mL for the entire one-week dosing interval. The first cohort (n=3) has been successfully completed, and a subject with AML remains on study having received 8 doses to date. Recruitment of the next dose cohort is ongoing. Pharmacokinetic data and tumor expression profiles will be presented. Disclosures:Hagey:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Lancet:KaloBios Pharmaceuticals, Inc.: Research Funding. Palath:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Wei:KaloBios Pharmaceuticals, Inc.: Research Funding. Lackmann:KaloBios Pharmaceuticals, Inc.: Research Funding. Cortes:KaloBios Pharmaceuticals, Inc.: Research Funding. Boyd:KaloBios Pharmaceuticals, Inc.: Research Funding. Shochat:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yarranton:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bebbington:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Leff:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership.

Abstract LB-45: High aldehyde dehydrogenase activity is a marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia: novel therapeutic implications.

Abstract Only a minority of cells, the leukemic stem cells (LSC), within AML are responsible for tumor growth and maintenance. Many patients experience relapse after therapy which originates from outgrowth of therapy resistant LSC. Therefore, eradication of LSC is necessary to cure AML. Both the normal hematopoietic stem cells (HSC) and LSC co-exist in the bone marrow (BM) of AML patients and success of anti-LSC strategies relies on specific elimination of LSC while sparing HSC. LSC are contained within the CD34+CD38-, the side population (SP) and the high aldehyde dehydrogenase (ALDH) activity compartments. ALDH is a detoxifying enzyme responsible for oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as cyclophosphamide. It has been shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blasts. In view of applicability of LSC specific therapies the detoxification by ALDH is clinically very important. A difference in ALDH activity between HSC and LSC might be used to preferentially kill LSC while sparing HSC. To establish ALDH activity differences between HSC and LSC it should be possible to discriminate between them. We have shown that LSC can be identified and discriminated from HSC using stem cell-associated cell surface markers, such as CLL-1, lineage markers (CD7, CD19, CD56) and recently CD34/CD45 expression and cell size characteristics (Terwijn, Blood 111: 487, 2008). This offers the opportunity to identify co-existing LSC and HSC in the AML BM. We now show that, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that HSC that co-exist with LSC in BM of AML patients have a higher ALDH activity as compared to their malignant counterparts. We have analyzed ALDH activity in HSC and LSC, both present in the BM from 18 AML patients. In nine BM AML samples, defined as CD34negative (&amp;lt;1%CD34+ blasts), the CD34+ compartment contained only normal CD34+CD38− HSC. The ALDH activity in these CD34+ HSC, is a factor 4,4 (range 1,7–18,9) higher than in LSC. In nine BM AML samples, defined as CD34positive AML, the CD34+CD38- HSC have a 7,7 fold (range 1,73–29,2 fold) higher ALDH activity as compared to putative LSC. In both CD34-positive and CD34-negative AML, we confirmed the identity of HSC and LSC by screening for molecular aberrancies present in AML blasts. The level of the ALDH activity of HSC within the AML BM is similar to that of HSC in NBM of healthy donors. In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype) at diagnosis. Consequently, AML patients with high ALDH activity in HSC might benefit from treatment with agents that will be converted by ALDH enzymes, such as cyclophosphamide, whereby the difference between the activity in LSC and HSC will define the therapeutic window. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-45. doi:10.1158/1538-7445.AM2011-LB-45

Targeting Signal Pathways active in Cancer Stem Cells to Overcome Drug Resistance.

Over the last several decades, although there have been advances in the treatment of diseases such as children leukemia, Hodgkin's disease, and testicular cancer, however, the survival of patients with the most common malignancies such as lung, breast, liver, and colon cancers has not changed signifi cantly. Most of patients will relapse and ultimately died of the disease. Th e scarce effi cacy of current treatments indicates the resistance of cancer cells to cytotoxic agents, radiation, and even immunotherapy and survival from the treatment without major injure. It is believed that tumors is initiated and maintained by a rare population of cancer cells with stem cell properties known as cancer stem cell (CSC). Th e concept that a rare population of the tissue stem cells maybe the cellular origin of cancer was proposed almost 150 years ago. Approximately 50 years ago the concept that only a small subpopulation of so-called leukemic stem cells (LSCs) may be connected to the maintenance and evolution of myeloid leukemia emerged. Conclusive evidences for the existence of LSCs come from the function assay using SCID-leukemia and NOD/SCID-leukemia xenotransplantation models in which mice were transplanted with leukemic cells from the bone marrow and peripheral blood of AML patients. Th ese studies demonstrated that the leukemic graft s were highly representative of the original patients disease and the SCID/leukemia initiating cell presented at a frequency of 0.2-100/10 mononuclear cells. More recently, this principle has also been extended to other tumors, such as breast, brain, prostate, pancreas, colon, lung, liver, and head and neck tumors. CSCs have been reported to be the only tumorigenic population and play a central role in relapse because of the failure of current chemotherapy to eradicate them. The existence of CSCs highlights the critical need for the new therapeutic strategies to directly target the CSC population for ultimately curing cancer. Based on the solid evidences that cancers are stem cell diseases, the view of drug resistance change. It is believed that CSCs are naturally resistant to conventional chemotherapy and serve as the main mediators of drug resistance. Moreover, it is accepted that drug resistance is governed by the mutations that confer protection mechanism through modulation of cell survival factors. To that end, a number of signal pathways involved in CSCs viability and survival, namely the Hedgehog, Ras, FLT3, PI3K/AKT, NF-κB, mTOR are aberrantly regulated in CSCs. Because of their wide-ranging biological eff ects, deregulation one or more of these pathways may give rise to a failure of current chemotherapy. Others and we have long been interested in exploring the mechanisms of drug resistance of CSCs infl uenced by these cell survival pathways and molecular interaction networks. Thus we can determine the critical elements and the general rules driving the network to guide the use of specifi c inhibitors of a given pathway. Th is review will focus on the drug resistance of CSCs and the signal pathway and their potential cross-talk (Fig 1).

Targeting Multiple Active Kinase Pathways In 11q23 Translocated Pediatric Acute Myeloid Leukemia Using a VEGFR2 Antibody Together with a MEK Inhibitor.

AbstractAbstract 3626One of the major pathways involved in the angiogenic process is the VEGF/VEGFR signaling pathway. VEGFA was found to be an independent prognostic factor for therapeutic outcome in AML (de Bont et al, 2002;Aguayo et al, 1999). Moreover, it has been shown that after chemotherapy the expression level of VEGFR2 is restored to normal in the bone marrow of AML patients in complete remission (Padro et al, 2002). Both VEGF and its receptors Flt-1(VEGFR1) and KDR (VEGFR2) are expressed by leukemic blasts which results in both a paracrine and an autocrine VEGF/VEGFR2 pathway in AML (Dias et al, 2001). In response to VEGF stimulation, VEGFR2 has been shown to transmit intracellular signals leading to activation of multiple downstream signaling pathways including the mitogen-activated protein (MAP) kinases, JAK)STAT, and Phosphoinositide 3-kinases (PI3 kinases). In AML it has been described that simultaneous activation of these pathways results in a poor prognosis (Kornblau et al, 2006). Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene at locus 11q23 are associated with a poor outcome. These MLL translocations represent 15–20% of pediatric acute myeloid leukemia's. In this study we show that VEGFR2 expression is significantly higher on blasts of 11q23 translocated acute myeloid leukemia's compared to blasts of AML patients with another karyotype. In addition, using peptide arrays and proteome profiler arrays we have found that the VEGF receptor signaling pathway and part of the MAP kinase signaling pathway are active and or phosphorylated in blasts of 11q23 translocated patients (Fig. 1). Proteins from these active pathway are potential targets in the treatment of pediatric 11q23 translocated AML. To further investigate these potential targets we have performed ex vivo drug target assays using an inhibitor and an antibody against two key proteins from the major signal transduction pathways, VEGFR2 antibody (IMC1121b) and MEK inhibitor (U0126). No effect of VEGFR2 inhibition was measured in two AML cell lines (HL-60 and THP-1). Whereas, MEK inhibition had an effect on both cell lines, LC50 8.7 μM and 7.5 μM for HL-60 and THP-1 respectively. Cell survival of the THP-1 cell line (containing a 11q23 translocation) was substantially inhibited after addition of a combination therapy with an inhibitor against MEK and a antibody against VEGFR2 (LC50 2.9 μM). In contrast, the VEGFR2 antibody in combination with the MEK inhibitor had only a marginal effect on the HL-60 cell line (LC50 7.7 μM). Future experiments to investigate the effect of the combination therapy on primary patient material are underway. Overall these data show for the first time that targeting multiple active pathways in pediatric 11q23 AML could result in promising opportunities concerning future therapy options. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Polo-Like Kinase-1 (Plk-1) Inhibitor BI 2536 Induces Mitotic Arrest and Apoptosis in Vivo: First Demonstration of Target Inhibition in the Bone Marrow of AML Patients

Background: BI 2536 is a potent and selective inhibitor of Plk-1, which plays a crucial role in the regulation of mitosis. Inhibition of Plk-1 leads to mitotic arrest and apoptosis. Therefore, Plk-1 inhibitors are currently in clinical testing as anti-proliferative agents in cancer patients. BI 2536, the first specific Plk-1 inhibitor in clinical testing, demonstrated strong anti-proliferative effects on AML cell lines in vitro and in in vivo models. To investigate target inhibition in the malignant cell, bone marrow samples from patients who participated in a Phase I/II study of BI 2536 single-agent therapy in elderly patients with relapsed or refractory AML were analyzed.Methods: Pharmacodynamic analyses including immunocytochemistry (ICC) and flow cytometry (FACS) of bone marrow (BM) were performed to examine cell morphology, phosphorylation of histone H3 (phospho-H3), and induction of apoptosis. Samples were acquired before and 24 h after the first administration of BI 2536. FACS analysis included propidium iodide (PI)-FACS to determine the percentage of cells residing in various cell cycle stages (G0/1-, S- and G2/M-phases) and Annexin V-Cy5-staining for apoptosis. Immunocytochemistry included staining for phosphorylated histone H3 and TUNEL assay for apoptosis.Results: At the time of analysis, data from 28 patients treated at doses in the range of 50 to 400 mg BI 2536 were available. BM taken after BI 2536 administration showed an increase of phospho-H3 positive cells as compared to baseline prior to treatment. There was a trend toward a positive correlation between phospho-H3 increase and increase in dosage of BI 2536 (p=0.16) when treatment at low doses (50 to 60 mg) of BI 2536 were compared to treatment at higher doses (100 to 400 mg). Furthermore, trends were observed that an increase of phospho-H3 is positively correlated with both a higher percentage of cells in G2/M and an increase in apoptotic cells. The typical morphology of cells in mitotic arrest could be demonstrated by ICC. Interestingly, when patients with progressive disease after one cycle (PD) were compared to patients with disease stabilization or response (nonPD), a statistically significant positive correlation between PD and increase in phospho-H3 compared to baseline was found (p=0.03). Also, there was a clear trend for an increase in the percentage of cells in G2/M phase and apoptosis in patients with PD compared to patients with nonPD. Preliminary evaluation of other disease characteristics including karyotype (normal vs complex), secondary AML, complete response in previous treatments, baseline value for blasts in the bone marrow or in the peripheral blood, did not reveal any signs of correlation with the clinical response to BI 2536 treatment.Conclusion: BI 2536 treatment increases the number of cells in G2/M phase (as detected by FACS analysis and phospho-H3 staining) and the number of apoptotic cells within 24 h after administration. In line with the clinical observation of rapid blast reduction both in the BM and the peripheral blood, these findings indicate that BI 2536 induces mitotic arrest and apoptosis of the malignant target cells in AML patients. The biological meaning of the correlation between the BM findings and the clinical response to BI 2536 is unclear, but if substantiated by more data, these results may suggest a predictive value of BM examinations for the response to BI 2536.

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.

Effect of 4-hydroperoxycyclophosphamide on leukemic and normal human myeloid progenitor cells

The sensitivity of myeloid progenitor cells from normal subjects (N-CFU-GM) and from leukemic patients in complete remission (LR-CFU-GM) to 4-hydroperoxycyclophosphamide (4-HC) were compared to the sensitivity of leukemic progenitor cells (L-CFU) to this drug. The results were expressed as the dose of 4-HC needed to kill 90% (TD 90) of the progenitor cells. The mean TD 90 were respectively for N-CFU-GM : 59(± 11 S.E.M.) nM ml −1 and for L-CFU 79(± 6 S.E.M.) nM ml −1. Thus, L-CFU were equally sensitive to 4-HC as N-CFU-GM. Moreover, the mean TD 90 for LR-CFU-GM was 87 (± 5 S.E.M.) nM ml −1. Thus, the sensitivity of N-CFU-GM and LR-CFU-GM did not differ significantly from that of L-CFU. These results are not encouraging for the use of 4-HC in vitro to eliminate the residual leukemic cells from autologous bone marrow of AML patients in complete remission. The sensitivity of L-CFU was modified neither by previous cytoreductive therapy (different from cyclophosphamide) nor by the time elapsed since diagnosis of AML.