Characterization of Rare, Dormant, and Therapy-Resistant Cells in Acute Lymphoblastic Leukemia.

Tumor relapse is associated with dismal prognosis, but responsible biological principles remain incompletely understood. To isolate and characterize relapse-inducing cells, we used genetic engineering and proliferation-sensitive dyes in patient-derived xenografts of acute lymphoblastic leukemia (ALL). We identified a rare subpopulation that resembled relapse-inducing cells with combined properties of long-term dormancy, treatment resistance, and stemness. Single-cell and bulk expression profiling revealed their similarity to primary ALL cells isolated from pediatric and adult patients at minimal residual disease (MRD). Therapeutically adverse characteristics were reversible, as resistant, dormant cells became sensitive to treatment and started proliferating when dissociated from the in vivo environment. Our data suggest that ALL patients might profit from therapeutic strategies that release MRD cells from the niche.

Anti-leukemic effects of the V-ATPase inhibitor Archazolid A.

Prognosis for patients suffering from T-ALL is still very poor and new strategies for T-ALL treatment are urgently needed. Our study shows potent anti-leukemic effects of the myxobacterial V-ATPase inhibitor Archazolid A. Archazolid A reduced growth and potently induced death of leukemic cell lines and human leukemic samples. By inhibiting lysosomal acidification, Archazolid A blocked activation of the Notch pathway, however, this was not the mechanism of V-ATPase inhibition relevant for cell death induction. In fact, V-ATPase inhibition by Archazolid A decreased the anti-apoptotic protein survivin. As underlying mode of action, this work is in line with recent studies from our group demonstrating that Archazolid A induced S-phase cell cycle arrest by interfering with the iron metabolism in leukemic cells. Our study provides evidence for V-ATPase inhibition as a potential new therapeutic option for T-ALL.

An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.

TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses.

Activation of DNA damage response by antitumor therapy counteracts the activity of vinca alkaloids.

BACKGROUND/AIM: Anthracyclines have been proven able to reduce the activity of vinca alkaloids by induction of cell-cycle arrest. The present study aims at identifying the critical initiation steps of signal transduction which transduce the inhibitory effects on the cytotoxicity of vinca alkaloids. MATERIALS AND METHODS: Several new cytostatic drug classes were evaluated together with vincristine in tumor cell lines and patients' tumor cells. RNA interference was used for molecular analyses. RESULTS: Inhibition of vincristine was observed by all cytostatic drugs, which induced cell-cycle arrest. Knockdown of proteins of the DNA damage response ascribed the inhibitory effect to a common pathway involving Chk-1, p53 and p21. Upstream of Chk-1 signal transduction depended on both ATM and ATR for all drugs except methotrexate. CONCLUSION: We have identified critical signaling steps of the DNA damage response system activated by cytostatic drugs, which reduce the anti-tumor activity of vinca alkaloids. The obtained results encourage the development of novel therapeutic strategies to prevent pathway interactions based on the molecular understanding of drug action and drug-drug interactions.

Impact of the p53 status of tumor cells on extrinsic and intrinsic apoptosis signaling.

BACKGROUND: The p53 protein is the best studied target in human cancer. For decades, p53 has been believed to act mainly as a tumor suppressor and by transcriptional regulation. Only recently, the complex and diverse function of p53 has attracted more attention. Using several molecular approaches, we studied the impact of different p53 variants on extrinsic and intrinsic apoptosis signaling. RESULTS: We reproduced the previously published results within intrinsic apoptosis induction: while wild-type p53 promoted cell death, different p53 mutations reduced apoptosis sensitivity. The prediction of the impact of the p53 status on the extrinsic cell death induction was much more complex. The presence of p53 in tumor cell lines and primary xenograft tumor cells resulted in either augmented, unchanged or reduced cell death. The substitution of wild-type p53 by mutant p53 did not affect the extrinsic apoptosis inducing capacity. CONCLUSIONS: In summary, we have identified a non-expected impact of p53 on extrinsic cell death induction. We suggest that the impact of the p53 status of tumor cells on extrinsic apoptosis signaling should be studied in detail especially in the context of therapeutic approaches that aim to restore p53 function to facilitate cell death via the extrinsic apoptosis pathway.

Leukemia-initiating cells of patient-derived acute lymphoblastic leukemia xenografts are sensitive toward TRAIL.

Cancer stem cells represent the most important target cells for antitumor therapy. TRAIL (TNF-related apoptosis-inducing ligand) is a potential anticancer agent that induces apoptosis in a wide variety of tumor cells, but its ability to target cancer stem cells is currently unknown. Here we investigated whether TRAIL targets leukemia-initiating cells. Limiting dilution transplantation assays were performed on xenografts from pediatric patients with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) in NSG mice. In vitro treatment of xenograft cells with TRAIL significantly reduced and delayed their engraftment and procrastinated animal death from leukemia. Systemic TRAIL treatment of mice injected with patient-derived pre-B ALL xenograft cells abrogated leukemia in 3 of 5 mice in 1 sample. In conclusion, our data suggest that TRAIL targets leukemia-initiating cells derived from pre-B ALL xenografts in vitro and in vivo, and hence constitutes an attractive candidate drug for treatment of ALL.

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins.

The fresh water polyp Hydra belongs to the phylum Cnidaria, which diverged from the metazoan lineage before the appearance of bilaterians. In order to understand the evolution of apoptosis in metazoans, we have begun to elucidate the molecular cell death machinery in this model organism. Based on ESTs and the whole Hydra genome assembly, we have identified 15 caspases. We show that one is activated during apoptosis, four have characteristics of initiator caspases with N-terminal DED, CARD or DD domain and two undergo autoprocessing in vitro. In addition, we describe seven Bcl-2-like and two Bak-like proteins. For most of the Bcl-2 family proteins, we have observed mitochondrial localization. When expressed in mammalian cells, HyBak-like 1 and 2 strongly induced apoptosis. Six of the Bcl-2 family members inhibited apoptosis induced by camptothecin in mammalian cells with HyBcl-2-like 4 showing an especially strong protective effect. This protein also interacted with HyBak-like 1 in a yeast two-hybrid assay. Mutation of the conserved leucine in its BH3 domain abolished both the interaction with HyBak-like 1 and the anti-apoptotic effect. Moreover, we describe novel Hydra BH-3-only proteins. One of these interacted with Bcl-2-like 4 and induced apoptosis in mammalian cells. Our data indicate that the evolution of a complex network for cell death regulation arose at the earliest and simplest level of multicellular organization, where it exhibited a substantially higher level of complexity than in the protostome model organisms Caenorhabditis and Drosophila.