Lüder Hinrich Meyer

ETV6/RUNX1-positive relapses evolve from an ancestral clone and frequently acquire deletions of genes implicated in glucocorticoid signaling

Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P=.01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n=3), glucocorticoid receptor NR3C1 (n=4), and components of the mismatch repair pathways (n=3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P=.02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.

The Combination of the PARP Inhibitor Rucaparib and 5FU Is an Effective Strategy for Treating Acute Leukemias

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA. Mol Cancer Ther; 14(4); 889–98. ©2015 AACR.

Central Nervous System Involvement in Acute Lymphoblastic Leukemia Is Mediated by Vascular Endothelial Growth Factor

In acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement is a major clinical concern. Despite nondetectable CNS leukemia in many cases, prophylactic CNS-directed conventional intrathecal chemotherapy is required for relapse-free survival, indicating subclinical CNS manifestation in most patients. However, CNS-directed therapy is associated with long-term sequelae, including neurocognitive deficits and secondary neoplasms. Therefore, molecular mechanisms and pathways mediating leukemia-cell entry into the CNS need to be understood to identify targets for prophylactic and therapeutic interventions and develop alternative CNS-directed treatment strategies. In this study, we analyzed leukemia-cell entry into the CNS using a primograft ALL mouse model. We found that primary ALL cells transplanted onto nonobese diabetic/severe combined immunodeficiency mice faithfully recapitulated clinical and pathological features of meningeal infiltration seen in patients with ALL. ALL cells that had entered the CNS and were infiltrating the meninges were characterized by high expression of vascular endothelial growth factor A (VEGF). Although cellular viability, growth, proliferation, and survival of ALL cells were found to be independent of VEGF, transendothelial migration through CNS microvascular endothelial cells was regulated by VEGF. The importance of VEGF produced by ALL cells in mediating leukemia-cell entry into the CNS and leptomeningeal infiltration was further demonstrated by specific reduction of CNS leukemia on in vivo VEGF capture by the anti-VEGF antibody bevacizumab. Thus, we identified a mechanism of ALL-cell entry into the CNS, which by targeting VEGF signaling may serve as a novel strategy to control CNS leukemia in patients, replacing conventional CNS-toxic treatment.

Attenuated measles virus controls pediatric acute B-lineage lymphoblastic leukemia in NOD/SCID mice

Novel therapies are needed for pediatric acute lymphoblastic leukemia resistant to conventional therapy. While emerging data suggest leukemias as possible targets of oncolytic attenuated measles virus, it is unknown whether measles virus can eradicate disseminated leukemia, in particular pediatric acute lymphoblastic leukemia. We evaluated the efficacy of attenuated measles virus against a large panel of pediatric xenografted and native primary acute lymphoblastic leukemias ex vivo, and against four different acute lymphoblastic leukemia xenografts of B-lineage in non-obese diabetic/severe combined immunodeficient mice. Ex vivo, attenuated measles virus readily spread among and effectively killed leukemia cells while sparing normal human blood cells and their progenitors. In immunodeficient mice with disseminated acute lymphoblastic leukemia a few intravenous injections of attenuated measles virus sufficed to eradicate leukemic blasts in the hematopoietic system and to control central nervous system disease resulting in long-term survival in three of the four xenografted B-lineage leukemias. Differential sensitivity of leukemia cells did not require increased expression of the measles entry receptors CD150 or CD46 nor absence of the anti-viral retinoic acid-inducible gene I/melanoma differentiation associated gene-5 /interferon pathway. Attenuated oncolytic measles virus is dramatically effective against pediatric B-lineage acute lymphoblastic leukemia in the pre-clinical setting warranting further investigations towards clinical translation.

Leukemia reconstitution in vivo is driven by cells in early cell cycle and low metabolic state

In contrast to well-established hierarchical concepts of tumor stem cells, leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia have not yet been phenotypically identified. Different subpopulations, as defined by surface markers, have shown equal abilities to reconstitute leukemia upon transplantation into immunodeficient mice. Using a non-obese diabetes/severe combined immunodeficiency human acute lymphoblastic leukemia mouse model and cell cycle analysis annotating cells to distinct cycle phases, we functionally characterized leukemia-initiating cells and found that cells in all stages of the cell cycle are able to reconstitute leukemia in vivo, with early cycling cells (G1blow population) exhibiting the highest leukemia-initiating potential. Interestingly, cells of the G2/M compartment, i.e. dividing cells, were less effective in leukemia reconstitution. Moreover, G1blow cells were more resistant to spontaneous or drug-induced cell death in vitro, were enriched for stem cell signatures and were less metabolically active, as determined by lower levels of reactive oxygen species, compared to G2/M stage cells. Our data provide new information on the biological properties of leukemia-initiating cells in B-cell precursor acute lymphoblastic leukemia and underline the concept of a stochastic model of leukemogenesis.

Tight regulation of FOXO1 is essential for maintenance of B-cell precursor acute lymphoblastic leukemia

The FOXO1 transcription factor plays an essential role in the regulation of proliferation and survival programs at early stages of B-cell differentiation. Here, we show that tightly regulated FOXO1 activity is essential for maintenance of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Genetic and pharmacological inactivation of FOXO1 in BCP-ALL cell lines produced a strong antileukemic effect associated with CCND3 downregulation. Moreover, we demonstrated that CCND3 expression is critical for BCP-ALL survival and that overexpression of CCND3 protected BCP-ALL cell lines from growth arrest and apoptosis induced by FOXO1 inactivation. Most importantly, pharmacological inhibition of FOXO1 showed antileukemia activity on several primary, patient-derived, pediatric ALL xenografts with effective leukemia reduction in the hematopoietic, lymphoid, and central nervous system organ compartments, ultimately leading to prolonged survival without leukemia reoccurrence in a preclinical in vivo model of BCP-ALL. These results suggest that repression of FOXO1 might be a feasible approach for the treatment of BCP-ALL.

Overcoming Apoptosis Resistance In High Risk Acute Lymphoblastic Leukemia By Smac Mimetics In a Preclinical ALL Xenograft Model In Vivo

Defects in cell death signaling e.g. overexpression of “Inhibitor of Apoptosis” (IAP) proteins are associated with poor prognosis and might be one reason for treatment failure and relapse of acute leukemia. Therefore, IAP antagonists, so called SMAC mimetics (SMs), provide a promising novel treatment strategy for pediatric ALL. In this study we investigated the effects of the small molecule SM BV6 on 42 primary ALL samples. Intriguingly, 70% of all individual patient-derived leukemias showed cell death induction after BV6 treatment in a TNF-α dependent manner. Previously, we described that rapid engraftment of ALL cells in NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in ALL cells and indicative for early patient relapse. Importantly, ALL samples with a TTLshort/early relapse phenotype showed activation of the constitutive deficient apoptosis signaling pathway upon BV6-treatment, demonstrating that SMs induce apoptosis signaling in former apoptosis resistant primary ALL cells. We further evaluated the in vivo efficacy of BV6 on high-risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. Most interestingly, a profound reduction of tumor load and prolonged survival of animals was observed upon BV6 in vivo treatment alone which was even more pronounced in combination with multidrug chemotherapy. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death inducing effect of BV6, indicating that BV6 induced apoptosis involves signaling via TNF-α and thereby provides a potential biomarker for the identification of patients who would benefit from SM treatment. Taken together, we show that the small molecule SM BV6 induces cell death via a TNF-α loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high-risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL.