Sarah Mirjam Eckhoff

Intrinsic and chemo-sensitizing activity of SMAC-mimetics on high-risk childhood acute lymphoblastic leukemia

SMAC-mimetics represent a targeted therapy approach to overcome apoptosis resistance in many tumors. Here, we investigated the efficacy of the SMAC-mimetic BV6 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In ALL cell lines, intrinsic apoptosis sensitivity was associated with rapid cIAP degradation, NF-κB activation, TNF-α secretion and induction of an autocrine TNF-α-dependent cell death loop. This pattern of responsiveness was also observed upon ex vivo analysis of 40 primograft BCP-ALL samples. Treatment with BV6 induced cell death in the majority of ALL primografts including leukemias with high-risk and poor-prognosis features. Inhibition of cell death by the TNF receptor fusion protein etanercept demonstrated that BV6 activity is dependent on TNF-α. In a preclinical NOD/SCID/huALL model of high-risk ALL, marked anti-leukemia effectivity and significantly prolonged survival were observed upon BV6 treatment. Interestingly, also in vivo, intrinsic SMAC-mimetic activity was mediated by TNF-α. Importantly, BV6 increased the effectivity of conventional induction therapy including vincristine, dexamethasone and asparaginase leading to prolonged remission induction. These data suggest SMAC-mimetics as an important addendum to efficient therapy of pediatric BCP-ALL.

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.

Characterization and identification of leukemia initiating cells in pediatric acute lymphoblastic leukemia.

In acute lymphoblastic leukemia (ALL) leukemia initiating cells (LICs) have been considered to be organized in an hierarchical fashion, however recent findings demonstrated LIC-activity also in more committed cells supporting a stochastic stem cell concept. Thus, the nature of leukemia initiating cells in ALL still remains elusive. As an alternative approach to define LICs by expression of cellular markers as commonly employed, we addressed LIC activities in ALL by functional investigation of cellular subfractions of distinct cell cycle phases. Patient-derived xenograft BCP-ALL cells were sorted according to cell cycle stages (i.e. G0/G1 and G2/M) and subsequently transplanted onto NOD/SCID mice. All cell fractions led to engraftment indicating LIC activity of all leukemia cells. However, cells isolated from G0/G1 cell cycle phases led to early leukemia onset in contrast to cells from late cell cycle (G2/M) constantly showing lowest LIC activity. Strikingly, this difference in LIC activity was maintained upon secondary transplantation. In an alternative approach, we investigated metabolic activities in cellular leukemia subfractions and identified low metabolic activity in cells of early G0/G1 cell cycle phases compared to increased cellular metabolism in cells of late G2/M. To address LIC-capacities of ALL cells with distinct metabolic activities on a functional level, cellular fractions were sorted according to low or high ROS levels and subsequently transplanted onto NOD/SCID mice. Interestingly, a prolonged engraftment was observed upon transplantation of ROShigh cells in contrast to faster leukemia repopulation in recipients transplanted with ROSlow cells, showing that the metabolic activity is indicative for its leukemia initiating activity. In summary, we identified LIC-activity in all leukemia subpopulations. Importantly, our findings indicate that leukemia initiating cells in ALL are enriched in early cell cycle and characterized by low metabolic activity.

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.