Arjo P Rutten

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

Introduction Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. Results For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patients bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n = 88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n = 91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n = 91), which reflect the total neoplastic burden, revealed four patient groups with different survival. Conclusion and Perspective Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.

Primary acute myeloid leukemia cells with overexpression of EVI-1 are sensitive to all- trans retinoic acid

Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.

Abstract 2339: IGFBP7 eradicates leukemic stem and progenitor cells in acute myeloid leukemia

Despite high complete remission rates after chemotherapy treatment, only 30-40% of acute myeloid leukemia (AML) patients ( Since LSC and HSC share the same immunophenotype (CD34+CD38-), we and others identified markers including CLL-1 as well as scatter properties, to distinguish leukemic from normal stem cells. By these properties we purified LSC, HSC and progenitors fractions from AML bone marrow. Gene expression profiling revealed the insulin-like growth factor binding-protein-7 (IGFBP7) as differentially expressed between LSC and HSC and between LSC and the AML bulk. IGFBP7 is a secreted tumor suppressor and based on our expression data we hypothesized that IGFBP7 might induce the eradication of LSC. Using a knockdown strategy, we show that decreased levels of IGFBP7 in AML cell lines result in decreased sensitivity to chemotherapy. Moreover, we show that both IGFBP7 overexpression and recombinant human IGFBP7 (rhIGFBP7) reduces the survival of leukemic stem and progenitor cells from AML patients. Incubation with rhIGFBP7 showed decreased survival of leukemic blasts while sparing healthy cells. Most importantly, IGFBP7 significantly reduces human leukemic engraftment of primary AML cells in immune-deficient NOD/SCID-IL2γ knockout (NSG) mice. Altogether, LSC have reduced levels of IGFBP7 which might be responsible for their decreased sensitivity to chemotherapy. The eradication of LSC and leukemic progenitors can be accomplished by enhancing IGFBP7 levels, suggesting that AML patients might benefit from a combination of rhIGFBP7 and chemotherapy. This combination therapy might prevent relapse and improve AML outcome. Citation Format: Han Verhagen, Marjon Smit, David de Leeuw, Arjo Rutten, Mei-Ling Tsui, Fedor Denkers, Monique Terwijn, Patrick Celie, Gert Ossenkoppele, Gerrit Jan Schuurhuis, Linda Smit. IGFBP7 eradicates leukemic stem and progenitor cells in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2015-2339