Linda Smit

Normal hematopoietic stem cells within the AML bone marrow have a distinct and higher ALDH activity level than co-existing leukemic stem cells.

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDHbright). The ALDHbright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDHlow. In CD34-positive AML, LSC are also ALDHlow but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.

MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Purpose: Classification of acute leukemia is based on the commitment of leukemic cells to the myeloid or the lymphoid lineage. However, a small percentage of acute leukemia cases lack straightforward immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of acute leukemia that cannot be classified as either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). The lack of clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. Experimental Design: Here, we compared the microRNA (miRNA) expression profiles of 17 cases with acute leukemia of ambiguous lineage and 16 cases of AML, B-cell acute lymphoid leukemia (B-ALL), and T-cell acute lymphoid leukemia (T-ALL). Results: We show that leukemias of ambiguous lineage do not segregate as a separate entity but exhibit miRNA expression profiles similar to AML, B-ALL, or T-ALL. We show that by using only 5 of the most lineage-discriminative miRNAs, we are able to define acute leukemia of ambiguous lineage as either AML or ALL. Conclusion: Our results indicate the presence of a myeloid or lymphoid lineage-specific genotype, as reflected by miRNA expression, in these acute leukemias despite their ambiguous immunophenotype. miRNA-based classification of acute leukemia of ambiguous lineage might be of additional value in therapeutic decision making. Clin Cancer Res; 19(8); 2187–96. ©2013 AACR.

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.

MicroRNA-551b is highly expressed in hematopoietic stem cells and a biomarker for relapse and poor prognosis in acute myeloid leukemia

MicroRNA-551b is highly expressed in hematopoietic stem cells and a biomarker for relapse and poor prognosis in acute myeloid leukemia

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.

Specific Depletion of Leukemic Stem Cells: Can MicroRNAs Make the Difference?

For over 40 years the standard treatment for acute myeloid leukemia (AML) patients has been a combination of chemotherapy consisting of cytarabine and an anthracycline such as daunorubicin. This standard treatment results in complete remission (CR) in the majority of AML patients. However, despite these high CR rates, only 30–40% (<60 years) and 10–20% (>60 years) of patients survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with stem cell-like properties, often referred to as “leukemic stem cells” (LSCs). LSCs co-exist in the bone marrow of the AML patient with residual healthy hematopoietic stem cells (HSCs), which are needed to reconstitute the blood after therapy. To prevent relapse, development of additional therapies targeting LSCs, while sparing HSCs, is essential. As LSCs are rare, heterogeneous and dynamic, these cells are extremely difficult to target by single gene therapies. Modulation of miRNAs and consequently the regulation of hundreds of their targets may be the key to successful elimination of resistant LSCs, either by inducing apoptosis or by sensitizing them for chemotherapy. To address the need for specific targeting of LSCs, miRNA expression patterns in highly enriched HSCs, LSCs, and leukemic progenitors, all derived from the same patients’ bone marrow, were determined and differentially expressed miRNAs between LSCs and HSCs and between LSCs and leukemic progenitors were identified. Several of these miRNAs are specifically expressed in LSCs and/or HSCs and associated with AML prognosis and treatment outcome. In this review, we will focus on the expression and function of miRNAs expressed in normal and leukemic stem cells that are residing within the AML bone marrow. Moreover, we will review their possible prospective as specific targets for anti-LSC therapy.

VEGFC antibody therapy drives differentiation of AML

High expression of VEGFC predicts adverse prognosis in acute myeloid leukemia (AML). We therefore explored VEGFC-targeting efficacy as an AML therapy using a VEGFC mAb. VEGFC antibody therapy enforced myelocytic differentiation of clonal CD34+ AML blasts. Treatment of CD34+ AML blasts with the antibody reduced expansion potential by 30% to 50% and enhanced differentiation via FOXO3A suppression and inhibition of MAPK/ERK proliferative signals. VEGFC antibody therapy also accelerated leukemia cell differentiation in a systemic humanized AML mouse model. Collectively, these results define a regulatory function of VEGFC in CD34+ AML cell fate decisions via FOXO3A and serve as a new potential differentiation therapy for patients with AML.Significance: These findings reveal VEGFC targeting as a promising new differentiation therapy in AML. Cancer Res; 78(20); 5940-8. ©2018 AACR.

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

Abstract 3890: MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. The main cause for this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with a stem cell-like character. These so-called leukemic stem cells (LSC) are thought to be responsible for relapse. We hypothesized that success of novel anti-AML therapies relies on functional manipulation of genes including microRNAs, resulting in elimination of leukemic (stem) cells while sparing residual co-existing normal hematopoietic stem cells (HSC). We aimed at identification of microRNAs differentially expressed between HSC, LSC and the AML bulk obtained from the same AML bone marrow, taking into account the effects of the leukemic microenvironment. To that end, we described immunophenotypic markers that can distinguish LSC from HSC. Moreover, we identified that HSC have higher aldehyde dehydrogenase activity than LSC (Schuurhuis et al. Plos One 2013). Comparing the microRNA expression profile of LSC with that of HSC showed that microRNA-551b (miR-551b) is highly expressed in residual HSC in the AML bone marrow. To determine whether miR-551b is a HSC specific microRNA we purified stem and progenitor cell subsets from normal bone marrow and showed that miR-551b is the highest expressed in the two most primitive CD34+CD38- populations i.e. CD90+CD45RA- HSC and CD90-CD45RA- multipotent progenitors. To investigate whether the expression of miR-551b is of clinical importance in AML we determined its expression in AML bone marrow samples (n=154) and showed that high miR-551b is associated with lower complete remission (CR) rates after the first cycle of induction chemotherapy, shorter relapse free survival and a worse overall survival. In line with miR-551b being a stem cell miRNA, high expression in AML was associated with an undifferentiated morphology (FAB M0). To shed more light on the functional role of miR-551b in AML we correlated the expression of miR-551b with overall gene expression in a large panel of AML patients. Many of the genes that highly correlated with miR-551b like; MLLT3, INPP4B, HTR1F, HOPX, PROM1 and others, are also present in published HSC signatures. In conclusion, miR-551b is specifically expressed in normal stem and multipotent progenitor cells and high expression in AML is associated with poor prognosis. Currently, our research focuses on the function of miR-551b in AML. Citation Format: David C. de leeuw, Fedor Denkers, Peter Valk, Iris de Rink, Ron Kerkhoven, Gerrit Jan Schuurhuis, Gert J. Ossenkoppele, Linda Smit. MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2014-3890

Abstract LB-246: MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Classification of acute leukemia (AL) is based on commitment of the leukemic cells to either the myeloid or the lymphoid lineage. However, a small percentage of AL cases display features of both hematopoietic lineages and lacks immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). The lack of a clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have shown to be able to accurately discriminate AL of the lymphoid lineage from those of the myeloid lineage. Here, we compared the microRNA expression profiles of acute leukemias of ambiguous lineage with those of B-ALL, T-ALL and AML. MicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed. Unsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between cases of the myeloid and lymphoid lineages. The top differentially expressed microRNAs between AML and ALL were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. Subsequently, we compared the general miRNA expression profiles of AL of ambiguous lineage with those of the AML, B-ALL and T-ALL cases thereby showing that these leukemias of ambiguous lineage do not segregate as a separate entity. Moreover, unsupervised clustering of all the AL samples using the top 10 percent of most variable microRNAs resulted in clustering of leukemias of ambiguous lineage with either AML, B-ALL or T-ALL. Furthermor, qRT-PCR expression analysis of the five most discriminative microRNAs between ALL and AML was able to define AL of ambiguous lineage as either AML or ALL. Thus, our results indicate the presence of a lymphoid or myeloid lineage specific microRNA expression profile in AL cases of ambiguous lineage, despite their mixed immunophenotype. At diagnosis, the classification of AL of ambiguous lineage by microRNA expression analysis might be of additional value in therapeutic decision making. Citation Format: Dave C. de Leeuw, Willemijn van den Ancker, Fedor Denkers, Rene X. Menezes, Theresia M. Westers, Gert J. Ossenkoppele, Arjan A. van de Loosdrecht, Linda Smit. MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-246. doi:10.1158/1538-7445.AM2013-LB-246