Targeting integrin linked kinase and FMS-like tyrosine kinase-3 is cytotoxic to acute myeloid leukemia stem cells but spares normal progenitors
Introduction
Among the malignant blast cells in patients with acute myeloid leukemia (AML) are progenitors that exhibit the capacity for self-renewal of their own numbers and a much greater proliferative capacity than the majority of leukemic cells [1]. These AML progenitors can be demonstrated by their capacity to engraft and proliferate in immunodeficient mice (NOD/SCID mouse leukemia-initiating cells or NOD/SL-IC) and to initiate long-term malignant hematopoiesis in tissue culture (long-term suspension culture-initiating cells or SC-IC) [2], [3], [4]. Cell surface phenotypes which enrich for progenitors that are detected in both assays are often similar to each other and to those exhibited by primitive normal progenitors [3], [4], [5]. It seems possible that such progenitors are important for maintenance of leukemia in patients.
Aberrant cell signaling is thought to play a role in maintenance of the leukemic clone in AML by providing a proliferative advantage to malignant cells and allowing them to escape mechanisms that lead to cell death or apoptosis. The FMS-like tyrosine kinase 3 (FLT3) and phosphatidylinositol-3-kinase (PI3K)-dependent pathways are two candidate signaling pathways thought to play such roles [6], [7], [8].
The FLT3 receptor is a member of the type III receptor tyrosine kinase (RTK) subfamily which also includes c-kit, c-FMS and PDGF [9]. Activation of FLT3 normally occurs when FLT3 ligand binds to the receptor, inducing formation of a homodimer which in turn activates the kinase domain of the receptor, resulting in signaling to downstream pathways such as Ras, and PI3K [10], [11], [12]. FLT3 is mutated in approximately one third of AML samples, the majority of these mutations being internal tandem duplications (ITD) of the juxtamembrane region of the receptor although other mutations exist which can, like the ITD, lead to aberrant and constitutive activation of the receptor tyrosine kinase and downstream signaling pathways [13], [14].
The PI3K-dependent signaling pathway controls cell growth and proliferation via activation of Akt and downstream targets, and is activated in a large number of AML samples including those with poor prognostic features [8], [15]. Inhibition of this pathway by compounds targeting various intermediates is cytotoxic to AML blasts. Integrin linked kinase (ILK) is an ankyrin-repeat containing serine/threonine kinase involved in phosphorylation of Akt (ser473) and GSK3. ILK is overexpressed or constitutively active in a large number of cancers, and is ubiquitously expressed in AML blast samples [16]. Inhibition of ILK by siRNA-mediated approaches or small molecule inhibitors in solid tumors results in apoptosis and/or impaired invasion of the cancer cells [17], [18], [19].
Here we demonstrate that specific inhibition of ILK using siRNA is toxic to primary human AML CFC progenitors, suggesting ILK as a relevant target for AML therapy. In addition, the expression of FLT3, ILK and phosphorylated GSK3 (as a marker of activation of Akt) was measured in subpopulations of AML blasts enriched for NOD/SL-IC and SC-IC as well as quiescent and cycling leukemic cells. Furthermore, inhibition of these targets with QLT0267, a small molecule inhibitor of both ILK and FLT3 [16], is shown to inhibit the survival of both SC-IC and NOD/SL-IC but to have relatively little effect on normal bone marrow SC-IC, and NOD/SCID repopulating cells (RC). The combined effect on AML CFC of QLT0267 with conventional chemotherapy drugs often used in the treatment of AML (cytarabine or daunorubicin) was also investigated. In total, these data suggest that ILK and FLT3 expression are more important to the maintenance of leukemia-initiating cells in AML than to normal primitive progenitors and that their combined inhibition may be useful in eradicating the leukemic clone while sparing normal hematopoiesis.
Section snippets
AML and normal cells
Peripheral blood (PB) cells and bone marrow samples were obtained from 11 newly-diagnosed AML patients and normal bone marrow donors after informed consent and with the approval of the Clinical Research Ethics Board of the University of British Columbia (Supplementary Table 1). Among the 11 AML patients ranging in age from 28 to 72 years, 10 had intermediate risk bone marrow cytogenetics (7 normal karyotype) and one (sample 3) good risk (inv(16)) cytogenetics at diagnosis according to Medical
siRNA inhibition of ILK is toxic to primary human AML CFC
Previous investigation had determined that ILK was ubiquitously expressed in AML blasts [16]. siRNA targeting ILK was used to determine if downregulation of this expression would affect AML CFC growth from four patient samples (Fig. 1A). 72 h exposure to ILK siRNA reduced AML CFC growth, as compared to control, non-targeting siRNA, from all 4 patient samples (n = 3). ILK mRNA expression was quantified using qRT-PCR and ILK knockdown was determined as compared to ILK expression in untreated cells.
Discussion
The PI3K pathway is frequently constitutively active in many human malignancies including AML [8], [15], [34], [35], [36], [37]. Although this pathway is necessary for normal cell proliferation, differentiation and survival its downregulation has shown selective toxicity for malignant rather than normal cells [15], [16]. It has been suggested that this differential sensitivity is a form of ‘oncogene addiction’ in which malignant cells become abnormally dependent on pathways that provide them
Conflict of interest
The authors have no conflict of interest to declare with regards to this work.
Acknowledgements
The authors thank QLT Inc. for their gift of QLT0267, Virginia Gray and Gitte Gerhard for technical assistance and Christine Kelly for help with manuscript preparation. This work was supported by grants from the Cancer Research Society of Canada (DEH) and the Leukemia and Lymphoma Society of Canada (DEH).
Contributors: A.M.: concept and design, collection and/or assembly of data, data analysis, manuscript writing; S.D.: concept and design, final approval of manuscript; D.H.: concept and design,
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