Identification and Characterization of an Activating TrkA Deletion Mutation in Acute Myeloid Leukemia

Abstract

In this study, we utilized retroviral transfer of cDNA libraries in order to identify oncogenes that are expressed in acute myeloid leukemia (AML). From screens using two different cell types as targets for cellular transformation, a single cDNA encoding a variant of the TrkA protooncogene was isolated. The protein product of this protooncogene, TrkA, is a receptor tyrosine kinase for nerve growth factor. The isolated transforming cDNA encoded a TrkA protein that contains a 75-amino-acid deletion in the extracellular domain of the receptor and was named ⌬TrkA. ⌬TrkA readily transformed fibroblast and epithelial cell lines. The deletion resulted in activation of the tyrosine kinase domain leading to constitutive tyrosine phosphorylation of the protein. Expression of ⌬TrkA in cells led to the constitutive activation of intracellular signaling pathways that include Ras, extracellular signal-regulated kinase/mitogen-activated protein kinase, and Akt. Importantly, ⌬TrkA altered the apoptotic and growth properties of 32D myeloid progenitor cells, suggesting ⌬TrkA may have contributed to the development and/or maintenance of the myeloid leukemia from which it was isolated. Unlike Bcr-Abl, expression of ⌬TrkA did not activate Stat5 in these cells. We have detected expression of ⌬TrkA in the original AML sample by reverse transcriptase PCR and by Western blot analysis. While previous TrkA mutations identified from human tumors involved fusion to other proteins, this report is the initial demonstration that deletions within TrkA may play a role in human cancers. Finally, this report is the first to indicate mutations in TrkA may contribute to leukemogenesis.

Effects of the leukemia-associated AML1-ETO protein on hematopoietic stem and progenitor cells

“…Such mutations occur most commonly in AML, M3, but they are also found in B9% of t(8;21) þ AML (Kottaridis et al, 2001). Activating point mutations in the c-kit tyrosine kinase receptor have also been found in t(8;21) (and in inv(16) AML) (Care et al, 2003) and, less commonly, activating mutations in the M-CSF receptor (c-fms) or in the TrkA and TrkC receptors (which bind nerve growth factor and neurotrophin ligands, respectively) have been reported (Reuther et al, 2000;Abu-Duhier et al, 2003). AML cells express IL-3 receptors and respond relatively uniquely to this cytokine for growth (Jordan et al, 2000).…”

Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression

“…An example includes the TrkA deletion mutation that was identified in a retroviral cDNA library derived from a patient with leukemia. 41 These types of modifications would not be detected by other genome wide approaches, such as cDNA chip technologies, the latter of which also does not determine causal events vs caused events. The selection process utilized in this study involved repetitive rounds of IL-3 withdrawal followed by recovery of the remaining viable cells by the readdition of IL-3.…”

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway

“…A stably transfected human MCF10A cell line was engineered to express a constitutively active form of human TrkA containing a 75-amino-acid extracellular deletion (MCF10A-TrkA-Δ) originally isolated from an AML patient (7). A cellular EC 50 value of 1.2 ± 0.7 nmol/L was determined for AZ-23, a result corroborated through immunoblotting for phosphorylated TrkA protein ( Fig.…”

“…Trk receptors have also been shown to be potent oncogenes with roles in malignant transformation, metastasis, and survival signaling in human tumors (3)(4)(5). Independent mechanisms of Trk pathway activation, including constitutive oncogenic fusions, autocrine signaling, and point mutations, have been described in medulloblastoma; neuroblastoma; acute myelogenous leukemia (AML); and thyroid, pancreatic, breast, lung, and prostate cancers, suggesting that the Trk pathway may be more broadly involved in carcinogenesis and tumor cell survival (6)(7)(8)(9)(10)(11)(12).…”