Novel Approaches to Target Mutant FLT3 Leukaemia

Abstract

Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinases (RTK) and is involved in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Oncogenic mutations in the FLT3 gene resulting in constitutively active FLT3 variants are frequently found in acute myeloid leukaemia (AML) patients and correlate with patient's poor survival. Targeting FLT3 mutant leukaemic stem cells (LSC) is a key to efficient treatment of patients with relapsed/refractory AML. It is therefore essential to understand how LSC escape current therapies in order to develop novel therapeutic strategies. Here, we summarize the current knowledge on mechanisms of FLT3 activity regulation and its cellular consequences. Furthermore, we discuss how aberrant FLT3 signalling cooperates with other oncogenic lesions and the microenvironment to drive haematopoietic malignancies and how this can be harnessed for therapeutical purposes.

Keywords: FMS-like tyrosine kinase 3 (FLT3); acute myeloid leukaemia (AML); cancer cell vulnerability; haematopoietic niche; oncogenic signaling; re-sistance development.

Figure 1

Activating ITD in the juxtamembrane domain and point mutations in the tyrosine kinase domain (TKD) of FLT3. (a) Inactive, autoinhibited conformation of FLT3 (1RJB.pdb). Residues involved in ATP-binding and catalysis (K644, D829) and residues subjected to mutation in leukemia are indicated. (b) Model of active FLT3 kinase domain demonstrating the region for ITD insertions and known frequent TKD point mutations resulting in activation of FLT3. Homology modelling of FLT3 kinase domain was based the active conformation of the CSF-1 receptor (3LCD.pdb) using MODELLER within the UCSF Chimera 1.14 software package. The activation segment of the kinase domain is marked in pink, the juxtamembrane domain is marked in blue. N676K [10,11], ITD reviewed in [9]. References: D835Y [12,13,14,15], D835H [12,13,14], D835E [13,14], D835N [13], D835V [13], I836 del. [13,16], I836T [13], S840G [17], S840insGS [17], Y842C [18,19], Y572C [19,20], N841I [21], N841K [19,22].

Figure 2

Biosynthesis and major signalling pathways activated by FLT3 wildtype (WT) (a) and FLT3 with internal tandem duplication (ITD) mutation (b). (a) Wildtype (WT) FLT3 is synthesized and processed in the endoplasmic reticulum (ER) and the GOLGI and reaches the plasma membrane (PM) as inactive monomer. Binding of the FLT3 ligand (FL) induces dimerization, autophosphorylation and induction of downstream signalling. This comprises activation of the Ras/mitogen activated protein kinase (MAPK) pathway and the phosphoinositol-3 kinase (PI3K)/AKT pathway from the PM and to some extent STAT5 phosphorylation from endosomes (not shown). Consequently, FLT3 WT signalling induces proliferation and differentiation of haematopoietic progenitor cells. FLT3 WT is inactivated via dephosphorylation by protein tyrosine phosphatases (PTPs) such as the transmembrane PTPRJ but also cytoplasmic PTPs such as SHP-1. (b) FLT3 with internal tandem duplication (ITD) mutations in the juxtamembrane region is constitutively and ligand-independently active at the PM and preferentially at endomembranes such as ER and endosomes (not shown). In particular, STAT5 is aberrantly activated at endomembranes. FLT3 ITD induces reactive oxygen species (ROS) production via activation of NADPH oxidase (NOX) 4. As a consequence, PTPRJ gets inactivated via oxidation of catalytic active cysteine residues. Dimerization of PTPRJ also reduces its catalytic activity. FLT3 ITD signalling can be blunted by the use of specific tyrosine kinase inhibitors (TKI) that are in clinical use. However, secondary mutations in FLT3 can cause inhibitor-resistance. Potential novel therapeutics comprise inhibitors of STAT5, ROS quencher and peptides that prevent dimerization of PTPRJ, thereby enhancing its activity.

Figure 3

Interaction of haematopoietic stem cells (HSC) and FLT3 ITD-positive blast cells with the haematopoietic bone marrow niche. (a) Under physiological conditions, HSC are recruited and immobilised via the chemokine CXCL12, produced by bone marrow stromal cells in the perivascular niche. On HSC CXCL12 binds to and activates the G-protein coupled chemokine receptor CXCR4. HSC give rise to more restricted progenitor cells that fuel differentiated peripheral blood cells. (b) FLT3 ITD⁺ leukaemic blast cells shape the bone marrow haematopoietic niche. Through activation of the cytoplasmic serine/threonine kinase Pim1, FLT3 ITD enhances CXCR4 signalling and mediates leukaemic blast recruitment to the perivascular niche. Through the induction of tumour necrosis factor (TNF)-expression in endothelial cells, leukaemic blast cells drive TNF-mediated decay of stroma cells and in consequence, impaired normal haematopoiesis. (c) The perivascular niche, in particular stromal cells contribute to tyrosine kinase inhibitor (TKI)-resistance through (I) secretion of cytokines and growth factors that enable bypassing of inhibited FLT3 ITD and (II) CYP3A4-mediated catabolism of TKI in stromal cells.

Figure 4

Targeting novel vulnerabilities in FLT3 mutant cells as novel therapeutic strategy. (a) FLT3 ITD induces expression of activating transcription factor (ATF) 4 that enhances the unfolded protein response (UPR) and triggers autophagy via autophagy related protein (ATG) 12. Export from the ER and degradation of misfolded proteins via the proteasome is part of the UPR. FLT3 ITD⁺ blasts are susceptible to proteasome inhibition via e.g. the clinically approved Bortezomib. Inhibtion of ER-to-Golgi trafficking via Flustatin further enhances UPR and UPR-mediated cell death in these cells. (b) FLT3 ITD signalling reduces Ku86 expression and consequently DNA damage repair by non-homologues end-joining. As a consequence, FLT3 ITD⁺ blasts rely on poly (ADP-ribose) polymerase (PARP) 1-mediated DNA damage response, making them vulnerable for PARP inhibitors such as Olaparib. (c) TKI-mediated inhibition of FLT3 ITD reduces expression of Glucose transporter Glut3 and lactate dehydrogenase (LDHA). As a consequence, ITD⁺ blasts rely on Glutamine as carbone source for the tricarboxylic acid cycle (TCA) cycle, making them vulnerable for glutaminase (GLS)-inhibitors such as the drug candidate CB-839 (d) Tyrosine kinase inhibitor (TKI)-mediated inhibition of FLT3 ITD signalling in leukaemic blast cells can be bypassed by expression and cytokine-mediated activation of the receptor tyrosine kinases Axl (by its ligand GAS6) or granulocyte colony stimulating factor receptor G-CSFR, or by activation of the interleukin (IL)-3 receptor complex. Signalling via Axl is regulated via ADAM17-mediated limited proteolysis. Simultaneous inhibition of FLT3 and Axl enhances therapeutic response. PM, plasma membrane; ER endoplasmatic reticulum.