Mechanisms of resistance against PKC412 in resistant FLT3-ITD positive human acute myeloid leukemia cells

Abstract

Treatment of acute myeloid leukemia (AML) remains challenging with many patients harboring unfavorable prognostic parameters such as FLT3 internal tandem duplication (FLT3-ITD) mutations leading to a constitutively activated FLT3-receptor tyrosine kinase (RTK). Activation of proteins by phosphorylation of tyrosine residues is a common mechanism in leukemia development. Therefore, specific tyrosine kinase inhibitors (TKI) have been developed for AML therapy and are currently under investigation. The staurosporine derivate PKC412 (Midostaurin) was found to be an effective inhibitor of the FLT3-RTK and is currently undergoing clinical trials for FLT3-mutated AML patients. Since resistance towards TKIs has been observed in vitro and in clinical trials, we have generated a PKC412-resistant clone (MV4-11r) of the human myelomonoblastic cell line MV4-11, which carries a homozygous FLT3-ITD mutation. MV4-11r displayed higher vitality after addition of PKC412 compared with MV4-11 with a pronounced reduction of apoptotic cells. Cytogenetic characterization revealed the acquisition of additional aberrations in the resistant cell line such as clonal alterations at chromosome 13q with additional FLT3 signals. Microarray analysis revealed significant expression changes in several genes prior to and after incubation with PKC412. The expression status of candidate genes being regulated by FLT-ITD like JAG1, p53, MCL-1, C-KIT, and FLT3/-L was confirmed by real-time PCR. In summary, resistance against PKC412 appears to be mediated by upregulation of anti-apoptotic genes and down-regulation of proapoptotic signals as well as genes that are involved in normal and malignant hematopoiesis.

FLT3 as a therapeutic target in AML: still challenging after all these years

“…89,90 In clinical trials, it has been shown that FLT3-TKI treatment and myelosuppressive chemotherapy may induce an increase in FL expression and/or FLT3 cell surface expression. 52, 91 An alternative mechanism of resistance is activation of compensatory pathways rendering FLT3-mutated cells independent of FLT3 signaling.…”

Drug resistance in mutant FLT3-positive AML

“…In addition, blocking FLT3 ligand with an FLT3 ligand-neutralizing antibody was observed to enhance the proapoptotic activity of ABT-869 against ABT-869-resistant cells (Zhou et al, 2009). Another study of resistance in MV4-11 cells made resistant to PKC412 revealed acquisition of clonal alternations at chromosome 13q, additional FLT3 signaling, and changes in gene expression before and after treatment with PKC412, including that of FLT3 ligand (Stolzel et al, 2010).…”

“…Aberrant expression of antiapoptotic proteins is another mechanism of resistance to FLT3 inhibitors. For example, increased protein levels of the inhibitor of apoptosis (IAP), survivin, as well as activation of STAT1, STAT3 and STAT5, were observed in ABT-869-resistant MV4-11 cells (Zhou et al, 2009), and antiapoptotic proteins were upregulated in PKC412-resistant MV4-11 cells (Stolzel et al, 2010). Similarly, overexpression of antiapoptotic proteins of the BCL2 family has been found to mediate resistance to FLT3 inhibition in hematopoietic cells with activating FLT3 mutations Mechanisms of resistance to FLT3 inhibition E Weisberg et al (Kohl et al, 2007) and the antiapoptotic protein, MCL-1, was observed to be induced by a non-juxtamembrane ITD that has integrated into the b-2 sheet of the first kinase domain (FLT3_ITD627E) (Brietenbuecher et al, 2009).…”

FLT3 inhibitors for acute myeloid leukemia: a review of their efficacy and mechanisms of resistance

“…Mutations or expression modification of genes regulating apoptosis, such as BCL2, MCL-1, and NF-jB, can render leukemic cells independent of FLT3 signaling [86,[95][96][97]. In an in vitro model, midostaurin resistance appeared to be in part governed by up-regulation of anti-apoptotic signals and down-regulation of pro-apoptotic pathways [98].…”