Mechanisms of Resistance to the FLT3-Tyrosine Kinase Inhibitor PKC412 in Patients with AML.
Authors: Heidel F. , Breitenbuecher F. , Kindler T. , Solem F. K. , Carius B. , Thiede H.-M. , Cohen P. S. , Roesel J. , Estey E. H. , Feldman E. , Ehninger G. , Schiller G. J. , Klimek V. , Nimer S. , DeAngelo D. , Giles F. , Stone R. M. , Huber C. , Fischer T. 
Published: April 2020

Abstract
The FLT3 receptor tyrosine kinase is expressed in 70-90% of cases of AML. Up to 35% of patients with AML show mutations in the JM-region or kinase domain of FLT3. These lead to autophosphorylation promoting ligand-independent cell proliferation and inhibition of apoptosis. Treatment with FLT3 tyrosine kinase inhibitors (TKI) is a promising tool in therapy of AML. Preliminary results investigating the FLT3-TKI PKC412 in patients with relapsed/refractory AML revealed that 11/15 patients (73%) with mutated FLT3 and 16/46 patients (35%) with WT FLT3 showed a &gt;50% blast response in peripheral blood (Estey E et al. Blood.2003; 102:919a). Despite its remarkable efficacy in reducing the leukemic clone, remissions upon single agent therapy using PKC412 tend to be short and secondary resistance occurs rather rapidly. So far, very little is known on the mechanisms of resistance to therapy using FLT3-TKIs. Therefore, we sought to characterize the molecular mechanisms involved. Overall PB and BM samples from 5 patients undergoing single agent therapy with PKC412 were analyzed. Three patients revealed reactivation of FLT3-kinase at relapse. Unchanged protein levels of Flt3 as detected by Western blotting before therapy and at relapse showed that amplification of FLT3 does not appear to play a role in development of secondary resistance. PKC412 plasma levels, as measured by HPLC, were similar in patients with and without reactivation of the kinase. Therefore, we examined the bioactivity of plasma obtained at relapse to inhibit phosphorylation of FLT3 in MV4-11 cells harboring FLT3-ITD. In two patients, plasma inhibited FLT3-tyrosine phosphorylation at the time of remission as well as at relapse, while in one patient, plasma was not able to inhibit FLT3-phosphorylation. Therefore, in this patient, an inhibitory activity in serum appeared to be operating. A potential candidate for this inhibitory activity is alpha1-glycoprotein (AGP) that was shown previously to mediate resistance to STI571 treatment. Our experiments showed that AGP is indeed able to inhibit bioactivity of PKC412 in-vitro. However, AGP-levels monitored in PB samples from patients included in this analysis did not show elevated plasma levels at relapse. Ex-vivo treatment of primary blasts with PKC412 at relapse showed that in one patient, blasts were still sensitive to PKC412. In two other patients, blasts showed resistance using a wide dose range of PKC412. Therefore, in these patients, cDNA sequencing of FLT3 was performed. In one patient, we identified a point mutation leading to an aminoacid exchange within the N-lobe of the FLT3-kinase domain at relapse. This mutation was not present at start of PKC412 therapy. Experiments analyzing the functional role of the novel mutation in the hematopoietic cell line 32D are in progress and will be presented. In conclusion, we have preliminarily identified examples of kinase-dependent and independent mechanisms operating in clinical resistance to PKC412.