TTT-3002 Is Active Against Relapsed/Refractory AML Patient Bone Marrow With FLT3 TKI-Resistance Mutations At Residue D835

Over 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FLT3, either internal tandem duplication (ITD) or point mutations (FLT3/PM). FLT3/ITD mutations in particular confer a poor prognosis, and thus several FLT3 tyrosine kinase inhibitors (TKIs) have been developed and are currently in clinical trials. However, many have failed due to insufficient achievement of FLT3 kinase inhibition in vivo. This is thought to be due to insufficient potency, reduced activity against FLT3/PM, and/or selection for resistance-conferring point mutations in FLT3/ITD. Therefore, the search for novel FLT3 TKIs that overcome some of these resistance mechanisms that result in persistent FLT3 activation is necessary to improve the cure rate for this disease. We have previously reported on TTT-3002, a novel TKI that possesses the most potent activity against FLT3 phosphorylation, with IC₅₀s of 100-250pM. Here we characterize the activity of this compound against TKI-resistant AML.

The phenomenon of drug resistance associated with selection of FLT3 point mutations is being observed with increasing frequency as higher levels of inhibition by FLT3 TKIs are achieved in clinical trials of FLT3/ITD AML patients. In order to predict the ability of TTT-3002 to successfully treat some of these resistance mutations, we used site-directed mutagenesis to generate a series of FLT3/ITD TKI resistant cell lines. We directly compared the activity of TTT-3002 to other FLT3 TKIs currently in clinical trials including CEP-701, sorafenib, AC220 and PKC412. TTT-3002 remained the most potent inhibitor of all six FLT3/ITD TKI resistance mutants screened, with viability IC₅₀ values of less than 1nM (with the single exception of Ba/F3-G697R/ITD, IC₅₀=11nM). It was thus able to effectively inhibit FLT3 with TKI resistance mutations against which other TKIs are ineffective. Therefore, this compound’s broad spectrum of activity against FLT3/ITD TKI resistance mutations may enable it to successfully treat FLT3/ITD AML patients who have become resistant to other FLT3 TKIs.

To examine this directly, human AML samples were obtained from patients that developed resistance to sorafenib or AC220 while on clinical trials and presented with a dual D835/ITD mutation or D835 point mutation alone at the time of relapse. These blasts were evaluated for their ex vivo sensitivity to TTT-3002, sorafenib and AC220, with a diagnostic FLT3/ITD⁺ patient sample for comparison. Encouragingly, we observed significant effects on proliferation, induction of apoptosis, and cell signaling when cells were treated with TTT-3002. AC220 was moderately active against FLT3/ITD⁺ blasts and one patient sample with a D835/ITD mutation, and sorafenib was active against only the diagnostic FLT3/ITD sample, but none of the three relapse samples. Therefore, TTT-3002 maintains activity against human AML patient samples that are resistant in clinical trials to the FLT3 TKIs sorafenib and AC220.

To account for the patterns of drug resistance observed for different FLT3 TKIs in our study, we modeled the positions of staurosporine-like (TTT-3002, CEP-701 and PKC412) and sorafenib-like inhibitors (sorafenib and AC220) bound to FLT3. Examination of amino-acid substitutions in FLT3 known to confer resistance to TKIs from clinical trials shows that substituting arginine for glycine at position 697 likely leads to steric clashes with FLT3 TKIs. It has been proposed that the F691 residue forms a stabilizing π-π stacking interaction with AC220, and thus a mutation to a Leu accounts for reduced binding affinity for this TKI. TTT-3002 is predicted to bind FLT3 without making direct contact with the F691 residue, and thus is unaffected by mutations at this site accounting for its continued potency. The closer proximity of F691 and N676 to sorafenib compared to staurosporine likely underlies the trend for substitutions at these sites to confer resistance to sorafenib-like but not staurosporine-like inhibitors.

In summary, TTT-3002 has the potential for increased clinical applicability due to its ability to target not only FLT3/ITD and FLT3/PM AML patients but also patients who have developed resistance to other FLT3 TKIs through the selection of resistance mutations within the FLT3/ITD allele. These preclinical studies further support the entry of TTT-3002 into human clinical trials.