FLT3 TKI Are Differentially Affected By Both Plasma Binding and Stromal Survival Factors
Authors: David Young J , Ma Hayley S , Small Donald 
Published: October 2019

Abstract
The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML). As such it represents an attractive target for tyrosine kinase inhibitors (TKI) for the treatment of AML, a number of which have entered clinical trials. However, to date, these trials have demonstrated limited clinical efficacy. Evidence indicates that failure to achieved adequate inhibitory activity against FLT3, at least partially as a result of plasma protein binding, as well as survival factors provided by stroma, are among the reasons for the limited efficacy of FLT3 TKI.
In this study we have characterized the effects of plasma binding and stromal survival factors on TTT-3002, a novel TKI that has demonstrated potent inhibition of the internal tandem duplication (FLT3-ITD) as well as a large panel of clinically described kinase mutants (FLT3-KD), and compared it to the effects these same conditions have on other FLT3 TKI.
Using plasma from pediatric/young adult patients of a variety of ages (n = 24, median 7.2 years, 4.1-36.9 years), the impact of human plasma binding on the activity of TTT-3002 against FLT3-ITD dependent proliferation. 50% human plasma results in a moderate decrease in activity of TTT-3002 (IC50 45.2 nM in human plasma, 2.4 nM in media with 10% FCS). This is a markedly smaller shift than several TKIs currently or previously in clinical trials including sorafenib, CEP-701, PKC-412, and AC-220 (50 to >500-fold increase in IC50). The data demonstrate a clear linear relationship between plasma alpha-1 acidglycoprotein (AGP), an acute-phase reactant, and fold-increase in IC50 of TTT-3002 (1.7 fold-increase per 10 mg/dL AGP). The data also demonstrate a wide variability in patient plasma AGP concentration that is dependent upon age, but also appears to be significantly impacted by position in treatment (median 117 mg/dL, 58-354 mg/dL). Plasma AGP strongly inhibits several FLT3 inhibitors including CEP-701 and PKC-412 (>100-fold increase in IC50); whereas other FLT3 inhibitors such as sorafenib and AC-220 are inhibited by human plasma in an AGP-independent manner. These data indicate the presence of several human plasma proteins whose binding leads to drug inhibition that TTT-3002 is nonetheless resistant to, in comparison to other clinically relevant FLT3 inhibitors. The identification of these proteins is ongoing.
Using primary human bone marrow stromal cell co-culture with two FLT3-ITD cell lines increases the IC50 of TTT-3002 but to a lower extent than that of AC-220 (MV4-11: 1.1-1.9 fold versus 2.3-2.7 fold, MOLM-14: 2.2-2.3 fold versus 3.5-3.6 fold). The data are consistent across stromal cells cultured from several different pediatric patients (n = 5).
These data suggest that TTT-3002 may be more resistant to some of the inhibitory effects that have led to disappointing results with other similar FLT3-targeted TKIs. As such, TTT-3002 represents an appealing candidate for clinical studies.

Disclosures
No relevant conflicts of interest to declare.