TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with activity against FLT3-associated leukemias in vitro and in vivo

Abstract

Key Points• A novel TKI is discovered with potent and selective activity against FLT3-mutant cell lines and primary patient samples.• TTT-3002 is effective in vivo in several mouse tumor models of FLT3/ITDassociated AML with minimal toxicity.More than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC 50 ) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC 50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML. (Blood. 2014;123(10):1525-1534

BET Protein Antagonist JQ1 Is Synergistically Lethal with FLT3 Tyrosine Kinase Inhibitor (TKI) and Overcomes Resistance to FLT3-TKI in AML Cells Expressing FLT-ITD

“…These include the protective effects of stromal niche cells (41), increased levels and activity of FLT3 ligand on FLT3 TK (15,17), trans-phosphorylation and increased activity of FLT3 by other kinases such as PIM-1 and SYK (42,43), and the collateral dependency on other important pro-growth and pro-survival signaling mechanisms (5,6,12,13). Other studies have focused on developing selective and more potent FLT3-TKI to overcome primary resistance to the first and second generation FLT3-TKI already in the clinic (44,45). Here, we document for the first time that co-treatment with the BET protein antagonist (BA) JQ1 and FLT3 inhibitor quizartinib or ponatinib synergistically induced apoptosis of not only cultured but also of the primary AML BPCs expressing FLT3-ITD.…”

All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo

“…[30][31][32] For details on transplantation, drug administration, and experimental analysis, see supplemental Methods (available on the Blood Web site).…”

FLT3 Kinase Inhibitor TTT-3002 Overcomes Both Activating and Drug Resistance Mutations in FLT3 in Acute Myeloid Leukemia

“…Cells were cultured in the presence of inhibitor for 1 hour at 37°C, and FLT3 and phospho-FLT3 expression was analyzed by performing immunoprecipitation of whole cell extracts for FLT3 (S-18), followed by SDS-PAGE and Western blotting as described previously (24). Other proteins were detected from whole cell lysates using the indicated antibodies and a horseradish peroxidase conjugated goat anti-rabbit secondary antibody followed by ECL.…”

“…We have previously reported that TTT-3002 is the most potent FLT3 inhibitor discovered to date, with picomolar IC 50 values against FLT3/ITD phosphorylation (24). Here we assess the activity of TTT-3002 against a broad spectrum of known FLT3/PMs, as well as a number of TKI resistance mutations within the FLT3/ITD allele.…”