Inhibition of USP10 Induces Degradation of Oncogenic FLT3: A Novel Approach to Therapy of Leukemia

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Overall, the survival with current chemotherapy is only 20-40%, declining steadily with advancing age. Approximately 30% of AML patients have mutations that constitutively activate the FLT3 gene. The most common FLT3 mutation results in tandem duplications within the juxtamembrane domain, observed in 20-25% of AML patients (internal tandem duplication, ITD), associated with markedly decreased survival. FLT3 kinase domain inhibitors, including SU11248, SU5416, CEP-701 and PKC412 (midostaurin), have been shown to induce partial, and usually brief, remissions in clinical trials of relapsed AML patients when administered as single agents. In a large trial in newly diagnosed patients, however, midostaurin was shown to increase survival when combined with standard chemotherapy.[1] This study supports the notion that inhibition of FLT3 may be important, at least in patients with mutations in the FLT3 gene. Since drug resistance develops in some patients with newly diagnosed AML and virtually all patients with advanced disease, additional strategies to target FLT3 would be of value.

We discovered that the deubiquitinating enzyme (DUB) ubiquitin specific protease 10 (USP10) removes a degradative ubiquitin tag from mutant FLT3 thereby contributing to high levels of the oncogenic protein in AML (Fig 1a). Screening of our preclinical DUB inhibitor library for ability to selectively kill growth factor-independent FLT3-ITD-positive Ba/F3 cells over IL-3-dependent parental Ba/F3 cells identified HBX19818, a reported USP7 inhibitor, as the top hit. The effects are not unique to the Ba/F3 system: when profiled against a panel of 7 leukemia cell lines, HBX19818 conferred a substantial growth suppressive effect only to those expressing the FLT3-ITD oncoprotein (Fig 1b). As an initial assessment of the mechanism of HBX19818 we confirmed that it does promote ubiquitin-mediated degradation of FLT3-ITD (Fig 1c) and that the effect is selective as HBX19818 does not impact protein levels of wt FLT3. HBX19818 is published as an irreversible USP7 inhibitor,[2] however DUBome selectivity profiling data we generated identifies USP10 as the most potently inhibited DUB of the compound (USP10 IC₅₀ = 14 µM). We went on to validate USP10 as the DUB that stabilizes FLT3-ITD using a combination of small molecule and genetic experiments. Notably, HBX19818 binds and inhibits USP10 in cells (data not shown), small hairpin knockdown of USP10 phenocopies the antiproliferative and FLT3 degradation effects of HBX19818 (Figure 1d and data not shown), and a direct interaction between USP10 and FLT3-ITD is observed in co-immunoprecipitation experiments (Fig 1e). Additionally, SAR studies reveal correlation among USP10 IC₅₀, FLT3-ITD degradation and anti-proliferative effects for the HBX19818 chemical series, and we identified a second chemotype that phenocopies its effects. In support of the translational potential of USP10 inhibition for FLT3 mutant AML, we observed that both USP10 inhibitor series synergize with FLT3 kinase inhibitors, suppress growth of mutant FLT3-expressing primary AML cells and primagraft AML cells and, importantly, display the ability to overcome the FLT3 inhibitor-resistant mutant FLT3-ITD-F691L among other FLT3 kinase inhibitor-resistant mutants (Fig. 1f and data not shown).

Overall, our data strongly support degradation of mutant FLT3 as an alternative approach to therapeutically target FLT3. This approach, which focuses on targeting USP10, could prove more efficacious than kinase inhibitors by simultaneously blocking both enzymatic and scaffolding functions of FLT3, and blocking compensatory increases in FLT3 protein or resistant point mutations associated with some kinase inhibitors. Importantly, this is the first demonstration of stabilization of an AML mutant driver protein by a DUB enzyme and introduces a novel therapy for FLT3 mutant-positive AML.

References:

1. Stone, R.M., ASH, 2015.

2. Reverdy, C., et al., Chem Biol, 2012. 19, 467-77.

Figure 1.

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Figure 1.

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