Abstract 2034: Co-treatment with NPM1 antagonist and FLT3 inhibitor or pan-histone deacetylase inhibitor exerts superior efficacy against cultured and primary human AML cells co-expressing mutant NPM1 and FLT3-ITD.
Authors: Fiskus Warren , Balusu Ramesh , Peth Karissa , Hembruff Stacey , Abhyankar Sunil , McGuirk Joseph , Bhalla Kapil N. 
Published: August 2014

In approximately 50% of the normal-karyotype (NK) AML, a mutant (mt) NPM1 is localized in the cytoplasm (NPM1c+), which is pathogenetic for AML. Co-expression of FLT3-ITD with mtNPM1 results in poor outcome in AML. We have previously reported that in the cultured (OCI-AML3) cells carrying mtNPM1, shRNA-mediated knockdown of both the wild-type and mtNPM1 induced p53, p21, p27 and CEBPα, while attenuating the levels of HOXA9 and Meis1. This was associated with the morphologic features of myeloid differentiation, apoptosis, as well as a marked inhibition of the colony growth of OCI-AML3 cells (p&lt;0.01). Additionally, knockdown of NPM1 sensitized OCI-AML3 cells to all-trans retinoic acid (ATRA) (0.25 to 2.0 μM)-induced differentiation and apoptosis (p&lt;0.01). Findings of our present studies demonstrate that, as compared to the shRNA-mediated knockdown of total NPM1 (wild-type and mtNPM1), selective knockdown of mtNPM1 alone induced less differentiation and apoptosis of OCI-AML3 cells, as well as caused less inhibition of the leukemogenic potential of OCI-AML3 cells in NOD/SCID mice. The small molecule inhibitor NSC348884 is known to disrupt the oligomerization of wild-type and mtNPM1. NSC348884 dose-dependently (1.0 to 3.0 μM) induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA-induced apoptosis (p&lt;0.01). Patient-derived primary AML cells co-expressing mtNPM1 and FLT3-ITD, as well as OCI-AML3 cells transfected with FLT3-ITD lentivirus (OCI-AML3/FI cells) that co-expressed NPM1c+ and FLT3-ITD were relatively resistant to NSC348884 and/or ATRA. Notably, combination with NSC348884 (2.0-5.0 μM) and the FLT3 antagonists ponatinib (0.5 to 1.0 μM), AC220 (0.2 to 1.0 μM) or midostaurin (100 to 500 nM) induced more apoptosis than either agent alone against OCI-AML3/FI and primary AML cells co-expressing mtNPM1 and FLT3-ITD (p&lt;0.01). Importantly, treatment with the clinically achievable levels of pan-histone deacetylase inhibitor panobinostat (PS, 10 to 50 nM) depleted NPM1 levels and induced differentiation of OCI-AML3, OCI-AML3/FI, and primary AML cells co-expressing mtNPM1 and FLT3-ITD (40 to 70%). Additionally, as compared to treatment with each agent alone, co-treatment with NSC348884 and PS induced significantly more apoptosis in these cells (p&lt;0.01). This was associated with reduction in the levels of p-FLT3, p-AKT, p-STAT5 and NPM1, as well as induction of p53 and p21 levels. Therefore, co-treatment with an NPM1 antagonist and PS or FLT3 inhibitor is highly active against AML cells co-expressing mtNPM1 and FLT3-ITD. These findings support the rationale for in vivo testing of these combination therapies against AML with co-expression of mtNPM1 and FLT3-ITD.

Citation Format: Warren Fiskus, Ramesh Balusu, Karissa Peth, Stacey Hembruff, Sunil Abhyankar, Joseph McGuirk, Kapil N. Bhalla. Co-treatment with NPM1 antagonist and FLT3 inhibitor or pan-histone deacetylase inhibitor exerts superior efficacy against cultured and primary human AML cells co-expressing mutant NPM1 and FLT3-ITD. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2034. doi:10.1158/1538-7445.AM2013-2034