Genetic and Pharmacologic Notch4 Inhibition Synergizes with FLT3 Tyrosine Kinase Inhibition in Vitro to More Effectively Eliminate FLT3/ITD-Positive Leukemia Cells

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. Mutated in approximately 1/3 of patients, FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML). The presence of FLT3 internal tandem duplication (ITD) mutations, which renders FLT3 constitutively active, confers a particular poor prognosis. While several FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling, the clinical success of these drugs is limited because they fail to achieve frequent complete durable responses, despite achieving high levels of FLT3 inhibition. These limited clinical responses suggest that monotherapy is unlikely to be curative. Current clinical trials of FLT3 inhibitors are administered in combination with chemotherapy to achieve success in inducing complete remissions. While there is hope that FLT3 inhibition in combination with chemotherapy will increase cure rates, it would be optimal to achieve cures without chemotherapy altogether and its inherent toxicities. Towards this goal it is necessary to uncover critical genes/pathways that collaborate with FLT3 mutations to transform cells. In recent years, the use of whole genome sequencing has greatly expanded the list of genes mutated in AML. However, many of these mutations do not represent a practical therapeutic target because they are present in a low frequency in the patient population, making molecular targeted therapy for each of these improbable to achieve. We hypothesize that these mutated genes likely funnel into a more limited number of signaling pathways, with some pathways being more important than others in contributing to FLT3/ITD AML. Identifying FLT3/ITD cooperative pathways whose inhibition might synergize with FLT3 inhibition could hold promise for greatly improving the cure rate in FLT3 mutant AML.

We performed a loss of function RNAi screen in FLT3/ITD+ AML cell lines (Molm14 and MV411) to uncover genes and pathways whose inhibition combined with FLT3/ITD inhibition to more effectively kill FLT3 mutant AML cells. Notch4 signaling was identified as one such potential target. We found Notch4 to be overexpressed in FLT3/ITD+ AML cells lines as well as in the Lin^-Sca⁺Kit⁺ fraction of bone marrow isolated from FLT3/ITD+ mice. Stable, tetracycline-inducible Notch4 knockdown cell lines were established in Molm14 and MV411 cell lines. While Notch4 knockdown alone slightly decreased cell growth, Notch4 knockdown in combination with FLT3 TKI demonstrated a significant decrease in proliferation compared to FLT3 TKI treated cells expressing a scrambled shRNA control. Additionally, colony forming unit assays revealed that Notch4 knockdown cells treated with FLT3 TKIs exhibited a decrease in clonogenicity compared to FLT3 TKI treated control cells. Pharmacologic inhibition of Notch4 by treatment with gamma secreatse inhibition (GSI) was also tested. Molm14, MV411, and THP-1 (FLT3 wild type AML) cells were treated with FLT3 TKIs (sorafenib, CEP-701, or AC220) alone, GSI alone, FLT3 TKI plus GSI, or vehicle control and assessed for anti-proliferative and apoptotic effects by MTT, and annexin V/7-AAD staining, respectively. In both assays, the combination of FLT3 TKI and GSI exhibited synergy with combination index (CI) values <1. Furthermore, Western blot analysis showed that while treatment of Molm14 and MV411 cells with GSI had little effect on FLT3 signaling (pSTAT5, pAKT, pMAPK activation), the combination of GSI plus FLT3 TKI inhibited FLT3 signaling significantly more than treatment with FLT3 TKI alone. The results of these experiments identify Notch4 signaling as a potential pathway whose inhibition can synergize with FLT3 inhibition to more effectively kill FLT3/ITD+ AML cells. Combining FLT3 inhibition with Notch4 inhibition may have potential for improving the cure rate of patients with FLT3 mutant AML.