Targeting the NRF2/HO-1 Antioxidant Pathway with Consequences for Notch Signaling in FLT3-ITD Positive AML: A Novel Therapeutic Approach

Approximately 30% of patients with acute myeloid leukemia (AML) harbor activating mutations in the fms-like tyrosine kinase receptor 3 (FLT3).  Such mutations are associated with increased propensity to relapse and dramatically decreased survival. FLT3 tyrosine kinase inhibitors (TKI), such as quizartinib, have shown modest clinical effects as single agents, and resistance remains a significant clinical problem.    In addition to constitutive activity of the FLT3 receptor, AML cells with an internal tandem duplication of FLT3 (FLT3-ITD) exhibit elevated levels of reactive oxygen species (ROS).  Antioxidants such as heme-oxygenase 1 (HO-1) are often upregulated in conjunction with increased ROS levels. In addition to its role as an antioxidant, HO-1 also has known proliferative and anti-apoptotic functions in some cell types.  A study in non-small cell lung carcinoma recently suggested that HO-1 inhibits the Notch pathway through binding the Notch receptor.  As Notch signaling exerts pro-apoptotic effects in AML, targeting HO-1 may induce Notch signaling, representing a potential therapeutic approach. 

Our data show that HO-1 is particularly elevated in FLT3-ITD expressing AML cells. Interestingly, HO-1 protein was found to be further elevated in AML cells with acquired resistance to FLT3-directed TKI compared to TKI-sensitive cells.  Importantly, knockdown of HO-1, or inhibition of HO-1 catalytic activity with zinc protoporphyrin (ZnPP), leads to decreased survival and proliferation, demonstrating a critical pro-survival role of HO-1.  We have determined that expression of HO-1 is under the control of NRF2, a transcriptional regulator of the antioxidant response, in FLT3-ITD positive AML cells.  Inhibition of NRF2 with brusatol resulted in increased sensitivity to quizartinib in TKI-sensitive cell lines.  Further, brusatol restored sensitivity to low nanomolar concentrations of quizartinib in TKI-resistant cells. Together, our work shows that combined inhibition of FLT3 and NRF2 using quizartinib and brusatol, respectively, exhibits synergy in the context of FLT3-ITD AML. 

Given the reported interaction between HO-1 and Notch, we sought to determine whether this interaction occurs in AML.  Using the TCGA AML database, we found significant co-occurrence of HMOX1 (HO-1) and NOTCH2 mRNA expression in 200 patient samples. In vitroimmunoprecipitation of the Notch2 receptor revealed that HO-1 directly interacts with Notch2 in FLT3-ITD positive AML cells.  Importantly, brusatol treatment resulted in activation of the Notch pathway as demonstrated by cleavage of the Notch receptor and increased transcript levels of HES1, a canonical downstream target of Notch signaling. Given that HO-1 interacts with Notch2 and is under NRF2 control, we propose that decreasing HO-1 levels with brusatol treatment will modulate the Notch pathway such that its anti-leukemic effects can be exerted. 

In conclusion, inhibition of HO-1, directly or through NRF2 inhibition, demonstrates synergistic effects with FLT3 inhibition in both TKI-sensitive and –resistant AML.  Furthermore, NRF2 inhibition induces Notch activation, providing a potential mechanism for this synergistic combination.  Our data suggest that antioxidant modulation is a potential therapeutic approach for FLT3-ITD positive AML.