Investigators: Houda Alachkar; Andrew Mackay; George Yaghmour
Acute myeloid leukemia (AML) remains the most common acute leukemia with poor outcome. Currently less than 30% of patients achieve 5-year overall survival. Leukemia stem cells (LSCs) play a major role in AML initiation, growth and relapse. Unfortunately, available therapeutic strategies do not effectively target LSCs, as evident by high relapse rates. Among patients with AML, those carrying FLT3-ITD mutations have even worse clinical outcomes and higher incidence of relapse compared with the FLT3 wild type allele (FLT3-WT). The FLT3-ITD mutation occurs in 30% of patients with cytogenetically-normal AML. This mutation alters structure in the juxtamembrane domain of the FLT3 receptor, which leads to constitutive activation and proliferation of AML cells. Targeted therapies against FLT3-ITD have long been investigated and recently two FLT3 inhibitors received FDA approval for treating FLT3-ITD+ patients. Midostaurin is approved for the treatment of pre-transplant patients in combination with standard therapy. Still half of these patients die of their disease within 4 years. Gilteritinib is approved for relapsed and refractory AML in patients with FLT3-ITD. Yet, the Phase III ADMIRAL Trial showed that only 37% of treated patients survived longer than one year. Altogether, these data paint a grim picture of the treatment options and clinical outcome facing patients with FLT3-ITD+ AML and challenge us to design and develop better therapeutic approaches. Our team recently identified CD99 as a novel therapeutic target that is upregulated in AML in comparison with normal hematopoietic cells. We discovered that targeting CD99 with a loss of function or an antiCD99 antibody reduced cell viability and induced apoptotic cell death in AML cell lines and primary blasts from AML patients. We found that CD99 ectopic expression promoted leukemia growth and colony formation. Importantly, we discovered that FLT3-ITD mutation was associated with high CD99 expression. Targeting FLT3 with the FLT3 inhibitor midostaurin reduced CD99 protein levels. Further, immunoprecipitation revealed that CD99 binds both FLT3-WT and FLT3- ITD receptors. Taken together, our data suggest interplay between the two receptors and that targeting the CD99/FLT3 interaction is a novel therapeutic strategy. Based on these conclusions, we in collaboration with Dr. Mackay’s laboratory, developed anti-CD99 and anti-FLT3 single chain variable fragments (scFv) fused at the amino terminus of an elastin-like peptides (ELP) linked by a thrombin cleavage site peptide. We have characterized and demonstrated pharmacological activities of these antibodies using in vitro and in vivo preclinical models of AML. An important question to explore is whether these antibodies alone or combined are effective in eliminating LSCs. Here we hypothesize that dual targeting of CD99 and FLT3 is more effective in eradicating FLT3-ITD leukemic stem cells than antibodies targeting a single receptor; furthermore, we propose that FLT3 inhibitors plus anti-CD99/anti-FLT3 antibodies will further enhance CD99-targeted therapy efficacy against LSCs. Two objectives of this proposal are: i) Demonstrate activity of anti-CD99 plus anti-FLT3 scFvs in eradicating leukemia stem cells using preclinical models. ii) Assess the therapeutic benefit of combining FLT3 inhibitors (midostaurin or gilteritinib) with CD99/FLT3-targeted therapy. This proposal extends our research effort supported by a prior Ming Hsieh grant (2018), which resulted in the development of the antiCD99 and anti-FLT3 scFV-ELP fusions and the submission of a paper to the journal of controlled release. The successful completion of this study will establish a novel therapeutic strategy and support our application for NIH-R01 (submitted Feb-2019, Oct-2019) grant with the data needed to bring it to a fundable score. The ultimate goal of this multidisciplinary project (Alachkar, MacKay, Yaghmour) is to bring anti-CD99/anti-FLT3 ELP therapeutic strategy from preclinical investigation into an FDA-approved therapy that improves the outcome of patients with AML.