All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD⁺ leukemia stem cells in vitro and in vivo

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)⁺ LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD⁺ cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD⁺ cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD⁺ LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD⁺ patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drug's apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD⁺ LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.