Targeting Critical Kinases and Anti-Apoptotic Molecules Overcomes Steroid Resistance in Infant MLL-Rearranged Leukemia
Authors: De Groot Anne Pieta , Saito Yoriko , Kawakami Eiryo , Hashimoto Mari , Aoki Yuki , Ono Rintaro , Ogahara Ikuko , Fujiki Saera , Kaneko Akiko , Watanabe Takashi , Takagi Masatoshi , Tomizawa Daisuke , Koh Katsuyoshi , Eguchi Mariko , Ishii Eiichi , Ohara Osamu , Shultz Leonard , Mizutani Shuki , Ishikawa Fumihiko 
Published: November 2019

Acute lymphoblastic leukemia (ALL) with rearrangement of the mixed-lineage leukemia (MLL) gene frequently affects infants and is associated with a poor prognosis. Standard treatment protocol for infant MLL-rearranged ALL (MLL-ALL) includes glucocorticoids (GCs). However, resistance to GCs remains a major problem. Therefore, it is important to find new treatment strategies that overcome GC resistance in MLL-ALL.
To identify novel therapeutic targets to overcome GC resistance in MLL-ALL, we compared transcriptional profiles of normal cord blood CD34+CD38- hematopoietic stem and progenitor cells and GC-resistant infant MLL-ALL leukemia-initiating cells (LICs). We found enrichment of the Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathways in the LICs. We hypothesized that activation of these kinases may contribute to GC resistance in MLL-ALL cells. Using our previously developed infant MLL-ALL patient-derived xenograft (PDX) models (Aoki et al., Blood, 2015), we recapitulated human GC-resistance in vivo, characterized by increased hCD45+ chimerism in the peripheral blood after treatment with dexamethasone (% peripheral blood human MLL-ALL at pre-treatment: 34.1±5.2% vs. post treatment 51.3±6.6%, n=26). In order to investigate if inhibition of SFKs and FLT3 can overcome GC-resistance, we treated the MLL-ALL PDX models with our previously developed FLT3-SFK multiple kinase inhibitor RK-20449 (Saito et al., Science Translational Medicine, 2013). Combination treatment with dexamethasone and RK-20449 successfully eliminated human MLL-ALL cells from the peripheral blood (dexamethasone alone: 47.2±7.2% vs. combination: 5.6±2.8% hCD45+ cells, p=6.91E-7), bone marrow (dexamethasone alone: 72.2±4.6% vs. combination: 19.5±4.8% hCD45+ cells, p=1.79E-10), and spleen (dexamethasone alone: 52.6±4.8% vs. combination: 11.7±3.5% hCD45+ cells, p=3.75E-9). In addition, combination treatment eliminated infiltrated MLL-ALL from kidney and liver of the MLL-ALL PDX models. Bcl-2 homology domain 3 (BH3) profiling demonstrated that MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic Bcl-2 protein for their survival. Additional inhibition of Bcl-2 by ABT-199 led to complete elimination of MLL-ALL cells in vitro and in vivo in all MLL-ALL patient cases (Figure1).
Taken together, in this study we demonstrated that inhibition of SFKs and FLT3 by RK-20449 overcomes GC-resistance in MLL-ALL. Further, we identified Bcl-2 dependence as a mechanism of treatment resistance in MLL-ALL. Therefore, we believe that the combined inhibition of kinase and anti-apoptotic pathways may lead to effective treatment options for highly resistant infant MLL-ALL.
Aoki, et al., Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood, 2015: p. 967-980.
Saito, Y., et al., A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo. Science Translational Medicine, 2013: p. 1-15.

Disclosures
No relevant conflicts of interest to declare.