Dual Inhibition Of PI3K and mTOR Shows Preferential Antileukemic Activity In MLL-Rearranged AML
Authors: Sandhöfer Nadine , Metzeler Klaus H. , Hinrichsen Tanja , Tiedt Sebastian , Zellmeier Evelyn , Groiß Volker , Wachter Oliver , Klein Hanns-Georg , Hiddemann Wolfgang , Jeremias Irmela , Spiekermann Karsten 
Published: October 2019

Abstract

Background
Despite the recent advances and better understanding of the biology of acute myeloid leukemia (AML), only little improvement was achieved in treatment and cure rates. One of the key signaling pathways known to be frequently deregulated in AML is the PI3K/AKT/mTOR signaling axis. In 50-70% of AML patients, its abnormal activation indicated by constitutive phosphorylation of AKT and mTOR, can be observed. Thus, this pathway is seen as a promising target in cancer treatment. In this study we examined the relevance of the PI3K/AKT/mTOR signaling axis in AML by using small molecule inhibitors of AKT, mTOR or both PI3K and mTOR in in vitro and in vivo models.

Results
With primary AML patient samples we performed propidium iodide (PI) staining and FACS apoptosis analysis. A wide spectrum of cytotoxic activity was observed for single small molecule inhibitors of AKT (MK-2206), mTOR (rapamycin) and a dual PI3K/mTOR inhibitor (BEZ-235) after treatment for 48-72 hours. Dual inhibition of both PI3K and mTOR led to a higher apoptosis induction than single inhibition of AKT or mTOR alone. We could show that patient samples carrying a MLL-translocation, MLL-partial tandem duplication (MLL-PTD) or growth factor signaling (GFS) activating mutations (FLT3, RAS) have a higher sensitivity towards PI3K pathway inhibitors compared to other patient samples (for BEZ-235 median 40.8% vs. 18.8% specific apoptosis). Higher sensitivity in this subgroup was also observed in human-derived AML cell lines. According to their response in apoptosis and proliferation assays we subdivided six AML cell lines into two groups with one group carrying a MLL-AF9 fusion gene plus additional GFS related mutations. The biological response of dual PI3K and mTOR inhibition was well correlated to pathway activity as could be determined by Western Blot and FACS analysis. With 38 MLL-AF9 rearranged patient samples we performed a targeted next generation sequencing approach to further study the relationship between MLL and activating mutations targeting GFS pathways. In 76% of all analyzed patient samples we detected mutations in GFS signaling related genes such as KRAS, NRAS, FLT3 and CBL. KRAS and NRAS mutations were observed much more frequently in MLL-rearranged AML (in 42% and 18% of patients, respectively) than reported in other AML subgroups. Because of the high incidence of RAS mutations we further performed combination treatment studies with a MAPK inhibitor in THP-1 as a model cell line for this patient cohort (carrying a MLL-AF9 fusion and the NRAS G12D mutation). Effects after combination treatment were highly synergistic in apoptosis and proliferation assays (combination index value: 0.05). In vivo activity of PI3K and mTOR inhibiton by BEZ-235 was investigated by using an AML xenograft mouse model derived from MOLM-13 cells, carrying a MLL-AF9 fusion and a FLT3 mutation. To enable in vivo monitoring of the cells after injection, cells were engineered to express GFP and enhanced firefly luciferase (eFFLuc). Treatment led to significantly delayed tumor progression and prolonged overall survival. The tumor load was quantified by in vivo imaging and post mortem FACS analysis and was lower in BEZ-235 treated than in vehicle treated mice. Six hours after administration of the drug, S6rp phosphorylation in leukemic cells was significantly decreased by 50% compared to untreated mice but reached its basal level again 24 hours after administration.

Conclusions
Our data implicate a possible therapeutic benefit of PI3K/AKT/mTOR inhibition in AML especially in the MLL-rearranged and GFS signaling mutated subgroup. Combination of PI3K/mTOR inhibitors with drugs inhibiting rescue pathways such as the MAPK pathway and Jak2/STAT5 or combination with drugs promoting cell death could improve the therapeutic efficacy of targeted therapies in AML.

Disclosures:
No relevant conflicts of interest to declare.