Temsirolimus, An Allosteric mTORC1 Inhibitor, Is Synergistic with Clofarabine in AML and AML Leukemia Initiating Cells
Authors: Chiarini Francesca , Grimaldi Cecilia , Ricci Francesca , Tazzari Pierluigi , Iacobucci Ilaria , Martinelli Giovanni , Pagliaro Pasqualepaolo , McCubrey James , Amadori Sergio , Martelli Alberto M 
Published: October 2019

Abstract
Abstract 2596
Although intensive combination chemotherapy is effective in inducing complete remission in most patients with AML, the majority of responders subsequently relapse and ultimately die of the disease. This is particularly true of elderly (>60 years old) patients. The development of new therapies is therefore vital and, in this regard, targeting signaling pathways that are upregulated in AML represents a rapidly expanding research field. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway is a common finding in AML and preclinical studies have highlighted a critical role for PI3K and its downstream effectors, Akt and mammalian target of rapamycin (mTOR), for leukemic cell survival. Especially mTOR complex 1 (mTORC1) represents a highly attractive target for cancer therapy, as it controls cap-dependent mRNA translation, a process frequently deregulated in tumor cells and that strongly contributes to oncogenesis. Preclinical data suggest that allosteric mTORC1 inhibition with rapamycin impairs leukemia initiating cell (LIC) function in AML. While PI3K/Akt inhibitors are just beginning to be tested in the clinic, mTORC1 inhibitors have already been investigated as antileukemic agents. Recently, a phase II clinical trial combining temsirolimus (an mTORC1 inhibitor) and clofarabine has been performed in elderly patients with relapsed or refractory AML (NCT007755903). Clofarabine is a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. Here, we report the results of our pre-clinical evaluation of the efficacy of such a drug combination on AML cells.
The combination of clofarabine and temsirolimus (CLO-TOR; drug concentration range: 6.125 nM–100 nm; ratio 1:1) was cytotoxic to a panel of AML cell lines (U937, OCI-AML3, HL60, THP1), as documented by MTT assays. The combination was highly synergistic, with combination indexes (CIs) ranging from 0.05 at the lowest drug concentration to 0.79 at the highest concentration. Treatment with CLO-TOR induced a G1 phase cell cycle arrest and apoptotic cell death of leukemic cells, as demonstrated by Annexin V/propidium iodide staining and cytofluorimetric analysis. Western blot analysis documented a dose-dependent cleavage of caspase-3, -8, -9. The combined treatment inhibited phosphorylation of Ser 473 p-Akt and of mTORC1 downstream targets (S6RP and 4E-BP1) more than either drug alone. Moreover, CLO-TOR affected the MEK/ERK pathway, as documented by dephosphorylation of p-ERK 1/2 and 90-kDa ribosomal S6 kinase. The effectiveness of the CLO-TOR treatment was not influenced by overexpression of 170-kDa P-glycoprotein (P-gp, one of the main determinants of drug-resistance), as documented by MTT assays performed on drug-sensitive and drug-resistant (i.e. overexpressing P-gp) CEM leukemic cells. The CLO-TOR combination, when employed at the same concentrations as for AML cell lines, was also cytotoxic to primary cells from AML patients (CIs: 0.11–0.25). CLO-TOR was highly effective in inducing apoptosis in an AML patient cell subpopulation (CD34+/CD38−/CD123+) which is enriched in putative LIC, as documented by quadruple staining and flow cytometric analysis of Annexin V-positive cells. The percentage of apoptotic cells in the CD34+/CD38−/CD123+ cell subset treated with CLO-TOR ranged between 90% and 97%, and was much higher than in samples treated with TOR alone (about 45%) or CLO alone (about 70%). The combined treatment also resulted in Akt, S6RP, and 4E-BP1 dephosphorylation in this cell subpopulation. In summary, the CLO-TOR combination could represent a valuable innovative treatment for AML patients, also in light of its efficacy against putative LIC.

Disclosures:
No relevant conflicts of interest to declare.