Targeting mTOR by a New Generation of Kinase Inhibitors Sensitizes Leukemia Cells for Chemotherapy Via Suppressing FANCD2 Expression
Authors: Li Jie , Guo Fukun , Sipple Jared , Kozma Sara , Thomas George , Zheng Yi , Pang Qishen 
Published: October 2019

Abstract
Abstract 1514
The mammalian target of rapamycin (mTOR) is a key regulator of nutrient metabolism, cell growth and proliferation. Inhibition of mTOR signaling by rapamycin or rapamycin in combination with antineoplastic agents has been shown to block cancer cell proliferation and cancer angiogenesis. Second-generation pharmacological mTOR inhibitors, which inhibit both mTORC1 and mTORC2 by directly targeting the ATP-binding site of mTOR, have recently shown improved activity in tumor suppression and are under clinical development for cancer therapy. Indeed, it is found that the mTOR kinase inhibitor PP242 inhibits cell proliferation more effectively than rapamycin in pre-clinical models, suggesting the additive contributions of mTORC1 and mTORC2 to cancer cell proliferation and survival. In the present study, we have explored the therapeutic value of PP242 in sensitizing tumor suppression by anti-cancer drugs. We found that the combination of PP242 with Cytarabine (AraC) or Etoposide induced significant higher apoptosis than single-agent treatment in several human lymphoma and leukemia cell lines including K562, Molt-Luc2, and K562-Luc2. Specifically, using Molt-Luc2 cells, the percentages of apoptosis for combined PP242-AraC and PP242-Etoposide treatments were 76.3±4.2% and 78.2+5.9%, respectively, in comparison with 52.7±6.3% and 38.2±4.5%, respectively, in AraC- and Etoposide-treated cells; the basal level of apoptosis in these leukemic cells was 5–8%. Further, PP242, but not Rapamycin, sensitized the leukemia and lymphoma cells to DNA damage induced by AraC or Etoposide, evidenced by a marked increase in g-H2AX foci (94±5% cells in PP242-AraC group vs 25±3% cells in AraC-alone group or 98±4% cells in PP242- Etoposide group vs 36±3% cells in Etoposide-alone group), as well as DNA-strand breaks (comet-tailed value of 25.4±1.2% in PP242-AraC group and 31.2±3.2% in PP242-Etoposide group compared to 9.8±1.2% in AraC-alone and 11.4±2.8% Etoposide-alone groups, respectively). This increased DNA damage response can be attributed to a suppression of the expression of FANCD2, a critical DNA damage repair component of the Fanconi pathway, by PP242, in both normal lymphoblasts and leukemic cells. Significantly, the effect of PP242 on Fanconi gene expression was FANCD2-specific as PP242 had no effect on the expression of other Fanconi proteins such as FANCA and FANCC, and forced expression of FANCD2 by a viral promoter completely abolished the sensitizing effect of PP242 on drug-induced leukemia cell death. We are currently using a mouse xenotransplant model to explore the in vivo effect of the combination of PP242 with AraC for human leukemia cells. Our findings suggest that the mTOR kinase inhibitor PP242 enhances antitumor activity of conventional chemo-drugs by suppressing FANCD2 and associated DNA damage response and consequently augmenting DNA damage leading to apoptosis. Therefore, PP242 combined with chemotherapy could represent a novel strategy for the treatment of hematopoietic malignancies.

Disclosures:
No relevant conflicts of interest to declare.