Combined Inhibition of mTOR and DNA-PK Blocks Survival, Adhesion, Proliferation and Chemoresistance in Primary Chronic Lymphocytic Leukemia (CLL) Cells
Authors: Thijssen Rachel , van Bochove Gregor , Derks Ingrid AM , ter Burg Johanna , de Rooij Martin FM , Spaargaren Marcel , Kersten Marie Jose , Eldering Eric , Kater Arnon P. 
Published: October 2019

Abstract
CLL progression and chemoresistance can result from signals from the lymph node (LN) microenvironment and from acquired aberrations in the DNA damage repair (DDR) pathway. Clinical targeting of kinases upstream in the B cell receptor (BCR) activation pathway, such as Btk or PI3Kδ results in egress of cells from the LN microenvironment [1,2]. Such prolonged lymphocytosis during kinase-inhibitor treatment appears to pose no clinical disadvantage [3]. However, it enhances the chance of accumulating resistance-inducing mutations, and therefore drugs that combine LN egress with direct cytoxicity could provide an improved therapeutic strategy for CLL.
The mTOR complex, consisting of mTOR1 and 2, is the main downstream kinase of the PI3K/Akt pathway and contributes to proliferation and survival. DNA-PK is a kinase required for non-homologous end joining (NHEJ) of the DNA repair pathway. Inhibitors of crucial components of the DDR pathway might be active in CLL, especially in patients harboring mutations in DNA repair molecules such as Ataxia telangiectasia mutated (ATM). In this study the potency of a novel dual mTOR1,2 and DNA-PK inhibitor (CC-115) was studied in primary CLL samples of different prognostic subgroups with respect to induction of cytotoxicity, and inhibition of adhesion, CD40-mediated chemoresistance and proliferation.
In vitro, CC-115 inhibited mTOR1 and 2 and also affected the DDR reflected by inhibition of irradiation-induced γH2AX, not only in ATM-mutated but also in ATM-wild type CLL cells. CC-115 showed induction of caspase-dependent cell killing (IC50=0.625µM) which was more robust than selective kinase inhibitors (table 1), irrespective of p53 or ATM status. This cytotoxic effect was not observed in the T cells from CLL patients. BCR-mediated adhesion to fibronectin [4] was inhibited by CC-115 to a similar extent as PI3Kδ inhibitor (idelalisib) (table 1). CD40-mediated chemoresistance [5] could be reverted completely by CC-115 while more specific inhibitors had only modest effects. CLL proliferation induced by CD40L+IL-21 treatment [6] was completely blocked by both CC-115 and a dual mTOR1,2 inhibitor but not by inhibitor of the more upstream kinase PI3Kδ (table 1).
In conclusion, these data show that CC-115 induces direct cytotoxicity and inhibits several clinically relevant biological features of CLL, and provide a rationale for clinical trials with CC-115 in CLL patients.
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Table 1. The effect of the mTOR1,2 + DNA-PK inhibitor (CC-115), mTOR1,2 inhibitor (CC-214), DNA-PK inhibitor (NU7441) and PI3Kδ inhibitor (idelalisib) on CLL cells in functional assays. CC-115 CC-214 NU7441 Idelalisib (CAL-101) Target mTOR1,2 DNA-PK mTOR1,2 DNA-PK PI3Kδ Cytotoxicity (IC50) 0.625 µ M >10µM >10µM >10µM Inhibition of adhesion 40%** 0% 22% 48%** Activation Inhibition of CD40-mediated resistance to fludarabine (6.25 µ M) 92%** 42%* n.d. 21% Inhibition of CD40L+IL21 induced proliferation 98%** 98%* 28% 51%** n.d. = not done The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01

Disclosures
Kersten: Celgene: Research Funding. Kater:Celgene: Research Funding.