Targeting Nedd8 Activating Enzyme Induces DNA Damage and Cell Cycle Arrest and Sensitizes Chronic Lymphocytic Leukemia (CLL) B-Cells to Alkylating Agents

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-kappaB (NFκB) signaling in the CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that stromal-mediated NFκB pathway activity and CLL cell survival may be successfully abrogated in vitrousing MLN4924. MLN4924 is an investigational agent that inhibits the NEDD8-activating enzyme (NAE) and thereby prevents neddylation of Cullin-RING ubiquitin ligases (CRL), resulting in stabilization of their protein substrates, including inhibitor of NFκB. However, targeting NAE also slows degradation of other proteins and thus full biological consequences are tissue-dependent. In adherent solid tumor cell lines, MLN4924 induced expression of Cdt1, a DNA replication licensing factor, followed by DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B-cells has not been determined. Here we show that, under the growth conditions, targeting NAE induces DNA damage and cell cycle arrest in primary CLL B-cells.

B-cells obtained from patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. CLL cells were incubated in the presence or absence of 25 ng/mL IL-21. MLN4924 was provided by Millennium Pharmaceuticals, Inc.

CD40L-expressing stromal co-cultures rapidly induced anti-apoptotic BCL2 family proteins MCL1 and BCLX in peripheral blood CLL cells, leading to protection from spontaneous apoptosis and chemoresistance. Prolonged (72 h) co-cultures induced cell cycle progression in ~5% of the CLL cells. IL-21 further enhanced proliferation and sensitized CLL cells to MLN4924. Thus, treatment with 1 μM MLN4924 induced apoptosis in 44.6±5.4% of the CD40L-stimulated, and in 63.7±2.6% of the CD40L/IL-21-stimulated CLL cells at 24 h (p=0.00004). CD40-stimulated CLL cells treated with MLN4924 showed Cdt1 accumulation, DNA re-replication, activation of DNA damage response pathways as evidenced by accumulation of pRPA, pChk1, pChk2 and gH2AX which preceded PARP cleavage, and G2 arrest. DNA damage and G2 arrest were more prominent in IL-21-treated cells. Targeting NAE also resulted in early accumulation of the endogenous cyclin-dependent kinase inhibitors p21 and p27, both CRL targets. By contrast, retinoblastoma protein hypophosphorylation occurred late and did not precede apoptosis. siRNA-mediated knockdown of Cdt1, but not p21 or p27 prevented MLN4924-mediated DNA damage response and CLL cell apoptosis. Finally, MLN4924 did not lead to accumulation of Cdt1, p21 or p27 in CLL cells cultured off stroma.

Since alkylating agents target primarily cycling cells, we further determined whether they would cooperate with MLN4924 to induce DNA damage in CLL. While either chlorambucil or bendamustine exhibited little toxicity against the CD40L-stimulated CLL cells, MLN4924 sensitized CLL cells to those agents. MLN4924 further augmented bendamustine toxicity in the presence of IL-21. MLN4924-mediated DNA damage response was augmented in the presence of bendamustine. Interestingly, this combination proved ineffective in the CLL samples with del(17p), suggesting that bendamustine effect depends on the intact p53 pathway.

Here we characterize DNA damage response pathway in CLL, where it remains understudied. Targeting NAE in cycling CLL cells induces Cdt1 accumulation, DNA damage and cell cycle arrest. This data provide additional insights into the biological consequences of targeting NAE in CLL B-cells as well as preclinical rationale to study the potential clinical activity of MLN4924 in CLL, alone or in combination with bendamustine or chlorambucil.