Relationships Between Aberrant Activity of the NF-κB Subunits and Outcome In Chronic Lymphocytic Leukemia: The Dual Role of DNA Damage Sensor Enzymes
Authors: Mulligan Evan A , Hunter Jill E , Baird Arabella EG , Elliott Sarah , Summerfield Geoffrey P , Hamlen Karen , Lowe Chris , Wallis Jonathan , Veuger Stephany , Durkacz Barbara W , Willmore Elaine 
Published: October 2019

Abstract 3588

Poor prognosis patients with chronic lymphocytic leukaemia (CLL) can be identified by cytogenetic abnormalities such as del(17p), and del(11q) and corresponding mutations in TP53 and ATM (ataxia telangiectasia mutated kinase) respectively, which are associated with chemoresistance by virtue of defects in the DNA damage response pathway. We have demonstrated that overexpression of DNA-dependent protein kinase (DNA-PK), which mediates non-homologous end joining, is also associated with poor prognosis CLL1. Recent data show that constitutive activation of the p65 subunit of the transcription factor, NF-κB, confers poor survival in CLL. Since the repertoire of genes activated by NF-κB includes anti-apoptotic and pro-survival genes, NF-κB therefore represents an attractive target for therapeutic intervention. The parthenolide analogue, LC-1, has been shown to be synergisitic with fludarabine in ex vivo studies on CLL cells2, and has validated the concept of targeting NF-κB and prompted clinical trials in CLL patients. Previous data from our laboratory using cell line models demonstrated that DNA damage-induced NF-κB activation requires the base excision repair protein, poly(ADP-ribose) polymerase (PARP), to confer radioresistance3. The DNA damage sensor, ATM, also mediates the response to DNA damage, via phosphorylation of IKK-γ (Inhibitory κ-Kinase/NEMO). In fact, we demonstrated that radio-sensitization by an ATM inhibitor is mediated via NF-κB, rather than by the inhibition of double strand break repair4. Since these observations identify key DNA damage response proteins as regulators of NF-κB activity, we hypothesized that inhibitors of PARP, ATM and DNA-PK, could chemosensitize CLL cells via inhibition of NF-κB.

Here, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding (measured by ELISA) in an unselected CLL cohort. Constitutive activation of the p65 and p50 subunits correlated closely (P= 0.001, n= 57), and predicted shorter time to first treatment (TTFT) and overall survival (OS). Importantly, higher activation occurred in del(17p) (e.g. p50; P= 0.05, n= 49) cases and in those cases that had received treatment. p52 and c-Rel activation correlated with p65 and p50 activation. However, although high p65 and p50 activation predicted shorter OS and TTFT, increased p52 and c-Rel activation were associated with a longer TTFT (up to 40 months) demonstrating the complex crosstalk between the NF-κB subunits in CLL.

We were able to correlate p50 and p65 subunit activation with ex vivo resistance to fludarabine and chlorambucil in 12 cases: the most chemoresistant cases had higher p50 and p65 DNA binding activity. Preliminary data indicates that levels of activated NF-κB binding seen in the ELISA correlates with protein expression in nuclear extracts isolated from CLL cells (P= 0.0013, n=26). Here we make the novel observation that the increase in DNA damage-induced NF-κB activation is linked to increased DNA-PK activation. DNA-PK catalytic subunit levels were significantly higher in patients with high p65 activation (P= 0.02, n= 30), regardless of treatment status. Furthermore, a selective inhibitor of DNA-PK, NU7441, increased mitoxantrone-induced cytotoxicity in CLL cells (up to 50-fold) and reduced p65 and p50 DNA binding, indicating a direct link between DNA-PK and NF-κB activation. Ongoing studies are investigating this mechanistic link further, using chromatin immunoprecipitation (ChIP) and using siRNA knockdown. CLL cells were radiosensitized by either the DNA-PK inhibitor, NU7441 or the pan IKK inhibitor, BAY 11–7082. Strikingly, a combination of these two inhibitors had no further effect on radiosensitization, suggesting that DNA-PK and NF-κB act in a common pathway. We have demonstrated that inhibition of ATM sensitizes CLL cells to DNA damage, and future work will assess the impact of ATM and PARP inhibition on NF-κB activity in CLL cells. These data present a novel role for DNA-PK in the regulation of NF-κB and highlight important new therapeutic avenues for the use of DNA-PK inhibitors, which may prove useful in overcoming NF-κB mediated therapeutic resistance in CLL.

Willmore E, et al, Clin Cancer Res. 2008. Hewamana S, et al, Clin Cancer Res. 2008. Hunter JE, et al, Proceedings of the AACR. 2009. Veuger SJ et al, Under review, DNA repair. 2010.

Disclosures:

No relevant conflicts of interest to declare.