P B S-1086, a “Pan-Rel” Inhibitor, Decreases Viability of Chronic Lymphocytic Leukemia Cells

Abstract 867

The NF-κB signaling pathway is constitutively activated in chronic lymphocytic leukemia (CLL). Aberrant activation of the p65 (RelA) subunit of NF-κB is associated with chemoresistance, a shorter lymphocyte doubling time and poor overall survival (OS) in CLL. The transcription factor, NF-κB, regulates anti-apoptotic and pro-survival genes, and therefore represents a drug target for therapeutic intervention. Several studies have shown that targeting NF-κB decreases CLL cell survival, induces apoptosis and inhibits transcription of key pro-survival genes. In CLL, poor response to therapy and poor outcome are closely related to cytogenetic abnormalities. Loss of p53 function (del(17p) and/or TP53 mutation) confers chemo-resistance due to a defective DNA damage response pathway. Loss of the ATM (ataxia telangiectasia mutated) kinase (del(11q) and/or mutation of ATM) is also associated with chemo-resistance. We hypothesized that blocking NF-κB may circumvent the requirement for an active DNA damage response pathway, and that NF-κB inhibition may be efficacious independent of cytogenetic abnormalities in CLL. We tested the hypothesis with PBS-1086, a novel small molecule “pan-rel” inhibitor that has shown activity against multiple myeloma in vivo.

For target validation, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding and rel expression in nuclear extracts prepared from patient-derived CLL cells. Levels of activated NF-κB binding correlated with rel protein levels. We confirmed that constitutive activation of the p65 subunit predicted shorter overall survival (p= 0.03, n=80, HR (hazard ratio) 2.4). We also found that the p50 and RelB subunits were associated with poor survival (p = 0.04, n = 80, HR = 2.5 and p=0.17, n=50, respectively). Whereas p65 and p50 (activated by the canonical pathway through IKKβ), were present at high levels in the majority of cases, RelB (activated by the non-canonical pathway) was found to be variable among patients, with levels ranging from very high to undetectable. We found that patients with del(17p) had higher levels of p65 and p50 DNA binding activity, whereas patients with del(11q) had higher levels of RelB activity. We examined the effect of PBS-1086, a “pan-rel” inhibitor, on viability of patient-derived CLL cells and compared it with BAY 11–7082, an IKKβ inhibitor that affects p65 and p50. PBS-1086 was more potent at decreasing CLL viability in a concentration-dependent manner than BAY 11–7082 (mean LC₅₀ 6.4 +/− 1.9 μM, and 14.7 +/− 1.7 μM, respectively, n=20). LC₅₀ values of PBS-1086 and BAY 11–7082 derived from individual CLL samples correlated significantly, suggesting that they acted by inhibiting the same pathway (p=0.02, n=20). PBS-1086 LC₅₀ values also correlated inversely with the DNA binding activity of p65 (p=0.01, n=17), suggesting that these CLL cells were heavily reliant on p65 for survival, and were therefore exquisitely vulnerable to compounds inhibiting p65. There was no significant difference in sensitivity to PBS-1086 or BAY 11–7082 among patients with different cytogenetic abnormalities, or Binet stage A or stage B/C cases, or between cases with or without IGVH gene mutations. Patients who had received prior chemotherapies at the time of sample collection showed a trend to be more resistant to ex vivo treatment with Fludarabine than those naïve to chemotherapies, however treatment status had no impact on ex vivo sensitivity to PBS-1086. Submaximal concentrations of Fludarabine and PBS-1086 were additive in decreasing CLL cell viability (n=3). Mechanistic studies showed that PBS-1086 inhibited p65 and p50 DNA binding activity in CLL cells (n=5). In one example case that had constitutive expression of all of the NF-κB subunits, PBS 1086 inhibited the DNA binding activity of all rel subunits. Both PBS-1086 and BAY 11–7082 induced apoptosis in a concentration-dependent manner in CLL cells (Annexin V staining, n=4) that paralled the inhibitor-induced loss of viability.

These data demonstrate that PBS-1086 reduced survival of CLL cells ex vivo through inhibiting the DNA binding activity of rel subunits concomitantly with induction of apoptosis. Such effects of PBS-1086 were independent of poor prognosis cytogenetics, and of Binet stage or IGVH status. Thus, PBS-1086 is promising as a therapeutic agent by itself and also as a chemo-sensitizer to overcome NF-κB-mediated chemo-resistance in CLL.