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T. Lewis et al.
alkylating agents. This raises the possibility that they can selectively inhibit transcription factors,16 so this study set out to determine the in vitro and in vivo biological effects of a series of novel C8-linked PBD-benzofused hybrids.
Initially library screening identified three lead com- pounds. All three PBD showed high potency in five dif- ferent multiple myeloma cell lines with LD50 values in the low nanomolar range. Subsequently, the PBD showed similar high potency in a cohort of 46 primary CLL sam- ples and significantly lower toxicity in normal age- matched B- and T-lymphocytes. The most cytotoxic PBD, DC-1-192, showed 2.4-fold and 4.6-fold differential toxi- city in CLL cells suggesting that this compound has a pos- itive therapeutic index. We went on to show that DC-1- 192 was well tolerated in a systemic in vivo xenograft model of myeloma and significantly prolonged the sur- vival of the mice.
Subset analysis of the CLL cohort data revealed that DC-1-192 was equipotent in poor prognostic groups including IGHV unmutated cases (P=0.96). Furthermore, samples derived from patients with BIRC3 or NOTCH1 mutations showed significantly increased sensitivity to DC-1-192. These mutations are known to cause aberrant activation of NF-κB signaling and are associated with resistance to chemoimmunotherapy and inferior clinical outcome.28-32 Although these mutations are linked with non-canonical NF-κB activation, here we showed that nuclear expression of the canonical p65 subunit was a predictor of in vitro sensitivity to DC-1-192.
Given these findings, we plotted the previously pub- lished NF-κB index for each of the myeloma cell lines23
Activation of NF-κB has also been implicated in the development of resistance to chemotherapeutic drugs in myeloma and CLL.35 Several DNA-damaging agents, including melphalan and fludarabine, have been shown to induce the activity of NF-κB, thereby contributing to cellular resistance to the cytotoxic effects of these treat- ments.6,36 In myeloma, bortezomib has been shown to re- sensitize malignant cells to the effects of chemotherapy.37 However, the emergence of bortezomib-resistant sub- clones ultimately leads to relapse in many patients.38 One putative mechanism of bortezomib resistance is the con- stitutive expression of NF-κB. Although bortezomib can prevent de novo activation of the canonical pathway, it has no significant effect on constitutive NF-κB activity.27 In this study, we showed that direct competitive inhibition of NF-κB at the site of transcription led to the re-sensiti- zation of multiple myeloma cells to the effects of borte- zomib. This synergistic effect is likely to be multifactori- al, but indicates that bortezomib and the PBD have differ- ent molecular targets.
Similarly, in CLL Bruton tyrosine kinase (BTK) has been shown to be a critical downstream mediator of B-cell receptor signaling that is often constitutively activated in CLL patients. The targeting of this kinase with the BTK inhibitor, ibrutinib, has shown notable effects in patients with relapsed CLL39,40 and this is mediated, at least in part, by the distal inhibition of NF-κB.41 However, emerging evidence of resistance mechanisms to ibrutinib strongly implicate NF-κB.42 Here, we show that the combination of DC-1-192 with ibrutinib produced cytotoxic synergy suggesting that the PBD and ibrutinib target NF-κB through different mechanisms and/or that they have other, non-overlapping, molecular targets. Furthermore, synergy was enhanced when primary CLL cells were co- cultured on CD40L-expressing fibroblasts in order to mimic the lymph node microenvironment. This suggests that PBD may be particularly useful in targeting tissue- resident tumor cells.
In summary, the novel PBD compunds evaluated in this study showed low nanomolar toxicity in both primary CLL cells and myeloma cell lines. In addition, primary CLL cells carrying BIRC3 or NOTCH1 mutations were preferentially sensitive to the cytotoxic effects of DC-1- 192 suggesting that this agent may be a potential thera- peutic option for these poor-risk subsets. Mechanistically, the PBD demonstrated promising dual inhibitory proper- ties on both the canonical and non-canonical NF-κB path- ways, a characteristic that has been previously linked to significant antitumor effects in multiple myeloma.43 Furthermore, the PBD showed in vitro synergy with borte- zomib and ibrutinib in multiple myeloma and CLL, respectively, providing a strong rationale for the use of these agents in the treatment of relapsed/refractory B-cell neoplasms.
Disclosures
This work was supported by a grant (n. 12-1263/JGATCBR) from Worldwide Cancer Research (formerly AICR) to DET, KMR and CP in 2012. This support included a PhD stu- dentship held by DBC, which enabled all of the novel medicinal chemistry. All of the authors declare that they have no material conflict of interests.
Contributions
TL performed experiments, analyzed data and drafted the
against their respective LD
cell lines showed an inverse relationship between their
NF-κB index and DC-1-192 LD 50
50
for DC-1-192. Four of the five
value suggesting that response to DC-1-192 was influenced by how NF-κB- dependent the cell lines were. JJN3 cells were the excep- tion to this rule; these cells had a high NF-κB index (10.8) but were relatively resistant, in comparison to the other four cell lines, to the cytotoxic effects of DC-1-192 (mean
= 6 nM). The reasons for this are likely to be multiple and may be unrelated to NF-κB, but it is worthy of note that JJN3 cells possess a cytoplasmic EFTUD2-NIK fusion gene, which may alter p100 processing to p52. Indeed, when we assessed the impact of the PBD on nuclear NF- κB subunit DNA binding in JJN3 cells, all three com- pounds showed significant inhibition of the p65 and p50 canonical subunits as well as the non-canonical subunit RelB after 4 h. In contrast, no significant change in p52 was observed following treatment with the PBD. We sub- sequently repeated the experiments using the U266 cell line, which has a TRAF3 mutation leading to the cyto- plasmic accumulation of NF-κB inducing kinase (NIK).23,24 These cells showed a significant reduction in all four NF- κB subunits including p52 following short-term treatment with PBD.
LD
50
The rapid reduction in nuclear NF-κB subunit expres- sion indicates that NF-κB inhibition precedes apoptosis in these cells and may contribute to the efficacy of the PBD. Given the DNA binding characteristics of these com- pounds, it seems possible that they compete for NF-κB binding motifs, thereby inhibiting the transcription of NF-κB target genes. The reduction in nuclear NF-κB sub- units observed in this study may have been caused by the shuttling of unbound NF-κB back to the cytoplasm and/or targeted degradation.33,34
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haematologica | 2021; 106(4)