Abstract
Introduction: Chronic lymphocytic leukemia (CLL) is an incurable, heterogenetic disease dependent on B cell receptor (BCR) signaling with subsequent nuclear factor-kappa B (NF-κB) activation resulting in the evasion of apoptosis and enhanced malignant B cell growth. Targeted therapies such as ibrutinib (IBR; BTK inhibitor) and venetoclax (VEN; BCL2 antagonist) have revolutionized the management of CLL, however ~20% of patients relapse, signifying the urgent need for novel therapeutics for CLL patients especially those with refractory/relapse (ref/rel) disease. Additionally, various tumor microenvironment (TME) stimuli fuel CLL growth and contribute to drug resistance through the activation of numerous signaling pathways (BCR, CD40R, TLR, BAFFR) and consequential sustained NF-κB activation. Currently, there are no FDA approved drugs that effectively target the NF-κB protein family. Herein we introduce 36-286 (N3), a novel spirocyclic dimer which displays NF-κB inhibitory activity and elicits potent anti-leukemic properties. N3 is a dimer of a spirocyclic α-methylene-γ-butyrolactone analog that covalently binds to surface exposed cysteine residues on NF-κB proteins (IKKβ and P65) (Rana S et al, 2016). Our study aims to investigate N3's mode of action (MOA) and to establish its anti-leukemic effects in CLL including drug-resistant disease, thereby introducing a novel therapeutic option for rel/ref disease.
Methods: Cell growth via MTS proliferation assay was determined following treatment with N3 (0.125 - 2 μM) in a panel of malignant B cell lines [CLL (HG3, MEC1, OSUCLL), diffuse large B cell lymphoma (Pfeiffer, RC, RIVA), mantle cell lymphoma (Jeko1)], and in patient derived CLL cells stimulated with CpG ODN 2006 (CpG; 3.2 μM). Viability testing of normal B cells isolated from healthy donors was conducted following N3 treatment. Anti-tumor properties of N3 (1 - 2 μM; 4h) in the HG3 and OSUCLL cell lines were further confirmed under conditions mimicking different TME stimuli such as α-IgM (10 μg/mL), CD40L (100 ng/mL), BAFF (50 ng/mL) or CpG (3.2 μM). Protein expression of oncogenic MYC, select NF-κB pathway proteins (IKKα, IKKβ, P65, IκBα, RelB) and the anti-apoptotic protein MCL1 was determined following treatment with N3 (0.25 - 2 μM; 4h) by immunoblot (IB). Next, we induced IBR resistance in HG3 cells by prolonged exposure to increasing IBR concentrations (~10-15 fold its IC 50 in parental cells). Cell proliferation via MTS was determined following treatment with N3 on these resistant cells. To gain insight on the potential MOA of N3 in CLL, we adapted a proteomics-based approach (TMT labeled mass spectrometry) and conducted RNA-seq in OSUCLL cells treated with N3 (1 - 2 μM) for up to 24 h. Subsequent pathway analysis was performed to identify the top factors modulated by N3.
Results: N3 showed remarkable efficacy (IC 50 < 0.6 μM) across all the malignant B cell lines evaluated while sparing normal B cells. In CpG stimulated primary CLL, N3 resulted in marked anti-leukemic effects (0.125 μM) comparable to IBR (1 μM). N3 induced cell apoptosis in CLL cell lines in a dose-dependent manner with marked PARP cleavage. Furthermore, our IB analyses of N3 treated CLL cell lines showed reduced levels of NF-κB pathway proteins, MYC and MCL1. Notably, N3 was effective in reducing levels of the above-mentioned proteins in the presence of the various TME stimuli. Strikingly, N3 maintained its cytotoxic effects in ibrutinib resistant HG3 cells. Studies to confirm N3's cytotoxicity in VEN resistant CLL cells are ongoing. Top ten pathways from both proteomics and RNA-seq analyses revealed an upregulation of the unfolded protein response (UPR) and inhibition of cap-dependent protein translation. IB analyses of select factors related to UPR (CHOP, XBP1, PERK, IRE1) and protein translation (eIF2α, 4E-BP1, PDCD4) in N3 treated CLL cells validated our omics' findings. Efforts to identify the proteome wide direct targets of N3 in CLL cells are currently underway.
Conclusion: N3 is a novel pre-therapeutic lead that targets multiple survival and proliferation pathways through the inhibition of NF-κB activity and upregulation of UPR. We show that its highly cytotoxic in tumor B cells while sparing normal B cells. Moreover, N3 sustained its anti-tumor properties under different TME stimuli and in IBR resistant cells, indicating the potential use of this compound in rel/ref patients following evaluation in murine CLL models.
No relevant conflicts of interest to declare.
