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posted on 2024-05-22, 14:20 authored by Alexandria P. Eiken, Audrey L. Smith, Sydney A. Skupa, Elizabeth Schmitz, Sandeep Rana, Sarbjit Singh, Siddhartha Kumar, Jayapal Reddy Mallareddy, Aguirre A de Cubas, Akshay Krishna, Achyuth Kalluchi, M. Jordan Rowley, Christopher R. D'Angelo, Matthew A. Lunning, R. Gregory Bociek, Julie M. Vose, Amarnath Natarajan, Dalia El-Gamal SpiD3 inhibits proliferation and induces apoptosis in malignant B cells. A, Chemical structures of analog 19 (monomer), SpiD3 and SpiD7 (dimers of analog 19). Full synthesis details reported elsewhere. B, Mitochondrial activity of CLL cell lines, HG-3 and OSU-CLL, was determined by MTS assay following treatment with increasing concentrations of analog 19, SpiD3, and SpiD7 for 72 hours (n = 3–4 independent experiments/cell line). C, The IC50 values (mean ± SEM) of a panel of B-cell malignancy cell lines following SpiD3 treatment (72 hours) was determined by MTS assay (n = 3–5 independent experiments/cell line). CLL: chronic lymphocytic leukemia, ABC-DLBCL: activated B-cell-like diffuse large B-cell lymphoma, GC-DLBCL: germinal-center-like diffuse large B-cell lymphoma, MCL: mantle cell lymphoma. #Indicates a DH lymphoma or a TH lymphoma. On the right is a table of the IC50 values (mean ± SEM). D, Percent apoptosis of HG-3 and OSU-CLL cell lines was determined by Annexin V/PI viability assay following 24 hours treatment with SpiD3 (n = 4 independent experiments/cell line). E, Representative immunoblot analyses of total and cleaved PARP in OSU-CLL and HG-3 cells following 4 hours treatment with SpiD3 or DMSO vehicle (VEH). GAPDH serves as the loading control (n = 6 independent experiments/cell line). F, MTS assay of OSU-CLL cells (n = 3 independent experiments) treated for 24 hours with increasing concentrations of SpiD3 (blue) with or without cocurrent z-VAD-FMK treatment (50 µmol/L, tan). G, Representative immunoblot analyses of total and cleaved PARP in OSU-CLL cells treated with SpiD3 for 4 hours in the presence or absence of z-VAD-FMK pretreatment (1 hour, 50 µmol/L). GAPDH served as the loading control (n = 4 independent experiments). Error bars and IC50 values are shown as mean ± SEM. Asterisks denote significance versus VEH: *, P < 0.05; **, P < 0.01.
Funding
HHS | NIH | National Cancer Institute (NCI)
HHS | NIH | National Institute of General Medical Sciences (NIGMS)
History
ARTICLE ABSTRACT
Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NFκB signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacologic agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the Eµ-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NFκB signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its antileukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated Eµ-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NFκB signaling and the UPR, culminating in profound antitumor properties independent of TME stimuli.
SpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NFκB signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins, SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall, SpiD3 exploits critical CLL vulnerabilities (i.e., the NFκB pathway and UPR) highlighting its use in drug-resistant CLL.
