The Effect of a Novel Preclinical Agent, SpiD3, on Drug-resistant Chronic Lymphocytic Leukemia

B-cell receptor (BCR) signaling is a central driver in chronic lymphocytic leukemia (CLL), along with activation of pro-survival pathways (e.g., NF- κB) and aberrant anti-apoptotic (e.g., BCL2), culminating to CLL cell survival and drug-resistance. Front-line targeted therapies such as ibrutinib (IBR) and venetoclax (VEN) have radically improved CLL management. Yet, persisting CLL cells lead to relapse in ~20% of patients, signifying the need for alternative therapeutics with novel approaches to CLL cell elimination and overcoming resistance mechanisms. SpiD3 is a novel spirocyclic dimer of analog 19 displaying NF-κB inhibitory activity. Recently, we have shown that SpiD3 inhibits CLL proliferation and induces cytotoxicity, by promoting futile activation of the unfolded response pathway (UPR) and generation of reactive oxygen species (ROS), resulting in insurmountable endoplasmic reticulum stress. RNA-sequencing analysis of IBR- and VEN-resistant CLL cell lines revealed ferroptosis, UPR signaling, and oxidative stress among the top pathways modulated by SpiD3 treatment. By examining SpiD3 induced protein aggregation, ROS production, and ferroptosis in preclinical models of CLL, our data demonstrates marked SpiD3-induced anti-leukemic properties and CLL cell cytotoxicity, including in cell lines resistant to current front-line therapeutics, substantiating the development of SpiD3 as a novel therapeutic approach to management of relapse/refractory CLL disease. To investigate the gene changes in ibrutinib-resistant HG-3 CLL cell line compared to their wild-type counterpart. Additionally, assess how SpiD3 modulated those genes in both the wild-type and resistant cells. We then performed gene expression analysis on wild-type HG-3 CLL cells and ibrutinib-resistant HG-3 CLL cells treated in triplicate with either vehicle, SpiD3, or ibrutinib.