Fusion Toxin BLyS-Gelonin Inhibits Growth of Malignant Human B Cell Lines In Vitro and In Vivo

Abstract

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC50 values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic “payloads” to malignant B cells.

The pathophysiological significance of PPM1D and therapeutic targeting of PPM1D-mediated signaling by GSK2830371 in mantle cell lymphoma

“…Both GSK2830371 and bortezomib target the p38 MAPK signaling pathway [ 33 – 35 ], and activation of p38 MAPK has been found to cause MCL cell death [ 35 ]. As shown in Supplementary Figure S5 , GSK2830371 and bortezomib synergistically increased levels of phosphorylated p38 MAPK in a p53-independent manner.…”

“…Most PPM1D substrates either initiate or contribute to cellular stress signals and the removal of activating phosphorylation has been thought to liberate cells from cellular stress [ 39 ]. p38 MAPK is generally a pro-apoptotic and stress-related protein that is regulated by PPM1D and the proteasome [ 33 – 35 ] and its activation has been found to cause MCL cell death [ 35 ]. Our data, which indicate that the pharmacologic inhibition of p38 significantly reduced GSK2830371/bortezomib lethality in both p53 wild-type and mutated MCL cells, suggest that this stress pathway contributes functionally to apoptosis.…”

Anti-tumor activities and apoptotic mechanism of ribosome-inactivating proteins

“…Luster et al [ 25 ] have reported that treatment with rGel-BLyS, rGel fused to a B-lymphocyte stimulator, rapidly reduced the tumor burden and markedly prolonged survival in xenograft mouse models of spread lymphoma or leukemia; in this setting, cell death was not induced by caspase activation but rather was partially mediated by the ribotoxic stress response. Furthermore, the rGel-BLyS fusion toxin combined with the proteasome inhibitor bortezomib restrained lymphoma growth and down-regulated nuclear factor kappa B (NF-κB) activity, which is critical for cellular proliferation and survival [ 26 ].…”

Impact of Endocytosis Mechanisms for the Receptors Targeted by the Currently Approved Antibody-Drug Conjugates (ADCs)—A Necessity for Future ADC Research and Development

“…Dose–response curves of BLyS-Gel with and without latrunculin A pretreatment were superimposable and therefore suggested that internalization did not involve an actin-cytoskeleton-dependent endocytic mechanism. Nonetheless, Luster et al reported that BLyS-Gel was localized to lysosomes after internalization [ 165 ]. They showed that when cells were treated with chloroquine, a drug that accumulates in acidic endosomes/lysosomes, leading to their rupture, the fusion toxins were more efficacious.…”