Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation by NSC 644221.

PURPOSE: The discovery and development of small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1) is an attractive, yet challenging, strategy for the development of new cancer therapeutic agents. Here, we report on a novel tricyclic carboxamide inhibitor of HIF-1alpha, NSC 644221. EXPERIMENTAL DESIGN: We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1alpha. RESULTS: NSC 644221 inhibited HIF-1-dependent, but not constitutive, luciferase expression in U251-HRE and U251-pGL3 cells, respectively, as well as hypoxic induction of vascular endothelial growth factor mRNA expression in U251 cells. HIF-1alpha, but not HIF-1beta, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1alpha protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1alpha as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [35S]methionine. In contrast, NSC 644221 decreased the rate of HIF-1alpha translation relative to untreated controls. Silencing of topoisomerase (topo) IIalpha, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1alpha. The data presented show that topo II is required for the inhibition of HIF-1alpha by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, gammaH2A.X, and G2-M arrest in the majority of cell lines tested, it only inhibited HIF-1alpha in a distinct subset of cells, raising the possibility of pathway-specific "resistance" to HIF-1 inhibition in cancer cells. CONCLUSIONS: NSC 644221 is a novel HIF-1 inhibitor with potential for use as both an analytic tool and a therapeutic agent. Our data provide a strong rationale for pursuing the preclinical development of NSC 644221 as a HIF-1 inhibitor.

A novel iron chelator that does not induce HIF-1 activity.

Human skin cells (FEK-4) have been shown to undergo an immediate and transient release of low molecular mass iron (LMrFe) when subjected to UVA (320-380 nm) irradiation and this iron may act as a pro-oxidant and increase tissue injury. In order to decrease this transient release of LMrFe, cells were treated with the iron chelators desferrioxamine (DFO) and salicylaldehyde isonicotinoyl hydrazone (SIH). However, although the iron pool decreased, an increase in the DNA binding activity of the hypoxia inducible factor-1 (HIF-1) was observed when DFO and SIH were administered to normal growing FEK-4 cells. The induction of HIF-1 activates the expression of several genes associated with hypoxia and iron homeostasis. HIF-1 induction has also been associated with protection against certain forms of oxidative stress. Therefore, it is difficult to use a conventional HIF-1 activating iron chelator (such as DFO) for mechanistic studies of protection against iron-mediated oxidative stress since any protection observed could be a consequence of either the chelation of LMrFe or the induction of protective genes associated with the hypoxic response. In order to observe the effect of iron chelation on cell function without the induction of hypoxia responsive genes, cells were treated with the novel iron chelator N-(2-hydroxybenzyl)-L-serine (HBSer). Although this compound is an effective iron chelator under the conditions employed in this experiment, it does have a lower iron-binding constant than either DFO or SIH. This may be the major determinant of the observation that the compound does not induce HIF-1 binding or activate HIF-1 responsive transcriptional promoters.

Synergystic induction of HIF-1alpha transcriptional activity by hypoxia and lipopolysaccharide in macrophages.

Hypoxia Inducible Factor-1 (HIF-1) is activated by a variety of stimuli, including inflammatory mediators. In this report we investigated the role that bacterial lipopolysaccharide (LPS) and hypoxia play in the regulation of HIF-1-dependent gene expression in macrophages. We report that murine macrophages stimulated with low concentrations of LPS (1-10 ng/ml) expressed significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA when cultured under hypoxic compared to normoxic conditions. Functional studies of the iNOS promoter demonstrated that the synergistic interaction between LPS and hypoxia was mediated, at least in part, by the NFkappaB and the HIF-1 binding sites. In addition, transient transfection experiments using a Hypoxia Response Element (HRE)-containing plasmid showed that LPS and hypoxia synergistically induced HIF-1-dependent transcriptional activity. Interestingly, LPS did not significantly affect HIF-1alpha protein levels or HIF-1 DNA binding activity relative to hypoxic induction. HIF-1alpha, but not HIF-2alpha, was critical for the synergistic induction of HRE-dependent transcriptional activity in macrophages, as indicated by experiments using siRNA targeting HIF-1alpha or HIF-2alpha. Addition of ROS-scavengers completely abrogated the synergistic induction of HIF-1 transcriptional activity by LPS and hypoxia, but neither inhibited HIF-1 transcriptional activity induced by hypoxia alone nor affected HIF-1alpha protein levels or HIF-1 DNA binding induced by hypoxia alone or hypoxia plus LPS. Taken together, our results demonstrate that LPS and hypoxia act synergistically to induce HIF-1alpha-transcriptional activity and they emphasize the existence of a cross talk between hypoxic and non-hypoxic signaling pathways in the regulation of macrophages gene expression.