Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors

Abstract

Summary Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.

Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity

“…As seen in Figure 3a, increasing concentrations of BRD9876 increased the K M ATP but had negligible effect on the V max , consistent with the drug being a competitive inhibitor of ATP binding with a K I of 3.8 ± 1.5 nM (see also Figure S4). This result differs from previous work showing that BRD9876 is an ATP-noncompetitive inhibitor of Eg5 36 , but it is consistent with biochemical results from other inhibitors that bind at the α4–α6 interface 31–35 . Molecular docking simulations of BRD9876 binding to apo-Eg5 predicted that BRD9876 binds at the same site on Eg5 as another well-characterized rigor inhibitor PVZB1194 (Figure S5), consistent with the mutagenesis prediction 36 .…”

“…Using a phenotypic screening approach, Chattopadhyay et al 36 found that, similar to monastrol 25 and STLC 23 , BRD9876 treatment resulted in enhanced spindle monopolarity and G2/M arrest in multiple myeloma 1S cells. Analysis of phospho-mimetic Eg5 mutants in cells was used to argue that BRD9876 preferentially binds to and inhibits MT-bound Eg5, and mutagenesis was used to argue that the compound binds near the junction of helices α4 and α6, similar to other rigor inhibitors 33,34 .…”

“…The thiazole FCPT 31,32 is thought to act as an ATP competitive inhibitor by binding directly to the nucleotide binding site. In contrast, mutagenesis and structural analysis revealed that biaryl compounds GSK-1 33 , PVZB1194 34,35 act as ATP-competitive inhibitors but bind near the α4–α6 interface 33,35,36 ; thus they behave as allosteric competitive inhibitors of ATP binding. A related compound, BRD9876, was recently identified as an Eg5 inhibitor that binds to the α4–α6 interface, but was reported to be ATP non-competitive, and to preferentially bind to MT-bound Eg5 36 .…”

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

“…Our strategy led to the identification of a drug that inhibits both cell-autonomous and non-cell-autonomous pathways, acting in vitro and in vivo, while sparing normal HSCs. We propose that recapitulating tissue-like conditions (75)(76)(77) represents a promising avenue in the quest for new cancer therapies in other tumors.…”

Small molecule regulation of normal and leukemic stem cells

“…These include the use of cytokine combinations [9], improved bioreactor-based methodologies [10], mesenchymal stromal cell cocultures [11][12][13] and immobilized biologicals such as delta 1 notch ligand [14]. New methodologies to identify potent small molecules active on human HSPCs were pioneered by Boitano et al [15].…”