(P 446) SYNERGISTIC TARGETING OF KIF11 AND AURKA TO IMPROVE OUTCOMES FOR EWING SARCOMA

Objective: Ewing sarcoma (EWS) is a rare and aggressive pediatric bone sarcoma. The pathological EWS/ETS gene fusion results in a driver oncogenic transcription factor, that remains undruggable. Standard of care treatment options for these children and young adults consists of multiple cycles of chemotherapy that often lead to toxicity, secondary malignancies, and co-morbidities. Although chemotherapy has improved the overall survival (OS) for localized disease, patients with metastatic disease have poor prognosis with less than 30% OS. To identify new therapies for this neglected disease, we conducted in silico and drug re-purposing screens (PMID: 27863422; 31025088). We identified proteins essential for mitotic spindle formation and cell cycle progression, including Kinesin Family Member 15 (KIF15), its binding partner, Xklp2 (TPX2), and Aurora Kinase A (AURKA) which are upregulated by EWS-FLI1, the primary oncogenic driver of EWS. Importantly, KIF15/TPX2 cooperates with KIF11 (Eg5), a molecular motor protein which cross-links anti-parallel microtubules in the mitotic spindle to maintain spindle bipolarity. We hypothesize that targeting key and redundant proteins essential for mitotic progression would provide a therapeutic vulnerability for Ewing sarcoma by inducing G2/M arrest and mitotic catastrophe. Given the lack of clinical grade KIF15 inhibitors, we targeted the protein upstream by inhibiting AURKA (VIC-1911; VITRAC Therapeutics) to block phosphorylation of KIF15S1169 and its subsequent targeting to the spindle. Thus we assessed co-targeting of KIF11 (SB-743921) and AURKA (VIC-1911) to exploit the chances for better efficacy, decreased toxicity, and reduced development of drug resistance. To advance our hypothesis, we evaluated the drug combination for synergy in vitro and tumor efficacy in vivo using EWS models

Methods: Drug Synergy assay:500 cells/well were plated in triplicates in 96-well plates in 8x8 matrix and incubated for 72 hours post drug treatment and cell viability was assessed by CellTiter-Glo reagent and synergy was calculated using SynergyFinder tool.
Cell cycle analysis: Cells were harvested and fixed in 70% ethanol overnight, post-drug treatment and then stained with FxCycle™ PI/RNase Staining Solution. DNA content was measured using the Attune™ NxT flow cytometer.
Capillary Western Blot (Wes): ProteinSimple Wes System was used to determine protein expression.1:50 dilution was used for primary antibodies.
Tumor xenograft and drug treatment studies: Six-week-old female NSG mice were inoculated subcutaneously with a suspension of TC-71 cells (2 × 106). Mice were randomized into four groups as follows: 1) Vehicle control; 2) SB-743921 only (2.5 mg/kg), 3) VIC-1911 (37.5 mg/kg), and 4) combination of SB-743921 and VIC-1911.

Results: The top fifteen genes that we identified to be essential for EWS progression are listed (Figure 1A). Further, in silico analysis of RNA expression data of solid cancers indicates that KIF15 expression levels are the highest in EWS and that KIF11 expression is the second highest in EWS after synovial sarcomas (Figure 1B). SB-743921 and VIC-1911 were tested across different EWS fusion bearing cells namely EWS-FLI Type II (SKES-1), EWS-FLI Type III (CHLA-258), and EWS-ERG (COG-E-352), and we observed synergistic drug activity with the combination treatment irrespective of fusion type (Figure 2). Combination treatment led to an increase in G2/M arrest and apoptosis (Figure 3A). Quantification of differences in cell-cycle phases indicate a greater percentage of cells in G2/M phase for combination treatment of VIC-1911 (10 nM) + SB-743921 (2.5 nM) (53.1%) compared to single drugs [VIC-1911, 10 nM (34.3%), SB-743921, 5 nM (43.7%)], or the untreated control (29.2%). Wes analysis was done to determine protein expression following drug treatment and we observed increases in 1) KIF11 and AURKA expression in the combination group, and 2) c-PARP and p-KIF11Thr926 levels indicating apoptosis and delay in mitotic exit (Figure 3B). In vivo mouse xenograft studies using TC-71 demonstrate complete tumor regression with the combination treatment (Figure 4A) and significant effect on overall survival of the mice compared to control and single drug treatment groups (Figure 4B).

Conclusion: We have identified a novel combination of mitotic inhibitors targeting KIF11 and KIF15 via AURKA that is highly synergistic in inhibiting the growth of an aggressive tumor such as Ewing sarcoma. Our findings are highly relevant, timely and clinically translatable given the lack of proper therapies for this rare and orphaned disease