(P 247) HARNESSING EWING SARCOMA'S REPLICATION STRESS BY TARGETING DDK AND WEE1 IN COMBINATION

Objective: Ewing sarcoma is an aggressive pediatric cancer for which chemotherapy is very intense and often results in acute toxicities. Here, we have found that the combined inhibition of CDC7 kinase (DDK) and WEE1 kinase forces high levels of mitotic errors and synergistic cell death, specifically in Ewing sarcoma cells. Furthermore, when DDK and WEE1 inhibitors are used with irinotecan in murine models, the combination results in impressive reduction of tumor burden. This therapeutic strategy has the potential to provide a highly efficacious and minimally toxic treatment strategy for patients with Ewing sarcoma.

Methods: A673 (Ewing; ATCC, catalog no. CRL-1598, RRID:CVCL_0080) and RD-ES (Ewing; ATCC catalog no. HTB-166, RRID:CVCL_2169) cell lines were obtained through ATCC. TC32 (Ewing; RRID:CVCL_7151) were obtained through Children's Oncology Group. A673 cells were cultured in DMEM, RDES, and TC32 in RPMI1640. All media was supplemented with 10% FBS and 1% antibiotic-antimycotic (Gibco Life Technologies). All cell lines were grown at 37°C and 5% CO2 and were used within 10 passages of receipt from ATCC or COG. Cells were tested for Mycoplasma and all Mycoplasma testing was conducted by PCR through IDEXX BioAnalytics and was negative. Cell lines authentication was performed via short tandem repeat (STR) profiling.

Cells were treated with 300 nmol/L TAK-931 (DDK inhibitor), 1 μmol/L MK1775 (WEE1 inhibitor) or combination for 24 hours. Cells were then fixed and stained for DNA content (PI) and pHH3 and analyzed via FACS. Premature mitotic cells were identified as cells that stained positive for pHH3 that contain < 4N DNA content. Quantification of percentage of premature mitotic cells as a proportion of total mitotic cells [(premature mitotic cells/total mitotic cells) * 100%] (n = 3 biological replicates, two-way ANOVA, ****, P < 0.0001).

Mice were housed in the Laboratory Animal Shared Resource (LASR) at Roswell Park in a limited access barrier facility. LASR is an AAALAC International Accredited Animal Program. Female NSG mice at 6 to 8 weeks of age received subcutaneous injection of 1 million TC32 cells in the left flank. Two times weekly calipers used to measure change in tumor growth. Treatments started once tumors reached 200 mm3. And were performed for 3 weeks. In the second experiment, mice were rechallenged with another 3 weeks of treatment at Week 6. Mice were treated with either no therapy (vehicle control – 5 mice), MK1775 (Wee1 inhibitor; 50 mg/kg PO daily Days 1-5) + TAK-931 (DDK inhibitor; 60 mg/kg PO daily Days 1-3) (DDKi + WEE1i - 10 mice), Trabectedin (0.15 mg/kg IV or IO weekly x 3 weeks) (10 mice), Irinotecan (15 mg/kg IV weekly) + Temozolomide (40 mg/kg IP weekly) (standard relapse comparator - 10 mice), Irinotecan + MK1775 (10 mice), Cyclophosphamide (40mg/kg IV) + Doxorubicin (3.3mg/kg IV) + Vincristine (0.5mg/kg IV) given on Day 1 once on day 1 of week 1 (standard up-front regimen – 10 mice). Irinotecan + MK1775 + TAK-931 (novel triplet - 10 mice). Of note, the DDK and WEE1 inhibitors were dosed BID (twice daily) in the first experiment and then changed to daily in the second experiment. If tumors reached volume greater than 2000 mm3, the mice were euthanized as per Roswell Park Institutional Animal Care and Use Committee (IACUC) guidelines.

The survival data were summarized by treatment group using Kaplan-Meier curves, where estimates of the median time were obtained with 95% confidence intervals. Associations were assessed using the log-rank test. All analyses were conducted in SAS v9.4 (Cary, NC) at a significance level of 0.05.

Results: The inhibition of DDK in Ewing sarcoma cells results in a dramatic reduction in the rate of DNA replication, significantly delaying replication completion and forcing a mitotic arrest to maintain viable levels of genomic integrity. The mitotic arrest is highly dependent on the activity of the checkpoint kinase WEE1. This stress places a heavy burden onto WEE1, making it a prime target for therapeutic intervention in this context. Our in vitro data confirms that the combined inhibition of WEE1 and DDK results in the unscheduled mitotic entry of Ewing cells with underreplicated DNA (Figure 1) that results in gross mitotic catastrophe, emergence of potentially immune-stimulatory micronuclei and synergistic cell death.

Our follow-up in vivo data demonstrates inhibition of tumor growth with DDK in combination with WEE1 inhibition that is improved with the addition of irinotecan. The frequency of DDK and WEE1 was reduced to once daily due to some evidence of weight loss with BID dosing and, while this mitigated toxicity, it also led to reduced efficacy (Figures 2-4). Unfortunately, trabectedin proved excessively toxic in the first mouse experiment and was not pursued further, although future combinations are of interest. The week 6 rechallenge in the second experiment appeared to indicate that the combination of DDK and WEE1 inhibition was needed, as the therapy overlapped for the first three days and then WEE1 inhibitor was given alone for Days 4-5. Intriguingly, only the triplet of the two novel inhibitors plus irinotecan had mice surviving past Week 6.

We are now planning experiments that will build upon these preliminary experiments but optimize the timing of DDK and WEE1 inhibition (alternating DDK and WEE1 inhibition). We will try treating the mice for longer than 3 weeks at a time to see if cure can be achieved. We are also looking to test the combination in additional cell lines, including a PDX model that is parafemoral.

Conclusion: The novel combination of two orally bioavailable inhibitors of DDK and WEE1 already separately in single-agent study in human patients, TAK-931 and MK1775, plus irinotecan may prove to be a promising combination to treat patients with Ewing sarcoma based on our preliminary data. To our knowledge, the combination of DDK and Wee1 inhibition has not been tested in humans to date but would be a logical next step.