Immunomodulation of Ewing sarcoma to enhance allogenic NK cell therapy

While the introduction of the combination of intensive chemotherapy and multi-modal While the use of chemotherapy, surgery and irradiation have improved outcomes in Ewing sarcoma (ES), such about 70% of patients are cure, progress has plateaued for over three decades and there have been essentially no new advances. Furthermore, for patients with metastatic disease at diagnosis, or those who have relapsed after initial therapy, the five-year disease-free survival is 20-30%. These realities highlight a major unmet clinical need for sarcoma patients. The overarching hypothesis of this proposal is that drug-priming can unmask Ewing sarcoma cells to become more sensitive to immune therapy with natural killer (NK) cells. It is well accepted that chemotherapy exerts its beneficial (and toxic) effects through the alteration of cellular pathways. Such pathways include those involved in mRNA or protein expression. Likewise, cancer cell sensitivity to immune recognition is a complex process that is tightly controlled both at the mRNA and protein level. Considering these two concepts together, we hypothesize that concurrent administration of drugs have the potential to either augment or inhibit the desired anti-cancer immune reactions. To test this hypothesis, we have performed a high-throughput drug screening of 2,569 drug FDA-approved drugs for drugs for their ability to enhance NK cell killing of ES cell lines. We have identified 56 drugs which do not directly kill ES cells, but when added to ES cells, increase the sensitivity of NK cell killing. Based on these results, our aims are SA1: to identify how these 56 drugs increase NK cell killing of ES cells, and SA2: to determine whether they work in mice engrafted with sarcoma. We anticipate that the drugs are acting on the sarcoma cells to either make them more “visible” to the immune system or to make the sarcoma cells more sensitive to NK cell mediated killing. In our initial screen, we used a single, clinically achievable, dose of drug. Here we will test a range of doses for the identified compounds to better understand to better know the optimal drug dose. In the drugs that are safest and have the most augmentation of NK killing, we will characterize the mechanism of enhanced killing by interrogating the ES cell for the expression of surface molecules, known to be important in NK cell recognition of sarcoma. We will also interrogate the genetic pathway that are activated and repressed by these promising drugs. Finally, we will simulate combination therapy by using our patient derived xenograft mouse models, where ES tumors are engrafted into mice, and they will be treated with the drug alone, NK cells alone and combinations of the two. At the conclusion of this project, we anticipate that we have identified key pathways that can modulate ES cells to be better killed by NK cells using drugs that are already FDA approved, and thus could be rapidly incorporated into sarcoma treatment trials.