Administrative Supplement: Mechanistic and Pharmacologic Studies of Selective Mithramycin Analogues Targeting EWS-FLI1 in Ewing Sarcoma

The effect of MTM analogues on hematological cancers and anti-tumor immunity: The LLS grant was focused on one analogue of mithramycin (MTM), designated MTM23. In the context of this supplement, studies will be performed to test other analogues of MTM developed and characterized in Aims 1 and 2 (RO1 CA 243529) for their ability to modify growth of lymphomas and leukemias and enhance anti-tumor T-cell responses. We have cell lines representing leukemias that develop in children and adults as well as different types of lymphoma (follicular and diffuse large B-cell lymphomas). The LLS application was based our novel finding that MTM23 suppressed IL-10 production by leukemia cells and in preliminary studies enhanced anti-tumor immunity. This is unique since most chemotherapy agents inhibit both the cancer cells and immune cells. The analogues developed in Aims 1 and 2 will be tested for their ability to inhibit secretion of IL-10, a cytokine that suppresses T-cell responses required for anti-tumor immunity. We have in vivo animal models for leukemia and lymphoma to test the efficacy of the MTM analogues that either inhibit the growth of lymphoma/leukemia or only inhibit IL- 10 secretion without any effect on the proliferation of lymphoma/leukemia. These include Eì-Tcl1 mouse leukemia and Mec-1 human leukemia cells that can grow in the immunodeficient NOD-Scid.IL-2Rã-/- (NSG) mice. Tumor specific T cells are generated by two methods. 1. T-cells are primed in BL/6 mice with the tumor cells for ten days and subsequently purified by magnetic beads and injected into the NSG mice with tumor cells. The in vivo models are designed to test the ability of the analogues to enhance T-cell immunity to the cancer cells. Doses of analogues determined to be optimal based on their pharmacokinetic evaluation in Aim 3 of the NCI funded RO1 grant will be used. 15-color flow cytometry will be used to define the changes in specific T-cell subsets, with an emphasis on effector or memory cell status to understand the effects of the analogues on anti-tumor immunity. IL-10 levels will also be monitored. 2. CD19 specific chimeric antigen receptor (CAR) expressing T cells will be prepared by lentiviral transfection of anti-CD3+anti-CD28 activated T cells. Mice will be injected with tumor cells, CAR-T-cells with and without selected MTM analogues. Leukemia growth will be monitored as above. We have already obtained the lentiviral constructs and have successfully transduced T cells as shown by the reporter gene expression. All the adoptive transfer conditions for leukemia cell growth and monitoring have already been established in the laboratory (Alhakeem et al 2018). Subsequently the analogues will be tested for their ability to enhance effects of checkpoint blockade therapy that has found great success in several cancers such as melanoma and lung cancer. The experiments will consist of injecting the selected MTA analogue and antibody to PD-1 or PD-L1 or CTLA-4 into mice bearing the tumor and tumor primed T cells or CAR T-cells. NSG mice will be injected with leukemia cells with or without the selected MTM analogue in combination with or without checkpoint blockade antibody to PD-1. Only one of the three checkpoint molecules will be investigated during this time period. Other checkpoint molecules will be tested in the subsequent period of the fellowship. Parent Grant Admin. Supplement Rationale for the Supplement Aim 3. Determine selectivity, cytotoxicity, pharmacology and efficacy (Leggas) of MTM analogues in cells, mice and tumor models of Ewing sarcoma Assess if MTM analogues affect non-Ewing cancer cells with an emphasis on hematological cancers and if they enhance anti-tumor immunity. One MTM analogue was found to suppress IL-10 production by cancer cells. IL-10 inhibits anti-cancer immunity. We will test if other MTM analogues have dual role in cancer by directly inhibiting cancer cells and enhancing anti-tumor immunity. 1. Alhakeem, S., M. K. McKenna, K.Z. Oben, S.K. Noothi, J. R. Rivas, G.C. Hildebrandt, R.A. Fleischman, V. M. Rangnekar, N. Muthusamy, and S. Bondada. Chronic lymphocytic leukemia derived interleukin-10 suppresses anti-tumor immunity. J. Immunol. 200(12):4180-4189 (2018) PMID:29712773