1. ACTIONS OF L-THYROXINE (T4) AND NANO-DIAMINO-TETRAC (NDAT, NANOTETRAC) ON PD-L1 IN CANCER CELLS Paul J. Davis, Hung-Yun Lin, Shaker A. Mousa Albany Medical College, Albany, NY, USA; Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences; Taipei Medical University, Taipei, Taiwan

2. The PD-1 (programmed death- 1)/PD-L1 (PD-ligand 1) checkpoint is a critical regulator of activated T cell-cancer cell interactions, serving to defend tumor cells against (T cell-mediated) immune destruction. Pharmaceutical interest is high in PD-L1 antibody use in solid tumor chemo-therapy to render cancer cells susceptible to host killer T cell action. We have developed a non- immunological strategy for downregulation of PD-L1 gene expression and PD-L1 protein content in tumor cells.

3. The non-immunologic strategy is based on pharmacologic regulation of a target on the extracellular domain of plasma membrane integrin  v  3. This target is a thyroid hormone-tetraiodothy- roacetic acid (tetrac) receptor that controls—from the cell surface— the expression of a panel of cancer cell defense genes, including PD- L1.

4. O CH 2 -CH-COOH NH 2 I - - I - - I - - I - - 3’ 5’5 3 Thyroxine (T 4 ) O HO CH 2 -CH-COOH NH 2 I - - I - - I - - 3’ 5’5 3 3,5,3’-Triiodothyronine (T 3 ) HO O CH 2 --COOH I - - I - - I - - I - - 3’ 5’5 3 Tetrac HO Low-grade thyromimetic within cells TH antagonist at integrin  v  3 TH receptor

5. PLGA nanoparticle Figure 1 In Nanotetrac, shown here, tetrac is covalently bound to a linker (ether bond) which, in turn, is amide-bonded to a PLGA nanoparticle. The action of tetrac is limited in this formulation to the thyroid hormone-tetrac receptor on the extra- cellular domain of integrin  v  3.

6. Human triple-negative breast cancer (MDA-MB-231) cells and human colon cancer (HCT116. HT- 29) cells were cultured in DMEM (breast) or RPMI-1640 (colon), each with 10% FBS. Two days prior to study of cells, 0.25% charcoal-stripped serum replaced 10% FBS. Cells were treated with L-thyroxine (T4, 10 -7 M total hormone, 10 -10 M free), NDAT (10 -7 M tetrac equivalent) or both for 24 h.

7. Tumor cell RNA was harvested and PD-L1 mRNA quantitated by qPCR. PD-L1 protein was measured by western blotting.

8. Figure 2 A. B. MDA-MB 231 cell mRNA abundance

9. Figure 3 MDA-MB 231 cell PD-L1 protein content ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - B.A. 2.7-fold increase 25-35% decrease in content with NDAT

10. Figure 4 HCT116 cell mRNA A. B.

11. Figure 5 HCT116 cell PD-L1 protein ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - B. ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - A. 25-60% decrease in basal or stimulated content with NDAT

12. Figure 6 HT-29 cell mRNA A. B.

13. Figure 7 HT-29 cell protein ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - B.A. 40% decrease in basal or stimulated content with NDAT

14. Figure 8 ◄ PD-L1 ◄ GAPDH 50 kDa - 36 kDa - + PD98059- PD98059 NDAT (10 -7 M) T 4 (10 -7 M) + - - - - ++ + + - - - - ++ + Dependence on MAPK of induction by T 4 of PD-L1 in cultured HCT116 cells

15. SUMMARY In MDA-MB-231 breast cancer cells, T 4 significantly stimulated PD- L1 gene expression by 40% and increased PD-L1 protein 2.7-fold; these effects were blocked by NDAT. In HCT116 and HT-29 colon carcinoma cells, T 4 significantly increased PD-L1 gene expression by 20-60% and protein abundance by 25-65%; these effects were blocked by NDAT. Basal levels of mRNA and protein were also reduced by NDAT. MAPK mediates the T 4 effects.

16. CONCLUSIONS The PD-1/PD-L1 defensive tumor cell checkpoint is T 4 - responsive. Host patient T 4 supports this cancer cell defense. Hormonal effects vary among cell lines. NDAT eliminates the contribution of T 4 to the checkpoint and also variably reduces basal levels of PD-L1.