ACTIONS OF LTHYROXINE T 4 AND NANODIAMINOTETRAC NDAT

ACTIONS OF L-THYROXINE (T 4) AND NANO-DIAMINO-TETRAC (NDAT, NANOTETRAC) ON PD-L 1 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

The PD-1 (programmed death 1)/PD-L 1 (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-L 1 antibody use in solid tumor chemo-therapy to render cancer cells susceptible to host killer T cell action. We have developed a nonimmunological strategy for downregulation of PD-L 1 gene expression and PD-L 1 protein content in tumor cells.

• The non-immunologic strategy is based on pharmacologic regulation of a target on the extracellular domain of plasma membrane integrin avb 3. This target is a thyroid hormone-tetraiodothyroacetic acid (tetrac) receptor that controls—from the cell surface—the expression of a panel of cancer cell defense genes, including PD-L 1.

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

I O O N H I O O I I OH PLGA nanoparticle O N H H N I O O I I I O OH In Nanotetrac, shown here, tetrac is covalently bound to a diaminopropane linker which, in turn, is amide-bonded to a PLGAnanoparticle. The action of tetrac is limited in this formulation to the thyroidhormone-tetrac receptor on the extracellular domain of integrin avb 3. Figure 1

• Human triple-negative breast cancer (MDA-MB-231) cells and human colon cancer (HCT 116. 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 (T 4, 10 -7 M total hormone, 10 -10 M free), NDAT (10 -7 M tetrac equivalent) or both for 24 h.

• Tumor cell RNA was harvested and PD-L 1 m. RNA quantitated by q. PCR. • PD-L 1 protein was measured by western blotting.

MDA-MB 231 cell m. RNA abundance A. B. Figure 2

MDA-MB 231 cell PD-L 1 protein content A. B. 50 k. Da - ◄ PD-L 1 36 k. Da - ◄ GAPDH 36 k. Da - 50 k. Da - 2. 7 -fold increase 25 -35% decrease in content with NDAT Figure 3 ◄ PD-L 1 ◄ GAPDH

HCT 116 cell m. RNA A. B. Figure 4

HCT 116 cell PD-L 1 protein A. B. 50 k. Da - ◄ PD-L 1 36 k. Da - ◄ GAPDH 25 -60% decrease in basal or stimulated content with NDAT Figure 5

HT-29 cell m. RNA A. B. Figure 6

HT-29 cell protein A. 50 k. Da - 36 k. Da - B. ◄ PD-L 1 50 k. Da - ◄ PD-L 1 36 k. Da - ◄ GAPDH 40% decrease in basal or stimulated content with NDAT Figure 7

Dependence on MAPK of induction by T 4 of PD-L 1 in cultured HCT 116 cells NDAT (10 -7 M) T 4 (10 -7 M) - - PD 98059 + - - + + + - + PD 98059 + - - + + + 50 k. Da - ◄ PD-L 1 36 k. Da - ◄ GAPDH Figure 8

SUMMARY • In MDA-MB-231 breast cancer cells, T 4 significantly stimulated PDL 1 gene expression by 40% and increased PD-L 1 protein 2. 7 -fold; these effects were blocked by NDAT. • In HCT 116 and HT-29 colon carcinoma cells, T 4 significantly increased PD-L 1 gene expression by 20 -60% and protein abundance by 25 -65%; these effects were blocked by NDAT. • Basal levels of m. RNA and protein were also reduced by NDAT. • MAPK mediates the T 4 effects.

CONCLUSIONS • The PD-1/PD-L 1 defensive tumor cell. 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-L 1.

CONCLUSIONS 2 • Immunologic reduction in tumor cell production of PD-L 1 and attendant side effects can be obviated by hormonal/chemical strategies.