Paul J. Davis, Hung-Yun Lin, Shaker A. Mousa 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

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.

The non-immunologic strategy is based on pharmacologic regulation of a target on the extracellular domain of plasma membrane integrin avb3. 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.

3,5,3’-Triiodothyronine (T3) NH2 3’ 3 HO O CH2-CH-COOH 5’ 5 I - I - Thyroxine (T4) I - I - NH2 3’ 3 HO O CH2-CH-COOH 5’ 5 I - 3,5,3’-Triiodothyronine (T3) I - I - 3’ 3 HO O CH2--COOH 5’ 5 I - I - Tetrac Low-grade thyromimetic within cells TH antagonist at integrin avb3 TH receptor

In Nanotetrac, shown here, tetrac is PLGA nanoparticle 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 avb3. Figure 1

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.

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

MDA-MB 231 cell mRNA abundance Figure 2

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

HCT116 cell mRNA A. B. Figure 4

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

HT-29 cell mRNA A. B. Figure 6

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

Dependence on MAPK of induction by T4 of PD-L1 in cultured HCT116 cells + - + - + - NDAT (10-7 M) + - - T4 (10-7 M) + + - - + + 50 kDa - ◄ PD-L1 36 kDa - ◄ GAPDH Figure 8

SUMMARY In MDA-MB-231 breast cancer cells, T4 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, T4 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 T4 effects.

CONCLUSIONS The PD-1/PD-L1 defensive tumor cell. Host patient T4 supports this cancer cell defense. Hormonal effects vary among cell lines. NDAT eliminates the contribution of T4 to the checkpoint and also variably reduces basal levels of PD-L1.

CONCLUSIONS 2 Immunologic reduction in tumor cell production of PD-L1 and attendant side effects can be obviated by hormonal/chemical strategies.