Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models

Abstract

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. β-catenin is the key transcriptional regulator of WNT/β-catenin signaling. Evidence for β-catenin-mediated immune evasion is found in 13% of all cancers, 42% of primary cutaneous melanoma and a mouse melanoma model. Currently, there are no therapeutic strategies available for targeting WNT/β-catenin signaling to counteract checkpoint inhibitor resistance in melanoma. Here we report that a specific small-molecule tankyrase inhibitor, G007-LK, attenuates WNT/β-catenin and YAP signaling pathways in the syngeneic murine B16-F10 melanoma model enabling sensitivity to anti-PD-1 immune checkpoint therapy. RNA sequencing of 18 tankyrase inhibitor-treated human melanoma cell lines and B16-F10 cells revealed a transcriptional response profile for a subpopulation. This cell line sub-group displayed elevated baseline YAP signaling activity and was susceptible to reduce melanocyte inducing transcription factor (MITF) expression upon tankyrase inhibition.

Identification of Response Signatures for Tankyrase Inhibitor Treatment in Tumor Cell Lines

“…YAP signaling target gene expression was reduced in all cell lines. Together, these observations are in line with recent reports that TNKSi induced accumulation of nuclear YAP correlating with reduced YAP target gene expression (Kierulf-Vieira et al, 2020;Waaler et al, 2020b). However, the observations are at odds with earlier reports that TNKSi induced a reduction of nuclear YAP leading to reduced YAP target gene expression (Wang et al, 2015(Wang et al, , 2016.…”

“…To evaluate the effect of G007-LK on YAP signaling, the selected cell line panel was first examined by Western blot analysis. Treatment of each cell line with G007-LK stabilized AMOT, AMOTL1, and AMOTL2 proteins in both cytoplasmic and nuclear extracts (Figures 5B and S5A), consistent with earlier reports using HEK293T cells (Wang et al, 2015), Nuclear YAP accumulation was enhanced in UO-31, OVCAR-4, and ABC-1 cells, similar to recent observations (Kierulf-Vieira et al, 2020;Waaler et al, 2020b), while no change in nuclear YAP levels were observed in COLO 320DM or RKO cells. Moreover, cytoplasmic YAP was not affected in any cell lines following TNKSi (Figure S5A).…”

“…Similar to our previous protocol (Waaler et al, 2020b), cells grown on coverslips pre-coated with poly-Llysine (sc-286689, Santa Cruz Biotechnology) were fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich) for 15 minutes at room temperature and permeabilized with 0.1% Triton-X100/PBS (T8787, Sigma-Aldrich, 15 minutes at room temperature). Incubation with primary antibodies (24 hours at 4 C) was followed by incubations with secondary antibodies (1 hour at room temperature), both diluted in PBS with 4% bovine serum albumin.…”

“…The therapeutic effect can be enhanced and broadened by combining tankyrase inhibition (TNKSi) with inhibitors of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), epidermal growth factor receptor, or mitogen-activated protein kinase against colorectal cancer xenografts (Schoumacher et al, 2014;Solberg et al, 2018). Recently, combining TNKSi with antibody-based inhibition of programmed cell death 1 has shown effect in syngeneic melanoma mouse models (Waaler et al, 2020b). TNKS1/2 are members of the poly (ADP-ribose) polymerase (PARP) family of enzymes that control protein turnover and activities by catalyzing the post-translational modification poly-ADP-ribosylation (Haikarainen et al, 2014;Smith et al, 1998).…”

B2M gene expression shapes the immune landscape of lung adenocarcinoma and determines the response to immunotherapy

“…MC38 cells are murine colon carcinoma cells, and syngeneic mice subcutaneously transplanted with these cells are widely used in preclinical immunotherapy studies because they are sensitive to PD‐1 inhibition [36]. On the other hand, the CT26 murine colon carcinoma model [37‐39] and the B16 murine melanoma model [40‐42] are reportedly less sensitive to PD‐1 or PD‐L1 blockade. Interestingly, we found that MC38 cells expressed higher levels of B2M than the other two lines, as indicated by the mean fluorescence intensities of B2M expression in the FACS results (Figure 3h).…”