Abstract 5543: TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α
Authors: Miret Juan J. , Luster Troy A. , Lizotte Patrick , Wu Min , Nzikoba Sarah , Taus Luke Taus J. , Gokhale Prafulla C. , Kirschmeier Paul , Barbie David , Paweletz Cloud P. 
Published: September 2020

Abstract
The clinical successes achieved by different immunotherapies have resulted in a paradigm shift in treatment modalities. Despite these significant advances, not all patients benefit from the use of these therapies, creating a need to develop additional approaches to enhance and broaden their clinical application. To identify genes whose products can increase or decrease the sensitivity of tumor cells to the immune system, we used a CTL assay to screen a whole genomic CRISPR library. We co-culture a mouse cell line, ID8, expressing a model antigen (Ova) with transgenic CD8 T cells (OT-I) recognizing this antigen. A set of controls that enhance or decrease CTL activity behaved as expected. Comparison of the CRISPR score identified several hits that increased or decreased the sensitivity of the tumor cells to CTL killing. Subsets of these hits belong to two pathways involved in CTL-mediated killing: the IFN-γ and the TNF-α signaling pathways. We evaluated which of these hits would be amenable to therapeutic modulation, and decided to focus on the kinase TAK1 for confirmation and validation studies. A TAK1 deficient cell line was more sensitive to CTL killing, which was prevented by expression of TAK1, confirming the role of TAK1 in this process. A TAK1 gene carrying an inactivation mutation K63W did not rescue the effects of TAK1 KO, indicating that TAK1 enzymatic activity was necessary. Several pathways mediate CTL killing: Perforin/Granzyme B, IFN-γ, TNF-α, Fas & TRAIL pathways. To determine TAK1 MOA, we studied the effects of a Perforin/Granzyme B inhibitor CMA. CMA inhibited CTL activity in a dose-dependent manner on WT cells, but did not inhibit CTL activity on TAK1 deficient cells, indicating TAK1 effects are independent of this pathway. We then tested the sensitivity of TAK1 KO cells to TNF-α. TAK1 KO cells were more sensitive to TNF-α mediated killing, and similar results were observed with several additional cell lines (MC38, EMT6, KP). TNF-α can activate the JNK, p38, and NF-κB pathways, and the apoptosis extrinsic pathway to regulate cell growth and cell death. Kinetics studies monitoring pathway activity upon TNF-α stimulation showed that TAK1 KO cell lines induced cFLIP degradation before observing PARP cleavage, and that the NF-κB pathway, which has been observed to mediate cFLIP synthesis, was not activated. We proceeded to evaluate the effects of TAK1 deficiency in a mouse syngeneic model. TAK1 deficiency resulted in reduced growth and increased survival in the MC38 in vivo model. In summary, by screening a CRISPR library against a CTL assay, we identified TAK1 as a novel potential target for immunotherapies. TAK1 deficiency enhances CTL killing and results in decreased tumor growth and increased survival in vivo. This results support the development of TAK1 inhibitors to enhance the anti-tumor action of the immune system.
Citation Format: Juan J. Miret, Troy A. Luster, Patrick Lizotte, Min Wu, Sarah Nzikoba, Luke Taus J. Taus, Prafulla C. Gokhale, Paul Kirschmeier, David Barbie, Cloud P. Paweletz. TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5543.