2830 - Impact of TWIST1 Transactivation Domain towards Oncogene-Induced Senescence Suppression in Non-Small Cell Lung Cancer

A. Lafargue^1,2, H. Wang^3,4, S. Thiruganasambandam³, R. P. Gajula³, A. C. Shetty², Y. Song², B. W. Simons³, T. M. Nguyen^1,3, N. Connis³, D. D. Chowdhury², J. H. Chang¹, D. N. Council¹, K. Taparra³, M. Rezaee⁵, N. Zachara³, Z. S. Morris⁶, C. McFarland⁷, S. A. Abdulkadir⁸, C. L. Hann³, and P. T. Tran^1,3; ¹University of Maryland Baltimore, School of Medicine, Baltimore, MD, ²Department of Radiation Oncology, Division of Translational Radiation Sciences, University of Maryland Baltimore, School of Medicine,, Baltimore, MD, ³Johns Hopkins University, School of Medicine, Baltimore, MD, ⁴GenoImmune Therapeutics, Wuhan, China, ⁵Department of Radiation Oncology and Molecular Radiation Sciences, School of Medicine, Johns Hopkins University, Baltimore, MD, ⁶University of Wisconsin School of Medicine and Public Health, Madison, WI, ⁷Case Western Reserve University, School of Medicine, Cleveland, OH, ⁸Northwestern University Feinberg School of Medicine, Chicago, IL

Purpose/Objective(s): Non-small cell lung carcinoma (NSCLC) is a major cause of cancer mortality. High expression of the epithelial-to-mesenchymal transition transcription factor TWIST1 is strongly associated with metastatic cancers and treatment resistance. Additionally, TWIST1 can upregulate O-GlcNAcylation which (1) is required to suppress fail-safe programs such as oncogene (KRas^G12D)-induced senescence (OIS) to accelerate tumorigenesis in primary NSCLC tumors, and (2) is a potential modulator of DNA repair/radiation response. We hypothesized that the transactivation function of TWIST1 and downstream target programs are critical in the promotion of tumorigenicity and radioresistance. Materials/

Methods: We created a novel genetically engineered mouse model (GEMM) allowing tetracycline-inducible expression in the lung epithelium (via lung specific CCSP-reverse tetracycline transactivator (C)) of KRas^G12D (R) with Twist1^wt (T) or with Twist1^F191G transactivation-null mutant (F). We also created non-cancer Human Bronchial Epithelial Cell (HBEC) co-expressing HRas^G12V oncogene with human TWIST1^wt (HBEC-HRas^G12V-TWIST1^wt) or transactivation-null TWIST1^F187G mutant (HBEC-HRas^G12V-TWIST1^F187G).
Results: CRT mice had shorter tumor-free survival and more aggressive tumors compared to CR/CRF mice indicating that the Twist1 transactivation domain is required for Twist1-dependent tumorigenesis acceleration. Also, Twist1^wt expression promoted radioresistance in cell lines and GEMMs. Contrary to CRT, CRF showed a progressive loss of TWIST1^F191G expression over time suggesting no functionality/no selective advantage. CRT lung tumors had higher proliferation (Ki67) and lower cell-cycle arrest (p16) compared to CR/CRF suggesting that the transactivation domain of Twist1 is important for OIS suppression. Supporting these data, we observed in HBEC that the co-expression of TWIST1^wt could suppress HRas^G12V-induced senescence while TWIST1^F187G mutant could not. HBEC-HRas^G12V-TWIST1^wt also sustained tumorigenic/invasive programs. Interestingly, we observed that O-GlcNAcylation inhibition rescued OIS in HBEC-HRas^G12V-TWIST1^wt while O-GlcNAcylation stimulation in HBEC-HRas^G12V-TWIST1^F187G suppressed OIS. Importantly, TWIST1^wt modulated MYC downstream targets, and MYC activity inhibition in HBEC-HRas^G12V-TWIST1^wt using the novel MYC inhibitor MYCi975 also rescued OIS induction.
Conclusion: Altogether, these results suggest that TWIST1 may suppress OIS via MYC signaling and nominate MYCi975 as a means to activate latent OIS programs. MYC inhibiting strategies could serve as a therapeutic sensitizer for TWIST1-expressing NSCLC. This work and our future studies on TWIST1 on the control of OIS, O-GlcNAcylation, and radioresistance mechanisms may help to identify new potential NSCLC therapeutic strategies.