Radiation and Cancer Biology

SS 33 - Biology 6 - Radiation Biology and Radiation Sensitizers

244 - In Vivo shRNA Screening Reveals Differential Radiosensitization Within HPV+ and HPV- Head and Neck Squamous Cell Carcinomas

Wednesday, October 24
8:45 AM - 8:55 AM
Location: Room 004

In Vivo shRNA Screening Reveals Differential Radiosensitization Within HPV+ and HPV- Head and Neck Squamous Cell Carcinomas
L. E. Colbert1, M. Kumar2, L. Yang2, D. Molkentine2, K. Bridges2, J. N. Myers3, T. Xie3, M. J. Frederick4, C. Pickering3, and H. D. Skinner2; 1Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 2Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 3Department of Head and Neck Surgery, University of Texas MD Anderson Cancer Center, Houston, TX, 4Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine, Houston, TX

Purpose/Objective(s): Human papillomavirus (HPV) related head and neck squamous cell carcinomas (HNSCC) are known to be exquisitely radiosensitive compared to HPV negative HNSCC. Even within HPV+ HNSCC, there is variable radiation response that is not well understood. The purpose of this analysis was to identify novel targets for radiosensitization in the future.

Materials/Methods: In-vivo screening was performed utilizing a barcoded shRNA library focused upon DNA damage repair containing 342 unique targets. A total of 6 HNSCC cell lines (3 HPV positive and 3 HPV negative) were transduced with the shRNA library and allowed to form subcutaneous flank tumors, which were then treated with 2 Gy/day for 3-5 days depending upon the cell line to achieve an approximate 20% reduction in tumor volume. The tumors were then harvested, barcodes were sequenced and compared to reference cells to identify shRNAs that are enriched or depleted. A modified RSA p-value was used to account for cell line heterogeneity and multiple comparisons. Genes were ranked within cell lines according to radiation sensitivity and RSA p-value. A threshold for hits was set for each individual cell line based on RSA p-value and rank. A stricter RSA p-value and rank threshold was used to compare genes with differential expression between HPV+ and HPV- lines. Paired Wilcoxon test was used to compare RSA p-values for each gene for these between HPV+ and HPV- lines. Unsupervised hierarchical clustering was performed for all cell lines.

Results: 37 overall targets associated with radiosensitization were identified for both HPV+ cell lines and HPV- cell lines. All targets identified within the HPV- lines were accounted for within HPV+ lines, but an additional 17 hits were identified within the HPV+ lines; including UBE2V2, DDB1, RPL13A, RAD51 and RUVBL2. Using the stricter threshold, HPV positive specific top hits included TP53BP1, CDK1 and RFC5 (RSA p-value<-1.25). Gene expression of these targets within the TCGA HNSCC cohort showed higher levels of expression in HPV positive versus HPV negative tumors (CDK1 p=5x10-23, TP53BP1 p=1x10-3 and RFC5 p=4x10-15). Unsupervised clustering of all targets resulted in three unique clusters. Cluster 1 was strongly associated with stronger RSA p-value within HPV positive lines, cluster 2 was associated with stronger RSA p-value within HPV negative lines, and cluster 3 demonstrated similar RSA p-values between cell lines.

Conclusion: Overall, this in vivo screen identified several novel genes associated with increased radiation sensitivity within HPV positive and HPV negative tumors. These serve as unique targets for radiosensitization and prognostication and warrant further pre-clinical and clinical investigation.

Author Disclosure: L.E. Colbert: None. M. Kumar: None. L. Yang: None. D. Molkentine: None. K. Bridges: None. T. Xie: None. M.J. Frederick: None.

Heath Skinner, MD, PhD

Disclosure:
No relationships to disclose.

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