25th EHA Congress Travel Award 受賞レポート　石尾　崇

名前：石尾 崇【北海道大学大学院医学研究院 血液内科学教室】
発表形式：e-Poster

Title:

Genome-Wide Crispr Library Screening identifies genetic vulnerabilities and therapeutic targets in adult T-cell leukemia/lymphoma

Authors:

Takashi Ishio¹, Sarvesh Kumar², Joji Shimono¹, Yuquan Lin², Emmanuel Bachy², Michael N. Petrus², Yandan Yang², Michiyuki Maeda³, Hideki Goto¹, Satoshi Hashino⁴, Takanori Teshima¹, Thomas A. Waldmann², Louis M. Staudt², Masao Nakagawa¹

Affiliations:

1. Hokkaido University Faculty of Medicine, Sapporo, Japan
2. National Cancer Institute, NIH, Bethesda, United States
3. Institute for Virus Research, Kyoto University, Kyoto, Japan
4. Health Care Center, Hokkaido University, Sapporo, Japan

Abstract:

Background: Adult T-cell leukemia/lymphoma (ATLL) is associated with the retrovirus human T-cell lymphotropic virus type I with dismal prognosis. Previous studies have recently utilized next generation sequencing technology for the identification of mutated genes that may be pivotal in the pathogenesis of ATLL. However, the identification of indispensable genes for the proliferation and/or survival of ATLL cells still remains unknown due to the complexity of genomic/epigenetic alterations in the ATLL genome.

Aims: The aim of this study was to identify previously undescribed genetic vulnerabilities and therapeutic targets in ATLL by a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening.

Methods: Three ATLL cell lines were transduced with lentiviral construct for Cas9 nuclease, and subsequently with lentiviral delivery of the human CRISPR Brunello pooled library (Addgene 73178) of 76,441 single-guide RNAs targeting 19,114 protein-coding genes.

Results: Compared with the control cell lines, 23 essential genes, including novel genes (CDK6, JUNB, STAT3, and CCND2) were identified to be involved in ATLL cell proliferation and/or survival. Among these, CDK6 (cyclin-dependent kinase 6), a serine/threonine kinase that forms heterodimers with D-type cyclins, had the best score. This complex regulates E2F transcription factors through the phosphorylation of Rb, resulting in G1-S transition of the cell cycle. Utilizing publicly available microarray data from patients, we demonstrated the higher expression of CDK6 in ATLL than that of the other subtypes of T-cell lymphomas. In confirmatory experiments, two sgRNAs targeting the coding sequences of CDK6 exhibited strong toxicity in five ATLL cell lines in a temporal fashion, which was mediated by G1 cell arrest and partially through apoptosis. Collectively, the data showed CDK6 is essential for cellular proliferation and survival in ATLL.

We extended our analysis to evaluate the pharmacological inhibition of CDK6 in ATLL cells. CDK4/6 inhibitor Palbociclib was toxic in 11 ATLL cell lines and in four primary ATLL cells but the range of IC50 values were relatively broad (9-6500 nM) among them. In our CRISPR-Cas9 screening, we also identified that the genes in mTOR complex 1 pathway (MTOR, RPTOR, MLST8, EIF4E and RPS6), which function as a nutrient/energy/redox sensor and controller of protein synthesis, were essential for the proliferation and/or survival in the ATLL cell lines. Importantly, the combination of palbociclib with mTOR inhibitor (Everolimus or AZD8055) showed additive toxicity in ATLL cell lines, which significantly decreased in the level of phosphorylated Rb compared to single agent with palbociclib. As a further confirmation of our in vitro findings, treatment of an ATLL cell line-xenograft mouse model with the combination of palbociclib and everolimus showed significant inhibition of tumor growth without systemic toxicity, which resulted in improving overall survival.

Summary/Conclusion: Through the findings of this study, we have provided evidence that CDK6 can serve as a novel therapeutic target for ATLL. We propose that the combination of CDK4/6 and mTOR inhibitor is worth to be evaluated as a therapeutic strategy for ATLL in future clinical study.