Adult T-cell leukemia/lymphoma (ATLL) is an aggressive hematological malignancy associated with the retrovirus human T-cell lymphotropic virus type I. Clinical outcomes of currently available chemotherapies are generally inferior with extremely poor 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 remains a formidable challenge due to the complexity of genomic/epigenetic alterations in the ATLL genome.
To investigate previously undescribed therapeutic targets in ATLL, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening to identify genetic vulnerabilities in ATLL cells. Three ATLL cell lines were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of the human CRISPR Brunello pooled library (Addgene 73178) of 76,456 single-guide RNAs targeting 19,144 protein-coding genes to cause DNA double-stranded cleavage by the Cas9 nuclease and loss-of-function of the respective genes. Compared with the control cell lines, 23 essential genes, including BATF3 (which we previously discovered by shRNA library screening; Nakagawa et al., Cancer Cell. 34:286-297. 2018) and 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 critical regulatory serine/threonine kinase that forms heterodimers with D-type cyclins, had the best score. The CDK6/D-type cyclin complex regulates E2F transcription factors through the phosphorylation of Rb (retinoblastoma protein), resulting in G1-S transition of the cell cycle.
Utilizing publicly available microarray data from peripheral T-cell lymphoma patients, we demonstrated the higher expression of CDK6 in ATLL than that of the other subtypes of T-cell lymphomas, which prompted us to focus on CDK6 as a therapeutic molecular target in ATLL. 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. We confirmed on-target effect of the sgCDK6 by successfully rescuing cells from toxicity using retroviruses expressing sgRNA-resistant CDK6 cDNA in two ATLL cell lines. The knockout of CDK6 and decrease in the level of phosphorylated Rb were confirmed by immunoblot of sgCDK6-transduced ATLL cell lines. Collectively, the data showed an essential role for CDK6 in cellular proliferation and survival in ATLL.
Of the 19,144 genes examined, CDK6 was considered the best vulnerable target for ATLL; therefore, we extended our analysis to evaluate the pharmacological inhibition of CDK6 in ATLL cells. Palbociclib, FDA-approved CDK4/6 inhibitor for breast cancer, 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 ATLL lines. Because aproximately 20% of ATLL patients carry genetic alteration in a cell cycle/apoptosis regulator TP53 gene, we hypothesized that TP53 alteration may affect the sensitivity of ATLL cells to palbociclib.
First, we assessed TP53 status of ATLL cell lines by Sanger sequencing and immunoblotting and showed that six TP53-altered ATLL cell lines exhibited significantly higher IC50 for palbociclib compared with five TP53-intact ATLL cell lines (p<0.05). Notably, CRISPR-mediated TP53 knockout in a TP53-intact ATLL cell line, which exhibited increased resistance to palbociclib, supported the notion that TP53 is a potential molecular biomarker for palbociclib in ATLL. As a further confirmation of our in vitro findings, treatment of a TP53-intact ATLL cell line-xenograft mouse model with palbociclib showed significant inhibition of tumor growth without systemic toxicity. Through the findings of this study, we have provided evidence that CDK6 can serve as a novel therapeutic target for ATLL. We proposes that CDK6 inhibition with CDK4/6 inhibitor palbociclib, together with the biomarker anaylsis including TP53 status, is worth to be evaluated as a therapeutic strategy for ATLL in future clinical study.
Teshima:Novartis: Honoraria, Research Funding. Waldmann:Bioniz: Membership on an entity's Board of Directors or advisory committees. Staudt:Nanostring: Patents & Royalties. Nakagawa:akeda Pharmaceutical Company Limited: Research Funding.
Asterisk with author names denotes non-ASH members.