Mutational Landscape of Aggressive Natural Killer Cell Leukemia and Drug Sensitivity Profiling Reveal Therapeutic Options in Natural Killer Cell Malignancies
Authors: Dufva Olli , Kankainen Matti , Kelkka Tiina , Awad Shady Adnan , Sekiguchi Nodoka , Yadav Bhagwan , Eldfors Samuli , Kuusanmäki Heikki , Andersson Emma I , Malani Disha , Pietarinen Paavo , Saikko Leena , Kovanen Panu E , Suzumiya Junji , Suzuki Ritsuro , Ko Young Hyeh , Kim Won Seog , Chuang Shih-Sung , Loughran Thomas P. , Chan Wing Chung , Ohshima Koichi , Aittokallio Tero , Ishida Fumihiro , Mustjoki Satu 
Published: October 2019

Abstract
INTRODUCTION
Natural killer (NK) cell malignancies are rare aggressive neoplasms that are classified by the WHO as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK-cell leukemia (ANKL). Recently, genome and exome level studies in NKTCL have shed light on the mutational spectrum of the disease. However, somatic mutations in ANKL have not been characterized. Here, we identified somatic mutations in 14 cases of ANKL to further clarify the genetic landscape underlying malignant NK cell proliferation. We compared the discovered variants to those detected in NKTCL to understand whether the two diseases harbor common molecular alterations. Moreover, we used high-throughput drug screening and RNA sequencing on NK cell lines derived from ANKL, NKTCL and other malignant NK cell proliferations to identify therapeutically actionable drivers of malignant NK cell growth.
METHODS
We performed whole-exome sequencing on genomic DNA extracted from peripheral blood or bone marrow samples of 14 ANKL patients. To compare the mutational profiles in ANKL and NTKCL, we re-analyzed the published whole-exome NKTCL data from Jiang et al. (Nat Genet 2015) using our somatic variant calling pipeline. For profiling of drug responses, we used a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs to screen the ANKL cell lines IMC-1, KHYG-1 and NK-92, NKTCL cell lines NK-YS and SNK-6 as well as DERL-7, KAI3, NKL, YT and IL-2-stimulated NK cells from healthy donors. All drugs were tested in 384-well format in 5 concentrations over a 10,000-fold concentration range for 72 h, cell viability was measured and normalized dose response curve values were used to calculate a drug sensitivity score (DSS) for each drug. Finally, we performed amplicon sequencing of known cancer driver genes and RNA sequencing on the cell lines and healthy NK cells to identify driver mutations and integrate gene expression profiles with drug sensitivity patterns.
RESULTS
We identified recurrent somatic activating mutations in STAT3 in 21% (3 of 14) of ANKL patients. Other mutated genes included RAS-MAPK pathway molecules (BRAF, NRAS, KRAS), protein phosphatases regulating JAK-STAT and PI3K-AKT-mTOR pathways (PTPRT, PTPRK, INPP5D) as well as several epigenetic modifiers (TET2, ARID2, KDM2B, SETD7, SETD2) and the tumor suppressor TP53. Interestingly, we detected mutations in genes recurrently mutated in NKTCL, such as the RNA helicase DDX3X and the cell surface receptor FAS. Re-analysis of the published NKTCL data revealed a high frequency of missense mutations in receptor type and non-receptor type protein phosphatases (e.g. PTPRC, PTPRR, PTPRT, PTPN1, PTPN2, PTPN3), many of which have established roles as negative regulators of JAK-STAT signaling. These findings potentially expand the subset of NK cell tumors where the JAK-STAT pathway is somatically activated and implicate deregulated JAK-STAT signaling as a major driver in these diseases.
The malignant NK cell lines used in drug sensitivity profiling frequently harbored mutations in same genes and pathways, including STAT3 (N=3), STAT5B (N=1), DDX3X (N=2), KRAS (N=1), FAS (N=2) and several epigenetic modifiers, thus validating these cell lines as relevant disease models. The drug sensitivities in NK cell lines showed a high correlation and the cell lines formed a distinct group from other lymphoid and myeloid leukemia cell lines in unsupervised hierarchical clustering, suggesting an NK-cell specific drug response pattern. The most effective targeted drugs across all NK cell lines included HDAC inhibitors, inhibitors of Aurora and Polo-like kinases, JAK inhibitors, HSP inhibitors and CDK inhibitors as well as the Bcl-2 inhibitor navitoclax. Compared to other leukemia and lymphoma cell lines, JAK inhibitors, navitoclax and methotrexate emerged as the most NK-cell specific compounds.
CONCLUSIONS
Our genetic data demonstrate extensive heterogeneity in the mutational spectrum of ANKL and implicate JAK-STAT and RAS-MAPK signaling as well as disruption of epigenetic modifiers in the disease pathogenesis. Integrated drug sensitivity and gene expression profiling corroborates the JAK-STAT pathway as a major therapeutically actionable driver of malignant NK cell proliferation and identifies other potential novel targeted therapy options such as Bcl-2 inhibition in NK cell malignancies.

Disclosures
Suzumiya: Chugai: Honoraria, Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko kirin: Research Funding; Astellas: Research Funding; Eisai: Honoraria, Research Funding; Takeda: Honoraria. Ohshima:Chugai: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.