A Novel Aggressive NK Cell Leukemia Cell Line and Mouse Model for Evaluation of Therapeutic Candidates
Authors: Zhao Xiaoxian , Astbury Caroline , Lin Feng , Phillips Darren C. , Hsi Eric D. 
Published: November 2021

Introduction: Aggressive nature killer-cell leukemia (ANKL) is a rare, highly lethal and disseminated malignancy derived from NK cells. Patients are most commonly young to middle aged adults who present with constitutional symptoms and often have organomegaly, hemophagocytosis, and multiorgan failure. Most cases are refractory to therapy and the median survival is less than 2 months despite multiagent chemotherapy. Development model systems and novel targeted therapeutic agents remains an urgent issue. In this study we established a cell line and a NOD-scid IL2Rgammanull(NSG) mouse model of ANKL, which represented the primary malignant cells. We characterized the molecular features of this model and explored new targeted agents against it.

Methods: Primary tumor cells were cultured ex vivo in the presence of human IL-2 and then injected into a NSG mouse. Flow cytometry, immunohistochemistry (IHC), CISH-EBER were used to confirm the engraftment and to compare the pathologic features of engrafted tumor cells with the primary sample. Karyotyping and oligonucleotide/single nucleotide polymorphism microarray analyses were performed to compare the established cell line CCANKL with primary tumor cells. Selective inhibitors targeting anti-apoptotic BCL-2 family members and a CD6-targeted antibody drug conjugate (ADC) were tested for their treatment efficacies in this model.

Results: Leukemic peripheral blood from a 31-year old woman with relapsed ANKL demonstrated a hyperleukocytosis in which NK cells accounted for 88% of cells. Immunophenotyping showed the malignant cells expressed CD2, 7, 38, 45 and 56, and lacked CD3, 4, 5, 8, 10, 11b, 13, 14, 16, 19, 20, 22, 33, 34, 57, 64, 65, 117, HLADR and Kappa or Lambda immunoglobulin light chain. IHC of an involved lymph node showed the presence of EBV (EBER in situ hybridization) and a proliferative index of 70%. Isolated PBMC were cultured ex vivo and serially passaged as a cell line (CCANKL) in the presence of human IL2. CCANKL cells successfully engrafted in NSG mice and displayed splenomegaly. IHC and Immunophenotyping data confirmed both CCANKL cells and engrafted cells in NSG mice represented primary tumor cells. Both the primary PBMC and CCANKL cells had similar, abnormal karyotypes of 46,XX,dup(1)(q21q42), del(6)(q15q25)[1]/46,idem,der(20)t(7;20)(q11.2;q13.1)[9] and 46,XX,del(6)(q15q25),der(20)t(7;20)(q11.2;q13.1),add(22)(p11.1)[10], respectively. Microarray analysis of both primary tumor cells and CCANKL confirmed some of the karyotypic findings, but also revealed additional abnormalities, in particular for the CCANKL cell line. There were also some differences for the copy number changes and regions of loss of heterozygosity observed between the primary tumor cells and CCANKL. Abnormalities involving 1q, 6q and 7p have been reported in association with aggressive NK cell leukemia.

Both immunoblotting and IHC revealed expression of anti-apoptotic proteins of BCL-xL, MCL-1 and pro-apoptotic BIM in CCANKL cells and NSG-engrafted tumors, while BCL-2 was positive in cultured cells by Western blotting but not detectable in engrafted tissue via IHC. Expression level of BCL-xl was stable but there were enhanced expression of MCL-1 and BIM during 72-hour starvation of IL2 in cultured CCANKL cells. Single agent of venetoclax, BCL-xl inhibitor (A-1331852), and CDK9 inhibitor (A-1592668), at range of 1 - 1000 nM, had no detectable (BCL-2), mild (BCL-xL), and strong (CDK9) killing activities against CCANKL cells.

We further confirmed expression of CD6, a surface glycoprotein expressed on most T-cells and a subset of NK cells, by IHC and flow cytometry in CCANKL and also tested a novel CD6-targeted ADC. Compared to naked anti-CD6 IgG and isotype control, CD6-ADC was capable of inducing apoptosis in CCANKL cells. Administration of either anti-CD6 IgG or CD6-ADC (4 mg/kg) on the same day as inoculation of CCANKL cells in NSG mice blocked engraftment of these malignant cells in NSG mice. The 2 nd ANKL case tested by IHC showed positive of CD6 in malignant cells.

Conclusion: A novel aggressive NK cell leukemia cell line and a NSG mouse model were established. The in vitro data support further investigation of the expression features of BCL-2 family proteins in ANKL cases and in vivo evaluation for the efficiency of anti-apoptotic protein inhibitors in this aggressive disease. Further studies targeting CD6 in ANKL is warranted.

Disclosures

Lin: Takeda Pharma: Consultancy, Research Funding. Phillips: AbbVie Inc.: Current Employment, Current equity holder in publicly-traded company. Hsi: AbbVie Inc, Eli Lilly: Research Funding.