Epigenetic Modulation of CD48 By NPM-ALK Promotes Immune Evasion in ALK+ ALCL
Authors: Wu Rui , Ivan Elena , Sahasrabuddhe Anagh Anant , Shaw Timothy , Mullighan Charles G. , Leventaki Vasiliki , Elenitoba-Johnson Kojo S. J. , Lim Megan S. 
Published: November 2019

Background: NPM-ALK is the prototypical oncogenic fusion characteristic of anaplastic lymphoma kinase anaplastic large cell lymphoma (ALK+ ALCL). While NPM-ALK promotes lymphomagenesis through deregulation of multiple of cellular processes, its role in immune evasion is largely unknown. Significantly, 30% of ALK+ ALCL patients develop resistance to current treatment. Therefore, the identification and development of novel immune modulatory therapies remains an opportunity. Using unbiased mass spectrometry-based N-glycoproteomic analysis, we identified preferential loss of proteins (CD48, CD2 and CD244) that regulate cell-mediated target cell lysis in five ALK+ ALCL cell lines compared with other T cell lymphoma-derived cell lines. We hypothesized that NPM-ALK deregulates the expression of key cell surface molecules that function in immune evasion via an epigenetic mechanism to attenuate nature killer (NK) cell-mediated cytotoxicity against neoplastic ALK+ ALCL cells.
Methods: We performed global N-glycoproteomic analysis using a hydrazide-based peptide enrichment chemistry followed by tandem mass spectrometry. We also examined CD48 expression in 5 ALK+ ALCL cell lines by qRT-PCR, flow cytometry and immune-blotting (IB). Immunohistochemistry was used to assess the expression of CD48 in tissue microarray (TMA) of ALK+ ALCL, Peripheral T Cell Lymphoma Not Otherwise Specified (PTCL, NOS) (n=8 and 69 respectively). Cases with more than 15% positive neoplastic cells were scored as positive for CD48. RNA-seq-based gene expression study of 32 ALK+ALCL patients was used to assess the correlation between expression of ALK and CD48, CD2, CD58 and CD244. To assess the role of NPM-ALK in CD48 downregulation in ALK+ ALCL, we stably expressed NPM-ALK in primary CD4+T cells (NA119 and NA149). CD48 promoter methylation was correlated with ALK expression in 27 ALK+ALCL patients by RNA-seq and DNA methylation arrays. The interaction between STAT3 and CD48 promoter was examined by chromatin immunoprecipitation (ChIP). We assessed the effect of Crizotinib (ALK inhibitor), S31-201 (STAT3 inhibitor) and 5-aza-2' deoxycytidine (DNMT1 inhibitor) on the expression of CD48 and attendant NK cell-mediated cytotoxicity using WST-1 assay on ALK+ ALCL cell lines. To assess the functional significance of CD48, ALK+ ALCL cell lines (DEL, Karpas299) were stably transduced with CD48 and NK-cell mediated cytotoxicity was determined.
Results: We confirmed the aberrant loss of CD48 in 5 ALK+ ALCL cell lines through qRT-PCR, flow cytometry and IB. In contrast, activated peripheral blood T cells expressed abundant CD48 by IB. In TMAs, ALK+ ALCL demonstrated significantly higher percentage of CD48 negative cases (62.35%) compared with PTCL, NOS (8.7%, p&lt;0.001). Gene expression analysis of ALK+ALCLs showed a negative correlation between ALK and CD48 (R=-0.5537), CD2 (R=-0.5381), and CD244 (R=-0.4177) but not CD58 (R=0.0064). Ectopic expression of NPM-ALK in primary CD4+T cells led to downregulation of CD48. Inhibition of ALK (Crizotinib), STAT3 (S31-201) as well as DNMT1 (5-aza-2'-deoxycytidine) increased CD48 transcript and protein levels in Crizotinib sensitive (Karpas299, NA149) ALK+ ALCL cell lines. DNA methylation analysis of ALK+ ALCL tissue samples showed that the methylation status of the CD48 promoter (1500bp upstream of transcriptional start site) was positively correlated with ALK levels (R= 0.60). ChIP analysis demonstrated that STAT3 directly binds to the CD48 promoter (at 445-0 bp upstream of CD48 transcriptional starting site). NK cell-mediated cytotoxicity against malignant cells was also increased following ALK (DMSO 22.75% vs Crizotinib 55.9%, p=0.0009) and STAT3 (DMSO 22% vs S31-201 55.1%, p&lt;0.001) inhibition. Exogenous expression of CD48 in DEL and Karpas299 cells led to significant increase in the percentage of NK cell-mediated cytotoxicity (DELEV 20.7% vs DELCD48 32.7%, p&lt;0.0313; Karpas299EV 15.5% vs Karpas299CD48 26.9%, p&lt;0.0055).
Conclusion: Expression of cell surface molecules such as CD48, CD2 and CD244 that function in immune surveillance is negatively regulated by NPM-ALK in a kinase-dependent manner and lead to immune evasion. Further, the loss of CD48 expression in ALK+ ALCL is mediated by epigenetic mechanisms which maybe reversible and have therapeutic significance in ALK+ ALCL.

Disclosures
Mullighan: AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.