Specific PML Isoforms and SATB1 Affect HLA Expression In Classical Hodgkin Lymphoma
Authors: Liu Yuxuan , Van Den Berg Anke , Rutgers Bea , Veenstra Rianne , de Jong Debora , Kortman Gertrud , van Imhoff Gustaaf , Visser Lydia , Diepstra Arjan 
Published: October 2019

Abstract

Introduction
Hodgkin Reed-Sternberg (HRS) cells are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I and class II are expressed in most EBV+ cHL cases, with an even enhanced expression in some cases. The mechanisms underlying enhanced or lost HLA expression are unknown. Promyelocytic leukemia protein (PML) is the main component of nuclear bodies (NBs) and it organizes the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. The PML transcript can be processed into seven different isoforms. Downregulation of PML isoform III or V results in a reduced HLA-A and HLA-G expression in Jurkat T cells, whereas PML enhances HLA class II expression by upregulating the class II transactivator (CIITA). Special AT-rich region binding protein 1 (SATB1) has been shown to be associated with PML-NBs in the HLA region. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G and HLA-H.

Aim
To determine if specific PML isoforms and SATB1 regulate HLA expression in cHL cell lines.

Methods
We stained HLA class I, HLA class II, PML-NBs and SATB1 in 54 EBV+ cHL cases and in 27 EBV- cHL cases. We analyzed HLA class I, HLA class II, PML isoforms I to V and SATB1 mRNA expression levels by qRT-PCR in cHL cell lines (n=6). Next, we inhibited SATB1 and PML expression in the cHL cell line L1236 using shRNA constructs. The effects of the shRNA constructs on HLA protein expression levels were assessed by Western blot and flow cytometry.

Results
We observed a normal HLA class I membrane expression in 40% of EBV+ and 20% of EBV- cases. There was a stronger than normal HLA class I protein expression in approximately 40% of EBV+ cHL and not in EBV- cHL patients. The number of PML-NBs was positively correlated to the level of HLA class I expression (p<0.01), whereas the percentage of SATB1 positive cells was inversely correlated with the level of HLA class I expression in EBV+ cHL cases (p<0.05). We observed normal HLA class II expression in 50% of EBV+ and 50% of EBV- cases. A stronger than normal HLA class II expression was observed in 4 EBV+ and 1 EBV- cHL patients. The number of PML-NBs and the percentage of SATB1 positive cells were not correlated with classical HLA class II expression in EBV+, EBV- or the total cHL patient group.
We further investigate the association of PML, SATB1 and HLA class I and II in six cell lines. At the mRNA level, significant positive correlations were found between PML-III and HLA-C and between PML-III and β2M. Borderline significant positive correlations were found between PML-V and CIITA and between PML-V and HLA-DPB1. Suggestive, though not significant, correlations were observed of various PML isoforms and SATB1 for several HLA genes. The limited number of cell lines, precluded more definitive conclusions for the other comparisons.
To further study the role of specific PML isoforms and SATB1 in regulation of HLA gene expression, we inhibited PML-III, PML-V or SATB1 in L1236 by shRNA-based knockdown. The PML-III shRNA induced a ∼80% reduction at the PML-III mRNA level, but did not affect membranous HLA class I expression and possibly affected HLA class II expression levels in L1236. The shRNA for PML-V was not effective and needs to be optimized and experiments with a pan-PML shRNA are ongoing. The SATB1 shRNA1 induced a ∼75% at the SATB1 mRNA level and a ∼80% reduction at the protein level, while SATB1 shRNA2 showed a ∼75% reduction and a ∼25% reduction, respectively. Inhibition of SATB1 by shRNA1 significantly upregulated HLA class II expression (P=0.02) and no significant effect on HLA class I expression. Inhibition of SATB1 by shRNA2 showed no significant effect on HLA class I and II expression.

Conclusion
PML and SATB1 protein levels are associated with HLA class I in HRS cells. Various negative associations of SATB1 with HLA genes at the mRNA level support a possible regulatory effect. This was indeed observed upon inhibition of SATB1 in L1236 that induced enhanced HLA-DR/DP/DQ expression. The PML regulation appears to be complex with different PML isoforms showing different associations with specific HLA genes.

Disclosures:
No relevant conflicts of interest to declare.