Whole Transcriptome Sequencing Identifies Novel Pathways Associated with Paroxysmal Nocturnal Hemoglobinuria- Increased CXCR2 Expression in PNH Granulocytes
Authors: Hosokawa Kohei , Kajigaya Sachiko , Keyvanfar Keyvan , Townsley Danielle M. , Dumitriu Bogdan , Desierto Marie , Rios Olga , Weinstein Barbara , Feng Xingmin , Young Neal S. 
Published: October 2019

Abstract
Background. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder that arises from hematopoietic stem cells (HSCs). PNH is caused by a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A), responsible for a deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). PNH is a clonal disease that originates from HSCs, as the originating PIGA mutation is present in cells of multiple lineages, including myeloid, erythroid, and lymphoid cells. However, a critical question regarding PNH that has yet to be fully explained despite several decades of research is the mechanism responsible for clonal expansion of PIGA -mutant cells in bone marrow failure. Using RNA-seq, we identify pathways, coding and non-coding RNA transcripts, splice variants, or single nucleotide variants and other alterations that may relate to the selective advantage of PNH clone.
Method. Blood samples were obtained after informed consent from patients with 14 PNH and 18 age-matched healthy donors. From PNH patients and healthy donors, 4 samples were used for RNA sequencing 6 samples were used for validation by flow cytometry. The liquid FLAER method was used for the detection of PNH-type granulocytes. For RNA extraction, granulocytes were sorted for CD11b+ FLAER+ granulocytes, CD11b+ FLAER- granulocytes. For bone marrow staining, cells not expressing lineage markers were separated into five subpopulations: Long-term hematopoietic stem cells (LT-HSC; Lin- CD34+ CD38- CD90+), short-term hematopoietic stem cells (ST-HSC; Lin- CD34+ CD38- CD90-), common myeloid progenitors (CMP; Lin- CD34+ CD38+ CD123+ CD45RA-), granulocyte-monocyte progenitors (GMP; Lin- CD34+ CD38+ CD123+ CD45RA+) and megakaryocyte-erythrocyte progenitors (MEP; Lin- CD34+ CD38+ CD123- CD45RA-).
Results and Discussion. First, RNA expression levels in CD11b+ FLAER+ and CD11b+ FLAER- populations of PNH patients were analyzed using RNA sequencing. Expression levels of 7 mRNAs (CSF2RB, ACSL1, FCGR3B, IL1RN, CXCR2, TREM1, and TNFRSR10C) were significantly upregulated (> 3 FC, P < 0.01) in CD11b+ FLAER- cells of PNH patients compared with CD11b+ FLAER+ cells. To validate the differential expression observed in GPI-AP- granulocytes from PNH patients, protein expression levels of CSF2RB, FCGR3B, CXCR2, TREM1, and TNFRSF10C were assessed by flow cytometry. In CD11b+ FLAER- granulocytes of 6 PNH patients, increased expression of CXCR2 was validated, whereas decreased expression of FCGRB and TNFRSF10C were validated compared with CD11b+ FLAER+ granulocytes and that of healthy controls. Low expression FCGRB and TNFRSR10C in CD11b+ FLAER- granulocytes were considered to be reasonable, as these were GPI-APs. Next, we examined whether increased CXCR2 expression in PNH-type cells was validated in different peripheral blood cell populations. Increased CXCR2 expression in PNH-type cells was confirmed in granulocyte and monocyte populations, not in T cell or B cell population. We checked the expression levels of CXCR1 and CXCR2, which are closely related receptors that recognize CXC chemokines. CXCR2 expression was significantly different between normal and PNH-type cells in granulocytes and monocytes, and CXCR1 expression was significant only for granulocytes. To address the difference of CXCR2 expression levels between normal and PNH-type cells in more undifferentiated cells, we next examined the CXCR2 expression levels in bone marrow hematopoietic stem cells. Expression of CXCR2 was weakly expressed in hematopoietic stem cells and progenitors, both in normal and PNH-type cells, suggesting that difference of CXCR2 expression between normal and PNH-type cells is evident only in differentiated myeloid cells, not in hematopoietic stem cells or lymphoid cells.
Conclusion. We provide evidence for increased expression of CXCR2 in PNH-type granulcoytes and monocytes by RNA-seq and flow cytometry. The differential expression of CXCR2 might partly explain the dominance of PNH clones in myeloid cells in patients. CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML (Schinke C, et al, Blood, 2015). Understanding the mechanism of increased CXCR2 expression in PNH-type cells may offer new therapeutic strategies and novel mechanistic insight into the pathophysiology of PNH.

Disclosures
Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.