Abstract
BackgroundThe aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle.MethodsRaji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization.ResultsWe found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair.ConclusionsAs a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.
Highlights
The aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies
As a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy
These results indicate that HPRT has substantial presence on the surface of Raji cells and has insignificant presence on the surface of their normal counterparts
Summary
The aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. A disadvantage of utilizing this biomarker target is that patients’ healthy B cell populations decrease because CD19 is not specific to cancer cells. Another disadvantage of targeting CD19 is that some tumors experience antigen loss which confers resistance to CD-19-targeted immunotherapy, and approximately 10–20% of patients relapse following treatment with CD19-CAR therapy [19, 20]. To aid in reducing antigen loss, researchers seek to identify new immunotherapy biomarkers that can be targeted to eliminate B cell malignancies. New targets such as CD22, CD20, and ROR1 have all shown promise in eliminating certain B cell malignancies, but further research is needed to expand targetable antigens on the surface of malignant cells [21,22,23,24]
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