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Download 1Department of Hematology, 2Department of Dermatology, Leiden
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LOSS OF PIGH EXPRESSION FREQUENTLY RESULTS IN A GPI-NEGATIVE SUBCLONE LACKING CD52 MEMBRANE EXPRESSION, CONFERRING ALEMTUZUMAB RESISTANCE TO B CELL ACUTE LYMPHOBLASTIC LEUKEMIA In a previous study (Nijmeijer et al, 2010), outgrowth of CD52 negative escape variants after alemtuzumab treatment was shown in a mouse model engrafted with human B-ALL. These variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression due to loss of GPI anchor expression The aim of the current study was to further unravel the mechanism underlying the loss of CD52/GPI anchor expression in adult B-ALL GPIneg and GPIpos B-ALL cultures Cell cultures generated from two B-ALL patient samples were phenotypically highly comparable to the original patient sample Patient VBK Patient MHX CD52 PIGH Leiden BVALL Leiden HPALL PBGD • • • Frequency of CD52/GPI anchor negative cells was analyzed in blood and bone marrow samples from treatment naïve B-ALL, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and hairy cell leukemia (HCL) patients and healthy donors by flow cytometry with a fluorescently labeled GPI-specific aerolysin FLAER and CD52 and CD19 counterstaining (detection limit 0.01%) [email protected] CD52/GPI negative cell populations were present in 10 out of 18 B-ALL samples, but not in other B cell malignancies such as CLL (n=5), MCL (n=5), HCL (n=6), or in healthy donors (n=5) KYE9115 GPI positive and GPI negative B-ALL populations were purified by fluorescence activated cell sorting (FACS) and from these populations mRNA was isolated, cDNA synthesized, and expression of the 26 genes that comprise the GPI anchor biosynthesis pathway was analyzed by conventional PCR using specific primers and visualized by gel electrophoresis Two different B-ALL samples were cultured, and subsequently divided using FACS into pure GPI negative and GPI positive subcultures to perform retroviral transduction studies with constructs encoding PIGH, PIGA or an empty vector Flaer (GPI anchor) GPI negative and positive subcultures were used to compare the promoter CpG methylation by bisulfite sequencing. GPI negative subculture was used for a demethylation assay using 5-Azacitidine in a 14 day culturing regimen wtPIGH restores GPI expression Restored GPI anchor expression in the GPI negative B-ALL subcultures upon retroviral transduction with the PIGH construct, but not PIGA or empty vector NGFR (marker) T2A PIGH, PIGA, or empty Construct used for transductions encodes for a markergene coupled to PIGH or PIGA via the autocleaving T2A peptide Flow cytometric analysis of the transduced GPI negative cell subcultures HP-ALL GPIneg BV-ALL GPIneg Example of a flow cytometric analysis of CD52/GPI-anchor negative cells within the CD19+ B cell compartment in a B-ALL sample Sample nr Patient ID Sample type** # B-cells GPI negative screened (%) 1 HRG7504 PB 385,018 4.98 2 MEP6179 PB 274,959 2.14 3 KYE9115 PB 36,783 0.91 4 PGZ0747 BM 769,559 0.65 5 JHO8262 BM 25,465 0.28 6 EBG6102 BM 938,627 0.18 7 HBZ8516 PB 31,171 0.11 8 MHJ8932 PB 331,127 0.04 9 AOR6995 BM 628,534 0.03 10 HBP6230 PB 795,546 0.03 11 LOW9484 BM 40,682 0.00 12 LMJ5903 PB 226,151 0.00 13 ASC5968 PB 103,698 0.00 14 CPH6760 BM 356,142 0.00 15 JWK9219 BM 45,701 0.00 16 MGR9751 PB 21,305 0.00 17 MRQ3306 PB 514,778 0.00 18 PMX8367 BM 674.114 0.00 ** BM, Bone marrow; PB, Peripheral blood Detailed analysis of the GPI negative and GPI positive ALL subcultures showed: • Loss of PIGH mRNA was not caused by genetic aberrations • Higher level of promoter methylation in GPI negative subcultures • Demethylation using azacytidine resulted in GPI anchor reexpression Promoter methylation assay by bisulfite sequencing CpG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 TSS HP GPI+ L L L L L L L L L L L L L L L L HP GPI- L L L M M L M L L L L L L L L L + PIGA MOCK 25 BV GPI+ L L L L L L L L L L L L L L L L BV GPI- L M L M M M M L L L M M L L L L 20 Healthy B-cells U U U U U U U U U U U U U U U U Demethylation assay U Unmethylated 15 10 BV 0.5nM azacytidine 5 0 Flaer L Partially methylated HP 0.5nM azacytidine HP 0nM azacytidine Highly or fully M methylated No PIGH expression in cells Recurrent loss of PIGH mRNA expression, but of none of the other genes involved in GPI anchor biosynthesis, in purified GPI negative B-ALL cells DPM1 DPM2 DPM3 GPAA1 PIGA PIGB PIGC PIGF PIGH PIGK PIGL PIGM PIGN PIGO PIGP PIGQ PIGS PIGT PIGU PIGV PIGW PIGX PIGY PIGZ PGAP1 PGAP2 PGAP3 SL15 GPI: + -- + -EBG 6102 KYE 9115 HRG 7504 MEP 6179 PGZ 0747 JHO 8262 AOR 6995 Example of a B-ALL sample screened for mRNA expression for the 26 GPI genes (n=3) Epigenetic downregulation of PIGH + PIGH GPI positive and negative subcultures were generated from the Leiden-ALL cell cultures by FACS. mRNA expression analysis showed that the GPI negative subcultures also lack expression of PIGH. Flaer B-ALL cells at diagnosis neg GPI DOI: 10.3252/pso.eu.21EHA.2016 In contrast, alemtuzumab which targets the glycophosphatidylinositol (GPI) anchored CD52 protein has shown modest clinical efficacy, which could be caused by the outgrowth of CD52 negative B-ALL escape variants neg GPI CD52 The development of novel antibody based therapies targeting CD19 or CD20 antigens has improved treatment outcomes in B cell acute lymphoblastic leukemia (B-ALL) Inge of Dermatology, Leiden University Medical Center, The Netherlands. Material and methods • Constantijn J.M. 1 Jedema PIGH mRNA expression analysis in B-ALL samples, with GAPDH as a loading control Conclusions In the majority of B-ALL patients CD52/GPI negative cell populations were present at diagnosis These cells lost CD52/GPI expression due to loss of PIGH mRNA expression, a key component in GPI anchor synthesis The CD52/GPI negative phenotype of B-ALL cell subcultures was stable, but could be corrected by enforced PIGH expression by retroviral transduction Loss of PIGH mRNA was not due to DNA mutations, but possibly due to epigenetic down regulation by methylation of the promoter region Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI negative escape variants in B-ALL Inge JEDEMA Background J.H.Frederik 1 Halkes , Poster presented at EHA 21 on: 10th June 2016 of Hematology, 2Department Maarten H. 1 Falkenburg , Acute lymphoblastic leukemia - Biology 2 1Department Wim H. 2 Vermeer , BV 0nM azacytidine 0 5 10 15 162-P H.M. Esther van 2 Zoutman , % GPI positive cells Kevin 1 Egmond , NGFR (marker gene) Floris C 1 Rijs , Time (days) There are no conflicts of interest to disclose 21eha 1 Loeff ,