Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster I
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Myeloid Malignancies
Methods: Either one or both CD33 isoforms were overexpressed in a CD33 KO background in THP-1, HL-60, and Kasumi-1 cell lines. Fluorescent-labeled antibody bound CD33 internalization was measured via flow cytometry and live cell imaging over a 2-4 hour duration. Cell viability post GO treatment was assessed using CellTiter-Glo 2.0. All animal experiments were carried out in accordance to the guidelines of the Institutional Animal Care and Use Committee (IACUC). NSG-SGM3 (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ) mice were treated with 1 Gy radiation 24h before transplant as a preconditioning regime and injected with HL60 cells overexpressing CD33 D2 via tail vein. 3-4 weeks post-transplant, these mice were injected with HL2451-PE antibody and in vivo targeting to CD33-D2 in bone marrow cells was assessed by flow cytometry 1 h later.
Results: While P67.6-bound-CD33-FL internalized as expected in AML cell lines expressing CD33-FL only, we show for the first time that HL2541-bound-CD33-D2 also internalizes upon antibody binding in cell lines expressing CD33-D2 isoform only. In all AML cell lines co-expressing both CD33-FL and CD33-D2 isoforms, a >2.5-3 fold decrease in this antibody-bound internalization was observed for each isoform. In response to GO exposure for 48 hours, cells co-expressing both CD33-FL and CD33-D2 isoforms had 7-9.5-fold higher IC50values (233 nmol and 277 nmol) compared to cells expressing only CD33-FL (33.19 nmol and 29 nmol). As expected, cells expressing CD33-D2 only did respond to GO. HL2451-PE detected CD33 in vivo in femur bone marrow cells from NSG-SGM3 mice transplanted with HL-60 CD33D2, but not from mice injected with PBS only. PE-stained cells were also detected in humerus, tibia, L spine and extramedullary tissues like spleen and tumors/chloromas from lower abdomen tissue, suggesting localization of CD33-D2 not only in skeletal system but also in extramedullary disease.
Discussion: Our findings that CD33-D2 internalizes upon antibody binding in vitro and that CD33 internalization is compromised in a system mimicking heterozygous expression provide a mechanistic insight on the previous clinical observation showing lack of benefit from GO in patients heterozygous for the splicing SNP. Additioanlly, we have also established for the first time a CD33 D2 AML mouse model to show that the disease develops first in the bones before progressing and entering blood to spreads to other tissues. Ongoing and future studies are also focused on the development of novel imaging modalities like CD33-D2 isoform immunoPET imaging to determine treatment outcomes and disease relapse non-invasively.
The study was supported by Leukemia Lymphoma Society, UFHealth Cancer Center, and the Frank A. Duckworth Endowment.
Disclosures: No relevant conflicts of interest to declare.
See more of: Oral and Poster Abstracts