Unraveling Subcellular Localization of CD33 Splicing Polymorphism Variant
Authors: Shastri Vivek M. , Hylkema Tiffany A. , Gbadamosi Mohammed O , Meshinchi Soheil , Lamba Jatinder K. 
Published: November 2021

Abstract
Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcome. The standard AML therapy has been intense chemotherapy and stem-cell transplantation (SCT). However, the addition of targeted immunotherapeutic agents such as gemtuzumab ozogamicin (GO) to this regimen has shown promise in trials, such as AAML03P1 (NCT00070174) and AAML0531 (NCT00372593) (Cooper et al., 2012; Gamis et al., 2014). Since these trials, GO has been added to the National Comprehensive Cancer Network (NCCN) practice guidelines for use in AML therapy for newly diagnosed and relapse patients, both in combination with chemotherapy and as monotherapy (NCCN., 2018). GO is a humanized monoclonal antibody conjugated to calicheamicin and targets the cell surface antigen CD33, observed on most AML blasts (Bernstein, 2002; Walter et al., 2012). Upon binding to GO, CD33 is internalized and the subsequent release of calicheamicin mediates cytotoxicity. CD33 is a member of the SIGLEC family containing two extracellular domains: IgV and IgC (Andrews et al., 1983). The IgV domain is coded by CD33 exon 2 and is the binding site for GO. A modulatory splicing single nucleotide polymorphism (SNP) rs12459419 (C&gt;T; A14V) in exon 2 results in a shorter isoform (CD33-D2) and has been previously reported to be significantly associated with CD33 abundance on cell surface as well as clinical benefit from GO addition to standard chemotherapy. Patients with CC genotype (~50% of the study cohort) expressed full length CD33 (CD33-FL) and benefitted significantly from GO. However, heterozygous CT patients did not derive the same benefit and were shown to have intermediate CD33 levels (Lamba et al., 2017). The biological rationale for this discrepancy is poorly understood, with a major barrier being the lack of antibodies binding the IgC domain, and by extension the only extracellular domain of CD33-D2. To address this, we generated novel antibodies directed against the IgC domain and have recently shown that CD33-D2 is variably expressed on the surface of AML cell lines as well as primary cells (Gbadamosi et al., 2021). In this study, we have further explored the subcellular localization of both CD33-D2 and CD33-FL. Using pMXs retroviral particles encoding CD33-FL or CD33-D2, we engineered CRSIPR/Cas mediated CD33 KO AML cell lines to overexpress either of the two CD33 isoforms. Cells were then screened and confirmed for expression using either P67.6 or HL2541, which recognizes CD33-D2 (Fig. 1). We used immunofluorescence microscopy to determine subcellular localization of CD33 isoforms in these cell lines with the following organelles and their specific markers: endoplasmic reticulum (calreticulin), peroxisomes (PMP70), or lysosomes (LAMP1). Maximum projections from Z-stacks show that both CD33-FL and CD33-D2 localize only to peroxisomes in AML cells under normal, unperturbed conditions (Fig. 2). With this, our group has shown the cell surface and subcellular localization of CD33-D2 for the first time in AML cells. Ongoing studies are focused on determining the differences in CD33 glycosylation patterns as well cytokine signaling in AML cells expressing these variants. Our finding adds to the growing knowledge of CD33-D2 biology and sets the platform for further functional studies to gain mechanistic insights into its role in response to AML immunotherapy. The study was supported by Leukemia Lymphoma Society and St Baldrick's Foundation.
Figure 1 Figure 1.

Disclosures
Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company.