Identification and Phenotypic Characterization of a Subpopulation of Acute Myelogenous Leukemia (AML) Cells with Increased Plastic Adherence.
Authors: Greenfield Katharine , Mokhtari Saloomeh , Haug Melissa , Porada Christopher D , Almeida-Porada Graca 
Published: October 2019

Abstract
Abstract 2556
The high incidence of relapse in acute myelogenous leukemia (AML) patients has been attributed to the existence of a small population of leukemic stem cells (LSC) that current therapies are unable to eradicate. LSC, in similarity to normal hematopoietic stem cells (HSC), are able to engraft, self-renew, and interact with cells within the bone marrow (BM) niche. During leukemogenesis, changes within the BM microenvironment promote LSC survival and expansion, and shelter leukemic cells from chemotherapy. Therefore, displacing these cells from the BM niches prior to chemotherapy may decrease drug resistance and prevent relapse of the disease. The binding of α4β1 integrins (VLA-4 or CD49d/CD29) to the stromal extracellular matrix and cell surface ligands is a key component in homing and trafficking, specifically in the migration of HSC beneath marrow stromal cells, and VLA-4 has been shown to be crucial for the persistence of minimal residual disease in AML. KG1a is a differentiation-resistant AML cell line containing a very primitive population of CD34+CD38- cells. Upon culture, the majority of KG1a cells remain in suspension, while a small percentage adheres to the tissue culture plastic (Adh-KG1a). Here, we hypothesized that characterization of adhesion molecules that were either unique or significantly altered in this subset could lead to identification of putative therapeutic targets for dislodging AML cells from microenvironmental niches. KG1a and Adh-KG1a populations were evaluated by flow-cytometry for the presence of CD49d and CD29.The FACSort results were then analyzed using FlowJo7.6 software. No statistically significant (p>0.05) differences were found in the percentage of Adh-KG1a and KG1a that expressed CD49d (91.6±3% vs. 86.2±5%) or CD29 (95.0±2% vs. 91.8±1.8%). However, we found that there is a small and unique population of adherent CD29+ cells, with lower fluorescent intensity (MFI=326), that is not present in the non-adherent population (MFI=435), suggesting that this low level of surface expression is unique to the adherent fraction. Analysis of CD11a, CD44, CD18, CD106, CD105, CD34, CD107 and CD38 showed that none of these molecules were expressed at significantly different levels between KG1a and the Adh-KG1a fraction. Addition of anti-CD44 or anti-CD29 antibodies to the cells in culture did not result in decreased numbers of Adh-KG1a cells/flask, but resulted in induction of heterotypic aggregation of Adh-KG1a. Cell cycle analysis did not show significant differences between the 2 populations. However, analysis of CD54 expression demonstrated that 12.2% more of the Adh-KG1a were positive for CD54 than the non-adherent cells. Furthermore, characterization of gangliosides on KG1a and Adh-KG1a showed that the non-adherent fraction contained significantly more GM3 than the Adh-KG1a. Given prior reports showing that: 1) high levels of VLA-4 (CD49d/CD29) are associated with better prognosis in AML; 2) high levels of CD54 correlate with low relapse-free survival probability in AML; and 3) ganglioside composition has been shown to exert a pronounced effect on drug-resistance/sensitivity in AML, our findings of an adherent population of primitive hematopoietic stem/progenitor cells within KG1a that are CD29dim, express high levels of CD54, and contain lower levels of GM3, suggest that this unique subpopulation may play an important role in relapse in AML, and could represent a target for novel therapeutics that could better eradicate the elusive LSC and promise an improved disease-free survival rate in AML.

Disclosures:
No relevant conflicts of interest to declare.