Deciphering the Role and Therapeutic Potential of Macrophage Marker CD68 in Pediatric Acute Myeloid Leukemia

Introduction:

Pediatric acute myeloid leukemia (pedAML) is a heterogeneous hematological malignancy in childhood with a relapse rate of around 30%. Targeting the micro-environment has shown to exhibit a synergistic therapeutic effect when combined with conventional chemo- or immunotherapy. Leukemia-associated macrophages (LAMs) make the leukemic blasts and stem cells less sensitive to the immune system and to standard therapies. CD68 is a macrophage marker, expressed in LAMs, with a role in clearing cell debris, regulating phagocytosis, and recruiting macrophages. CD68 has also been proposed as a universal monitoring marker of minimal residual disease (MRD) in adult AML and is the most commonly expressed marker in myeloid sarcoma. In this study, we want to investigate the expression of CD68 in (ped)AML and its possible role in the biology of AML. By evaluating the expression in normal hematological counterparts we want to predict hematological toxicity when using CD68-targeted therapy.

Methods:

Using the TARGET database, association analyses of CD68 expression with clinical and molecular AML characteristics were performed, as well as survival analyses using the Kaplan-Meier method and univariate and multivariate Cox regression analyses (including age, sex, white blood cell count at diagnosis, cytogenetic abnormalities, molecular aberrations, and MRD at the end of course 1). Single-cell data of Van Galen et al (2019) were used to compare CD68 expression in malignant to normal hematological cell fractions. CD68 mRNA and protein expression were evaluated in hematological cell lines using RT-qPCR, Western Blot, and flow cytometry. Intracellular and membrane protein expression of CD68 was evaluated in pedAML and cord blood samples through flow cytometry. Knockdown (KD) and overexpression (OE) models were generated in THP-1 (high CD68 expression) and HL-60 (low CD68 expression), and proliferation, chemosensitivity, and cell cycle assays were performed. Through RNA sequencing differentially expressed genes were identified in the KD and OE models and a gene set enrichment analysis was done.

Results:

In silico analyses showed a significant correlation between high CD68 transcript levels and the presence of KMT2A rearrangement and inversion 16. Survival analyses of the TARGET pedAML patient cohort showed a significantly worse overall and event-free survival in patients with higher compared to lower CD68 expression, although not confirmed in multivariate analysis. Analysis of single-cell data of an adult AML patient cohort of Van Galen et al showed higher expression of CD68 in malignant hematopoietic stem cells and progenitor cells compared to their normal counterparts. RT-qPCR, Western blot, and flow cytometry confirmed higher expression of CD68 in AML cell lines compared to other leukemic cell lines. Flow cytometry showed higher intracellular expression of CD68 in 6 out of 8 pedAML samples compared to the cord blood blasts. CD68 KD THP-1 cells showed a decrease in proliferation and higher sensitivity to cytarabine compared to the THP-1 non-targeting control, while CD68 OE HL-60 cells were more resistant and showed a higher percentage of cells in the G1 phase and a lower percentage in the S phase in comparison to the HL-60 mock control. RNA sequencing revealed upregulation of multiple genes in CD68 KD THP-1 cells, suggesting a regulatory role of CD68 on the transcript level, with mostly transcription factors and genes involved in RNA polymerase complexes. CD68 OE showed up and downregulation of genes involved in migration and differentiation, as well as proliferation and apoptosis.

Conclusion:

Macrophage marker CD68 is highly expressed in AML cell lines and in pedAML samples compared to normal counterparts. As our functional analyses suggest a proto-oncogenic role of CD68 and CD68 is expressed in LAMs, targeting CD68 as single or adjuvant therapy might be beneficial for both pediatric and adult AML patients.

No relevant conflicts of interest to declare.