High expression of signal regulatory protein beta 2 marks a favourable prognostic AML subgroup and associates with increased sensitivity to phagocytosis

Abstract

Acute myeloid leukaemia is an aggressive hematologic malignancy that remains exceedingly difficult to treat despite recent advancements. High expression of CD47 on leukemic blasts interacts with the inhibitory receptor SIRP-alpha (SIRP-α) on innate immune cells, resulting in a strong "don't eat me" signal. Therefore, identifying AML patients who could benefit from immune-targeted therapies is crucial SIRP-β2 is predominantly expressed in myeloid cells and positively regulates innate anticancer immunity. Furthermore, endogenously expressed SIRP-β2 potentiates cancer cell trogocytosis by granulocytes. Here, we delineate the role of SIRP-β2 in AML. High expression of SIRP-β2 is independently associated with favorable overall survival (OS) and event free survival (EFS) independent of the ELN intermediate risk group. SIRP-β2 is more prevalent in the more committed FAB M4 and M5 subgroups. SIRP-β2 is also expressed on normal myeloid cells in patient samples, with higher expression on tumor-suppressive M1 macrophages than on adverse prognostic and tumor-supportive M2 macrophages. In line with this, co-culture of macrophages/neutrophils with ectopically expressed SIRP-β2 tumor cells results in an increased phagocytosis/trogocytosis treated with anti-CD47. These data indicate that AML patients with high SIRP-β2 AML expression could significantly benefit from innate immune-targeting therapies such as CD47 immune checkpoint inhibitor.

Keywords: AML; Innate immune targeting; Phagocytosis; SIRP-ß2.

Fig. 1

Expression of CD47 and SIRP-α do not, but SIRP-β2 does correlate with patient’s overall survival. A. SIRP-β2 expression on CD34, NBM and AML of the GSE6891 set (n = 234). B. Kaplan meier plot of the OS and EFS (C) of AML patients with the upper two (SIRP-β2^High) and lower two (SIRP-β2^Low) quartiles (n = 234). D. Multivariate cox regression of OS and EFS (E) on SIRP-β1, SIRP-β2, SIRP-α and CD47. F. SIRP-β2 expression in ELN risk groups: favourable, intermediate and adverse risk groups. G. Kaplan meier plot of the OS in the ELN intermediate risk group of AML patients with the upper two (SIRP-β2^High) and lower two (SIRP-β2^Low) quartiles. H. Up or downregulation of SIRP-β2 expression in various karyotypes and mutations of AML. I. SIRP-β2 expression divided amongst FAB classification (M0-M6). J. Multivariate of OS survival probability in M5 AMLs in CD47, SIRP-α and SIRP-β1. K. Univariate of OS probability in FAB classification. All boxplots compare the individual groups versus the other groups, t-test and Mann Whitney U test where used for parametric and non-parametric statistical analysis. p values are indicated as: ***p < 0.001, **p < 0.01, and *p < 0.05

Fig. 2

Ectopic expression of SIRP-β2 in hematopoietic cancer cell lines augments their macrophage-mediated phagocytic removal upon anti-CD47 antibody treatment. A. Protein expression of CD47, SIRP-α, SIRP-β1 and SIRP-β2 as determined by mass spectrometry analysis of membrane extracts of 44 primary AML samples. B. SIRP-β2 expression on blasts cells and macrophages derived from AML patients (n = 8), determined by flow cytometry. C. SIRP-β2 expression on M1 and M2 macrophages of 6 AML samples, determined by flow cytometry D. Percentages of M1 and M2 on the total macrophage compartment of 6 AML samples, determined by flow cytometry. E. Uniform manifold approximation projection (UMAP) based on flow cytometry data, projecting the different types of macrophages of 6 AML samples. Two UMAPs projecting the florescent intensity of SIRPB2 and CD163 of the macrophages. F. Quantification of SIRP-β2 in overexpressed AML cell lines (U937, THP-1, OCI-AML3 and HL-60), using RT-qPCR. G. Ectopic expression of SIRP-β2 of THP-1, determined by flow cytometry. H. Illustrative images of phagocytosis of THP-1.EV and THP-1.SIRP-β2 cells after 3 h co-cultured with monocyte-derived macrophages, including medium control and co-treatment with B6H12. I. Gating strategy of monocyte-derived macrophages in combination with THP-1.SIRP-β2, using flow cytometry. J. Quantification of monocyte-derived macrophage phagocytosis of THP-1.EV/THP-1.SIRP-β2, OCI-AML3.EV/OCI-AML3.SIRP-β2, HL-60.EV/HL-60.SIRP-β2 and U-937.EV/U-937.SIRP-β2. K. Quantification of granulocyte trogocytosis of THP-1.EV/THP-1.SIRP-β2 and OCI-AML3.EV/OCI-AML3.SIRP-β2. Student’s t-test: p values are indicated as: **p < 0.01, and *p < 0.05