2038

Signal regulatory protein-α (SIRPα) is a transmembrane receptor selectively expressed on myeloid and neuronal cells. The broadly expressed cell surface CD47 molecule acts as a major extracellular ligand. The SIRPα cytoplasmic tail contains ITIM motifs that upon CD47 binding mediate the recruitment and activation of the cytosolic tyrosine phosphatases SHP-1 and SHP-2. SIRPα signaling negatively regulates many signaling pathways leading to reduced tumor migration, survival and cell transformation. Indirect evidence has suggested that SIRPα may be downregulated in acute myeloid leukemic (AML).

We studied SIRPα mRNA levels in a micro-array data set of 285 AML samples and found a strong association with AML subtypes. (Minimal/poorly) differentiated myeloblastic and promyelocytic AMLs expressed low SIRPα mRNA levels. mRNA expression was higher in myelomonocytic/monocytic AMLs (comparable to normal bone marrow). These findings were confirmed by Western blotting on representative AML cell lines and 25 AML patient samples. SIRPα protein expression was low in AML cell lines KG1a (FAB M0) and Kasumi-1(FAB M2), while HL60 (FAB M3), U937 and THP1 (FAB M5) cells expressed higher protein levels. In the patient samples, SIRPα protein expression was absent/low in FAB M1/2 cases and high in FAB4/5 cases.

We investigated whether SIRPα down-regulation in specific AML subsets, such as t(8;21) FAB M2 AML, resulted from epigenetic promoter hypermethylation. Although exposure to demethylating agents and HDAC inhibitors did increase SIRPα expression in t(8;21) Kasumi-1 cells, only limited methylation of the SIRPα gene promoter in these cells and primary t(8;21) AML patient samples was observed.

In addition, we studied the functional significance of SIRPα by retroviral reconstitution of Kasumi-1 cells and incubation with an agonistic SIRPα antibody (ED9; 10 μg/ml for 7 days). Daily cell counting and annexin V/7-AAD FACS staining demonstrated significant growth inhibition (p=0.02) and promoted apoptosis (p=0.005). No effect was seen in Kasumi-1 empty vector control cells. Finally, we determined the efficacy of ED9 in combination with 6 concentrations of cytarabine (ara-C; 0.625-0.00061 μg/ml), daunorubicin (DNR; 2.56-0.0025 μg/ml) and etoposide (VP16; 10-0.0098 μg/ml) in these Kasumi-1 cells, by 4-day MTT assay. Calcusyn analysis demonstrated synergism between ED9 and ara-C (CI=0.46±0.32), DNR (CI=0.74±0.06) and VP16 (CI=0.60±0.05).

In conclusion, SIRPα expression appears to be reduced in specific AML subsets. SIRPα-derived signals can directly control myeloid cell growth and induce apoptosis in AML M2 t(8;21) Kasumi-1 cells. Moreover, agonistic SIRPα triggering synergized with conventional chemotherapeutic agents. Our results create a rational basis for the design of anti-leukemic therapies targeting SIRPα.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA