Selective Targeting of Leukemic Over Normal Stem Cells by the Serotonin Receptor Antagonist SB-216641

• Published in: Blood 2011-11, Vol.118 (21), p.1884-1884
• Main Authors: Kahn, Alissa R., Hartwell, Kimberly A., Miller, Peter G., Ebert, Benjamin L., Golub, Todd R., Moore, Malcolm A.S.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V118.21.1884.1884

Abstract 1884 Acute myeloid leukemia (AML) is a common and aggressive hematologic malignancy affecting both children and adults which continues to have high mortality rates as well as high morbidity from toxic therapies. New treatments are needed to improve cure rates and decrease morbidity. A niche-based high throughput screen done in a murine system identified candidate small molecules potentially toxic to leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell KA, Miller, PG et al., in preparation). One such compound, SB-216641, demonstrated dose-dependent activity against leukemia in both a cell autonomous and non-autonomous manner, by modifying niche–based support. SB-216641 is a selective serotonin receptor antagonist specific for the 5-HT1B receptor, highlighting a pathway not previously investigated in the context of AML or leukemia stem cell biology. We examined the effects of this candidate small molecule on 7 human primary AML samples. CD34+ cells were isolated from these samples with immunomagnetic beads. Using the colony forming assay to assess kill of progenitor cells, all samples had ≥99% cell kill at 25 μM (10 times the IC-50 found in the murine system). We then assessed the compound’s effect on LSCs using the cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. The leukemic cells were pulse treated for 18 hours and washed to remove residual SB-216641 prior to placement on MS-5 murine stroma and therefore only the direct effect on the leukemic cells was measured in this assay. CAFCs were read out at week 5, or week 2 when the sample was FLT3-ITD+ (Chung KY et al, Blood 2005, Vol 105, 77–84). We first tested five samples at 25 μM. All samples formed cobblestone areas in the control setting (46–200 CAFCs/106 cells plated). Four samples had no CAFC formation with SB-216641 and the remaining sample had >95% decrease in CAFC formation. We then performed serial dilutions using the CAFC assay in the human primary samples as well as in HSCs derived from cord blood to obtain the IC-50 for human AML and to ensure that our differential cell kill of LSCs versus normal HSCs held true in the human samples. IC-50 for the human primary leukemias was found to be 630 nanomolar and at 10 μM all leukemic samples were fully killed with 100% survival of normal human HSCs [see figure 1]. As a confirmatory study, using HL60 and U937 human AML cell lines transduced with GFP-lucifer