3102 – HIGH-THROUGHPUT SCREEN ON PRIMARY HUMAN ACUTE MYELOID LEUKEMIA STEM CELLS (LSCS) IDENTIFIES NOVEL ANTI-LSC COMPOUNDS

Abstract

Acute myeloid leukemia (AML) is an aggressive form of blood cancer. Despite the use of cytotoxic standard-of-care drugs, patients often succumb to the disease partially due to the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies targeting the unique biology of LSCs are needed to improve outcomes. High-throughput screens of human AML LSCs are generally not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. To overcome these challenges, we first optimized the conditions for a 4-week in vitro large-scale expansion (>600 million bulk) and enrichment of the CD34+ LSC-containing fraction (>90% purity) for a primary human AML sample (OCI-AML-8227). Next, we performed a high-throughput screen of 11,140 chemical molecules in 3 stages. First, the viability after treatment of AML CD34+ cells and healthy cord blood (CB) CD34+ cells was read out using a CellTiter Glo assay. 61 compounds had >70% inhibition of 8227 CD34+ cells and < 30% inhibition on CB CD34+ cells. Next, we determined the dose response of each compound and refined the hits to 33 potent compounds with LC50 < 1 μM, including novel compounds and classes previously shown to target bulk and leukemic stem cells in AML. Finally, we determined the LC50 specifically in the CD34- (blast) and CD34+CD38- (LSC-enriched) OCI-AML-8227 populations using flow cytometry. We identified 15 novel anti-LSC compounds with high efficacy against CD34+CD38- AML cells. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs to be able to determine the broad applicability of these compounds. Further, we will determine the mechanisms of action of the top candidates. Acute myeloid leukemia (AML) is an aggressive form of blood cancer. Despite the use of cytotoxic standard-of-care drugs, patients often succumb to the disease partially due to the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies targeting the unique biology of LSCs are needed to improve outcomes. High-throughput screens of human AML LSCs are generally not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. To overcome these challenges, we first optimized the conditions for a 4-week in vitro large-scale expansion (>600 million bulk) and enrichment of the CD34+ LSC-containing fraction (>90% purity) for a primary human AML sample (OCI-AML-8227). Next, we performed a high-throughput screen of 11,140 chemical molecules in 3 stages. First, the viability after treatment of AML CD34+ cells and healthy cord blood (CB) CD34+ cells was read out using a CellTiter Glo assay. 61 compounds had >70% inhibition of 8227 CD34+ cells and < 30% inhibition on CB CD34+ cells. Next, we determined the dose response of each compound and refined the hits to 33 potent compounds with LC50 < 1 μM, including novel compounds and classes previously shown to target bulk and leukemic stem cells in AML. Finally, we determined the LC50 specifically in the CD34- (blast) and CD34+CD38- (LSC-enriched) OCI-AML-8227 populations using flow cytometry. We identified 15 novel anti-LSC compounds with high efficacy against CD34+CD38- AML cells. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs to be able to determine the broad applicability of these compounds. Further, we will determine the mechanisms of action of the top candidates.