Identification of Compounds That Target Acute Myeloid Leukemia Stem Cells Using a Scalable Next-Generation Screening Platform

The main barrier to curing acute myeloid leukemia (AML) is disease relapse, which occurs due to therapy resistance and persistence of leukemic stem cells (LSCs) after conventional induction chemotherapy. LSCs possess stem cell properties such as quiescence and self-renewal, as well as a transcriptional signature that resembles that of normal hematopoietic stem and progenitor cells. While patient-derived xenograft (PDX) models are the most stringent way to examine the properties of LSCs in human AML, this labor-intensive assay is not amenable to high throughput approaches to identify druggable vulnerabilities.

Although current therapies effectively reduce tumor burden, achieving lasting event-free survival remains a challenge in the management of patients with AML. Current drug discovery efforts are focused on finding agents that eradicate bulk tumor, but not necessarily LSCs. Here, we report a next-generation screening strategy as a novel paradigm that allows the direct elucidation of molecules which antagonize LSC properties. This screening workflow is based on two scalable models of LSCs. The first is the gene expression profile of a core set of 104 genes (LSC104) that are differentially expressed between LSC+ and LSC‒ fractions obtained from primary AML samples and validated in xenotransplantation assays (Ng et al, Nature 2016). This LSC gene expression signature is strongly associated with overall survival and chemotherapy response in independent cohorts of AML patients comprising all subtypes. The second is a continuous AML culture system derived from a patient with relapsed AML that maintains, and allows the prospective enrichment of, a rare population of quiescent, self-renewing LSCs restricted to the CD34+CD38‒ fraction. These LSCs, but not the bulk tumor cells in this system, demonstrate leukemia-initiating capacity both in vitro and in PDX models. Similarly, the LSC fraction of this system, but not the bulk cells, has a LSC104 expression profile that correlates strongly with that of LSC-enriched fractions from patient samples. Together, these components form a clinically-relevant scalable model system to identify compounds with activity against LSC stemness properties, based on alterations in the proportion of LSCs in the continuous AML culture system as well as in the core LSC104 gene expression profile. To this end, we assembled a collection of 1200 curated bioactive small molecules, including 150 metabolic inhibitors and 35 targeted epigenetic probes, and performed a high-dimensional throughput screen using flow-cytometry to examine the effect of these compounds on the LSC-enriched CD34+CD38‒ fraction in our AML culture system. Candidate hits were then tested for their effects on the LSC104 profile. Rigorous analyses unveiled a number of candidate compounds with the potential to antagonize LSC properties, including several already in clinical use or in clinical trials against AML, such as cytarabine, and inhibitors of FLT3, CDK, PLK1, and aurora kinase, as well as several classes of compounds not previously described against AML, targeting NAMPT, BRPF1B, CHK1, and KSP, among others.

In conclusion, we describe a next-generation scalable throughput approach that integrates stem cell biology to uncover modulators of stemness in AML. This multi-parametric screening strategy serves as a platform for the deeper understanding of druggable vulnerabilities of LSCs, and serves as a starting point to achieve lasting event-free survival in AML patients.