Summary Patient-derived xenografts (PDXs) are the most valuable tool for preclinical drug testing because they retain the genetic diversity and phenotypic heterogeneity of the original tumor. Acute myeloid leukemia (AML) remains difficult to engraft in immunodeficient mice. This is particularly true for long-term frozen patient specimens. This protocol is designed to establish PDXs of human AML with improved engraftment rates. The optimized approach increases the viability of patient cells before implantation, efficiently monitors in vivo engraftment, and maximizes bone marrow collection. For complete details on the use and execution of this protocol, please refer to Salik et al. (2020) and Lynch et al. (2019) .

Acute myeloid leukemia (AML) is a heterogenous malignancy, where the persistence of chemo-resistant leukemia stem cells (LSCs) contributes to disease relapse. We have previously demonstrated the clinical significance of WNT/β-catenin signaling in driving AML LSCs (Science, 327:1650-1653, 2010; Cancer Cell, 38:1-16, 2020). In this study, we uncover that GADD45a (growth arrest and DNA-damage inducible protein) is an essential regulator of β-catenin signaling pathway and its loss promotes LSC function and leukemia progression. Transgenic knockout of Gadd45a led to a progressive increase in aberrant self-renewal and leukemogenesis in vivo. Gadd45a-/- leukemic cells developed a more aggressive leukemia with a shorter latency than Gadd45a+/+ cells in mice, indicating the involvement of Gadd45a loss in AML initiation and progression. Subsequent serial transplantation experiments showed that Gadd45a deletion enhanced LSC self-renewal in vivo. In agreement with our findings in murine LSCs, deletion of GADD45a by CRISPR/Cas9 in AML patient-derived xenograft (PDX) cells revealed increased engraftment and tumor burden in NSG mice. Consistent with our phenotypic observations, knockout of GADD45a increased βcatenin activity and key WNT/self-renewal target genes in human AML cells. In addition, our cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) data showed that GADD45a deletion in patient-derived LSCs was associated with cell metabolism, reactive oxygen species and tumor progression, as well as poor patient outcomes in AML. Further studies are being conducted to evaluate transcriptional mechanisms discovered by our single-cell sequencing. Taken together, this study is the first to demonstrate that GADD45a loss promotes LSC potential and consequently enhances tumor growth in murine and PDX models of AML, thus showcasing GADD45a as a promising therapeutic target in AML. References: Wang Y, Krivtsov AV, Sinha AU, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327:1650-1653. Salik B, Yi H, Hassan N, et al. Targeting RSPO-LGR4 signaling for leukemia stem cell eradication in acute myeloid leukemia. Cancer Cell. 2020; 38:1-16. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a difficult-to-treat blood cancer. A major challenge in treating patients with AML is relapse, which is caused by the persistence of leukemia stem cells (LSCs). Self-renewal is a defining property of LSCs and its deregulation is crucial for re-initiating a new leukemia after chemotherapy. Emerging therapeutic agents inhibiting aberrant self-renewal pathways, such as anti-RSPO3 monoclonal antibody discovered in our recent study, present significant clinical potential that may extend beyond the scope of leukemogenesis. In this chapter, we provide an overview of normal and malignant hematopoietic stem cells, discuss current treatments and limitations, and review key self-renewal pathways and potential therapeutic opportunities in AML.