Session: 617. Acute Myeloid Leukemia: Biology, Cytogenetics, and Molecular Markers in Diagnosis and Prognosis: Identification of Key Molecules and Pathways in Leukemia Stem Cells and Leukemogenesis
Hematology Disease Topics & Pathways:
AML, Diseases, Animal models, cellular interactions, Biological Processes, Technology and Procedures, Study Population, Clinically relevant, gene editing, Myeloid Malignancies, hematopoiesis, flow cytometry, NGS, molecular interactions
The screen ranked CXCR4 as the most critical cell surface receptor on AML stem cells with all five sgRNAs targeting Cxcr4 depleting more than 4-fold in vivo. Cxcr4 has previously been shown to be critical in several types of cancers including hematological malignancies. Moreover, CXCR4 has been shown to be important for homing of normal and leukemia stem cells in the bone marrow. To validate the role of CXCR4 in a microenvironment and cell intrinsic manner in AML, we disrupted Cxcr4 in c-Kit+ MLL-AF9 AML cells using lentiviral vectors expressing sgRNAs targeting Cxcr4 and GFP as a marker gene. Whereas Cxcr4 disruption did not affect the growth and survival of the AML cells in in vitro cultures with standard cytokines, a 15-fold depletion (p = 0.006) of Cxcr4 sgRNA-expressing AML cells was observed 12 days post-transplantation in vivo. We next studied whether the loss of CXCR4 on the surface of leukemic cells would translate into a survival difference in vivo. Notably, the mice receiving sorted leukemia cells expressing Cxcr4 sgRNAs did not develop AML, demonstrating that CXCR4 is essential for LSCs in vivo. To investigate if this effect was due to a homing defect, we transplanted Cxcr4 sgRNA-expressing cells into sublethally irradiated mice and measured the number of AML cells in the bone marrow after 24 hours. A 2.5-fold reduction (p = 0.03) of leukemia cells expressing Cxcr4 sgRNA compared to the control sgRNA was observed in the bone marrow, suggesting that the depletion of leukemic cells in vivo was partially due to a homing defect.
CXCL12, the most studied ligand of CXCR4, is a homeostatic chemokine widely expressed by several cell types in the bone marrow and exists both in a membrane and soluble form. Although the CXCR4-CXCL12 interaction is critical for the retention of normal hematopoietic stem and progenitor cells in the bone marrow, the role of CXCL12 in AML is less characterised. To study the role of different CXCL12-expressing bone marrow cell populations in AML development, we first transplanted c-Kit+ MLL-AF9 leukemia cells into Cxcl12f/f-Tek-Cre+ mice, which are devoid of Cxcl12 expression in endothelial cells, and in Cxcl12f/f–Prx1-Cre+ mice, which lack expression of Cxcl12 in mesenchymal stem cells. No reduction in leukemia levels in the blood and bone marrow was observed with either of these mouse strains as recipients, indicating that Cxcl12 expression by endothelial cells and MSC is not critical for AML development. Next, we repeated the experiment using Cxcl12f/f-Ubc-Cre+ mice, which lack Cxcl12 expression in all cells. Intriguingly, there was no reduction in leukemia levels in these mice. Instead, a significant 2-fold increase in leukemia levels in the peripheral blood (p=0.011) and a 1.5-fold increase in leukemia levels in the bone marrow (p=0.043) were observed 12 days after transplantation. These findings suggest that the CXCR4-CXCL12 interaction is dispensable for AML development and progression.
Taken together, we here established an in vivo pooled CRISPR/Cas9 drop-out screen using murine LSCs and identified Cxcr4 as an essential regulator for LSCs. Further validations identified that CXCR4 has a previously uncharacterized CXCL12-independent essential role for MLL-AF9 AML cells. Ongoing studies are aimed at identifying the ligand that is essential for CXCR4 in AML.
Disclosures: Ebert: Celgene: Research Funding; GRAIL: Consultancy.
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