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4137 Genetic Determinants of Dependency on Regulated Protein Synthesis in Leukemia Stem Cells

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Myeloid Malignancies, Biological Processes, pathogenesis
Monday, December 11, 2023, 6:00 PM-8:00 PM

Yuanyuan Ji, Ph.D.*, Jebrail Dempsey, B.S.*, Toby Thomas, B.S.*, Jacob Lark, B.S.*, Albert Son, B.S.* and Stephen Chung, M.D.

Division of Hematology/Oncology, Department of Internal Medicine, Children's Research Institute, University of Texas Southwestern, Dallas, TX

Acute myeloid leukemia (AML) is initiated and sustained by rare self-renewing leukemia stem cells (LSCs). LSCs do not resemble HSCs, but rather more mature hematopoietic progenitor cells that usually have no self-renewal potential. However, during leukemogenesis, LSCs become aberrantly self-renewing, but the mechanisms that allow for this are poorly understood. We recently used models of AML driven by AML-ETO9a and MLL-AF9 to show that LSCs may co-opt from HSCs a dependence on regulation of protein synthesis mediated by CD99 (ASH 2021, #2222), which we identified as a therapeutic target highly expressed in LSCs (PMID: 28123069). However, it remains unclear if LSCs from specific subtypes of AML are particularly dependent on regulated protein synthesis. In an analysis of transcriptome data from the ECOG 1900 cohort, we found that certain mutations (KIT, PTEN, AML-ETO, IDH1/2 and FLT3-ITD) were associated with the highest levels of CD99, raising the possibility that such AML subtypes are more dependent on CD99. In the TCGA AML cohort, CD99 expression was significantly higher in AML patients with FLT3-ITD mutations but not NRAS mutations (Fig. 1A).

To test if LSCs from AMLs driven by mutations in FLT3 or NRAS differ in their dependence on regulation of protein synthesis by CD99, we crossed Cd99-/- (CD99 KO) mice with two mouse models of AML driven by these alleles to generate Cd99-/-;Mx1-Cre;Tet2fl/fl;Flt3ITD/+ and Cd99-/-;Mx1-Cre;Tet2fl/fl;NrasLSL-G12D/+ mice (CD99 KO TET2/FLT3 and CD99 KO TET2/NRAS). TET2 was chosen as a cooperating allele given its neutral association with CD99 in the above human datasets, and it additionally co-occurs frequently with mutations in FLT3 or NRAS. To generate AML in both models, Tet2 was deleted with polyinosine-polycytidylic acid injections, followed by transplant of 4 million bone marrow (BM) cells into irradiated wild-type (WT) mice. At 16 weeks, recipients of CD99 WT TET2/FLT3 BM developed an expansion of aberrant donor-derived Mac-1+ c-kit+ cells in the peripheral blood (PB), and BM aspirates revealed an accumulation of LSK CD48+CD150- cells that harbor LSC activity in this model (Fig. 1B). In contrast, recipients of CD99 KO TET2/FLT3 BM exhibited significant depletion of these aberrant populations (P=0.0055 and P=0.019 respectively, n=10 for each group). Recipients of CD99 WT TET2/FLT3 BM also exhibited increased leukocytosis and thrombocytopenia, which were attenuated in recipients of CD99 KO TET2/FLT3 BM (P=0.046 and P=0.042, respectively). In contrast, there was no difference between recipients of CD99 WT TET2/NRAS and CD99 KO TET2/NRAS BM in emergence of the aberrant Mac-1+ c-kit+ cells and thrombocytopenia (P=0.3237 and P=0.9752, respectively, n=10 for each group). These data suggest that loss of CD99 may impair leukemogenesis in TET2/FLT3 but not TET2/NRAS AMLs.

To determine if CD99 is required for LSC function, we performed secondary transplants of one million BM cells from primary recipients into lethally irradiated WT recipients. Mice transplanted with CD99 KO TET2/FLT3 BM, compared with those transplanted with CD99 WT TET2/FLT3 BM, demonstrated fewer donor-derived cells in the PB. While CD99 WT TET2/FLT3-transplanted mice succumbed to AML at a median of 11 weeks, all CD99 KO TET2/FLT3-transplanted mice survived beyond 16 weeks. PB smears from CD99 KO TET2/FLT3 mice demonstrated leukocytes with condensed chromatin and lobulated nuclei, indicative of restored hematopoietic maturation. Finally, in vivo O-propargyl-puromycin assays showed that loss of CD99 resulted in increased protein synthesis in LSCs (LSK CD48+CD150-) (fold-change=1.69±0.18, n=3 for each group). In contrast, mice secondarily transplanted with CD99 KO TET2/NRAS BM exhibited similar engraftment, survival, and morphologic evidence of AML as those transplanted with CD99 WT TET2/NRAS BM.

In conclusion, our data demonstrate that LSC function in AML driven by mutated FLT3, but not mutated NRAS, is dependent on maintenance of regulated protein synthesis by CD99. We speculate that this differential dependency may reflect a different cell-of-origin, as recent work has shown that AMLs associated with FLT3-ITD exhibit particularly primitive cellular hierarchies (PMID: 35618837). Finally, our results suggest that certain high-risk AML sub-types associated with elevated CD99 expression may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs.

Disclosures: No relevant conflicts of interest to declare.

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