Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
MPN, Chronic Myeloid Malignancies, Diseases, Myeloid Malignancies
We isolated lineage-/low Sca1+c-Kit+ (LSK) cells from BM of donor mice expressing a conditional allele of mutant Jak2 from its endogenous locus (Jak2fl-V617F) in combination with an inducible Cas9-IRES-GFP allele and CreER-fusion allele, both from the Rosa26 locus (Rosa26CreER/lsl-Cas9-IRES-GFP). LSKs were transduced with lentivirus expressing a sgRNA targeting exon 4 of Trp53 (sgTrp53), located in the DNA-binding domain of Trp53. These transfected sgTrp53 LSKs, along with control groups transduced with either empty vector (EV) or a sgRNA targeting Catsper1 (sgCatsper1, off target cutting control), were transplanted into lethally irradiated recipients. Successful engraftment and CreER activity, induced by a 2-week period of Tamoxifen chow, were confirmed by flow cytometry.
Validation of Trp53-editing was performed using amplicon sequencing, revealing the most common mutations were frameshift mutations. A subset of these frameshift mutations exhibited an extremely high frequency, conferring a selective advantage within the cell populations in sgTrp53 mice. We further examined the levels of p53 induced by nutlin-3a (an MDM2 inhibitor) in edited BM cells from three groups of mice and found that Trp53 editing leads to a reduction in p53 levels and prevents the loss of viability in response to MDM2 inhibition. This indicates that CRISPR-Cas9 mediated Trp53 mutations result in a loss of function.
SgTrp53 mice recapitulated the evolution of leukemic transformation of MPN, showing a PV phenotype for 8 weeks post CreER activation, characterized by elevated hematocrit but normal leukocytes and platelet counts, consistent with the phenotype exhibited by the EV and sgCatsper1 recipients. Subsequently, after 12 weeks, this MPN phenotype transformed to acute leukemia. This disease progression was associated with significantly shortened survival of sgTrp53 recipients, compared to the sgCatsper1 or EV control groups. Histopathologic analysis further supported these findings, showing leukocytosis and the presence of immature blast cells in the PB, spleen and BM of sgTrp53 mice, whereas such cells were absent in the control groups. Notably, the frequency of blast cells was correlated with the frequency of Trp53 frameshift mutations, consistent with what has been reported in MPN patients.
Additionally, CRISPR-Cas9 mediated Trp53-loss primarily affected the erythroid lineage, driving expansion of erythroblasts, megakaryocyte-erythroid progenitors (MEP), and Lineage-/low Sca1-c-Kit+ in sgTrp53 mice. The erythroblasts showed aberrant co-expression of CD41 and CD71. Through secondary transplants with unfractionated spleen cells, whole BM cells, or sorted MEP cell populations, we confirmed that the leukemia driven by CRISPR-cas9 mediated Trp53-loss in the Jak2V617F-driven PV is transplantable, and that the MEP compartment in leukemic sgTrp53 mice contains the leukemia initiation population.
In total, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice, faithfully replicating the process of clonal expansion and evolution observed in the leukemic transformation of MPN. These mouse models of secondary AML are valuable for testing new therapies targeting disease initiating stem cells. Moreover, our models represent the distinct stages of MPN progression, offering a platform to evaluate preventive and therapeutic interventions at various stages of this disease.
Disclosures: No relevant conflicts of interest to declare.