Modeling and therapeutic targeting of t(8;21) AML with/without TP53 deficiency

Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1);RUNX1-ETO is one of the most common subtypes of AML. Although t(8;21) AML has been classified as favorable-risk, only about half of patients are cured with current therapies. Several genetic abnormalities, including TP53 mutations and deletions, negatively impact survival in t(8;21) AML. In this study, we established Cas9+ mouse models of t(8;21) AML with intact or deficient Tpr53 (a mouse homolog of TP53) using a retrovirus-mediated gene transfer and transplantation system. Trp53 deficiency accelerates the in vivo development of AML driven by RUNX1-ETO9a, a short isoform of RUNX1-ETO with strong leukemogenic potential. Trp53 deficiency also confers resistance to genetic depletion of RUNX1 and a TP53-activating drug in t(8;21) AML. However, Trp53-deficient t(8;21) AML cells were still sensitive to several drugs such as dexamethasone. Cas9+ RUNX1-ETO9a cells with/without Trp53 deficiency can produce AML in vivo, can be cultured in vitro for several weeks, and allow efficient gene depletion using the CRISPR/Cas9 system, providing useful tools to advance our understanding of t(8;21) AML.

RUNX1-ETO alone is not sufficient for leukemogenic transformation and requires additional genetic alterations for progression to full blown AML [1,3].Studies have identified the collaborative genetic alterations, including mutations in KIT, ASXL1, ZBTB7A, NRAS, CBL, and TP53 genes in t(8;21) AML [5,6].Some of these mutations, such as those in KIT, ASXL1, and TP53, negatively affect survival.In particular, loss of the TP53 response pathway has been shown to be associated with drug resistance and disease progression in RUNX1-ETO leukemia [7], while RUNX1-ETO itself was shown to activate the p53 pathway that sensitizes leukemia cells to DNA damage [8].Therefore, new approaches for the treatment of t(8;21) AML patients with TP53 mutations or deletions need to be developed.
In addition to the full-length RUNX1-ETO, alternatively spliced isoforms of the RUNX1-ETO transcript have been identified in t (8;21) patients.RUNX1-ETO9a [9], a short isoform of RUNX1-ETO, encodes a C-terminally truncated RUNX1-ETO protein with a stronger leukemogenic potential than full-length RUNX1-ETO.Because RUNX1-ETO9a can induce AML without cooperating mutations in a mouse retroviral transduction-transplantation model, it has been widely used experimentally as mouse models of t(8;21) AML.
In this study, we developed novel mouse models for t(8;21) AML using RUNX1-ETO9a, Trp53 (the mouse homolog of TP53)-deficient mice and Cas9 knockin mice.The established Cas9 + , RUNX1-ETO9a-expressing AML cells with/without Trp53 deficiency will be useful tools for the development of effective therapeutic strategies for t(8;21) AML.

Mice
C57BL/6 mice (Ly5.1)obtained from Sankyo Labo Service Corporation, Tokyo, Japan, were employed in bone marrow transplantation assays.Trp53 −/− mice were sourced from the RIKEN BioResource Center in Ibaragi, Japan [10].Rosa26-LSL-Cas9 knockin mice were procured from The Jackson Laboratory (#024857) [11].To generate Trp53 −/− -Cas9 mice, Trp53 −/− mice were bred with Cas9 knockin mice.All animal experiments were granted approval by the Animal Care Committee of the Institute of Medical Science at the University of Tokyo (PA21-67) and were carried out in accordance with the Regulation on Animal Experimentation at the University of Tokyo, following the International Guiding Principles for Biomedical Research Involving Animals.
cSAM cells were previously generated using a murine transplantation model.Briefly, a C-terminally truncated form of ASXL1 and SETBP-D868N were transduced into mouse bone marrow progenitor cells, and transplanted into sublethally irradiated recipient mice.Leukemic cells were isolated from the bone marrow of the moribund mice and their leukemogenic activity was confirmed by serial transplantation [12,13].The cSAM cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1 ng/ml IL-3.

Cell growth assay
The cytotoxic effects of DS-5272, Cytarabine, Dexamethasone, and Decitabine against RUNX1-ETO9a cells with/ without Trp53 deficiency or cSAM cells were assessed using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) following the manufacturer's instructions.Cells were plated in 96-well plates at a density of 5 × 10 3 cells/well in 0.1 ml medium and treated with various concentrations of each compound.After 72 h of incubation at 37 °C, 8 μl of the Cell Counting Kit-8 was added to each well.Following a 1-h incubation at 37 °C, the absorbance at 450 nm was measured using a microplate reader (CLARIOstar Plus, BMG LABTECH, Ortenberg, GER).Relative cell viability was expressed as the ratio of the absorbance in each treatment group to that of the corresponding untreated control group.The data are presented as means ± standard deviation (SD) from more than three independent experiments.IC50 values were calculated using GraphPad Prism software.

Statistical analyses
GraphPad Prism 10 was employed for all statistical analyses.Pairwise comparisons of significance were carried out utilizing ordinary two-way ANOVA.Survival curve comparisons were performed using the log-rank (Mantel-Cox) test.Animal experiments were not subjected to blinding or randomization.Sample sizes were determined based on prior experience rather than a predetermined statistical method.All data are presented as mean ± SD.
To confirm the leukemogenic potential of the RUNX1-ETO9a-expressing cells, we then transplanted 1 × 10 6 spleen cells collected from the moribund primary recipient mice into sublethally irradiated (5.25 Gy) secondary recipient mice.Again, mice transplanted with RUNX1-ETO9a-Trp53 −/− -Cas9 + cells developed AML more rapidly than those receiving RUNX1-ETO9a-Cas9 + cells with intact Trp53 (Fig. 1E).We observed the accumulation of c-Kit + cells in the spleens of the secondary recipient mice, similar to the primary recipient mice (Fig. 1F).Thus, RUNX1-ETO9a alone can initiate AML development, and its combination with Trp53 deficiency significantly accelerates disease progression.
We then enriched leukemia stem cell activity of these RUNX1-ETO9a cells through tertiary and quaternary transplantation, finally resulting in the generation of aggressive AML cells capable of producing leukemia in 10 days even in non-irradiated recipient mice (Fig. 1G).

Distinct impact of RUNX1 depletion on Trp53-intact or deficient RUNX1-ETO9a cells
Next, we examined if the RUNX1-ETO9a cells can be cultured in vitro.We first cultured the RUNX1-ETO9a-Trp53 −/− -Cas9 + cells and RUNX1-ETO9a-Cas9 + cells obtained from moribund mice in RPMI-1640 medium with murine SCF, IL-3, and IL-6 for 5 days, and then reduced the concentrations of SCF and IL-6 gradually.The RUNX1-ETO9a cells with/without Trp53 deficiency grew well in the medium containing only IL-3 for at least 2 weeks.However, we observed a significant decline in the proliferative capacity of cells after approximately 20 days of in vitro culture.Both the Trp53-intact or deficient RUNX1-ETO9a cells were differentiated into mature myeloid cells, as evidenced by the reduced c-Kit expression and a remarkable increase of CD11b + cells at day 25 (Fig. 2A).Thus, RUNX1-ETO9a cells were not immortalized in vitro, but could be cultured for up to three weeks, which is sufficient for most in vitro experiments.

Discussion
Although t(8;21) AML has been classified as a favorable risk AML, a significant proportion of patients, especially those with specific co-operating mutations, often relapse and eventually die.TP53 is one of the genes whose mutations are associated with poor prognosis in t(8;21) AML [5].In this study, we established a novel murine model for t(8;21) AML with/without Trp53 deficiency using RUNX1-ETO9a, a short isoform of RUNX1-ETO with stronger leukemogenic potential [9].The RUNX1-ETO9a cells are able to generate AML in vivo and can be cultured in vitro for up to three weeks.In addition, the RUNX1-ETO9a cells established in this study express Cas9.Therefore, any gene of interest can be efficiently depleted in these cells.
Previous experimental studies have shown that loss of TP53 promotes disease progression and therapy resistance in RUNX1-ETO leukemia [7,8].Consistent with these findings, we showed that Trp53 deficiency accelerates the development of AML driven by RUNX1-ETO9a.However, it should be noted that Trp53 was already deleted in cells prior to RUNX1-ETO9a transduction in all these Trp53deficient t(8;21) AML models.Given that TP53 mutations are typically detected as secondary somatic mutations in t(8;21) AML, and that acute and chronic inhibition of TP53 sometimes show opposing effects [27], the effect of late Trp53 depletion in the established RUNX1-ETO9a leukemia warrants further investigation.The Cas9 + RUNX1-ETO9a cells established in this study will be useful for this purpose.Furthermore, our mouse t(8;21) AML models will provide ideal platforms to perform the in vivo CRISPR/Cas9 library screening to identify key regulators that promote or suppress the development of RUNX1-ETO leukemia, particularly in vivo.
Using these RUNX1-ETO9a cells with or without Trp53 deficiency, we showed that targeting RUNX1 is only effective in Trp53-intact RUNX1-ETO9a cells.Previous studies have shown that RUNX1 has a dual role in leukemogenesis  [28].RUNX1 acts as a tumor promoter by promoting the survival of AML cells [20,21], in part through activation of TP53-mediated pro-apoptotic signaling [19,29].On the other hand, RUNX1 also acts as a tumor suppressor by inhibiting myeloid maturation [20,30].Therefore, it is likely that the tumor suppressor role of RUNX1 is more pronounced in the Trp53-deficient RUNX1-ETO cells.Thus, our data together with previous findings strongly suggest that the antileukemic effect mediated by RUNX1 depletion requires functional TP53.
Various novel therapeutic strategies for treating RUNX1-ETO leukemia have demonstrated promise in either clinical or experimental investigations [1].These include a KIT inhibitor dasatinib [31], JAK inhibitors [15,32], HDAC inhibitors [33], and glucocorticoid drugs such as dexamethasone.In this study, we found that RUNX1-ETO9a cells were particularly sensitive to dexamethasone regardless of Trp53 status.While glucocorticoids are widely used to treat lymphoid malignancies [26], they are generally not deemed beneficial in the context of AML.However, several previous reports have repeatedly shown that glucocorticoids are effective in suppressing the growth of t(8;21) AML cells at low doses, but not in other subtypes of AML [34,35].These findings, together with our data, provide a rational basis for clinical testing of glucocorticoid drugs, such as dexamethasone, against t(8;21) AML including those with TP53 alterations.
In summary, we established novel murine Cas9 + RUNX1-ETO9a cells with intact or deficient Trp53.These cells allow testing the effect of novel drugs in vitro and in vivo, enable genetic screens using sgRNA libraries, and will provide valuable information on the role of TP53 in the development of t(8;21) AML in future studies.