Oncogenic cooperation between TCF7-SPI1 and NRAS(G12D) requires β-catenin activity to drive T-cell acute lymphoblastic leukemia

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the β-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/β-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/β-catenin interaction.

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

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Software and code
Policy information about availability of computer code For Figure 4B, the primary conditional bone marrow transplant with the TCF7-SPI1 construct was 5 mice -carried out in a single cohort. For the TCF7-SPI1_P2A_NRAS G12D and NRASG12D only there are 9 mice in total and were two independent cohorts of (n=4, n=5 for TCF7-SPI1_P2A_NRAS G12D; n=5, n=4 for the NRASG12D). For the secondary transplant recipients there are 5 mice for the NRAS only, and there were 6 recipients for TCF7-SPI1_P2A_NRAS G12D, where each was a single cohort. No power analysis was carried out for these experiments. For figure 5B, these are the recipients for a single bone marrow transplant into 5 or 6 recipients, as a single cohort experiment side-by-side. In Figure 4B, 2 mice were excluded for the TCF7-SPI1_P2A_NRAS G12D primary transplants (2 mice from cohort 1 and 1 mouse from cohort 2 as there was no engraftment e.g. because of missed injection) For the NRAS only, one mouse was excluded due to no engraftment. In figure  4C, it was not possible to weigh all spleens/thymus' if a recipient mouse died in the cage overnight. For example, there are only 7 thymus weights for the NRAS only mice. As noted in the methods, the mouse was considered "event" when WBC >20,000. However, for NRAS only mice, the WBC rarely rose to this level and the surrogate marker was rapid weight loss in 24-36 hours likely due to the enlarged thymus blocking the oesophagus.
The bone marrow transplants in figure 4 concerning the TCF7-SPI1_P2A_NRAS G12D and NRAS G12D are two independent replicates with independent donor/recipient mice being used (i.e. two independent cohorts). The remainder and the secondary transplants are a single independent experiment with multiple mice. For the in vitro luciferase work, all data is presented in figure 5 where data represents technical replicates with biological replicates for these experiments presented in the supplementary figure as described in the manuscript. The replication for Figure 6B luciferase data is also provided in supplementary data. Cell growth experiments in figure 3, data represents three

Antibodies
Antibodies used wells for each construct followed over time and each well measured once for each data collection point. For the inducible mouse model studies for doxycycline chow induced knockdown of the fusion undertaken during the revision period, a total of 12 mice were used in the experiment with 8 mice receiving dox-chow in each cohort. This experiment in total is a a single biological replicate with 8 technical replicates. Figure 5B is a single bone marrow transplant where the recipients were technical replicates all receiving the same transduced hematopoietic and stem progenitor cells. For Figure 6, the inducible knockdown experiment was a single transplant where PDX cells were injected into 12 mice (technical replicates) for each shRNAmir. This experiment was not independently repeated.
Mice were allocated retroviral constructs on a cage per cage basis. There was no formal randomization. For the bone marrow transplants presented in Figure 4 and Figure 5, no formal randomization was undertaken and not relevant for these experiments. Covariates such as sex, age and mean weights were controlled for across the cages. For Inducible mouse experiments (Figure 6), mice were allocated per group based on percentage engraftment was normalised across Dox-Chow and Normal Chow groups to ensure no difference in engraftment occurred between the two groups.
There was no blinding carried out during the study. To complete the analysis of the in vitro cell work correctly, there was no blinding of growth curves or luciferase expression vector experiments to complete the analysis, with sample information and corresponding treatments known to investigators. For in vivo studies, documentation outlining what mice received which retroviral construct is mandated as part of the ethics and monitoring process. All scientists were therefore aware of which mice had which construct. Similarly, for the doxycyline treatment of mice, this chow is provided on a cage per cage basis preventing investigators being blinded to the treatments.
We have provided an independent All used antibodies were commercially available conjugated antibodies which were validated by the manufacturer for the intended use in flow cytometry; validation statements are available on the website of the manufacturer on the product page of each antibody. These are summarised below from the manufacturer's websites for each antibody:.
CD1a Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.
BV-421 Mouse anti-Human CD8a, Clone HIT8a, BD 740078. This antibody has been tested by flow cytometric analysis of mouse thymocytes. (https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-humanantibodies/cell-surface-antigens/bv421-mouse-anti-human-cd8-hit8a/p/740078) PE-cyanine7 Mouse anti-human CD117, Clone 104D2, Lot#2162008. This antibody has been pre-titrated and tested on human peripheral blood cells. (https://www.bdbiosciences.com/us/applications/research/stem-cell-research/cancer-research/human/ pe-cytrade7-mouse-anti-human-cd117-104d2/p/339195) Mel888 cells were a gift from Lionel Larue, Normal and Pathological development of melanocytes lab, Institut Curie, France. They are also known as 888-Mel and were originally purchased from ATCC. The 293T and Ba/F3 cells were from DSMZ. The Ba/F3-Cre line is a stable derivative of Ba/F3 cells that were transduced with MSCV-Cre recombinase-Puromycin for the stable expression of Cre-recombinase. These were made in house within the laboratory of Prof Jan Cools. The Pro-T primary cell line is made in house from bone marrow stem cells as outlined in the methods section.
The Mel888 cells were not authenticated. The 293T and Ba/F3 cells were authenticated using STR analysis.
No commonly misidentified cell lines were used in this study C57BL/6 female mice as recipients for bone marrow transplants. Male CD2-Cre on a C57BL/6 background was used as a donor mouse for the bone marrow transplant experiments. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice or (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJArc 9NRG) mice were used for the PDX models and were always female. In all cases the mice used as donors for bone marrow transplants were aged between 6-8 weeks at time of harvest. Recipient mice for bone marrow transplants were between 8-11 weeks. For NRG and NSG mice, at time of injection ages were between 8-11 weeks. For housing, mice were held under PC2 conditions in individually ventilated caging with a maximum of 6 or a minimum of 2 mice per cage. Dimensions for mice cages: 369 X 156 X 132 mm, with a floor area of 440 cm2. The mice were maintained in a protected and controlled environment. The animal facility was barrier protected with the air HEPA filtered and the room maintained at positive pressure and at a temperature 22 +/-1 degree Celsius. Sterile feed and water were provided ad libitum. The light in the animal facility was on a 12h cycle. All experimental work was conducted in biosafety or cytotoxic cabinets to further protect the animals from microbiological threat.
No wild animals were used in the study