Infectious stimuli promote malignant B-cell acute lymphoblastic leukemia in the absence of AID

The prerequisite to prevent childhood B-cell acute lymphoblastic leukemia (B-ALL) is to decipher its etiology. The current model suggests that infection triggers B-ALL development through induction of activation-induced cytidine deaminase (AID; also known as AICDA) in precursor B-cells. This evidence has been largely acquired through the use of ex vivo functional studies. However, whether this mechanism governs native non-transplant B-ALL development is unknown. Here we show that, surprisingly, AID genetic deletion does not affect B-ALL development in Pax5-haploinsufficient mice prone to B-ALL upon natural infection exposure. We next test the effect of premature AID expression from earliest pro-B-cell stages in B-cell transformation. The generation of AID off-target mutagenic activity in precursor B-cells does not promote B-ALL. Likewise, known drivers of human B-ALL are not preferentially targeted by AID. Overall these results suggest that infections promote B-ALL through AID-independent mechanisms, providing evidence for a new model of childhood B-ALL development.


Supplementary Figure 1: Aid expression in preleukemic precursor B cells. a) Aid expression during natural infection driven mouse B-ALL.
Relative expression of Aid in BM preleukemic precursor pro-pre-B cells sorted from control wild-type (WT), Pax5-het, Sca1-ETV6-RUNX1 either housed under SPF conditions or exposed to natural infections (conventional facility). WT total spleen of an immunized mouse was used as a positive control. Precursor propre-B cells sorted from Aid-KO mice were used as a negative control. Error bars represent the mean ± SD of three replicates. b) Aid expression in murine preleukemic Pax5-het, and Sca1-ETV6-RUNX1 pro-B cells after in vitro stimulation with TLR ligands. Fold induction of Aid expression on RNA levels of murine pro-B cells of WT, Sca1-ETV6-RUNX1 and Pax5-het mice after stimulation with different TLR ligands (TLR2/4 -LPS, TLR3 -polyIC, TLR7/8 -R848 and TLR9 -CpG) in vitro. Experiments were performed in 3 replicates, condition Pax5-het + LPS was performed in 2 replicates. Error bars represent the mean ± SD of three replicates. The expression levels were normalized to their unstimulated control. **p<0.01, ***p<0.001; Student´s two tailed t-test.

Supplementary Figure 2: Flow cytometric analysis of diseased Pax5het/Aid-het mice and Pax5-het/Aid-KO mice.
Representative plots of cell subsets are shown and compared to wild-type mice.

Supplementary Figure 3: Haematoxylin and eosin staining of tumourbearing Pax5-het/Aid-het mice. H-E staining of WT mice and tumour-bearing
Pax5-het/Aid-het mice showing infiltrating blast cells in spleen, liver, and lymph nodes. Loss of normal architecture resulting from effacement with cells morphologically resembling lymphoblast can be shown. Images are representative of 3 replicates. Scale bar represents 500 µm (=100X) for large panels and 100 µm (=400X) for inset.

Supplementary Figure 4: Haematoxylin and eosin staining of tumourbearing Pax5-het/Aid-KO mice.
H-E staining of WT mice and tumour-bearing Pax5-het/Aid-KO mice showing infiltrating blast cells in spleen, liver, and lung. Loss of normal architecture resulting from effacement with cells morphologically resembling lymphoblast can be shown. Images are representative of 3 replicates. Scale bar represents 500 µm (=100X) for large panels and 100 µm (=400X) for inset.

Supplementary Figure 5: Comparison of blast cell percentages in Pax5het/Aid-het and Pax5-het/Aid-KO mice.
The results showed that there are not statistical differences between both groups (Mann Whitney test). Error bars represent the mean ± SD of three replicates. Figure 6: Immunoglobulin clonality in Pax5-het/Aid-het and Pax5-het/Aid-KO B-ALL. PCR analysis of immunoglobulin heavy-chain gene rearrangements in infiltrated BM of diseased Pax5-het/Aid-het mice and Pax5-het/Aid-KO mice. Thymocytes (T cells) were included as a negative control, and sorted CD19 + B-cells (B cells) from the spleens of healthy mice were included as a control for polyclonal rearrangements (indicated by numbers, 1-4) within the mature B-cell population. It can be seen that infiltrated tissues shown an increased clonality within their immunoglobulin repertoire (coloured squares). Source data are provided as a Source Data file.

Query= CVD001 Length=359
Score E Sequences producing significant alignments:  Alignments      The first cut-off that provide us with a set of overexpressed genes corresponds to a delta value of 1.241685 and a false discovery rate of 0.348 which is clearly beyond an acceptable statistical error.  The table shows the pathogens tested to monitor the health status of the animals housed in the conventional facility during the time the animals have been studied. Indicated are pathogens to which the mice were exposed when transferred to the conventional animal facility.

Supplementary
Supplementary Table 3. List of cancer-related genes mutated in Pax5het/Aid-het and Pax5-het/Aid-KO B-ALL, their mutational context highlighting if they are located in AID hotspots or not.
Supplementary Table 4: List of human B-ALL drivers 1 and the mutational status of their mouse orthologs in Pax5-het/Aid-het and Pax5-het/Aid-KO B-ALL. First column indicates whether these driver genes are AID off-targets in mouse 2 .