Signaling Pathway Mutations Cooperate with the PICALM/MLLT10 Fusion in a Knock-in AML Mouse Model

The PICALM/MLLT10 (or CALM/AF10; C/A) is a rare but recurring fusion gene detected in patients with T-cell acute lymphoblastic leukemia, malignant lymphoma and acute myeloid leukemia (AML). Our lab has previously established a C/A knock-in mouse model, where the fusion gene preceded by a loxP-flanked transcriptional stop cassette was inserted into the Rosa26 locus (the R26LSLCA strain). To achieve tissue-specific expression of C/A, R26LSLCA mice were crossed with three different Cre-inducer lines, namely the Vav-Cre, MB1-Cre and CD19-Cre. In the Vav-Cre strain, Cre recombinase is expressed in the entire hematopoietic system, while in the MB1-Cre and CD19-Cre lines, Cre is expressed in the B-cell compartment. Using this model, Dutta et al. showed that C/A leads to leukemia only when expressed in all hematopoietic cells, including hematopoietic stem cells and does not lead to induction of leukemia when expressed in B-cells alone (Dutta et al., Leukemia 2016).

To further characterise leukemias driven by the C/A fusion, we established new R26LSLCA and Vav-Cre crosses. Similar to what was found previously, the Vav-Cre/R26LSLCA mice developed leukemia with a long median latency of 1 year (366.5 days; N=24) and complete penetrance. On post-mortem analysis, the Vav-Cre/R26LSLCA mice were found to have splenomegaly and leucocytosis. Expression of the fusion gene was confirmed in these mice by RT-PCR on the cDNA from bone marrow (BM) cells. Immunophenotyping analysis of the BM cells and splenocytes of these mice revealed a predominantly myeloid phenotype.

To assess the effect of expression of C/A on the transcriptome, we performed RNA-seq analysis using BM cells from 17 leukemic mice and 12 healthy controls. In accordance with reports on C/A patient samples, the Hoxa genes Hoxa7 and Hoxa9, as well as the Hox-cofactor Meis1 were significantly upregulated in our C/A expressing mice. In addition, genes involved in B-cell development, such as Cd19, Pax5 and Ikzf3, and Gata1, which is essential for the development of erythrocytes, were downregulated. To prove that the Vav-Cre/R26LSLCA mice were indeed leukemic and had the potential to propagate the disease, we serially transplanted BM cells from 4 Vav-Cre/R26LSLCA mice into congenic recipients. The secondary (N=20) and tertiary (N=19) transplanted mice developed leukemia with a significantly shorter median latency of 21 and 17 days, respectively.

Our group and others have shown that a single genetic lesion is in most cases not sufficient to cause leukemia. Thus, to determine if additional somatic mutations were acquired by the C/A knock-in mice, we performed whole exome sequencing on the DNA from BM cells of the 4 knock-in mice, some of their daughter secondary and tertiary leukemias as well as the corresponding germline DNA samples (N=19). We identified between 2-32 mutations per leukemia exome, including mutations in genes and/or members of gene families that have been implicated in human leukemias. Mutations were detected in the transcription factors Sfpi1, Zeb1, and Sox9; Aff2, a member of the Af4/Fmr2 gene family; and Sf3a2, a component of the RNA-splicing machinery, among others. In addition, in two C/A sister secondary leukemias and the corresponding tertiary leukemias, we detected a missense mutation at amino acid 507 (G507A) of the Ptpn11 gene, which is homologous to G507 of human PTPN11, a known hotspot mutation site in AML and juvenile myelomonocytic leukemia. Interestingly, Dutta et al. also reported a G507 mutation (G507V) in Ptpn11 in a C/A knock-in mouse. PTPN11 regulates the RAS/MAPK signaling pathway. This finding is reminiscent of what we have previously reported in a C/A driven murine bone marrow transplantation model, where we detected somatic mutations in several AML-associated signal transduction genes, including Flt3, Cbl and Kras (Desai et al., Leukemia 2020).

Taken together, our results strongly suggest that mutations in genes involved in signaling cascades, particularly those in the RAS/MAPK pathway, cooperate with the C/A fusion and lead to complete malignant transformation. We aim to functionally validate these findings by establishing mouse models that harbor the C/A fusion as well as mutations in Ptpn11 and other signal transduction genes. These murine models will be immensely valuable for gaining a deeper insight into PICALM/MLLT10-mediated leukemogenesis, studying cooperating mutations and for testing new targeted therapies.