Probing Co-Operating Somatic Mutations in MLL-ENL Driven Leukemogenesis

Missense mutations and other chromosomal perturbations underlie the heterogeneity of acute leukemia. While Mixed lineage leukemia-1 (MLL1/KMT2A) translocations are recurrent in human acute leukemia, most if not all MLL fusions are permissive for either acute myeloid (AML), acute lymphoid (ALL) or mixed/biphenotypic leukemia (MLL). At the same time, previous work have suggested that MLL rearranged leukemias require few additional mutations to induce transformation(1, 2).

The MLL-ENL translocation is apart from being associated with AML also found in human acute T cell leukemia (T-ALL). Despite this, but consistent with most previous observations in mice, we failed to observe development of ALL even when inducing MLL-ENL in multiple early hematopoietic progenitors(3). We hypothesized that this could be caused by a compromised T cell generation induced by the MLL-ENL translocation product. Culturing bone marrow Granulocyte-Monocyte-Lymphoid Progenitor (GMLPs) on OP9-DL1 stromal cells revealed that MLL-ENL blocked differentiation at a stage that phenotypically corresponds to a primitive DN1 stage. In vivo, short-term induction of MLL-ENL led to deregulation of T cell differentiation, with an evident block at the DN2/3 stage. As such a compromise in T cell generation could omit T-ALL development from immature T cell progenitors in the bone marrow, we next assessed the leukemic capacity of defined lymphoid progenitor cells at different stages of development. The latent myeloid potential of DN1 cells and early B lymphoid progenitors (BLPs), a property lost upon further differentiation of these cells, was sufficient to confer potent leukemia initiating activity for AML. By contrast, mice transplanted with of later stages of T cell and B cell progenitors failed to associate with development of disease.

The discrepancy of lineage assignment between MLL translocations in established human leukemia and attempts to mimic these in the mouse might be due to a requirement of necessary co-mutations. Although MLL fusions typically associate with few secondary mutations, previous studies have established that activating somatic mutations in the RAS pathway are frequently co-occurring with MLL rearranged leukemias. Therefore, we next investigated whether mutation order might influence on the developing murine leukemia. Co-expression of KRAS^G12D in GMLPs led to a significant reduction in disease latency compared to MLL-ENL alone. However, disease was still restricted to the myeloid lineage. In strikingly contrast, but in agreement with previous reports, mice receiving KRAS^G12D expressing GMLPs in the absence of MLL-ENL expression developed T-ALL with a median latency of 143 days and with roughly a 30% penetrance. The onset of MLL-ENL expression 5 weeks post transplantation in KRAS^G12D expressing GMLPs also gave rise to T-ALL, but with a shorter mean latency (111 days) and higher (50%) penetrance. The sequence of acquisition of somatic mutations therefore can influence not only on disease latency, but also on the lineage assignment of the developing leukemia.

Finally, to identify novel somatic mutations occurring during MLL-ENL induced leukemogenesis, we generated paired leukemic samples by transplanting leukemia initiating cells from individual donor mice into separate cohorts of recipients, followed by whole genome and exome sequencing upon development of AML. While MLL-ENL induced AML associated with very few secondary mutations, targeted re-sequencing of a panel of variants validated a few single nucleotide variants from leukemic samples, including amongst others driver mutations in PTPN11 and RAS known from studies of human leukemia. Further studies are now undertaken to obtain functional insights into the mode of action of the identified somatic mutations.

References

1. Andersson AK, Ma J, Wang J, Chen X, Gedman AL, Dang J, et al. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nature genetics. 2015;47(4):330-7.

2. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England journal of medicine. 2013;368(22):2059-74.

3. Ugale A, Norddahl GL, Wahlestedt M, Sawen P, Jaako P, Pronk CJ, et al. Hematopoietic stem cells are intrinsically protected against MLL-ENL-mediated transformation. Cell reports. 2014;9(4):1246-55.