Abstract
The MLL-AF9 fusion protein occurring as a result of t(9;11) translocation gives rise to pediatric and adult acute leukemias of distinct lineages, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and mixed phenotype acute leukemia (MPAL). The mechanisms underlying how this same fusion protein results in diverse leukemia phenotypes among different individuals is not well understood. Given emerging evidence from genome-wide association studies (GWAS) that genetic risk factors contribute to MLL-rearranged leukemogenesis, here we tested the impact of genetic background on survival and phenotype of a well-characterized Mll-AF9 knockin mouse model. We crossed this model to five distinct inbred strains (129, A/J, C57BL/6, NOD, CAST), and tested their F1 hybrid progeny for dominant genetic effects on Mll-AF9 phenotypes. We discovered that genetic background altered peripheral blood composition, with Mll-AF9 CAST F1 demonstrating significantly increased B lymphocyte frequency while the remainder of the strains exhibited myeloid-biased hematopoiesis, similar to the parental line. Genetic background also impacted overall survival, with Mll-AF9 A/J F1 and Mll-AF9 129 F1 having significantly shorter survival, and Mll-AF9 CAST F1 having longer survival, compared to the parental line. Furthermore, we observed a range of hematologic malignancies, with Mll-AF9 A/J F1, Mll-AF9 129 F1 and Mll-AF9 B6 F1 developing exclusively myeloid cell malignancies (myeloproliferative disorder (MPD) and AML) whereas a subset of Mll-AF9 NOD F1 developed MPAL and Mll-AF9 CAST F1 developed ALL. This study provides a novel in vivo experimental model to evaluate the underlying mechanisms by which MLL-AF9 results in diverse leukemia phenotypes and provides definitive experimental evidence that genetic risk factors contribute to survival and phenotype of MLL-rearranged leukemogenesis.
Introduction
Chromosomal rearrangements involving the mixed-lineage leukemia 1 (MLL1) gene, also known as Lysine [K]-specific methyltransferase 2A (KMT2A), generate fusion proteins causing aggressive acute leukemias in infants, children and adults. MLL-rearranged leukemias comprise ∼10% of acute leukemias across all age groups1. Patients with MLL-rearranged leukemias generally have a poor prognosis, with high-risk treatment options and frequent relapse. This underscores an unmet need for novel therapeutic approaches to improve outcomes in MLL-rearranged leukemia.
Emerging evidence from GWAS studies suggest that heritable genetic polymorphisms can modify the risk of MLL-rearranged leukemia2-4. To definitively test causation and build upon these findings toward development of novel therapeutic targets, use of in vivo mouse models of MLL-rearranged leukemia is ideal. However, the vast majority of genetically engineered mouse models of human leukemia are studied on a single inbred genetic background, C57BL/6, despite genetic variability having been recognized as an important modifier of leukemogenesis in mouse models5-8.
The first MLL fusion protein to be modeled as an endogenous knockin allele in mice was Mll-AF9 (t(9;11))9. After an early myeloproliferative phase, Mll-AF9 mice primarily succumb to AML, and only in rare cases to ALL10,11. This is notably distinct from human disease, where MLL-AF9 is found in both B-cell ALL (B-ALL) and AML in infants and children, and AML in adults12. Here, we have utilized this well-characterized Mll-AF9 knockin mouse model to test the extent to which dominant genetic alleles modify Mll-AF9-driven leukemogenesis using genetically diverse mouse strains13,14.
Results and Discussion
To determine the role of genetic diversity in MLL-AF9 leukemia, we crossed the Mll-AF9 knock-in mouse model9 with the five distinct inbred strains A/J, C57BL/6 (B6), 129S1/SvlmJ (129), NOD/ShiLtJ (NOD) and CAST/EiJ (CAST). We studied F1 hybrid mice heterozygous for Mll-AF9 from these crosses versus the parental genetic background (Fig. 1A). The MLL-AF9 parental strain has been maintained as it was historically, on a mixed B6 and 129 background. While the parental Mll-AF9 strain has previously been demonstrated to develop leukemia around ∼6 months of age, detectable myeloid proliferation has been observed by 8 to 10 weeks of age9,11. Consistent with this observation, analysis of peripheral blood (PB) of parental Mll-AF9 mice compared to MllWT littermates at 8 weeks of age showed significantly increased myeloid cell frequency with concomitant reduction in T cell frequency (Fig. 1B). This same phenotype was observed in Mll-AF9 A/J F1, Mll-AF9 B6 F1, Mll-AF9 129 F1 and Mll-AF9 NOD F1 mice. In contrast, myeloid cell frequency in Mll-AF9 CAST F1 mice was not significantly different from MllWT mice but instead a significant increase in B cell frequency and reduction in T cell frequency were observed. To determine whether this observation was based on baseline differences in the CAST genetic background, we examined PB composition in wild-type CAST mice versus the other strains used in this study. We found that wild-type CAST mice have no differences in PB composition compared to the other strains (Supp. Fig. 1), suggesting that this phenotype is a direct consequence of Mll-AF9 expression.
Monitoring PB composition until pathology developed revealed that Mll-AF9 CAST F1 mice maintained significantly reduced frequency of myeloid cells and increased frequency of B cells with aging compared to the parental Mll-AF9 strain (Fig. 2A). In concordance with previous studies11, median survival in the parental Mll-AF9 strain was 225 days. In contrast, Mll-AF9 CAST F1 had a longer median survival (361 days; P = 0.079) and Mll-AF9 A/J F1 and Mll-AF9 129 F1 that had significantly shorter median survival (172 days; P = 0.0071, and 178 days; P = 0.0179, respectively) (Fig. 2B). This data suggests that genetic background can alter the development and progression of leukemia caused by Mll-AF9.
Characterization of the hematologic malignancies that developed in these strains also revealed genetic background-dependent distinctions. Consistent with previous studies9,11, parental Mll-AF9 mice developed myelomonocytic AML with 100% penetrance (Fig. 3A) characterized by leukocytosis and thrombocytopenia (Fig. 3B), splenomegaly (Fig. 3C), >20% blasts in the BM (Fig. 3D), and abundant myeloid cell infiltration into the spleen and liver (Fig. 3D,E). Strains with shorter median survival (Mll-AF9 A/J F1 and Mll-AF9 129 F1) developed either AML or an early and aggressive MPD-like disorder characterized by splenomegaly, <20% immature or blast-like cells in the BM, and high frequency of Gr1-expressing granulocytes in the BM (Fig. 3E). In the Mll-AF9 CAST F1 strain exhibiting the longest median survival, 50% of mice were found to have ALL characterized by leukocytosis, splenomegaly, spleen and liver infiltration, and high frequency of B220lo c-Kit+ blast cells in the bone marrow and spleen. While CAST mice have not been broadly studied in the context of leukemia or other cancer development, CAST F1 mice do have increased tumor growth in a model of neuroendocrine prostate carcinoma15, suggesting that the increased survival we have observed is not due to a general tumor-resistant genetic background. Of note, one individual Mll-AF9 NOD F1 mouse was found in our study to develop MPAL characterized by leukocytosis, thrombocytopenia, spleen and liver infiltration, and bi-phenotypic B220+ CD11b+ blast cells in the bone marrow. As NOD mice are a polygenic model for autoimmune type 1 diabetes and exhibit aberrant immunophenotypes it is interesting to speculate how this influences the development of a bi-phenotypic leukemia.
By introducing genetic variation into the MLL-AF9 knockin mouse model, our work has identified that disease latency and leukemia phenotype is significantly affected by heritable genetic variants segregating among common inbred strains, and suggests the presence of specific, dominant-acting modifier alleles in one or more strains. These findings support that genetic background differences may play a role in how and why leukemogenesis resulting from a common fusion oncogene can result in distinct etiology among different individuals. As epigenetic dysregulation is a critical driver of MLL-rearranged leukemia16,17, we posit that altered survival and leukemia phenotypes may be related to differences in epigenetic or chromatin state in genetically diverse mice. This is also supported by GWAS identification of single nucleotide polymorphisms in ARID5B, encoding part of the histone H3K9me2 demethylase complex, that modify risk for MLL-rearranged early childhood leukemia4. More broadly, apart from the presence of the fusion oncogene, data from our lab and others support that other factors strongly influence the specific outcome, including the cell type-of-origin18-20, the developmental stage and context in which the chromosome translocation occurs21,22, and the individual’s genetic background. Importantly, this study included five of the eight founder strains of the Collaborative Cross (CC) and Diversity Outbred (DO) mouse populations23,24, complementary resources that enable one to model human genetic diversity and map genetic modifiers that underlie phenotype differences in the population. Future studies will take advantage of these powerful tools to map the genetic determinants of leukemia susceptibility and phenotype, with the goal of identifying novel gene targets for the development of new therapies for MLL-rearranged leukemia.
Methods
Experimental animals
Kmt2atm2(MLLT3)Thr/KsyJ (referred to as Mll-AF9, stock no: 009079) mice were obtained from, and aged within, The Jackson Laboratory. The Mll-AF9 model was created on a 129P2/OlaHsd background, crossed with C57BL/6NCrl females for four generations, and has been maintained at The Jackson Laboratory since 2012 on a mixed C57BL/6 and 129S1/SvlmJ background by breeding with B6129PF1/J. The Mll-AF9 original strain was crossed to the A/J, C57BL/6 (B6), 129S1/SvlmJ (129), NOD/ShiLtJ (NOD) and CAST/EiJ (CAST) strains to create F1 generation experimental mice. Male and female F1 progeny from each strain cross were included in the studies and monitored from 8 weeks of age until moribund. Female and male mice were analyzed for PB CBC data at 6 months of age. The Jackson Laboratory’s Institutional Animal Care and Use Committee (IACUC) approved all experiments.
Peripheral blood analysis
PB was collected from mice via retro-orbital sinus and red blood cells were lysed before staining mature lineage markers: B220 (clone RA3-6B2), CD3e (clone 145-2C11), CD11b (clone M1/70), Gr-1 (clone RB6-8C5). Stained cells were analyzed on an LSRII (BD) and populations were analyzed using FlowJo V10. Differential blood cell counts were obtained from PB using an Advia 120 Hematology Analyzer (Siemens)
Analysis of moribund mice
Moribund mice identified by declining health status were euthanized and PB, spleen, liver, and BM harvested. Single-cell suspensions of PB, spleen, and BM were analyzed by flow cytometry for mature lineage markers and c-Kit (clone 2B8), using an LSRII (BD) and populations were analyzed using FlowJo V10. Differential blood cell counts were obtained from PB using an Advia 120 Hematology Analyzer (Siemens). Cytospin preparations of whole BM MNCs were stained with May–Grunwald–Giemsa stain. Liver and spleens were fixed for 24 h in 10% buffered formalin phosphate, embedded in paraffin, and sections were stained with H&E. Histological images of stained BM, liver, and spleen were captured on a Nikon Eclipse Ci upright microscope with SPOT imaging software (v.5.6).
Statistical analysis
Overall survival, Log-rank (Mantel–Cox) test was performed on Kaplan–Meier survival curves. Statistical analysis of non-survival data was performed by Brown-Forsythe one-way ANOVA test followed by Dunnett’s multiple comparisons test. All statistical tests, including evaluation of normal distribution of data and examination of variance between groups being statistically compared, were assessed using Prism 8 software (GraphPad).
Acknowledgements
This work was supported by National Institutes of Health (NIH), NCI Cancer Core Grant P30CA034196, and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) T32HD007065 (K.Y.). This work was also supported by The V Foundation V Scholar award (J.J.T.) and grants from the Maine Cancer Foundation (J.J.T.). K.Y. is supported by an American Society of Hematology (ASH) Scholar Award and the Pyewacket Fund at The Jackson Laboratory. We thank Steve Munger, Jennifer SanMiguel, and members of the Trowbridge laboratory for helpful discussion and critical comments, and Rebecca Bell for experimental and laboratory support.