Unraveling How Treatment Shapes Clonal Evolution and Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal KrasG12D

Our understanding of how individual mutations, whether present in all or just a fraction of the leukemia cells, affect cellular responses to therapy is limited. Leukemia mouse models provide unique possibilities to explore how therapy affects the evolution of genetically distinct clones and to identify mechanisms of resistance allowing transfer to human disease. Herein we investigated how different therapies influenced survival, clonal evolution, and resistance patterns in mouse KMT2A::MLLT3 leukemia with subclonal Kras^G12D.

Bone marrow (BM) from a leukemia expressing KMT2A::MLLT3-mCherry in all cells and a de novo Kras^G12D in 38% of cells, were re-transplanted to sublethally irradiated recipients¹. Upon engraftment, treatment was started with either chemotherapy mimicking the induction regimen given for AML patients (cytarabine for 5 days + doxorubicin for 3 days, n=4), or the MEK inhibitor Trametinib either alone for 28 days (n=7), or after chemotherapy (n=6); controls received vehicle.

Our results show that each treatment significantly prolonged survival with a median latency of 25 days for chemotherapy, 27 days for Trametinib alone and 33.5 days for chemotherapy followed by Trametinib, versus 20 days for the controls. At the time of leukemia development, most of the cells in the BM expressed KMT2A::MLLT3-mCherry, and mice displayed splenomegaly and leukocytosis.

Next, we investigated how treatment impacted the evolution of the Kras^G12D-mutated KMT2A::MLLT3 cells, through targeted sequencing. In control mice receiving vehicle, the Kras^G12D-containing leukemia cells had a competitive advantage over cells expressing KMT2A::MLLT3-alone and increased to clonal dominance in all mice. However, upon chemotherapy treatment the frequency of KMT2A::MLLT3+Kras^G12D cells stable at around 40%, indicating that chemotherapy suppressed their expansion, but could not eradicate these cells. However, tertiary transplantation followed by repeated chemotherapy treatment caused the Kras^G12D-containing leukemia cells to expand to clonal dominance indicating a competitive advantage. Across the other treatment combinations, most mice relapsed with the Kras^G12D-mutated cells expanding to clonal dominance, except for mice receiving chemotherapy followed by Trametinib where two mice relapsed with either reduced or maintained frequency of Kras-mutated cells.

In 37% of the mice, the variant allele frequency (VAF) of the Kras^G12D-mutation was above 0.5, in line with allelic imbalance at the Kras locus, with mice of those mice receiving targeted treatment suggesting target-dependent mechanisms of resistance and a selective pressure for increased Ras-levels during MEK-inhibition. In line with this, we have previously shown that upon secondary transplantation of BM cells from the Kras^G12D-donor mouse¹, there is strong selective pressure for additional Kras^G12D with 40% of the cells, acquiring an additional chromosome 6, where Kras is located, or a Robertsonian translocation involving two chromosomes 6. We will now investigate if the same mechanism occur in these mice and link that to the VAF and the individual treatments.

Next, to evaluate how the specific treatments affected gene expression patterns and to determine how resistance occur, RNA sequencing (n=19) revealed 3 major clusters, containing either 1) the untreated mice, 2) mice that received Trametinib either alone or with chemotherapy and 3) mice that received chemotherapy. However, resistance upon treatment with chemotherapy was characterized by an up-regulation of fatty acid metabolism and ROS signaling pathways whereas resistance upon treatment with Trametinib was characterized by up-regulation of signaling pathways such as JAK/STAT, TP53, and TNFα suggesting different mechanisms of resistance and ways to target the resistant cells.

Taken together, the different treatments given impacted latency and how the Kras^G12D-mutated cells expanded and targeted sequencing highlighted that allelic imbalance at the Kras-locus occur upon resistance with Tramatenib. Finally, the transcriptomic analysis proposed deregulation of distinct pathways depending on the specific treatment providing means for targeting of the resistant cells.

Reference:

1. Hyrenius-Wittsten, A., et al., De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia. Nat Commun, 2018. 9(1): p. 1770.