Modeling the Cellular Origin of EVI1 ⁺ MLL-AF9-Driven Acute Myeloid Leukemia (AML)

We previously reported that transplantation of hematopoietic stem cells (HSC) expressing a doxycycline (DOX)-regulated AML-associated MLL-AF9 fusion transgene can induce an invasive and chemoresistant disease in mice formed by tumour cells that express the transcription factor EVI1, a known marker of poor prognosis in AML and some solid cancers. To better understand the association of EVI1 and the cellular origin of the disease we analyzed Evi1-IRES-GFP reporter mice (female, n=8, 8-10 weeks old) and found that not only the, quiescent long-term hematopoietic stem cell (LT-HSC : lineage marker-negative (lin ^-) cKit ⁺Sca1 ⁺(= LSK), CD34 ^-CD135 ^-CD150 ⁺CD48 ^-, 24±3.7%, n=8) compartment, but also more proliferating multipotent progenitors such as MPP1 (LSK, CD34 ⁺CD135 ^-CD150 ⁺CD48 ^-, 23±4.6%, n=8), MPP2 (LSK, CD34 ⁺CD135 ^-CD150 ⁺CD48 ⁺, 6±2.3%, n=8) and MPP3 (LSK, CD34 ⁺CD135 ^-CD150 ^-CD48 ⁺, 2±1%, n=8) contain a significant number of cells that express abundant Evi1 ("Evi1 ^high") at steady state. Notably, we did not observe any significant changes in numbers of Evi1 ⁺ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments 5 days after DOX-mediated induction of the iMLL-AF9 fusion. To address the impact of Evi1 on clonogenic growth of iMLL-AF9-expressing LT-HSC, we plated Evi1 ^high and Evi1 ^low naïve cells in methylcellulose (MC) and found that upon addition of DOX, Evi1 ^high cells formed more colonies with an invasive morphology ("type IV") compared to Evi1 ^low cells (n=11, p<0.05). Immunophenotypically, cells from Evi1 ^high cell-derived colonies retained a more immature phenotype, reflected by higher cKit ⁺Sca1 ⁺ expression (n=11, p<0.05). Plated Evi1 ^high cells formed Evi1 ⁺ colonies whereas Evi1 ^low lost Evi1 expression (n=11, p<0.05). To address the differential transformation susceptibility in vivo, we transplanted identical numbers of naïve steady-state Evi1 ⁺ iMLL-AF9 LT-HSC and MPP1 into irradiated syngeneic recipients. While recipients of Evi1 ⁺ MPP1 cells developed an invasive AML earlier than Evi1 ⁺ LT-HSC-transplanted mice (n=27, median latency: 96.5 vs. 146.5d, n.s.), very similar disease phenotypes were observed. In contrast, transplants of Evi1 ^- MPP1 or LT-HSC resulted in a significantly delayed disease induction (n=31, median latency: >200d; LT-HSC: n.s.; MPP1: p<0.05). Although Evi1 ⁺ cell-induced disease did present with more extensive organ infiltration by leukemic blasts than Evi1 ^- AML the phenotypes were similar. We also wondered whether modulation of the HSC compartment by exogenous factors may change Evi1 expression and affect AML induction. We found that 2 days after a single injection (200mg/kg) of recombinant mouse thrombopoietin (mTPO) the number of LT-HSC (n=23; 647 vs. 1165/10 ⁶ lin ^- cells, p<0.05), but not of MPP1, significantly increased. Similarly, a single application of the synthetic mTPO receptor agonist Romiplostim (RP, 200mg/kg) resulted after 48h in an increase of LT-HSC (n=14; 647 vs. 1459/10 ⁶ lin ^- cells, p<0.0005). Likewise, a single dose (10mg/kg) of polyinosinic:polycytidylic acid (pI:pC) also significantly increased the number of LT-HSC (n=9; 460 vs. 2300/10 ⁶ lin ^- cells, p<0.005) but not of MPP1 after 24h. In contrast, 5-Fluorouracil (5-FU; 150mg/kg) did not significantly change the number of LT-HSC and MPP1, 3- and 6-days post-injection. However, only mTPO and RP but not pI:pC or 5-FU significantly increased the fraction of Evi1 ^high expressing LT-HSC (23 vs. 50%, 23 vs. 49%; n=29, p<0.0001) and MPP1 (22 vs. 47%, 22 vs- 48%; n=29, p<0.0001). Transplantation of identical numbers of iMLL-AF9 LT-HSC and MPP1 isolated 2 days after mTPO application to the donors into irradiated syngeneic recipients resulted in a significantly faster induction of Evi1 ⁺ AML than controls (n=19, MPP1: 35 vs. 96.5d, p<0.0001; LT-HSC: 41 vs 146.5d, p<0.0001). Currently ongoing single-cell RNA sequencing experiments of LT-HSC and MPP1 with and without in vivo mTPO stimulation conditionally expressing the iMLL-AF9 fusion should provide some mechanistic insights into increased susceptibility for EVI1 ⁺ AML. Our results so far demonstrate that the dynamics of the HSC compartment critically affects the cellular origin and biology of MLL-AF9 driven AML.