Modeling the Cellular Origin of EVI1 + MLL-AF9-Driven Acute Myeloid Leukemia (AML)
Authors: Chatel-Soulet Hugues-Etienne , Juge Sabine , Bagger Frederik Otzen , Tzankov Alexandar , Kurokawa Mineo , Séguin Jonathan , Schwaller Juerg 
Published: November 2021

Abstract
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&lt;0.05). Immunophenotypically, cells from Evi1 high cell-derived colonies retained a more immature phenotype, reflected by higher cKit +Sca1 + expression (n=11, p&lt;0.05). Plated Evi1 high cells formed Evi1 + colonies whereas Evi1 low lost Evi1 expression (n=11, p&lt;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: &gt;200d; LT-HSC: n.s.; MPP1: p&lt;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&lt;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&lt;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&lt;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&lt;0.0001) and MPP1 (22 vs. 47%, 22 vs- 48%; n=29, p&lt;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&lt;0.0001; LT-HSC: 41 vs 146.5d, p&lt;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.

Disclosures
Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau.