Cell-Intrinsic Depletion of AML1-ETO-Positive Pre-Leukemic HSCs By Kras Activating Mutation
Authors: Di Genua Cristina , Norfo Ruggiero , Rodriguez-Meira Alba , Drissen Roy , Booth Christopher , Povinelli Benjamin , Repapi Emmanouela , Gray Nicki , Carrelha Joana , Kettyle Laura , Jamieson Lauren , Neo Wen Hao , Nerlov Claus , Mead Adam J 
Published: April 2021

Abstract
Mutations in acute myeloid leukemia (AML) are acquired hierarchically. Pre-leukemic mutations, such as AML1-ETO, are acquired within the hematopoietic stem cell (HSC) compartment as an early event, whereas signaling mutations, such as KRAS, are late events acquired within transformed leukemic progenitors and rarely detectable within the HSC compartment (PMID 24550281). This raises the possibility that signaling pathway mutations are detrimental to clonally expanded pre-leukemic HSC. To explore this hypothesis, we generated mice carrying A ml1ETO/+ or KrasG12D/+ conditional knock-in alleles, either individually or combined (AEK), crossed to an Mx1- Cremouse line. Due to previously described spontaneous recombination in primary KrasG12D/+ mice with Mx1- Cre, competitive transplantation was performed using 250,000 fetal liver (FL) cells and 1x106wild-type (WT) bone marrow (BM) competitor cells into lethally irradiated recipients. Mutations were induced with polyI:C 4 weeks post-transplantation, and mice were culled 8 weeks post-polyI:C to assess the hematopoietic phenotype.
Transplantation of Aml1ETO/+ FL was insufficient to induce a hematological malignancy in primary recipient mice. In contrast, recipients of KrasG12D/+ FL developed a fatal myeloproliferative neoplasm (MPN) including leukocytosis (white blood cell count 50±22 x109/L), anemia (11.5±0.7 g/dL), splenomegaly (564±71mg) and an increase in the Mac1+Gr1lo myeloid cells in the peripheral blood (fc = 10.04, p &lt; 0.0001) and spleen (fc = 48.56, p &lt; 0.0001) compared to WT. Strikingly, transplantation of AEK FL cells did not result in a more aggressive disease but led to the amelioration of key features of the MPN phenotype associated with KrasG12D/+, including restoration of normal white blood cell count and hemoglobin levels, reduction in spleen size (fc = 0.69, p &lt; 0.05) and a decrease in the number of Mac1+Gr1lo myeloid cells in the peripheral blood (fc = 0.54, p &lt; 0.05) and spleen (fc = 0.51, p &lt; 0.05) compared to KrasG12D/+ .
We reasoned that the amelioration of the MPN phenotype in AEK FL recipients might be due to disease propagating HSCs being functionally compromised by both mutations in combination. Numbers of phenotypic donor HSCs (CD45.2 Lin-Sca1+cKit+Flt3-CD150+) were increased in Aml1ETO/+ FL recipients compared to WT (fc = 5.17, p &lt; 0.0001) and were normal in recipients of KrasG12D/+ FL. In comparison with Aml1ETO/+ FL recipients, numbers of HSCs were markedly reduced in AEK FL recipients (fc = 0.30, p &lt; 0.0001) supporting the idea that KrasG12D/+ has a specific detrimental effect on Aml1ETO/+ HSCs. Secondary transplantation revealed an increase in myeloid reconstitution (fc = 3.37, p &lt; 0.01) and HSC number (fc = 9.64, p &lt; 0.05) in Aml1ETO/+ mice compared to WT. In contrast, BM from primary recipients of KrasG12D/+ or AEK FL showeda lack of engraftment in secondary recipients and markedly reduced HSC number. These results indicate that in the absence of a signaling pathway mutation, Aml1ETO/+ confers a clonal advantage to HSCs. However, KrasG12D/+ has a marked detrimental effect on Aml1ETO/+ expressing pre-leukemic HSCs leading to a clonal disadvantage due to loss of phenotypic and functional HSCs.
RNA-sequencing of WT, Aml1ETO/+, KrasG12D/+ and AEK HSCs revealed an enrichment for E2f (p &lt; 0.01) and Myc (p &lt; 0.01) targets in AEK HSCs compared to Aml1ETO/+ HSCs, indicating an increase in cell cycle activation. This was confirmed by flow cytometry as we found a marked decrease in AEK HSCs in G0 compared to Aml1ETO/+ (fc = 0.16, p &lt; 0.001). Several candidate genes have been identified, including Gzmb (log2FC = -2.21, p &lt; 0.0001) and Gja1 (log2FC = -3.65, p &lt; 0.0001) which were both down-regulated in Aml1ETO/+, but up-regulated in AEK HSCs (Gzmb log2FC = 0.96, p &lt; 0.0001; Gja1 log2FC = 2.76, p &lt; 0.0001). Knock-outs of Gzmb and Gja1 have been shown to increase HSC reconstitution (PMID 24752302) and cause an expansion of the Lin-Sca1+cKit+ compartment (PMID 16531325) respectively, indicating a potential role of the down-regulation of these genes for pre-leukemic HSC expansion.
Our data provides evidence that KrasG12D/+ has a marked detrimental impact on Aml1ETO/+ pre-leukemic HSCs, associated with distinct transcriptional signatures, helping to explain why signaling mutations such as KRAS mutations are not observed within pre-leukemic HSCs in AML patients.

Disclosures
Mead: BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.