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K-Ras mutation depletes pre-leukemic HSC
KM mice compared to CON, likely reflecting increased mobilization associated with myeloproliferation. The increase in LMPP was reversed in AKM mice (Online Supplementary Figure S3). Together, these data demonstrate that when Aml1-ETO is co-expressed with K-RasG12D the myeloproliferative phenotype caused by K-RasG12D is ameliorated rather than enhanced.
K-RasG12D reverses the hematopoietic stem cell expansion associated with Aml1-ETO
As the co-expression of Aml1-ETO and K-RasG12D was insufficient to induce acute leukemic transformation, this provided an ideal model to study the impact of these mutations on pre-leukemic HSC. Here, expression of the mutations from their endogenous loci (rather than through viral transduction) is crucial in order to ensure faithful expression level of the mutations within the hematopoietic hierarchy, including the HSC compart- ment. We reasoned that the myeloproliferative phenotype may have been ameliorated when Aml1-ETO and K- RasG12D were co-expressed due to loss of disease propa- gating HSC. We therefore analyzed LSKCD150+Flt3– phe- notypic HSC eight weeks post-poly(I:C). The SLAM marker CD48 was not used as it was previously reported that CD48 expression is dysregulated in Aml1 deficient HSC.32
We observed an expansion of HSC expressing Aml1- ETO compared to CON (5-fold increase, P<0.0001) (Figure 3A and B). There was no significant difference in HSC number expressing K-RasG12D compared to CON. However, when K-RasG12D was co-expressed with Aml1-ETO the HSC expansion caused by Aml1-ETO was reversed (3-fold decrease, P<0.0001) (Figure 3A and B).
To determine if K-RasG12D is detrimental to the func- tion of Aml1-ETO-expressing HSC we performed serial transplantations. In secondary recipients, Aml1-ETO- expressing cells showed an increase in myeloid reconstitu- tion over time and a 10-fold increase in phenotypic HSC number compared to CON (Figure 3C and D), indicating a competitive advantage, possibly due to an enhanced self- renewal capacity. In contrast, secondary transplant with K-RasG12D-expressing cells, or in combination with Aml1-ETO, showed markedly decreased myeloid recon- stitution and HSC number (Figure 3C and D). The lack of engraftment following bulk BM transplantation from AKM mice also supports the concept that AKM progenitor cells (which would be included in bulk BM transplants) do not acquire aberrant self-renewal capacity. Furthermore, Aml1-ETO-expressing LSK cells showed increased replat- ing potential in vitro (Figure 3E), in keeping with previous reports.16 In contrast, the additional expression of K- RasG12D abrogated this enhanced replating potential (Figure 3E). Collectively, these results support the concept that Aml1-ETO expression is associated with increased self-renewal of HSC. But in the additional presence of K- RasG12D, HSC are at a competitive disadvantage, consis- tent with functional impairment of AKM HSC.
K-RasG12D expression induces loss of quiescence in Aml1-ETO-expressing hematopoietic stem cells
To gain molecular insight into the functional impair- ment of HSC expressing K-RasG12D, we carried out RNA sequencing of AM, KM, AKM and CON CD45.2 LSKCD150+Flt3– cells from competitively transplanted recipients eight weeks post-poly(I:C) (n=5 replicates per
genotype). Gene set enrichment analysis (GSEA) revealed a marked enrichment in E2F, Myc, and G2M checkpoint associated gene expression in AKM compared to AM, likely indicating higher levels of cell cycle activity. In keep- ing with the observed functional impairment of HSC (Figure 4A-C). Cell cycle activation was confirmed by flow cytometry, showing a 4-fold decrease in quiescent (G0) AKM, compared to AM, HSC (Figure 4D and E). This was accompanied by loss of HSC self-renewal-associated gene expression and an acquisition of gene expression associated with granulocyte-macrophage progenitors (GMP) that lack Gata1 expression that give rise to neu- trophils and monocytes (Figure 4F-H).33 This is consistent with phenotypic HSC from AKM mice showing transcrip- tional signatures of myeloid progenitor cells rather than HSC.
To identify genes that may be involved in the loss of quiescence and HSC function, we performed differential gene expression analysis. Aml1-ETO expression caused an up-regulation of 52 genes and down-regulation of 36 genes in phenotypic HSC compared to CON (Figure 5A- C). Among the down-regulated genes were Gja1 and Gzmb (Figure 5D and E). K-RasG12D caused more exten- sive disruption of gene expression, with up-regulation of 389 genes and down-regulation of 526 genes compared to CON (Figure 5A and B). Among the up-regulated genes were MAPK pathway genes such as Etv4 and Ccnd1 (Figure 5F and G). Genes that were down-regulated by KRAS activation were down-regulated in KM versus CON and AKM versus CON HSC (Online Supplementary Figure S4A and B), in keeping with activation of this signaling pathway by activated K-RasG12D in both Aml1-ETO positive and negative cells.34
Hematopoietic stem cells co-expressing Aml1-ETO and K-RasG12D showed up-regulation of 319 genes and down-regulation of 482 genes compared to CON (Figure 5A and B). Many of the up- and down-regulated genes in AKM HSC overlapped with KM HSC (Figure 5A and B), indicating that KRAS confers some of the same transcrip- tional changes in both Aml1-ETO positive and negative HSC. This was confirmed in a principal component analy- sis (PCA) and hierarchical clustering of the HSC from all four genotypes which demonstrated that AKM HSC clus- tered closely with KM HSC indicating that K-RasG12D is driving the separation in gene expression in AKM HSC from CON and AM HSC (Online Supplementary Figure S4C-F). However, the majority of these genes were only dysregulated in the presence of both mutations (Figure 5A and B), indicating that the two mutations together collab- orated to induce a distinct pattern of gene expression that only partially overlapped with K-RasG12D or Aml1-ETO regulated genes (Figure 5A-C). Gja1 and Gzmb were up- regulated in HSC co-expressing Aml1-ETO and K- RasG12D when compared to Aml1-ETO (Figure 5C-E).
Gene set enrichment analysis showed an enrichment of oxidative phosphorylation and loss of genes associated with hypoxia in AKM HSC compared to AM (Figure 5H and I and Online Supplementary Table S3). Interestingly, GzmB causes reactive oxygen species (ROS) produc- tion,35,36 which can lead to apoptosis and cell death of HSC, suggesting GzmB expression may lead to increased levels of ROS and apoptosis in AKM HSC. Genes associated with the p53 pathway were also down-regulated in AKM HSC compared to AM (Figure 5J). Loss of Gja1 has been shown to increase p53 levels;37 therefore, increased
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