The Role of Histone H3-K27M Mutation in Preleukemic HSC Expansion

Mutations affecting the epigenetic landscape are frequent in acute myeloid leukemia (AML) and can cause transcriptional reprogramming and influence cell fate. Our team and others discovered that mutations in histone H3 genes (H3-K27M/I), previously found in aggressive childhood glioma, can also occur in AML (Boileau et al., Nat Commun, 2019; Lehnertz et al., Blood, 2017). These are gain-of-function mutations that inhibit the polycomb repressive complex 2 (PRC2), thus decreasing the repressive H3-K27 trimethylation mark and enhancing global gene expression. We observed that these mutations can occur as an early step in leukemogenesis and are present in preleukemic cells. We also determined that H3-K27M mutation increases the frequency of HSCs, results in a skewed myeloid differentiation, and increases the aggressiveness of AML cells (Boileau et al., Nat Commun, 2019). To better understand how the alterations of HSCs unfold following the acquisition of H3-K27M, we sought to investigate the kinetics of HSC self-renewal and expansion in vivo and examine the transcriptional profile of AML patients carrying H3-K27M. To this end, human CD34+CD38- cells derived from cord blood (CB) were isolated and transduced with either Histone H3-K27M mutant or H3-wt control lentiviral vectors, as done previously. Vectors have a hybrid bidirectional promoter (SFFV/minimalCMV) to drive the expression of both the marker GFP and the insert gene. Four days following transduction, cells were injected intrafemorally into 20 NSG mice per condition. Every 4 weeks, five mice were sacrificed per condition at 4, 8, 12 and 16-week time-points. Cells were then extracted from the femurs, tibias and pelvis as well as the spleen, and grafts were analyzed by flow cytometry. LT- and ST-HSCs, lineage committed progenitors as well as mature lymphoid and myeloid cells were quantified using established CD markers. Our xenotransplantation data revealed that the H3-K27M mutation, but not H3-wt, leads to a large expansion of the HSC, MPP, CMP, GMP and MEP populations that progressively increase after 8 weeks post transplantation, indicating better overall HSCs maintenance and self-renewal. To gain insights into the molecular mechanisms of HSC quiescence, we examined our unpublished single-cell RNA-seq (scRNA-seq) dataset of human CD34+ cells overexpressing Histone H3-K27M (n=21605) or H3-wt control (n=17954), collected at week-16 post transplantation. Our data showed an increased quiescence signature for H3-K27M HSCs, compared to H3-wt. In addition, we performed a horizontal meta-analysis of the TCGA, Leucegene, and beat-AML RNA-seq datasets of bulk AML (969 samples) to identify transcriptional signatures enriched in H3-K27M/I patients (6 samples). Our analysis revealed that primary H3-K27M/I samples are enriched in known HSC and quiescence signatures as well as in genes that we found upregulated in the H3-K27M HSC cluster from our scRNA-seq dataset. This suggests that the transcriptional reprogramming granted by H3-K27M during early leukemogenesis can be maintained after the progression to full-blown AML. Overall, our data indicate that H3-K27M mutation drives preleukemic HSC expansion by activating self-renewal and quiescence programs. This will enable the deciphering of new molecular mechanisms driving the transformation of preleukemic HSCs into leukemic stem cells and provide new insights to improve therapeutic options.

No relevant conflicts of interest to declare.