A Novel in Vitro Model of Pre-Leukemic Competition Reveals “Super-Competitive” TET2-Mutant Hematopoietic Stem Cell Clones

Pre-leukemic disorders, such as MDS or MPN, are well-established models for characterizing disease evolution and transformation into leukemia, allowing the study of clinically-relevant key genetic events such as loss-of-function TET2 mutations. However, the mechanisms by which a TET2-mutant clone gains competitive advantage over its wild-type (WT) counterparts remains unclear due to challenges in modelling cell competition efficiently at scale.

Here, we report a novel in vitro model of single-cell derived clonal competition, demonstrating the existence of TET2-mutant competitor clones that impair wild-type expansion. Leveraging the recently reported PVA-based expansion cultures (Nature, 2019), we developed a cell competition culture protocol which allows single cells to be grown and the resulting clones to be split.

To validate the system, single EPCR⁺ CD45⁺ Sca-1^high CD150⁺ CD48^- (ESLAM Sca⁺) WT cells were expanded for 14 days, after which clones were split and distributed in equivalent cell numbers across multiple wells. 14 days later, flow cytometric analysis and in vivo functional assays showed that the behavior of the original clone was maintained.

Using the CD45.1/CD45.2 congenic system to discriminate between WT (CD45.1) and TET2 clones (CD45.2) we set up clonal competition cultures: Single ESLAM Sca⁺ cells from WT and TET2 knockout mice (Tet2^-/-) were isolated and expanded for 14 days. Clones were split and distributed across 36 competition wells, each with 50% WT cells from one clone and 50% Tet2^-/- cells from another clone, creating a matrix of 3 x 3 competition cultures alongside a WT and Tet2^-/- control for each clone. Flow cytometry analysis of clonal composition revealed the presence of a previously unidentified type of TET2 “super competitor” clone. These super competitors impaired the growth and expansion of wild-type clones, and in some instances, completely depleted their WT counterparts. Albeit rare (1 super competitor identified in approximately every 12 analyzed TET2 competitor clones, n = 192), TET2 super competitors consistently impair or completely suppress the expansion of their wild-type counterparts across all the competition wells.

Interestingly, the data showed that the HSC frequency within the TET2 super competitor clones was lower than that in WT and TET2 competitor clones, denoting that suppression of WT competitors might be attributed to extrinsic factors, rather than being driven by the HSCs themselves. Transcriptomic analysis of the different clone types highlighted a molecular signature of inflammation and iron metabolism.

To further explore this phenomenon, we harvested media from the 28-day competition cultures, and performed Data-Independent Acquisition Mass Spectrometry (DIA-MS). In agreement with the RNA-seq data, DIA-MS analysis revealed the secretion of inflammatory molecules into the media by enhanced TET2 super competitor clones, including Ifit3m, Cd14, and C3, which are central to the innate immune response and macrophage function.

Thus, our data have demonstrated that, while the proportion of phenotypic HSCs within TET2 super competitor clones is reduced, secretion of inflammatory molecules potentiates the expansion of such clones and hinders the self-renewal potential of the WT rival clones.

In conclusion, following the development of a novel in vitro clonal level competition model, we have identified a novel type of pre-leukemic competitor clone. This enables the investigation of molecular and cellular mechanisms with clinical relevance, to better understand how malignant HSCs enhance self-renewal and suppress normal HSC clones in blood cancers.

No relevant conflicts of interest to declare.