HSC/MPP-like Signature Predicts Inferior Outcome of Multiple Pediatric Leukemias
Authors: Zhang Jingliao , Zong Suyu , Duan Yongjuan , Zhao Bei-bei , Li Jun , Chen Xiaoli , Yang Wenyu , Wu Peng , Zhu Xiaofan , Zhang Yingchi 
Published: December 2024

Acute leukemia (AL) as the most common childhood malignancy widely encompasses acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and Acute leukemias of ambiguous lineage (ALALs). The pathogenesis of AL is mainly attributed to the malignant transformation of hematopoietic stem/progenitor cells (HSPCs) into leukemic stem cells (LSCs) and the consequent blockage of normal hematopoietic differentiation. Currently, the mechanism of heterogeneous LSCs contributing to relapse and resistance to therapies remains to be challenging issues.

Following the designed sorting strategies to enrich leukemic cells and healthy bone marrow mononuclear cells (BMMCs) or stem/progenitor cells, we carried out high-throughput droplet-based 5′-single-cell RNA-sequencing (scRNA-seq) from four subtypes (B-ALL, T-ALL, AML, and MPAL) of pediatric leukemia at multiple stages and healthy donors, and identified manually curated pediatric hematopoietic differentiation landscape as HSC/MPP, erythroid, myeloid, and lymphoid) subclusters referring to expression patterns of marker genes. By projecting leukemic signatures from 23 pediatric acute leukemia patients at diagnosis onto this reference profiling of hematopoietic differentiation landscape. Notably, B-ALL and T-ALL blasts were dominantly projected onto LMPP-like and CLP-like subclusters, and the AML leukemic cells were broadly partitioned into granulocyte-monocyte progenitor-like and monocyte-like subclusters. Intriguingly, we observed significantly higher frequencies of HSC/MPP-like signatures in 4 of the 6 T-ALL, 2 of the 5 MPAL, and all of the 4 AML leukemic blasts, raising the lack of HSC/MPP signatures in our B-ALL cohort. With molecular heterogeneities in progenitor subclusters of MPALs, it was noteworthy that HSC/MPP-like signatures in T/Myeloid MPALs were presented with higher frequencies in contrast to those in B/Myeloid MPALs and the KMT2A-rearanged MPAL.

To understand the dynamics in proportional leukemic subclusters under chemotherapeutic pressure, we mapped transcriptomic signatures of longitudinal leukemia for the KMT2A-rearranged MPAL patient, who followed a standardized chemotherapy regimen for ALL and subsequently underwent umbilical cord blood transplantation due to progressive residual leukemia. Against a backdrop of continuous increases in measurable residual diseases (MRD) during hematopoiesis reconstitution, the LMPP-like subclusters demonstrated a decrease, while HSC/MPP-like subclusters strikingly emerged and gradually expanded over the course of treatment. Though not attaining dominance, the persistent growth of HSC/MPP-like signatures stood out as a salient observation within this dynamic context, inciting speculation of a potential correlation with chemoresistance.

Further assessment of differential expressed genes intersection within HSC/MPP subclusters across the three leukemia subtypes enriched 146 up-regulated genes and 122 down-regulated genes, with 24 genes exhibited ≥ 2-fold expression differences in the context of upregulated genes. We applied Cox proportional-hazards model to interrogate core biomarkers representing stemness that were also correlated with long-term outcomes across multiple pediatric leukemia, yielding to an ultimate 9 genes with robustness across leukemic subtypes. The predictive power of the 9-genes stemness-score in chemoresistance and long-term outcome was further validated from internal and external leukemic cohorts.

In this study, we utilized scRNA-seq to establish a differentiation landscape of normal HSPCs from HD and mapped multiple subtypes of leukemic cells on the hematopoietic differentiation roadmap. Notably, we found that HSC/MPP-like cells were commonly present in three out of the four leukemic subtypes. With longitudinal tracing, we discovered the gene expression signature of those leukemic cells was closely associated with leukemic molecular behaviors as well as clinical outcomes. Furthermore, the HSC/MPP-like signature was validated to be prominently correlated with chemoresistance and inferior long-term outcomes, warranting further mechanistic investigations.