Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Abstract

Background Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.

Comprehensive multi-omics single-cell data integration reveals greater heterogeneity in the human immune system

“…3A and 3B). Of note, SuPERR-seq matched the B-cell subclusters to the different stages of their developmental pathway 33 (Fig. 5).…”

IGF2BP3 Associates with Proliferative Phenotype and Prognostic Features in B-Cell Acute Lymphoblastic Leukemia

“…However, no association with outcome was evident for the IGF2BP3 protein in our population-based biopsy cohort, possibly because of its relatively small size or low number of events. A recent single cell analysis of early therapy response in B-ALL showed that patients with high proliferative features are more sensitive to induction chemotherapy, and that therapy-resistant clones are more likely among the quiescent cell populations [ 37 ]. Hence, IGF2BP3 expression is associated with cell populations that are actively dividing and more are likely to be killed by chemotherapy, which is reflected in the overall outcome of patients.…”

Single-Cell Sequencing: Biological Insight and Potential Clinical Implications in Pediatric Leukemia

“…Mehtonen et al used scRNA-seq for investigating cell states and transcription factor activities during normal B-cell differentiation and compared those to leukemic cell profiles of six patients with ETV6/RUNX1 B-ALL at diagnosis and during induction therapy (day 15) [ 58 ]. Their results suggested that ETV6/RUNX1 -positive leukemic cell states are most similar to a pro-B state, differ between patients in cell cycle activity, express genes that reprogram the immune microenvironment, and that the induction therapy may modulate leukemic cells towards a pre-B state.…”

“…However, the clinical significance of these findings needs further clarification, as it is yet unknown whether blasts at various developmental changes or with variable maturation potential have indeed fitness advantage during chemotherapy. In ETV6 - RUNX1 + ALL, scRNA-seq revealed elevated activity of B-lineage differentiation transcription factors (TFs), also including the former leukemia genome-wide association study hit ETS domain-containing protein Elk-3 (ELK3) [ 58 ]. Leukemic TF activities persist during chemotherapy and thus provide potentially exploitable vulnerability option to overcome resistance.…”

“…In line with this, leukemia samples as well as lymphoma cells circulating in the peripheral blood were the subjects in hematology-focused projects of early days [ 26 , 27 ], with acute myeloid leukemia (AML) being the most frequently published oncohematological entity [ 28 ]. To date, SCS datasets have been generated for a wide range of blood cancers, including chronic myeloid leukemia [ 29 ], myeloproliferative neoplasms [ 30 , 31 ], myelodysplastic syndrome/acute myeloid leukemia [ 21 , 27 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ], acute lymphoblastic leukemia (ALL) [ 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ], chronic lymphocytic leukemia [ 59 , 60 , 61 ], mantle cell lymphoma [ 61 , 62 , 63 ], follicular lymphoma [ 61 , 64 , 65 , 66 ], diffuse large B-cell lymphoma [ 61 ], multiple myeloma [ 26 , 67 ], Hodgkin lymphoma […”