Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Genomic analyses have redefined the molecular subgrouping of pediatric acute lymphoblastic leukemia (ALL). Molecular subgroups guide risk-stratification and targeted therapies, but outcomes of recently identified subtypes are often unclear, owing to limited cases with comprehensive profiling and cross-protocol studies. We developed a machine learning tool (ALLIUM) for the molecular subclassification of ALL in retrospective cohorts as well as for up-front diagnostics. ALLIUM uses DNA methylation and gene expression data from 1131 Nordic ALL patients to predict 17 ALL subtypes with high accuracy. ALLIUM was used to revise and verify the molecular subtype of 281 B-cell precursor ALL (BCP-ALL) cases with previously undefined molecular phenotype, resulting in a single revised subtype for 81.5% of these cases. Our study shows the power of combining DNA methylation and gene expression data for resolving ALL subtypes and provides a comprehensive population-based retrospective cohort study of molecular subtype frequencies in the Nordic countries.

The oncofetal protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) belongs to a family of RNA-binding proteins involved in localization, stability, and translational regulation of target RNAs. IGF2BP3 is used as a diagnostic and prognostic marker in several malignancies. Although the prognosis of pediatric B-cell acute lymphoblastic leukemia (B-ALL) has improved, a subgroup of patients exhibits high-risk features and suffer from disease recurrence. We sought to identify additional biomarkers to improve diagnostics, and we assessed expression of IGF2BP3 in a population-based pediatric cohort of B-ALL using a tissue microarray platform. The majority of pediatric B-ALL cases were positive for IGF2BP3 immunohistochemistry and were associated with an increased proliferative phenotype and activated STAT5 signaling pathway. Two large gene expression data sets were probed for the expression of IGF2BP3—the highest levels were seen among the B-cell lymphomas of a germinal center origin and well-established (KMT2A-rearranged and ETV6-RUNX1) and novel subtypes of B-ALL (e.g., NUTM1 and ETV6-RUNX1-like). A high mRNA for IGF2BP3 was associated with a proliferative “metagene” signature and a high expression of CDK6 in B-ALL. A low expression portended inferior survival in a high-risk cohort of pediatric B-ALL. Overall, our results show that IGF2BP3 shows subtype-specificity in expression and provides prognostic utility in high-risk B-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.

Glucocorticoids are a mainstay in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). The glucocorticoid receptor (GR), a ligand-activated transcription factor (TF), mediates their actions. Chromatin occupancy, chromatin-protein networks (chromatomes) and gene programmes of GR are regulated by SUMOylation, a post-translational modification with therapeutic implications in other hematomalignancies. To unravel the GR-SUMOylation crosstalk in B-ALL, we induced hypoSUMOylation in NALM6 B-ALL cells with a SUMOylation inhibitor (SUMOi, ML-792). Genome-wide profiling of GR and SUMO chromatin-binding and chromatin accessibility revealed that hypoSUMOylation augmented GR chromatin occupancy and altered chromatin openness. Association with transcriptome data indicated that the hypoSUMOylation-induced GR-binding sites predominantly repressed genes associated with cell cycle and DNA replication. Consistently, hypoSUMOylation potentiated glucocorticoid-induced cell cycle arrest and growth suppression. Moreover, our proteomic analyses revealed that the protein network of chromatin-bound GR is tightly intertwined with SUMO2/3 and that SUMOylation modulates the stability of the network. The chromatome contained several B-cell TFs with cognate binding motifs found on GR-adjacent chromatin sites, indicating their simultaneous occupancy on chromatin. In sum, our data imply potential for targeting SUMOylation to increase sensitivity to glucocorticoids in B-ALL, supported by ex vivo data of glucocorticoid and SUMOi TAK-981 combination-treated B-ALL patient samples.

ETV6::RUNX1 leukemia is the second most common subtype of childhood B cell acute lymphoblastic leukemia (B-ALL). Although it generally has a low relapse risk, a significant proportion of B-ALL relapses occur within this subtype due to its relatively high incidence. Measurable residual disease at the end of induction therapy is a well-established biomarker predicting treatment outcomes, while no genomic biomarkers are routinely applied in clinics. In this study, we used multiomic data from ETV6::RUNX1 leukemias to identify genomic features predictive of therapy response at disease presentation. In the deeply characterized sub-cohort we discovered that fast-responding cases frequently exhibited the APOBEC mutational signature and the gene expression signature of high cell cycle activity. In contrast, rearrangements of IGK genes were more frequent in slow responders. Additionally, response-related mutations were identified in transcriptional regulators and tumor suppressor genes (INTS1, NF1, TP53). Copy number analysis revealed that fast responders harbored more frequent deletions of chr12 p-arm, leading to transcriptomic changes affecting genes associated with induction therapy response (KRAS, FKBP4), while a shorter gain in chr12 was more common in slow responders. The identified genetic and transcriptomic markers of treatment sensitivity pave the way for improved disease classification at presentation, potentially improving clinical outcomes.