Single-cell genomics details the maturation block in BCP-ALL and identifies therapeutic vulnerabilities in DUX4-r cases.

B cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy, driven by multiple genetic alterations that cause maturation arrest and accumulation of abnormal progenitor B cells. Current treatment protocols with chemotherapy have led to favorable outcomes but are associated with significant toxicity and risk of side effects, highlighting the necessity for highly effective, less toxic, targeted drugs, even in subtypes with a favorable outcome. Here, we used multimodal single-cell sequencing to delineate the transcriptional, epigenetic, and immunophenotypic characteristics of 23 childhood BCP-ALLs, belonging to the BCR::ABL1-positive, ETV6::RUNX1-positive, high hyperdiploid, and recently discovered DUX4-rearranged (DUX4-r) subtypes. Projection of the ALL cells along the normal hematopoietic differentiation axis revealed a diversity in the maturation pattern between the different BCP-ALL subtypes. Whereas the BCR::ABL1-, ETV6::RUNX1-positive, and high hyperdiploidy cells mainly showed similarities to normal pro-B cells, the DUX4-r ALL cells also displayed transcriptional signatures resembling mature B cells. Focusing on the DUX4-r subtype, we found that the blast population displayed multilineage priming toward non-hematopoietic cells, myeloid, and T cell lineages, but also an activation of PI3K/AKT signaling that sensitized the cells to PI3K inhibition in vivo. Given the multilineage priming of the DUX4-r blasts with aberrant expression of the myeloid marker CD371 (CLL-1), we generated chimeric antigen receptor T cells, which effectively eliminated DUX4-r ALL cells in vivo. These results provide a detailed characterization of BCP-ALL at the single-cell level and reveal therapeutic vulnerabilities in the DUX4-r subtype with implications for the understanding of ALL biology and new therapeutic strategies.

Interleukin 4 promotes phagocytosis of murine leukemia cells counteracted by CD47 upregulation.

Cytokines are key regulators of tumor immune surveillance by controlling immune cell activity. Here, we investigated whether interleukin 4 (IL4) has antileukemic activity via immune-mediated mechanisms in an in vivo murine model of acute myeloid leukemia driven by the MLL-AF9 fusion gene. Although IL4 strongly inhibited leukemia development in immunocompetent mice, the effect was diminished in immune-deficient recipient mice, demonstrating that the antileukemic effect of IL4 in vivo is dependent on the host immune system. Using flow cytometric analysis and immunohistochemistry, we revealed that the antileukemic effect of IL4 coincided with an expansion of F4/80+ macrophages in the bone marrow and spleen. To elucidate whether this macrophage expansion was responsible for the antileukemic effect, we depleted macrophages in vivo with clodronate liposomes. Macrophage depletion eliminated the antileukemic effect of IL4, showing that macrophages mediated the IL4-induced killing of leukemia cells. In addition, IL4 enhanced murine macrophage-mediated phagocytosis of leukemia cells in vitro. Global transcriptomic analysis of macrophages revealed an enrichment of signatures associated with alternatively activated macrophages and increased phagocytosis upon IL4 stimulation. Notably, IL4 concurrently induced Stat6-dependent upregulation of CD47 on leukemia cells, which suppressed macrophage activity. Consistent with this finding, combining CD47 blockade with IL4 stimulation enhanced macrophage-mediated phagocytosis of leukemia cells. Thus, IL4 has two counteracting roles in regulating phagocytosis in mice; enhancing macrophage-mediated killing of leukemia cells, but also inducing CD47 expression that protects target cells from excessive phagocytosis. Taken together, our data suggest that combined strategies that activate macrophages and block CD47 have therapeutic potential in acute myeloid leukemia.

Sister chromatid cohesion defects are associated with chromosomal copy number heterogeneity in high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploid acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. The main driver event of this disease is a nonrandom aneuploidy consisting of gains of whole chromosomes but without overt evidence of chromosomal instability (CIN). Here, we investigated the frequency and severity of defective sister chromatid cohesion-a phenomenon related to CIN-in primary pediatric ALL. We found that a large proportion (86%) of hyperdiploid cases displayed aberrant cohesion, frequently severe, to compare with 49% of ETV6/RUNX1-positive ALL, which mostly displayed mild defects. In hyperdiploid ALL, cohesion defects were associated with increased chromosomal copy number heterogeneity, which could indicate increased CIN. Furthermore, cohesion defects correlated with RAD21 and NCAPG mRNA expression, suggesting a link to reduced cohesin and condensin levels in hyperdiploid ALL. Knockdown of RAD21 in an ALL cell line led to sister chromatid cohesion defects, aberrant mitoses, and increased heterogeneity in chromosomal copy numbers, similar to what was seen in primary hyperdiploid ALL. In summary, our study shows that aberrant sister chromatid cohesion is frequent but heterogeneous in pediatric high hyperdiploid ALL, ranging from mild to very severe defects, and possibly due to low cohesin or condensin levels. Cases with high levels of aberrant chromosome cohesion displayed increased chromosomal copy number heterogeneity, possibly indicative of increased CIN. These abnormalities may play a role in the clonal evolution of hyperdiploid pediatric ALL.

Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells.

Dysregulation of cytokines in the bone marrow (BM) microenvironment promotes acute myeloid leukemia (AML) cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the BM niche in a screening setting is challenging. Here, we developed an ex vivo cytokine screen using 11 arrayed molecular barcodes, allowing for a competitive in vivo readout of leukemia-initiating capacity. With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 AML mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using Tnfsf13-/- recipient mice, we confirmed that TNFSF13 supports leukemia initiation also under physiological conditions. TNFSF13 was secreted by normal myeloid cells but not by leukemia mouse cells, suggesting that mature myeloid BM cells support leukemia cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner by binding TNFRSF17 and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease. Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of AML-initiating cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.

The complement receptor C3AR constitutes a novel therapeutic target in NPM1-mutated AML.

Mutated nucleophosmin 1 (NPM1) is the most common genetic alteration in acute myeloid leukemia (AML), found in ∼30% of cases. Although mutations in this gene are considered favorable according to current risk stratification guidelines, a large fraction of patients will experience relapse, demonstrating the urgent need for new treatment options. Therefore, we aimed to identify cell surface proteins specifically expressed on NPM1-mutated AML cells, allowing for potential targeting with antibody-based therapies. Herein, we report on an arrayed flow cytometry-based screen directed to 362 cell surface markers. In comparing the cell surface expression on NPM1-mutated AML cells with primitive (CD34+ CD38-) normal bone marrow cells, we identified the complement receptor C3AR as being specifically expressed in NPM1-mutated AML. By flow cytometry and single-cell RNA sequencing, we further show that normal hematopoietic stem and progenitor cells lack detectable C3AR gene and protein expression, making it particularly suitable as a target for antibody therapy. We also demonstrate that C3AR in combination with GPR56 distinguishes the leukemic stem cells (LSCs) in NPM1-mutated AML from the normal hematopoietic stem cells, defining the LSC population, as shown by transplantation into immunodeficient mice. Mechanistically, the stimulation of C3AR-expressing cells with C3a, the ligand of C3AR, leads to the activation of ERK1/2 and increased survival of AML cells, suggesting that this is an important signaling axis in this subtype of AML. Finally, we show that antibodies directed against C3AR efficiently elicit natural killer cell-mediated killing of primary AML cells ex vivo, highlighting C3AR as a candidate therapeutic target in NPM1-mutated AML.

Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity.

Acute myeloid leukemia (AML) is initiated and propagated by leukemia stem cells (LSCs), a self-renewing population of leukemia cells responsible for therapy resistance. Hence, there is an urgent need to identify new therapeutic opportunities targeting LSCs. Here, we performed an in vivo CRISPR knockout screen to identify potential therapeutic targets by interrogating cell surface dependencies of LSCs. The facilitated glucose transporter type 1 (GLUT1) emerged as a critical in vivo metabolic dependency for LSCs in a murine MLL::AF9-driven model of AML. GLUT1 disruption by genetic ablation or pharmacological inhibition led to suppression of leukemia progression and improved survival of mice that received transplantation with LSCs. Metabolic profiling revealed that Glut1 inhibition suppressed glycolysis, decreased levels of tricarboxylic acid cycle intermediates and increased the levels of amino acids. This metabolic reprogramming was accompanied by an increase in autophagic activity and apoptosis. Moreover, Glut1 disruption caused transcriptional, morphological, and immunophenotypic changes, consistent with differentiation of AML cells. Notably, dual inhibition of GLUT1 and oxidative phosphorylation (OXPHOS) exhibited synergistic antileukemic effects in the majority of tested primary AML patient samples through restraining of their metabolic plasticity. In particular, RUNX1-mutated primary leukemia cells displayed striking sensitivity to the combination treatment compared with normal CD34+ bone marrow and cord blood cells. Collectively, our study reveals a GLUT1 dependency of murine LSCs in the bone marrow microenvironment and demonstrates that dual inhibition of GLUT1 and OXPHOS is a promising therapeutic approach for AML.

CXCR4 Signaling Has a CXCL12-Independent Essential Role in Murine MLL-AF9-Driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is defined by an accumulation of immature myeloid blasts in the bone marrow. To identify key dependencies of AML stem cells in vivo, here we use a CRISPR-Cas9 screen targeting cell surface genes in a syngeneic MLL-AF9 AML mouse model and show that CXCR4 is a top cell surface regulator of AML cell growth and survival. Deletion of Cxcr4 in AML cells eradicates leukemia cells in vivo without impairing their homing to the bone marrow. In contrast, the CXCR4 ligand CXCL12 is dispensable for leukemia development in recipient mice. Moreover, expression of mutated Cxcr4 variants reveals that CXCR4 signaling is essential for leukemia cells. Notably, loss of CXCR4 signaling in leukemia cells leads to oxidative stress and differentiation in vivo. Taken together, our results identify CXCR4 signaling as essential for AML stem cells by protecting them from differentiation independent of CXCL12 stimulation.

Clonal competition within complex evolutionary hierarchies shapes AML over time.

Clonal heterogeneity and evolution has major implications for disease progression and relapse in acute myeloid leukemia (AML). To model clonal dynamics in vivo, we serially transplanted 23 AML cases to immunodeficient mice and followed clonal composition for up to 15 months by whole-exome sequencing of 84 xenografts across two generations. We demonstrate vast changes in clonality that both progress and reverse over time, and define five patterns of clonal dynamics: Monoclonal, Stable, Loss, Expansion and Burst. We also show that subclonal expansion in vivo correlates with a more adverse prognosis. Furthermore, clonal expansion enabled detection of very rare clones with AML driver mutations that were undetectable by sequencing at diagnosis, demonstrating that the vast majority of AML cases harbor multiple clones already at diagnosis. Finally, the rise and fall of related clones enabled deconstruction of the complex evolutionary hierarchies of the clones that compete to shape AML over time.

Agonistic targeting of TLR1/TLR2 induces p38 MAPK-dependent apoptosis and NFκB-dependent differentiation of AML cells.

Acute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34+CD38- cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in MLL-AF9-driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine Trp53-/-MLL-AF9 AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine AML1-ETO9a-driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to MLL-rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the MLL-AF9 AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.