Key Points• TLR1 is upregulated on primitive AML cells.• Agonistic targeting of TLR1/TLR2 induces apoptosis and differentiation of primitive AML cells in vivo.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 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.

Cytokines provide signals that regulate immature normal and acute myeloid leukemia (AML) cells in the bone marrow microenvironment. We here identify interleukin 4 (IL4) as a selective inhibitor of AML cell growth and survival in a cytokine screen using fluorescently labeled AML cells. RNA-sequencing of the AML cells revealed an IL4-induced upregulation of Stat6 target genes and enrichment of apoptosis-related gene expression signatures. Consistent with these findings, we found that IL4 stimulation of AML cells induced Stat6 phosphorylation and that disruption of Stat6 using CRISPR/Cas9-genetic engineering rendered cells partially resistant to IL4-induced apoptosis. To evaluate whether IL4 inhibits AML cells in vivo, we expressed IL4 ectopically in AML cells transplanted into mice and also injected IL4 into leukemic mice; both strategies resulted in the suppression of the leukemia cell burden and increased survival. Notably, IL4 exposure caused reduced growth and survival of primary AML CD34+CD38− patient cells from several genetic subtypes of AML, whereas normal stem and progenitor cells were less affected. The IL4-induced apoptosis of AML cells was linked to Caspase-3 activation. Our results demonstrate that IL4 selectively induces apoptosis of AML cells in a Stat6-dependent manner—findings that may translate into new therapeutic opportunities in AML.

Highlights d In vivo CRISPR screening identifies CXCR4 as a key regulator of AML stem cells d CXCL12 expression in the bone marrow is dispensable for AML development d CXCR4 signaling protects AML cells from oxidative stress and differentiation

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.

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.

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.

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 of 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 suggests that combined strategies that activate macrophages and block CD47 have therapeutic potential in AML.

Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting. The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation. Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production. To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136). Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. Disclosures No relevant conflicts of interest to declare.