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IL1RAP is expressed by tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC). Signaling by IL1 through the IL1R1/IL1RAP complex promotes cancer progression and contributes to the immune suppressive microenvironment in PDAC. The IL1RAP-blocking antibody nadunolimab blocks the signaling of both IL-1a and IL-1b and is currently evaluated in a phase I/IIa clinical study for PDAC (NCT03267316). Cancer-associated fibroblasts (CAFs) are a primary constituent of the PDAC stroma and has previously been shown to be regulated by IL-1. The aim of this study was to explore the functional consequences of nadunolimab treatment on the crosstalk between tumor cells and CAFs. Co-cultures of the PDAC cell line BxPC3 and pancreatic CAFs induced major changes in gene expression of both cell types as determined by RNA sequencing, indicating an extensive communication between the two cell types. Inclusion of nadunolimab to the co-cultures resulted in only 6 differentially expressed genes (padj Citation Format: Nils Hansen, Pablo Peña, Finja Hansen, Petter Skoog, Susanne Larsson Faria, Karin von Wachenfeldt, Carl Högberg, Camilla Rydberg Millrud, David Liberg, Marcus Järås. The IL1RAP-blocking antibody nadunolimab disrupts pancreatic cancer cell and fibroblast crosstalk, reduces recruitment of myeloid cells and inhibits tumor growth [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C055.

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.

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.

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.

Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors, but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Interleukin-1 receptor accessory protein (IL1RAP; IL1R3) is a coreceptor of interleukin-1 receptor type 1 and has been found upregulated on CML stem cells. Here, we show that primitive (CD34+CD38-) CML cells, in contrast to corresponding normal cells, express a functional interleukin-1 (IL-1) receptor complex and respond with NF-κB activation and marked proliferation in response to IL-1. IL1RAP antibodies that inhibit IL-1 signaling could block these effects. In vivo administration of IL1RAP antibodies in mice transplanted with chronic and blast phase CML cells resulted in therapeutic effects mediated by murine effector cells. These results provide novel insights into the role of IL1RAP in CML and a strong rationale for the development of an IL1RAP antibody therapy to target residual CML stem cells.

Dysregulation of cytokines in the bone marrow microenvironment promotes acute myeloid leukemia cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the bone marrow niche in a screening setting is challenging. Herein, 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 acute myeloid leukemia 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 bone marrow 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 acute myeloid leukemia-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.

Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

SummaryFarnesyl pyrophosphate (FPP) is an intermediate in cholesterol biosynthesis, and it has also been reported to activate platelet LPA (lysophos-phatidic acid) receptors. The aim of this study was to investigate the role of extracellular FPP in platelet aggregation. Human platelets were studied with light transmission aggregometry, flow cytometry and [35S]GTPγS binding assays. As shown previously, FPP could potentiate LPA-stimulated shape change. Surprisingly, FPP also acted as a selective insurmountable antagonist to ADP-induced platelet aggregation. FPP inhibited ADP-induced expression of P-selectin and the activated glycoprotein (Gp)IIb/IIIa receptor. FPP blocked ADP-induced inhibition of cAMP accumulation and [35S]GTPγS binding in platelets. In Chinese hamster ovary cells expressing the P2Y12 receptor, FPP caused a right-ward shift of the [35S]GTPγS binding curve. In Sf9 insect cells expressing the human P2Y12 receptor, FPP showed a concentration-dependent, although incomplete inhibition of [3H]PSB-0413 binding. Docking of FPP in a P2Y12 receptor model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP. In conclusion, FPP is an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12 receptor. It could be an endogenous antithrombotic factor modulating the strong platelet aggregatory effects of ADP in a manner similar to the use of clopidogrel, prasugrel or ticagrelor in the treatment of ischaemic heart disease.

Background: The citric cycle intermediate succinate has recently been identified as ligand for the G-protein coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signalling pathways of this receptor in platelets. Methods and Results: Using RT-PCR, we could demonstrate that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y(1) receptor. Light transmission aggregation experiments showed a dose-dependent aggregation induced by succinate reaching a maximum response at 0.5mM. The effect of succinate on platelet aggregation was confirmed with flow cytometry showing increased surface expression of activated GPIIb/IIIa, and P-selectin. Intracellular SUCNR1 signalling was found to result in decreased cAMP levels, Akt phosphorylation mediated by PI3Kβ activation and receptor desensitisation. Further, succinate-induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A(2) and ATP release. The platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y(12) receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate-induced platelet aggregation depends on thromboxane A(2) generation, ATP release and P2Y(12) activation. (Less)

Acute myeloid leukemia (AML) is an aggressive disease associated with poor prognosis, hence, new therapeutic strategies are needed. The AML cells reside in the bone marrow niche and depend on external signals such as cytokines for their growth and survival. In contrast to providing a supporting role, cytokines can act as negative regulators of AML cells. We previously identified that interleukin 4 (IL-4) induces apoptosis of AML cells in a Stat6-dependent manner and demonstrated strong anti-leukemic activity of IL-4 in a murine AML model driven by MLL-AF9 (Peña-Martínez et al., Leukemia 2017). Ectopic expression of IL-4 in AML cells resulted in prolonged survival in C57BL/6 mice, with mice almost devoid of leukemia cells in bone marrow and spleen upon the time of sacrifice. However, the IL-4-induced apoptosis of AML cells did not fully explain the strong antileukemic activity of IL-4 in vivo. To investigate whether the anti-leukemic effect of ectopic IL-4 expression in vivo was microenvironment dependent, we used IL-2 receptor gamma(-/-) immunodeficient mice, which lack a functional IL-4 receptor type I complex, as recipients for transplantation of murine MLL-AF9 AML cells. Notably, there was no antileukemic activity of IL-4 in these mice, suggesting that the in vivo anti-leukemic effect of IL-4 was predominantly microenvironment-dependent. To identify the cell population that was responsible for the antileukemic effect of IL-4 stimulation, we performed flow cytometry analysis of bone marrow and spleen cells in mice exposed to high IL-4 levels. We found that the anti-leukemic activity of IL-4 was associated with a significant expansion of F4/80+ macrophages in both the bone marrow and spleen, which was confirmed by immunohistochemistry. To determine whether macrophages were responsible for the anti-leukemic effect of IL-4, we depleted macrophages with clodronate liposomes in mice receiving IL-4-secreting AML cells. Depletion of macrophages partially rescued the anti-leukemic effect of IL-4, indicating that macrophages stimulated with IL-4 were responsible for the antileukemic activity. Consistent with these findings, in macrophage differentiation assays with either murine or human monocytes, IL-4 enhanced the phagocytosis of leukemia cells. To characterize the IL-4-induced macrophages, we performed RNA sequencing of F4/80+ macrophages from animals exposed to high IL-4 levels. Gene expression analysis revealed IL-4-induced M2 polarization of the macrophages, as key markers of M2 activation such as Arg1, Retnla and Chil3 were upregulated. Moreover, consistent with the in vitro findings, gene set enrichment analysis revealed an enrichment of phagocytosis signatures in the macrophages stimulated with IL-4. Strikingly, we found that exposure of AML cells to IL-4 resulted in strong upregulation of CD47, a 'don't eat me signal', which protects cells from macrophage-mediated phagocytosis. By CRISPR/Cas9-mediated disruption of Stat6 in IL-4 treated AML cells, we found that the CD47 upregulation was Stat6 dependent, recognizing a previously unknown link between IL-4 signalling and CD47 upregulation. Consistent with this finding, combined antibody blockade of CD47 on leukemia cells and IL-4 stimulation of macrophages resulted in enhanced phagocytosis of AML cells. Taken together, this study shows that IL-4 has anti-leukemic activity by activating macrophages, findings that might translate into new immunotherapeutic opportunities in AML and possibly other malignancies. Disclosures No relevant conflicts of interest to declare.

The biology of acute myeloid leukemia (AML) is largely dictated by the complex combinations of mutations that initiate and propagate the disease. It is therefore critical that models accurately reflect the mutation patterns of the specific leukemias under investigation. In recent years, patient-derived xenografts have emerged as one of the most faithful models to study AML and evaluate potential treatments in vivo. However, little is still known about how leukemias are affected by transplantation and consecutive passaging in vivo. To address this issue, we characterized the mutational patterns and clonal dynamics in 19 AML patient samples and 65 corresponding xenografts from primary and secondary recipients by whole-exome sequencing. We found that xenografts from each patient generally displayed the same mutational profile but with striking differences from the corresponding patient sample as a result of extensive clonal competition. Only 1 of 19 patient samples (5%) retained its mutation pattern in vivo and an additional 3 of 19 samples (16%) displayed gains or losses of non-recurrent presumed passenger mutations. However, the remaining 15 of 19 samples (79%) were shown to contain multiple clones with different recurrent AML mutations that engrafted according to one of the four identified patterns of clonal dynamics: loss, expansion, burst and stable. In 5 of 19 samples (26%), we observed a loss or reduction of subclones containing either NRAS, KRAS, CDKN2A, RUNX1T1 or del(19)(p13), giving way to the respective parental clones, despite allele frequencies in the diagnostic samples suggesting these mutations to be present in the entire cell populations. Seven of 19 samples (37%) showed an expansion of a minor subclone in the patient sample to the point where it made up the entire xenograft. This was paralleled by the complete loss of parental clones as well as sibling clones carrying other recurrent AML mutations such as BCOR, NRAS and WT1. Furthermore, two of the expanding subclones (with TET2 loss of heterozygosity and monosomy 7) were present but not detected by sequencing (100x) of the diagnostic samples, showing that xenografts can unmask unexpected clonal complexity and rare subclones in patient samples. Notably, certain expanding subclones were defined by mutations in NRAS and CDKN2A, which were both lost in other patient samples, suggesting that the interplay between driver mutations is complex and that engraftment cannot be predicted solely based on single mutations. In an additional 2 of 19 samples (11%), a subclonal burst was observed. In these cases, subclones with mutations in SMC3 and PTPN11, respectively, expanded in primary recipients but were lost upon secondary transplantation, indicating that subclones may differ not only in their ability to initiate but also to sustain leukemia in vivo. This notion is further supported by the observation that certain subclones that decreased in vivo only did so in secondary recipients. Notably, only 1 of 19 patient samples (5%) displayed a pattern of stable clonal dynamics, where a subclone was retained at its original frequency throughout primary and secondary passaging. In conclusion, patient-derived xenografts are highly representative of the corresponding patient samples, in that the cells maintain their mutation patterns and gain very few, if any, novel driver mutations throughout consecutive passaging in vivo. However, most AML patient samples comprise multiple clones that differ in their ability to initiate and propagate leukemia in vivo. This leads to xenografts with clonal compositions and mutation patterns that are significantly different from the patient samples, demonstrating the need for genetic profiling of individual xenografts for precise studies of AML biology and treatment. Disclosures Fioretos: Cantargia: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Objectives The platelet ADP P2Y 12 receptor which is a target for the antithrombotic drug clopidogrel is also distributed on vascular smooth muscle cells and stimulate contraction. This study investigates whether AZD6140, in contrast to clopidogrel, can inhibit ADP-mediated arterial contractions. Methods Mice were treated with clopidogrel, 50 mg/kg, 24 and 2 h before experiment. Thoracic aorta ring segments from both clopidogrel-treated ( n =5) and untreated ( n =4) mice were mounted in myograph baths. Contractions of human left internal mammary arteries (IMA) and small arteries were studied in an identical manner. Results Clopidogrel treatment per os did not inhibit contractions by the stable ADP analogue 2-MeSADP (10 µM). However, addition of 1 µM AZD6140 in vitro inhibited ADP contraction (% of maximal contraction by 60 mM K + ) both in the clopidogrel-treated, from 64% to 32% ( P =0.002) and in the untreated group, from 59% to 33% ( P =0.015). 2-MeSADP contractions in human IMA and small arteries were inhibited by AZD6140. Conclusions The antiplatelet drug AZD6140 blocks the contractile effects of ADP in both murine and human vasculature. These effects of AZD6140 could be beneficial in the management of conditions in which vasospasm may play a role.

Patient-derived xenografts have emerged as an attractive model to faithfully recapitulate acute myeloid leukemia (AML) in vivo and to test the therapeutic efficacy of new treatment regiments. The most efficient host for AML cells appears to be the NSGS mouse strain engineered to express the human cytokines SCF, GM-CSF and FLT3L. However, little is known about how the underlying genomics affect engraftment in this model and how the genomics and clonality of the patient samples are affected by the successional passaging in vivo. To address these questions, we transplanted cells from 27 AML patients to NSGS mice. First, we correlated engraftment to leukemia-associated mutations determined by whole-exome sequencing. The frequency of engraftment in primary recipients for the most common mutations in AML were as follows; FLT3-ITD: 29% (2/7), NPM1: 75% (9/12), DNMT3A: 55% (6/11), IDH1/2: 54% (7/13), TET2: 100% (5/5), all samples: 54% (13/24). Although samples carrying the FLT3-ITD mutation engrafted with relatively low frequency, they generated more prolific disease, with cell numbers several fold higher than for any other patient. Samples transplanted to multiple mice showed strikingly similar characteristics. Next, we determined changes in mutation patterns and clonal composition by whole-exome sequencing of human myeloid cells sorted from primary and secondary recipient mice. For all the 11 patients analyzed, the variant allele frequencies (VAF) of the mutations found in the patient sample were increased to or maintained at around 50% by the first passage in vivo. This corresponds to a heterozygous mutation being present in the whole cell population and indicates that the xenotransplantation model enriches the leukemic cells. Importantly, no novel mutations in known AML-associated genes were detected after either the first or second passage in mice, demonstrating that the genotype of the patient sample is preserved during expansion in vivo. We then studied the clonal evolution in the 3 patients who presented with multiple clones. Notably, all 3 cases displayed drastic changes in the allele frequencies of specific mutations. One of the patients had what appeared to be 2 clones at diagnosis, a major clone with 8 AML-associated mutations (VAF 30-55%) and a minor clone with an additional NRAS mutation (VAF 5%). After one passage in vivo, the BCOR mutation in the major clone had disappeared (VAF 52% to 0%), while the other mutations in the major clone were maintained or slightly increased and the NRAS mutation had increased drastically (VAF 5% to 46%). This shows that the minor clone containing the NRAS mutation must have branched from a cell with all the presumed major clone mutations except that in BCOR and that this subclone completely outcompeted the major clone in vivo. This clonal composition also remained upon secondary transplantation. The second patient presented with a major clone carrying 9 mutations (VAF 35-55%) and a small subclone containing an SMC3 mutation (VAF 2%). Also in this case, the small subclone vastly outgrew the presumed founding clone in multiple mice to a VAF of 25-35%. The third patient carried mutations in 11 genes at diagnosis. Upon in vivo passaging, the lowest-frequency mutation, in IDH2, had markedly increased (VAF 10% to 36%), whereas the 3 second-lowest frequency mutations had completely disappeared (VAF 20-23% to 0%), while the high-frequency mutations remained at close to 50%. This development reveals 3 subclones at diagnosis; a founding clone that had given rise to 2 independent subclones, the largest of which was lost upon transplantation and the smallest of which vastly expanded. Hence, for all 3 patients with multiple clones, the smallest subclone drastically expanded in vivo at the expense of the others. This may reflect the biology in the patient, where subclones can only reach detectable levels by expanding much more rapidly than the founding clone. We show that this process continues in the xenografts and may thus model the evolution from diagnosis to relapse. In conclusion, our results suggest that AML patient cells generally maintain their genotype during passaging in vivo but that clonal competition drastically alters the mutational landscape, emphasizing the need for genetic characterization of patient-derived xenografts. Disclosures Fioretos: Cantargia: Equity Ownership.

Farnesyl pyrophosphate (FPP) is an intermediate in cholesterol biosynthesis, and it has also been reported to activate platelet LPA (lysophosphatidic acid) receptors. The aim of this study was to investigate the role of extracellular FPP in platelet aggregation. Human platelets were studied with light transmission aggregometry, flow cytometry and [35S]GTPγS binding assays. As shown previously, FPP could potentiate LPA-stimulated shape change. Surprisingly, FPP also acted as a selective insurmountable antagonist to ADP-induced platelet aggregation. FPP inhibited ADP-induced expression of P-selectin and the activated glycoprotein (Gp)llb/llla receptor. FPP blocked ADP-induced inhibition of cAMP accumulation and [35S]GTPγS binding in platelets. In Chinese hamster ovary cells expressing the P2Y12 receptor, FPP caused a right-ward shift of the [35S]GTPγS binding curve. In Sf9 insect cells expressing the human P2Y12 receptor, FPP showed a concentration-dependent, although incomplete inhibition of [3H]PSB-0413 binding. Docking of FPP in a P2Y12 receptor model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP. In conclusion, FPP is an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12 receptor. It could be an endogenous antithrombotic factor modulating the strong platelet aggregatory effects of ADP in a manner similar to the use of clopidogrel, prasugrel or ticagrelor in the treatment of ischaemic heart disease.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34(+) cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML.

Acute myeloid leukemia (AML) is a fatal disease that contains rare immature cells with self-renewal and leukemia-initiating capacity, known as leukemia stem cells (LSCs). Because current therapies are inefficient in eradicating LSCs, new therapies are warranted that efficiently target this cell population. One strategy being explored towards new therapies is identification of novel therapeutic targets on the cell surface of AML stem cells. To identify a cell surface protein upregulated on LSC, we used flow cytometry to measure the expression level of 13 cell surface proteins on immature leukemic cells from 18 AML patients and bone marrow cells from 7 healthy controls. We identified Toll-like receptor 1 (TLR1) as significantly upregulated in the immature CD34+CD38-compartment compared to corresponding normal cells that were almost devoid of TLR1 expression. These findings are consistent with elevated TLR1 mRNA levels observed in MDS patients (Wei et al, Leukemia, 2013). To evaluate the role of Tlr1 on immature leukemic cells, we performed shRNA-mediated inhibition of Tlr1 in MLL-AF9-expressing murine c-Kit+ leukemic cells. By using lentiviral vectors expressing the Tlr1-shRNAs along with a puromycin resistance gene, we identified two unique shRNAs that successfully suppressed the Tlr1 transcript and protein expression in comparison to a shRNA control. We next co-expressed the two Tlr1-shRNAs along with GFP in leukemia cells and monitored the percentage of GFP positive cells over time. Expression of the Tlr1-shRNAs resulted in strong depletion of the leukemic cells both in vitro and in vivo relative to the control shRNA. These findings suggest that TLR1 is important for the growth and survival of leukemic cells. To further address the role of Tlr1 on leukemic cells, we stimulated the leukemic cells with Pam3CSK4, a specific Tlr1/2 agonist. Pam3CSK4 alone was added to in vitro cultures of leukemic cells for three days, leading to increased survival and a slight increased number of leukemic cells. However, flow cytometric analysis revealed a differentiation shift of cells stimulated with Pam3CSK4 indicated by a decreased expression of the immature cell surface marker c-Kit and an increased expression of the myeloid linage marker Mac-1. To evaluate how Pam3CSK4 affects LSCs, we added Pam3CSK4 to ex vivo-cultures of leukemic cells for 3 days and then transplanted the cells into sublethally irradiated mice. Blood samples after two weeks showed a decreased leukemic burden in mice receiving Pam3CSK4-stimulated cells compared to controls. These findings suggest that enforced TLR1/TLR2-signaling causes differentiation of LSCs. In summary, this study demonstrates that TLR1 is upregulated on AML-stem cell enriched patient cells and that TLR1 expression is finely balanced to maintain LSCs. More specifically, our data suggest that the leukemic cells require Tlr1-expression for survival, but enhanced Tlr1/Tlr2-activation force the LSCs into differentiation. Hence, our study suggests that approaches aiming either for inhibition or enforced activation of TLR1 in AML should be explored further towards a potential new AML therapy. Collectively, we here identify TLR1 as a novel and promising candidate therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.

Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To ultimately achieve a cure for CML, there is a need to establish improved disease models allowing increased understanding of disease pathogenesis and to evaluate novel therapeutics. Aim: In this study, we investigated if retroviral expression of BCR-ABL1 in cord blood (CB) CD34+ progenitor cells transplanted into NOD/SCID IL2–receptor gamma deficient (NSG) mice would recapitulate features observed in CP CML. Methods: CD34+ progenitor cells from human CB were transduced with a retroviral vector expressing BCR-ABL1 and subsequently transplanted into NSG mice. Mice were sacrificed at signs of illness and bone marrow (BM) and spleen cells were analyzed using flow cytometry, immunohistochemistry, FISH and RNA sequencing. Secondary transplants were also performed. BM mononuclear cells (MNC) from CP CML patients were analyzed using flow cytometry and FISH. Results: For the majority of the BCR-ABL1 transplanted (BA) mice, signs of disease were evident within 100 days post transplantation. Spleens were enlarged and both control and BA mice showed robust human engraftment in both BM and spleen. Expression of BCR-ABL1 also induced expansion of the transduced cells. In addition to the previously described features of this humanized transplantation model such as a mild expansion of human myeloid cells, BCR-ABL1 expression induced an inflammatory-like state in the BM and spleen of the BA mice as evident by an increase of human macrophages/histiocytes and T cells. In addition, an expansion of aberrant mast cells in BA mice was observed. Secondary transplantations failed to induce a similar disorder as in the primary mice, but long-term engraftment was achieved, showing only mast cells in secondary BA mice. These mast cells aberrantly expressed CD25 (IL2RA), a cell surface receptor normally only expressed on neoplastic mast cells. This was in contrast to secondary control mice where the long-term engraftment was dominated by B cells. Because CP CML can progress into lymphoid BC, we next explored if BCR-ABL1 expression in CB CD34+ cells would show a disturbance also of the B cell lineage. We observed a striking block at the pre B cell stage, resulting in an accumulation of pre B cells in the BM of BA mice. RNA sequencing of sorted BCR-ABL1-expressing pre B cells from BA and control mice revealed that BCR-ABL1 expression results in a significant de-regulation of about 700 genes. Among the up-regulated genes, many were shown to be involved in inflammation and apoptosis. Finally, by sorting BM MNCs from patients with CP CML into different populations of B cell progenitors and performing FISH for BCR-ABL1, we confirmed that a similar block in B cell differentiation is present in CP CML patients. Conclusion: This humanized mouse model of CML reveals previously unexplored features of CP CML, including expansion of aberrant mast cells and a block at the pre B cell stage, and should provide a valuable model for future studies of mechanisms involved in the disease pathogenesis of CML. Disclosures No relevant conflicts of interest to declare.

Background Mild hypothermia is currently standard of care for cardiac arrest patients in many hospitals and a common belief is that hypothermia attenuates platelet aggregation. We wanted to examine the effects of clopidogrel on platelet aggregation during hypothermia. Methods Platelet reactivity at 37°C and 33°C was evaluated by light transmission aggregometry and vasodilator-stimulated phosphoprotein (VASP) in blood from healthy volunteers before, and 24 hours after, a 600 mg loading dose of clopidogrel. Results Collagen, 5-HT, epinephrine, U46619 and ADP-induced platelet aggregation was unaltered or even increased by hypothermia. After clopidogrel, there was a significant increase in platelet aggregation for 5 and 20 μM ADP at 33°C compared to 37°C (46 ± 5 vs. 34 ± 5% and 58 ± 4 vs. 47 ± 4%, p < 0.001, n = 8). Hypothermia also increased ADP-induced aggregation after pretreatment with the P2Y1 antagonist MRS2500. The decreased responsiveness to clopidogrel during hypothermia could be overcome by addition of the reversible P2Y12 antagonist AZD6140. ADP-induced inhibition of VASP-phosphorylation was unaffected by hypothermia both in the presence and absence of clopidogrel. A dose-response curve for ADP-induced platelet aggregation revealed increased potency for ADP during hypothermia with no difference in efficacy. Conclusion Mild hypothermia did not attenuate platelet aggregation, instead it even increased ADP-stimulated platelet aggregation after clopidogrel treatment. Dual platelet inhibition with aspirin and a P2Y12 receptor antagonist is probably needed for patients with acute coronary syndromes treated with mild hypothermia, and it is possible that future ADP blockers could be of benefit.

Myeloid malignancies, including chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML), display upregulation of IL1RAP at the cell surface of the primitive (CD34+CD38-) leukemic cells (Järås et al. PNAS, 2010; Barreyro et al. Blood, 2012; Askmyr et al. Blood 2013). IL1RAP is an essential co-receptor for the interleukin-1 (IL1R1) and the interleukin-33-receptors (IL33R, ST2) to convey signals upon binding by their ligands IL1 and IL33, respectively. IL1RAP has previously mainly been studied in the context of inflammation and little is known about its role in normal hematopoiesis and if it’s upregulation in malignant hematopoiesis is of pathogenetic importance. In this study, we first characterized normal hematopoiesis in Il1rap(-/- ) mice, and discovered that these mice displayed a reduction in myeloid cells in the peripheral blood (9.8x10^5 vs. 1.68x10^6 myeloid cells per mL, p=0.0076), suggesting that Il1rap is involved myelopoiesis. To test whether Il1rap regulates normal hematopoietic stem cells (HSC), we performed competitive stem cell transplantations, and demonstrated that Il1rap-/- HSC gave rise to equal donor contribution as Il1rap+/+ HSC, even in secondary transplantations, suggesting that Il1rap is dispensable for normal HSC function. As IL1RAP is upregulated in myeloid neoplasms but it has been unclear whether this upregulation is functionally involved in disease biology, we next explored if IL1RAP upregulation alone is sufficient to cause features of a myeloid neoplasm. To this end, we retrovirally expressed IL1RAP along with GFP in cord blood (CB) CD34+ cells and transplanted the cells to NSG mice. At 200 days post transplantation, a striking myeloid lineage skewing (67% N=6, vs. 34% N=3, CD33+ among GFP+ cells in bone marrow, p=0.031), accompanied by a reduction of CD19+ cells (21% vs. 53%, p=0.041), was observed in mice that had received IL1RAP overexpressing (IL1RAP+) cells. In addition, mice that had received IL1RAP+ cells displayed enlarged spleens (102 mg vs. 71mg, p=0.034) and showed a myeloid cell expansion when compared to MIG control mice (10% vs. 3.7% CD33+ among GFP+ cells, p=0.035). Having demonstrated that IL1RAP is involved in steady-state myelopoiesis and that IL1RAP upregulation leads to myeloid lineage skewing, we next investigated if primary CML primitive cells display a different sensitivity for cytokines (IL1B and IL33) that signal through IL1RAP-associated receptors. CB and CML cells were stimulated with either single cytokines or cytokines in combination with SCF, in serum free liquid cultures. Whereas IL1B did not affect the in vitro proliferation of normal CB CD34+ cells, primary CML CD34+ cells, and particularly CML CD34+CD38- cells, showed a strong response to IL1B stimulation with more than 10-fold higher cell counts compared to unstimulated CML CD34+CD38- cells, following 7 days of culture (5.6x103 vs. 7.5x104 cells p= 0.006), while IL33 had minor effects (5.6x103 vs. 8.5x103 p=0.040), suggesting that IL1RAP upregulation may render primitive malignant myeloid cells hypersensitive to IL1B stimulation. In summary, these findings demonstrate that IL1RAP is involved in steady state myelopoiesis and that enforced IL1RAP expression, in cord blood CD34+ cells, alone is sufficient to induce features of a myeloproliferative disorder in mice. Primitive CML cells with upregulation of IL1RAP at the cell surface were more sensitive to IL1B compared to corresponding normal cells, which were unaffected. Collectively, these observations suggest that IL1RAP upregulation may contribute to the pathogenesis of myeloid neoplasms. Disclosures: Richter: Cantargia: Consultancy, Equity Ownership. Järås:Cantargia: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors (TKIs) but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Thus, there is still a need for novel treatment strategies in CML. We have previously shown that Interleukin 1 receptor accessory protein (IL1RAP), a co-receptor of IL1R1, is highly expressed on primitive CML cells and that a polyclonal IL1RAP antibody can direct natural killer (NK) cells to specifically target and destroy CD34+CD38- CML cells in an in vitro-based antibody dependent cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). The aim of the present study was to investigate the consequences of IL1RAP expression on primitive CML cells and the in vivo therapeutic efficacy of monoclonal IL1RAP antibodies against CML cells. Primary chronic phase (CP) CD34+ CML cells were cultured in medium supplemented with cytokines known to signal through receptor complexes involving IL1RAP. The addition of IL1 to the cultures resulted in a marked cellular expansion specifically for the primitive CD34+CD38- CML cells. Moreover, the CD34+CD38- cells showed phosphorylation of the downstream mediator of IL1-signaling NFKB. RNA-sequencing confirmed the activation of NFKB and of genes involved in cell cycling, indicating that IL1 stimulation of CD34+CD38- CML cells induced proliferation. Upon addition of an IL1RAP antibody capable of blocking IL1-signaling to the suspension cultures, the IL1-induced expansion and NFKB phosphorylation of CD34+CD38- CML cells was suppressed. Interestingly, both the IL1RAP expression and the response to IL1 as measured by NFKB phosphorylation was retained during TKI treatment of the cells. To assess the in vivo effects of IL1RAP antibodies in CML models, we first engrafted NOD/SCID mice with BCR/ABL1 expressing BV173 cells and treated the mice with the monoclonal IL1RAP antibody mAb81.2. Mice receiving treatment with mAb81.2 displayed a prolonged survival compared to controls, accompanied by reduced levels of leukemic cells in the BM. In vitro studies showed that mAb81.2 lacked a direct effect on cellular expansion or apoptosis. Instead, the IL1RAP antibody could direct NK cells to elicit killing of the leukemic cells, thereby suggesting effector cell mediated mechanisms to be an important in vivo mode-of-action. To validate the in vivo effects on primary CML cells, we next engrafted CP or blast phase (BP) CML cells into immunodeficient mice. Following engraftment of CP CD34+ CML cells into NSG mice and subsequent treatment with mAb81.2, a reduction of human myeloid cells was observed, suggesting that the treatment targeted the leukemic graft. Importantly, mAb81.2 treatment also reduced the levels of candidate CD34+CD38-IL1RAP+ CML stem cells. Finally, BP CML cells were engrafted into NOD/SCID mice that have a more intact effector cell function compared to NSG mice. Following treatment with mAb81.2 a significant reduction of leukemic cells in the BM as well as in the periphery was observed compared to control mice. Importantly, secondary transplantations revealed a therapeutic effect also on the BP CML stem cells. In vitro ADCC assays confirmed that CML BP cells, including a sample with the highly TKI-resistant T315I mutation, could be targeted and killed using mAb81.2. We conclude that IL1RAP antibodies can suppress IL1-induced expansion of primitive CML cells and that in vivo administration of IL1RAP antibodies in CML xenograft models has anti-leukemic effects that extend to the CML stem cells. These results show that an antibody-based therapy against IL1RAP can be used to efficiently target CML stem cells. Disclosures Richter: BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Järås:Cantargia AB: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership.

SUMMARY 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., In Brief In an in vivo CRISPR screen, Ramakrishnan et al. identify CXCR4 as a critical regulator of AML stem cells. Although the CXCR4 ligand CXCL12 is dispensable for leukemia development, CXCR4 signaling is essential for AML cells because it protects them from differentiation., Graphical Abstract