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Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator IRF2BP2 as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene-expression studies demonstrated that IRF2BP2 represses IL1β/TNFα signaling via NFκB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival.Significance:This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death.See related commentary by Puissant and Medyouf, p. 1617.This article is highlighted in the In This Issue feature, p. 1599

Supplementary Table from Unleashing Cell-Intrinsic Inflammation as a Strategy to Kill AML Blasts

Clonal hematopoiesis of indeterminate potential (CHIP) is a state in which somatic mutations in hematopoietic stem cells lead to clonal expansion of blood cells in individuals without hematologic malignancy. The mutated genes, including TET2, DNMT3A, ASXL1, TP53, JAK2, and SF3B1, are also recurrently mutated in myeloid malignancies. Individuals with CHIP have an increased risk of developing a hematologic cancer. Moreover, individuals with CHIP have an elevated risk of all-cause mortality that is significantly attributable to cardiovascular disease, independent of traditional risk factors. The mechanism for this increased risk is likely linked to increased inflammation driven by mutated macrophages, in part through inflammasome activation. This has broadened our understanding of how chronic diseases are influenced by CHIP and of the mechanistic role of inflammation in these disorders.

Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator IRF2BP2 as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene-expression studies demonstrated that IRF2BP2 represses IL1β/TNFα signaling via NFκB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. Significance: This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death. See related commentary by Puissant and Medyouf, p. 1617. This article is highlighted in the In This Issue feature, p. 1599

Leukemic blasts are immune cells gone awry. We thus hypothesized that dysregulation of inflammatory pathways can maintain a leukemic state. In contrast to traditional cancer immunotherapy, we exploited inflammatory signaling within AML blasts as cell-intrinsic, self-directed immunotherapy. Corroborating the hypothesis that AML cells depend on proper regulation of inflammatory networks, we identified an AML subgroup enriched for inflammatory pathways, associated with a monocytic lineage signature. To discover AML selective, immune-modulating vulnerabilities, we integrated data from the Cancer Dependency Map on 789 cancer cell lines with independent genome-wide screens, identifying Interferon regulatory factor 2 binding protein 2 (IRF2BP2). We validated AML cell dependency on IRF2BP2 with orthogonal genetic approaches in vitro and in vivo and studied acute IRF2BP2 degradation. Perturbation of IRF2BP2 resulted in cell death with hallmarks of apoptosis. To decipher how IRF2BP2 relates to inflammatory signaling, we studied IRF2BP2 localization on chromatin. We found genome-wide IRF2BP2 binding in promoter and in enhancer regions. Global gene expression studies following degradation of IRF2BP2 showed an increase in expression of the majority of IRF2BP2 bound genes, supporting a role for IRF2BP2 as a transcriptional repressor. Gene set enrichment analyses identified NF-κB-related immune response signatures as the most significantly altered leading us to hypothesize that IRF2BP2 represses NF-κB-mediated TNFα signaling that, when acutely perturbed, leads to leukemia cell death. Indeed, we confirmed an activation of NF-κB-signaling, an increase in nuclear RELA protein, and gain in RELA chromatin binding following degradation of IRF2BP2. Moreover, a mutant “super-repressor” allele of IκBα rescued the impaired cell growth upon IRF2BP2 perturbation, supporting cell death associated with IRF2BP2 loss being mediated through activation of NF-κB signaling. In addition, we identified IL-1ß as an enhancer of the inflammatory response repressed by IRF2BP2. Using patient-derived xenograft models, we demonstrated a significant reduction in leukemia burden and an increase in median survival in mice that had received patient-derived AML cells with IRF2BP2-targeting CRISPR guides compared to control guides. Importantly, loss of IRF2BP2 in normal bone marrow-derived hCD34+ cells had no effect on colony forming capacity. In summary, we demonstrate that IRF2BP2 represses IL-1ß/TNFα signaling via NF-κB, and IRF2BP2 perturbation results in hyperinflammation leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for cell death, and motivate the exploration of alternative immune-mediated therapies in cancers that have yet to reap the benefits of the immunotherapy revolution. Citation Format: Jana M. Ellegast, Gabriela Alexe, Amanda Hamze, Shan Lin, Hannah J. Uckelmann, Philipp J. Rauch, Maxim Pimkin, Linda Ross, Neekesh V. Dharia, Amanda L. Robichaud, Amy Conway Saur, Delan Khalid, Mark Wunderlich, Lina Benajiba, Behnam Nabet, Nathanael S. Gray, Stuart H. Orkin, Kimberly Stegmaier. Unleashing cell-intrinsic inflammation as a strategy to kill AML blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB076.

Summary Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P 1 signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Favorable-risk human acute myeloid leukemia (AML) engrafts poorly in currently used immunodeficient mice, possibly because of insufficient environmental support of these leukemic entities. To address this limitation, we here transplanted primary human AML with isolated nucleophosmin (NPM1) mutation and AML with inv(16) in mice in which human versions of genes encoding cytokines important for myelopoiesis (macrophage colony-stimulating factor [M-CSF], interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) were knocked into their respective mouse loci. NPM1mut AML engrafted with higher efficacy in cytokine knock-in (KI) mice and showed a trend toward higher bone marrow engraftment levels in comparison with NSG mice. inv(16) AML engrafted with high efficacy and was serially transplantable in cytokine KI mice but, in contrast, exhibited virtually no engraftment in NSG mice. Selected use of cytokine KI mice revealed that human M-CSF was required for inv(16) AML engraftment. Subsequent transcriptome profiling in an independent AML patient study cohort demonstrated high expression of M-CSF receptor and enrichment of M-CSF inducible genes in inv(16) AML cases. This study thus provides a first xenotransplantation mouse model for and informs on the disease biology of inv(16) AML.

Although the molecular pathways that cause acute myeloid leukemia (AML) are increasingly well understood, the pathogenesis of peripheral blood cytopenia, a major cause of AML mortality, remains obscure. A prevailing assumption states that AML spatially displaces nonleukemic hematopoiesis from the bone marrow. However, examining an initial cohort of 223 AML patients, we found no correlation between bone marrow blast content and cytopenia, questioning the displacement theory. Measuring serum concentration of thrombopoietin (TPO), a key regulator of hematopoietic stem cells and megakaryocytes, revealed loss of physiologic negative correlation with platelet count in AML cases with blasts expressing MPL, the thrombopoietin (scavenging) receptor. Mechanistic studies demonstrated that MPLhi blasts could indeed clear TPO, likely therefore leading to insufficient cytokine levels for nonleukemic hematopoiesis. Microarray analysis in an independent multicenter study cohort of 437 AML cases validated MPL expression as a central predictor of thrombocytopenia and neutropenia in AML. Moreover, t(8;21) AML cases demonstrated the highest average MPL expression and lowest average platelet and absolute neutrophil counts among subgroups. Our work thus explains the pathophysiology of peripheral blood cytopenia in a relevant number of AML cases.

IL-1b signaling augments continued splenic monocyte supply during acute inflammation., Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity’s critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.

Clonal hematopoiesis of indeterminate potential (CHIP) was recently identified as a major risk factor for development of both hematologic malignancies and atherosclerotic cardiovascular disease in humans. The most commonly mutated gene in CHIP, DNMT3A, is a de novo DNA methyltransferase. The second most commonly mutated gene is TET2, an enzyme which can lead to loss of DNA methylation, and thus is thought to have an opposing biochemical function to DNMT3A. Surprisingly, mutations in both genes lead to convergent phenotypes, such as clonal expansion of mutated stem cells, increased risk of malignant transformation, and increased risk of coronary heart disease. A molecular mechanism linking CHIP and cardiovascular disease has been explored only for loss of function mutations in the Tet2 gene (Jaiswal et al., NEJM 2017; Fuster et al., Science 2017). Here we tested the ability of null mutations in Dnmt3a to contribute to atherosclerosis in hypercholesteremic mice. We further explored the biological basis for this association through gene expression analyses and single-cell RNA sequencing. To model cardiovascular disease associated with DNMT3A-mutated CHIP, atherosclerosis-prone Ldlr-/- mice received bone marrow from Dnmt3a+/+ mice (WT), or from Dnmt3a-/- mice (KO) and WT mice in a 1:9 ratio to mimic a typical variant allele fraction observed in human CHIP. Mice then consumed a high-fat, high-cholesterol diet (HFD), and underwent assessment of atherosclerosis. At 9 weeks, mice that had received 10% Dnmt3a-/- bone marrow displayed an average lesion size that was 40% larger compared to mice receiving control marrow only (p=0.04). The increase in lesion size resembles that we previously observed in mice receiving Tet2-/- marrow (Jaiswal et al., NEJM 2017). De novo DNA methylation by Dnmt3a can alter gene expression. To elucidate how such changes may accelerate atherosclerosis, we first performed transcriptome analysis using bulk RNA sequencing of cholesterol-stimulated bone marrow derived macrophages (BMDM) from either WT or KO mice. BMDMs lacking Dnmt3a showed significantly augmented expression of genes belonging to the CXC chemokine cluster, Cxcl1, Cxcl2 and Cxcl3, as well as increases in mRNAs encoding canonical pro-inflammatory cytokines Il1b and Il6. These changes mirrored those we saw in macrophages lacking Tet2 (Jaiswal et al., NEJM 2017). We next asked how transcriptomic changes observed using the ex vivo BMDM system translated into the in vivo lesional environment. Single-cell RNA sequencing (10X Genomics) was performed on atherosclerotic aortae from mice that had been competitively transplanted with WT, Dnmt3a-/-, or Tet2-/- marrow at a 1:9 ratio. Clustering demonstrated broad changes in lesional immune cell composition in mice harboring CHIP. Lack of either Tet2 or Dnmt3a substantially expanded the myeloid compartment, containing cells that drive atherogenesis. A reciprocal reduction mainly affecting T lymphocyte populations accompanied this expansion. Within the myeloid cell compartment, Dnmt3a-/- or Tet2-/- donor cells, but not WT donor cells, gave rise to a distinct lesional macrophage population. These cells expressed markers associated with tissue-resident macrophages (Mrc1, Lyve1, F13a1), but also highly expressed several inflammatory mediators (Cxcl1, Pf4, Ccl2, Ccl7, Ccl8), and a characteristic set of transcription factors (Jun, Fos, Egr1). Overall, the present study reveals broad changes to the lesional cellular composition and transcriptome induced by the most common CHIP mutations, and provides novel insight into the mechanisms by which CHIP accelerates atherosclerosis. Despite exerting opposite catalytic functions, lack of Dnmt3a or of Tet2 function lead to a myriad of similar downstream transcriptomic and cellular changes. These results indicate that mutations in Dnmt3a and Tet2 accelerate atherosclerosis through convergent mechanisms. Disclosures No relevant conflicts of interest to declare.

Semaphorins and their receptors, plexins, have emerged as important regulators of a multitude of biological processes. Plexin-B3 has been shown to be selectively expressed in postnatal oligodendrocytes. In contrast to the well-characterized Plexin-A family and the Plexin-B family members Plexin-B1 and -B2, no data are available on the functional role of Plexin-B3 in the central nervous system in vivo. Here we have elucidated the functional significance of Plexin-B3 by generating and analyzing constitutive knock-out mice. Plexin-B3-deficient mice were found to be viable and fertile. A systematic histological analysis revealed no morphological defects in the brain or spinal cord of mutant animals. In detailed behavioural analyses of locomotor activity, motor coordination, motor learning, and anxiety levels Plexin-B3-deficient mice were indistinguishable from wild-type controls. Thus we conclude that under physiological conditions Plexin-B3 is not essential for the development and function of the central nervous system.

Favorable-risk acute myeloid leukemia (AML) constitutes up to 40% of newly diagnosed AML cases. However, only 66% of younger and 33% of older patients are alive 3 years post diagnosis. Studying the biology of these entities in vivo has been inherently difficult: in fact, faithful xeno-engraftment of human AML in immunodeficient mice has been limited to higher-risk AML. To address this limitation we hypothesized that a human myelopoiesis-supportive environment may permit xeno-engraftment of favorable-risk AML: we transplanted primary human AML with isolated NPM1 mutation and AML with inv(16) into knock-in (KI) mice with human versions of genes encoding M-CSF, IL-3, GM-CSF and Thrombopoietin. Newborn MISTRG mice (Rongvaux et al. Nat Biotechnol. 2014) were sub-lethally irradiated, intra-hepatically injected with AML blasts and analyzed 16-24 weeks post transplantation. Flow cytometric analysis of bone marrow (BM) and blood of cytokine KI versus NSG mice revealed profound differences: Average engraftment was 36% in the BM of cytokine KI mice compared to only 10% in NSG mice across all patient samples transplanted. In line with engraftment levels, engraftment efficacy was higher in cytokine KI mice. Importantly, average engraftment in cytokine KI mice approached BM and peripheral blast count observed in human AML patients, while NSG engraftment levels were significantly lower. AML engraftment in cytokine KI mice was found to be independent of expression of the SIRPα transgene in mice expressing all four cytokines. NMP1mut AML showed a trend towards higher BM engraftment in MI(S)TRG versus NSG mice. Expression of the NPM1mutallele was more than 3-fold higher in MI(S)TRG mice compared to NSG mice. Performing targeted next generation sequencing in human and engrafted murine BM, we found comparable allelic frequency ranging from 38% to 51% for NPM1 mutation in a patient's leukemia and engrafted cytokine KI mice. Of note, even the composition of NPM1 mutation subclones was constant between patient and cytokine KI recipients. In contrast to NPM1mut, a striking difference in quantitative inv(16) AML engraftment was observed between cytokine KI and NSG mice, both by aggregate and split-sample analysis. Serial transplantation further validated engraftment of an inv(16) leukemia-initiating cell population in cytokine KI mice. To dissect the contribution of respective single human KI cytokines to inv(16) AML propagation, we analyzed engraftment in Rag2-/-gc-/- mice carrying, in addition to a human SIRPα transgene, further KI genes for human thrombopoietin (STRG), M-CSF and Thrombopoietin (MSTRG), and M-CSF, IL-3, GM-CSF and Thrombopoietin (MI(S)TRG). STRG mice, as NSG mice, did not support engraftment. In contrast, addition of M-CSF in MSTRG mice led to robust engraftment of inv(16) AML, while addition of IL-3 and GM-CSF in MI(S)TRG mice resulted in only marginal, if any, improvement over MSTRG mice. Of note, knock-in of human M-CSF has previously been shown to be sufficiently cross-reactive to rescue the murine M-CSF knock-out phenotype (Rathinam et al. Blood 2011). We thus reasoned that the engraftment advantage observed in MSTRG mice was likely due to human M-CSF effects on blasts and not due to reduction in mouse macrophages. Together, this establishes a central role for M-CSF in maintenance of inv(16) AML. When profiling cytokine receptor expression for TPO, M-CSF, IL-3 and GM-CSF on inv(16) AML blasts in comparison to NPM1mut AML and an aggregate of non-favorable-risk AML cases, using microarray data from an international cohort study (Wouters et al. Blood 2009), inv(16) AML showed significantly higher expression of each of the 4 receptors compared to NPM1mut AML, and significantly higher expression of M-CSFR and GM-CSFR compared to non-favorable-risk AML; the overall highest difference in expression was observed for M-CSFR. Moreover, gene set enrichment analysis demonstrated enrichment of M-CSF-inducible genes in inv(16) AML compared to NPM1mutAML and non-favorable-risk AML cases, confirming increased signaling through the M-CSF pathway. In sum, we here describe the first faithful and highly efficacious xenotransplantation model of favorable-risk, and specifically, inv(16) AML. We further demonstrate a strong dependency of this subtype on M-CSF, a finding that might provide a basis for therapy optimization studies informed by an improved understanding of favorable-risk AML biology. Disclosures No relevant conflicts of interest to declare.

Brain metastases treated with stereotactic radiosurgery may show delayed enlargement on post-treatment imaging that is of ambiguous etiology. Histopathologic interpretation of brain specimens is often challenging due to the presence of significant radiation effects admixed with irradiated residual tumor of indeterminate viability. The purpose of this study was to assess the impact of histologic findings on clinical outcomes following resection of these lesions. Between 2004 and 2010, 690 patients with brain metastases were enrolled in a prospective gamma knife data repository, and lesions requiring excision were identified. Tissue specimens were divided into four groups based on the ratio of treatment related inflammatory changes (TRIC) to tumor cells, and subsequently patient outcomes were assessed. Of 2,583 metastases treated, 36 were excised due to symptomatic enlargement. Only TRIC, without residual evidence of tumor, was seen in 36 % (13/36) of specimens. Resection of these lesions resulted in 100 % local control in follow-up. Of the remaining 23 lesions that contained any viable-appearing tumor within the resected specimen, 8 recurred after resection. Lesions that enlarged in the first 6 months were more likely to contain higher amounts of residual tumor cells. Patients with even

Progressively enlarging encephalopathic changes are now well-documented effects of gamma knife radiosurgery (GKRS) occurring ~3-30 months after treatment of both benign and malignant brain lesions. These changes can be variably associated with inflammatory demyelination and necrosis and/or recurrent tumor. While radiographic differentiation between encephalopathic changes and recurrent tumor is of high clinical relevance, confident interpretation of post-radiosurgery imaging changes can be challenging or even impossible in some cases. Gadolinium-enhanced MRI of these lesions reveals variable amounts of enhancing and non-enhancing components within these lesions that have not been clearly correlated with structural-pathologic change. The goal of this study is to characterize the histopathological changes associated with enhancing versus non-enhancing regions of GKRS-treated lesions. MRI images of patients with progressive, etiologically ambiguous brain lesions following GKRS were reviewed prior to explorative neurosurgery. Chosen for this study were lesions in which distinct areas of enhancement and non-enhancement of at least 5 mm in size could be identified (n = 16). Distinctly enhancing and non-enhancing areas were separately biopsied and histologically evaluated. Only cases with uniform histological results are presented in this study. Enhancing and non-enhancing areas in post GKRS lesions represent separate pathological changes. Radiographically enhancing areas correlate either with recurrent tumor growth or inflammatory demyelinating changes. Lack of radiographic enhancement correlates with coagulative necrosis if the sample is taken from the center of the lesion, or with reactive astrocytosis if the sample is taken from the periphery. Separate biopsy of enhancing and non-enhancing regions of post-GKRS encephalopathy was able to confirm that the pathologies in these areas are distinct. These findings allow for better-informed correlation of histological and radiological changes and a better understanding of post-treatment tissue pathology.

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b + Ly-6C hi monocytic and CD11b + Ly-6G hi granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

Immune Sentinels A classic paradigm in immunology holds that the immune response occurs in two waves: Rapidly responding cells of the innate immune system help to contain the invading pathogen and alert lymphocytes. These cells of the adaptive immune system then help to clear the infection and go on to form long-lasting memory. However, some specialized populations of lymphocytes can also respond quickly to an infection and carry out functions that overlap with the innate immune system. Now, Rauch et al. (p. 597 , published online 12 January) describe one such cell type—innate response activator (IRA) B cells. IRA B cells recognize bacterial liposaccharide through Toll-like receptor 4 and, in response, produce the cytokine GM-CSF, which activates other innate immune cells. Deletion of IRA B cells in mice impaired their ability to clear a bacterial infection and promoted septic shock.

Background— Atherosclerotic lesions are believed to grow via the recruitment of bone marrow–derived monocytes. Among the known murine monocyte subsets, Ly-6C high monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here, we hypothesized that the bone marrow outsources the production of Ly-6C high monocytes during atherosclerosis. Methods and Results— Using murine models of atherosclerosis and fate-mapping approaches, we show that hematopoietic stem and progenitor cells progressively relocate from the bone marrow to the splenic red pulp, where they encounter granulocyte macrophage colony-stimulating factor and interleukin-3, clonally expand, and differentiate to Ly-6C high monocytes. Monocytes born in such extramedullary niches intravasate, circulate, and accumulate abundantly in atheromata. On lesional infiltration, Ly-6C high monocytes secrete inflammatory cytokines, reactive oxygen species, and proteases. Eventually, they ingest lipids and become foam cells. Conclusions— Our findings indicate that extramedullary sites supplement the hematopoietic function of the bone marrow by producing circulating inflammatory cells that infiltrate atherosclerotic lesions.

Acute myeloid leukemia (AML) induces profound impairment of healthy hematopoiesis. The production deficit in the bone marrow (BM) leads to development of peripheral anemia, thrombocytopenia and neutropenia, which is a major cause of AML-associated morbidity and mortality. Despite much progress in understanding of AML biology, the mechanisms by which AML blasts interact with elements of normal hematopoiesis to cause cytopenia are unclear. Conventional wisdom has it that blasts infiltrate the marrow and displace normal hematopoiesis. If this concept were to be true, there should be a strong correlation between BM blast count and peripheral cytopenia. Surprisingly, analysis of 223 patients with newly diagnosed AML at a tertiary referral center revealed lack of correlation between initial BM blast count [% of cellularity] and hemoglobin level (ρ=-0.11, P=0.12), platelet count (ρ=-0.00, P=0.53) and absolute neutrophil count (ρ=0.13, P=0.06). This indicates that mechanisms other than displacement of normal hematopoiesis dictate the severity of cytopenia in AML patients. Hematopoiesis is tightly regulated by cytokines. Among them, thrombopoietin (TPO) acts through its receptor c-Mpl as the master regulator of megakaryopoiesis, but also exerts upstream effects on hematopoietic stem and progenitor cells (HSPC). TPO levels are controlled by receptor-mediated scavenging by cells carrying c-Mpl on the surface, with platelets representing the lion's share in a healthy organism. This negative feedback loop results in strong negative correlation between serum TPO concentration and platelet count in the steady state. When we examined this relationship in our AML cohort, TPO levels did not follow the expected negative correlation with platelet counts (ρ=-0.10, P=0.59). Comparison with historic controls with thrombocytopenia induced by chemotherapy for non-hematopoietic malignancy revealed that the lack of correlation was driven by AML cases with severe thrombocytopenia that had lower than expected levels of TPO in the serum. As HSPC are known to express c-Mpl, we hypothesized that HSPC-derived AML blasts may also express the receptor and cause insufficiency of hematopoiesis by means of receptor-mediated TPO scavenging. To test this hypothesis, we compared c-Mpl expression on blasts in AML cases with severe thrombocytopenia and low TPO concentration (potential scavenger cases) to cases with TPO levels adequate for the degree of cytopenia. Both surface flow cytometry and qPCR demonstrated higher c-Mpl expression in potential scavenger cases (3.1-fold, P=0.02). To determine whether this difference in expression translates into increased serum TPO clearance, we incubated AML blasts with high (c-Mpl+) and low (c-Mpl-) receptor expression in serum containing recombinant human TPO at a concentration of 100 pg/mL. After 2h, TPO clearance reached 45 pg per 106 cells in wells with c-Mpl+ blasts, compared to only 4 pg per 106 cells in wells with c-Mpl- blasts (P=0.02). This confirms the hypothesis that AML blasts can lower TPO levels by virtue of their c-Mpl expression. Validation studies in an independent, multi-center Dutch-Belgian-Swiss cohort of 437 AML cases confirmed lack of correlation between initial BM blast count and cytopenia. Ranked gene list correlation analysis of whole genome microarray data proved significant enrichment of the MPL transcript in patients with severe thrombocytopenia when compared to patients with average platelet counts (rank 27/20'589, FDR Lastly, we asked if MPL expression was related to cytogenetic or molecular AML subtype: indeed, microarray analysis showed higher MPL expression in cases of AML with t(8;21) than in any other subtype (P In summary, our study demonstrates that cytopenia in AML is independent of BM blast count, but strongly correlated with c-Mpl expression on blasts. We show that c-Mpl+ blasts clear TPO, causing insufficient TPO levels and contributing to development of thrombocytopenia and neutropenia. The work may have important ramifications for treatment of AML-induced cytopenia, especially in the relapsed or refractory setting. Disclosures No relevant conflicts of interest to declare.

this study examines whether peer-teaching, in the setting of a three-day revision course in anatomy, is effective in preparing medical students for their national anatomy exam.the anatomy course was designed for candidates taking the first part of the German national medical exam. Increase of knowledge during the course was assessed by tests before and after the course (group A). To test equivalence, two control groups participated in the pre-test (group B) or in the course and in the post-test (group C). Participants anonymously rated 14 feedback items on a five-point Likert scale ranging from 1 (full agreement) to 5 (full disagreement).group A students' performance improved significantly during the course with a mean increase of 7.15 points (11.9% improvement; p0.001). Equivalence testing showed that performance of group A students in the pre-/post-tests was equal to those of group B pre-tests and group C post-tests, respectively. Agreement on the 14 feedback items was highly significant (p0.001 for all items), with a global median of 1.this study shows that a three-day anatomy revision course is effective and highly appreciated by medical students in their preparation for the national exam. Moreover, peer-teaching is reliable at this stage of the medical curriculum.

Results: Forty-nine men and 72 women were identified. Fourteen patients who had previous non-MLC-based SRS were excluded. The mean nidus volume was 3.95 cm (range: 0.19-33.00) and median dose was 17.5 Gy (range: 12-22.5). Forty-six percent of patients had a grade 3 AVM (range: 2-5). Median follow-up for patients followed by MRI and DSAwas 20 mos (range: 12-114) and 30 mos (range: 12-103), respectively. AR of OBL by MRI and DSA at 5 years from SRS were 80% and 76% respectively. PreSRS ICH was the only secondary endpoint affecting OBL (HR: 2.40; p Z 0.049, CI Z 1.01-5.77). There was a 6.8% risk of post SRS ICH with the highest probability (19%) at 2 years. The median time to RN was 24 mos (range: 4-48) while the CR of symptomatic RN was 14%. LRA revealed that RN at 2 years strongly correlated with OBL (Odds Ratio: 4.21, p Z 0.01, CI Z 1.41-12.62). Higher V12 and lower EI significantly predicted RN (p Z 0.001 and 0.003, respectively). A cross-validated RN model showed that V12 predicted RN at 24 mos better than other single or conjoined variables (74.8% accuracy, SE, 0.3%). Conclusion: Our OBL rates at 5 years are at least comparable if not better than other series. Interestingly, we were able to achieve these OBL rates with a median dose of 17.5 Gy, which is lower than most other series. Presence of RN at 2 years predicted OBL arguing that normal tissue radiation sensitivity correlates with AVM radiation sensitivity. Pre-SRS ICH status correlated significantly with OBL. Lastly, optimizing EI or reducing the V12 may decrease the incidence of RN. Author Disclosure: E. Marchan: None. J. Hanfelt: None. N.M. Oyesiku: None. A. Dhabban: None. S. Hsieh: None. M.K. Khan: None. W.J. Curran: None. H.K. Shu: None. I.R. Crocker: None.