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Acute myeloid leukemia (AML) patients refractory to induction therapy or relapsed within 1 year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in preclinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single-cell RNA sequencing using prospectively acquired patient specimens identified IFN response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well tolerated, with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies.Significance:This study demonstrates a favorable safety profile and promising clinical activity of ficlatuzumab and cytarabine in high-risk AML, thus supporting further investigation of this combination in a randomized trial. It also shows the utility of a novel application using multiplexed single-cell analyses to detect on-target activity and identify biomarkers of response.This article is highlighted in the In This Issue feature, p. 403

Supplementary Data from Inhibition of MET Signaling with Ficlatuzumab in Combination with Chemotherapy in Refractory AML: Clinical Outcomes and High-Dimensional Analysis

Despite the clinical benefit associated with gilteritinib in relapsed/refractory acute myeloid leukemia (AML), most patients eventually develop resistance through unknown mechanisms. To delineate the mechanistic basis of resistance to gilteritinib, we performed targeted sequencing and scRNASeq on primary FLT3-ITD-mutated AML samples. Co-occurring mutations in RAS pathway genes were the most common genetic abnormalities, and unresponsiveness to gilteritinib was associated with increased expression of bone marrow-derived hematopoietic cytokines and chemokines. In particular, we found elevated expression of the TEK-family kinase, BMX, in gilteritinib-unresponsive patients pre- and post-treatment. BMX contributed to gilteritinib resistance in FLT3-mutant cell lines in a hypoxia-dependent manner by promoting pSTAT5 signaling, and these phenotypes could be reversed with pharmacological inhibition and genetic knockout. We also observed that inhibition of BMX in primary FLT3-mutated AML samples decreased chemokine secretion and enhanced the activity of gilteritinib. Collectively, these findings indicate a crucial role for microenvironment-mediated factors modulated by BMX in the escape from targeted therapy and have implications for the development of novel therapeutic interventions to restore sensitivity to gilteritinib.

Background Mantle cell lymphoma (MCL) is a rare, highly heterogeneous type of B-cell non-Hodgkin’s lymphoma. The sumoylation pathway is known to be upregulated in many cancers including lymphoid malignancies. However, little is known about its oncogenic role in MCL. Methods Levels of sumoylation enzymes and sumoylated proteins were quantified in MCL cell lines and primary MCL patient samples by scRNA sequencing and immunoblotting. The sumoylation enzyme SAE2 was genetically and pharmacologically targeted with shRNA and TAK-981 (subasumstat). The effects of SAE2 inhibition on MCL proliferation and cell cycle were evaluated using confocal microscopy, live-cell microscopy, and flow cytometry. Immunoprecipitation and orbitrap mass spectrometry were used to identify proteins targeted by sumoylation in MCL cells. Results MCL cells have significant upregulation of the sumoylation pathway at the level of the enzymes SAE1 and SAE2 which correlated with poor prognosis and induction of mitosis associated genes. Selective inhibition of SAE2 with TAK-981 results in significant MCL cell death in vitro and in vivo with mitotic dysregulation being an important mechanism of action. We uncovered a sumoylation program in mitotic MCL cells comprised of multiple pathways which could be directly targeted with TAK-981. Centromeric localization of topoisomerase 2A, a gene highly upregulated in SAE1 and SAE2 overexpressing MCL cells, was lost with TAK-981 treatment likely contributing to the mitotic dysregulation seen in MCL cells. Conclusions This study not only validates SAE2 as a therapeutic target in MCL but also opens the door to further mechanistic work to uncover how to best use desumoylation therapy to treat MCL and other lymphoid malignancies.

Richter’s Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.

Setting nutritional standards for larval zebrafish (Danio rerio) that maximize growth, survival, and reproductive success is challenging. We evaluated the effects of different feeding regimens on larval zebrafish by comparing Gemma Micro 75 pelleted diet and live-type L rotifers (Brachionus plicatilis) in 3 feeding regimens starting at 9 days postfertilization (dpf): bolus feeding of live diet (BL), continuous feeding of live diet (CL), and pelleted diet (PD). Animals in the PD and CL groups were longer than the BL group at 4–5 weeks postfertilization. The PD group was also greater in body depth than both live diet groups. There was no significant difference in weight between the groups. There were also no significant differences in fecundity or sex ratios indicating that all feeding methods successfully promote growth of a useful breeding stock of fish. In addition, we quantified the equipment, consumable, and labor costs associated with these methods, and found that the PD regimen was superior to both live diet regimens. These data suggest that providing a high nutrient-density pelleted diet to larval and juvenile zebrafish is an effective means to increase early growth and to decrease cost and labor associated with nursery care.

Acute myeloid leukemia (AML) patients refractory to induction therapy or relapsed within 1 year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in preclinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single-cell RNA sequencing using prospectively acquired patient specimens identified IFN response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well tolerated, with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies. Significance: This study demonstrates a favorable safety profile and promising clinical activity of ficlatuzumab and cytarabine in high-risk AML, thus supporting further investigation of this combination in a randomized trial. It also shows the utility of a novel application using multiplexed single-cell analyses to detect on-target activity and identify biomarkers of response. This article is highlighted in the In This Issue feature, p. 403

INTRODUCTION AND OBJECTIVE:Recently single-cell RNA sequencing (scRNA-Seq) has increased our depth of understanding of genomic features of distinct cellular populations. To reveal the dynamics of b...

Hematopoietic stem cells (HSCs) are functionally and genetically diverse and this diversity decreases with age and disease. Numerous systems have been developed to quantify HSC diversity by genetic barcoding, but no framework has been established to empirically validate barcode sequences. Here we have developed an analytical framework, Selection of informative Amplicon Barcodes from Experimental Replicates (SABER), that identifies barcodes that are unique among a large set of experimental replicates. Amplicon barcodes were sequenced from the blood of 56 adult zebrafish divided into training and validation sets. Informative barcodes were identified and samples with a high fraction of informative barcodes were chosen by bootstrapping. There were 4.2 ± 1.8 barcoded HSC clones per sample in the training set and 3.5 ± 2.1 in the validation set (p = 0.3). SABER reproducibly quantifies functional HSCs and can accommodate a wide range of experimental group sizes. Future large-scale studies aiming to understand the mechanisms of HSC clonal evolution will benefit from this new approach to identifying informative amplicon barcodes.

There are limited data describing incidence of symptomatic venous thromboembolism (VTE) in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) patients receiving peg-asparaginase.Single-institution retrospective analysis of 44 AYA ALL patients treated with peg-asparaginase. Rates of VTE and proposed risk factors were assessed.18 patients (41%) had a symptomatic VTE following peg-asparaginase. The cumulative incidence rate was 25% (95% CI: 13-38%) within 30 days of the initial dose. Personal history of thrombosis was statistically significantly associated with an increased risk of VTE with HR of 2.73 (95% CI: 1.40-5.33, p = 0.003) after adjusting for gender.These data indicate a high rate of VTE in the AYA ALL population following treatment with peg-asparaginase.

Introduction: The development of Bruton tyrosine kinase inhibitors (BTKi) and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Ibrutinib and other second generation BTKi as monotherapies generally do not produce minimal residual disease negative (MRD-) complete remissions even with extended therapy. The reason for lack of continued elimination of CLL to a MRD- status over time is unknown, and we hypothesized that biological differences in the CLL cells or immune microenvironment might make them resistant to elimination. Methods: Samples were obtained from patients on continuous ibrutinib who hadn't relapsed at time points of 3 years on treatment and 5 years on treatment; and pre-ibrutinib. Isolated CLL cells were subject to B-cell receptor (BCR) sequencing using NEBNext Immune Sequencing Kit by New England Biolabs (NEB, Inc., USA). In a separate cohort, 10X VDJ+5'-sequencing was performed on peripheral blood mononuclear cells. Flow cytometry and ELISA were used to identify alterations in immune cell subtype and identify immune profiles associated with MRD positive (MRD+) status. Results: To identify the clonal pattern in MRD+, we performed deep sequencing of the BCR repertoire on samples from 13 patients with 3 time points each. We found that dominant clones tended to remain constant, but new clones appeared in later time points (Figure 1). MiXCR (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique productive and nonproductive CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Two out of three patients (patients 1 and 3) showed significant change in the clone over time. In patients 1 and 2, we saw that heavy chain clones emerge at later time points. In patient 3 alone, we observed that at 5 years there are two dominant clones. Our findings suggest that each patient shows a diverse repertoire of CLL clones and that the dominant clone does not change significantly across time points. To identify cell populations based on gene expression patterns, we performed 10X VDJ+5'-seq. Based on the expression of known markers, we identified CLL cells and other immune cell subtypes. We identified differentially expressed genes (DEGs) for CLL cells in each time points. Over time, we observed upregulation of CD79a, LTB, TAGLN2, and LGALS, genes typically associated with leukemic cell survival. Suggesting differential expression of pro-survival genes contribute to continued presence of MRD over time. T cells are known to be dysfunctional in CLL and have not previously been extensively studied in the setting of long term BTKi. We performed flow cytometry to determine the repertoire and function of T cells at 3 and 5 years of ibrutinib therapy. We found that the percentage of CD3+ T cells increases at later time points in all the 8 patients (p Conclusion: Extended ibrutinib treatment yields a subset of patients who become MRD- whereas a large majority remain MRD+. Our findings suggest that BCR repertoire in CLL MRD might change in long term ibrutinib therapy and induce necessary genes for its survival in the microenvironment. Although T cells and NK cells are non-functional at later time points, better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells. Differentiating the biology of why certain patients attain MRD- status on BTK inhibitor is of high interest as it could provide rationale for therapy discontinuation or add on approaches. Figure 1 Figure 1. Disclosures Rogers: AbbVie Inc.: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Innate Pharma: Consultancy; Pharmacyclics LLC: Consultancy; Janssen Pharmaceuticals, Inc: Research Funding; ovartis Pharmaceuticals Corporation: Research Funding. Bhat: Beigene: Consultancy; AstraZeneca: Consultancy; Aptitude Health: Honoraria; Onclive: Honoraria. Kittai: Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Woyach: AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. Disclosures Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

Introduction The FDA has recently approved several oral targeted therapies for Acute Myeloid Leukemia (AML). These therapies have been approved in patients with relapsed/refractory disease and as frontline therapy in patients ineligible for intensive induction chemotherapy. These agents are also being increasingly utilized as frontline therapy in patients of all ages and fitness levels on clinical trials and through off label prescribing. The decision to use intensive chemotherapy vs targeted therapy is particularly relevant in patients aged 60-75 due to the heterogeneous nature of this population with respect to disease characteristics, performance status, and comorbidities. However, the relative survival impact of intensive chemotherapy vs targeted therapy in this patient population has not been extensively studied. We conducted a retrospective analysis to compare survival outcomes of these treatment approaches and to determine the relative impact of treatment intensity compared to other variables that are known to affect survival in AML patients. Methods In this single-center, retrospective study, patients aged 60-75 diagnosed with AML from 2016-2020 were included if they received treatment with high intensity chemotherapy (HiC), low intensity targeted therapy (LITT), or both during the course of treatment and prior to transplant. HiC was defined as a regimen containing cytarabine + an anthracycline given on a "7+3" based schedule. Patients treated with liposomal cytarabine-daunorubicin were excluded. LITT was defined as venetoclax, gilteritinib, enasidenib, or ivosidenib alone or in combination with a hypomethylating agent. Between-group analysis was conducted for patients who had received HiC at any point during treatment (any HiC) vs patients who had not (LITT only). Overall Survival (OS) was analyzed by Kaplan-Meier method with log-rank test used for between-group comparisons. Cox regression model was used to associate risk factors with OS. Univariable models were first fit, then a multivariable model was built using backward selection. Transplant status was included as a time-dependent variable. Results A total of 141 patients were included, 80 received any HiC and 61 received LITT only. Compared with the any HiC group, patients in the LITT only group demonstrated older age, a higher percentage of secondary AML, a lower percentage of FLT3 ITD mutations, a higher percentage of IDH1 and IDH2 mutations, a lower white blood cell count, and a trend toward higher ELN risk classification at baseline (Table 1). Median OS was significantly longer in the any HiC group (21.8 months vs 13.6 months). A significantly higher percentage of patients receiving any HiC underwent allogeneic stem cell transplantation, but post-transplant OS was not significantly different between the two groups (Table 2). On univariable analysis, receipt of HiC, lower ELN risk classification, and receipt of transplant were all significantly associated with superior OS. Age, performance status, secondary AML, and white blood cell count at diagnosis notably did not have a significant association with OS in this cohort. On multivariable analysis, treatment intensity was no longer found to have an independently significant impact on survival after accounting for ELN risk (hazard ratio (HR) for unfavorable 3.02, p Discussion The results of this study show that baseline disease characteristics and receipt of transplant were the most important predictors of survival in this cohort of AML patients aged 60-75. These factors were notably more impactful than treatment intensity and chronological age. These findings support the use of transplant in this patient population regardless of treatment intensity, especially in those with higher risk disease. Limiting factors in this study include the retrospective design and relatively small sample size. Ultimately, larger trials with more patients will be needed to confirm these findings including prospective, randomized trials comparing intensive chemotherapy to lower intensity targeted therapy in patients who are transplant-eligible at baseline. Figure 1 Figure 1. Disclosures Bhatnagar: Novartis: Honoraria; Pfizer: Honoraria; Kite: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Astellas: Honoraria; Cell Therapeutics: Honoraria, Research Funding. Blachly: INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; KITE: Consultancy, Honoraria. Borate: Genentech: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicine: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharma: Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rampal: Membership on an entity's Board of Directors or advisory committees; Galecto, Inc.: Consultancy; Promedior: Consultancy. Mims: Glycomemetics: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Aptevo: Research Funding; Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Xencor: Research Funding; Kartos Pharmaceuticals: Research Funding; Genentech: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Kura Oncology: Consultancy; Syndax Pharmaceuticals: Consultancy; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding. Vasu: Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Novartis: Other: clinical trial support; Geron: Other: clinical trial support; Newave: Other: clinical trial support.

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin -CD34 -CD117 +/-CD94 +NKp80 -CD16 -CD57 - stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 + stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 - stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR + cells among CD200R1 - stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin -CD34 -CD117 +/-CD94 +NKp80 +CD16 -CD57 -) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 - stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 + stage 4A cells represent NK cell precursors, whereas the CD200R1 - stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 + and CD200R1 - stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 + NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 +) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 + stage 4A clones contained ILC3s (CD94 -NKp44 +), significantly fewer clones from CD200R1 - stage 4A cells produced ILC3s (7 of 26 CD200R1 - clones vs. 20 of 23 CD200R1 + clones; p = 0.000587). Moreover, none of the CD200R1 - stage 4A-derived clonal cultures that contained KIR + NK cells contained ILC3s, suggesting that the majority of CD200R1 - stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

BackgroundMyeloid-derived suppressor cells (MDSC) are an immunosuppressive immature population of myeloid cells that are elevated in cancer patients. Increased levels of MDSC has been linked to dysregulated anti-tumor responses and reduced efficacy of immune checkpoint therapies thus making them an attractive target. MDSC express Bruton's tyrosine kinase (BTK) and can be depleted using ibrutinib, an FDA-approved irreversible inhibitor of BTK. BTK inhibition leads to reduced MDSC expansion/function in murine models and significantly improved activity of anti-PD-1 antibodies. In this study, single cell RNA-seq (scRNA-seq) was used to characterize the gene expression of MDSC from different cancer types and the effect of ibrutinib on MDSC gene expression.MethodsPeripheral blood mononuclear cells were isolated from patients with melanoma (n=2), head & neck (n=1), and breast cancer (n=1). MDSC were isolated via fluorescence activated cell sorting. MDSC isolated from melanoma patients (n=2) were treated in vitro for 4h with 1 uM ibrutinib or DMSO and scRNA-seq was performed using the Chromium 10x Genomics platform. ScRNA-seq samples were analyzed using the standard integrative workflow of Seurat v3, which addresses the sample heterogeneity. Cell clusters were identified using Seurat and annotated using SingleRversion3.12. Identification of gene markers for each cell cluster and cell-cluster-specific differential expression analyses were conducted using Seurat.ResultsBaseline gene expression of MDSC from patients with breast and head & neck cancer revealed similarities among the top expressed genes (S100A8, VCAN, and LYZ). In vitro ibrutinib treatment of MDSC from patients with melanoma resulted in significant changes in gene expression within the MDSC cluster compared to DMSO treatment. GBP1(-1.72 log fold change), IL 1β(-1.27 log fold change), and CXCL8(-0.63 log fold change) were among the top downregulated genes (pConclusionsAnalysis via scRNA-seq revealed similar gene expression patterns for MDSC from different cancer patients. There was downregulation of multiple genes and pathways important to MDSC function and migration after BTK inhibition.Ethics ApprovalThe study obtained ethics approval. IRB# 1999C0348

B ackground Premature death from opioid-related causes imposes an enormous public health burden across the United States. Between 2001 and 2016, the number of opioid-related deaths in the United States increased by 345%, from 9489 to 42 245 deaths (33.3 to 130.7 deaths per million population. Moreover, opioids may have immunosuppressive properties independent of their psychotropic effects; opioid use has been associated with increased invasive pneumococcal disease in a nested case-control study of 1233 Medicaid patients from Tennessee. In liver transplant recipients, opioid use disorder has been associated with increased mortality after transplant. The impact of opioid use disorder on patients receiving blood and marrow transplant (BMT) remains to be defined. Methods We performed a retrospective analysis of all consecutive adult patients who had BMT (autologous and allogeneic) from 1/1/2008 through 1/1/2018 at the James Comprehensive Cancer Center. Overall survival (OS) was measured from the date of transplant to the date of death, censoring at date of last follow up if alive. Progression free survival (PFS) was measured from the date of transplant to the date of disease progression or the date of death, whichever occurred first, censoring at last follow up if no event. OS and PFS were estimated by the Kaplan-Meier method and compared using the log-rank test. Opioid use (OU) was defined as a binary yes/no variable if an opioid was prescribed upon discharge from the hospital after BMT. The impact of OU, along with other patient, disease, and BMT related factors, on PFS/OS, was analyzed using Cox regression method. Results A total of 1585 patients were included in the analysis (Table 1). The median age at BMT was 58 (range=18-79) years; 59% were males; 60% had autologous transplants; and 58% were prescribed opioids upon discharge from the hospital. OU was significantly more in patients who were younger, have had allogeneic transplant, reduced intensity conditioning, had acute myeloid leukemia (AML), or higher BMT comorbidity index (CMI). On univariable analysis, OU was not associated with cumulative incidence of relapse (CIR) or PFS however it was associated with inferior OS; hazard ratio (HR)=1.25, 95% CI: 1.06-1.49; p=0.01 (Figure-1). There were no differences in CIR, PFS, or OS when autologous and allogeneic transplants were analyzed separately. Upon multivariable analysis of OS, OU lost statistical significance after controlling for age, diagnosis, type of transplant, intensity of conditioning regimen, CMI, and disease risk index (DRE). Of interest, OU independently predicted for superior OS at 100 days and 365 days post-BMT; HR=0.29, 95% CI 0.16-0.50 (p= Conclusion: Our results suggest that opioid use (OU) may have a long term negative impact on survival in BMT patients. The apparently protective effects of OU early on after BMT is elusive but may be possibly related immunomodulatory effects of opioids. A major limitation of our study is that OU is analyzed at a single time point at hospital discharge after BMT. We plan to undertake a more detailed analysis of ongoing OU after discharge and its impact on survival outcomes after BMT. Disclosures Brammer: Celgene Corporation: Research Funding; Seattle Genetics, Inc.: Speakers Bureau. Efebera:Celgene: Research Funding; Ohio State University: Current Employment; Pharmacyclics: Research Funding; Takeda: Honoraria, Speakers Bureau. Mims:Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy; Novartis: Speakers Bureau. Chaudhry:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bumma:Sanofi: Speakers Bureau; Amgen: Speakers Bureau. Khan:Amgen: Consultancy; Janssen: Consultancy. Devarakonda:Janssen: Consultancy. Jaglowski:Novartis: Consultancy, Research Funding; CRISPR: Consultancy; Juno: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding. William:Kyowa Kirin: Consultancy, Honoraria; Merck: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Guidepoint Global: Consultancy; Dova: Research Funding; Celgene: Consultancy, Honoraria.

Background Haplo-HCT emerged as a reliable option for patients with high risk hematological malignancies with no HLA-matched related or unrelated donors available. Retrospective registry data suggest comparable outcomes to HLA-matched donors; however, relatively little is known regarding predictors of disease relapse following haplo-HCT. We hypothesize that attainment of full-donor CD3 and CD33 chimerism at day 30 (D30) and 100 (D100) is associated with lower incidence of relapse after haplo-HCT. Methods We undertook a retrospective analysis of 78 patients who underwent haplo-HCT at the Ohio State University James Cancer Hospital between January 2013 and December 2017. The associations between patient characteristics including CD3/CD33 donor chimerisms and the cumulative incidence rate (CIR) of relapse were evaluated using proportional sub-distribution hazards model, treating death without relapse as the competing risk. The models were built adopting a landmark analysis approach at D30 and D100. Progression-free survival (PFS) was estimated using the method of Kaplan-Meier. Results Median age at haplo-HCT was 56 (20-74) years and 68% were males. 50% had acute myeloid leukemia/myelodysplastic syndrome, and 54% had intermediate disease risk index (DRI). Most patients (85%) received reduced-intensity conditioning and 50% received bone marrow (BM) grafts. Complete (100%) CD3 donor chimerism was observed in 63 (84%) patients at D30, and subsequently 59 (95%) patients at D100. Complete CD33 donor chimerism was observed in 67 (89%) patients at D30, and subsequently 56 (88%) patients at D100. In univariable D30 landmark analysis, the CIR of relapse was significantly lower in patients who achieved complete CD3 (and CD33) chimerism compared with patients with less than complete chimerism (Figure 1A; p Conclusions Complete CD3 and CD33 donor chimerisms at D30 and D100 predict a patient's risk for relapse following haplo-HCT. We observed a strong association between CD3 chimeric status at D30 with DRI, as well suggestive that high risk disease may be associated with impaired immune reconstitution after transplant and perhaps a less robust graft vs disease effect.

e19026 Background: The USFDA has recently approved several oral targeted therapies for the treatment of acute myeloid leukemia (AML). The relative survival impact of these agents vs standard induction chemotherapy in the frontline setting is unknown, especially in patients who undergo allogeneic hematopoietic cell transplant (HCT). Methods: This is a retrospective, single-institution study of 125 patients with AML diagnosed 2015-2020 who received HCT. Patients were included if induced with high intensity chemotherapy (HiC) or lower intensity targeted therapy (LITT). HiC was defined as cytarabine + anthracycline given on a “7+3” based schedule. LITT was defined as venetoclax, gilteritinib, enasidenib, or ivosidenib alone or combined with hypomethylating agent. Patients receiving liposomal cytarabine-daunorubicin were excluded. Outcomes of interest were overall survival (OS), non-relapse mortality (NRM), and cumulative incidence of relapse (CIR). Between-groups analysis was conducted comparing receipt of any HiC to LITT only. OS was analyzed by Kaplan-Meier method and compared using log-rank test; NRM and CIR was estimated through cumulative incidence function and compared by Fine-Gray test. Results: Of 125 HCT recipients, 108 received any HiC and 17 LITT only. Two patients received HiC after suboptimal response to LITT. Median age at HCT was 61 years, 48% were female, and 90% were in complete remission at HCT. Population characteristics and outcomes as well as between-groups comparison are presented in the table. LITT only group was older with longer time to HCT and exclusively received RIC. With median follow-up of 23 months, median OS had not been reached. There were no statistically significant differences in median OS or one-year NRM or CIR between groups. Conclusions: Despite differences in age, conditioning regimen, and time to HCT in patients receiving any HiC vs LITT only, we have shown similar OS, CIR, and NRM. Difference in age is in contrast to lack of difference in HCT-CI and KPS, but is partially explained by limited access to LITT for younger patients. Longer time to HCT among LITT only recipients represents a major opportunity for pre-HCT optimization of recipient health. Prospective study from time of diagnosis will be important to understanding the appropriate use of LITT in HCT-eligible patients.[Table: see text]

e19004 Background: Acute Myeloid Leukemia (AML) has traditionally been treated frontline with intensive induction chemotherapy in patients fit enough for this treatment. The FDA has approved several oral targeted therapies for AML in recent years. The survival impact of these agents vs induction chemotherapy is unknown. Methods: In this single-center, retrospective study, patients diagnosed with AML from 2015-2020 were included if they received treatment with either high intensity chemotherapy (HiC) or lower intensity targeted therapy (LITT). HiC was defined as a regimen containing cytarabine + anthracycline given on a “7+3” based schedule. Patients treated with liposomal cytarabine-daunorubicin were excluded. LITT was defined as venetoclax, gilteritinib, enasidenib, or ivosidenib alone or in combination with a hypomethylating agent. Patients fell into four groups: HiC only, LITT only, HiC followed by LITT, and LITT followed by HiC with assignment censored at transplant. Overall survival (OS) was estimated using Kaplan-Meier method and patients receiving any HiC vs LITT only were compared using log-rank test. Results: A total of 332 patients were included: 177 received HiC only, 116 LITT only, 32 HiC before LITT, and 7 LITT before HiC. Baseline characteristics and OS data are outlined in the table. The any HiC group had a lower median age and more patients with WBC >10 K/µL at diagnosis, as well as more patients receiving allogeneic hematopoietic cell transplant (HCT). OS was superior in the any HiC group vs LITT only group. Receipt of any HiC remained predictive of OS after adjusting for age (HR 0.65, 95% CI 0.44-0.96, p = 0.03); however, was no longer predictive of OS after adjusting for age and receipt of HCT. Conclusions: While HiC was associated with superior OS compared to LITT only treatment in univariable analysis, the survival benefit was no longer apparent after adjusting for age and receipt of HCT. The results suggest that intensity of AML treatment is less impactful on prognosis than ability to receive HCT. Differences in age were likely confounded by clinical trial eligibility and prescribing information specifically affecting patients receiving LITT. In the era of LITT, prospective randomized studies of intensity of AML therapy, particularly in non-favorable risk disease, are imperative to striking a balance between toxicity and cure for patients of all ages.[Table: see text]

Blaser et al. use live imaging of the zebrafish hematopoietic niche to show that cxcl8/cxcr1 signaling positively regulates HSPC engraftment by increasing HSPC-niche interactions, HSPC mitotic rate, niche size, and expression of cxcl12a in a niche-autonomous manner., The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.

The sinusoidal endothelial cell niche is a component of the hematopoietic microenvironment with an important role in regulating hematopoietic stem cell (HSC) fate. Mechanisms by which the niche control the long-term functional and phylogenetic diversity of the hematopoietic system are poorly understood. We performed gene expression profiling on FACS-purified endothelial cells from the zebrafish caudal hematopoietic territory (CHT), the sinusoidal endothelial niche equivalent to the mammalian fetal liver. Kdrl:GFP+lyve1b:DsRed+ CHT endothelial cells were compared to kdrl:GFP+lyve1b:DsRed- non-CHT endothelial cells by bulk RNA-seq and 106 genes were downregulated in the CHT with q Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Cellarity: Consultancy; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder.

Single cell RNA sequencing (scRNA-seq) is a powerful method to understand gene expression changes in specific subsets of heterogeneous cell populations and to provide novel insight into mechanisms of drug resistance. However, substantial cost and interpatient variability have limited its application as a correlate for prospective clinical trials. To overcome these challenges, we employed a genetic polymorphism-based multiplexing approach to assay peripheral blood samples prospectively collected from patients with relapsed/refractory AML treated on a Phase 1b study using the anti-hepatocyte growth factor monoclonal antibody, Ficlatuzumab (NCT02109627). Ficlatuzumab was administered on study days 1, 15, 29 and 42-44 in combination with high-dose cytarabine. Peripheral blood was collected from 11 subjects on study days 0, 1, 2, 3 and 42-44. N = 7 subjects achieved a complete response to therapy and N = 4 did not respond to therapy. A peripheral blood sample from a healthy volunteer was collected and processed in parallel. 57 samples were multiplexed into 5 pools of 11 to 12 samples using a Latin square design to maximize genetic diversity within each pool and minimize batch effects. Each pool was assayed in duplicate on a 10X Chromium single cell isolation system using 3'-capture technology, targeting 40,000 cells per replicate pool. After initial bioinformatic processing, 175,667 unique cell barcodes were recovered. Genotype-free demultiplexing was performed using Freemuxlet (https://github.com/yelabucsf/popscle). Single nucleotide variants were annotated within the aligned reads from each pool using the 1000 Genomes variant database. A Bayesian clustering algorithm was applied to assign each cell to a group of cells with like genotype. Reference sample genotypes were determined using the Infinium OmniExpressExome-8 BeadChip (Illumina) and these were used to map each cluster to known subject identity. This approach unambiguously assigned 105,363 cells (60.0%) to single subjects and 60,787 (34.6%) to inter-sample doublets. 103,415 cells were retained after removal of intra-sample doublets and low-quality cells. Dimensionality reduction and clustering identified 3 groups of cells that expressed AML blast markers including CD33, CD34, KIT and HLA-DR. We hypothesized that induction of HGF expression by AML blasts might be a mechanism of resistance to anti-HGF therapy. AML blasts were stratified by treatment day and multivariable regression was performed using cluster assignment as a potential confounder. This showed that non-response to study therapy was independently correlated with higher HGF levels on study D0 (q = 0.008), D1 (q = 0.02), and D42-44 (q = 5x10-7). To identify additional biomarkers of resistance to anti-HGF therapy, we performed unsupervised gene module analysis and found 20 groups of genes that were co-regulated within the dataset. One module was strongly induced in non-responders compared to responders at D0 (q = 0), D1 (q = 1x10-278), and D42-44 (q = 0). This module was highly enriched for genes related to type-1 interferon signaling and antiviral response including IL6, IRF7, IRF2, STAT1, STAT2 and APOBEC3A. Unsupervised hierarchical clustering based on gene module scores segregated non-responders from complete responders and showed that the normal control cells were most similar to complete responders at D42-44. These data suggest that multiplexing using genetic polymorphisms increases the number of scRNA-seq samples it is feasible to analyze in the context of a prospective clinical trial. Gene module analysis is able to identify pathways as potential biomarkers of drug resistance in an unsupervised way. These approaches will aid studies in which interpatient variability is a challenge to interpretation of scRNA-seq data. Disclosures Andreadis: Merck: Research Funding; Gilead/Kite: Consultancy; Karyopharm: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Research Funding; Genentech: Consultancy, Current equity holder in publicly-traded company; BMS/Celgene/Juno: Honoraria, Research Funding; Incyte: Consultancy.

BackgroundA significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC.MethodsImmunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein.ResultsWe found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/− MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (pConclusionsThese results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.

Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro–derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro–derived HSCs as a therapeutic modality.

Hematopoietic stem cells (HSCs) are functionally and genetically diverse. This diversity decreases as a function of age and with the development of myeloid neoplasia. Numerous systems have been developed to genetically "barcode" HSCs and quantify HSC clone numbers in the setting of native hematopoiesis, but no experimental and computational framework has been established to perform this analysis on a large number of biological replicates. Here we have employed the transgenic zebrafish system, Genome Editing of Synthetic Target Arrays for Lineage Tracing (GESTALT), to develop a framework scaled to permit the investigation of putative oncogenes or niche factors with moderate to small effect sizes. The GESTALT line carries a single copy of an array of 10 tandem CRISPR target sites that is highly variable after being repaired by non-homologous end joining. GESTALT embryos were injected with sgRNAs and Cas9 protein to generate genetic barcodes at the GESTALT locus. Fifty-six zebrafish were raised to adulthood and GESTALT variants were read from a sample of peripheral blood by amplicon sequencing. In a training set of 35 fish we developed a computational model to discriminate informative barcodes from uninformative GESTALT variants in each sample. This model identifies variants shared among samples and excludes them as uninformative. Experimentally-defined thresholds were used to minimize inter-sample correlation (mean Pearson Correlation Coefficient = 0.0021) while maximizing the number of retained informative barcodes in each sample. Bootstrapping and k-means clustering were used to classify samples with a high or low fraction of informative (FI) barcodes (FI cutoff = 0.65). 24 samples within the training set were classified in the High-FI group (mean FI = 0.93 ± 0.02, compared to 0.40 ± 0.04 in the Low-FI group). We then evaluated 3 methods of quantifying GESTALT HSC clones from all sequenced barcodes in the High-FI samples and found that counting barcodes with a VAF>0.02 produced the most normal distribution across the training set (Shapiro-Wilk test for normality, W = 0.95, p = 0.21). 4.3 ± 0.38 GESTALT HSC clones were detected in the training set. In an independent validation set of 21 samples, the number of GESTALT HSC clones was 3.4 ± 0.55, (p = 0.2). This study provides the largest reference dataset to date for HSC genetic barcoding experiments in the zebrafish and addresses the validity of barcode sequences, sample selection and clone enumeration in a systematic, objective way. The GESTALT barcoding system and our analysis framework compare favorably with other methods of studying HSC clonal diversity under conditions of native hematopoiesis in terms of scale, cost, labor and barcode validity. Future studies aiming to understand the mechanisms of HSC clonal evolution in the context of health, ageing and disease will benefit from this framework for enumerating functional HSC clones. Disclosures No relevant conflicts of interest to declare.

We currently have little understanding of the mechanisms by which hematopoietic stem and progenitor cells (HSPCs) gain a selective advantage in patients with clonal hematopoiesis and other myeloid neoplasms. The chemokine CXCL8 is elevated in a subset of patients with myeloid neoplasms. Our previous work in zebrafish has discovered a novel role for cxcl8 and its receptor, cxcr1, in supporting colonization of HSPCs within the sinusoidal endothelial cell niche of the embryonic zebrafish known as the caudal hematopoietic tissue (CHT). We hypothesized that mosaic overexpression of cxcl8 in a population of HSPCs during development would alter HSPC-niche interactions, selectively favor HSPCs expressing cxcl8 and lead to their expansion in adults. To test this hypothesis, we microinjected DNA constructs encoding cxcl8-2A-GFP or GFP alone under the control of the HSPC-specific Runx1+23 enhancer into zebrafish embryos at the single-cell stage. Time lapse fluorescence video microscopy and single-cell tracking was performed on HSPCs within the CHT. Overexpression of cxcl8 nearly doubled the amount of time HSPCs resided within the CHT when compared to expression of GFP alone as a control (cxcl8: 4.94 ± 0.86 h vs GFP: 2.54 ± 0.18 h, p=0.01, N=142 tracked cells). Substitution of WT cxcl8 with a mutant cxcl8 construct lacking the ELRCXC motif required for receptor binding reduced these effects (WT cxcl8: 6.6 ± 0.48 h vs ELRCXC-cxcl8: 5.3 ± 0.33 h, p=0.02, N=355 tracked cells). To observe HSPC-niche interactions, kdrl:mCherry endothelial cell reporter zebrafish were microinjected with Runx1+23:cxcl8-2A-GFP or Runx1+23:GFP DNA constructs. The percent of time individual HSPCs spent closely interacting with a single group of CHT endothelial cells (endothelial cell cuddling) was quantified over the period from 52 to 72 hours post-fertilization. Overexpression of cxcl8 by HSPCs increased HSPC-endothelial cell cuddling time by 30% (cxcl8: 87% vs GFP: 57%, p=0.001). To directly test competition between wild type and cxcl8 overexpressing HSPCs, zebrafish embryos were microinjected with a 1:1 molar ratio of Runx1+23:cxcl8-2A-mCherry and Runx1+23:clover DNA. Single cxcl8-2A-mCherry+ and clover+ competitor cells were tracked by time-lapse fluorescence confocal microscopy. HSPCs expressing cxcl8 resided longer within the CHT than competitor HSPCs when quantified over the period from 72 to 96 hours post-fertilization (cxcl8: 4.0 ± 0.20 h vs competitor: 2.5 ± 0.25 h, p=2.0 x 10-6, n=426 tracked cells). Single cell RNA-sequencing (scRNA-seq) of zebrafish embryos with mosaic expression of cxcl8 in HSPCs showed upregulation of cxcl12a in endothelial cells compared to endothelial cells from control embryos (p=5.19 x 10-3), suggesting a possible mechanism to explain the increased CHT residency time. Zebrafish with mosaic expression of Runx1+23:cxcl8 were raised to adulthood and the kidney marrow cells were analyzed by flow cytometry. Compared to clutchmate controls, Runx1+23:cxcl8 mosaic transgenics had a higher hematopoietic progenitor/precursor to lymphocyte ratio, suggesting a mild differentiation block and possible lineage skewing (cxcl8: 2.0 ± 0.15 vs control: 1.6 ± 0.10, p=0.048, N=25 animals). Taken together, these data support a model in which pre-malignant HSPC clones aberrantly express cxcl8 and acquire a selective advantage over normal clones through enhanced interactions with the endothelial cell niche. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

Patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) have poor treatment outcomes when treated with traditional chemoimmunotherapy. Superior outcomes have been observed with the 2 CAR-T cell products approved by the US FDA; tisagenlecleucel (CTL109) and axicabtagene ciloleucel (Axi-cel) with 12 months overall survival rates of 49% and 59% respectively. The 2 major acute toxicities of these therapies are cytokine release syndrome (CRS) and neurotoxicity. The incidence of grade 3 or higher neutropenia, anemia, and thrombocytopenia was 78%, 43%, and 38% in patients treated with axi-cel. Little is known regarding the patterns and outcomes of these cytopenias. Here we present our preliminary experience on the patterns and outcomes of cytopenias in 32 patients who received commercially-available CAR-T cell therapy at our institution. We retrospectively reviewed all DLBCL patients receiving either CTL109 or axi-cel between January and September 2018 at our institution. All patients received a conditioning regimen of low-dose cyclophosphamide and fludarabine, followed by CAR-T infusion. Persistent cytopenias were defined as incomplete absolute neutrophil count ( The median age of patients was 59 years (range, 23-80); 63% were male, 4 patients (13%) received CTL109 and 28 (87%) received axi-cel. Twenty-two patients (69%) had a 3 month follow up imaging for assessment of response, of whom 7 patients (32%) achieved complete response (CR), 6 patients (27%) achieved partial response (PR), and 9 patients (41%) had progressive disease (PD). Persistent neutropenia was observed in 3 patients (9%), while persistent thrombocytopenia was seen in 21 cases (65%) and persistent anemia was the most common with a frequency of 72% (23/32 cases). Median time to neutrophil, platelet and Hb recoveries were 11 days (range, 5-218 days), 59.5 days (range, 4-241 days), and 76 days (range, 0-218 days), respectively. Filgrastim was used in 15 (47%) patients to facilitate neutrophil recovery. Twenty-three patients (72%) experienced neurotoxicity, of whom 11 (34%) had grade 3-4 toxicity. The frequency of CRS was 94% (29 out of 32 patients), with grade 3-4 CRS observed in 4 cases (12.5%). We observed no significant association between CRS and persistent cytopenias. In our preliminary series of 32 patients, persistent anemia and thrombocytopenia were more common than neutropenia after CAR-T cell therapy. However, most of these patients required growth factor support for neutrophil recovery. Longer duration of follow up is necessary to determine the impact of persistent cytopenias on outcomes and survival after CAR-T cell therapy.

Introduction Autologous stem cell transplant patients (ASCT) are at risk for bacterial. Antibacterial prophylactic strategy is variable among centers. Fluoroquinolone prophylaxis in ASCT patients has not been found to add a survival benefit. We performed a retrospective review of patients undergoing ASCT with and without bacterial prophylaxis to compare endpoints of interest. Methods At our institution, patients undergoing ASCT for multiple myeloma (MM) receive levofloxacin 500 mg daily as prophylaxis, while lymphoma patients do not. We retrospectively examined MM or lymphoma patients undergoing ASCT between July of 2015 and 2018 for bacteremia episodes. MM patients received melphalan and lymphoma patients received BEAM. All patients received growth factor support. Clinical and microbial data was recorded and analyzed using SPSS. Results In total, 172 patients underwent ASCT for lymphoma and 343 for MM. Seventeen percent (30/172) of lymphoma patients and 5.5% (19/324) of MM patients had febrile neutropenic episodes associated with bacteremia. Relative risk was 3.1 [1.83 – 5.43, p Discussion Fluoroquinolone prophylaxis resulted in less blood stream infection episodes but more breakthrough infections with resistant isolates. There was no statistically significant difference between the rates of CDI infections despite a trend to lower incidence in the prophylaxis group. Bacteremia was more difficult to treat for the resistant isolates. There were two infection-related mortalities in the prophylaxis group. In the era of antimicrobial resistance, further reevaluation of the utility of fluoroquinolone prophylaxis should be carefully weighed against the risks.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

Background: Haploidentical hematopoietic cell transplantation (haplo-HCT) is an advancing and potentially curative therapy for patients with hematologic malignancies when the availability of HLA-matched donors are limited. The common source for haplo-HCT is donor bone marrow, but peripheral blood (PB) haplo-HCT is an emerging and less invasive option with similar survival outcomes. However, multiple series showed an increased incidence of culture negative fever early after haplo-HCT, which has been attributed to cytokine release syndrome (CRS) and more commonly observed with PB haplo-HCT. In this study, we investigated the incidence and impact of CRS on clinical outcomes and financial toxicity in PB haplo-HCT patients treated at a single center. Methods: A total of 40 patients who underwent PB haplo-HCT in the Ohio State University between Jan 2015 - Dec 2017 were included. CRS was defined and graded based on the criteria proposed by Lee, et al (Blood, 2014). Symptoms occurring before day +14 were included, patients with a documented infection (e.g. positive culture data) and clinical suspicion of sepsis were excluded. Overall survival (OS) was defined as the time from transplantation until death. Transplant-related mortality (TRM) was defined as death before day +28 from any cause and patients who died after day +28 with no evidence of disease. Results:. The median age at HCT was 53 years (range, 19 to 74), 63% were male. The most common diagnosis was acute myeloid leukemia (47.5%), followed by lymphoma (22.5%), acute lymphoblastic leukemia (7.5%) and myelodysplastic syndrome (7.5%). Two patients had undergone previous autologous HCT. At the time of haplo-HCT, 62% of patients were in complete remission, 38% had relapsed/refractory disease. Donor was the child in 58%, sibling in 24% and parent in 18% with an antigen match ratio of 5/10 in 50%, 6/10 in 30%, 7/10 in 12.5%, 8/10 in 7.5%. Mismatch for sex and ABO blood group were seen in 50% and 45%, respectively. Twenty percent of patients received myeloablative conditioning with fludarabine/busulfan/thiotepa, while 80% received reduced-intensity regimen with fludarabine/cyclophosphamide/total body irradiation. The incidence of CRS was 85%, including grade 1-2 CRS in 77.5% and grade 3-4 CRS in 7.5%. Time to the onset of CRS was Conclusions: CRS is commonly observed after PB haplo-HCT and usually manifests with fever within 48 hours of cell infusion. We observed favorable survival outcomes in patients who experienced mild CRS, which may suggest an association between mild CRS and graft-versus-leukemia effect. A minority of patients developed severe CRS requiring intensive care unit support, which was associated with poor OS, high TRM, prolonged hospital stay and increased financial toxicity. Further studies investigating biomarkers that can predict the development of severe CRS might enable the incorporation of CRS prophylaxis (e.g. with tocilizumab) into standard conditioning regimens for PB haplo-HCT. Disclosures Vasu: Boehringer Ingelheim Inc: Membership on an entity's Board of Directors or advisory committees. Mims:Novartis: Consultancy; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Devine:Kiadis Pharma: Consultancy. Jaglowski:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Consultancy.

7070Background: Cxcl8 is an angiogenic chemokine that is highly expressed in a subset of patients with MPN or clonal hematopoiesis of indeterminate potential (CHIP), a finding which is associated w...

IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Rα–chain to cells expressing the IL-15Rβγ complex, we postulated that certain IL-15–bearing cells must be required for NK cell homeostasis. Using IL-15WT/WT and IL-15−/− mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11chi dendritic cells (DCs), we demonstrate that ablation of the resting CD11chi DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11chi DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11chi DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.

Entry into the cell cycle is mediated by cyclin-dependent kinase 4/6 (CDK4/6) activation, followed by CDK2 activation. We found that pharmacologic inhibition of the Flt3 internal tandem duplication (ITD), a mutated receptor tyrosine kinase commonly found in patients with acute myelogenous leukemia (AML), led to the down-regulation of cyclin D2 and D3 followed by retinoblastoma protein (pRb) dephosphorylation and G1 cell-cycle arrest. This implicated the D-cyclin-CDK4/6 complex as a downstream effector of Flt3 ITD signaling. Indeed, single-agent PD0332991, a selective CDK4/6 inhibitor, caused sustained cell-cycle arrest in Flt3 ITD AML cell lines and prolonged survival in an in vivo model of Flt3 ITD AML. PD0332991 caused an initial cell-cycle arrest in well-established Flt3 wild-type (wt) AML cell lines, but this was overcome by down-regulation of p27Kip and reactivation of CDK2. This acquired resistance was not observed in a Flt3 ITD and a Flt3 wt sample from a patient with primary AML. In summary, the mechanism of cell-cycle arrest after treatment of Flt3 ITD AML with a Flt3 inhibitor involves down-regulation of cyclin D2 and D3. As such, CDK4/6 can be a therapeutic target in Flt3 ITD AML but also in primary Flt3 wt AML. Finally, acquired resistance to CDK4/6 inhibition can arise through activation CDK2.

Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.

Natural killer (NK) cells contribute to host immunity, including tumor surveillance, through the production of interferon gamma (IFN-γ). Although there is some knowledge about molecular mechanisms that induce IFN-γ in NK cells, considerably less is known about the mechanisms that reduce its expression. Here, we investigate the role of the Hlx transcription factor in IFN-γ production by NK cells. Hlx expression is induced in monokine-activated NK cells, but with delayed kinetics compared to IFN-γ. Ectopic Hlx expression decreases IFN-γ synthesis in primary human NK cells and IFN-γ promoter activity in an NK-like cell line. Hlx protein levels inversely correlate with those of STAT4, a requisite factor for optimal IFN-γ transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation and proteasome-dependent degradation of the active Y693-phosphorylated form of STAT4. Thus, Hlx expression in activated NK cells temporally controls and limits the monokine-induced production of IFN-γ, in part through the targeted depletion of STAT4.

The "holy grail" of allogeneic stem cell transplantation is to preserve the graft-versus-tumor (GVT) effect while eliminating graft-versus-host disease (GVHD). Endogenous donor-derived interleukin 15 (IL-15) has been implicated in the pathogenesis of acute GVHD, yet the mechanism by which it impacts this lethal process remains unclear. Using the well-described and clinically relevant C57BL/6 --> B6D2F1 murine model of acute GVHD, we demonstrate that in trans presentation of IL-15 by donor bone marrow-derived cells is required for the rapid onset of acute GVHD. Recipients of IL-15-/- C57BL/6 bone marrow cells show diminished type 1 polarization of T cells, yet there is no decrease in donor T-cell reconstitution. A molecular basis for these findings is provided with the observation that expression of T-bet, the master control gene for type 1 T-cell functions, is necessary for IL-15-mediated acute GVHD lethality. Finally, we demonstrate that in the absence of donor-derived IL-15, the GVT effect is maintained. These findings thus establish a mechanism by which endogenous donor-derived IL-15 impacts the pathobiology of acute GVHD and GVT activity.

There have been substantial advances in understanding the events that regulate gene expression at the cellular and molecular level; however, there has been limited progress integrating this information to understand how biological systems function in vivo. For example, the anti-inflammatory cytokine IL-10 is thought to down-regulate the effects of the pro-inflammatory cytokine IFN-gamma on monocyte activation following LPS stimulation. However, the often-postulated reciprocal regulation of IL-10 gene expression by IFN-gamma has not been studied in vivo. Here we demonstrate that the regulation of IL-10 gene expression has at least two phases following challenge with LPS or a gram-negative organism. In C57BL/6 mice, early IL-10 induction occurs independently of STAT-1, while a delayed active repression of IL-10 gene expression is critically dependent on STAT-1, but only partially dependent upon IFN-alpha/beta and IFN-gamma. This in vivo IL-10 production comes from blood monocytes, but not tissue macrophages, and cannot be reproduced in vitro. This study provides new insights into the regulation of IL-10 following challenge with a gram-negative organism, and highlights the importance of studying these cytokine regulatory pathways in vivo.

SummaryThe oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.

Efforts at harnessing the antitumour activity of natural killer (NK) cells have been investigated for the immunotherapy of human cancer for over two decades. Initial trials, focusing on the use of ex vivo-generated lymphokine activated killer (LAK) cells or activated NK cells, or in vivo cytokine therapy to expand and activate NK cells against autologous tumours, have yielded only modest success. Recent understanding of the means by which NK cells kill target cells through a complex set of activating and inhibitory receptors recognising corresponding ligands on tumour cells has paved the way for the design of improved strategies for NK cell-based immunotherapy. The net balance of activating and inhibitory signals through NK cell receptors determines whether an NK cell becomes activated or not. Successful therapeutic strategies should now focus on manipulating the balance in favour of activating receptor signalling. In the case of autologous cancers, such strategies may include the use of monoclonal antibodies with cytokines to better direct NK cells to their tumour targets through the process of antibody-dependent cellular cytotoxicity (ADCC) or the in vivo blocking of inhibitory interactions between NK receptors (NKRs) and ligands on tumour cells. Alternatively, allogeneic NK cells can be used whenever there is mismatching of inhibitory NK cell receptors and ligands. Finally, methods to modulate expression of NK cell receptors and their ligands on tumour cells by cytokines and other agents should be explored. In this review, the impact of NKR biology on the development of novel strategies for the use of NK cells in the treatment of human cancer is discussed.

The vascular niche is an important regulator of hematopoietic stem and progenitor cell (HSPC) function during development and in response to non-physiologic stress. The zebrafish caudal hematopoietic territory (CHT) is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). We have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT) during late embryogenesis. This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p=0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p=0.004). To understand whether CXCR1 acted directly on the vascular niche or indirectly via secreted factors or circulating cells, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p=0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as "donors" and WT embryos as "recipients". The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p=0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in HSPC biology and they provide a new example of how innate immune signaling pathways are important for interactions between stem and progenitor cells and the niche. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

CXCL8 (IL-8) is a chemokine with pleiotropic roles in host defense, angiogenesis and tumor metastasis. CXCL8 and its specific receptor, CXCR1, are broadly expressed within the hematopoietic and vascular systems. In an effort to identify novel secreted factors with effects on hematopoietic stem and progenitor cell (HSPC) function, we have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT). The CHT is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p = 0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p = 0.004). These studies did not show whether CXCR1 acted directly on the vascular niche or whether CXCR1 expression in endothelial cells might induce expression of soluble factors or activate circulating cells that then cause expansion of the niche through indirect mechanisms. To address this, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p = 0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as “donors” and WT embryos as “recipients”. The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p = 0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in angiogenesis and HSPC biology and they provide a new example of how innate immune signaling pathways are important for stem cell function. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Citation Format: Bradley W. Blaser, Jessica L. Moore, Elliott Hagedorn, Brian Li, Vera Binder, Owen Tamplin, Leonard I. Zon. CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B035.

An increased incidence of cardiovascular complications has been documented in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Despite this, little is known about the risk factors for hyperlipidemia or the role of lipid-lowering therapy early after transplantation. We performed a retrospective analysis of all patients who underwent allogeneic HSCT at the Dana-Farber Cancer Institute from 1998 to 2008 and who survived more than 100 days. The incidence of hypercholesterolemia and hypertriglyceridemia in the first 2 years after transplantation was 73.4% and 72.5%, respectively. In multivariable analysis, the development of acute graft-versus-host disease was independently associated with both hypercholesterolemia (odds ratio [OR] = 1.62) and hypertriglyceridemia (OR = 1.54) after transplantation. Statin use was instituted in 29% of patients and was associated with a significant net reduction in total cholesterol (65 mg/dL, P < .0001), triglyceride (118 mg/dL P < .0001), and LDL levels (59 mg/dL P < .0001) without any significant adverse effects. These data suggest that hyperlipidemia is common in the first 2 years after allogeneic transplantation when most patients remain under the care of the transplantation physician and lipid-lowering therapy may be underutilized. Given the cardiovascular risk associated with hyperlipidemia and the tolerability of statins, further prospective evaluation of lipid abnormalities and their treatment seems well warranted.

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell–mediated lysis of HLA-C–expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3–positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

Murine models of disease are vital to the understanding of pathogenesis and the development of novel therapeutics. We have previously established interleukin (IL)-15 transgenic (tg) mice that demonstrate rapid proliferation of natural killer (NK) and T cells, followed by spontaneous transformation to lethal leukemia. Herein, we have characterized this model, which has many features in common with the aggressive variants of NK and T large granular lymphocyte leukemia (LGLL) in humans. The LGLL blasts are cytolytic and produce IFN-gammaex vivo. Cytogenetic analysis revealed trisomy of chromosome 17 and/or 15. This model should provide opportunities to develop effective standard therapies for this fatal disease.

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL(+) leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BCCD34+ and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34(+) and CD34(+)/CD19(+) bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL) (T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.