Expansion Of Myeloid Cells Research Articles

Abstract A117: Tumor-produced IL-6 and IL-8 are associated with MDSC accumulation and correlate with long-term clinical outcomes in melanoma patients

Abstract Background: Recruitment and expansion of immunosuppressive myeloid cells present a significant barrier to the successful treatment of melanoma. We aimed to identify tumor-secreted factors that altered the frequency of MDSCs and correlated with clinical outcomes in advanced melanoma patients. We hypothesized that production of IL-6 and IL-8 by melanoma tumors would lead to expansion and accumulation of MDSCs and correlate with long-term clinical outcomes. Methods: Expression of IL-6 and IL-8 in melanoma tumors as well as the plasma concentration of IL-6 and IL-8 were measured and compared with the frequency of circulating MDSCs in a total of 52 stage IV melanoma patients. These measures were correlated with tumor burden, BRAF status, lactic acid dehydrogenase (LDH) levels and with clinical outcomes. Samples were collected beginning in January 2011 and clinical follow-up was collected through January 2018. Results: The plasma concentration of both IL-6 and IL-8 correlated with tumor burden (p < 0.0001 and p= 0.0122, respectively). Patients with high plasma concentrations of either IL-6 (40%) or IL-8 (63%) had worse median overall survival (MOS) (7.5 months, p < 0.0001 and 9.2 months, p = 0.0015, respectively) compared to patients with low concentrations, where MOS was not reached. Patients with high plasma concentrations of both IL-6 and IL-8 (35%) had even worse MOS (6.7 months) than patients with low levels of both cytokines (MOS not reached, 31%) p < 0.0001. We observed that patients with lower circulating concentrations and lower tumoral expression of IL-6 and IL-8 showed decreased frequencies of circulating MDSCs. Furthermore, patients with low frequencies of MDSCs (MOS not reached) had better overall survival than patients with high frequencies of these cells (MOS 11.9 months) p = 0.0224. Multivariable analysis showed that when adjusting for variables BRAF status and LDH in the model, the hazard for the event of death for subjects with both high IL-6 and IL-8 is 4.46 times (95% CI, 1.4, 14.2) that of the hazard for the subjects with low IL-6 and IL-8 (p = 0.01). Additionally, the hazard of death for subjects with higher total MDSCs is 3.3 times (95% CI, 1.2, 9.0) that for subjects with low total MDSCs. Conclusions: The durability of the nearly 7-year follow-up time for these advanced melanoma patients further strengthens the implied importance of IL-6 and IL-8 in the accumulation of MDSC’s in melanoma patients. These data provide clues for potential therapeutic targets in advanced melanoma patients. Citation Format: Richard P. Tobin, Kimberly R. Jordan, Dana Davis, Victoria M. Vorwald, Kasey Couts, Dexiang Gao, Derek E Smith, William A Robinson, Virginia Borges, Martin D McCarter. Tumor-produced IL-6 and IL-8 are associated with MDSC accumulation and correlate with long-term clinical outcomes in melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A117.

Exploiting epigenetically mediated changes: Acute myeloid leukemia, leukemia stem cells and the bone marrow microenvironment.

Acute myeloid leukemia (AML) derives from the clonal expansion of immature myeloid cells in the bone marrow, and results in the disruption of normal hematopoiesis and subsequent bone marrow failure. The bone marrow microenvironment (BME) and its immune and other supporting cells are regarded to facilitate the survival, differentiation and proliferation of leukemia stem cells (LSCs), which enables AML cells to persist and expand despite treatment. Recent studies have identified epigenetic modifications among AML cells and BME constituents in AML, and have shown that epigenetic therapy can potentially reprogram these alterations. In this review, we summarize the interactions between the BME and LSCs, and discuss changes in how the BME and immune cells interact with AML cells. After describing the epigenetic modifications seen across chromatin, DNA, the BME, and the immune microenvironment, we explore how demethylating agents may reprogram these pathological interactions, and potentially re-sensitize AML cells to treatment.

Bendamustine with Total Body Irradiation Limits Murine Graft-Versus-Host Disease in Part Via Myeloid-Derived Suppressor Cells

BackgroundGvHD remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An under-investigated strategy to reduce GvHD is the modification of the preparative conditioning regimen. Cyclophosphamide (CY) and total body irradiation (TBI) is a commonly utilized myeloablative conditioning regimen, but is associated with GvHD. Bendamustine (BEN) is an alkylating agent that has been applied recently in nonmyeloablative chemotherapy-based conditioning regimens, demonstrating reduced myelosuppression, transplant related mortality, and acute graft versus host disease (GvHD). We have recently published that BEN can effectively replace CY when given following murine haploidentical bone marrow transplantation. We therefore hypothesized that BEN can also have advantages over CY when given in conjunction with TBI as a myeloablative preparative regimen.MethodsWe used a major histocompatibility mismatched murine BMT model to evaluate GvHD following BEN-TBI and CY-TBI conditioning. BALB/c (H-2d) mice received equivalent doses of BEN or CY (based on maximum tolerated dose), followed by TBI (400 cGy) and infusion of bone marrow (BM) cells with splenocytes (SC) or purified T-cells from C57BL/6 (H-2b) mice. GvHD morbidity and mortality and engraftment were monitored and MDSC frequencies were assessed by flow cytometry and immunofluorescence.ResultsWe demonstrate that BEN-TBI conditioning, while facilitating complete donor chimerism, results in significantly less GvHD and improved survival compared to CY-TBI. In BEN-TBI conditioned mice, suppressive CD11b+Gr-1high myeloid cells are increased in the blood, bone marrow (BM), and spleen, with no evident differences in suppressive function. The ratio of Gr-1+ cells to T-cells is significantly increased in BEN-TBI mice in the intestines. When Gr-1high cells are depleted prior to transplant, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of G-CSF, which promotes CD11b+Gr-1+ myeloid cell expansion, trends towards an increase in survival in BEN-TBI mice.ConclusionsThese findings indicate a potential role of granulocytic myeloid-derived suppressor cells (MDSCs) in the mechanism by which BEN allows engraftment with reduced GvHD. BEN-TBI conditioning reduces GvHD morbidity and mortality compared to CY-TBI and may present a safer alternative to CY-TBI as a myeloablative conditioning regimen in allogeneic HCT. DisclosuresNo relevant conflicts of interest to declare.

The HACE1-NRF2 Axis a Novel Target in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). Despite this genetic heterogeneity, AML patients share Leukemia associated oncogenes such as NF-E2-related factor 2 (Nrf2) (Rushworth SA et al.). NRF2 is a transcription factor that activates genes with antioxidant response elements (ARE)-containing promoters and protects cancer cells from apoptosis. Inhibition of NRF2 or antioxidant defense increases the level of Radical Oxygen Species (ROS), leading to tumor supression (Chio IIC et al.). Recently, the E3 Ubiquitin-Protein Ligase HACE1, a tumor suppressor in solid tumors, was demonstrated to promote the expression of NRF2 in Huntigton disease (Rotblat B et al.). Thus, we hypothesized a role for HACE1 as an oncogenic factor acting through NRF2 activation in myeloid malignancies and provide first data supporting the HACE1-NRF2 axis to be a novel target in acute myeloid leukemias.Material and methodsThe mRNA expression data from AML patients (296 samples) vs normal Hematopoietic Stem Cells (HSC) (6 samples) were exported from the bloodSpot database. HACE1 mRNA and protein expression was measured by q-RT-PCR and western blot in 12 commercially available Myeloid Malignancies cell lines. The HACE1 inducible knock down (KD) was carried out by Sleeping Beauty Transposon system in U937 and NOMO-1 cell lines. The cell viability was analyzed by Cell Titer Glo Luminescent assay. Apoptosis was measured by Annexin V (AV)/Propidium Iodide (PI) assay.Results and discussionHACE1 mRNA is downregulated in AML patients compared to HSC (***p<0.001, Bloodspot database). However mRNA and HACE1 protein expression do not correlate in AML cell lines, suggesting post translational modifications. High HACE1 protein expression was observed in most AML cell lines. HACE1 KD reduced drastically the cell viability of U937 cells through caspase activation and NRF2 degradation. However, no effect on cell viability was observed in NOMO-1 cells. Recently, non-programmed cell death necroptosis induction has been described by TNFR1 activation in HACE1 knock out Mouse Embrionic Fibroblast cells (Tortola L et al.). In line with this study, we observed that TNF induces strong cell death in HACE1 KD NOMO-1 cells within 48 hours.In addition HACE1 KD promotes autophagy through p62 degradation (late autophagy marker) in U937 cells. Autophagy has recently been described to contribute to the differentiation and death of AML cells, and to the promotion of immunostimulatory signals activating immune responses against cancer cells (Chen L et al.; Pietrocola F et al.). Thus HACE1 might be a potential target to induce autophagy, providing a novel therapeutical target in the treatment of myeloid malignancies.Finally, HACE1 KD in our hands promoted sensitization of U937 and NOMO-1 cells to cytatarabine, the backbone therapy in AML patients. This treatment promotes HACE1 protein expression at 24 and 48 hours in NOMO-1 cells, which may explain the better response rates of HACE1 KD cells to cytarabine.Taken together, we provide first evidence of HACE1 being a novel oncogene in AML and that the HACE1-NRF2 axis is a promising target in the treatment of Acute Myeloid Leukemias. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Azacitidine and Ascorbate Inhibit the Competitive Outgrowth of Human TET2 Mutant HSPCs in a Xenograft Model of Pre-Leukemia

The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine.To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in “Hit & Run,” we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis.First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p<0.02). This underscores the importance of TET2 in promoting erythroid differentiation and suggests TET2 mutations can exert a myeloid lineage skewing sensitive to ascorbate.Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p<0.0001; HDR, n=3 independent experiments, p<0.0001) and absence of erythroid BFU-E. Interestingly, analysis of indels in Hit & Run colonies showed that serial replating enriched for a 65 base pair deletion that results in a null allele, suggesting that TET2-disrupted cells outcompete normal HSPCs in vitro.Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease.Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14).Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. DisclosuresNo relevant conflicts of interest to declare.

Recurrent Genomic Aberrations of D-Type Cyclins Are Therapeutic Targets of CDK4/6 Inhibitors in t(8;21) and MLL-Rearranged Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease, characterized by expansion of undifferentiated myeloid precursor cells. The outcome for AML has improved through optimal treatment protocols, new drugs, and better supportive care; however, relapse remains common and patients with relapsed AML have poor prognosis. Recent genome-wide analyses revealed several recurrently mutated genes in AML, however, few of these driver mutations have been developed as therapeutic targets to date. In AML, t(8;21) and MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape is limited.Patients and Methods:The AML-05 study is a Japanese nationwide multi-institutional study of children (age < 18 years old) with de novo AML, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). The trial was registered with the UMIN Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm; number UMIN000000511) and conducted in accordance with the principles set down in the Declaration of Helsinki, and approved by the Ethics Committees of all participating institutions. All patients, or their parents / guardians, provided written informed consent. For whole-exome sequencing (WES), whole-exome capture was accomplished by liquid phase hybridization of sonicated genomic DNA using a bait cRNA library (SureSelect Human ALL Exon V5 or V5 Inc RNA 5 kit), following the manufacturer's protocol. Massively parallel sequencing of the captured targets was performed using a HiSeq 2000/2500 (Illumina) with the paired-end 126-133 bp read option. For targeted sequencing, target enrichment was performed using a SureSelect custom kit (Agilent) designed to capture all coding exons of the 338 genes. Similarly, CCND1, CCND2, and CCND3 were also captured and sequenced in t(8;21) AML samples. For cell cycle analysis, cell lines were treated with DMSO, palbociclib (500 nM), or abemaciclib (500 nM) for 24 h. Then, cells were stained with propidium iodide and analyzed using a FACS Canto II flow cytometer (BD Biosciences).Results and Discussion:First, we analyzed paired AML tumor and germline samples from nine pediatric MLL-rearranged AML patients by WES. In total, 52 mutations (mean, 5.8 mutations/patient) were identified, including known mutational targets in AML, such as FLT3, BRAF, SETD2, BCORL1, and WT1. Moreover, novel CCND3 mutation was detected in one patient. Next, we analyzed 56 samples from patients with pediatric MLL-rearranged AML enrolled in the JPLSG AML-05 study, using targeted sequencing. We selected 338 genes, among which were previously reported and putative driver genes, including CCND3, for targeted sequencing. We identified eight mutations in CCND3 in five pediatric MLL-rearranged AML patients (8.9%). All mutations were clustered in the PEST domain. Four of the eight mutations were R271fs, which is a known hot-spot mutation in lymphoid malignancies. Mutations of the other D-type cyclins (CCND1, CCND2) have been reported in t(8;21) AML (Leukemia, 2017); therefore, we also searched for mutations in CCND1, CCND2, and CCND3 by targeted sequencing of samples from pediatric t(8;21) AML patients (n=105). CCND1 (n=3, 2.9%) and CCND2 (n=8, 7.6%) mutations were detected; however, no mutations of CCND3 were detected. By contrast, there were no mutations of CCND1 or CCND2 in MLL-rearranged AML (n=56), suggesting that mutations of D-type cyclins exhibit a subtype-specific pattern in AML. A recent study demonstrated that genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4/6 inhibitor (Cancer Cell, 2017), therefore, we also examined the effects of CDK4/6 inhibitors (abemaciclib and palbociclib) on two t(8;21) AML cell lines (Kasumi-1 and SKNO-1) and five MLL-rearrangement AML cell lines (ML-2, MV4-11, MOLM-13, THP-1, and NOMO-1) were analyzed. All cell lines described above exhibited impaired proliferation after treatment with CDK4/6 inhibitors. Furthermore, treatment of these cell lines with CDK4/6 inhibitors resulted in detection of lower frequencies of S/G2/M phase cells by flow cytometry, suggesting that cells were arrested in G1 phase via CDK4/6 inhibition. These data provide further insights into the genetic basis of, and potential therapeutic strategies in t(8;21) and MLL-rearranged acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Development of an Automated Tool for Assessing Response in Patients with Polycythemia Vera

Myeloproliferative neoplasms (MPNs) are a group of hematologic malignancies characterized by the clonal proliferation of one or more hematopoietic cell lineages demonstrated by terminal myeloid cell expansion into the peripheral blood. The most commonly diagnosed Philadelphia chromosome negative (Ph-) MPNs include essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF).Assessing various categories of response to a treatment is determined according to multiple factors, including laboratory values; transfusion dependence; degree of splenomegaly; mutational status and variant allelic frequency (VAF); cytogenetic abnormalities; and bone marrow morphological features, such as myeloblasts, fibrosis, and the volume ratio of hematopoietic stem cells in bone marrow [1]. While many of these components exist in electronic health records (EHRs), they are documented with varying degrees of structure. Some terms are recorded as tabular values, whereas others are captured purely in free text (Table 1). However, nearly all clinical trials and reviews studying patients with MPNs rely on expert adjudication of response as determined by these data points.The current process for determining response involves manual review of the patient's EHR data, an arduous task requiring extensive human effort. To alleviate these efforts, the Richard T. Silver Myeloproliferative Neoplasm Center at Weill Cornell Medicine (WCM) worked in tandem with the Architecture for Research Computing in Health (ARCH) program to develop a method for assessing response in patients with MPNs. A research data repository (RDR) containing data from both outpatient and inpatient EHRs was designed to allow for computational assessment of response.Structured data elements, including laboratory values, mutational data, and VAF, were extracted from the EHR. A natural language processing (NLP) pipeline using the Leo framework was developed [2] to extract data on cellularity, reticulin fibrosis, and myeloblast count from bone marrow biopsy pathology reports (Figure 1). Other data points, including splenomegaly and symptom burden, continue to require interpretation and manual collection by trained research personnel, who enter these data points into REDCap (Research Electronic Data Capture), a WCM-provisioned secure web application for managing clinical databases. These manual data are then pivoted and loaded into a Microsoft SQL Server environment [3]. Data generated by the MPN RDR were frequently reviewed for quality control, which drove subsequent iterations designed to minimize any identified errors.After acceptable confidence levels had been achieved, the MPN RDR was queried to provide data that were used to contribute to response assessments [1] for a retrospective review studying PV patients. Fully automated response parameters included hematocrit, platelet, and white blood cell count values; cellularity; reticulin fibrosis; and JAK2V617F VAF. Partially automated response assessments included rates of phlebotomy. Manually collected response criteria included symptom burden, degree of palpable splenomegaly, and indications of hemorrhagic/thrombotic events. Extracted data were merged with manually collected data within clinically justified temporal windows and applied to PV response criteria. The process provided valuable insight on potential modifications to the extraction process.Future steps include extending these processes to criteria still dependent on manual collection. Efforts are currently under way to apply NLP to hepatosplenomegaly and cytogenetics reports. This process will also be used to assist in response assessments for the spectrum of other MPN subtypes. While the feasibility of a fully comprehensive computable approach to assessing response in MPNs may not be entirely feasible nor even advisable, it is rather the objective that clinical data collection, which has been historically onerous, become automated to the furthest extent possible so as to allow research personnel to focus on the extraction of data elements that do require manual adjudication. Adaption of a similar workflow may help other institutions expand their ability to assess response in patients with MPNs and, potentially, additional hematologic malignancies. DisclosuresNo relevant conflicts of interest to declare.

Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses

Mice engrafted with components of a human immune system have become widely-used models for studying aspects of human immunity and disease. However, a defined methodology to objectively measure and compare the quality of the human immune response in different models is lacking. Here, by taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we provide an in-depth comparison of immune responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells promotes transcriptomic responses akin to those of human vaccinees. These enhanced transcriptomic profiles correlate with the development of an antigen-specific cellular and humoral response to YFV-17D. Altogether, our approach provides a robust scoring of the quality of the human immune response in humanized mice and highlights a rational path towards developing better pre-clinical models for studying the human immune response and disease.

Zebrafish miR-462-731 regulates hematopoietic specification and pu.1-dependent primitive myelopoiesis

MicroRNAs (miRNAs) play significant roles in both embryonic hematopoiesis and hematological malignancy. Zebrafish miR-462-731 cluster is orthologous of miR-191-425 in human which regulates proliferation and tumorigenesis. In our previous work, miR-462-731 was found highly and ubiquitously expressed during early embryogenesis. In this study, by loss-of-function analysis (morpholino knockdown combined with CRISRP/Cas9 knockout) and mRNA profiling, we suggest that miR-462-731 is required for normal embryonic development by regulating cell survival. We found that loss of miR-462/miR-731 caused a remarkable decrease in the number of erythroid cells as well as an ectopic myeloid cell expansion at 48 hpf, suggesting a skewing of myeloid-erythroid lineage differentiation. Mechanistically, miR-462-731 provides an instructive input for pu.1-dependent primitive myelopoiesis through regulating etsrp/scl signaling combined with a novel pu.1/miR-462-731 feedback loop. On the other hand, morpholino (MO) knockdown of miR-462/miR-731 resulted in an expansion of posterior blood islands at 24 hpf, which is a mild ventralization phenotype resulted from elevation of BMP signaling. Rescue experiments with both BMP type I receptor inhibitor dorsomorphin and alk8 MO indicate that miR-462-731 acts upstream of alk8 within the BMP/Smad signaling pathway and functions as a novel endogenous BMP antagonist. Besides, an impairment of angiogenesis was observed in miR-462/miR-731 morphants. The specification of arteries and veins was also perturbed, as characterized by the irregular patterning of efnb2a and flt4 expression. Our study unveils a previously unrecognized role of miR-462-731 in BMP/Smad signaling mediated hematopoietic specification of mesodermal progenitors and demonstrates a miR-462-731 mediated regulatory mechanism driving primitive myelopoiesis in the ALPM. We also show a requirement for miR-462-731 in regulating arterial-venous specification and definitive hematopoietic stem cell (HSC) production. The current findings might provide further insights into the molecular mechanistic basis of miRNA regulation of embryonic hematopoiesis and hematological malignancy.

Progesterone treatment enhances the expansion of placental immature myeloid cells in a mouse model of premature labor

Introductionimmature-myeloid cells (IMCs) are proangiogenic bone marrow (BM)-derived cells that normally differentiate into inflammatory cells such as neutrophils, monocytes and dendritic cells (DCs). We characterized placental IMCs comparing their gene expression and subpopulations to tumor IMCs, and tested our hypothesis that progesterone that inhibits preterm labor, may affect their abundance and differentiation. Methodsdifferences between IMC-subpopulations in subcutaneous tumors versus placentas in C57BL/6 or ICR (CD-1) mice were analyzed by flow cytometry and gene expression was detected by microarrays. BM- and placental cells were incubated with or without progesterone and IMC subpopulations were analyzed. For preterm labor induction pregnant mice pretreated or not with progesterone were or were not treated with Lipopolysaccharide (LPS). Resultswe detected enrichment of granulocytic-IMCs in placentas compared to tumors, paralleled by a decrease in monocytic-IMCs. mRNA expression of placenta- versus tumor IMCs revealed profound transcriptional alterations. Progesterone treated BM-CD11b+ cells ex-vivo induced enrichment of granulocytic-IMCs and a decrease in monocytic-IMCs and DCs. LPS treatment in-vivo led to an increase in BM-IMCs in both progesterone pretreated or non-pretreated mice. In the placenta LPS decreased the IMC population while progesterone led to complete abrogation of this effect. Discussionplacental IMCs differ from tumor-IMCs in both subpopulations and gene expression. Progesterone enhances the proliferation of placenta-specific granulocytic IMCs ex-vivo and LPS induced labor is accompanied by a decrease in placental IMCs only in progesterone non-pretreated mice. We thus speculate that the protective effect of progesterone in preventing preterm labor may be explained at least in part by this specific anti-inflammatory effect.

Knock-in of the Wt1 R394W mutation causes MDS and cooperates with Flt3/ITD to drive aggressive myeloid neoplasms in mice.

Wilms tumor 1 (WT1) is a zinc finger transcriptional regulator, and has been implicated as both a tumor suppressor and oncogene in various malignancies. Mutations in the DNA-binding domain of the WT1 gene are described in 10–15% of normal-karyotype AML (NK-AML) in pediatric and adult patients. Similar WT1 mutations have been reported in adult patients with myelodysplastic syndrome (MDS). WT1 mutations have been independently associated with treatment failure and poor prognosis in NK-AML. Internal tandem duplication (ITD) mutations of FMS-like tyrosine kinase 3 (FLT3) commonly co-occur with WT1-mutant AML, suggesting a cooperative role in leukemogenesis. The functional role of WT1 mutations in hematologic malignancies appears to be complex and is not yet fully elucidated. Here, we describe the hematologic phenotype of a knock-in mouse model of a Wt1 mutation (R394W), described in cases of human leukemia. We show that Wt1+/R394W mice develop MDS which becomes 100% penetrant in a transplant model, exhibit an aberrant expansion of myeloid progenitor cells, and demonstrate enhanced self-renewal of hematopoietic progenitor cells in vitro. We crossbred Wt1+/R394W mice with knock-in Flt3+/ITD mice, and show that mice with both mutations (Flt3+/ITD/Wt1+/R394W) develop a transplantable MDS/MPN, with more aggressive features compared to either single mutant mouse model.

Bendamustine with Total Body Irradiation Limits Murine Graft-versus-Host Disease in Part Through Effects on Myeloid-Derived Suppressor Cells

Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11b+Gr-1high myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1high cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11b+Gr-1+ myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Systemic Candidiasis and TLR2 Agonist Exposure Impact the Antifungal Response of Hematopoietic Stem and Progenitor Cells.

We have previously demonstrated that Candida albicans induces differentiation of hematopoietic stem and progenitor cells (HSPCs) toward the myeloid lineage both in vitro and in vivo in a TLR2- and Dectin-1-dependent manner, giving rise to functional macrophages. In this work, we used an ex vivo model to investigate the functional consequences for macrophages derived from HSPCs in vivo-exposed to Pam3CSK4 (a TLR2 agonist) or C. albicans infection. Short in vivo treatment of mice with Pam3CSK4 results in a tolerized phenotype of ex vivo HSPC-derived macrophages, whereas an extended Pam3CSK4 treatment confers a trained phenotype. Early during candidiasis, HSPCs give rise to macrophages trained in their response to Pam3CSK4 and with an increased fungicidal activity; however, as the infection progresses to higher fungal burden, HSPC-derived macrophages become tolerized, while their fungicidal capacity is maintained. These results demonstrate that memory-like innate immune responses, already described for monocytes and macrophages, also take place in HSPCs. Interestingly, extended Pam3CSK4 treatment leads to an expansion of spleen HSPCs and myeloid cells, and drastically reduces the fungal burden in the kidney and spleen during systemic C. albicans infection. This protection against tissue invasion is abrogated by immunodepletion of HSPCs, suggesting their protective role against infection in this model. In addition, HSPCs produce in vitro cytokines and chemokines in response to C. albicans and Pam3CSK4, and these secretomes are capable of inducing myeloid differentiation of HSPCs and modulating peritoneal macrophage cytokine responses. Taken together, these data assign an active role for HSPCs in sensing pathogens during infection and in contributing to host protection by diverse mechanisms.

Abstract PR09: Breaking down barriers restricting myeloid cell differentiation and infiltration in the tumor microenvironment with a first-in-class antibody targeting semaphorin4D, and rational combination therapies

Abstract Purpose: We expand mechanistic findings in preclinical studies to demonstrate that antibody blockade of semaphorin 4D (SEMA4D, CD100) reduces expansion of MDSC and shifts the balance of myeloid cells within the TME to facilitate tumor rejection. Efficacy is further enhanced when combined with various immunotherapies. Design of phase 1/2 combination trials of VX15/2503, a humanized IgG4 antibody targeting SEMA4D, with immune checkpoint inhibition will be presented. Methods: Anti-SEMA4D antibodies were evaluated alone and in combination with other immunotherapies in various preclinical models. Antitumor activity and immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine, and gene expression analysis. A phase I trial for single agent VX15/2503 was completed, and several 1b/2 combination immunotherapy trials are planned. Results: SEMA4D restricts migration of monocytes and promotes expansion of suppressive myeloid cells in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo restricts the infiltration and modulates polarization of leukocytes in the TME. Antibody blockade of SEMA4D facilitated recruitment of activated DCs and T lymphocytes in preclinical models. MDSCs were significantly reduced in tumor and blood following treatment, and new data characterizing MDSC function in preclinical models will be described. A significant shift towards increased Th1 cytokines (IFNg, TNFa) and CTL-recruiting chemokine CXCL9, with concurrent reduction in Treg- , MDSC- , and M2-macrophage promoting chemokines (CCL2, CXCL1, CXCL5) was also observed. Accordingly, Teff:Treg ratio (3x, p&amp;lt;0.005) and CTL activity (4x, p&amp;lt;0.0001) were increased. These coordinated changes in the tumoral immune context are associated with durable tumor rejection and immunologic memory in murine colon, breast, and melanoma models. Importantly, anti-SEMA4D treatment can further enhance activity of coadministered immunotherapies and chemotherapy. For example, combinations with immune checkpoint inhibitor anti-LAG3 or anti-CTLA-4 cause complete tumor regression in 90% or 100% of mice, as compared to ~20% with monotherapy (p&amp;lt;0.01). New data presented include synergistic activity of combinations of anti-SEMA4D with anti-LAG3 and additional studies of combinations with epigenetic modulators, including treatment of established tumors. Conclusions: SEMA4D blockade represents a novel mechanism to promote functional immune infiltration into the tumor and enhance immunotherapy. VX15/2503 treatment was well tolerated in a phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Plans for several clinical trials will be presented, including a phase 1b/2 of combination therapy with avelumab in immunotherapy-naïve NSCLC, and combination with anti-PD-1 or ipilimumab in various indications. A neoadjuvant trial of VX15 with anti-PD-1 in patients with metastatic colorectal and pancreatic cancers will be described, as well as a phase 1/2 trial of VX15 in pediatric and osteosarcoma patients. This abstract is also being presented as Poster B01. Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Cathie Foster, Alan Howell, Leslie Balch, John E. Leonard, Terrence L. Fisher, Clint Allen, Paul Clavijo, Siwen Hu-Leiskovan, Antoni Ribas, Ernest S. Smith, Maurice Zauderer. Breaking down barriers restricting myeloid cell differentiation and infiltration in the tumor microenvironment with a first-in-class antibody targeting semaphorin4D, and rational combination therapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR09.

ASXL2 regulates hematopoiesis in mice and its deficiency promotes myeloid expansion.

Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.

3114 - Pyrimido-Indole Derivative, UM171 Promotes Differentiation and Expansion of Myeloid Progenitors and NK Cells From Human Pluripotent Stem Cells

Advances in hematopoietic differentiation from human pluripotent stem cells (hPSCs) open up new opportunities for the development of technologies for manufacturing patient-customized blood products. Scaling up production of blood cells from hPSCs would be the next step toward manufacturing and clinical translation of hPSC-based therapies for blood diseases. The pyrimidoindole derivative UM171 was recently identified as a chemical compound that can robustly expand hematopoietic stem cells (HSCs), including HSCs with long-term repopulation potential. Here we evaluated the effect of UM171 on CD43 hematopoietic progenitors (HPs) produced from hPSCs in vitro. hPSCs were differentiated for 9 days in chemically-defined 2D cultures, to induce the formation of CD43+ HPs. CD43+ HPs were then expanded in Serum Free Expansion Medium (SFEM), in the presence of SCF, TPO, FLT3, IL6, and IL3, and with UM171 (35 nM) or DMSO (control), and assayed for phenotype and colony forming cell (CFC) potential. We found that UM171 treatment resulted in a significant increase in the frequency and number of total and myeloid biased CD34+CD43+ HPs, and a significant increase in myeloid CFC number relative to DMSO, after up to 7 days of expansion. Interestingly, UM171 treatment resulted in the maintenance and expansion of an intermediate CD45+235a+/-41alo42b- population with significant myeloid (CFC-GM and CFC-G) potential. In contrast, DMSO expanded cells showed enrichment of a 235a+/-41ahi population that was positive for expression of the mature megakaryocytic lineage marker, CD42b, and had no CFC potential, suggesting megakaryocytic lineage restriction. In addition, we found that UM171 significantly and robustly enhances CD56+ NK cell generation from hPSC-derived CD43+ cells. Overall, our studies revealed that UM171 promotes the expansion of myeloid and NK cell progenitors from hPSCs. Delineating the molecular, cellular, and functional mechanisms behind these effects will be critical in elucidating the clinical applications of this molecule. and in gaining new insight into hematopoietic progenitor cell biology, particularly expansion, lineage commitment, and differentiation.

3109 - BM-Mscs From Risk-Stratified AML Patients Support Normal Hematopoiesis and are Anti-Inflammatory in in Vivo Preclinical Models of Hematopoietic Reconstitution and Severe Colitis

Acute myeloid leukemia (AML) represents a heterogeneous group of disorders characterized by the rapid expansion of immature myeloid cells (blasts) in the bone marrow (BM). There is wide disease heterogeneity and patient risk-stratification principally relies on cytogenetic-molecular data. BM mesenchymal stem/stromal cells (BMSC) are key components of the hematopoietic niche thought to contribute to leukemia pathogenesis. We recently reported a correlation between behaviors of AML-derived BMSCs (AML-BMSCs) in vitro and overall survival in AML patients. To elaborate on this finding, we investigated the capacity of AML-BMSCs from risk-stratified de novo AML patients to regulate the homeostasis of healthy CD34+ hematopoietic stem/progenitor cells (HSPC), and to exert anti-inflammatory effects in vivo in a pre-clinical model of severe acute colitis. We report that regardless of risk-group, AML-BMSCs support, similar to HD-BMSCs, the survival, proliferation, differentiation and clonogenecity of CD34+ cells in vitro, and the in vivo immune deficient mice repopulating assays. Additionally, AML-BMSC were capable of reversing the inflammatory phenotype in preclinical models of acute severe colitis, showing a greater anti-inflammatory capacity compared to HD-BMSC. Collectively, these data indicate that AML-BMSCs retain the capacity to support healthy HPSC and can suppress inflammation in vivo.

Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin β subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.

Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.