ALBERT

Description: Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage− Sca-1+ c-Kit+ CD150+ CD48− HSPC subset (LSK CD150+ CD48− cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48− cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

Description: Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell...

Description: Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.

Description: Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted. Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL). We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P

Description: PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)–restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)–like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [KD] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSCs) but not normal leukocytes (P 〈 .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P 〈 .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.

Description: Abstract 2090 We have shown that cytotoxic T lymphocytes (CTL) with specificity for the cyclin E (CCNE) derived HLA-A2-restricted peptide CCNE144-152 (ILLDWLMEV) specifically lyse myeloid and lymphoid leukemia in proportion to CCNE overexpression. Full length (FL) CCNE is also overexpressed in most solid tumors, including breast cancer where it is a poor prognostic factor. In addition, leukemia and many breast cancers express tumor-specific low molecular weight (LMW) isoforms of CCNE that result from post-translational processing of the FL protein. Neutrophil elastase (NE), derived from the primary granules of neutrophils, cleaves FL CCNE into LMW forms and NE has been identified in breast cancer tissue. Therefore, we hypothesized that CCNE may also be a breast cancer tumor antigen because the CCNE144-152 peptide is contained within the overexpressed FL and LMW forms, and that effective T cell immunity could be amplified by increased availability of LMW forms within tumor cells exposed to NE. The link between innate immunity, inflammation, and tumor immunity is poorly understood, and this mechanism could explain a role for tumor-infiltrating inflammatory cells in breast cancer. To test this, we elicited CCNE-CTL from peripheral blood lymphocytes of HLA-A2+ healthy donors by weekly stimulation with CCNE-pulsed T2 cells and low-dose IL-2. After 21 days, cytotoxicity of target cells by the lymphocytes was tested with a standard 4-hour calcein AM-based assay. The CCNE-CTL specifically lysed T2 cells pulsed with CCNE (30%) but not non-pulsed T2 cells (0%) at an effector:target (E:T) ratio of 20:1 (p 〈 0.01). Next, we tested whether CCNE-CTL killed HLA-A2+ MDA-MB-231 (231) breast cancer cells. First, we confirmed that FL CCNE and LMW forms were expressed in 231 cells, while low expression of FL CCNE and no expression of the LMW forms was observed in benign epithelial cells by Western blot. Next, we showed that CCNE-CTL mediated 49% lysis of 231 breast cancer cells at an E:T ratio of 20:1. To look for in vivo evidence of CCNE recognition, we studied peripheral blood lymphocytes from breast cancer patients by flow cytometry with CCNE/HLA-A2 tetramers and anti-CD8 antibodies. In 3 of 4 breast cancer patients we identified CCNE-CTL, with no detectable CCNE-CTL in healthy controls. Together, these results confirm that CCNE is also a tumor antigen in breast cancer. NE, which cleaves CCNE and is expressed in breast cancer tissue, may be produced endogenously by breast cancer cells, or exogenously by inflammatory cells in the tumor microenvironment. Therefore, we studied 231 cells and three other breast cancer cell lines (MDA-MB-453, MCF-7, and HER18) for NE expression. RT-PCR performed with NE-specific primers showed that none of the cells expressed NE mRNA and Western blot showed no NE protein expression, suggesting that NE in tumor tissue derives from neutrophils or other inflammatory cells. To determine whether NE is taken up by breast cancer cells, we used flow cytometry to show that 231 cells pulsed with soluble NE took up an increasing amount of NE and was maximal by 24 hours when intracellular NE expression in 231 cells was comparable to that of HL60 leukemia cells that express high levels of NE. In addition, LMW isoforms of CCNE were increased in NE-pulsed 231 cells, by Western blot. Importantly, CCNE-CTL specific lysis of NE-pulsed 231 cells was 2-fold higher compared to nonpulsed 231 (46% versus 22% at E:T 10:1, p = 0.01). Taken together, these data show that overexpressed CCNE144-152 is a novel breast cancer peptide antigen. Furthermore, exogenous NE is taken up by breast cancer cells, increasing LMW forms of CCNE and enhancing the susceptibility of breast cancer cells to CCNE-CTL-mediated cytolysis. This study links a specific enzyme secreted by neutrophils in the innate immune response to tumors to a specific adaptive immune response against breast cancer, and it suggests that immunotherapy targeting CCNE is warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4301 The leukemia associated antigens (LAA) proteinase-3 (P3) and neutrophil elastase (NE) are serine proteases found within neutrophil azurophil granules and are aberrantly expressed by leukemia. The human leukocyte antigen (HLA)-A2 restricted nonomeric peptide PR1, derived from P3 and NE, has been detected on leukemia cell surface in association with HLA-A2. PR1-specific cytotoxic T lymphocytes (CTL) were detected in patients following hematopoietic stem cell transplantation and were associated with clinical remission. Furthermore, in a phase I/II clinical trial, PR1-vaccine showed immunologic and clinical responses in patients with MDS and AML. Antigen cross presentation is a mechanism by which exogenous antigens are taken up by antigen presenting cells (APC) and presented in association with major histocompatibility (MHC)-I molecule. Cross presentation is thought to be the primary mechanism by which tumor antigens are presented to elicit tumor specific immune responses. Cross presentation by dendritic cells (DC), which express co-stimulatory molecules, leads to immune priming, while antigen presentation by B cells is associated with immune tolerance. Since cross presentation of leukemia antigens has been associated with anti-leukemia immune responses, we investigated whether P3 and NE are cross presented by DCs and B cells, and whether the source of P3 and NE (soluble vs. leukemia-cell associated) determines priming of the anti-PR1 immune response. We have previously reported that serum P3 levels are significantly higher in leukemia patients, and we now also report higher serum NE levels in patients with AML (median±standard deviation (SD)= 1236 ±572 ng/ml; n=15) and CML (median ± SD = 941 ± 494 ng/ml; n=15), in comparison with healthy donor blood (median ± SD = 65 ± 177 ng/ml; n=5) (p