ALBERT

Description: Introduction The involvement of the bone marrow microenvironment (BMME) into disease progression and therapeutic response of myelodysplastic syndromes (MDS) is indisputable. Hereby, mesenchymal stromal cells (MSCs) play an important role for both the support of the leukemic clone and the remaining healthy hematopoietic stem and progenitor cells (HSPCs). The extracellular matrix (ECM) secreted by MSCs regulates stem cell fate through the modulation of cytokine and growth factor delivery and may also be targeted by clinically available drugs such as luspatercept, a novel recombinant fusion protein containing modified extracellular domain of activin receptor IIB. Luspatercept is a first-in-class erythroid maturation agent with promising results in lower-risk MDS patients with red blood cell transfusion dependency. Aim To shed light on the largely unknown composition and function of the MSC-derived ECM, we have characterized ECM from MDS patients vs. healthy controls and elucidate how luspatercept may modulate their functional characteristics. Methods Bone marrow-derived MSCs from patients with lower-risk MDS and age-matched healthy donors (HD) were treated with RAP-536, a murine homologue of luspatercept harboring the same activin receptor IIB domain. MSCs of three patients were treated with RAP-536 and RNA sequencing was carried out. Gene expression and pathway analyses were performed using the Reactome tool (https://reactome.org). Candidate genes were validated by quantitative real-time PCR (qPCR). For the generation of ECM, MSCs were seeded on poly-octadecene-alt-maleic anhydride and human fibronectin coated glass slides in the presence or absence of RAP-536. To yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning electron microscopy (SEM), sulfated glycosaminoglycan (GAG), fibronectin and collagen staining as well as GAG quantification (Blyscan assay). Moreover, purified HD CD34+ HSPCs were cultured on ECM scaffolds for 6 and 9 days, respectively. Subsequently, expansion of adherent and supernatant cells was determined and the phenotype was analyzed by flow cytometry. Results RNA sequencing of MDS MSCs after six days of RAP-536 treatment revealed a total of 58 significantly regulated genes, thereof 24 up- and 34 down-regulated genes. Gene enrichment and pathway analyses revealed a striking involvement in ECM organization, collagen biosynthesis and formation. Moreover, integrin cell surface interaction genes showed significantly differential expression. Focusing on collagens as important ECM components, we identified Col7A1 and Col4A2 to be down-regulated. Indeed, both collagen mRNAs were significantly decreased by 46% and 25%, respectively, in MSCs after RAP-536 treatment compared to untreated controls. SEM characterization and immunofluorescence staining of the ECM showed a more compact fiber network produced by MDS MSCs. Moreover, MDS ECM contained higher levels of collagen and GAGs. Blyscan assay confirmed the latter observation, showing significantly higher sulfated GAG concentrations in MDS ECM. Interestingly, trapping of TGFβ superfamily ligands, such as GDF-11, by RAP-536 clearly reduced Col4 staining intensity in MDS MSC ECM. Structural and compositional ECM differences had functional impact on the expansion of HSPCs cultured on the matrices. Significant higher total cell numbers were detected on healthy ECM (18.3-fold vs. 12.1-fold expansion, *p〈 0.05) but not on MDS ECM (12.9-fold) after 9 days of culture. The number of adherent cells increased 8.5-fold on healthy and 4.3-fold on MDS ECM and could be further increased after RAP-536 treatment of MSCs. Using flow cytometry, we found a 3.1-fold increased proportion of CD90+ HSPCs in the adherent fraction on healthy but only 1.8-fold on MDS MSC ECM. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be significantly higher expressed in the adherent HSPC fraction. RAP-536 treatment resulted in up to 20% higher expression of both CD90 and integrin subunits. Summary We demonstrate an association between induced collagen abundance and reduced hematopoietic support in ECM derived from MDS MSCs and conclude that compact MDS ECM structure induced by TGFβ superfamily members may alter the cytokine environment for HSPCs. Consequently, TGFβ ligand trapping by RAP-536/luspatercept leads to ECM re-organization and thus an improved hematopoietic support. Disclosures Stoelzel: Shire: Consultancy, Other: Travel funding; Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Description: Introduction: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis due to genetic and functional abnormalities of hematopoietic stem and progenitor cells (HSPCs). Accumulating evidence now also points to the bone marrow microenvironment (BMME) as a key mediator of MDS pathophysiology. Increased levels of transforming growth factor beta (TGF-β) superfamily ligands, including growth differentiation factor 11 (GDF-11), in the bone marrow have been linked to ineffective erythropoiesis and activation of SMAD2/3 signalling in MDS. Luspatercept (ACE-536) is a novel recombinant fusion protein containing modified activin receptor type IIB linked to the fragment crystallisable (Fc) domain of human immunoglobulin G1 and is a first-in-class erythroid maturation agent. Luspatercept binds to selected TGF-β superfamily ligands, including GDF-11 and activin B, restoring late-stage erythropoiesis in MDS mice and patients. Whether luspatercept modulates the BMME is unknown. Aim: We investigated the potential impact of luspatercept on the biology of mesenchymal stromal cells (MSCs). Methods: MSCs from patients with either high-or low-risk MDS and age-matched healthy donors (HD) were treated with GDF-11 in the presence or absence of RAP-536, a homologue of luspatercept harboring the same activin receptor IIB domain. Subsequently, Smad signaling pathway activation was analyzed by Western blot. Moreover, MSC phenotype, stromal derived factor-1 (SDF-1/CXCL12) expression and secretion as well as the osteogenic differentiation potential were recorded. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with GDF-11 ± RAP-536 before freshly isolated CD34+ HSPCs were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly. After four weeks, the HSPCs were seeded in methylcellulose medium to perform a CFU-GEMM assay. Further, engraftment and migration potential of co-cultured HSPCs was analyzed in zebrafish embryos in vivo. Results: GDF-11/RAP-536 treatment did not impact viability, proliferation, and growth pattern of MSCs. The osteogenic differentiation was significantly improved by RAP-536 treatment which was shown by a 2.3-fold increase of ALP activity. GDF-11 induced Smad2/3 phosphorylation in MSCs which was inhibited by RAP-536 to about 50% of activity. The MSC phenotype characterized by the expression of CD73, CD90, CD105, CD146, CD29 and CD44 was not significantly influenced by RAP-536 treatment. Interestingly, the chemokine SDF-1 which plays an important role for the interaction and support of HSPCs was significantly up-regulated in MDS and HD MSCs by RAP-536 at both the mRNA (1.9-fold) and the protein level (2.1-fold; 313.0±35.0 vs. 649.7±76.8 pg/ml, *p〈 0.05, n=3). This restored SDF-1 secretion of MSCs resulted in functional effects of HSPCs when co-cultured with MSCs. The number of CAF-C was significantly higher after two, three, and four weeks of HSPCs cultured on pre-treated MSC layers (12.0-fold / 3.4-fold / 1.75-fold). The phenotypical analysis of HSPCs demonstrated increased expression levels of CXCR4 in the adherent fraction. Addition of the CXCR4 antagonist AMD3100 blocked enhanced colony formation by RAP-536 confirming the previous observation on SDF-1 modulation. Clonal growth of CAF-C-derived HSPCs after four weeks of co-culture on un- or pre-treated MSC monolayers was analyzed in a secondary colony-forming cell assay (CFU) for an additional 14 days. The frequency of CFUs was increased in all lineages with significant differences in total colony numbers (15±5.8 vs. 35.8±14.7, *p〈 0.05, n=6). These results were validated in vivo in a zebrafish model. HSPCs were co-cultured with GDF-11 and RAP-536 pre-treated MDS-MSCs for one week before injection into the zebrafish circulation. One and two days post-injection, a significantly higher cell proportion could be detected in the caudal hematopoietic (CHT) region as a result of RAP-536 pre-treatment (Fig.1). Conclusion: These data provide first evidence that RAP-536 has also the capacity to modulate MSCs which might contribute to the restoration of hematopoiesis in MDS. Disclosures Platzbecker: Celgene: Research Funding.

Description: Introduction: Flow cytometry (FCM) is considered as a co-criterion in MDS diagnostics if the main diagnostic methods are not sufficient to clearly diagnose or rule out MDS. The iMDSFlow working group of the European LeukemiaNet worked on recommendations regarding the harmonization of preanalytics, data analysis, as well as of appropriate diagnostic scores. The aims of the present study were (1) to test whether the parameters and reference ranges applied for FCM diagnostics in MDS do have an impact on prognosis; and (2) whether the incorporation of computational algorithms in data mining could further improve the prognostic information of FCM. Methods: FCM diagnostics was performed in bone marrow (BM) of 303 patients cytomorphologically classified as MDS using a Lyse-wash-method and measuring on a FACSCantoII cytometer. For FCM data evaluation, commonly used diagnostic flow-scores - FCSS (Wells et al. 2003), Ogata-score (Ogata et al. 2009), new iFS-score (Cremers et al. 2017), ELN-Red-score (Westers et al. 2017), Red-score (Mathis et al. 2013) - were applied, including the analysis of progenitor cells, granulopoiesis, monopoiesis, and nucleated erythropoiesis. Thus, 55 FCM parameters necessary to assess the mentioned flow-scores and 33 additional FCM parameters have been evaluated. Clinical variables (n=11) including IPSS-R (vLR+LR: 163 pts., Int: 81 pts., HR+vHR: 59 pts.) have also been recorded. Median follow-up time was 28 months (1.5 - 84 mo). Overall survival (OS) was assessed in uni- and multivariate Cox proportional hazards regression analysis using log-rank likelihood test. Reference ranges for FCM parameters have been assessed before using BM of 49 healthy donors. In addition, in order to improve prognostic output, a computational approach was devised that tests every possible combination of binary, ternary, and quaternary marker strata segregation to identify the thresholds that bring about optimum separation of hazard strata in each model based on log-likelihood p-value. Finally, it was assessed whether the FCM variables correlate with IPSS-R and also have independent predictive value in MDS. Results: First, MDS flow-scores and IPSS-R have been tested for OS resulting in a significant association of higher flow-scores with shorter survival, Ogata: median OS: 51 mo vs. not reached (NR), hazard ratio (HR)=2.1, p=0.00084; FCSS: 70 mo vs. NR, HR=2.1, p=0.013; new iFS: 70 mo vs. NR, HR=1.9, p=0.02; IPSS-R: 37 vs. 55 vs. 74 mo; HR=2.4/1.4, p=0.0045. Second, we wanted to explore whether single FCM parameters inherit prognostic information using the diagnostic reference ranges. Of note, 19 of all FCM parameters were significantly associated with OS, e.g. decreased side scatter (granulopoiesis; 37 mo vs. NR, HR=2.4, p=0.000047), decreased % of B-lymphatic progenitors (lyPC; 52 mo vs. NR, HR=2.3, p=0.0013). As a third step, a computational learning algorithm was applied. In 7/19 parameters already showing significant differences in OS while using the diagnostic reference ranges, the algorithm could add information. Moreover, the algorithm optimized thresholding for 17 additional FCM parameters, resulting in clearer survival differences, e.g. decreased side scatter (myPC, 33 vs. 74 mo, HR=2.6 p=0.00002), aberrant CD5 expression (granulopoiesis; 32 vs. 74 mo, HR=2.5 p=0.000045). Next, we performed an independent multivariate Cox model with lasso penalty, considering 14 FCM parameters which in univariate analysis showed a clearer survival difference compared to IPSS-R. Of note, this identified the following as best predictors of OS: IPSS-R, decreased side scatter (granulopoiesis), CD45 MFI (ratio of CD45 MFI lymphocytes : myPC), and decreased % B-lymphatic progenitors. Conclusions: In addition to the known importance of FCM in MDS diagnostics, we provided evidence that FCM has also an important prognostic impact in relation to IPSS-R even in this heterogeneous group of MDS patients. Thus, in univariate analysis a computational learning algorithm was able to refine thresholding resulting in an improved separation of OS curves. Importantly, even in multivariate analysis FCM proved its significance for patient outcome. Further studies should evaluate, whether a flow-score including the most influential variables could add prognostic information to IPSS-R. Disclosures Kordasti: Celgene: Research Funding; Novartis: Research Funding; Boston Biomed: Consultancy; API: Consultancy. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Description: Lymphoma cell survival and progression are putatively dependent on a specific microanatomic localization within secondary lymphoid organs. Despite compelling data correlating homeostatic chemokine receptor expression and human lymphoma pathogenesis, genetic models that either mimic lymphoma dissemination or dissect a crosstalk of lymphoma and stromal cells are missing. Applying the genetically tractable Eμ-Myc transgenic mouse model, we show that the chemokine receptor CCR7 regulates Eμ-Myc lymphoma homing to lymph nodes and distinctive microanatomic sites of the spleen. CCR7-controlled access of lymphoma cells to the splenic T-cell zone led to a significant survival advantage compared with CCR7-deficient lymphoma cells, which were excluded from this zone. Within the niche, lymphoma cells stimulated a reciprocal cross-talk with gp38+ fibroblastic reticular cells. This reciprocal cooperation program was mediated by lymphoma B cell–presented lymphotoxin, which acted on lymphotoxin-β–receptor-bearing stromal cells followed by alteration of stromal cellular composition. Cross-talk inhibition by lymphotoxin-α deletion and using a lymphotoxin-β receptor-immunoglobulin fusion protein impaired lymphoma growth. Thus, abrogation of CCR7-governed migration and of sustained lymphotoxin signaling could provide new targets in lymphoma therapy.

Description: Background Regulatory T cells (Tregs) are a non-redundant, suppressive population of CD4+ T cells that are essential for the prevention of autoimmune diseases in humans. They may also contribute to overall immune suppression in malignancies and prevent effective immune surveillance against malignant cells. The correlation between the increased number of Tregs and higher-risk MDS has been shown before (Kordasti, Blood 2007). Expansion of effector T cells (Teff) also signifies the lower-risk disease (Kordasti, BJH, 2009). Using deep phenotyping and unbiased automated clustering we have previously identified novel populations of Tregs with distinct functional properties (Kordasti, Blood 2016), including a CD161-expressing subpopulation (Povoleri, Nature Imm, 2018). The aim of this study was to interrogate Treg and Teff subpopulations in more depth and identify transcriptomic profiles of Tregs and Teff in bone marrow (BM) and peripheral blood (PB) of MDS patients, and potential differences between PB and BM residing cells. Single-cell RNA sequencing (scRNAseq) in combination with oligonucleotide-tagged antibodies and an in-house pipeline utilizing Seurat for QC, data integration and analysis were used for this purpose. Methods PB and BM were collected from a low (del(5q)) and a high-risk (monosomy 7) MDS patient. These samples were magnetically enriched for CD4+ T cells, followed by flow sorting of Tregs (CD4+, CD25high, and CD127low) and Teff (CD4+, CD25low, and CD127high). In total, 8 cell fractions were used for downstream scRNAseq and concomitant protein expression profiling using the BD Rhapsody Single-Cell Analysis System (Becton Dickinson, CA, USA). Cell fractions were first labelled by oligo-tagged antibodies (CD95, CD45RO, CD38, CTLA-4, PD-1, HLA-DR) and a sample tag (for sample multiplexing). After labelling, all samples were combined for cartridge-based single-cell capture and molecular indexing of mRNA transcripts on magnetic barcoded beads. Following bead retrieval and on-bead cDNA synthesis, parallel targeted mRNA (259 T cell-related genes) and Abseq sequencing libraries were generated. Deep sequencing was performed using the HiSeq400 platform. Nonlinear dimensionality reduction was carried out using uniform manifold approximation and projection (UMAP), which produced UMAP layouts and heatmaps to identify populations. Tregs were further stratified according to high/low expression of the CD95 protein, CD161, and CD150 mRNA expression. The Wilcoxon Rank Sum test was used for statistics followed by Bonferroni corrections. The Gene Set Variation Analysis (GSVA) package was used for pathway enrichment analysis. Results and discussion Unbiased analysis by UMAP was able to clearly separate the high- and low-risk patients based on the Treg and Teff signature in BM, which was not the case in PB (Figure 1a and 1b). At protein level, BM Tregs from the high-risk patient had a significantly higher expression of CD95 (p = 7.23 x 10-17), CD45RO (p = 1.06 x 10-51), CTLA4 (p = 4.47 x 10-83) PD-1(p = 5.42 x 10-81), and CD38 (p = 4.16 x 10-63) (Figure 1c), suggesting a more functional Treg profile. GSVA analysis of transcriptomes showed significant changes between high- and low-risk patients, for both Tregs (102 pathways p≤0.05) and Teffs (89 pathways p≤0.05) in BM. Specifically, Tregs from high-risk MDS were more enriched with non-TCR-mediated activation pathways, including leukocyte differentiation, Jak-STAT signaling, and positive regulation of leukocyte cell-cell adhesion (Figure 1d). A deeper investigation of Tregs showed that subsets of interest, in particular the IL-17 secreting CD161+ Tregs and the hematopoietic stem cell quiescence-maintaining CD150+ Tregs (Hirata, 2018) were found in low abundance in the BM of both high- and low-risk patients; 16% and 10%; and, 1% and 7%, respectively. In summary, we have developed an analytical pipeline and provide evidence for the ability of simultaneous single-cell transcriptomics and protein expression to identify different BM-specific T cell signatures in high- versus low-risk MDS. Interestingly, Tregs from high risk MDS BM show higher expression of Treg-related proteins and are more enriched in non-TCR-specific activation pathways, which may reflect a myeloid derived inflammatory environment. Overall, these findings suggest a bone marrow-specific Treg signature in MDS, which could distinguish low- and high-risk disease. Disclosures McLornan: Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Platzbecker:Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Kordasti:Celgene: Research Funding; Novartis: Research Funding; Boston Biomed: Consultancy; API: Consultancy.