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Introduction: Daratumumab (Dara) is an anti-CD38 monoclonal antibody representing a novel treatment agent for multiple myeloma (MM). Nonetheless, several studies have reported a Dara-related impairment of CD34+ hematopoietic stem cell (HSC) mobilization and post-autologous stem cell transplantation (ASCT) complications, including low yields of mobilized HSCs and delayed neutrophil engraftment. Impact of Dara on the mobilization process and HSCs remains poorly understood even though sufficient yields of CD34+ cells are necessary for a successful ASCT and subsequent patient recovery. Aims: To compare the effect of the Dara-containing (Dara-Bortezomib-Dexamethasone [D-VCd]) and conventional (Bortezomib-Thalidomide-Dexamethasone [VTd]) therapy on CD34+ HSCs. Methods: Transplant eligible MM patients were treated with D-VCd or VTd induction regimen followed by a cyclophosphamide + G-CSF mobilization and a high-dose melphalan D -1 before ASCT. Flow cytometry (FCM) screening of CD34+ subsets was performed in the bone marrow (BM) or apheresis product (AP) at three consecutive time points: 1) diagnostic BM (DG), 2) mobilization AP (MOB), 3) a day prior ASCT BM (D-1). Furthermore, RNA sequencing (RNAseq) of sorted CD34+ cells was performed on total RNA with ribo-depletion protocol in AP after the induction. D-VCd samples had lower RNA yields thus the D-VCd or VTd groups were processed as independent batches. Results: Clinical data revealed no significant differences in mobilization (p >0.050) likely due to a small cohort sizes (D-VCd n=5 vs VTd n=9), though a trend towards worse performance in D-VCd was observed. Median CD34+ cell yield was 3.08 vs 10.56 x 10 6/kg. Platelet recovery of >20x10 9/L was D+14 vs D+12 (range: 11-18 vs 10-16). Neutrophil recovery of >0.5x10 9/L was D+12 in both groups (range: 11-17 vs 11-12). In FCM analysis, DG (n=14), MOB D-VCd (n=5) vs VTd (n=9), D-1 D-VCd (n=7) vs VTd (n=15) were compared. CD34+ frequency (Fig. 1A) difference in MOB D-VCd vs VTd was insignificant (median: 1.15% vs 1.89%), whereas CD34+ fraction dropped in D-1 D-VCd (median: 0.52% vs 0.72%, p=0.027), albeit there was no significant reduction in D-1 D-VCd vs initial DG (median: 0.52% vs 0.45%). Differences in the distribution of certain HSC subsets were detected in the CD34+ pool (Fig. 1B-E). Frequency of multipotent progenitors (MPPs) (Fig. 1B) was increased in MOB D-VCd (median: 82.1% vs 66.2%, p=0.004). Frequency of lympho-myeloid-primed progenitor + granulocyte-monocyte progenitor (LMPP+GMP) (Fig. 1C) subset was reduced in D-VCd in both MOB (median: 1.7% vs 16.9%, p=0.042) and D-1 (median: 5.3% vs 14.0%; p=0.026). Erythro-myeloid progenitors (EMPs) (Fig. 1D) were reduced in MOB D-VCd (median: 10.7% vs 19.5%, p=0.042), while the frequency of EMPs increased in D-1 D-VCd (median: 20.8% vs 12.4%, p=0.045). No considerable differences were found in the expression of adhesion molecules CD44/HCAM or CD184/CXCR4. CD38 was strongly diminished in the whole D-VCd CD34+ fraction of MOB and D-1. To understand whether the differences in the mobilization efficacy after D-VCd induction were reflected in the expression profile of mobilized CD34+ cells, differential expression analysis was performed. Overall 133 significantly deregulated genes (p(-)1) between cohorts (D-VCd n=5 vs VTd n=5) were revealed (Fig. 2). Pathway analysis showed cellular response and localization as the most deregulated categories. The list of deregulated genes contained 25% of non-coding RNAs, some of which were linked to a protein localization in the cell (RN7SL1/2). The expression of adhesion molecules was inspected independently. Out of 59 HSC hallmark genes, only 8 were significantly altered in D-VCd. Interestingly, the main homing molecule CXCR4 seemed to be downregulated in D-VCd, while integrins A3 and B4 were upregulated. Conclusions: Despite the limited cohort sizes, a prospective trend of delayed neutrophil and platelet recovery was observed after D-VCd therapy. FCM analysis revealed a significant reduction of CD34+ subsets responsible, among others, for a reconstitution of neutrophils and megakaryocytes. A strong signal in transcriptome data which would potentially explain differential mobilization in D-VCd cohort was not detected, nevertheless, several genes with adhesive/homing and stem cell differentiation function were indeed altered. The results warrant further investigation. Figure 1 Figure 1. Disclosures Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

I ntroduction Extramedullary disease (EMD) is a less frequent manifestation of multiple myeloma (MM), where MM plasma cells become independent of the bone marrow (BM) microenvironment and infiltrate other tissues and organs. The incidence of EMD is increasing and is associated with worse prognosis and drug resistance. The specific and efficient treatment is lacking. Therefore, a better understanding of EMD pathogenesis is desperately needed. Aims To identify biological pathways leading to EMD development and to evaluate therapeutic targets in EMD plasma cells with further focus on EMD tumor microenvironment to reveal presence of effector immune cells that are crucial for immunotherapy. Methods To identify EMD specific genes, FACS/MACS sorted aberrant plasma cells were collected from: i) fresh 11 EMD relapse tumors for which we had ii) 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis (NDMM), iii) 9 unpaired fresh NDMM without EMD confirmed by PET-CT and iv) 6 unpaired fresh relapsed MM (RRMM). For library preparation, we used total RNA with rRNA depletion protocol and Illumina sequencing. Residual rRNA was filtered out by SortMeRNA. Differential expression analysis was performed using Salmon for read mapping and quantification and Deseq2 package. For single-cell RNAseq we used 10x Genomics technology for sequencing and CellRanger and Seurat for data processing and analysis. Results To better understand the aggressive nature of EMD, we have analyzed bulk RNA samples (7 EMD samples plus 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis). Our preliminary analysis revealed a unique EMD profile (Fig 1A) with 423 up-regulated and 421 down-regulated genes in EMD samples (adjusted p-value < 0.1; absolute fold change > 1.5), with G2M checkpoint proteins being the most enriched hallmark pathways pointing to higher proliferation of EMD cells. EMD down-regulated genes mainly belong to genes of the adaptive immune response which together with lower immunoglobulin production suggest loss of mature plasma cell function. Among the top genes uniquely overexpressed in EMD (versus RRMM or NDMM) were SCD and ELOVL6 that regulate crucial steps in unsaturated fatty acids synthesis. Also their transcription factor SREBF1 was significantly up-regulated. The importance of these genes in EMD pathogenesis can be supported by the involvement of SREBP1 in stem cell differentiation and mediation of bortezomib resistance by ELOVL6 (Yi et al. 2018, Lipchick et al. 2021). Our dataset also revealed several deregulated lncRNA in EMD compared to NDMM. MALAT1 was highly expressed, however, we did not confirm results by Handa et al. 2017 showing lncRNA MALAT1 as upregulated in EMD. Furthermore, we aimed to evaluate expression of known immunotherapy MM targets being currently in use or under investigation. We compared the information about expression level in EMD vs paired NDMM, with unpaired NDMM without EMD lesion confirmed by PET/CT, and with RRMM. The analysis revealed a decrease in the expression of several antigens commonly used in anti-MM immunotherapy (e.g. CD38, SLAMF7, BCMA or PDL1) on EMD PCs (Fig 1B). Intriguingly, our data show EMD specific elevated expression of EZH2 gene being promising target in preclinical MM investigation which can prove efficient especially for the aggressive MM stage - EMD. Effective immunotherapy depends on the presence of effector immune cells. Therefore, we have evaluated immune cell types and their proportion in EMD tumors. Using flow cytometry we identified T and NK cells as the only immune cell subsets present in EMD tumors (median 0.9% and 0.5%, respectively). Single-cell RNAseq analysis of two EMD samples supported these findings. Conclusions Here, we present up to our knowledge the worldwide largest cohort of 11 EMD samples (including 7 longitudinal pre-EMD/EMD samples) analysed using RNAseq with focus on biological pathways and dysregulation of particular genes leading to EMD development. Drop of expression of several known drug targets may suggest limited efficacy of the modern treatment in EMD as already presented by Jelinek et al., 2021. Importantly, we are also providing the initial insight into the microenvironment (including single-cell RNA analysis) of EMD tumors, where we detected presence of T cell and NK cells in very limited numbers. Figure 1 Figure 1. Disclosures Hajek: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

In arthropod community ecology, species richness studies tend to be prioritised over those investigating patterns of abundance. Consequently, the biotic and abiotic drivers of arboreal arthropod abundance are still relatively poorly known. In this cross-continental study, we employ a theoretical framework in order to examine patterns of covariance among herbivorous and predatory arthropod guilds. Leaf-chewing and leaf-mining herbivores, and predatory ants and spiders, were censused on > 1000 trees in nine 0.1 ha forest plots. After controlling for tree size and season, we found no negative pairwise correlations between guild abundances per plot, suggestive of weak signals of both inter-guild competition and top-down regulation of herbivores by predators. Inter-guild interaction strengths did not vary with mean annual temperature, thus opposing the hypothesis that biotic interactions intensify towards the equator. We find evidence for the bottom-up limitation of arthropod abundances via resources and abiotic factors, rather than for competition and predation. publishedVersion

Silene latifolia is a dioecious plant species with chromosomal sex determination. Although the evolution of sex chromosomes in S. latifolia has been the subject of numerous studies, a global view of X chromosome structure in this species is still missing. Here, we combine X chromosome microdissection and BAC library screening to isolate new X chromosome-linked sequences. Out of 8 identified BAC clones, only BAC 86M14 showed an X-preferential signal after FISH experiments. Further analysis revealed the existence of the Athila retroelement which is enriched in the X chromosome and nearly absent in the Y chromosome. Based on previous data, the Athila retroelement belongs to the CL3 group of most repetitive sequences in the S. latifolia genome. Structural, transcriptomics and phylogenetic analyses revealed that Athila CL3 represents an old clade in the Athila lineage. We propose a mechanism responsible for Athila CL3 distribution in the S. latifolia genome.