The IL32/BAFF axis supports prosurvival dialogs in the lymphoma ecosystem and is disrupted by NIK inhibition

Aggressive B-cell malignancies, such as mantle cell lymphoma (MCL), are microenvironment-dependent tumors and a better understanding of the dialogs occurring in lymphoma-protective ecosystems will provide new perspectives to increase treatment efficiency. To identify novel molecular regulations, we performed a transcriptomic analysis based on the comparison of circulating MCL cells (n=77) versus MCL lymph nodes (n=107) together with RNA sequencing of malignant (n=8) versus normal B-cell (n=6) samples. This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of interleukin-32 beta (IL32β), whose expression was confirmed in situ within MCL lymph nodes by multiplex immunohistochemistry. Using ex vivo models of primary MCL cells (n=23), we demonstrated that, through the secretion of IL32β, the tumor was able to polarize monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. We highlighted that while IL32β-stimulated macrophages secreted several protumoral factors, they supported tumor survival through a soluble dialog, mostly driven by BAFF. Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract microenvironment-dependent induction of IL32β and BAFF-dependent survival of MCL cells. These data uncovered the IL32β/BAFF axis as a previously undescribed pathway involved in lymphoma-associated macrophage polarization and tumor survival, which could be counteracted through selective NIK inhibition.


Supplemental Files
The IL32/BAFF axis supports prosurvival dialogs in the lymphoma ecosystem and is disrupted by NIK inhibition

Genome editing using the CRISPR-Cas9 system
To generate IL32 -/-MCL cells, we infected MINO and NTS3 cells with lentiviruses encoding for Cas9-mcherry (Addgene plasmid # 70182), and mCherry positive cells were sorted using FACSAria Cell sorter (Cytocell, SFR Bonamy, Nantes). Sorted cells were then infected with lentiviruses containing GFP and doxycycline-inducible sgRNA (Addgene plasmid # 70183) directed against IL32. The designed crRNA targeted the following sequence in IL32 gene: 5'-GGCCGCCATGTGCTTCCCGA-3'. GFP positive cells were sorted and sgRNA expression was induced. Cells were cloned by limiting dilutions from the bulk prior characterization of IL32 knockout by DNA sequencing and protein analysis ( Figure S7).

Secretome quantification
Monocytes were differentiated with CSF1 (50 ng/mL) with or without (rh)IL32β (100 ng/ml) during 3 days, supernatant was collected and soluble factors were quantified by cytokine array (Proteome Profiler Human Cytokine Array Kit, R&D Systems). Pixel densities were analyzed by using the Image Lab Software (Bio-Rad). Regarding IL32, concentration was determined in MCL cell line supernatants using Human IL32 duoset Elisa (R&D systems).

Bioinformatics analysis
Gene Expression Profiling (GEP) .CEL files were downloaded and processed in R-3.6.1 using the affy package, optical noise/background correction was performed by gcrma with standard options, and expression batches were finally normalized by quantiles using the limma package. Differential gene expression was assessed with limma package (up regulated gene: log2Fc>0.5; adjusted p<0.05) and we used the GAGE package (KEGG pathway analysis) for gene set enrichment analysis.
Full-length RNA-seq: Total RNA was extracted with RNeasy Mini kit (Qiagen) and quantified using a Nanodrop®ND-1000 spectrophotometer (Thermo Scientific). Quality and integrity of RNA samples were assessed using the 2100 Bioanalyzer and RNA 6000 Nano LabChip kit series II (Agilent Technologies). Library construction was performed from 500ng of total RNA with SureSelect  3 . Reads were aligned against human hg19 reference genome with tophat2.0.10 4 , reads count and differential analysis was realized with Gfold 5 .
Relative abundance of 9 known transcripts of IL32 was performed with Pennseq 6 using refseq annotation downloaded from UCSC.

IL32 locus-specific methylation analysis
Genomic DNA was extracted with QIAamp DNA Blood Mini kit (Qiagen) and treated with bisulfite (Active-Motif). The converted DNA was amplified by specific nested PCR for promoter and CpG island of IL32 (Table S3). The cloning step was realized with StrataClone PCR Cloning Kit (Stratagene). Extracted DNA from the selected colonies (Miniprep Plasmid DNA purification, Macherey Nagel) was sequenced and analyzed (n=8 clones per sample).

Other methods
Viability assays (Annexin-V staining) as well as real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR, control gene RPL37A) and immunoblot protocols have been previously described 9 . Statistical analyses were performed using two-sided Mann-Whitney, Wilcoxon-matched pairs signed-rank, or t-tests as stated in the Figure legends.
Analyses were performed using Graph-Pad Prism and R statistical softwares and all tests were considered statistically significant at p < 0.05.

Supplemental Tables
Supplemental Table S1 Patient samples and cell lines information.  Table S3 IL-32 promoter and CpG island nested PCR primers for methylation analysis  Figure 1B (n=4584 genes).

Supplemental Figure Legends
The x-axis represents the normalized enrichment score (NES) as described by Joly et al. 11 .
(C) Enrichment plots of the NFkB, NIK and BCR signatures (as described by Saba and colleagues 12 ) or LN signature (100 most strongly upregulated genes in LN compared to PB MCL in vivo) analyzed using GSEA and reflecting the relevance of our ex vivo culture model previously described 9 . (D) KEGG pathway enrichment analysis of the "Tumor-specific" (n=1269 genes) and the "Shared with NBC" (n=1948 genes) gene sets as define in Figure   1B. The x-axis represents the normalized enrichment score (NES).    treated with (rh)IL32β, as described in Figure 5 (D) Common KEGG pathway enrichment found in both macrophages and monocytes treated with (rh)IL32β, as described in Figure 5.
The x-axis represents the normalized enrichment score (NES).