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Work Package 2

Lead Beneficiary NHRF

Functional studies in biological fluids, cells and animals

Objectives
1. Elucidation of molecular features associated with MGUS/sMM progression to active MM
2. Assessment of drugs in MM cell and animal models

Description

Description of work:

This WP concerns the application of novel technologies in MM, aiming (a) at producing new scientific knowledge on the molecular and cellular pathways implicated in myelomagenesis, i.e., the transformation process from normal cell to symptomatic MM, and (b) to translate this knowledge into novel, selective and effective tools and strategies for the early diagnosis and prognosis of MM as well as the outcome of MM therapy.

For this, the following experimental systems will be used:

  1. A large collection of human MM cell lines (HMCLs; n=45) developed by CNRS, including HMCLs that are dependent on the addition of external growth factors for their proliferation and survival as well as drug-resistant HMCLs to melphalan (n=6), lenalidomide (n=6), bortezomib (n=7), dexamethasone (n=4) and HDACi (n=4)

  2. PBMCs and bone marrow plasma cells (BMPCs) representing the different stages of transition from MGUS/ smoldering MM to symptomatic MM,

  3. Syngeneic and humanized patient-derived mouse models of MM.

The integration of clinical and omics datasets will yield putative therapeutic targets for MM that will be validated using flow cytometry and/or super resolution microscopy. Appropriate drugs that are already used in the clinical setting will be examined for repurposing in the context of MM treatment in multicellular BM/MGUS/sMM/symMM spheroids (static and under flow conditions) and immunocompetent syngeneic and humanized mouse models.

Task 2.1: Identification and functional characterisation of key-regulators associated with the onset and progression of MM by in vitro studies (NHRF, CNRS, UKW, M2-32)

DNA Damage Response (DDR) NHRF, UKW

Since the DDR network and the Immune System are implicated in both the onset and progression of MM the cell lines and the primary cells mentioned above we will investigate a) DDR-related pathways, including signaling pathways, cell-cycle checkpoints, DNA repair mechanisms, and cell death pathways, as well as b) Immune System-associated parameters, such as the presence of cytoplasmic DNA and micronuclei, the induction of the STING pathway, the type I interferon signaling and the NF-kB pathway using advanced methodologies in combination with biochemical and new imaging technology based on confocal microscopy. Moreover, since DDR deficiency has recently emerged as an important determinant of tumor immunogenicity, the interplay between the DDR network and the Immune System, including the DNA damage-induced Immunogenic Cell Death (ICD), the modulation of antigen presentation at the surface of the cancer cells, the modification of PD-1/PD-L1 expression and the expression of critical proinflammatory cytokines will be studied following treatment of cells with genotoxic drugs known to induce immune response, namely melphalan and oxaliplatin (NHRF).

The evaluation will also comprise examination of resistance towards genotoxic agents, as well as functional analyses, qPCR and deep sequencing and will be conducted using cell lines, PBMCs, Bone Marrow Plasma Cells and 3D BM/ PC/MM spheroids (UKW).

Inflammatory cytokines UNAV, CNRS

In a recent study that investigated serum immune markers in MGUS patients over time, UNAV has found for the first time a conversion from low- to high-risk MGUS based on changes in monoclonal proteins in up to 70% of patients with high-risk disease before progression . As an extension to this study, the levels of soluble inflammatory cytokines (IL1β, IL6, IL8, IL10, IL12p70 and TNF-α) using the Cytometric Bead Array Platform, and bone-derived markers (BALP, RANKL and OPG) using commercially available ELISA kits will be analyzed in the plasma of MGUS, sMM and MM patients (UNAV).

Key-regulators associated with the onset and progression of MM will also be analysed using the normal memory B cell to plasma cell (PC) differentiation model (CNRS). This model was characterized using Hi-C (a method that produces a quantitative map of 3D chromatin contacts), ChIP-seq (7 histone marks), RNA seq, miRnome, ERRBS (DNA methylation), epitranscriptomic analysis, sc-ATAC-seq and sc-RNA-seq (CNRS). We will investigate how deletion or overexpression of the key-regulators associated with the onset and progression of MM could affect normal B cell to plasma cell differentiation, transcriptional, epigenetic programs and biological functions.

Proteostasis NKUA

Proteostatic mechanisms, oxidative load, and autophagy will be assessed in Bone Marrow Plasma Cells from MGUS, sMM and MM patients as well as in MM cell lines. The analysis of proteostatic mechanisms will provide functional insights on molecular features associated with the onset and progression of MM. Selected proteostasis-related genes will be analysed in order to elucidate their functional role in MM pathogenesis and acquired drug resistance.

The activities of the main cellular degradation machineries, namely proteasome and lysosomal cathepsins peptidases activities will be assayed in Bone Marrow Plasma Cells using specific fluorogenic peptides. The produced fluorescence will be recorded in a multimode microplate reader (excitation 380 nm, emission 460 nm).

For the assessment of Reactive Oxygen Species (ROS) production, Bone Marrow Plasma Cells will be incubated with 10 μM CM-H2DCFDA dye in PBS for 30 min at 37°C in the dark. Following dye removal, cells will be incubated for 10 min with PBS and then, the produced fluorescence will be measured in a multimode microplate reader (excitation 490 nm, emission 540 nm).

Staining of aggresomes (an indication of accumulating intracellular protein aggregates) as well as of autophagic vacuoles and monitoring of autophagic flux will be performed in Bone Marrow Plasma Cells using commercially available kits according to manufacturer’s instructions. The levels of aggresomes and autophagic vacuoles will be assessed by Flow Cytometry and Confocal Laser Scanning Microscopy.

Genes involved in proteostatic pathways (proteasomal, autophagic, chaperones, antioxidant and ER-stress genes), that will be identified in WP3 by transcriptomics and proteomics as differentially expressed between MGUS/sMM and MM, will be further investigated in order to identify their functional role in MM pathogenesis. Thus, gene overexpression or RNA silencing experiments will be performed in MM cell lines. Additionally, gene expression modulating experiments will be combined with clinically used drugs aiming to sensitize drug resistance MM cells to chemotherapy.

Associated deliverables: D2.1

Task 2.2: Multiparameter flow cytometric profiling of PBMC and Bone Marrow Plasma Cells and PC/BM microenvironment (NKUA, UNAV, UKW M2-32)

Changes in cell surface expression occur during the transition process. However, these changes haven’t been monitored in greater detail. In this context, cryopreserved samples of high viability (>85%) cells will be analysed with multiparametric flow cytometry (MFC) in order to highlight different protein expression profiles in the bone marrow plasma cells of different disease stages stages (MGUS/sMM/active MM) as well as distinct BM microervironmental fingerprints during the natural history of the disease. Moreover, PBMCs will be analysed in all disease stages in an effort to highlight distinct immune signatures correlating with the clinical progression of MM. The analysis will include a minimum of 20 matched samples (BM and PBMC samples of the same patient at different disease stages) to highlight those dynamic changes associating with health to disease progression.

Specific isotypic controls will be applied in each analysis to minimize any analytical bias and avoid false positive results. The number of plasma cells expressing each protein biomarker will be recorded as a percentage (%) of total plasma cells together with the mean and median fluorescent intensity of this biomarker in the whole plasma cell population. For BM niche and PMMC immune profiling, the prevalence of each cell subset will be recorded as a (%) of total BM or PB nucleated cells.

The cytokine profiling analysis will be applied on each patient’s BM and/or PB serum for the quantitative analysis of various selected cytokines. The analysis will be performed with sensitive assays, which can simultaneously detect the levels of at least 7 different cytokines. The design of the appropriate multi-color panels (optimum combination of antibodies-fluorophores) is crucial and will be applied in a way that enables the recovery of the maximum information and minimizes background noise.

Associated deliverable: D2.2

Task 2.3: Characterization of the epigenetic changes in MGUS/sMM and MM cells and comparison with normal Bone Marrow Cells to PCs differentiation stages (CNRS, M6-36)

The histone modifications (ChIP-seq) will be integrated with gene expression data (RNA-seq) and other critical epigenetic chromatin features such as chromatin accessibility (ATAC-Seq) and DNA methylation (ERRBS). To map topological chromatin interactions in an unbiased manner across the genome, we will use Hi-C, a state-of-the-art technique combining classic chromosome conformation capture and high-throughput sequencing. A specific effort will be performed to integrate and visualize the topological data, RNA-seq data and epigenetic features into loci of candidate oncogenes of main drivers of tumorigenicity. Chromatin interaction analyses will be performed to investigate several levels of global genome organization: 1) at the megabase level, the goal will be to examine whether “chromosome compartments”, multi-megabase-sized chromosome domains that are enriched in either active or inactive genes, change in different samples and whether the changes conform to predictive signatures. Compartments are defined by the patterns of contacts that are made by these domains. 2) at the sub-megabase level we will analyze “topologically associated domains (TADs)”, chromosome domains that typically contain one or few genes and their upstream or downstream regulatory DNA elements (i.e., enhancers) and are delimited by strong boundaries enriched by binding sites for the CTCF DNA binding protein. In particular, we will analyze whether the position of the boundaries of TADs change in MM cells, MGUS/sMM cells compared to normal plasma cell counterparts. Shifts in boundaries might lead to inappropriate contacts between promoters and distal enhancers. Furthermore, we will analyze whether the strength of TAD boundaries decreases in MGUS/sMM cells, either locally or genome-wide, which might again lead to inappropriate gene regulation.

3) at the sub-TAD level, we will study “chromatin loops”, i.e. individual regulatory contacts, such as those linking gene promoters to their cognate enhancers. Chromatin loops might directly inform on gene regulatory contacts. We plan to identify if the recurrent cytogenetic or genomic events can drive epigenetic, transcriptional and structural changes within chromatin domain related to MGUS/sMM progression to MM.

Validation of genomic 3D features associated to MM biology

We expect to identify molecular and epigenetic features associated with MGUS/sMM progression to symptomatic MM. These candidates will be knocked out using lentiviral vectors or CRISPR/Cas9 technology in human MM cell lines. We will thus be able to assess whether the knock-out of the identified candidates has strong effects MM cell apoptosis, cell cycle and response to conventional treatments of MM cells. Cell growth and cell viability will be investigated using an ATP assay and cell Proliferation using BrdU incorporation and DAPI labelling. Cell Death patterns and changes in unfolded protein response will be analyzed. Changes in transcriptional profiles will be studied using RNA-sequencing. The response to conventional treatments will be also characterized. To investigate the role of these molecular and epigenetic features in transition from a healthy PC state to a malignant state, CNRS will use the in vitro human B to plasma cell differentiation model (Task 2.1). Inactivation or overexpression of the identified candidates will be completed using lentiviral vectors or specific inhibitors to decipher their role in PC differentiation, functions and tumorigenesis. We therefore expect to obtain a list of molecular, epigenetic and 3D genomic features involved in the MGUS/sMM progression to symptomatic MM that will be validated using primary MM cells from patients.

Associated deliverable: D2.3

Task 2.4: Investigation of surface antigens and internalization of receptors by super resolution microscopy (UKW, M26-40)

It has been shown that even small amounts of cell surface antigens can suffice to trigger immune responses. Task

2.4 will focus on the detection and quantification of novel surface receptors identified by clinical and omics datasets using dSTORM super-resolution microscopy to assess whether these change during transition and are suitable for immunotherapeutic exploitation. The evaluation will be conducted using cell lines, PBMCs and BMPCs.

Associated deliverables: D2.4

Task 2.5: Syngeneic and humanized mouse models for testing drug repurposing in MM (NHRF, M2-46)

We plan to use 2 different models, the 5T MM-Derived C57BL/KaLwRij Model and theVk*myc Transgenic Model. These two different models will provide us all the necessary preclinical data to evaluate our integrated omics results and suggest novel therapeutic and prognostic regimes. To be more specific, we will plan to analyze with flow cytometry the immune cell populations of each model, to identify changes in the numbers or the activation of the different cell types of the immune system, related to the progression or the aggravation of the disease. We next plan to analyze the cytokine profile that is in the blood circulation of these mouse models by analyzing their plasma at different timepoints of disease, using mesoscale™. This analysis will provide hints on the tight regulation of the alterations of the immune cell populations. Next, we plan to perform the same analysis to the patient’s samples to validate the similarity of the models with the human disease. Last but not least we will use our syngeneic models to analyze the effects of the different drug combinations to the immune system and provide the rationale of novel drug combinations that should be directly translated to the clinic (NHRF).

To better translate the key findings from our syngeneic mouse models to the clinic and compare them with clinical features derived from patients’ primary cells and patient derived cell lines, we will develop in house a true humanized patient derived mouse model. Due to the immune suppressive abilities of NOD-SCID and RAG-1 KO mice and the undisputed, but not well explored, role of the innate and adaptive immune to the progression of the disease, we will develop a clinically relevant model, based on a humanized mouse model of MM. For that purpose, we will use the NSG MHCI/II double Knock out mouse model to engraft the patient’s hematopoietic stem cells and generate a humanized immune system to the mouse. This deliverable is highly dependent on novel sample accrual from all the partners. We plan to generate different humanized patient’s derived mice from at least 3 different stages of the disease, MGUS, MM early stage and MM advanced stage, we will study our novel therapeutic regimes and combinations as if we have a true clinical trial. Besides studying the efficacy of different drug combinations or test the efficacy of newly repurposed drugs, flow cytometry and the cytokine profile analysis will be our main focus also regarding this model (NHRF).

Associated deliverables: D2.5

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