DE LA FORMACIÓN Y PERFECCIONAMIENTO DE LOS CONTRATOS A TRAVÉS DE INTERNET

Oncology research is often focused on the cancer cell as a single entity. However, the tumor microenvironment has a profound effect in the initiation and progression of cancer.81 _{This tumor niche consists of cancer cells and a supporting framework, including}

mesenchymal stromal cells (MSCs), fibroblasts, endothelial cells, pericytes and a variety of immune-regulatory cells.82,83 _{Leukemic cells reside in the bone marrow, where they}

hijack the normal hematopoietic stem cell niches to create a leukemic niche.84 _{In this thesis,}

the importance of the bone marrow niche for BCP-ALL cell survival was demonstrated by microenvironment-induced resistance to JAK inhibitors (Chapter 3). Unraveling the mechanism of protection may provide novel options for therapeutic intervention.

Tumor cells can take advantages of niche specific signaling, manipulate their microenvironment to promote survival, or cancer might even be the consequence of a disrupted microenvironment.85-87 _{In myeloid malignancies, dysfunction of the bone}

marrow has been reported to contribute to the development of the disease and vice versa leukemogenesis can induce abnormal bone marrow niches.88-92 _{Conflicting results}

have been reported concerning the genetic background of mesenchymal stromal cells (MSCs) in pediatric BCP-ALL.93-95 _{Therefore, we studied in Chapter 6 of this thesis whether}

MSCs obtained from pediatric BCP-ALL patients (leukemic-MSCs) are different compared to MSCs obtained from healthy pediatric controls (control-MSCs). Our results indicate that leukemic-MSCs are intrinsically not different compared to control-MSCs. Instead, BCP-ALL cells induce gene expression changes in MSCs, which coincide with an increased survival of BCP-ALL cells. Each BCP-ALL sample induced a characteristic gene expression change, which was consistent between different MSCs. This change in expression was only transient, as elimination of leukemic cells reversed gene expression changes. The interferon/STAT1 pathway was the most commonly induced pathway, suggesting a central role of STAT1 in the interaction between MSCs and BCP-ALL cells. However, knockdown of STAT1 in our MSCs did not reduce the survival benefit of BCP-ALL cells upon co-culture with MSCs. Residual expression levels of STAT1 or rescue via alternative pathways may have occurred and therefore the role of the interferon/STAT1 pathway in BCP-ALL remains elusive.96 _{Taken together, our finding indicate that MSCs are not actively involved in the}

initiation of pediatric BCP-ALL or a relapse. Rather BCP-ALL cells manipulate MSCs to support leukemogenesis. Targeting the direct interaction between BCP-ALL cells and MSCs or downstream factors of these processes might offer alternative treatment approaches. In order to support BCP-ALL cell survival and vice versa induce changes in the gene expression profile of MSCs, MSCs and BCP-ALL cells need to interact. Interaction can occur through soluble factors (e.g. chemokines and cytokines) and cellular projections (e.g. tunneling nanotubes).97 _{Tunneling nanotubes (TNTs) can transfer various cargo, e.g.}

mitochondria and autophagosomes, which will affect with ongoing cellular processes.97-99

Previously we showed that TNT signaling induces secretion of pro-survival cytokines. Like gene expression profiles, these cytokine secretion patterns are leukemia-specific, again suggesting that BCP-ALL cells, but not MSCs, are responsible for the active modulation of the tumor microenvironment.100 _{Combining results from this study and Chapter 6 indicate}

that there are at least three different approaches to interfere with the ALL-MSC interaction: 1) TNT signaling, 2) ALL-induced signaling pathways in MSCs, and 3) cytokine signaling. Results presented in this thesis show the variety of effects induced in MSCs by leukemic samples. Interference with a common mechanism of the ALL-MSC interaction, like TNT

inhibition, would be an attractive treatment approach. Unfortunately, no specific TNT inhibitors are available yet. Since our results show that MSCs are not actively involved in leukemogenesis, therapeutic interference should preferentially target leukemic cells and/ or factors that are released upon co-culture of BCP-ALL cells and MSCs.

Immunotherapy

Targeted treatment strategies will be of great importance to improve the prognosis and reduce sight effect in pediatric BCP-ALL patients. In this thesis, we show promising results for the implementation of JAK inhibitor therapy in the treatment of BCP-ALL, but also identified important limitations (accumulation of phosphorylated JAK2, tumor heterogeneity, micro- environmental induced resistance) of these type of inhibitors. These limitations should be taken into account during treatment and provide directions for future research.

Besides targeted therapies, immunotherapy includes a promising new treatment approach against leukemia. High remission rates were achieved in relapsed and refractory BCP-ALL patients by bispecific antibodies, immunotoxins, and chimeric antigen receptor T cell therapy.101-107 _{CD19 and CD22 are the main targets for these treatment strategies. Both}

proteins are expressed exclusively on B-cell lineage cells.101 _{Blinatumomab is a bispecific}

antibody that simultaneously binds to CD19-positive cells and to endogenous cytotoxic T cells. This binding enables recognition and elimination of CD19-positive cells by these cytotoxic T cells.101-103 _{Immunotoxins use the rapid internalization of CD22 upon antibody}

binding to mediate intracellular delivery of cytotoxic agents.104,108 _{Inotuzumab ozogamicin}

is an anti-CD22 monoclonal antibody that is conjugated to the cytotoxic antibiotic agent calicheamicin.109 _{The intracellular release of calicheamicin induces cells death}

in CD22-positive cells.109 _{Chimeric antigen receptor T cell therapy involves the ex vivo}

modification of cytotoxic T cells. Expression of chimeric antigen receptors against CD19 or CD22 directs these T cells towards the leukemic cells.105-107 _{Unfortunately, severe side}

effect are reported (e.g. severe cytokine release syndrome) and in time leukemic cells can also develop resistance against these immunotherapeutic approaches.103,105,106 _Leukemic

cells can evade these therapies via different mechanisms, e.g. loss of CD19 expression, increased regulatory T-cells, or immunosuppressive cytokines.110 _{Cells that acquired}

resistance due to loss of CD19 might be targeted by CD22-directed approaches.107 _In

addition, blockade of T cell activation through high expression levels of inhibitory ligands on tumor cells (i.e. PD-L1) was proposed to be one of the main contributors of resistance.111

Combined treatment with blinatumomab and antibodies that block the binding of PD-L1 on tumor cells to the PD-1 inhibitory T cell receptor (e.g. nivolumab) might increase the cytotoxicity of T cells. Indeed, promising results were obtained by combination therapy in a pediatric patient with refractory BCP-ALL.112 _{The potency of this combination therapy}

will be studied in more detail in a phase I study for relapsed or refractory CD19+ BCP-ALL (NCT02879695).

Taken together, targeted therapies and immunotherapy have a high potential in the treatment of leukemia. However, the ability of leukemic cells to acquire resistance against different types of therapies suggests that future treatment approaches may need to combine different strategies.

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