SummaryThe biological and clinical behaviors of hematological malignancies can be influenced by the active crosstalk with an altered bone marrow (BM) microenvironment. In the present study, we provide a detailed picture of the BM vasculature in acute myeloid leukemia using intravital two-photon microscopy. We found several abnormalities in the vascular architecture and function in patient-derived xenografts (PDX), such as vascular leakiness and increased hypoxia. Transcriptomic analysis in endothelial cells identified nitric oxide (NO) as major mediator of this phenotype in PDX and in patient-derived biopsies. Moreover, induction chemotherapy failing to restore normal vasculature was associated with a poor prognosis. Inhibition of NO production reduced vascular permeability, preserved normal hematopoietic stem cell function, and improved treatment response in PDX.

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. To this end, we studied a mouse model of human T cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting a stochastic mechanism underlies these processes. Yet, while T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells whilst perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function1. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, in order to combat the invasion by and survival of chemo-resistant T-ALL cells.

Impaired cell migration has been demonstrated in T cell acute lymphoblastic leukemia (T-ALL) cells upon calcineurin inactivation, among other phenotypic traits including increased apoptosis, inhibition of cell proliferation, and ultimately inhibition of leukemia-initiating cell (LIC) activity. Herein we demonstrate that the chemokine receptor CXCR4 is essential to the LIC activity of T-ALL leukemic cells both in NOTCH-induced mouse T-ALL and human T-ALL xenograft models. We further demonstrate that calcineurin regulates CXCR4 cell-surface expression in a cortactin-dependent manner, a mechanism essential to the migratory properties of T-ALL cells. Because 20%-25% of pediatric and over 50% of adult patients with T-ALL do not achieve complete remission and relapse, our results call for clinical trials incorporating CXCR4 antagonists in T-ALL treatment.

Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity. Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA-aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.

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Despite their initial efficient response to induction chemotherapy, relapse remains frequent in patients with T-cell acute lymphoblastic leukemia (T-ALL), an aggressive malignancy of immature T-cell progenitors. We previously reported sustained calcineurin (Cn) activation in human lymphoid malignancies, and showed that Cn inhibitors have antileukemic effects in mouse models of T-ALL. It was unclear, however, from these studies whether these effects resulted from Cn inhibition in leukemic cells themselves or were an indirect consequence of impaired Cn function in the supportive tumor microenvironment. We thus generated a Notch (intracellular Notch 1, ICN1)-induced T-ALL mouse model, in which conditional Cn genetic deletion is restricted to leukemic cells. Ex vivo, Cn deletion altered the adhesive interactions between leukemic cells and their supportive stroma, leukemic cell survival, proliferation, migration and clonogenic potential. In vivo, Cn activation was found to be critical for leukemia initiating/propagating cell activity as demonstrated by the failure of Cn-deficient leukemic cells to transplant the disease to syngeneic recipient mice. Importantly, combination of vincristine treatment with Cre-mediated Cn ablation cooperated to induce long-term remission of ICN1-induced T-ALL. These findings indicate that Cn is a promising target in T-ALL relapse prevention, and call for clinical trials incorporating Cn inhibitors during consolidation therapy.

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Abarrategi et al. summarize the methods used for human hematopoietic cell xenotransplantation and their milestones. They also discuss the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis.