The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin-SCL/TAL1-CD44 axis

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1 , the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1 T315I differ from BCR-ABL1 native ( BCR-ABL1 ) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1 T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1 T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1 T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

Vitamin K antagonists (VKAs) have been used in 1% of the world’s population for prophylaxis or treatment of thromboembolic events for 64 years. Impairment of osteoblast function and osteoporosis has been described in patients receiving VKAs. Given the involvement of cells of the bone marrow microenvironment (BMM), such as mesenchymal stem cells (MSCs) and macrophages, as well as other factors such as the extracellular matrix for the maintenance of normal hematopoietic stem cells (HSCs), we investigated a possible effect of VKAs on hematopoiesis via the BMM. Using various transplantation and in vitro assays, we show here that VKAs alter parameters of bone physiology and reduce functional HSCs 8-fold. We implicate impairment of the functional, secreted, vitamin K-dependent, γ-carboxylated form of periostin by macrophages and, to a lesser extent, MSCs of the BMM and integrin β3-AKT signaling in HSCs as at least partly causative of this effect, with VKAs not being directly toxic to HSCs. In patients, VKA use associates with modestly reduced leukocyte and monocyte counts, albeit within the normal reference range. VKAs decrease human HSC engraftment in immunosuppressed mice. Following published examples that alteration of the BMM can lead to hematological malignancies in mice, we describe, without providing a causal link, that the odds of VKA use are higher in patients with vs without a diagnosis of myelodysplastic syndrome (MDS). These results demonstrate that VKA treatment impairs HSC function via impairment of the BMM and the periostin/integrin β3 axis, possibly associating with increased MDS risk.

Lipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that absence of FLOT2 from leukemia- initiating cells (LIC) altered disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL-AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared to MLL-AF9+ cells. Downstream of CD44, BCR-ABL1-kinase-independent discrepancies were observed regarding expression, localization and activity of cell division control protein 42 homolog (CDC42) between wildtype and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML versus AML progression, with deficiency of FLOT2 impairing the development of CML.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance by release of small extracellular vesicles (sEV). In converse, we show here, that BMM-deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEV released by BMM-derived mesenchymal stromal cells (MSC). PLEKHM1-deficient MSC and their sEV carry increased amounts of syntenin and syndecan-1, resulting in a more immature B cell phenotype and increased number/function of leukemia-initiating cells (LIC) via focal adhesion kinase (FAK) and AKT signaling in B-ALL cells. Ex vivo pretreatment of LIC with sEV derived from PLEKHM1-deficient MSC led to a strong trend towards acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL-cell derived tumor necrosis factor (TNF) α or interleukin (IL)-1β condition murine and human MSC in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSC, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSC. In summary, our data reveal an important role of BMM-derived sEV for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared to BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.