Mast cells in Waldenstrom's macroglobulinemia support lymphoplasmacytic cell growth through CD154/CD40 signaling

Abstract Background: MYD88 mutations are present in over 95% of patients with Waldenstrom's Macroglobulinemia (WM), and promote Myddosome self-assembly that triggers NFκB-dependent survival through BTK and IRAK1/IRAK4 (Blood 122(7):1222-32). While current therapeutic strategies are aimed at blocking these downstream kinases, peptidomimetics that interfere with Myddosome self-assembly may offer a more targeted approach for blocking aberrant MYD88 signaling. Methods: We expressed by lentiviral transduction mini-peptides of MYD88 Toll/Interleukin-1 Receptor (TIR) or Death Domain (DD) sequences in mutated MYD88 WM and wild-type MYD88 control cells (Figure 1). We used phospho-flow analysis to evaluate for changes in pBTK, pIRAK1/IRAK4, and pNFKB, and determined cell growth and survival by Alamar Blue Assay, Annexin V, and cleaved Caspase 3 staining. Results: Transduction of TIR or DD mini-peptides in mutated MYD88 WM cells but not wild-type MYD88 control cells reduced NFKB activation and tumor cell growth, and prompted Annexin V and/or cleaved Caspase 3 staining. TIR interfering mini-peptides impacted BTK but not IRAK1/IRAK4 activation, whereas DD interfering mini-peptides showed an opposite effect. Conclusions: The findings demonstrate differences in BTK versus IRAK1/IRAK4 directed NFKB signaling in response to Myddosome self-assembly in MYD88 mutated WM cells. The feasibility of directly targeting MYD88 homodimerization to block aberrant MYD88 signaling was also recognized, and suitable peptide sequences for the development of peptidomimetics that interfere with Myddosome self-assembly and signaling were identified. The findings provide a framework for direct targeting of the Myddosome in MYD88 mutated WM disease. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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CD52 Is Expressed on Human Mast Cells and Is a Potential Therapeutic Target in Waldenström's Macroglobulinemia and Mast Cell Disorders

Clinical Lymphoma & Myeloma ◽

10.3816/clm.2006.n.029 ◽

2006 ◽

Vol 6 (6) ◽

pp. 478-483 ◽

Cited By ~ 30

Author(s):

Daniel Ditzel Santos ◽

Evdoxia Hatjiharissi ◽

Olivier Tournilhac ◽

Mariana Z.A. Chemaly ◽

Xavier Leleu ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Therapeutic Target ◽

Waldenström’S Macroglobulinemia ◽

Potential Therapeutic Target ◽

Human Mast Cells ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

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Lymphoplasmacytic Cells and Mast Cells Are Targets for Imatinib Mesylate (Gleevec, Glivec) in Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v104.11.4929.4929 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4929-4929 ◽

Cited By ~ 2

Author(s):

Olivier Tournilhac ◽

Daniel Ditzel Santos ◽

Lian Xu ◽

Evdoxia Hatjiharissi ◽

Yu Tsu ◽

...

Keyword(s):

Mast Cells ◽

Imatinib Mesylate ◽

Cell Lines ◽

Ex Vivo ◽

Pcr Analysis ◽

Rt Pcr ◽

Waldenström’S Macroglobulinemia ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract Recently, we demonstrated that in Waldenstrom’s macroglobulinemia (WM), clonal lymphoplasmacytic cells (LPC) appear to derive growth and survival signals from excess mast cells (MC) present in the bone marrow (BM). (Tournilhac et al, JCO 2004 22:571S). We therefore have sought agents which could target both LPC and MC. One such candidate is imatinib mesylate which has shown activity in certain mast cell disorders likely on the basis of its ability to target the tyrosine kinases, CD117 (Stem Cell Factor Receptor, c-kit), and Platelet Derived Growth Factor Receptor (PDGFa-R). By flow cytometric analysis, we demonstrated CD117 expression on sorted LPC from 17/22 (76.2%) WM patients, above 20% in 13 cases, whose expressionwe confirmed by RT-PCR analysis. Moreover, we also demonstrated expression of PDGFa-R along with its ligand PDGFa in BM LPC from 16/18 (88.9%) and 7/10 (70%) WM patients.. In addition to CD117, we found by RT-PCR analysis a PDGFa expression in LAD and HMC-1 MC lines and in KU the basophilic cell lines KU as well as in sorted BM MC (FcER1+ CD117+) from 3/10 (30%) WM patients. Importantly, co-culture of sorted LPC from WM patients along with 0.5% paraformaldehyde fixed LAD cells or ex vivo expanded (EVE) BM MC induced proliferation of sorted LPC from WM patients, which was inhibited at pharmacologically achievable levels of imatinib mesylate with an IC50 of 0.5–10 μmoles/L. Moreover, imatinib mesylate inhibited the LAD, HMC-1 and KU cell lines as well as EVE cord blood MC or EVE BM MC at an IC50 of 0.01–1 μmoles/L. Lastly, imatinib mesylate also inhibited proliferation of both MC and LPC when unfixed LAD or ex vivo MC were incubated with WM LPC with an IC50 of 1–10 μmoles/L. These studies therefore demonstrate that both LPC and MC are therapeutic targets for imatinib mesylate, and provide the framework for clinical studies evaluating its use in WM.

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CD52 Is Expressed on Human Mast Cells and Is a Therapeutic Target for the Anti-CD52 Monoclonal Antibody Campath-1H in Waldenstrom’s Macroglobulinemia and Mast Cell Disorders.

Blood ◽

10.1182/blood.v104.11.4885.4885 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4885-4885 ◽

Cited By ~ 1

Author(s):

Daniel Ditzel Santos ◽

Evdoxia Hatjiarissi ◽

Olivier Tournilhac ◽

Lian Xu ◽

Andrew Branagan ◽

...

Keyword(s):

Monoclonal Antibody ◽

Mast Cell ◽

Mast Cells ◽

B Cell ◽

Therapeutic Target ◽

Waldenström’S Macroglobulinemia ◽

Healthy Donors ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract Campath-1H is a monoclonal antibody used in the treatment of CD52 expressing B-cell malignancies. Recently, we and others have observed that Campath-1H shows unusually high activity in patients with relapsed/refractory Waldenstrom’s macroglobulinemia (WM), a B-cell malignancy with excess bone marrow mast cells (BMMC). Importantly, as we have recently shown, BMMC appear to provide direct support for WM tumor cell growth (Tournilhac et al, JCO 2004 22:571S), and are therefore a potential therapeutic target in WM. We therefore examined BMMC from patients with WM and other mast cell disorders for cell surface expression of CD52, and targeting by Campath-1H in preclinical studies. Multicolor flow cytometric analysis demonstrated CD52 expression on BMMC (FceRI+, CD117+) from 13/15 WM; 2/2 systemic mastocytosis (SM) patients; 2/4 healthy donors; as well as on the LAD and HMC MC lines. Moreover, RT-PCR analysis confirmed CD52 expression in sorted BMMC from 6/7 WM, along with 6/6 healthy donors. Importantly, Campath-1H induced high levels of antibody dependent cell mediated cytotoxicity (ADCC) actvity against LAD mast cells using activated NK effector cells. No direct cytotoxicity or antiproliferative activity by Campath-1H on LAD cells was observed. These studies demonstrate that CD52 is widely expressed on human MC and provide support for the use of Campath-1H in the treatment of WM and other systemic mast cell disorders.

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A Novel Functional Role for Soluble CD27 in the Pathogenesis of Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v106.11.4701.4701 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4701-4701 ◽

Cited By ~ 4

Author(s):

Allen W. Ho ◽

Xavier Leleu ◽

Evdoxia Hatjiharissi ◽

Olivier Tournilhac ◽

Lian Xu ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Tumor Cells ◽

Functional Role ◽

Flow Cytometric Analysis ◽

Waldenström’S Macroglobulinemia ◽

Flow Cytometric ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract The tumor necrosis factor (TNF) receptor family member, CD27, is a transmembrane co-stimulatory molecule present on primed T and B lymphocytes that also secrete a soluble form (sCD27). Recent evidence has suggested that interactions between CD27 and its TNF-like ligand, CD70, play a critical role in regulating B-cell activation and survival, though the detailed mechanism(s) by which this occurs remain unclear. Waldenstrom’s Macroglobulinemia (WM) represents a lymphoplasmacytic lymphoma characterized by a monoclonal IgM gammopathy and possesses a mast cell component that may contribute to its pathogenesis (Blood 104; 646a). Using ELISA assays, we observed that WM patients displayed significantly higher levels of sCD27 in their sera (median 7.45, range 0–19.42 U/ml) versus healthy donors (median 0, range 0–2.78 U/ml; p=2.5 x 10−7). CD27 was expressed in 7/7 patients using RT-PCR analysis, but was expressed on the cell surface of tumor cells in 5/12 patients using flow cytometric analysis. Conversely, CD70 expression was widely expressed on both tumor cells (6/6 patients) and mast cells (10/11 patients) using flow cytometric analysis. In order to define the functional role of sCD27 in WM, we cultured BCWM.1 (CD27−CD70+) WM cells, and LAD1 (CD27−CD70+) mast cells with sCD27 (0.1–50 ug/mL), and observed no effect on proliferation or induction of apoptosis. Culture of LAD1 cells with sCD27 did, however, result in marked upregulation of the TNF family ligands CD40L (CD154) and a proliferation induction ligand (APRIL), which previous work in our laboratory had implicated as mast cell proliferation and survival factors in WM. Taken together, these studies suggest a novel functional role for sCD27, and imply a pivotal role in the pathogenesis of WM.

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