The Dual Syk/JAK Inhibitor Cerdulatinib Antagonizes B-cell Receptor and Microenvironmental Signaling in Chronic Lymphocytic Leukemia

Abstract

Purpose: B-cell receptor (BCR)-associated kinase inhibitors, such as ibrutinib, have revolutionized the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative, and resistance is already emerging in a proportion of patients. IL4, expressed in CLL lymph nodes, can augment BCR signaling and reduce the effectiveness of BCR kinase inhibitors. Therefore, simultaneous targeting of the IL4- and BCR signaling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients.Experimental Design: PBMCs from patients with CLL were treated in vitro with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine, and cell signaling assay were performed and analyzed by flow cytometry, immunoblotting, q-PCR, and ELISA as indicated.Results: At concentrations achievable in patients, cerdulatinib inhibited BCR- and IL4-induced downstream signaling in CLL cells using multiple readouts and prevented anti-IgM- and nurse-like cell (NLC)-mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration-dependent manner, and particularly in IGHV-unmutated samples with greater BCR signaling capacity and response to IL4, or samples expressing higher levels of sIgM, CD49d⁺, or ZAP70⁺ Cerdulatinib overcame anti-IgM, IL4/CD40L, or NLC-mediated protection by preventing upregulation of MCL-1 and BCL-X_L; however, BCL-2 expression was unaffected. Furthermore, in samples treated with IL4/CD40L, cerdulatinib synergized with venetoclax in vitro to induce greater apoptosis than either drug alone.Conclusions: Cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease. Clin Cancer Res; 23(9); 2313-24. ©2016 AACR.

Figure 1. Regulation of Anti-IgM and Anti-IgD induced signaling by cerdulatinib.

CLL cells were treated with cerdulatinib, idelalisib (Idel) or ibrutinib (Ibr) at the stated concentrations for 1h and stimulated with; (A-D) bead immobilised (BI) (A) anti-IgM or (C) anti-IgD for 1.5hr or (B) soluble anti-IgM or (D) anti-IgD for 15min or 5min respectively. Levels of phosphorylated AKT (pAKT Ser473), ERK (pERK Thr202/Tyr204), S6kinase (pS6K Thr389) and S6 ribosomal subunit (pS6 Ser235/236) were assessed by immunoblotting. (E) CLL whole blood was treated in the presence or absence of increasing concentrations of cerdulatinib prior to activation with soluble anti-IgM and anti-IgD. Phosphorylated (p)ERK Y204, pSYK Y525/526 and pAKT S473 were assessed in CD19+ cells via phospho-specific flow cytometry. (F-G) CD19+ B cells from a CLL patient were treated with cerdulatinib for 60 minutes and stimulated with (F) soluble anti-IgM or (G) soluble anti-IgD and calcium flux assessed using flow cytometry as previously described. Bar graphs depict means ± SEM

Figure 2. Regulation of chemokine expression by cerdulatinib

CLL cells were stimulated with (A) soluble (n=5) or (B-C) immobilised (n=8) anti-IgM in the presence or absence of cerdulatinib (A, 2μM, B, 1μM) or ibrutinib (1μM) for 24h. CCL3 and CCL4 levels were quantified in the supernatant by enzyme-linked immunosorbant assays (ELISA). (D) CLL cells in co-culture with NLC were treated with cerdulatinib (2µM) for 24h followed by the quantification of CCL3/4 and CXCL13 in the culture supernatants (n=5). Bar graphs depict means ± SEM.

Figure 3. Regulation of STAT6 phosphorylation, and sIgM and CXCR4 expression by IL-4 and cerdulatinib

CLL cells were treated with cerdulatinib, idelalisib (Idel) or ibrutinib (Ibr) at the stated concentrations for 60mins then stimulated with IL-4 (10ng/ml) for a further 60mins. (A) Phosphorylated STAT6 (pSTAT6), STAT6 and actin were assessed using immunoblotting in a representative sample and (B) summarised (n=8). (C) CLL cells were treated with tofacitinib (JAK3 inhibitor) (CP), and SYK inhibitors fostamatinib (R406) and P505-15 (PRT) prior to IL-4 treatment and immunoblotting performed for pSTAT6, STAT6 and HSC70 in a representative sample. (D,E) CLL cells were incubated with cerdulatinib (1µM) or the JAK1/3 inhibitor tofacitinib (CP) (1µM) for 60mins then with IL-4 (10ng/ml) for a further 23hr. (D) Surface IgM and (E) surface CXCR4 were assessed using flow cytometry. Bar graphs depict means ± SEM

Figure 4. Cerdulatinib induces apoptosis in a concentration, time and caspase dependent manner

(A) CLL cells from 24 different patients were treated with cerdulatinib as indicated for 24, 48 and 72hrs and apoptosis assessed using PI/Annexin V by flow cytometry. Data is % of control (PI/Annexin V negative cells). (B-C) CLL cells were treated with cerdulatinib at the indicated concentrations in the presence or absence of 100µM pan caspase inhibitor ZVAD for 24hr and assessed for (B) PARP and caspase 3 cleavage by immunoblotting or (C) annexin V/PI analysis by flow cytometry. (D-G) Samples were characterised from analysis in (A) by; (D) IGHV mutational status, (E) ZAP-70, (F) CD49d and (G) IgM expression. Bar graphs depict means ± SEM

Figure 5. Cerdulatinib overcomes the protection of CLL cells by conditions which mimic the tumour microenvironment

(A) CLL cells were treated with cerdulatinib (2µM) for 24-48h in the presence or absence of soluble anti-IgM (10µg/ml). Cell viability was determined by propidium iodide and DiOC6 double staining by flow cytometry (n=12) (B) CLL cells were treated with bead immobilised (imm) anti-IgM in the presence or absence of 1 & 3µM cerdulatinib. Viability was assessed by PI/Annexin V staining using flow cytometry (n=8). (C) CLL cells were treated and assessed as indicated in (A) but in the presence or absence of nurse-like cells (NLC) (n=6). (D-E) CLL cells were treated with cerdulatinib or venetoclax in the presence or absence of (D) immobilised anti-IgM or (E) IL-4 (10ng/ml) and CD40L (300ng/ml) for 24h and protein expression of Bcl-X_L, Mcl-1, pSTAT6, HSC70 and Bcl-2 expression evaluated by immunoblotting. Bar graphs depict means ± SEM

Figure 6. Cerdulatinib synergises with Venetoclax to kill CLL cells

CLL cells were incubated with IL-4 (10ng/ml) and CD40L (300ng/ml) for 6h then treated with cerdulatinib and/or venetoclax (ABT-199) as indicated for a further 24hr. Viability was assessed using (A) flow cytometry (PI/Annexin V negative cells) and a representative flow cytometry plot is shown and (B) summarised (n=9) showing % of control (PI/Annexin V negative cells). (C) Synergistic interactions between cerdulatinib and venetoclax were evaluated as indicated and described in the Methods. XY line indicates observed survival=expected survival. Points beneath the line indicate synergistic interactions and points above the line indicate additive interactions.