Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma
- P. Balsas†1,
- A. Esteve-Arenys†1,
- J. Roldán1,
- L. Jiménez1,
- V. Rodríguez1,
- J. G. Valero1,
- A. Chamorro-Jorganes1,
- R. Puig de la Bellacasa2,
- J. Teixidó2,
- A. Matas-Céspedes1,
- A. Moros1,
- A. Martínez3,
- E. Campo1, 3,
- A. Sáez-Borderías4,
- J. I. Borrell2,
- P. Pérez-Galán1,
- D. Colomer1, 3 and
- G. Roué1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 29 December 2016
Accepted: 21 March 2017
Published: 31 March 2017
Pharmacological inhibition of B cell receptor (BCR) signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton’s kinase (Btk) and spleen tyrosine kinase (Syk) inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents.
We evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn), in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma.
IQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest.
These results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.
KeywordsB-NHL Btk Lyn Syk Cell migration Mouse model
The B cell receptor (BCR) regulates multiple cellular processes which are critical for maintenance and survival of B cells, including proliferation, differentiation, and cell migration . Antigen engagement to BCR extracellular domain leads to phosphorylation and activation of immunoreceptor tyrosine-based activation motifs located in the cytoplasmic portion and other proteins downstream the receptor. Within BCR signalosome, the Lck/Yes novel tyrosine kinase (Lyn) recruits and phosphorylates the spleen tyrosine kinase (Syk), triggering a proliferation and survival cascade signaling that involves the phosphorylation and activation of Brutons’ tyrosine kinase (Btk), which subsequently phosphorylates phospholipase Cγ2 (PLCγ2), leading to calcium mobilization and activation of several downstream pathways, including MAP kinases, Akt and NF-κB . In addition to tonic, ligand-mediated BCR signaling, chronic BCR activation can occur in the absence of antigen engagement , leading to aberrant, constitutive BCR activation in several B cell non-Hodgkin lymphoma (B-NHL) subtypes, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) [4–7]. In these entities, BCR signaling represents an important pro-survival stimulus that may be stronger than in normal B cells, supporting the recent emergence of several BCR-targeting therapies . But despite the promising results obtained with the first kinase inhibitors, such as fostamatinib and ibrutinib, specific for the Src-family kinases Syk and Btk , the design of new compounds is warranted to improve treatment efficacy and to by-pass the resistance appearing in primarily responsive patients [9–11]. In this context, we recently described the synthesis of a new family of 4-aminopyrido[2,3-d]pyrimidines with kinase inhibitory property and antitumoral activity in B lymphoid cells. Compound 19 (thereafter referred as IQS019) was identified as the most effective and specific molecule, with growth inhibitory 50 (GI50) doses in the low micromolar range. Docking studies and biochemical assays further showed that the compound inhibited the active site of the BCR kinases Syk, Lyn, and Btk with higher efficacy than the reference kinase inhibitors [12, 13]. Here, using an extended panel of B-NHL cell lines and primary samples, we describe the full mechanism of action of this compound and report its remarkable antitumoral activity in vitro and in distinct B-NHL xenotransplant mouse models.
Cell lines and patients samples
Sensitivity of B lymphoid cell lines to IQS019
B lymphoid subtypes
IQS019 cytotoxic effect (referred to untreated cells)
1 μM, 48 h
5 μM, 48 h
Kinase inhibition profiling
The kinase inhibition profile of IQS019 (0.1 and 10 μM) was evaluated at Proqinase (Freiburg, Germany) using a Kinase 400-Profiler Panel, according to previously described procedures . The residual activity (in %) for each compound well was calculated by using the following formula: Residual activity (%) = 100 x [(signal of compound–low control)/(high control–low control)].
Cell-based tyrosine kinase assay
In vitro inhibitory activity of IQS019 against BCR-related kinase was determined by Advanced Cell Dynamics (San Diego, CA, USA). Briefly, the Ba/F3 murine B lymphoid cell line was transfected with either a control vector or a vector containing the kinase domain of Btk, Syk, or Lyn, rending each cell line dependent upon activity of the recombinant kinase for survival. Cells were treated for 48 h with the indicated doses of IQS019 and cell viability was monitored via ATP concentration using CellTiter-Glo assay (Promega, Madison, WI, USA). IC50 values were determined using the GraphPad Prism software version 5.04 (San Diego, CA, USA)
Cell proliferation assay
Cells (4–6 x 105 cells/ml) were treated for the indicated times with IQS019 or ibrutinib (Selleck Chemicals, Munich, Germany) at doses ranging from 0.1 to 20 μM, and cell proliferation was determined by a modification of the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reduction method.
BCR stimulation and phospho-kinase detection
Cell lines (3–5 x 106 cells) and primary CLL samples (8–10 x 106 cells) were pretreated with 1 or 2.5 μM IQS019 for 90 min in FBS-free RPMI medium. Once starved, cells were incubated at 37 °C with 10 μg/ml of either anti-IgM (UPN-1, JVM-13, OCI-LY10 and primary CLL cells) or anti-IgG (DOHH-2) antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Based on preliminary experiments showing a cell type-dependent variation in the optimal duration of the stimulation, cells were exposed to their respective anti-Ig for 2 min (UPN-1 and OCI-LY10 cells), 30 min (DOHH-2 and JVM-13 cells), and 15 min (CLL primary cells). Detection of phospho-Syk, phospho-lyn and phospho-Btk was carried out by western blot and flow cytometry, respectively, as detailed in Additional file 1 Methods.
Cell lines and CLL primary cells were exposed as indicated to IQS019, with or without BCR ligation, and CXCL12-induced migration was evaluated using 24-well chemotaxis chambers containing 8 μm (cell lines) or 5 μm (primary cells) pore size inserts (Corning Life Science, Tewksbury, MA, USA), as previously described . To quantify CXCR4-dependent F-actin polymerization, cells (300.000–500.000) treated as above were fixed on poly-L-lysine–coated glass coverslips with 4% paraformaldehyde, washed in PBS, permeabilized for 10 min with a solution containing 0.1% saponin (in PBS), followed by a 30 min incubation with 50 μg/ml phalloidin-TRITC (Sigma-Aldrich). Then, coverslips were washed three times with saponin 0.03%, mounted on glass slides with DAPI-containing Fluoroshield mounting medium (Sigma-Aldrich), and visualized on a Nikon H5505 microscope by means of a 60X NA oil objective (Nikon, Amsterdam, Netherlands) with the use of Isis Imaging System v5.3 software (MetaSystems GmbH, Heidelberg, Germany).
Xenograft mouse models and immunohistochemical studies
For MCL xenotransplant model, CB17-SCID female mice (Janvier Labs, Le Genest-Saint-Isle, France) were inoculated subcutaneously with UPN-1 cells as previously described .Tumor-bearing mice were randomly assigned into equivalent cohorts and received a daily dose of 2 mg/kg, 10 mg/kg (i.p.), or 25 mg/kg (p.o.) IQS019-2MeSO3H or ibrutinib, or equal volume of vehicle, for 15 days, in a five/two (on/off) schedule. Animals were sacrificed and tumor samples were processed and stained for phospho-Histone H3 and cleaved caspase-3 as previously described . Detection of phospho-Syk, phospho-Lyn and phospho-Btk was carried out from OCT tumor section as explained in Additional file 1 Methods. For systemic FL model, 12 SCID mice were intravenously inoculated via tail vein with 1.5 x 107 DOHH-2 cells per mouse. One week later, animals were randomly assigned into two equivalent cohorts and treated intraperitoneally with 2 mg/kg IQS019-2MeSO3H or vehicle, as before. Mice were then sacrificed and immunodetection of phospho-BCR kinases was performed as detailed in Additional file 1 Methods.
Unless otherwise specified, the data are depicted as the mean ± SD of three independent experiments. Unpaired and paired T-tests were used to obtain the statistical analysis using Graph Pad Prism software 4.0. Results were considered statistically significant when p < 0.05 (*, **p < 0.01, ***p < 0.001).
Antitumor effect of the 4-aminopyrido[2,3-d]pyrimidine IQS019 in B lymphoid cell lines and primary samples
We further assessed the activity of the compound in vitro using a panel of 21 B-NHL cell lines representative of the CLL, MCL, FL and DLBCL subtypes (Table 1). We show that a 5 μM dose of the compound decreased cell proliferation in all the cell lines (range: 29–100%), being MCL and FL cells significantly more sensitive to the compound (mean cytotoxicity at 48 h: 67.2 ± 15%) than CLL cells and DLBCL cells of either activated B-cell (ABC) or germinal centre B-cell (GCB) subtype (mean cytotoxicity at 48 h: 42 ± 9%) (p = 0.0002). Based on these results, a set of 13 CLL primary cultures were exposed for 24 h to increasing doses of IQS019 and cell viability was measured by MTT assay. Although a high variability was observed among cases, the viability decreased in a dose-dependent manner in all the samples treated with the compound (Fig. 1d). The calculated IC50 was 6.1 μM in this set of samples, corresponding to the upper range of the values found in the cell lines. Similar responses were observed in FL and MCL primary cultures (data not shown). Of note, no association could be established between sensitivity to IQS019 and common cytogenetic alterations, TP53 mutation and/or deletion, or IGHV mutational status (Table 1, Additional file 1: Table S2 and Figure S2a). Of interest, a 24 h treatment with a 5 μM dose of the compound induced about 35% apoptosis increase in the representative cell lines UPN-1 and DOHH-2 (Additional file 1: Figure S2b). In CLL and primary cultures (n = 6) the average cell death induction reached 26% (range: 9.5–51.5%), as shown in the representative cases, CLL n.2 and CLL n.10 (Additional file 1: Figure S2b and data not shown). This phenomenon was completely abrogated in the presence of the pan-caspase inhibitor Q-VD-OPh. In parallel, the analysis of phospho-histone H3 levels as a surrogate of mitotic progression indicated a notable decrease of this marker in five out of six primary CLL cases treated with the compound (Additional file 1: Figure S2c). Thus, altogether these results demonstrate that IQS019 antitumor activity in B lymphoid cells involved both a blockade in cell proliferation and the induction of a caspase-dependent cell death.
IQS019 antagonizes constitutive and antigen-mediated BCR signaling
IQS019 inhibits CXCL12-mediated migration of malignant B cells
IQS019 is safe and impairs tumor outgrowth and malignant B cell homing to spleen in vivo
In the systemic DOHH-2 mouse model, mice dosing was initiated at day 7 post-inoculation, with a 2 mg/kg IQS019-2MeSO3H regimen, daily, for 15 days. Once inoculated, FL cells rapidly migrate to the spleen . Therefore, at the end of the procedure, entire spleens were processed, and the presence of malignant B cells was evaluated by labeling with anti-human CD45 antibody and tumor cell recounting on a flow cytometer. IQS019-2MeSO3H treatment induced a 52% reduction in tumor cell infiltration into the spleen, when compared to vehicle group (Fig. 5d, * p = 0.01). Accordingly, the r fluorescence values of phospho-Syk, phospho-Btk, and phospho-Lyn, decreased by 83, 57, and 33% in tumors B cells purified from IQS019-treated animals (Fig. 5e). Altogether, these results demonstrate that IQS019 is safe and exhibits in vivo efficacy against MCL and FL tumor burden, involving the inhibition of BCR signaling and the blockade of tumor cell homing to lymphoid compartment.
IQS019 shows superior anti-tumor activity than ibrutinib in vitro and in vivo
To unravel at the molecular level the mechanisms underlying this superior activity of IQS019 over the Btk inhibitor, we established an ibrutinib-resistant cell line designated UPN-IbruR, derived from the parental UPN-1 by repeated drug selection (Additional file 1 Methods). When compared with the parental cell line, UPN-IbruR presented approximately a 10-fold increase in the ibrutinib IC50 after 72 h of treatment (24.6 vs 2.4 μM for parental cells), with negligible difference in IQS019 IC50 (5.6 vs 2.3 μM for parental cells) (Additional file 1: Figure S5a).The ibrutinib resistance phenotype of UPN-IbruR cells was not associated to mutations in BTK or PLCG2 genes, which both harbored a wild type sequence (Additional file 1: Figure S5b), but may rather be associated to the activation of non-canonical NF-κB pathway, as suggested by the overexpression of p52 (Additional file 1: Figure S5c). While a similar dose-dependent decrease in phospho-Lyn and phospho-Btk levels was found in IQS019- and in ibrutinib-treated UPN-1 cells, the expression of phospho-Syk was almost completely lost only in cells exposed to 1 μM IQS019 (Fig. 6d,e). In sharp contrast, in UPN-IbruR cells, the Btk inhibitor failed to modulate the phosphorylation of the three kinases, while IQS019 showed significant inhibitory activity of phospho-Syk and phospho-Lyn at a dose as low as 1 μM (Fig. 6d). However, the compound was unable to downregulate phospho-Btk (Fig. 6e), suggesting that in ibrutinib-resistant cells, the capacity of IQS019 to inhibit Syk and Lyn may allow the compound to maintain a significant antitumoral activity independent of the expression of a non-druggable form of Btk. Altogether, these results point out a significant superior antitumoral activity of pleiotropic BCR kinase targeting by IQS019 over the sole inhibition of Btk, in in vitro and in vivo models of B-NHL.
BCR has recently emerged as a central oncogenic pathway that promotes growth and survival in various lymphoma subtypes . Constitutive activation of the three BCR-related kinases Syk, Lyn, and Btk have been well documented in CLL [18–20], MCL [21, 22], and FL  cells, while chronic BCR signaling has been reported in the ABC subtype of DLBCL . Consistently, BCR kinase inhibitors constitute promising therapeutic strategies in these different entities. Among these novel agents, the first-in-class Btk inhibitor ibrutinib has achieved high response rates (43–71%) in relapsed/refractory CLL, MCL and ABC-DLBCL patients, while its activity was less pronounced in FL patients (37% overall response rate) [24–27]. A small fraction of patients develop progressive disease after initial response to this agent [25, 27], in relation with the acquisition of mutations at the ibrutinib binding site (C481S) of Btk, or in the PLCγ2 gene [9–11]. Resistance to ibrutinib may also involve a lower dependency of malignant B cells toward Btk itself, than other downstream components of the pathway, like the Syk/Lyn-dependent kinase Erk . Accordingly, the Syk inhibitor fostamatinib and the Src inhibitor dasatinib have also shown efficacy in relapsed/refractory B-NHL [29, 30].
Following these observations, and in an effort to improve the therapeutic modulation of BCR signaling, we previously screened a library of compounds derived from pyrido[2,3-d]pyrimidines, for their capacity to bind to the active sites of Btk, Syk and/or Lyn . We identified IQS019 (compound 19) as a unique molecule with affinity for the three BCR kinases . In the present work, we confirm the inhibitory property of the compound against Btk, Syk and Lyn, as well as its selective antitumoral effect in B lymphoid cells, especially in MCL and FL cell lines, and independently of the response to ibrutinib. Our results suggest that IQS019 can counteract both chronic and tonic BCR signaling, as it shows similar antiproliferative activity in DLBCL cell lines from both the GCB and ABC subtype, which are respectively dependent for their survival on tonic (Syk/PI3K-mediated) and chronic (Syk/Btk-mediated) BCR signaling [8, 32–34]. This property might confer to IQS019 a greater activity than ibrutinib, which is preferentially active against tumors that rely on chronic active BCR signaling . Beside Btk, the direct inhibitory activity of IQS019 towards Syk and/or Lyn phosphorylation may also explain the capacity of the compound to activate apoptosis in vitro and in vivo, as pharmacological inhibition of Syk, has been reported to elicit the apoptotic cascade in preclinical models of DLBCL and CLL [35, 36]. Also, probably thanks to its apoptogenic property and specificity, IQS019 salt is found to be significantly active and safe at a dose of 2 mg/kg/day, which is much lower than the reported active concentrations of fostamatinib, dasatinib or ibrutinib in mouse models of lymphoid neoplasms [37–39], thus predicting a probable low incidence of secondary adverse effects of the compound.
Another downstream event regulated by Btk, Syk, and Lyn is the chemokine-mediated B cell migration, a process essential to tumor B cell survival . We show that IQS019 is able to impair in vitro cell migration towards CXCL12 in cell lines and primary samples, in both basal and anti-Ig-stimulated cultures. This property may be responsible, at least in part, for the reduced infiltration of tumor cell observed in FL-bearing mice dosed with the compound. Beside this effect, IQS019-mediated inhibition of Syk, Lyn, and Btk may further impair tumor maintenance and B cell homeostasis in vivo, which are largely dependent on the coordinated activity of the three kinases .
In summary, we describe IQS019 as a new and unique BCR kinase inhibitor able to counteract both constitutive and ligand-dependent activation of the BCR pathway in in vitro and in vivo models of B lymphoid neoplasms. Thanks to the unique capacity of the compound to inhibit the three upstream BCR kinases Lyn, Syk, and Btk, this study may offer a glimpse into possible application for the treatment of the most prevalent subtypes of B-NHL, including those low responders to current BCR kinase inhibitors.
A, G, and C protein kinase group
B cell receptor
B-cell non-Hodgkin lymphoma
Ca2+/calmodulin-dependent protein kinase
Chronic lymphocytic leukemia
Cyclin-dependent (CDKs), mitogen-activated, glycogen synthase and CDK-like protein kinase group
Diffuse large B-cell lymphoma
Fetal bovine serum
Germinal centre B-cell
- GI50 :
Growth inhibitory 50
Lck/Yes novel tyrosine kinase
Mantle cell lymphoma
Mitogen-activated protein kinase cascade component
Spleen tyrosine kinase
The authors gratefully acknowledge Pangaea Biotech for its involvement and support to the project and thank Sandra Cabezas for technical assistance.
This work was financially supported by Fondo de Investigación Sanitaria PI12/01847 and PI15/00102 (to G.R.), PI0110094 (to A.M.), European Regional Development Fund (ERDF) “Una manera de hacer Europa”, Ministerio de Ciencia e Innovación, SAF12/31242 (to D.C.), SAF11/29326 (to P.P.-G.), SAF2010-C21617-C02 (to JI.B.), Redes Temáticas de Investigación Cooperativa de Cáncer from the Instituto de Salud Carlos III (ISCIII) RD12/0036/0004 (to D.C.) and RD12/0036/0039 (to E.C.) and Generalitat de Catalunya 2014SGR346 (to D.C.) and 2014SGR795 (to E.C.). A.E.-A. and A.M.-C. were recipients of predoctoral fellowships from Ministerio de Ciencia e Innovación and A.M. hold an IDIBAPS intramural predoctoral fellowship. R.P. was supported by a grant within the Talent empresa 2009 program (2009 TEM 00128) of the Generalitat de Catalunya. AC-J holds a postdoctoral fellowship from Catalonian Agency for Management of University and Research Grants (AGAUR, Beatriu de Pinos program). This work was carried out at the Esther Koplowitz Center, Barcelona, under the CERCA Program (Generalitat de Catalunya).
Availability of data and materials
All relevant data and materials within this work are made available in this manuscript. Any additional information can be made freely available to any scientist on reasonable request.
PB and AE-A designed the study, performed the experiments, and analyzed data and co‐wrote the manuscript. JR and LJ performed the IQS019 sensitivity assays in the B-NHL cell lines. VR designed and performed the animal studies. RP performed the IQS019 synthesis. JT supervised the IQS019 synthesis, interpreted the results, and reviewed the manuscript. JGV, AC-J, AM-C, and AM provided support in the Western blot and flow cytometry analysis and in interpretation of the data. AM helped in designing the immunohistochemical and immunofluorescence assays. EC analyzed clinical data and reviewed the manuscript. AS-B supervised IQS019 kinase inhibition profiling and PK studies. JIB supervised IQS019 synthesis, interpreted the results, and reviewed the manuscript. PP-G analyzed data and co-wrote the manuscript. DC designed the study and reviewed the manuscript. GR conceived and designed the study, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.
A.S.-B. is an employee of Pangaea Biotech, SL. The remaining authors have no competing financial interests.
Consent for publication
Ethics approval and consent to participate
The manuscript involved the use of human and animal samples. The ethical approvals for this project, including the informed consent of the patients, the animal procedures and the handling of samples, were granted following the guidelines of the Hospital Clínic Ethics Committee (IRB, reg. num. 2012/7498) in compliance with the Animal Ethics Committee of the University of Barcelona (agreement #154/16).
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