Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

Background 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. Methods 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. Results 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. Conclusions 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

Additional file 1: of Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

Figure S1. IQS019 tyrosine kinase inhibitory profiling. Tyrosine kinase (TK) and tyrosine kinase-like (TKL) kinome tree was elaborated on the basis of residual in vitro kinase activity upon exposure to 100 nM or 1 μM IQS019, by means of Kinome Render software ( http://bcb.med.usherbrooke.ca/kinomerender.php ). Figure S2. Sensitivity of CLL primary cases to IQS019 is independent of IGHV mutational status and involves a caspase-dependent cell death process. (a) CLL primary cells, 9 of them with ummutated (UM) and 6 with mutated (M) IGHV gene, were treated with increasing concentrations of IQS019 for 24h. Cell viability was determined by MTT method. Shown are the median values from each CLL group (UM and M), referred to control, untreated cells. (b) IQS019 induces caspase-dependent cell death in MCL (UPN-1) and in FL (DOHH-2) cell lines, as well as in two representative CLL primary cultures. Cells were exposed for 24 hours to 5 μM IQS019, in the presence of absence of the pan-caspase inhibitor Q-VD-OPh (10 μM). Apoptosis was determined by simultaneous cytofluorimetric detection of Annexin-V and caspase-3/7 activity. (c) A set of 6 CLL primary cultures were treated with IQS019 as indicated, followed by Western Blot detection of phospho-histone H3 (p-H3), using β- actin as a loading control. Figure S3. Flow cytometry determination of CXCR4 membrane expression in B-NHL cell lines. Four representative cell lines were stained with a PE-labeled anti-CXCR4 antibody and analyzed on an Attune cytometer. CXCR4-specific signal (black curves) and isotypic control (grey filled curve) are represented. Figure S4. Safety and PK properties of IQS019-2MeSO3H in mice. (a) Twenty SCID mice (10 males and 10 females) received a single intravenous injection of IQS019-2MeSO3H at a 2 mg/kg, 10 mg/kg, or 50 mg/kg dose, or equivalent volume of vehicle, and animal weight was recorded at days 1, 3, 4, 7, 11, 14, 18 and 21 post-treatment. (b) Mean plasma concentration of IQS019-2MeSO3H in ICR mice over the time, after a single p.o. administration of a 25 mg/kg dose of the compound. Figure S5. Comparison of parental and ibrutinib-resistant derived B-NHL cell line. (a) Dose-response of the UPN-1 parental, and UPN-IbruR derived cell line exposed for 72 hours to increasing concentrations of ibrutinib or IQS019. (b) BTK and PLCG2 exon sequencing in UPN-IbruR cells. (c) Western blot detection of the alternative NF-κB pathway component, p52, in UPN-1 and UPN-IbruR cells. β-actin was used as a loading control. (DOC 3279 kb