Abstract 1207: SNS-062 demonstrates efficacy in chronic lymphocytic leukemia in vitro and inhibits C481S mutated Bruton tyrosine kinase

Abstract

Introduction: In order to address the issue of acquired resistance to ibrutinib, we sought to characterize the Bruton agammaglobulinemia tyrosine kinase (BTK) inhibitor SNS-062 in preclinical models of chronic lymphocytic leukemia (CLL). Methods: Primary CLL B cells were isolated from the whole blood of consented patients by ficoll density centrifugation and Rosette-Sep negative selection. Annexin V and propidium iodide flow cytometry was used to measure patient CLL cell viability and 7-AAD was used to measure viability in stromal co-culture. CD40 and CD86 expression was evaluated via flow cytometry subsequent to sustained 3.2uM CpG stimulation. BCR signaling in primary CLL cells was investigated by immunoblot following 1 hour treatment and following 1 hour or 24 hours of incubation with SNS-062 in XLA cell lines. ITK inhibition was investigated via immunoblot after stimulation with anti-CD3 and anti-CD28 and incubation with SNS-062 for 1 hour. SNS-062 was used at a concentration of 1uM in preclinical studies unless otherwise noted. Measurement of kinase activity in human recombinant WT BTK or C481S BTK was performed in a FRET kinase assay. Results: Immunoblots of BTK and ERK phosphorylation of XLA cells transfected with WT or C481S BTK demonstrated that SNS-062 inhibition is comparable to that of ibrutinib in WT BTK and greater than that of ibrutinib in C481S BTK. Using a recombinant kinase assay, we found the IC50 of SNS-062 against WT BTK to be 4.6nM and C481S BTK to be 1.1nM, suggesting that SNS-062 retains activity against the mutated BTK variant. Additionally, SNS-062 was found to be six times more potent than ibrutinib and greater than 640 times more potent than acalabrutinib against C481S BTK. SNS-062 demonstrates dose-dependent inhibition of BTK in primary patient CLL cells comparable to ibrutinib via immunoblot for BTK phosphorylation. The viability of primary patient cells treated with 0.1uM, 1.0uM, and 10.0uM SNS-062 for 48 hours was measured to be 96.7%, 96.1%, and 88.1%, respectively, that of the untreated condition. At 48 hours, SNS-062 decreased viability of primary CLL cells in the presence of HS5 stromal protection by 5.5%. SNS-062 was found to decrease CpG induced CD40 and CD86 expression by 8.7% and 15.7%, respectively. Using an in vitro kinase assay, SNS-062 inhibited ITK with an IC50 value of 24nM. An immunoblot of anti-CD3/CD28 stimulated Jurkat cells revealed that SNS-062 decreased the phosphorylation of ERK, implying inhibition of ITK. Conclusion: Unlike ibrutinib, SNS-062 inhibits BTK signaling in the presence of the C481S mutation and may address acquired resistance to covalent BTK inhibitors. SNS-062 decreases B cell activation markers, viability, and stromal cell protection in primary patient CLL cells and was shown to inhibit ITK, suggesting support of T cell mediated antitumor activities. These data support further investigation of this molecule and advancement into clinical trials. Citation Format: Catherine A. Fabian, Sean D. Reiff, Daphne Guinn, Linda Neuman, Judith A. Fox, Wendy Wilson, John C. Byrd, Jennifer A. Woyach, Amy J. Johnson. SNS-062 demonstrates efficacy in chronic lymphocytic leukemia in vitro and inhibits C481S mutated Bruton tyrosine kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2017-1207

AreBTKandPLCG2mutations necessary and sufficient for ibrutinib resistance in chronic lymphocytic leukemia?

“…One caveat here is that ARQ 531, like ibrutinib [54], is a nonspecific inhibitor of other Src and Tec family kinases [53], and thus any effects in BTK C481S mutant disease could also be due to effects on those other kinases. Vecabrutinib (SNS-062) is a reversible BTK inhibitor that blocks autophosphorylation of BTK in cells expressing either BTK or BTK C481S [55]. It has modest selectivity for BTK (IC 60 = 3nM); IC 50 values for ITK, TEC, BLK, LCK, SRC, and NEK11 are all also under 100nM [55].…”

“…Vecabrutinib (SNS-062) is a reversible BTK inhibitor that blocks autophosphorylation of BTK in cells expressing either BTK or BTK C481S [55]. It has modest selectivity for BTK (IC 60 = 3nM); IC 50 values for ITK, TEC, BLK, LCK, SRC, and NEK11 are all also under 100nM [55]. A phase 1a study has been performed in healthy subjects [56], and a phase 1b/2 dose escalation and cohort-expansion study in CLL patients is currently enrolling (NCT03037645).…”

Overcoming Ibrutinib Resistance in Chronic Lymphocytic Leukemia

“…Vecabrutinib is a novel reversible BTK and ITK inhibitor which has the ability to inhibit BTK, even in the presence of C481S mutations. Moreover, it seems to inhibit EGFR to a lesser extent than ibrutinib [115,116]. Recently, the preliminary results of the ongoing phase Ib/II study of vecabrutinib have been published (#NCT030376450) [117].…”

Advances in drug‐based therapies in chronic lymphocytic leukemia and future prospects

“…These mutations have been found to account for up to 85% of resistance mutations in one study . Vecabrutinib (SNS‐062), a noncovalent BTK inhibitor with the ability to inhibit signaling in the presence of the C481S mutation, has activity in vitro and studies in patients with CLL are underway (NCT03037645) . Similarly, ARQ531, a potent, reversible inhibitor of BTK with additional activity against Src family kinases and kinases related to ERK signaling, has activity in C481S and PLCG2 mutant CLL and is in a similar phase of development …”