ALBERT

Description: Sapphyrins are pentapyrrolic metal-free expanded porphyrins that localize to tumors. We have previously demonstrated that sapphyrins induce apoptosis in a variety of hematologic tumor cell lines including lymphomas (Ramos, DHL-4, HF-1), leukemias (Jurkat, HL-60), and myelomas (8226/S, 1-310, C2E3, 1-414). Through chemical modification of the parent compound, PCI-2000, a number of derivatives were generated and tested for induction of apoptosis in Ramos cells. PCI-2000 and one of the more potent apoptosis-inducing derivatives, PCI-2050, were injected into CD-1 nude mice bearing Ramos xenografts. Animals were sacrificed 48 hrs after injection and analyzed for drug uptake in the tumor, liver and spleen using flow cytometry. For PCI-2000, the relative uptake was spleen〉tumor〉liver. For PCI-2050 the relative uptake was tumor〉spleen〉liver, suggesting that PCI-2050 preferentially localizes in tumors compared to PCI-2000. Tumor cells isolated from PCI-2050 treated animals grew less well in culture and had more apoptotic cells than those derived from PCI-2000 or control animals. Uptake of PCI-2050 into xenograft tumor cells and tumor cell killing was dose dependent. PCI-2050 (10 umol/kg x 2 days in a row) was administered to Ramos xenograft bearing animals that were then monitored for tumor growth. In both minimal tumor (animals treated before tumor was palpable) and established tumor (palpable tumor) models, PCI-2050 reduced tumor growth by 60–75%. Alternative dosing strategies revealed that split dosing (allowing 1 or more days between doses) was more efficacious in tumor control than dosing 2 days in a row. At the doses used in this study, there was no myelosuppression or lymphosuppression, hepatic or renal abnormalities as assessed by complete blood count and comprehensive serum chemistry analysis, respectively. Our work demonstrates that PCI-2050 induces apoptosis in tissue culture and inhibits tumor growth in an animal tumor model while exhibiting minimal toxicity. PCI-2050 and other sapphyrin derivatives will be further evaluated as potential anti-cancer agents.

Description: There is increasing evidence indicating that B-cell-receptor (BCR) signaling is required for survival of non-Hodgkin’s lymphoma (NHL) cells. Bruton’s tyrosine kinase (Btk) is required for BCR signaling and mutations that inactivate human Btk cause X-linked-agammaglobulinemia, a B-cell immunodeficiency. Although Btk functions selectively in B cells, the Btk active site is structurally similar to the active site in several Src and Abl kinases and as a result, there have been few highly selective small molecule inhibitors of Btk. We have developed a series of covalent Btk inhibitors that target Cys-481 in Btk and this approach results in increased potency and selectivity over related kinases that lack a Cys residue at this position (ChemMedChem 15:58). PCI-32765 is a Cys-481 targeting Btk inhibitor that has been optimized for potency, selectivity and pharmacokinetics. In cellular assays, PCI-32765 inhibits BCR-stimulus induced phosphorylation of Phospholipase-C-gamma, a Btk substrate, as well as downstream phosphorylation of Erk (IC50 〈 100 nM). In addition, PCI-32765 induces apoptosis and inhibits proliferation in a subset of NHL cell lines including DHL-4, DHL-6, WSU-DLCL2, OCI-Ly10 and DOHH2 (IC50s = 0.6–1.6uM). We have used RNAi knockdown in DOHH2 cells as an independent method to confirm that Btk is required for lymphoma cell proliferation. In vivo, orally dosed PCI-32765 (50mg/kg) inhibits growth of DOHH2 and WSU-DLCL2 xenografts. PCI-32765 also prevents disease progression in a mouse collagen-induced arthritis model (12.5mg/kg PO), indicating that other B cell lineage diseases are sensitive to Btk inhibition. In order to further characterize the selectivity and in vivo potency of PCI-32765, we have developed PCI-33380, an active-site probe consisting of a covalent Btk inhibitor linked to the fluorophore Bodipy-FL. PCI-33380 binds to Btk and can be detected by flow cytometry or by denaturing gel electrophoresis of cell lysates. In cell lysates, the probe labels a single predominant band of the same molecular weight as Btk and this band is absent in cells from xid mice. Labeling of this band is inhibited (IC50=10nM) by a brief pre-treatment of cells with PCI-32765, indicating that the probe can be used to assess occupancy of Btk by a covalent inhibitor. We have used the probe to quantitate the inhibition of Btk by PCI-32765 in vivo. A single oral dose of PCI-32765 (10mg/kg) delivered to mice leads to rapid and complete inhibition of Btk in spleen. In addition, a single oral dose of PCI-32765 fully inhibits Btk in xenograft tumors and peripheral blood cells and this inhibition is maintained for up to 24hr. The Btk probe provides pharmacodynamic measurements that may allow optimization of dosing and schedule for in vivo studies and we are currently adapting the probe assays for use in monitoring the inhibition of Btk in human clinical trials.

Description: Abstract 4969 Bruton's tyrosine kinase (BTK), a Tec family kinase essential to signaling via the B-cell Receptor (BCR) has emerged as a very promising drug target in lymphoma. PCI-32765, a potent and selective covalently-bound small molecule inhibitor of BTK (Honigberg, Proc. Natl Acad. Sci. USA 107:13075, 2010), has demonstrated promising clinical activity in a number of Phase I and II clinical trials in patients with chronic lymphocytic leukemia (CLL) (Byrd et al., ASCO 2011 Annual Meeting Abstracts, JCO29 (15): 8012, 2010) and several subtypes of B-cell Non-Hodgkin's Lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL). Many cell lines derived from B-cell malignancies do not show in vitro growth inhibition by BTK inhibitors, perhaps because most established B-cell lines no longer depend on BCR stimulation for survival. However, some cell lines derived from the aggressive ABC (activated B-cell) subtype of DLBCL such as OCI-Ly10 and TMD8 are very sensitive to BTK inhibition by PCI-32765 (Davis et al, Nature463: 88, 2010). We show here that both of these cell lines are potently and irreversibly inhibited by PCI-32765 (EC50 continuous exposure 〈 10 nM and EC50 following a 1-hr pulse 〈 50 nM). Interestingly, 2 other ABC-DLBCL cell lines, OCI-Ly3 and HBL-1, in which the NF-κB pathway is activated by a mutation in CARD11 which is downstream of BTK, are not inhibited by PCI-32765. Both sensitive and resistant cells express Btk at similar levels, and the active site of BTK is fully occupied in the presence of low concentrations of the inhibitor as shown using a fluorescently-labeled affinity probe. We show that the sensitive TMD8 and OCI-Ly10 cells have chronically active BCR signaling to NF-κB which is dose-dependently inhibited by PCI-32765, as measured by comparing the phosphorylation and nuclear relocation of NF-κB p65 subunit. These cells also show inhibition of other major signaling pathways downstream of the BCR, including p-ERK, p-AKT, p-PLCγ and intracellular calcium flux, ultimately leading to NF-κB activation. The non-responsive OCI-Ly3 cells do not show inhibition of NF-κB p65 phosphorylation or nuclear relocation, although Btk is present and active, as shown by the probe assay and inhibition of p-AKT by PCI-32765. Furthermore, whole genome expression analysis reveals inhibition of mRNA expression of several NF-κB target genes in OCI-Ly10, particularly c-Myc, NF-κB subunits and several chemokines and cytokines, leading to downregulation of STAT3 signaling, NFAT signaling, cell cycle and cytokine-chemokine signaling pathways, none of which was observed in OCI-Ly3 cells. In vivo, PCI-32765 dose-dependently inhibited tumor growth in mice carrying OCI-Ly10 (and TMD8, ongoing) xenografts. Analysis of these tumors exhibited dose-dependent occupancy of the BTK active site as well as inhibition of signaling pathways. These results demonstrate the mechanistic basis of PCI-32765 inhibitory activity in ABC-DLBCL and support the ongoing clinical investigation of the therapeutic use of PCI-32765 in patients with this devastating disease. Disclosures: Balasubramanian: Pharmacyclics: Employment, Equity Ownership. Crowley:Pharmacyclics: Employment, Equity Ownership. Sirisawad:Pharmacyclics: Employment, Equity Ownership. Thiemann:Pharmacyclics: Employment, Equity Ownership. Chen:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.