Potent Btk Inhibitor Research Articles

Inside Cover: Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA‐Encoded Chemical Library (ChemBioChem 9/2017)

Inside Cover: Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA‐Encoded Chemical Library (ChemBioChem 9/2017)

High Major Response Rate, Including Very Good Partial Responses (VGPR), in Patients (pts) with Waldenstrom Macroglobulinemia (WM) Treated with the Highly Specific BTK Inhibitor Bgb-3111: Expansion Phase Results from an Ongoing Phase I Study

High Major Response Rate, Including Very Good Partial Responses (VGPR), in Patients (pts) with Waldenstrom Macroglobulinemia (WM) Treated with the Highly Specific BTK Inhibitor Bgb-3111: Expansion Phase Results from an Ongoing Phase I Study

The Bruton Tyrosine Kinase (BTK) Inhibitor ACP-196 Demonstrates Clinical Activity in Two Mouse Models of Chronic Lymphocytic Leukemia

Introduction: BTK is involved in B-cell receptor (BCR) signal transduction and is an established target for the treatment of chronic lymphocytic leukemia (CLL) (Byrd, NEJM, 2013). ACP-196 is a novel, potent second generation BTK inhibitor, which binds covalently to Cys481 in the ATP-binding pocket of BTK. IC50determinations on nine kinases with a cysteine in the same position as BTK showed ACP-196 to be more selective than the first-in-class BTK inhibitor, ibrutinib (Covey, AACR, 2015). We present data evaluating the anti-tumor effects of ACP-196 in established murine models of CLL.Methods: Two distinct murine models were used for these studies. In the TCL1 adoptive transfer model, leukemic cells from Eμ-TCL1 transgenic mice were transplanted into C57BL/6 mice, resulting in a CD5+/CD19+ leukemia with peripheral blood, spleen and nodal involvement. ACP-196 treatment in drinking water (0.16 mg/mL) commenced when recipient mice had > 10% CD5+/CD19+ leukocytes in the peripheral blood. Mice were followed for survival. Separate cohorts were sacrificed for pharmacodynamic analyses after 1 and 4 weeks of treatment. In the second model, NSG mice received primary human CLL cells. The xenografted human CLL cells have comparable tumor biology (including active BCR signaling) to activated human lymph node resident CLL cells (Herman, Leukemia, 2013). PBMCs harvested from CLL patients were adoptively transferred at 1 x 108 cells per mouse. ACP-196 was initiated on day -1 (at the time of busulfan priming) at multiple doses ranging from 0.006 to 0.3 mg/mL in drinking water.Results: In the TCL1 model, treatment with ACP-196 showed > 90% BTK occupancy of BTK after 1 and 4 weeks of therapy. ACP-196 inhibited BCR signaling as shown by decreased autophosphorylation of BTK and reduction in surface expression of the BCR activation markers CD86 and CD69. After 1 week of ACP-196 inhibited BCR signaling as shown by a 6-fold reduction of autophosphorylation of BTK in the presence of anti-IgM, and surface expression of the BCR activation markers CD69 and CD86 were decreased by 47% and 57% respectively. Inhibition of BTK and downstream BCR activation was maintained through at least day 28 of treatment. Most notably, ACP-196 treatment resulted in a significant increase in survival compared with mice receiving vehicle (median 81 vs 59 days, respectively; P =0.02). In the NSG xenograft model, ACP-196 at the times examined did not cause a significant treatment-induced lymphocytosis in the patients evaluated (n=6). After 4 weeks of treatment with ACP-196, the NSG mice were sacrificed, and BCR signaling activity and tumor burden in the spleen were evaluated. ACP-196 treatment showed decreases in phosphorylation of PLCγ2 and ERK (P <0.02) as expected with BTK inhibition. Additionally, a significant reduction was observed in the percentage of proliferating cells in mice treated with ACP-196 compared to vehicle control (as determined by Ki67 staining; median 18% (Intraquartile Range (IQR): 13-38) and 6% (IQR: 2-15), respectively, P =0.02). Lastly, a significant reduction in total tumor burden in the spleen was observed in the mice treated with ACP-196 (median reduction of 33%) compared with the vehicle treated mice (P =0.04).Conclusions: ACP-196 is a potent inhibitor of BTK as measured by inhibition of BCR activity, reduced tumor proliferation and increased survival. Overall, ACP-196 showed statistically significant efficacy in two murine models of CLL and is currently in Phase 3 trials for treatment-naive (ClinicalTrials.gov NCT0247568) and previously treated high-risk CLL (ClinicalTrials.gov NCT02477696).This work was supported by the Intramural Research Program of NHLBI, NIH, R01CA197870, K23 CA178183-02, and Acerta Pharma. DisclosuresGulrajani:Acerta Pharma: Employment. Krantz:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Lannutti:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Byrd:Acerta Pharma BV: Research Funding. Wiestner:Pharmacyclics: Research Funding. Johnson:Acerta Pharma: Research Funding.

Abstract C186: SNS-062 is a potent noncovalent BTK inhibitor with comparable activity against wild type BTK and BTK with an acquired resistance mutation

Abstract Background and purpose: BTK mediates B-cell receptor signaling and was validated as a target by the BTK inhibitor ibrutinib in several B-cell malignancies, including mantle cell lymphoma and chronic lymphocytic leukemia (CLL). However, patients may have or acquire resistance. Resistance mechanisms include mutation of the cysteine in the BTK active site that ibrutinib requires for covalent binding (C481). We identified and characterized SNS-062, a potent, noncovalent BTK inhibitor with activity towards BTK harboring resistance mutations that also inhibits ITK and may provide enhanced anti-tumor immune responses. SNS-062 shows restricted kinase selectivity and nonclinical pharmacology, pharmacokinetics (PK) and toxicology profiles distinct from ibrutinib and merits clinical investigation. Methods and Results: In in vitro kinase binding assays, SNS-062 bound to 9 kinases with Kd &amp;lt; 25 nM, including Tec kinase family members BTK (0.3 nM) and ITK (2.2 nM), but did not bind EGFR. Lack of EGFR inhibition by SNS-062 may offer safety advantages over ibrutinib, which has been associated with diarrhea and rash potentially related to its off-target effects on EGFR. Cellular inhibition of BTK was demonstrated by measuring inhibition of BTK auto-phosphorylation. SNS-062 inhibited pBTK in human whole blood with an average IC50 of 50 nM. A PK/ PD (pBTK) relationship was demonstrated in mice, with an in vivo IC50 for pBTK inhibition of 47 nM. Prolonged SNS-062-mediated in vivo inhibition of pBTK correlated with potent efficacy in a T cell-independent (type II) BTK dependent B cell mediated antibody response mouse model (ED50 11 mg/kg); efficacy was also observed in a type II collagen- induced arthritis (CIA) rat model (ED50 for summed knee and ankle histopathology 0.8 and 3.9 mg/kg). To assess the activity of SNS-062 and ibrutinib against BTK with acquired resistance mutations, inhibition of wild type (WT) and mutant C481S BTK was evaluated in direct kinase assays. SNS-062 inhibited WT and C481S BTK with similar IC50s (pBTK IC50s: WT BTK 2.9 nM, C481S BTK 4.4 nM) while ibrutinib potency was reduced 40-fold (pBTK IC50s: WT BTK 0.58 nM, C481S BTK IC50 25.7 nM). Similarly, ibrutinib showed a 100-fold loss of potency in C481S BTK expressing 293 cells (pBTK IC50s: WT BTK 0.016 μM, C481S BTK 1.7 μM) while SNS-062 activity was unaffected (pBTK IC50s: WT BTK 0.57 μM, C481S BTK 0.80 μM). SNS-062 had good oral bioavailability in rat and dog (%F ≥ 40%) and a terminal half- life of 5 to 6 hours. 28 day repeat-dose toxicology studies in rat and dog showed that SNS-062 was well tolerated with continuous drug levels and at exposures (AUC∞) much greater than those achieved for ibrutinib. These results suggest that SNS-062 plasma concentrations may be achieved that provide prolonged inhibition of both WT and mutant BTK. Conclusions: SNS-062 is a noncovalent BTK inhibitor that also inhibits ITK but not EGFR. SNS-062 activity against both WT and mutant C481S BTK was similar, while ibrutinib activity was decreased 40 to 100-fold. SNS-062 has a nonclinical PK/safety profile that will likely provide SNS-062 plasma concentrations for sustained inhibition of both WT and mutant BTK. These results support clinical development of SNS-062 for B cell malignancies, including CLL with acquired resistance to ibrutinib. Citation Format: Minke E. Binnerts, Kevin L. Otipoby, Brian T. Hopkins, Tonika Bohnert, Stig Hansen, Gene Jamieson, Pamela A. Howland, Eric H. Bjerkholt, Deborah A. Thomas, Judith A. Fox, Adam R. Craig. SNS-062 is a potent noncovalent BTK inhibitor with comparable activity against wild type BTK and BTK with an acquired resistance mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C186.

Abstract 14587: Proarrhythmic Effects of Ibrutinib, a Clinically Approved Inhibitor of Bruton’S Tyrosine Kinase (BTK) Used in Cancer Therapy

Introduction: Ibrutinib is a novel and potent BTK inhibitor which has been approved for several types of B-cell malignancies. Our meta-analysis of the published clinical trials finds that atrial fibrillation occurs in ~7% of patients after chronic ibrutinib exposure. Ventricular tachycardia has also been reported. Recently, multi-targeted tyrosine kinase inhibitors (TKIs) have been associated with arrhythmias; one implicated mechanism is increased late sodium current (INa-L) after chronic but not acute drug exposure. This effect appears to be mediated by inhibition of PI3Kinase since it is rescued by intra-pipette PIP3, a PI3K effector. Hypothesis: In this study, we tested the hypothesis that chronic exposure to ibrutinib exerts arrhythmogenic effects by this mechanism. Methods: Isolated adult mouse cardiac atrial/ventricular and rabbit ventricular myocytes as well as KCNH2 (HERG/Kv11.1)- or Kv4.3-transfected CHO cells were used for the electrophysiological studies pre- and post-ibrutinib (0.3 μ or 1 μM) acutely (5-10 min) and chronically (5 hrs in myocytes or 48 hrs in CHO cells). In some experiments, 1 μM PIP3 was added into the pipette solution. Results: Chronic but not acute ibrutinib prolonged cardiac action potentials (APs), triggered abnormal APs (EADs and DADs; Figure), and markedly increased cardiac INa-L (1.68±0.27% vs control 0.21±0.04% of peak current, P&lt;0.01) in mouse atrial cells. A similar INa-L increase was also seen in mouse and rabbit ventricular cells. The abnormal APs and increased INa-L were antagonized by adding 1 μM PIP3 into the pipette and by the INa-L blocker ranolazine (10 μM). Ibrutinib has minor and no effects on cardiac HERG and Kv4.3 currents. Conclusion: Ibrutinib, like other TKIs, increased cardiac INa-L, an effect reversed by intracellular PIP3. These data further reinforce the importance of PIP3 signaling as a proximate mechanism leading to arrhythmogenesis by emerging targeted oncology therapies.

Abstract 2599: Discovery and pharmacological characterization of the second generation of Btk inhibitors with improved target selectivity and enhanced in vivo efficacy

Abstract Bruton's tyrosine kinase (Btk) is an essential component of the B-cell receptor (BCR) signaling pathways regulating survival, activation, proliferation, and differentiation of B lymphocytes. The first Btk inhibitor, Ibrutinib, has demonstrated a significant clinical efficacy in a variety of B-cell malignancies. Through structure-guided approach, we have discovered two series of novel and potent Btk inhibitors, represented by EBI-1266 and EBI-1367, respectively. Both of these compounds showed high potency in inhibiting Btk kinase activity and growth of a number of aggressive lymphoma cell lines driven by aberrant BCR signaling, with IC50 value within low nanomolar range. Mechanistic studies examining BCR signaling pathway in related B cell lymphoma cell lines revealed that both EBI-1266 and EBI-1367 potently inhibited Btk phosphorylation and downstream Erk phosphorylation, similar to Ibrutinib. Furthermore, each compound showed unique and significant superiority over Ibrutinib: EBI-1266 had a cleaner selectivity profile against a panel of kinases with a cysteine residue in the conserved catalytic domain, and EBI-1367 exhibited &amp;gt;10-fold higher in vivo exposure in multiple preclinical species. In a tumor xenograft mouse model where tumor growth is dependent on Btk activity, both EBI-1266 and EBI-1367 showed significant oral anti-tumor activities, with EBI-1367 being more efficacious than Ibrutinib, consistent with a higher exposure of EBI-1367 in both blood and tumor tissues. No evidence of overt toxicities was observed in rodents with prolonged oral administration for two weeks with doses at least 3-fold above a highly efficacious dose. In summary, the pharmacological profiles of EBI-1266 and EBI-1367 indicate that these compounds have a great potential to become the next generation of Btk inhibitors, offering advantages in pharmaceutical development, enhanced in vivo efficacy and reduced toxicities. We are currently performing IND-enabling studies aiming to initiate phase I clinical trials for these Btk inhibitors. Citation Format: Jiayin Zhang, Dong Liu, Ru Shen, Yinfa Yan, Liuqing Yang, Minsheng Zhang, Jun Feng, Beibei Fu, Jerry Hu, Biao Lu, Hong Wan, Lei Zhang, Weikang Tao, Lianshan Zhang, Jingsong Cao. Discovery and pharmacological characterization of the second generation of Btk inhibitors with improved target selectivity and enhanced in vivo efficacy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2599. doi:10.1158/1538-7445.AM2015-2599

Abstract 2452: ONO-4059, a novel oral Bruton's tyrosine kinase (Btk) inhibitor that demonstrates potent pharmacodynamic activity through Phosphorylated Btk (P-Btk) inhibition, in addition to effective anti-tumour activity in a TMD-8 (DLBCL) xenograft model.

Abstract Purpose: The B-cell receptor (BCR) pathway plays a central role in signal transduction pathways that regulate survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is a key regulator of the BCR signaling pathway and abberant BCR signaling has been implicated in the survival of malignant B-cells. Recent studies indicate that targeting Btk may be effective in the treatment of B-cell malignancies. ONO-4059 is a highly potent and selective oral Btk inhibitor with an IC50 in the sub-nmol/L range. Previous studies with ONO-4059 (formerly known as ONO-WG-307; AACR 2012) demonstrated that Btk-mediated signaling through Akt and PKD is a critical role for the survival of activated B-cell-like (ABC) sub-type of diffuse large B cell lymphoma (DLBCL) cell lines [TMD-8]. It was also of interest to determine the extent of gene expression changes induced in TMD-8 cells after treatment with ONO-4059. To understand the effects of ONO-4059 on gene transcription, we analyzed the gene expression patterns of ONO-4059 in the TMD-8 xenograft model. Methods: TMD-8 cells were implanted subcutaneously into SCID mice and ONO-4059 was administered orally, twice a day (BID) in two groups of animals, at doses of 3 and 10 mg/kg. When the mean tumour volumes reached 100-200mm3, treatment was initiated daily for 14 days. After the final treatment of ONO-4059, the total RNAs were extracted from frozen re-sected tumour tissue specimens, which comprised of 10 samples at each dose, along with 10 vehicle tissue samples. Microarray analysis was conducted by Agilent technology. Results: The inhibitory level of P-Btk achieved indicates profound anti-proliferative activity of ONO-4059 in the TMD-8 model. Gene expression studies demonstrated that ONO-4059 affects a core set of genes which contains 9 down-regulated and 8 up-regulated genes in a dose-dependent manner. CXCL-10 is the prominent regulated gene that is down-regulated after ONO-4059 treatment and CXCL-10 has been shown to be involved in the pathological processes of human disorders, such as, infectious diseases, inflammatory and autoimmune diseases as well as cancer. From a publicly available microarray database, CXCL-10 expression has been shown to be up-regulated in lymphoma patients, suggesting that CXCL-10 may be a potential biomarker for ONO-4059 in the treatment of B-cell malignancies. Conclusion: Our data show that ONO-4059 has potent anti-tumour activity in a ABC-DLBCL xenograft model and that ONO-4059 achieves active tumor exposures as measured by P-Btk in vivo. ONO-4059 is currently being developed in a Phase I clinical trial for the treatment of B-cell malignancies. Additional work is now underway to determine whether the combining ONO-4059 with other targeted agents affects CXCL-10 expression. Citation Format: Tomoko Yasuhiro, Toshio Yoshizawa, Shingo Hotta, Yuko Ariza, Yoshiko Ueda, Ryohei Kozaki, Joseph Birkett. ONO-4059, a novel oral Bruton's tyrosine kinase (Btk) inhibitor that demonstrates potent pharmacodynamic activity through Phosphorylated Btk (P-Btk) inhibition, in addition to effective anti-tumour activity in a TMD-8 (DLBCL) xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2452. doi:10.1158/1538-7445.AM2013-2452

Abstract 923: Ibrutinib inhibits malignant cell adhesion and migration and reduces tumor burden in lymph node and bone marrow in a murine model of mantle cell dissemination and progression.

Abstract Introduction: Ibrutnib is a potent and selective Btk inhibitor that covalently modifies Cys481 in Btk and inhibits B cell antigen receptor (BCR) signaling. Ibrutinib is currently in a Phase II study in patients with relapsed or refractory mantle cell lymphoma (MCL). In MCL patients following 1-2 weeks of ibrutinib treatment, we observed a transient increase of MCL cells in peripheral blood which was associated with a rapid reduction of tumor burden in MCL patients suggesting mobilization of malignant cells from tissues to the periphery (Wanga; Changb). At the same time, levels of plasma chemokines CCL4, CCL22, CXCL13 were significantly decreased by 40-60% (p&amp;lt;0.05) (Changb). As Btk is an essential element in BCR- and chemokine-mediated adhesion and migration in B lymphocytes, we determined effects of Btk inhibition by ibrutinib in MCL primary cells co-cultured with stromal cells, and studied its effects in vivo on MCL homing in immunocompetent C57Bl/6 mice and lymphadenopathy in a disseminated human xenograft model of MCL in SCID mice. Results and Conclusions: Ibrutinib inhibited phosphorylation of Btk, PLCγ, Erk and JNK in MCL cell lines (Mino, Jeko, HBL2) and primary MCL cells stimulated by co-culture with stromal cells or anti-IgM. Furthermore, ibrutinib inhibited BCR- and chemokine (CXCL12, CXCL13)-induced adhesion and migration of MCL cells lines, and the migration of MCL underneath bone marrow-derived stromal cells in co-culture (pseudoemperipoleisis). In addition, ibrutinib significantly inhibited the production of chemokines CCL3, CCL4, CCL22, TARC and CXCL13 by 50-70% (p&amp;lt;0.01) in human MCL co-culture or following BCR activation in MCL cell lines or primary MCL cells. Gene expression studies of both MCL cells and patients treated with ibrutinib for either 24 hrs or 8 days resulted in over 80% inhibition of NF-κB, IL-6, CCL3, CCL4 gene expression. In vivo, ibrutinib treatment at 12 mg/kg inhibited homing of MCL cells into lymphoid tissues of C57Bl/6 mice by 70%. Ibrutinib at 3 and 12 mg/kg significantly and dose-dependently suppressed tumor burden in the mandibular/cervical lymph nodes, mesenteric lymph nodes/Peyer Patches and bone marrow by over 90% in a prevention model of MCL lymphadenopathy in SCID mice. In a treatment setting of the same model where tumor injected mice were treated with vehicle for 6 weeks prior to receiving 12 mg/kg of ibrutinib for 3 weeks, a 90% and 68% reduction of tumor cells in the lymph nodes and bone marrow, respectively, was observed. These results collectively demonstrate Btk inhibition mediated by ibrutinib, suppresses adhesion and migration of malignant MCL cells in vitro and reduces tumor burden in lymph nodes and bone marrow in vivo. aWang L et al., ASH 2011. bChang BY et al., ASH 2011. Citation Format: Betty Y. Chang, Michelle Francesco, Susanne Steggerda, Stella Chang, Padmaja Magadala, Lucy Jawed, Patricia Thiemann, Joseph J. Buggy. Ibrutinib inhibits malignant cell adhesion and migration and reduces tumor burden in lymph node and bone marrow in a murine model of mantle cell dissemination and progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 923. doi:10.1158/1538-7445.AM2013-923

Abstract 857: Development of a Bruton's tyrosine kinase (Btk) inhibitor - ONO-WG-307, a potential treatment for B-cell malignancies

Abstract Purpose: Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. ONO-WG-307 is a highly potent and selective Btk inhibitor with an IC50 in the sub-nmol/L range. We evaluated the inhibitory effect of ONO-WG-307 alone and in combination with the chimeric type I anti-CD20 antibody rituximab, using in vitro tumor growth assays and mouse xenograft models. Methods: Two types of tumor cell lines (follicular lymphoma (FL) and activated B-cell-like (ABC) sub-type of diffuse large B cell lymphoma (DLBCL)) wereplated on to micro-titration plates (at doses up to 200 umol/L for ONO-WG-307 and 100 ug/mL for rituximab) for in vitro cytotoxic assays. The same cells were also implanted subcutaneously (SC) into female SCID mice to explore ONO-WG-307 anti-tumor activity in vivo (orally at doses up to 50 mg/kg, bid for ONO-WG-307 and intraperitoneally at doses up to 10 mg/kg, q7d). The IC50 of the in vitro cytotoxic activity was determined using a MTS assay 72 and 96 hours after incubation. Anti-tumor activity was defined as the ratio of the median tumor volume of treatment groups versus control group. The determination of combination index (CI) was calculated by the median-effect method. The CI was used to express synergism (&amp;lt;1), additivity (=1) or antagonism (&amp;gt;1). Results: In the MTS assay, ABC-DLBCL cells were much more sensitive to ONO-WG-307 given as single agent compared with FL cell lines, however, the activity of rituximab single agent had no impact on ABC-DLBCL cell lines in contrast to its inhibitory activity against FL cell lines. When ONO-WG-307 was combined with rituximab, a moderate antagonism was observed in vitro on FL cell lines. In an ABC-DLBCL xenograft model, treatment with single agent ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth and also showed moderate anti-tumor activity in a FL xenograft model. Conclusion: ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in both the ABC-DLBCL and the FL cell lines and in xenograft models. These preliminary results suggest that ONO-WG-307 may be an effective therapy in the treatment of B-cell malignancies. To determine whether combination of ONO-WG-307 and rituximab is more efficacious, additional in vivo combination work is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 857. doi:1538-7445.AM2012-857

Abstract 2934: Targeting Bruton's tyrosine kinase with PCI-32765 blocks growth and survival of multiple myeloma and Waldenström macroglobulinemia via potent inhibition of osteoclastogenesis, cytokines/chemokine secretion, and myeloma stem-like cells in the bone marrow microenvironment

Abstract Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in osteoclastogenesis. Since Btk has not been characterized in multiple myeloma (MM) and Waldenström Macroglobulinemia (WM), we investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) milieu, as well as on MM and WM cancer cells. In CD14+ OC precursor cells, PCI-32765 abrogated RANKL/M-CSF-induced Btk activation and downstream PLCγ2, resulting in decreased number of multinucleated OC (&amp;gt;3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and inhibition of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It induced defective bone resorption activity, as evidenced by diminished pit formation on dentine slices. Lack of effect of Dexamethasone on osteoclastic activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 potently downregulates cytokine and chemokine secretion from OC cultures, i.e., MIP1α, MIP1α, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1-0.48 nM). It significantly decreased IL-6, SDF-1, MIP1α, MIP1α, and M-CSF in 2-week cultures of CD138-negative cells from active MM patients, associated with decreased TRAP staining. In MM and WM cells, immunoblotting analysis showed Btk expression in higher percentage of CD138+ myeloma cells from patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated increased expression of Btk and its downstream signaling components in WM cells than in CD19+ normal BM cells (p&amp;lt;0.001). Importantly, PCI-32765 blocked SDF-1-induced adhesion and migration of MM cell lines and patient MM cells via blockade of Btk signaling cascade. Quantigene analysis further showed PCI-32765-inhibited MIP1α mRNA in MM cells and many NF-κB-targeted transcripts in OC-lineages. PCI-32765 mitigated MM cell growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs. It blocked WM cell proliferation and induced apoptosis. Furthermore, myeloma stem-like cells from MM patients expressed Btk and PCI-32765 (10-100 nM) specifically blocked their abilities to form colonies in methylcellulose (n=5). In contrast, no toxicity was observed in Btk-negative BMSCs and OB. Oral administration of PCI-32765 (12 mg/kg) in mice significantly suppressed MM cell growth (p&amp;lt; 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results strongly support clinical trials of targeting Btk by PCI-32765 in the BM microenvironment to improve patient outcome in MM and WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2934. doi:1538-7445.AM2012-2934

The Bruton's Tyrosine Kinase (BTK) Inhibitor PCI-32765 Induces Durable Responses in Relapsed or Refractory (R/R) Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL): Follow-up of a Phase Ib/II Study

Abstract 983 Introduction:Btk is a central mediator of B-cell receptor signaling which is essential for normal B-cell development. PCI-32765 is an orally-administered irreversible inhibitor of Btk which induces apoptosis and inhibits cellular migration and adhesion in malignant B-cells. An early analysis of the phase Ib/II study PCYC-1102 showed PCI-32765 to be highly active and tolerable in patients with CLL (Byrd, ASCO 2011). Here we report longer-term follow-up of this multicenter phase Ib/II trial. Methods and Patients:Two cohorts of CLL patients (previously untreated ≥65 years old and relapsed/refractory [R/R] disease following at least 2 prior therapies, including fludarabine) were treated with oral PCI-32765 administered daily for 28-day cycles until progression of disease. Doses of 420mg (previously untreated and R/R) and 840mg daily (R/R) were examined. The patients with R/R disease are the subject of this report. Results:Sixty-one R/R CLL/SLL patients were enrolled (420mg cohort n=27, 840mg cohort n=34). The median follow-up time for the 420mg cohort is 10.2 months and for the 840mg cohort is 6.5 months. The median number of prior treatment regimens for the 420mg cohort was 3 (2–10) and for the 840mg cohort was 5 (1–12). Seventy-two percent of patients had at least one poor-risk molecular feature: del(17p) 31%, del(11q) 33%, IgVH un-mutated 57%. Treatment has been well tolerated. Two patients have discontinued for adverse events (AE); 6 patients have required reduction of PCI-32765 dose (420mg cohort 2/27, 840mg cohort 4/34). Grade 1 or 2 diarrhea, fatigue, nausea, and ecchymosis have been the most frequently reported AEs. Serious AEs (SAEs) have occurred in 38% of patients; SAEs considered potentially related to PCI-32765 have occurred in 10% of patients. Grade ≥3 AEs considered potentially related to PCI-32765 occurred in 21% of patients. A characteristic pattern of response, with a transient phase of lymphocytosis typically peaking within the first 2 months of Rx, followed by resolution over time, has been observed in the majority of patients. Objective response (ORR; PR + CR) by IWCLL criteria in the 420mg cohort cohort, previously reported as 48% with 6.2 months median follow-up (Byrd, et al ASCO 2011), is now 70% with 10.2 months median follow-up. ORR in the 840mg cohort is 44% at 6.5 months median follow-up. An additional 19%, and 35% of patients in these cohorts, respectively, have a nodal PR (>50% reduction in aggregate lymph node size) with residual lymphocytosis. ORR appears to be independent of molecular risk features. Eighty-two percent of patients (50/61; 420mg cohort 22/27, 840mg cohort 28/34) remain on PCI-32765. Only 8% (5/61) of patients have had progressive disease (PD); 6-month PFS is 92% in the 420mg cohort and 90% in the 840mg cohort. Treatment cessation not related to PD or AE includes: death (n=2) or investigator discretion (n=3). Conclusions:The potent Btk inhibitor PCI-32765 is well tolerated and is associated with high rates of 6-month PFS in R/R CLL/SLL. Phase III trials of PCI-32765 in CLL/SLL are planned. Disclosures:O'Brien:Pharmacyclics, Inc: Research Funding. Burger:Pharmacyclics, Inc: Research Funding. Blum:Pharmacyclics: Research Funding. Furman:Pharmacyclics, Inc: Research Funding. Coutre:Pharmacyclics, Inc: Research Funding. Sharman:Pharmacyclics, Inc: Research Funding. Flinn:Pharmacyclics, Inc: Research Funding. Grant:Pharmacyclics, Inc: Research Funding. Heerema:Pharmacyclics, Inc: Research Funding. Johnson:Pharmacyclics, Inc: Research Funding. Navarro:Pharmacyclics, Inc: Employment, Equity Ownership. Holmgren:Pharmacyclics, Inc: Consultancy. Hedrick:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics, Inc: Research Funding.

The Bruton's Tyrosine Kinase Inhibitor PCI-32765 Is Highly Active As Single-Agent Therapy in Previously-Treated Mantle Cell Lymphoma (MCL): Preliminary Results of a Phase II Trial

Abstract 442 Introduction:Bruton's tyrosine kinase (Btk) is a central mediator of B-cell receptor signaling which is essential for normal B-cell development. PCI-32765 is an orally administered irreversible inhibitor of Btk that induces apoptosis and inhibits cellular migration and adhesion in malignant B-cells. In a phase I trial of PCI-32765 in relapsed B-cell malignancies, objective responses were observed in seven of nine patients with MCL. Reported here are preliminary results of an ongoing phase II study of single-agent PCI-32765 in previously treated MCL. Methods and Patients:Patients with relapsed or refractory MCL who were either bortezomib-naïve or bortezomib-exposed (prior treatment with at least 2 cycles of bortezomib) were eligible for study PCYC-1104. PCI-32765 was administered orally at 560mg daily (in continuous 28-day cycles) until disease progression. Bortezomib-naive and bortezomib-exposed cohorts were evaluated separately. Tumor response was evaluated every 2 cycles and classified by 2007 NHL IWG criteria. Results:A total of 48 patients (29 bortezomib-naive, 19 bortezomib-exposed) have been enrolled on study PCYC-1104 between February 16, 2011 and July 20, 2011. The median age is 67 years (62–72). The median number of prior treatment regimens is 2 (1–5). Five patients (13%) had received prior autologous or allogeneic stem cell transplantation. Seven patients (15%) had bulky disease. Thirty-nine patients who have initiated treatment and have reported adverse event (AE) information are the subject of this preliminary report. Twenty-four patients (12 bortezomib-naive, 12 bortezomib-exposed) have undergone at least 1 follow-up tumor assessment and are evaluable for efficacy. Treatment has been well tolerated. No patients have discontinued treatment due to AEs. Grade 1 or 2 diarrhea, fatigue, and nausea have been the most frequently reported AEs. Grade >3 AEs considered potentially related to PCI-32765 have occurred in 4/39 patients (11%). Serious AEs (SAEs) have occurred in 8/39 patients (21%); 2 SAEs (1 rash, 1 febrile neutropenia) were considered potentially related to PCI-32765. One death, in a patient who was enrolled but did not receive PCI-32765 due to rapid disease progression, has occurred on study. The objective response rate (ORR) by IWG criteria is 67% (16/24); ORR is 58% (7/12) in the bortezomib-naive cohort and 75% (9/12) in the bortezomib-exposed cohort. To date, 35/39 patients remain on PCI-32765; reasons for discontinuation include progressive disease (n=3) and investigator decision (n=1). Conclusions:Preliminary data from a phase II trial suggests that the potent Btk inhibitor PCI-32765 is well tolerated and induces a high rate of objective responses in patients with relapsed or refractory MCL. More mature safety and efficacy data will be updated in the presentation. Phase III trials of PCI-32765 in MCL are planned. Disclosures:Wang:Pharmacyclics: Research Funding. Off Label Use: PCI-32765 in mantle cell lymphoma in a phase 2 clinical trial. Martin:Pharmacyclics: Research Funding. Blum:Pharmacyclics: Research Funding. Kahl:Pharmacyclics: Research Funding. Maeda:Pharmacyclics: Research Funding. Advani:Pharmacyclics: Research Funding. Williams:Pharmacyclics: Research Funding. Rule:Pharmacyclics: Research Funding. Rodriguez:Pharmacyclics: Employment, Equity Ownership. Pang:Pharmacyclics: Consultancy. Hedrick:Pharmacyclics: Employment, Equity Ownership. Goy:Pharmacyclics: Research Funding.