ALBERT

Description: Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α4β1-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

Description: Introduction: We previously demonstrated that engagement of the T-cell receptor (TCR) in malignant T-cells leads to ITK-dependent activation of the transcription factors NF-κB and GATA3, thus promoting chemotherapy resistance. TCR-mediated chemotherapy resistance was reversed upon knockdown or pharmacologic inhibition of ITK (Wang, 2017). ITK and resting lymphocyte kinase (RLK) are partially redundant TEC family kinase members involved in TCR signaling (Dubovsky, 2013). Here, we report results from preclinical studies evaluating the use of the ITK-specific inhibitor CPI-818 and the dual ITK/RLK inhibitor CPI-893 in the treatment of T-cell lymphoproliferative disorders. Methods/Results: Kinome screening was performed for CPI-818 and CPI-893. CPI-818 had high specificity for ITK over RLK (IC50 2.3 nM and 260 nM, respectively). In contrast CPI-893 had a high affinity for both ITK and RLK (IC50 0.36 nM and 0.4 nM, respectively). Normal and malignant T-cells from primary patient samples were evaluated for both ITK and RLK by western blot. As anticipated, normal CD3+ T-cells isolated from healthy donors expressed both ITK and RLK (n=3/3). In contrast, malignant T-cells preferentially expressed ITK in 3 of 3 cases and low levels of RLK in a single case. Tissue microarray analysis of cutaneous T-cell lymphoma and peripheral T-cell lymphoma cases further demonstrated ITK expression across multiple subtypes. To confirm on target effects of CPI-818 and CPI-893, Jurkat cells (which express ITK but not RLK) were stimulated with anti-CD3/CD28 beads for 5 minutes in the presence or absence of either agent. Auto-phosphorylation of ITK Y180 and PLC-γ Y783, an ITK substrate, were significantly reduced in the presence of either agent, confirming on target effects. We next evaluated the effect of both drugs on GATA3 expression. Cells were stimulated for 24 hours with anti-CD3/CD28 beads in the presence or absence of these agents (or vehicle control). ITK inhibition with either agent significantly reduced GATA3 expression in these experiments. We next evaluated the effect of CPI-818 and CPI-893 on cell viability in normal T-cells. TCR stimulation for 24 hours increased viability by 359% in the presence of vehicle control when normalized to unstimulated cells (n=3). Treatment with 1 µM CPI-893 reduced the cell viability to only 133% when compared to unstimulated controls (n=3, p = 0.0045). In contrast, treatment with the ITK-specific inhibitor CPI-818 at 1 µM had a numerically smaller, but still statistically significant effect (n=3, average viability 278%, p = 0.0056). We next quantified IL-10 expression by ELISA in cell culture supernatants. Consistent with the observed effect on cell proliferation and viability, only CPI-893 significantly reduced IL-10 production upon TCR stimulation (886 pg/mL vs 11.7 pg/mL, p

Description: Abstract 2716 Background Btk is a tyrosine kinase involved in B cell receptor (BCR) signal transduction. Recent studies indicate that chronic active BCR signaling is a pathogenic mechanism in ABC DLBCL that engages the classical NF-κB pathway. Mutations in the BCR pathway (CD79A/B and CARD11) and toll like receptor (TLR) pathway (MYD88) lead to constitutive NF-κB activation in ABC DLBCL. PCI-32765 kills ABC DLBCL cell lines with constitutive BCR signaling but has no effect on ABC and GBC DLBCL cell lines that do not rely on constitutive BCR signaling. PCI-32765 is an oral, well-tolerated and irreversible inhibitor of Btk. Study Design Patients with relapsed/refractory ABC DLBCL received PCI-32765 at a fixed dose of 560 mg po once daily × 35 days (1 cycle). Patients underwent CT and FDG-PET scanning pre-treatment and every 2 cycles. Where possible, pre-treatment and 48-hour post-treatment tumor biopsies were performed for gene expression profiling (GEP) and mutational analysis of CD79A/B, CARD11 and MYD88. Results Eight of 15 planned patients are enrolled. Characteristics include median (range) age 54 (40–79); LDH 〉 normal limits (63%); any extranodal site (75%) and stage 4 disease (63%). IPI distribution was 0–2 (25%) and 3–5 (75%). Patients received a median (range) of 3 (1–6) prior chemotherapy regimens. Best response by IWG criteria include CR: 2 (25%) for 11+ and 5 months; SD (stable disease) 3 (37%) for 4, 2 and 2 months; and PD (progressive disease) 3 (38%). One patient who was primary refractory achieved SD with PCI-32765, associated with a 25% tumor reduction, and is currently in CR following allogeneic BMT. PCI-32765 was well-tolerated without significant side effects. No patients discontinued PCI-32765 due to AE. Grade 〉3 AEs were reported in 4 patients, none were considered related to PCI-32765. One death has occurred on study, in a patient who received subsequent therapy following progression on PCI-32765. Toxicities that are possibly related to PCI-32765 include diarrhea (grade 1) in 2 patients, nausea (grade 1) in 2 patients and fatigue (grades 1–2) in 4 patients. CD79B mutations were uncovered in two patients, the patient with SD who achieved a 25% tumor response and one patient who achieved CR. Of note, the other patient who achieved CR did not have the CD79B mutation, suggesting that chronic active BCR signaling may occur in the absence of this mutation. None of the patients had MYD88 or CARD11 mutations. Comparison of the pre-treatment and on-treatment biopsy samples by gene expression profiling was completed on 5 patients (1 CR, 4 SD/PD). Gene expression signatures reflecting tumor infiltrating immune cells, including CD8+ T cells and macrophages, were diminished by PCI-32765 treatment in the one patient in CR and the one patient in SD who achieved a 25% tumor reduction, but not in the others. In these same two cases, a gene expression signature of interferon signaling was reduced upon PCI-32765 treatment, associated with a concomitant decrease in interferon gamma mRNA levels, again suggesting the loss of activated T cells in the responding tumors. One hypothesis to explain this observation would be cytokine modulation since chronic active BCR signaling promotes synthesis and secretion of IL-10 and multiple chemokines, including CXCL13, CXCL9 and CCL3, which were all down-modulated in the responding tumors. Conclusions The Btk inhibitor PCI-32765 has clinical activity in relapsed/refractory ABC DLBCL and modulates chronic active BCR signaling in responders. Thus, chronic active BCR signaling is a tractable therapeutic target in ABC DLBCL. Disclosures: Buggy: Pharmacyclics, Inc.: Employment. Hedrick:Pharmacyclics: Employment, Equity Ownership.

Description: Abstract 187 CLL patients with high-risk disease features have shorter remissions and a poor outcome with conventional chemo-immunotherapy, particularly in the relapsed disease setting. Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, thwarts B cell receptor (BCR) signaling and is a promising new targeted therapy for patients with mature B cell malignancies, particularly patients with CLL. Data from the Phase 1/2 trials demonstrated that high-risk CLL patients responded equally as well as low-risk patients to ibrutinib. Single-agent ibrutinib-treated CLL patients characteristically have delayed responses or stable disease due to persistent lymphocytosis, caused by re-distribution of tissue-resident CLL cells into the peripheral blood. To accelerate and improve responses and to expand upon the ibrutinib experience in high-risk CLL patients, we conducted a Phase 2 single-center clinical trial of ibrutinib plus rituximab, which accrued 40 patients between February and July, 2012. Methods: Patients were treated with ibrutinib 420 mg PO daily, in combination with weekly rituximab (375 mg/m2) for weeks 1–4 (cycle 1), then daily ibrutinib plus monthly rituximab until cycle 6, followed by daily single-agent ibrutinib. Study inclusion required high-risk disease (del17p or TP53 mutation [treated or untreated], patients with PFS 〈 36 months after frontline chemo-immunotherapy, or relapsed CLL with del11q. Results: Patient characteristics include a median age of 65 (range 35–82); median of 2 prior therapies, 14 female and 26 male patients. Median Rai stage was 4 (range 1–4), β2 microglobulin 4.2 mg/L (2.2 – 12.3), 31 patients had unmutated IGHV, only one patient mutated IGHV, the remaining patients had inconclusive IGHV results. 19 patients had del17p or TP53 mutation (4 without prior therapy), and 13 patients had del11q. At a median follow up of 4 months, 38 of 40 patients continue on therapy without disease progression. 1 patient died from an unrelated infectious complication, and one patient withdrew consent before starting therapy. Out of 20 patients evaluable for early response assessment at 3 months, 17 patients achieved a partial remission (PR) for an ORR of 85%, and three achieved a PR with persistent lymphocytosis. Interestingly, on this combination trial, the re-distribution lymphocytosis peaked earlier and the duration was shorter (see Figure) than with single-agent ibrutinib, presumably due to the addition of rituximab. Treatment was well tolerated, with only 13 cases of grade 3 (n=11) or grade 4 (n=2) toxicities, which were largely unrelated and transient, such as neutropenia, fatigue, pneumonia (n=1), insomnia, and bone aches. Questionnaires revealed an improved overall health and quality of life after 3 cycles of treatment in the evaluable patients (n=21). Conclusion: Ibrutinib in combination with rituximab is a safe, well tolerated regimen for high-risk CLL patients, which induces very high early response rates. These encouraging data, together with the Phase 1/2 data, emphasize the need for rapid further development of ibrutinib for high-risk CLL patients, given that current alternative treatment options for this patient population are not satisfactory. Disclosures: Burger: Pharmacyclics: Consultancy, Research Funding. Off Label Use: Ibrutinib in high-risk CLL. Faderl:Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding. James:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics: Employment, Equity Ownership. O'Brien:Pharmacyclics: Research Funding.

Description: Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here 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) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (〉3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p〈 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 982 Bruton's tyrosine kinase (Btk) is critical for B-lymphocyte function, due to its involvement in key functions in B-cells, e.g., maturation, activation, and trafficking. These functions are mediated by several receptors, including the B-cell antigen receptor (BCR) and the chemokine receptor CXCR4. Both BCR and CXCR4 play important roles in chronic lymphocytic leukemia (CLL), providing leukemic cells with survival and proliferation advantages. Initial studies in CLL suggest that inhibiting Btk with PCI-32765 (Pharmacyclics, Inc) is an effective therapeutic, reducing the size of solid lymphoid tissues leading initially to lymphocytosis and eventually to decreased blood absolute lymphocyte counts (ALCs). To understand the effect of blocking Btk-mediated signaling in CLL, we utilized an accelerated adoptive transfer CLL mouse model, injecting 5×106 TCL1 leukemia cells into SCID mice that succumb 5–6 weeks after cell transfer. Total 45 mice were injected, separated into 3 groups, and then treated with PCI-32765, at either 2, 3 or 4 weeks after cell transfer, with 5 mice from each group receiving either vehicle control or PCI-32765 (5 or 25 mg/kg/day) in daily drinking water. Mice were bled to track changes in ALCs. Animals treated at 2-weeks post cell transfer with the suboptimal (5mg/kg/day) and optimal (25mg/kg/day) doses exhibited a transient lymphocytosis at day 4, with a 7- and 10-fold increase in circulating TCL1 leukemia cells, respectively (p=0.002). By day 7, these levels had fallen to those of untreated mice. Until week 6, mice receiving the optimal dose of PCI-32765 at week 2 and 3 but not week 4, appeared healthy and had significantly reduced ALCs (p

Description: Abstract 3914 Inhibitors of histone deacetylases (HDACs) are currently in clinical testing for treating various cancers, and two have been recently approved by the US FDA for treating cutaneous T-cell lymphoma. Here we describe novel anti-inflammatory properties of the HDAC inhibitor PCI-24781 which is in clinical trials for multiple indications including lymphoma (Evens et al., Blood 114: 2726, ASH 2009 Annual Meeting Abstracts). Cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) have been shown to be involved in human inflammatory disorders, and an anti-IL-6 treatment was recently approved for rheumatoid arthritis (RA). Therefore, the effect of PCI-24781 on cytokine production by lipopolysaccharide (LPS)-stimulated human peripheral mononuclear blood cells (PBMC) as well as isolated monocytes was studied at the RNA expression level by microarrays and Taqman, and at the protein level by ELISA. PCI-24781 potently inhibits the production and secretion of several pro-inflammatory cytokines, including IL-6, TNF-a and interleukin-1beta (IL-1b), at both RNA and protein levels. In murine RAW macrophages as well, PCI-24781 inhibited LPS-stimulated IL-6 secretion at 20nM. PCI-24781 was most effective when given with or before LPS, but was still effective when given an hour after LPS. Similarly, PCI-24781 greatly attenuated in vivo pro-inflammatory cytokine production in LPS-treated Balb/c mice; the IC50 for IL-6 inhibition was 〈 5 mg/kg. Both the in vitro and in vivo IC50s for IL-6 inhibition are considerably less than the concentrations required to inhibit growth and induce apoptosis in tumor cells (0.2-0.5mM) and in xenograft models (60-80 mg/kg). The mechanism by which these cytokines are controlled involves attenuation of the LPS receptor TLR4 signaling at multiple levels, including acetylation of targets such as MKP-1 and NF-kB subunit p65 in the downstream MAPK and NF-kB pathways; other factors include reduced expression of proteasome, IKK and other NF-kB subunits. Interestingly, we observed a large reduction in levels of NOS2, which causes hypotension during sepsis by producing the inflammatory mediator nitric oxide (NO). Therefore the activity of PCI-24781 was tested in a model of sepsis where mice were treated with a lethal dose of 100 mg/kg LPS, an endotoxin known to be a major mediator of sepsis in humans. PCI-24781 was injected twice, first 16 h before LPS and then 2 h before LPS, in groups of 10 mice each. Control mice that did not receive any PCI-24781 all died within 2 days after LPS (mortality 100%). Pretreatment with PCI-24781 led to dose-dependent increase in survival with 60% of the mice surviving past 6 days with 2 doses of 50mg/kg PCI-24781. These data show that the HDAC inhibitor PCI-24781 protects mice from lethal endotoxemia. Thus, taken together, our data suggest that PCI-24781 has potent anti-inflammatory activities and may be useful to treat inflammatory disorders including RA and sepsis in humans. Disclosures: Balasubramanian: Pharmacyclics: Employment, Equity Ownership. Sirisawad:Pharmacyclics: Employment, Equity Ownership. Steggerda:Pharmacyclics: Employment, Equity Ownership. Cao:Pharmacyclics: Research Funding. Lowenstein:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership.

Description: Abstract 2569 Bruton's tyrosine kinase (BTK) is a member of the TEC family of non-receptor tyrosine kinases and is predominantly expressed in hematopoietic cells, except in T cells. BTK plays a prominent role in B cell receptor (BCR) signaling and several other pathways, including CXCR4 signaling, which is essential for lymphocyte homing. BTK activation downstream of the BCR leads to proliferation, differentiation, and survival of B cells. Functional BTK is necessary for normal B cell development, defective BTK result in a primary immunodeficiency called X-linked agammaglobulinemia (XLA). Because of the restricted expression and the B cell phenotype in BTK-deficient mice and XLA patients, BTK has become a promising therapeutic target in mature B cell malignancies. Ibrutinib is a selective, orally bioavailable, covalent BTK inhibitor currently studied in clinical trials in patients with Chronic Lymphocytic Leukemia (CLL) and other mature B cell malignancies. The importance of BTK in B-ALL is controversial. Initial study in childhood B-ALL revealed normal BTK protein levels, and gene expression profiling data from the St. Jude's group revealed BTK expression in B-ALL, but not in T-ALL. Other studies revealed abnormally spliced BTK mRNA and truncated BTK protein lacking kinase activity in some B-ALL samples. To explore the pre-clinical activity of ibrutinib in B-ALL, we used a panel of 14 B-ALL cell lines, representing pro-B (CD10+/−, TdT+, cyt Igμ-), pre-B (CD10+, TdT+, cyt Igμ+) and mature (CD10+/−, TdT-, surf IgM+) phenotypes. The panel included 4 Ph+/BCR-ABL1+ cell lines (Z-119, NALM-20, NALM-21, TOM-1). Western blot analysis revealed BTK expression in 12 out of 14 lines, while phospho-BTK (Y223) was present only in half of the cases. Effects of ibrutinib on B-ALL proliferation were tested in XTT assays, using increasing concentrations of ibrutinib (0.5 – 5 μM). All B-ALL cells responded to ibrutinib except for BTK-negative TANOUE cells. All other B-ALL cells displayed decreased proliferation with variable half maximal inhibitory concentrations of ibrutinib (IC50). The most sensitive cell lines (RCH-ACV, SMS-SB) had IC50 of 〈 1 μM; all BCR-ABL1+ cells showed IC50 〈 3.5 μM (see Figure). We also analyzed inhibitory effects of ibrutinib on B-ALL cell proliferation by serial automated cell counting, which confirmed the XTT assay data. B-ALL cells viability was determined after incubation with ibrutinib, which induced only minor decreases after 3 days of incubation. Preliminary data with primary B-ALL samples revealed BTK protein expression in 5 out of 5 samples. In co-culture with KUSA-H1 stromal cells, primary B-ALL cells showed moderate levels of apoptosis, ranging from 10 – 25% after 3 days of incubation with 1 μM ibrutinib, which is similar to levels of ibrutinib-induced apoptosis in CLL. Collectively, these data demonstrate that the BTK inhibitor ibrutinib can interfere with B-ALL cell proliferation and survival, providing a rationale for clinical testing of this novel, well-tolerated targeted agent in patients with relapsed B-ALL. Disclosures: O'Brien: Pharmacyclics: Research support Other. Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.