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Supplemental Figure 3. Mean (SD) TIRA plasma concentration. Cycle 1 Day 8 data are shown in patients receiving TIRA monotherapy with A) TIRA/IDELA, and B) TIRA/ENTO*

Figure S2 shows Hsp70 inhibition does not block clustering of acentrosomal spindle poles.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by failure of mature lymphocytes to undergo apoptosis. CLL cells are inherently resistant to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Pretreatment with histone deacetylase inhibitors (HDACi) sensitizes CLL cells to TRAIL-mediated apoptosis primarily via TRAIL-R1 and offers a novel approach for the therapy of CLL and other malignancies. Depsipeptide (romidepsin), a HDACi, did not enhance TRAIL binding to TRAIL-R1, TRAIL-R1 aggregation, or internalization of TRAIL-R1, but it enhanced Fas-associated death domain protein (FADD) recruitment to TRAIL-R1 in the death-inducing signaling complex. Cotreatment with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, dramatically inhibited the HDACi-mediated increase in FADD recruitment and sensitization to TRAIL-induced apoptosis and both of these were reversed by PKC inhibitors. Thus, enhanced FADD recruitment is a critical step in HDACi-mediated sensitization of CLL cells to TRAIL-induced apoptosis and this step is differentially affected by HDACi and phorbol 12-myristate 13-acetate. Using biotinylated TRAIL and streptactin-tagged TRAIL, we have identified several novel TRAIL receptor interacting proteins, including PKCβ, lymphocyte-specific protease-1, Lyn, and Syk. These molecules may play an as yet unappreciated role in TRAIL signaling in CLL cells and inhibition of one or more of these kinases/phosphatases may provide a novel target to overcome TRAIL resistance. [Mol Cancer Ther 2009;8(11):3088–97]

Supplementary Figures 1-5 from Enhanced Fas-associated death domain recruitment by histone deacetylase inhibitors is critical for the sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis

Supplemental Table 1 summarizes study inclusion and exclusion criteria. Supplemental Table 2 shows the doses and dose-escalation strategy used in this study. Supplemental Table 3 defines the dose-limiting toxicities that guided dose escalation. Supplemental Table 4 lists the number of patients by disease in the dose expansion phase. Supplemental Table 5 summarizes study drug exposure. Supplemental Table 6 lists TEAEs and laboratory abnormalities of interest that occurred during the study. Supplemental Table 7 shows responders and nonresponders according to biomarker risk groups. Supplemental Table 8 shows best overall response and gene aberration status as measured by FISH and NGS. Supplemental Table 9 provides tirabrutinib peak plasma concentrations. Supplemental Table 10 summarizes tirabrutinib, idelalisib, and entospletinib plasma exposure (AUC) and maximum plasma concentrations (Cmax). Supplemental Table 11 shows patients with free BTK below the lower limit of quantitation. Supplemental Figure 1 graphically illustrates the design of the study. Supplemental Figure 2 illustrates the disposition of patients over the course of the study. Supplemental Figure 3 shows line graphs illustrating tirabrutinib plasma concentrations over time. Supplemental Figure 4 shows box plots representing BTK levels at baseline and trough tirabrutinib concentrations.

Supplementary figure legends

Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6–Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785–96. ©2017 AACR.

Purpose: Inhibition of the antiapoptotic BCL2 family is one of the most promising areas of anticancer drug development. However, ABT-737, a specific BCL2 inhibitor, is neither orally bioavailable nor metabolically stable. To overcome these problems, the structurally related molecule ABT-263 was synthesized and recently entered clinical trials in hematologic malignancies, including chronic lymphocytic leukemia (CLL). Almost all laboratory studies have been carried out with ABT-737 rather than ABT-263, the drug being used in clinical trials. Currently there are no published data on the comparative effects of these inhibitors. To gain insight into the potential value or limitations of ABT-263 in the clinic, we assessed its ability to induce apoptosis in clinically relevant cellular models of CLL.Experimental Design: The susceptibility of freshly isolated primary CLL cells to these inhibitors was compared in standard culture conditions and in conditions that more closely mimic in vivo conditions in a whole blood assay system.Results: ABT-737 was more potent than ABT-263 at inducing apoptosis in CLL cells. In whole blood, ∼100-fold higher concentrations of both drugs were required to induce apoptosis. We found that ABT-263 was highly bound by albumin and that an increased albumin binding of ABT-263 as compared with ABT-737 accounted for the differential sensitivity of CLL cells.Conclusions: Our data indicate that the exquisite in vitro sensitivity of CLL cells to BCL2 inhibitors may be lost in vivo due to high cell densities and the albumin binding of ABT-263. Modification of ABT-263 may yield a BCL2 inhibitor with greater bioavailability and more favorable pharmacokinetics. Clin Cancer Res; 16(16); 4217–25. ©2010 AACR.

Supplemental Figure 2. Patient disposition

Purpose:Bruton tyrosine kinase (BTK) inhibition alone leads to incomplete responses in chronic lymphocytic leukemia (CLL). Combination therapy may reduce activation of escape pathways and deepen responses. This open-label, phase Ib, sequential dose-escalation and dose-expansion study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of the selective BTK inhibitor tirabrutinib alone, in combination with the PI3K delta (PI3Kδ) inhibitor idelalisib, or with the spleen tyrosine kinase (SYK) inhibitor entospletinib in patients with relapsed/refractory CLL.Patients and Methods:Patients received either tirabrutinib monotherapy (80 mg every day) or tirabrutinib 20–150 mg every day in combination with either idelalisib (50 mg twice a day or 100 mg every day) or entospletinib (200 mg or 400 mg every day).Results:Fifty-three patients were included. Systemic tirabrutinib exposure was comparable between monotherapy and combination therapy. No MTD was identified. Across all treatment groups, the most common adverse event was diarrhea (43%, 1 patient grade ≥3); discontinuation due to adverse events was uncommon (13%). Objective response rates were 83%, 93%, and 100%, and complete responses were 7%, 7%, and 10% in patients receiving tirabrutinib, tirabrutinib/idelalisib, and tirabrutinib/entospletinib, respectively. As of February 21, 2019, 46 of 53 patients continue to receive treatment on study.Conclusions:Tirabrutinib in combination with idelalisib or entospletinib was well tolerated in patients with CLL, establishing an acceptable safety profile for concurrent selective inhibition of BTK with either PI3Kδ or SYK. This small study did not establish a superior efficacy of the combinations over tirabrutinib alone. This trial is registered at www.clinicaltrials.gov (NCT02457598).

Supplementary Figure S1.

In healthy cells, pro- and anti-apoptotic BCL2 family and BH3-only proteins are expressed in a delicate equilibrium. In contrast, this homeostasis is frequently perturbed in cancer cells due to the overexpression of anti-apoptotic BCL2 family proteins. Variability in the expression and sequestration of these proteins in Diffuse Large B cell Lymphoma (DLBCL) likely contributes to variability in response to BH3-mimetics. Successful deployment of BH3-mimetics in DLBCL requires reliable predictions of which lymphoma cells will respond. Here we show that a computational systems biology approach enables accurate prediction of the sensitivity of DLBCL cells to BH3-mimetics. We found that fractional killing of DLBCL, can be explained by cell-to-cell variability in the molecular abundances of signaling proteins. Importantly, by combining protein interaction data with a knowledge of genetic lesions in DLBCL cells, ourin silicomodels accurately predictin vitroresponse to BH3-mimetics. Furthermore, through virtual DLBCL cells we predict synergistic combinations of BH3-mimetics, which we then experimentally validated. These results show that computational systems biology models of apoptotic signaling, when constrained by experimental data, can facilitate the rational assignment of efficacious targeted inhibitors in B cell malignancies, paving the way for development of more personalized approaches to treatment.

Bruton tyrosine kinase (BTK) inhibition is an effective therapy for many B-cell malignancies. Acalabrutinib is a next-generation, potent, highly selective, covalent BTK inhibitor. To characterize acalabrutinib tolerability, we pooled safety data from 1040 patients with mature B-cell malignancies treated with acalabrutinib monotherapy in nine clinical studies (treatment-naive: n = 366 [35%], relapsed/refractory: n = 674 [65%]; median [range] age: 67 [32–90] years; median [range] prior treatments: 1 [0–13]; median [range] duration of exposure: 24.6 [0.0–58.5] months). The most common adverse events (AEs) were headache (38%), diarrhea (37%), upper respiratory tract infection (22%), contusion (22%), nausea (22%), fatigue (21%), and cough (21%). Serious AEs (SAEs) occurred in 39% of patients; pneumonia (6%) was the only SAE that occurred in ≥2%. Deaths due to AEs occurred in 52 patients (5%); pneumonia (n = 8) was the only fatal AE to occur in ≥3 patients. AEs led to treatment discontinuation in 9%. Rates for the AEs of interest (all grades) included infections (67%), hemorrhages (46%), neutropenia (16%), anemia (14%), second primary malignancies (12%), thrombocytopenia (9%), hypertension (8%), and atrial fibrillation (4%). This pooled analysis confirmed acalabrutinib’s tolerability and identified no newly emerging late toxicities, supporting acalabrutinib as a long-term treatment for patients with mature B-cell malignancies.

8064 Background: Acala is a next-generation, highly selective, covalent Bruton tyrosine kinase inhibitor approved in the US for patients (pts) with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and previously treated mantle cell lymphoma (MCL). We evaluated the safety profile of acala monotherapy (monotx) in multiple B cell malignancies. Methods: Data from pts with activated B-cell diffuse large B-cell lymphoma, CLL, follicular lymphoma, MCL, multiple myeloma, prolymphocytic leukemia, Richter syndrome, SLL, or Waldenström macroglobulinemia treated with ≥1 dose of acala monotx in 9 studies were pooled. Acala was administered at 100 mg BID in most pts (100–400 mg total dose daily). Adverse events (AE) were assessed. Results: A total of 1040 pts were included (median age: 67 y [range: 32–90]; ECOG status ≤1: 93%; median exposure duration: 24.6 mo [range: 0–58.5]). A total of 360 (34%) pts discontinued acala, most commonly due to progressive disease (PD; 17%). AEs led to acala discontinuation in 97 (9%) pts; those in > 2 pts were pneumonia (n = 5) and thrombocytopenia (n = 4). Incidence of AEs, including the most common (any grade and grade ≥3), are shown in the Table. Events of clinical interest (ECIs) included atrial fibrillation (afib) of any grade in 46 (4%) pts and grade ≥3 in 13 (1%) pts; major hemorrhage (any grade) in 37 (4%) pts; grade ≥3 infection in 183 (18%) pts; hypertension (any grade) in 79 (8%) pts and grade ≥3 in 36 (4%) pts; and second primary malignancies (SPM) excluding non-melanoma skin cancer (NMSC; any grade) in 68 (7%) pts. Median (range) time to first onset in days for each ECI (any grade) was: afib, 522 (8–1280); major hemorrhage, 293 (4–1327); infections, 92 (1–1317); hypertension, 157 (2–1345); SPM excluding NMSC, 339 (7–1499). Death was reported in 139 (13%) pts, most commonly due to PD (6%) and AEs (5%). Conclusions: Acala monotx has a favorable tolerability profile with increased exposure across multiple mature B cell malignancies. Additional analyses will further explore the longitudinal characteristics of AEs. [Table: see text]

International audience; Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, welltolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Introduction: Tirabrutinib (TIRA; GS/ONO-4059) is a selective Bruton's tyrosine kinase (BTK) inhibitor. Entospletinib is a selective inhibitor of spleen tyrosine kinase (SYK). Both have single agent activity in lymphoma and updated results from the combination of TIRA+ENTO from this ongoing phase 1b study (NCT02457598) are reported here. Methods: Patients with previously treated non-germinal-center B-cell type (non-GCB) diffuse large B-cell lymphoma (DLBCL) or two prior lines of therapy for follicular lymphoma (FL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), or Waldenstrom's macroglobulinemia (WM) and no prior exposure to targeted inhibitors were eligible for enrollment. Patients were enrolled using a 3+3 dose escalation design with either ENTO 200mg or 400mg QD and doses of TIRA ranging from 20mg to 160mg QD. Cohorts were subsequently enrolled at multiple dose levels to evaluate disease-specific safety and efficacy. Results: As of March 5, 2018, 72 patients have enrolled on the combination. The median age was 67.5 years (range: 30-90) and the disease subtypes for patients enrolled were non-GCB DLBCL (n=32), FL (18), MZL (5), WM (7), SLL (2), MCL (8). No maximum tolerated dose and no dose-response relationship was observed with daily dosing of both agents at the dose levels evaluated. For patients with non-GCB DLBCL (n=32), the median number of prior therapies is 3 (range 1-7). The median duration of treatment is 8 weeks (range 2-98.1) with 6 patients still on treatment. 6/27 (22%) of the evaluable patients achieved a response; best overall response is shown in table 1. For patients with indolent NHL (n=40), the median number of prior therapies is 3 (range 1-6). The median duration of treatment is 36 weeks (range 0.1-116), with 29 patients still on treatment. 19/31 (61%) of the evaluable patients achieved a response with best overall response by indication shown in table 1. The most common treatment-emergent adverse events (AEs) are listed in table 2. Of the 71 patients who have received treatment on study, AEs led to treatment interruption or discontinuation of both study drugs in 10 and 1 patients, respectively. There have been 4 deaths on study, all due to disease progression. Conclusion: Once-daily dosing of TIRA up to 160 mg in combination with ENTO up to 400 mg QD was safe and well tolerated. Early results show activity in combination across all indications treated. Disclosures Salles: Novartis: Consultancy, Honoraria; Epizyme: Honoraria; Abbvie: Honoraria; Servier: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Pfizer: Honoraria; Merck: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Morphosys: Honoraria; Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Servier: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Dyer:Gilead Sciences, Inc.: Honoraria, Research Funding. Hodson:Gilead Sciences, Inc.: Research Funding. Ysebaert:Roche: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Cartron:Sanofi: Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche: Consultancy, Honoraria; Gilead Sciences: Honoraria. Davies:Acerta Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; ADC-Therapeutics: Research Funding; Janssen: Honoraria; Karyopharma: Membership on an entity's Board of Directors or advisory committees. Danilov:Aptose Biosciences: Research Funding; Gilead Sciences: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy; Astra Zeneca: Consultancy. Fegan:Roche: Honoraria; Abbvie: Honoraria; Gilead Sciences, Inc.: Honoraria; Napp: Honoraria; Janssen: Honoraria. Huang:Gilead Sciences, Inc.: Employment. Mitra:Gilead Sciences, Inc.: Employment. Rule:Roche: Honoraria; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, Inc.: Membership on an entity's Board of Directors or advisory committees; Celltrion: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Morschhauser:Janssen: Other: Scientific Lectures; Epizyme: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

7547Background: Acalabrutinib, a highly selective, potent, covalent BTK inhibitor, was assessed as monotherapy in pts with R/R de novo DLBCL. Methods: Eligible pts aged ≥18 y, with ECOG PS ≤2 and c...

GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.