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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.

Figure S1 shows protein expression of Hsp72, Hsc70, Nek6 and Nek7 in different cell types following different treatments.

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.