PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism by Roland B. Walter, Jason L. Pirga, Michelle R. Cronk, Sasha Mayer, Frederick R. Appelbaum, and Deborah E. Banker Blood Volume 106(10):3584-3593 November 15, 2005 ©2005 by American Society of Hematology

Nontoxic PK11195 doses can block efflux by Pgp, MRP, and BCRP drug transporters. Nontoxic PK11195 doses can block efflux by Pgp, MRP, and BCRP drug transporters. (A) To identify nontoxic Pgp-modulator doses, DOX40 cells were incubated across micromolar dose ranges with PK11195 (PK; 5 to 150 μM) and the established Pgp-modulator, CSA (0.2 to 5 μM). After 48-hour treatments, flow cytometry was performed in which “live” cell fractions contained cells with high DiOC63 staining (full mitochondrial membrane potential) and low PI fluorescence (high integrity), as illustrated in the top left panel. After other 48-hour treatments, 3H-Tdr was added for an additional 24 hours, and “live” cell fractions were measured as 3H-Tdr counts per minute relative to counts per minute in untreated wells. Treatment-specific live-cell fractions were calculated by subtracting untreated from treated fractions, and summary data from at least 3 assays are shown in the top right panel as means ± SEM. CSA (up to 1 μM) and PK11195 (up to 75 μM) did not significantly reduce live-cell fractions. Effects of CSA and PK11195 dose ranges were also measured in flow cytometry efflux assays after 1-hour MIT (20 μM) exposures. Representative histograms are shown in the bottom left panel. Efflux-blocking ratios were calculated as the MFI in the presence of test agent/MFI in the absence of test agent: “1” represents no efflux blocking, as denoted with a bold, horizontal line in the summary data from at least 3 assays that are shown in the bottom right panel. Also, 75 μM PK11195 blocked MIT efflux significantly more than any CSA dose in DOX40 cells. (B) Nontoxic doses of CSA (1 μM), MK-571 (MK; 25 μM), and GF120918 (GF; 1 μM) were used as Pgp, MRP, and BCRP modulators, respectively, along with dose ranges of PK11195, in MIT efflux assays of 7 additional AML and MM cell lines. Seventy-five μM PK11195 blocked MIT efflux significantly more than CSA in all cell lines tested and blocked MIT efflux significantly more than GF in BCRP-transduced HL60 cells. In all cases, efflux rations were calculated as described in panel A, and summary data from at least 3 assays are shown as means plus or minus SEM. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology

PK11195 can MIT-sensitize MDR cancer cells expressing Pgp, MRP, and/or BCRP. To determine the effects of nontoxic PK11195 (PK) doses on MIT cytotoxicity, DiOC63/PI flow cytometry assays were performed, as described for Figure 1. PK11195 can MIT-sensitize MDR cancer cells expressing Pgp, MRP, and/or BCRP. To determine the effects of nontoxic PK11195 (PK) doses on MIT cytotoxicity, DiOC63/PI flow cytometry assays were performed, as described for Figure 1. Cells were incubated with MIT doses that killed substantial fractions of isogenic parental, efflux-deficient cells, including RPMI 8226 (8226), HL60, and ML1 cells, as detailed in individual panel legends. Larger fractions of DOX40 (Pgp-expressing), MR20 (MR20; MRP- and BCRP-expressing), VCR (Pgp-expressing), AR (MRP-expressing), and ML1-BCRP (BCRP-transduced ML1) cells survived 24-hour MIT treatments relative to isogenic parental cells. KG1a (Pgp-expressing) cells were also relatively MIT resistant; 75 μM PK11195 cotreatments substantially decreased surviving fractions in ABC transporter-expressing cells. Summary data from at least 3 assays are shown as means plus or minus SEM. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology

PK11195 blocks MIT efflux in primary AML cells. PK11195 blocks MIT efflux in primary AML cells. To further assess the clinical relevance of our cell-line data, we performed efflux assays in primary AML cell samples. (A) Efflux blockade in Pgp-selective DiOC23 and ABC transporter-general MIT efflux assays was measured as increased dye retention. Efflux-blocking effects are presented as ratios, as in Figure 1. PK11195 and CSA effects were correlated in both assays, as noted in the figure. (B) In additional MIT efflux assays of 8 efflux-competent AML samples, MK-571 and Ko143 were used as specific MRP and BCRP modulators along with PK11195 and CSA. PK11195 again substantially blocked MIT efflux in all of these, and Ko143 measurably blocked MIT efflux in 4 samples, suggesting that these functionally expressed BCRP. MK-571 measurably blocked MIT efflux one, apparently MRP-expressing sample. PK11195 blocked efflux substantially more effectively than CSA in 3 samples. PK11195, CSA, and Ko143 also significantly increased MIT retention in repeated assays of normal CD34+CD38- hematopoietic progenitor cells (P = .01, P < .005, and P < .05, respectively), and PK11195 and CSA significantly increased MIT retention in more mature CD34+CD38+ normal cells (P = .05 in each case). Three immunolabeled NBM samples were analyzed, and summary data are shown as means ± SEM. KG1a cells were assayed along with primary cells in every assay; summary data from at least 5 assays are shown as means ± SEM. (C) 3H-PK11195 binding assays were performed in additional aliquots of 11 primary AML cell samples. Specific 3H-PK11195 binding was determined by competition with 1000-fold excess unlabeled PK11195, and competed counts are shown with means plus or minus SEMs for 7 samples (Pgp+ AMLs) in which both CSA and PK11195 increased DiOC23 retention and for 4 samples without efflux capacity (Pgp- AMLs). 3H-PK11195 binding was significantly higher in Pgp-positive AMLs than in Pgp-negative AMLs (P < .05), and Pgp-expressing DOX6 and DOX40 MM cells also bound very high levels of 3H-PK11195, but Pgp-expressing VCR and KG1a cells bound relatively low levels of PK11195 (means ± SEM are shown from at least 3 assays for each cell line). 3H-PK11195 binding was competed by excess of another pBR ligand, FGIN-1-27 (FGIN), but excess unlabeled CSA did not compete for 3H-PK11195 binding in any assay of Pgp-expressing cells. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology

pBR expression is not sufficient to produce efflux inhibition by PK11195. pBR expression is not sufficient to produce efflux inhibition by PK11195. (A) Jurkat lymphocytic leukemia cells were confirmed as pBR RNA-deficient in pBR-specific RT-PCR assays in which AML and MM cell lines served as positive controls (inset) and in ligand-binding assays in which DOX40 MM cells served as positive controls. Specific 3H-PK11195 binding was calculated by subtracting 3H-PK11195 counts per minute in the presence of 1000-fold excess unlabeled PK11195 from counts per minute in the absence of competitor. Lentiviral pBR transduction and flow sorting for GFP expression were used to produce J-pBR cells that stably expressed high levels of pBR as measured in 3H-PK11195 binding assays of whole-cell and mitochondrial lysates and as compared with positive control cells (DOX40) and negative controls (Jurkat, J-GFP, J-Neo). (B) Like parental Jurkat cells (white bars) and control J-GFP cells (gray bars), J-pBR cells (black bars) showed no MIT efflux that could be blocked by PK11195 (PK) or by known efflux inhibitors CSA, MK-571 (MK), or Ko143 (KO). Seventy-five micromolar PK11195 sensitized J-pBR cells to MIT significantly more than control J-GFP cells were affected, demonstrating functional pBR expression in J-pBR cells. (C) Lentiviral transduction was used to create K-pBR cells from Pgp-expressing, efflux-competent KG1a parental cells, as demonstrated in whole-cell PK11195 binding assays. K-pBR cells showed no more MIT efflux than control K-GFP cells when PK and CSA were used as efflux inhibitors. Cytotoxicity and efflux data and 3H-PK11195 binding data are presented as described for Figures 1 and 2, with summary data from 3 to 5 assays shown as means plus or minus SEM. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology

pBR expression is not necessary for efflux inhibition by PK11195 in MDR cells. pBR expression is not necessary for efflux inhibition by PK11195 in MDR cells. (A) pBR-deficient Jurkat-cell derivatives were produced that overexpress Pgp (J-MDR) as demonstrated with MRK16 anti-Pgp antibody staining. Representative flow cytometry data are shown as histograms, and summary data from at least 3 assays are shown as mean arbitrary fluorescence units (a.f.u.). Both CSA (▨) and PK11195 (▪) blocked DiOC23 and MIT efflux in J-MDR cells but not in parental Jurkat cells or control J-Neo cells, demonstrating specific efflux capacity in J-MDR cells and demonstrating PK11195's ability to block Pgp-mediated efflux in pBR-deficient cells. (B) Derivative Jurkat cells that overexpress BCRP (J-BCRP) were produced and characterized with BXP-34 anti-BCRP antibody staining. CSA (▨) and Ko143 (KO-143) (□) both increased MIT dye retention in J-BCRP cells, and PK11195 (▪) significantly blocked both MIT and Hoechst efflux in J-BCRP cells. Neither Hoechst 33342 nor MIT efflux was measured in control transporter-deficient cells, demonstrating specific efflux capacity in J-BCRP cells and demonstrating PK11195's ability to block BCRP-mediated efflux in pBR-deficient cells. Efflux data from at least 3 assays are presented, as described for Figure 1. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology

PK11195 apparently binds Pgp at PM sites distinct from CSA-binding sites. PK11195 apparently binds Pgp at PM sites distinct from CSA-binding sites. (A) Mitochondrial (MITO) and PM fractions were prepared from DOX40 MM cells by high-speed centrifugation. Fraction purities were confirmed in Western blot assays using antibodies recognizing mitochondrial-specific proteins (VDAC, COXIII), and the C219 antibody was used to recognize Pgp in PM fractions. 3H-PK11195 and 3H-CSA binding was measured in PM fractions and competed by PK11195 and CSA, respectively, but PK11195 and CSA did not cross-compete in these assays, further demonstrating that PK11195 binding is CSA independent. (B) PK11195 (▪) and CSA (▨) increased Rhodamine (RHO), Hoechst 33342 (Hoechst), and BODIPY-prazosin (PRAZ) retention in DOX40 cells, suggesting that both modulate multiple Pgp substrate-binding sites. PK11195 blocked RHO efflux significantly more than CSA, suggesting that PK11195 binds particular Pgp-binding sites more efficiently than CSA. Efflux data from at least 3 assays are presented, as in Figure 1. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology

PK11195 can activate Pgp-associated ATPase activity and alter Pgp conformation. PK11195 can activate Pgp-associated ATPase activity and alter Pgp conformation. To show that a nontoxic dose (75 μM) of PK11195 (PK) can specifically stimulate Pgp-associated ATPase activity, we performed assays in which treatment-specific (relative) ATPase activities were calculated (vanadate-sensitive ATPase activities in treated/untreated samples). Assays were performed with microsomal membranes (containing PMs and mitochondria) commercially prepared from Pgp-expressing SF9 insect cells (A), microsomes from Pgp-negative 8226 (gray bars) and Pgp-expressing DOX40 cells (black bars) (B), or PMs from DOX40 cells (C). Like VPL, PK specifically induced ATPase activity in all Pgp-positive models, with dose dependence in DOX40 PMs. Nontoxic doses of CSA (0.1, 1 μM) were ineffective in DOX40 models, but 1 μM CSA induced ATPase activity in Pgp-positive SF9 microsomes and 5 μM CSA induced activity in DOX40 PMs. Data from at least 3 assays are expressed as means plus or minus SEMs. (D) Specific effects of PK11195 on Pgp protein conformation were measured in 3 flow cytometry assays using the Pgp conformation-specific antibody, UIC2 (black bars), and the conformation-insensitive antibody, 15D3 (gray bars). PK11195 increased UIC2 immunoreactivity, but not 15D3 immunoreactivity, in live, Pgp-expressing DOX6, DOX40, KG1a, and VCR cells but not in Pgp-negative 8226 or HL60 parental cells. Roland B. Walter et al. Blood 2005;106:3584-3593 ©2005 by American Society of Hematology