Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL by Kallesh D. Jayappa, Craig A.

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Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL by Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Brian J. Capaldo, Stefan Bekiranov, Mark J. Axelrod, L. Kyle Brett, Julia D. Wulfkuhle, Rosa I. Gallagher, Emanuel F. Petricoin, Timothy P. Bender, Michael E. Williams, and Michael J. Weber BloodAdv Volume 1(14):933-946 June 13, 2017 © 2017 by The American Society of Hematology

Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology

IBR and VEN are variably cytotoxic in CLL and MCL patient PBMCs IBR and VEN are variably cytotoxic in CLL and MCL patient PBMCs. (A) Representative images showing cleaved PARP in CD5+/CD19+ cells in CLL (patient [Pt] 12) and MCL (Pt 22) patient PBMCs following ex vivo treatment with IBR (0.1 μM), VEN (25 nM), or IBR+VEN. IBR and VEN are variably cytotoxic in CLL and MCL patient PBMCs. (A) Representative images showing cleaved PARP in CD5+/CD19+ cells in CLL (patient [Pt] 12) and MCL (Pt 22) patient PBMCs following ex vivo treatment with IBR (0.1 μM), VEN (25 nM), or IBR+VEN. The concentrations of IBR and VEN that showed 20% to 30% cytotoxicity as single agents (measured by alamarBlue) in a CLL patient PBMC were selected for ex vivo treatment of patient samples (data not shown). (B-D) PBMCs from CLL (N = 24) and MCL (N = 8) patients were treated with IBR, VEN, or IBR+VEN. The cleaved PARP in CD5+/CD19+ cells in CLL samples showing synergy (B) or no synergy (C) with IBR+VEN and in MCL samples (D) is shown. Comparable results were also obtained by analyzing the dead cell staining of CD5+/CD19+ cells in CLL and MCL patient PBMCs: samples that exhibited synergistically high cleaved PARP also showed synergistic increases in dead cell staining following ex vivo treatment with IBR and VEN (supplemental Figure 2A-C). The time to achieve maximal synergistic cytotoxicity (∼6-18 hours) was chosen as the time point used for subsequent data analysis. Synergy was calculated using the Bliss model of independence, which generates interaction scores even where 1 drug has no effect. Data are expressed as means ± SD. Statistical significance was determined by ANOVA. *P < .05, **P < .01, ****P < .0001. NS, not significant; SSC, side scatter. Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology

Extrinsic microenvironmental agonists generate resistance to IBR+VEN. Extrinsic microenvironmental agonists generate resistance to IBR+VEN. (A) Diagram showing the patient PBMCs cocultured with stromal cells or preincubated with soluble agonists for 12 hours. Drugs as well as a second dose of agonists were added at 12 hours and cultured for an additional 12 hours, and cleaved PARP or dead cell staining in CD5+/CD19+ cells in CLL or MCL was analyzed by flow cytometry. (B) PBMCs from CLL patients (Pts 01, 03, and 12) were cocultured with HS-5 or HK cell line at a 10:1 ratio or preincubated with sCD40L (2 μg/mL), IL-10 (0.015 μg/mL), CpG-ODN (1.5 μg/mL), CXCL13 (0.5 μg/mL), IL-2 (1.54 ng/mL), BAFF (0.25 μg/mL), or IgM (25 μg/mL) and treated with IBR (0.1 μM) + VEN (25 nM) or DMSO as well as second doses of agonists for 12 hours as shown in panel A. Cleaved PARP in CD5+/CD19+ cells was analyzed by flow cytometry. (C-D) PBMCs from 22 CLL (Pts 1-3, 6-7, 12, 14, 21, 25-28, 33, 35, 39, 43, 46, 48, 51-52, 56, and 59) and 8 MCL (Pts 22-23, 38, 45, 49, 55, 60, and 66) patients and the Mino MCL cell line were preincubated with agonist mix (sCD40L [2 μg/mL] + IL-10 [0.015 μg/mL] + CpG-ODN [1.5 μg/mL]) for 12 hours. Samples were then incubated with IBR (0.1 μM) + VEN (25 nM) or DMSO for an additional 12 hours as well as a second dose of agonist mix. Cleaved PARP in CD5+/CD19+ cells in CLL samples (C) and MCL samples/Mino cell line (D) were analyzed by flow cytometry. (E) Ki67+/CD5+/CD19+ cells in CLL and MCL patient PBMCs (Pts 06, 35, 39, 43, and 45) preincubated with or without 2 doses of agonist mix at 12-hour intervals were analyzed by flow cytometry. (F) PBMCs from CLL patients (Pt 01 and 25) were preincubated with agonist mix for 12 hours and treated with increasing concentrations of IBR (0.1, 1, and 10 μM) and VEN (6.25, 12, 25, 100, 400 nM) for 12 hours along with a second dose of agonist mix. The cleaved PARP in CD5+/CD19+ cells was analyzed by flow cytometry. Data are expressed as means ± SD. The statistical significance was determined by ANOVA. *P < .05, ***P < .001, ****P < .0001. FACS, fluorescence-activated cell sorter. Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology

NF-κB pathway is activated in CLL and MCL cells preincubated with agonist mix. NF-κB pathway is activated in CLL and MCL cells preincubated with agonist mix. CLL and MCL patient PBMCs were preincubated with agonist mix for 12 hours. At the end of 12 hours, a second dose of agonist mix was added and incubated for another 6 hours. Cytoplasmic and nuclear localization of RelB or RelA in a minimum of 500 live/CD5+/CD19+ cells in CLL and MCL samples was analyzed in ImageStream using IDEAS ImageStream analysis software. (A-B) Representative images showing CD5 (red), CD19 (violet), RelB (green), 7AAD (orange), and 7AAD/RelB in CLL (A; top panel) and MCL (B; top panel) patient PBMCs or CD5 (red), CD19 (violet), RelA (green), 7AAD (orange), and 7AAD/RelA in CLL (A; bottom panel) and MCL (B; bottom panel) patient PBMCs. Original magnification ×60. (C-D) Percentage of nuclear localization of RelB and RelA in 16 CLL patient PBMCs (Pts 01-03, 7, 12, 21, 25-28, 33, 35, 43, 46, 51, and 52) (C) and 7 MCL patient PBMCs (Pts 23, 38, 45, 49, 55, 60, and 66) and Mino cells (D) preincubated with or without agonist mix. (E-G) CLL patient PBMCs (N = 3) were incubated with agonist mix for 12 hours and treated with BMS-345541 (16 μM) or IBR (0.1 μM) + VEN (25 nM) as well as a second dose of agonist mix for another 6 hours. The representative image of similarity score histograms showing shift in RelB (E) or RelA (F) colocalization with 7AAD. Percentage of nuclear localization of RelB and RelA in different treatment groups (G). (H-I) Mino cell line preincubated with agonist mix for 12 hours and treated with BMS-345541 (0-64 μM) and a second dose of agonist mix for another 6 hours. RelA, RelB, histone (H3), and tubulin proteins in cytoplasmic and nuclear fractions were analyzed by western blot (H). Bar graphs showing relative band densities of RelB and RelA proteins in the nuclear fractions (I). The data were average from 2 independent experiments. RelB and RelA in nuclear fractions were normalized to histone. Data are expressed as means ± SD. The statistical significance was determined by the paired Student t test. *P < .05, **P < .01, ****P < .0001. Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology

Antiapoptotic protein expression was upregulated in CLL and MCL cells preincubated with agonist mix. Antiapoptotic protein expression was upregulated in CLL and MCL cells preincubated with agonist mix. (A-C) Expression of antiapoptotic proteins Mcl-1, Bcl-xL, survivin, and Bcl-2 in 14 CLL (Pts 1, 3, 7, 12, 19, 21, 25-27, 48, 51-52, 54, and 59) and 6 MCL (Pts 22, 45, 55, 58, 60, and 66) patient PBMCs and Mino cell line incubated with 2 doses of agonist mix at 12-hour intervals was analyzed by flow cytometry. Representative histograms showing the expression of Mcl-1, Bcl-xL, survivin, and Bcl-2 in a CLL patient sample (Pt 03) treated with agonist mix (A). Antiapoptotic protein expression in CLL patient PBMCs (B) and MCL patient PBMCs/Mino cell line (C) was determined by calculating the geometric mean of fluorescence intensity (GM-FLI) of proteins in CD5+/CD19+ cells. Agonist mix treatment significantly upregulated the expression of Mcl-1 (2.14 ± 0.51 fold in CLL; 1.874 ± 0.604 fold in MCL) and Bcl-xL (1.47 ± 0.18 fold in CLL; 1.98 ± 0.44 fold in MCL), and survivin (1.3 ± 0.43 fold in CLL; 1.173 ± 0.164 fold in MCL) but not Bcl-2 (1.16 ± 0.11 fold in CLL; 1.091 ± 0.221 fold in MCL) in CLL and MCL cells. (D) A CLL patient PBMC (Pt 25) treated with agonist mix for 12 hours was incubated with BMS-345541 (BMS) (16 μM) or bortezomib (Bortez) (0.064 μM) for 9 hours along with a second dose of agonist mix, with or without IBR (0.1 μM) + VEN (25 nM) and antiapoptotic protein expression was analyzed by calculating GM-FLI of proteins in CD5+/CD19+ cells using flow cytometry. The data were normalized to DMSO control. Data are expressed as means ± SD. The statistical significance was determined by Student t test. *P < .05, **P < .01, ****P < .0001. Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology

Inhibition of NF-κB pathway or antiapoptotic proteins overcomes IBR+VEN drug resistance in CLL and MCL cells preincubated with agonist mix. Inhibition of NF-κB pathway or antiapoptotic proteins overcomes IBR+VEN drug resistance in CLL and MCL cells preincubated with agonist mix. (A-F) The NF-κB pathway inhibitor BMS-345541 (A) and Bortez (B) and antiapoptotic protein inhibitor UMI-77 (Mcl-1 inhibitor) (D), BH3I-1 (Bcl-xL inhibitor) (E), and YM155 (survivin) (F) were tested in increasing concentrations with or without IBR (0.1 μM) + VEN (25 nM) in CLL (Pts 01, 03, and 12) and a MCL (Pt 45) patient PBMC preincubated with agonist mix as shown in Figure 2A. The antiapoptotic protein inhibitor A1210477 (C) was similarly tested in 4 CLL patient samples (Pts 03, 33, 35, and 46). Cell viability was determined using an alamarBlue assay. The data were normalized to DMSO treatment control or IBR+VEN in triple drug treatment. The data presented as means ± SD. ↓ indicates drug concentration at which target kinase phosphorylation/signaling pathway/apoptotic protein was maximally inhibited, based on published values.56-59 Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology

Synergistic interaction of inhibitors of NF-κB or antiapoptotic proteins with IBR+VEN combination. Synergistic interaction of inhibitors of NF-κB or antiapoptotic proteins with IBR+VEN combination. Synergistic interaction of inhibitors of NF-κB (BMS-345541, Bortez, and Carfilzomib) and antiapoptotic proteins (A1210477(A121), BH3I-1, UMI-77, and YM155) was determined by calculating Bliss predicted and actual cytotoxicity of individual inhibitors with IBR (0.1 μM) + VEN (25 nM) combination. The statistical significance was determined by ANOVA. *P < .05, **P < .01. Kallesh D. Jayappa et al. Blood Adv 2017;1:933-946 © 2017 by The American Society of Hematology