The dual cell cycle kinase inhibitor JNJ-7706621 reverses ...
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The dual cell cycle kinase inhibitor JNJ-7706621 reverses resistance to CD37 targeted radioimmunotherapy in ABC-subtype Diffuse Large B Cell Lymphoma Cell Lines Disclosures Patzke: Nordic Nanovector ASA: Employment, Patent. Generalov, Melhus, Dahle: Nordic Nanovector ASA: Employment, Equity Ownership & Patents. Rødland, Syljuåsen: Instuonal research funds from Nordic Nanovector ASA, Patent. Bertoni: Instuonal research funds from Nordic Nanovector ASA. Acknowledgment Sylvia Kolenic: Nordic Nanovector ASA: Poster design and producon. References Rødland G, et al. (2019), Front. Oncol. 9:1301 | doi: 10.3389/fonc.2019.01301 Poster presented at 61 st American Society of Hematology (ASH) Annual Meeng and Exposion, Orlando, FL, USA, December 7-10, 2019. CONCLUSIONS INTRODUCTION RESULTS - LIBRARY SCREEN, VALIDATION, FUNCTIONAL ANALYSIS Figure 2. (A) Cells were treated for 18 hours with 177 Lu-lilotomab satetraxetan ranging from 0-2 µg/mL (specific acvity: 600 MBq/mg) or non-chelated lilotomab satetraxetan (1 µg/ml; HH-1-Dota), washed and plated in 96- well plates. Proliferaon was monitored between 72 and 144 h aſter seeding in micro-well ter plates ulizing a luminescent MT Cell Viability assay (RealTime-Glo). (B) Relave RLU ( 177 Lu-lilotomab satetraxetan to control) of data presented in (A). Error bars: Standard deviaon (STDEV) (n=5 for U-2932 and RIVA, n=3 OCI-Ly10). Inhibion of cell proliferaon on days 5 and 6 were significantly reduced compared to control (p<0.001, 1-way ANOVA) in U-2932 cells at doses ≥1 µg/mL, in RIVA at doses ≥0.25 µg/mL, and Oci-Ly10 at doses ≥0.1 µg/mL. (C) Experimental set- up for screening assay: U-2932 and RIVA cells were treated with 1 or 0.5 µg/mL 177 Lu-lilotomab satetraxetan (600 MBq/mg) for 18 hours, washed, and seeded onto 384-well plates pre-printed with the 384-compound Cambridge Cancer compound library sourced from Selleckchem at final concentraons of 10 nM, 100 nM or 1 µM. Untreated control cells were seeded on parallel plates. Gro Elise Rødland 1 , Katrine Melhus 2 , Roman Generalov 2 , Sania Gilani 1 , Francesco Bertoni 3 , Jostein Dahle 2 , Randi G. Syljuåsen 1 and Sebasan Patzke 1,2* 1 Instute for Cancer Research / Radiaon Biology, OUH Norwegian Radium Hospital, Oslo, Norway; 2 Research & Development, Nordic Nanovector ASA, Oslo, Norway, 3 Instute of Oncology Research, Università della Svizzera italiana, Bellinzona, Switzerland - RESULTS - SCREENING ASSAY PLATFORM Figure 5. Extended analysis of experiments shown in Figure 4. (A) Percentage of single cells with DNA content above 4n. (B) Median forward scaer (FSC) of live cells (a measure of cell size) normalized to pre-treatment mock control cells at 18 hour me point. (C) Percentage of dead cells as measured by Pacific blue staining. Bar diagrams show mean of two separate experiments, with individual data indicated with dots. Leſt panels: U-2932, right panels: RIVA Abstract 2574 A real-me proliferaon assay for screening for 177 Lu-lilotomab satetraxetan resistance- breaking compounds Combinatorial drug screen idenfies cell cycle kinase inhibitors as candidate drugs to overcome radioimmunotherapy resistance - The dual CDK1/2 and AURKA/B inhibitor JNJ-7706621 synergizes with 177 Lu-lilotomab satetraxetan Figure 3. (A, B) Plots showing the fracon of viable cells relave to untreated control at day 5, (A) U-2932 and (B) RIVA. X-axis: cells treated with drug alone; Y-axis: cells treated with drug combined with 177 Lu-lilotomab satetraxetan. Tested drug concentraons are indicated. Enriched hits are specified with name of common target and are in separate colors, the dual CDK1/2 and AuroraA/B inhibitor JNJ-7706621 is depicted in red. (C, D) Detailed dose-response analysis of JNJ-7706621 alone or in combinaon with indicated 177 Lu-lilotomab satetraxetan pre-treatment, (C) U-2932 and (D) RIVA (error bars: STDEV of triplicate samples). Right panels show Fa/CIs plots obtained by the Chou-Talaly method using the CompuSyn soſtware. The fracon affected (Fa) is the fracon of non-viable cells relave to untreated control. The Combinaon Index (CI) indicates whether the treatment with a specific combinaon is synergisc (<1) or antagonisc (>1). In blue are data from the same experiment as that shown in leſt panels, whereas red and yellow circles represent two independent experiments tesng alternave JNJ-7706621 concentraons (exp.2: 100, 266, 707, 1880, 5000 nM; exp.3: 200, 532, 1410, 3760 and 10 000 nM). Figure 4. A) Outline of experiment. U-2932 and RIVA cells were treated with indicated doses of 177 Lu-lilotomab satetraxetan. JNJ-7706621 was added to treated and untreated cells to a final concentraon of 500 nM. Samples were harvested for flow analysis before (pre) and 24, 72 and 144 hours aſter adding inhibitor. Cultures were replenished with medium (with or without inhibitor) aſter harvesng the 72 hour sample. Before fixaon, cells were stained with Pacific Blue in order to discriminate between live and dead cells. FxCycleFar Red was used to stain DNA. (B) DNA histograms showing cell cycle distribuon in U-2932 (leſt panels) and RIVA (right panels). Data shown are representave of two independent experiments. Increase in >4n populaon and cell size precedes cell death upon combined treatment with JNJ-7706621 and 177 Lu-lilotomab satetraxetan Combined treatment with 177 Lu-lilotomab satetraxetan and JNJ-7706621 induces growth delay and apoptoc death Figure 6: U-2932 cells were treated as in figure 4. (A) Bar diagram showing relave cell growth of cells between 24 and 72 hours aſter treatment (n=4; error bars represent SEM). (B) Bar diagram showing percentage of cells posive for cleaved PARP (n = 4; error bars represent SEM). (A,B) Stascal significance in differences between treatment groups were tested by One Way ANOVA: * = p<0.05, ** = p<0.01, *** = p<0.001. Figure 7: (A) Micrographs of control treated and combinaon therapy treated U-2932 and RIVA cells that were analyzed by flow cytometry (see Figures 4 and 5). Combinaon treatment promotes endoreduplicaon, mulnucleaon and aberrant mitoc figures (20x Phase2 NA0.8, DNA-Fx Farcycle Red). (B) Treatment with 177 Lu-lilotomab satetraxetan leads to DNA-damage induced G 2 –phase arrest and apoptoc cell death. Cells resistant to treatment adapt and recover from the arrest. Inhibion of CDK1 and AURKA/B interferes with bipolar- and mid- spindle assembly, causing chromosome congression and cytokinesis defects, respecvely. Combined treatment with JNJ-7706621 and 177 Lu-lilotomab satetraxetan reverses resistance likely by potenang the effect of persistent radiaon due to extended residence me in and fidelity of mitosis, the cell cycle phase in which repair capacity is low. Figure 1. CD37 is an internalizing transmembrane glycoprotein widely expressed on mature B-cells and B-cell malignancies. (A) The next generaon an-CD37 radioimmunoconjugate (RIC) 177 Lu-lilotomab satetraxetan (Betalun®), containing the beta-eming radionuclide luteum-177, is currently being tested as one-me injecon therapy in a clinical phase 2b trial for follicular lymphoma (FL) and a phase 1 trial for diffuse large B-cell lymphoma (DLBCL). (B) Five out of seven tested acvated B-cell like (ABC) DLBCL cell lines show high sensivity to 177 Lu-lilotomab satetraxetan in a dose-response screen (B; CyQuant proliferaon assay, day 6). (C) The double- hit/double-expressor ABC-DLBCL cell lines U-2932 and RIVA are resistant to CD37-targeng radioimmunotherapy, despite comparable expression of CD37 (RNAseq and mass spectrometry) and effecve cell surface binding of lilotomab to CD37 (flow cytometry). WT, wild-type; T, translocated; OE, overexpressed, Amp, amplified; RPKM, Reads per kilo base per million (Cascione et al. 2018; Tarantelli et al. 2018; Spriano et al. 2019). Conceptual model for synergisc acon of 177 Lu-lilotomab satetraxetan and the dual mitoc kinase inhibitor JNJ-7706621 in overcoming radioimmunotherapy resistance A B ABC-DLBCL cell line IC50 (CyQUANT) [µg/ml] CD37 mRNA [RPKM] Relave CD37 mRNA CD37 pro- tein MS [counts] Relave CD37 pr otein Relave CD37 surface HH1- DOTA binding TP53 MYC BCL2 Oci-Ly10 0,3 10,6 0,9 29.76 0.99 0,32 ± 0,04 inacve WT WT HBL1 0,6 11,3 1 30.83 01.02 n.d. inacve WT WT OCI-LY3 1,1 11,5 1 31.08 01.03 n.d. WT WT Amp TMD8 1,1 11 1 31.45 01.04 n.d. WT WT WT SUDHL2 2,5 9,6 0,8 29.34 0.97 n.d. WT WT WT RIVA 8,6 11,5 1 30.14 1 1 inacve T Amp U-2932 31,6 9,4 0,8 29.99 1.00 0,55 ± 0,02 inacve OE Amp A B C C B A JNJ-7706621 leads to extended G 2 /M-phase arrest and endoreduplicaon in 177 Lu- lilotomab satetraxetan treated cells • CD37-targeted radioimmunotherapy with 177 Lu-lilotomab satetraxetan (Betalun®) was effecve in five out of seven ABC-DLBCL cell lines. • Treatment resistance in two double-hit/double-expressor ABC-DLBCL cell lines is not due to loss of CD37 expression or 177 Lu-lilotomab satetraxetan (Betalun®) binding. • Drug library screening idenfied combinaons of 177 Lu-lilotomab satetraxetan (Betalun®) with mitoc kinase inhibitors or selected DNA targeng agents, such as topoisomerase or histone deacetylase inhibitors, to overcome treatment resistance. • Synergisc interacon of 177 Lu-lilotomab satetraxetan and the dual inhibitor of cyclin-dependent kinases CDK1/2 and Aurora kinases AURKA/B, JNJ-7706621, breaks treatment resistance by potenang inhibion of cell proliferaon and inducon of apoptosis. • Next steps is to study the combinaon of 177 Lu-lilotomab satetraxetan (Betalun®) and mitoc kinase inhibitors in aggressive DLBCL pre-clinical models. 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Transcript of The dual cell cycle kinase inhibitor JNJ-7706621 reverses ...
The dual cell cycle kinase inhibitor JNJ-7706621 reverses resistance to CD37 targeted radioimmunotherapy in ABC-subtype Diffuse Large B Cell Lymphoma Cell Lines
Disclosures Patzke: Nordic Nanovector ASA: Employment, Patent. Generalov, Melhus, Dahle: Nordic Nanovector ASA: Employment, Equity Ownership & Patents. Rødland, Syljuåsen: Institutional research funds from Nordic Nanovector ASA, Patent.Bertoni: Institutional research funds from Nordic Nanovector ASA.
Acknowledgment Sylvia Kolenic: Nordic Nanovector ASA: Poster design and production.
ReferencesRødland G, et al. (2019), Front. Oncol. 9:1301 | doi: 10.3389/fonc.2019.01301
Poster presented at 61st American Society of Hematology (ASH) Annual Meeting and Exposition, Orlando, FL, USA, December 7-10, 2019.
CONCLUSIONS
INTRODUCTION RESULTS - LIBRARY SCREEN, VALIDATION, FUNCTIONAL ANALYSIS
Figure 2. (A) Cells were treated for 18 hours with 177Lu-lilotomab satetraxetan ranging from 0-2 µg/mL (specific activity: 600 MBq/mg) or non-chelated lilotomab satetraxetan (1 µg/ml; HH-1-Dota), washed and plated in 96-well plates. Proliferation was monitored between 72 and 144 h after seeding in micro-well titer plates utilizing a luminescent MT Cell Viability assay (RealTime-Glo). (B) Relative RLU (177Lu-lilotomab satetraxetan to control) of data presented in (A). Error bars: Standard deviation (STDEV) (n=5 for U-2932 and RIVA, n=3 OCI-Ly10). Inhibition of cell proliferation on days 5 and 6 were significantly reduced compared to control (p<0.001, 1-way ANOVA) in U-2932 cells at doses ≥1 µg/mL, in RIVA at doses ≥0.25 µg/mL, and Oci-Ly10 at doses ≥0.1 µg/mL. (C) Experimental set-up for screening assay: U-2932 and RIVA cells were treated with 1 or 0.5 µg/mL 177Lu-lilotomab satetraxetan (600 MBq/mg) for 18 hours, washed, and seeded onto 384-well plates pre-printed with the 384-compound Cambridge Cancer compound library sourced from Selleckchem at final concentrations of 10 nM, 100 nM or 1 µM. Untreated control cells were seeded on parallel plates.
Gro Elise Rødland1, Katrine Melhus2, Roman Generalov2, Sania Gilani1, Francesco Bertoni3, Jostein Dahle2, Randi G. Syljuåsen1 and Sebastian Patzke1,2*
1Institute for Cancer Research / Radiation Biology, OUH Norwegian Radium Hospital, Oslo, Norway; 2Research & Development, Nordic Nanovector ASA, Oslo, Norway, 3Institute of Oncology Research, Università della Svizzera italiana, Bellinzona, Switzerland -
RESULTS - SCREENING ASSAY PLATFORM
Figure 5. Extended analysis of experiments shown in Figure 4. (A) Percentage of single cells with DNA content above 4n. (B) Median forward scatter (FSC) of live cells (a measure of cell size) normalized to pre-treatment mock control cells at 18 hour time point. (C) Percentage of dead cells as measured by Pacific blue staining. Bar diagrams show mean of two separate experiments, with individual data indicated with dots. Left panels: U-2932, right panels: RIVA
Abstract 2574
A real-time proliferation assay for screening for 177Lu-lilotomab satetraxetan resistance-breaking compounds
Combinatorial drug screen identifies cell cycle kinase inhibitors as candidate drugs to overcome radioimmunotherapy resistance - The dual CDK1/2 and AURKA/B inhibitor JNJ-7706621 synergizes with 177Lu-lilotomab satetraxetan
Figure 3. (A, B) Plots showing the fraction of viable cells relative to untreated control at day 5, (A) U-2932 and (B) RIVA. X-axis: cells treated with drug alone; Y-axis: cells treated with drug combined with 177Lu-lilotomab satetraxetan. Tested drug concentrations are indicated. Enriched hits are specified with name of common target and are in separate colors, the dual CDK1/2 and AuroraA/B inhibitor JNJ-7706621 is depicted in red. (C, D) Detailed dose-response analysis of JNJ-7706621 alone or in combination with indicated 177Lu-lilotomab satetraxetan pre-treatment, (C) U-2932 and (D) RIVA (error bars: STDEV of triplicate samples). Right panels show Fa/CIs plots obtained by the Chou-Talaly method using the CompuSyn software. The fraction affected (Fa) is the fraction of non-viable cells relative to untreated control. The Combination Index (CI) indicates whether the treatment with a specific combination is synergistic (<1) or antagonistic (>1). In blue are data from the same experiment as that shown in left panels, whereas red and yellow circles represent two independent experiments testing alternative JNJ-7706621 concentrations (exp.2: 100, 266, 707, 1880, 5000 nM; exp.3: 200, 532, 1410, 3760 and 10 000 nM).
Figure 4. A) Outline of experiment. U-2932 and RIVA cells were treated with indicated doses of 177Lu-lilotomab satetraxetan. JNJ-7706621 was added to treated and untreated cells to a final concentration of 500 nM. Samples were harvested for flow analysis before (pre) and 24, 72 and 144 hours after adding inhibitor. Cultures were replenished with medium (with or without inhibitor) after harvesting the 72 hour sample. Before fixation, cells were stained with Pacific Blue in order to discriminate between live and dead cells. FxCycleFar Red was used to stain DNA. (B) DNA histograms showing cell cycle distribution in U-2932 (left panels) and RIVA (right panels). Data shown are representative of two independent experiments.
Increase in >4n population and cell size precedes cell death upon combined treatment with JNJ-7706621 and 177Lu-lilotomab satetraxetan
Combined treatment with 177Lu-lilotomab satetraxetan and JNJ-7706621 induces growth delay and apoptotic death
Figure 6: U-2932 cells were treated as in figure 4. (A) Bar diagram showing relative cell growth of cells between 24 and 72 hours after treatment (n=4; error bars represent SEM). (B) Bar diagram showing percentage of cells positive for cleaved PARP (n = 4; error bars represent SEM). (A,B) Statistical significance in differences between treatment groups were tested by One Way ANOVA: * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 7: (A) Micrographs of control treated and combination therapy treated U-2932 and RIVA cells that were analyzed by flow cytometry (see Figures 4 and 5). Combination treatment promotes endoreduplication, multinucleation and aberrant mitotic figures (20x Phase2 NA0.8, DNA-Fx Farcycle Red). (B) Treatment with 177Lu-lilotomab satetraxetan leads to DNA-damage induced G2–phase arrest and apoptotic cell death. Cells resistant to treatment adapt and recover from the arrest. Inhibition of CDK1 and AURKA/B interferes with bipolar- and mid-spindle assembly, causing chromosome congression and cytokinesis defects, respectively. Combined treatment with JNJ-7706621 and 177Lu-lilotomab satetraxetan reverses resistance likely by potentiating the effect of persistent radiation due to extended residence time in and fidelity of mitosis, the cell cycle phase in which repair capacity is low.
Figure 1. CD37 is an internalizing transmembrane glycoprotein widely expressed on mature B-cells and B-cell malignancies. (A) The next generation anti-CD37 radioimmunoconjugate (RIC) 177Lu-lilotomab satetraxetan (Betalutin®), containing the beta-emitting radionuclide lutetium-177, is currently being tested as one-time injection therapy in a clinical phase 2b trial for follicular lymphoma (FL) and a phase 1 trial for diffuse large B-cell lymphoma (DLBCL). (B) Five out of seven tested activated B-cell like (ABC) DLBCL cell lines show high sensitivity to 177Lu-lilotomab satetraxetan in a dose-response screen (B; CyQuant proliferation assay, day 6). (C) The double-hit/double-expressor ABC-DLBCL cell lines U-2932 and RIVA are resistant to CD37-targeting radioimmunotherapy, despite comparable expression of CD37 (RNAseq and mass spectrometry) and effective cell surface binding of lilotomab to CD37 (flow cytometry). WT, wild-type; T, translocated; OE, overexpressed, Amp, amplified; RPKM, Reads per kilo base per million (Cascione et al. 2018; Tarantelli et al. 2018; Spriano et al. 2019).
Conceptual model for synergistic action of 177Lu-lilotomab satetraxetan and the dual mitotic kinase inhibitor JNJ-7706621 in overcoming radioimmunotherapy resistance
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Oci-Ly10 0,3 10,6 0,9 29.76 0.99 0,32 ± 0,04 inactive WT WT HBL1 0,6 11,3 1 30.83 01.02 n.d. inactive WT WT OCI-LY3 1,1 11,5 1 31.08 01.03 n.d. WT WT Amp TMD8 1,1 11 1 31.45 01.04 n.d. WT WT WT SUDHL2 2,5 9,6 0,8 29.34 0.97 n.d. WT WT WT RIVA 8,6 11,5 1 30.14 1 1 inactive T Amp U-2932 31,6 9,4 0,8 29.99 1.00 0,55 ± 0,02 inactive OE Amp
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JNJ-7706621 leads to extended G2/M-phase arrest and endoreduplication in 177Lu-lilotomab satetraxetan treated cells
• CD37-targeted radioimmunotherapy with 177Lu-lilotomab satetraxetan (Betalutin®) was effective in five out of seven ABC-DLBCL cell lines.
• Treatment resistance in two double-hit/double-expressor ABC-DLBCL cell lines is not due to loss of CD37 expression or 177Lu-lilotomab satetraxetan (Betalutin®) binding.
• Drug library screening identified combinations of 177Lu-lilotomab satetraxetan (Betalutin®) with mitotic kinase inhibitors or selected DNA targeting agents, such as topoisomerase or histone deacetylase inhibitors, to overcome treatment resistance.
• Synergistic interaction of 177Lu-lilotomab satetraxetan and the dual inhibitor of cyclin-dependent kinases CDK1/2 and Aurora kinases AURKA/B, JNJ-7706621, breaks treatment resistance by potentiating inhibition of cell proliferation and induction of apoptosis.
• Next steps is to study the combination of 177Lu-lilotomab satetraxetan (Betalutin®) and mitotic kinase inhibitors in aggressive DLBCL pre-clinical models.
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