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. 2020 Jun;34(6):1646-1657.
doi: 10.1038/s41375-019-0652-0. Epub 2019 Dec 11.

A novel CDK9 inhibitor increases the efficacy of venetoclax (ABT-199) in multiple models of hematologic malignancies

Darren C Phillips  1 , Sha Jin  2 , Gareth P Gregory  3   4 , Qi Zhang  5 , John Xue  2 , Xiaoxian Zhao  6 , Jun Chen  7 , Yunsong Tong  2 , Haichao Zhang  2 , Morey Smith  2 , Stephen K Tahir  2 , Rick F Clark  2 , Thomas D Penning  2 , Jennifer R Devlin  3   8 , Jake Shortt  4 , Eric D Hsi  6 , Daniel H Albert  2 , Marina Konopleva  5 , Ricky W Johnstone  3   8 , Joel D Leverson  9 , Andrew J Souers  2
Affiliations

A novel CDK9 inhibitor increases the efficacy of venetoclax (ABT-199) in multiple models of hematologic malignancies

Darren C Phillips et al. Leukemia. 2020 Jun.

Abstract

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.

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Conflict of interest statement

The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of this publication. DCP, SJ, JX, JC, YT, HZ, MS, SKT, RFC, TDP, DHA, JDL, and AJS are employees of AbbVie Inc. and are stock holders. GPG: Advisory board Takeda; speaker’s honoraria from Roche; research support from Amgen, Merck and AbbVie Inc.JS: Advisory boards, Celgene, Novartis, and BMS; speaker’s honoraria from Novartis, BMS, Roche, Amgen, and Takeda; research support from Amgen and Astex. RWJ: Research support from Abbvie Inc., Astra-Zeneca, BMS, Roche, and MecRX. MK: Advisory boards, F. Hoffman La-Roche and AbbVie; honoraria from Amgen, AbbVie, and Genentech; research funding from AbbVie, Genentech, Eli Lilly, Cellectis, Calithera, Stemline, Threshold, and Flexus; Reata Pharmaceuticals stock holder. EDH: Advisory board honoraria from Seattle Genetics and Celgene; research support from AbbVie Inc. and Eli Lilly. QZ, XZ, and JRD have nothing to disclose.

Figures

Fig. 1
Fig. 1
Chemical structures of A-1467729 and A-1592668.
Fig. 2
Fig. 2
A-1592668 induces on-target cell death in MCL-1 dependent tumor cell lines. H929 or MV4-11 cells were treated in vitro with A-1592668 at the indicated concentrations and times, and the impact on total and phosphorylated RNA polymerase-II (t-RNA pol-II and p-RNA pol-II respectively), MCL-1, Caspase-3, and PARP determined by western blot utilizing tubulin or β-actin as a loading control (a, b). Alternatively, MV4-11 or H929 cells were treated with A-1592668 for 16 h in the presence or absence of the pan-caspase inhibitor Q-VD-Oph (100 µM) and the percentage Annexin-V-positive cells determined by flow cytometry (c, d). Mice bearing MV4-11 tumors were treated with A-1592668 (5 mg/kg PO, twice a week for 3 weeks or the vehicle (n = 6 mice per group), and the effect on tumor growth determined as described in the materials and methods. The impact on weight was also assessed (e).
Fig. 3
Fig. 3
A-1592668 is active in an Eµ-myc mouse model of lymphoma. Eµ-myc mouse lymphoma cells (#4242) were treated with A-1592668 in vitro for 24 h and the percentage Annexin-V/PI-positive cells determined by flow cytometry. Data are presented as the mean ± s.e.m. of three independent experiments (a). Alternatively, Eµ-myc mouse lymphoma cells (#4242) were treated with A-1592668 (100 nM) in vitro for 3 h and the effects on Mcl-1 and p-RNA pol-II (Ser2/5) determined by western blot utilizing t-RNA pol-II and HSP90 as loading controls (b). C57Bl/6 mice bearing #4242 tumors were treated with A-1592668 (3 mg/kg PO, three times per week for two weeks, n = 10) or vehicle (n = 10) and the impact on animal survival (c), body weight (d), lymphocytes, hemoglobin, neutrophils, and platelets determined (e). Statistical difference was determined using an unpaired t-test, where p < 0.001 (***) was considered significant. In a separate cohort, untreated mice were allowed to establish bulky lymph nodal disease for 12 days before treatment with vehicle or a single dose of A-1592668 (3 mg/kg PO) for 3 h before tumors were excised and the impact on p-RNA pol-II (Ser2/5) and Mcl-1 determined by western blot using HSP90 as a loading control (f). C57Bl/6 mice bearing #4242 tumors were treated as in c in the presence or absence of venetoclax (50 mg/kg PO, every day for two weeks). Median survival rates for each treatment group are presented in Supplementary Table 2a (g). Eµ-myc mouse lymphoma cells overexpressing BCL-2 (#4242tmBcl-2) were treated with A-1592668 in vitro for 24 h and the percentage Annexin-V/PI positive cells determined by flow cytometry. Data are presented as the mean ± s.e.m of three independent experiments (h).
Fig. 4
Fig. 4
A-1592668 sensitizes NHL cells to venetoclax. SU-DHL-4 cells (a; n = 1), OCI-LY1 199R, or SC-1 199R (b) were treated with A-1592668 in combination with venetoclax in vitro for the indicated times and the impact on cell viability determined by CellTiterGlo. Data in b are presented as the mean ± s.e.m of three independent experiments. A-1592668 given orally at 1.5 mg/kg three times a week for 3 weeks either alone or in combination with venetoclax given orally at 50 mg/kg once a day for 3 weeks in the SU-DHL-4 flank xenograft model (n = 9 mice per treatment group). Data are presented as the mean tumor volume ± s.e.m. The impact on mouse weight is also shown (c). Primary DLBCL (blue) and FL (orange) patient samples were treated ex vivo with venetoclax in the presence or absence of A-1467729 (10 nM) for 6 h and the impact on total and phosphorylated RNA polymerase-II (t-RNA pol-II and p-RNA pol-II respectively), MCL-1, BCL-2, PARP cleavage, and β-actin determined by western blot (d), or the effect on cell viability determined using 7-AAD uptake (e).
Fig. 5
Fig. 5
A-1592668 sensitizes AML cells to venetoclax. A panel of AML cell lines was treated with A-1592668 (a; n = 3 independent experiments for each cell line), A-1210477 (b; n = 3 independent experiments for each cell line) or idarubicin (d; n = 2 independent experiments for each cell line) in combination with venetoclax for 6 h in vitro and the effect on cell viability determined by CellTiterGlo. Dose–response curves in the SKM-1 AML tumor cell line is shown, and the synergy assessed using the Bliss independence model, where Bliss Sums represent the cumulative Bliss scores across the combination matrix. Data are presented as the mean ± s.e.m. The impact of A-1592668 treatment in the presence or absence of venetoclax on total and phosphorylated RNA polymerase-II (t-RNA pol-II and p-RNA pol-II respectively), MCL-1, Caspase-3, and PARP determined by western blot utilizing GAPDH as a loading control (c). The SKM-1 flank xenograft model was given A-1592668 orally at 2.5 mg/kg twice a week for 3 weeks either alone or in combination with venetoclax orally at 50 mg/kg once a day for 3 weeks. Data are presented as the mean tumor volume ± s.e.m. of eight mice per treatment arm. The impact on mouse weight is also shown. ** 2× found dead and excluded from analysis on day 3, * 1× moribund and excluded from analysis on day 6 (e).
Fig. 6
Fig. 6
A-1592668 is active in PDX models of AML with added survival benefit achieved in combination with venetoclax. NSG mice bearing AML-PDX derived cells (PDX 4095636 and PDX 3899936) were treated A-1592668 orally at 4 mg/kg twice a week for 3 weeks either alone or in combination with venetoclax orally at 50 mg/kg once a day for 3 weeks and the impact on survival (n = 8 per treatment group; a, c) and engraftment in the periphery one week after treatment (day 7; b, d) determined. Levels of engraftment at the initiation of treatment are shown in Supplementary Fig. 6. A-1592668 provided a significant survival benefit over the vehicle treated mice in both PDX models (PDX 4095636, p < 0.0001; PDX 3899936, p = 0.0214) whereas venetoclax provided a significant survival benefit in PDX 3899936 (p = 0.0283) but not in PDX 4095636 (p = 0.3148). The combination of A-1592668 and venetoclax provided a significant survival benefit in PDX 4095636 over mice treated with vehicle or either agent alone (p < 0.0001). The combination of A-1592668 and venetoclax provided a significant survival benefit in PDX 3899936 over mice treated with vehicle (p < 0.0001), venetoclax alone (p = 0.0065), or A-1592668 alone (p = 0.0307). Median survival rates for each treatment group are shown in Supplementary Table 2b, c.
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