Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models

Abstract

Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

Figure 1

Anti-myeloma activity of PHA-767491. KMS-18 (A) and MM1S (B) were treated with different doses of PHA-767491 for 24 h and apoptosis was analysed by flow cytometry using AV staining. Data are presented as mean ± standard deviation of two independent repeats; (C,D) Purified mononuclear cells from two healthy donors were treated with different doses of PHA-767491 for 24 h and apoptosis was analysed by flow cytometry using AV staining. The M in the x axes defines Molarity.

Figure 2

PHA-767491 inhibits both CDC7 and CDK9 in KMS-18 cells. KMS-18 myeloma cells were incubated with 5 μM PHA-767491. Protein extracts were prepared at the indicated time points and analyzed by immunoblotting using the indicated antibodies.

Figure 3

Analysis of pro- and anti-apoptotic proteins in response to PHA-767491. KMS-18 and MM1S myeloma cells were incubated with 5 μM PHA-767491 for the indicated time. Protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies (A). In parallel samples, apoptosis (B) and DNA synthesis (C) were analysed by flow cytometry using AV staining and EdU incorporation assay respectively. Numbers in the gated regions represent the percentage of cells positive for either AV (B) or EdU (C) staining.

Figure 4

PHA-767491 does not affect BIM levels and causes caspase-independent MCL-1 downregulation. (A) KMS-18 and MM1S myeloma cells were incubated with 5 μM PHA-767491 for the indicated time. Protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies; (B) KMS-18 and MM1S cells were incubated with 30 μM QVD-OPH for 30 min and then treated with 5 μM PHA767491for 6 h (KMS-18) or 9 h (MM1S). After incubation, protein extracts were prepared and analyzed by immunoblotting using the indicated antibodies. F-L and C in the PARP lane indicate full length and cleaved respectively. Percentage of AV⁺ cells is indicated at the bottom.

Figure 5

PHA-767491 induces apoptosis in KMS-18 and MM1S in co-culture with HS5 cells. KMS-18 (A) and MM1S (B) cells were cultured with (open square) or without (black circle) HS5-H2B-GFP cells for 2 h and then treated with different doses of PHA-767491 for a further 24 h. Apoptosis was assessed by AV staining. In parallel samples, KMS-18 (C) and MM1S (D) cultured with (gray bars) or without (white bars) HS5-H2B-GFP cells for 2 h, were treated with 5 nM bortezomib and apoptosis was analysed 24 h after treatment using AV staining. Data are presented as mean ± standard deviation of two independent repeats. The M in the x axes defines Molarity.

Figure 6

Combination Analysis of PHA-767491 using Chou-Talalay Median-Effect Equation. MM1S cells were treated with PHA-767491 alone and in combination with Melphalan (Range 2 to 32 µM, A), Doxorubicin (Range 31 to 500 nM, B) or Bortezomib (Range 1.3 to 20 nM, C) in a non-constant ratio in a simultaneous fashion. Cell viability was examined by CellTiter Glo 48 h after drug treatment. Combination indices (CI) were calculated and the Log CI (circles) plotted in relation to the fraction of cells affected (Fa) at any given experimental point.

Figure S1

Sequential combination analysis of PHA-767491 with drugs constituting standard of care. MM1S cells were treated with PHA-767491 for 3 h followed by treatment with Melphalan (Range 2 to 32 µM, A) Doxorubicin (Range 31 to 500 nM, B) Bortezomib (Range 1.3 to 20 nM, C) in a non-constant ratio. Cell viability was examined by CellTiter Glo 48 h after drug treatment. Combination indices (CI) were calculated and the Log CI (circles) plotted in relation to the fraction of cells affected (Fa) at any given experimental point.

Figure S2

Sequential combination analysis of PHA-767491 with drugs constituting standard of care. MM1S cells were treated with Melphalan (Range 2 to 32 µM, A) Doxorubicin (Range 31 to 500 nM, B) Bortezomib (Range 1.3 to 20 nM, C) for 3 h followed by treatment with PHA-767491 in a non-constant ratio. Cell viability was examined by CellTiter Glo 48 h after drug treatment. Combination indices (CI) were calculated and the Log CI (circles) plotted in relation to the fraction of cells affected (Fa) at any given experimental point.