CDK2^AS is catalytically active and can be potently inhibited by the selective inhibitor 1NM-PP1. (A) Model demonstrates the engineered CDK2^AS with the new binding pocket generated by the F80G mutation and the corresponding chemical inhibitor, 1NM-PP1, to inhibit the kinase selectively. (B) Expression of HA-tagged CDK2^WT or CDK2^AS protein in HEK 293T cells. Western blot shows expression of tagged (arrow) and endogenous CDK2 proteins. (C) CDK2^WT was immunoprecipitated from HEK 293T cell lysates with anti-HA antibodies, and kinase activity toward the histone H1 substrate was assessed in the presence of increasing amounts of 1NM-PP1. (D) CDK2^AS was immunoprecipitated from HEK 293T cell lysates with anti-HA antibodies, and kinase activity toward the histone H1 substrate was assessed in the presence of increasing amounts of 1NM-PP1.

Small-molecule inhibition, rather than siRNA-mediated knockdown, of CDK2 in MEFs decreases cellular proliferation. (A) Growth curve of MEFs carrying either the WT or AS allele of the CDK2 gene. The cells were treated at day 0 with 5 μM 1NM-PP1 or DMSO, and the cell number was counted at each time point. Growth curves represent the average of five independent experiments. The error bars represent mean ± SEM. Proliferation of the CKD2^AS cells in the presence or absence of 1NM-PP1 on both days 3 and 4 (P < 0.00001; two-tailed t test) is shown. (B) Growth curve of MEF-CDK2^AS cells treated with either nontargeting control siRNA or CDK2-specific siRNA. The cells were transfected with designated siRNAs, and the cell number was counted at each time point. Growth curves represent the average of three independent experiments. The error bars represent mean ± SEM. The statistical significance applies to both days 4 and 6 (P < 0.00001; two-tailed t test). (C) Western blot analysis of CDK expression in MEFs treated with either control/CDK2 siRNA or with DMSO/1NM-PP1. The cells were treated with a given reagent on day 0 and collected at each time point. (D) Cell cycle profiles determined for MEFs containing either the WT or AS allele of CDK2 by FACS analysis with pulse-BrdU incorporation for 45 min, after treatment with DMSO or 5 μM PP1 for 72 h. Cells were dual-stained with an FITC-conjugated anti-BrdU antibody (y axis) and 7-AAD to look at DNA content (x axis). Gated populations represent dead (sub2n), G0/G1, S, and G2/M cell populations. The depicted results are representative of four different experiments. (E) Quantification of cell cycle profiles. The error bars represent mean ± SEM. *Statistical significance as determined by a paired two-tailed t test. (F) Rb Thr821 phosphorylation following 36 h of 1NM-PP1 treatment of MEF CDK2^WT or MEF CDK2^AS cells. The cells were first serum-starved for 90 h in the presence of 0.1% serum and subsequently released into and grown for an additional 36 h in the medium containing 10% (vol/vol) serum and DMSO or 1NM-PP1.

Diminished anchorage-independent growth and proliferation of HCT116 colon cancer cells following acute CDK2 inhibition. (A) Growth curve of HCT116 cells with either a WT or AS allele of CDK2 gene. The cells were treated on day 0 with 5 μM 1NM-PP1 or DMSO, and the cell number was counted at each time point. Growth curves represent the average of three independent experiments. The error bars represent mean ± SEM. Proliferation of the CKD2^AS cells in the presence or absence of 1NM-PP1 (P = 0.008; two-tailed t test) is shown. (B) Cell cycle profiles for HCT116 cells containing either the WT or AS allele of CDK2, determined by FACS analysis with pulse-BrdU incorporation for 45 min after treatment with DMSO or 5 μM PP1 for 72 h. Cells were dual-stained with an FITC-conjugated anti-BrdU antibody (y axis) and 7-AAD to examine DNA content (x axis). Gated populations represent dead (sub2n) and G0/G1, S, and G2/M cell populations. The depicted results are representative of four different experiments. (C) Quantification of cell cycle profiles. The experiment was repeated four times. *Statistical significance as determined by a paired two-tailed t test. The error bars represent mean ± SEM. (D) Anchorage-independent growth of HCT116 cells in soft agar. HCT116 cells were pretreated with DMSO or 5 μM 1NM-PP1 for 72 h. Following pretreatment, cells were plated in 0.32% soft agar containing DMSO or 5 μM 1NM-PP1. (Scale bar = 5 mm.) (E) Quantification of colony growth in soft agar after 21 d. The error bars represent mean ± SEM. (F) Tumorsphere formation of HCT116 cells. HCT116 cells were seeded into ultra-low-attachment plates in media containing DMSO or 5 μM 1NM-PP1. (Magnification: 40×.) (G) Quantification of tumorsphere formation after 14 d. The amount of live cells per well was quantified using a luminescence-based assay to determine levels of ATP. 1NM-PP1–treated wells were normalized to DMSO-treated wells for each cell line independently. The experiment was repeated five times. *Statistical significance as determined by a two-tailed t test. The error bars represent mean ± SEM. (H) Cell cycle profiles for tumorspheres formed from HCT116 cells after 10 d of growth in DMSO or 5 μM 1NM-PP1, determined by FACS analysis with pulse-BrdU incorporation for 24 h. Cells were dual-stained with an FITC-conjugated anti-BrdU antibody (y axis) and 7-AAD to examine DNA content (x axis). Gated populations represent G0/G1, S, and G2/M cell populations. The depicted results are representative of three different experiments. (I) Quantification of cell cycle profiles. The experiment was repeated three times. *Statistical significance as determined by a paired two-tailed t test. The error bars represent mean ± SEM.

CDK2 inhibition attenuates nonadherent colony formation but does not alter the growth of transformed MEFs in adherent conditions. (A) Western blot showing retroviral overexpression of the oncogenes MYC, RAS, and ABL in MEF-CDK2^WT or MEF-CDK2^AS cells. (B) Growth curve of the oncogene-transformed MEFs carrying either a WT or AS allele of CDK2 gene. The cells were treated at day 0 with 5 μM 1NM-PP1 or DMSO, and the cell number was counted at each time point. Growth curves represent the average of four independent experiments. The error bars represent mean ± SEM. Proliferation of the CKD2^AS cells in the presence or absence of 1NM-PP1 on days 3, 4, and 5 (P > 0.05) is shown. (C) Quantification of anchorage-independent growth in soft agar. The oncogene-transformed MEFs were grown in soft agar for 21 d in the presence of DMSO or 5 μM 1NM-PP1 and assessed for their sensitivity to CDK2 inhibition. The experiments were repeated at least three times in triplicate. The error bars represent mean ± SEM (P < 0.0001, two-tailed t test).

Nonadherent growth of oncogene-transformed MEFs results in the altered expression of cell cycle proteins and is associated with increased sensitivity to CDK2 inhibition. (A) 1NM-PP1 treatment of the oncogene-transformed MEFs alters nonadherent growth. The oncogene-transformed MEFs were subjected to the tumorsphere assay, and cell growth was assessed by quantitation of ATP abundance after 10–14 d of growth. 1NM-PP1 wells were normalized to DMSO-treated wells for each individual cell line. *Statistical significance as determined by a two-tailed t test. The error bars represent mean ± SEM. The experiment was repeated three times with Abl-overexpressing cells and five times each with MYC- and RAS-overexpressing cells. (B) Western blot analysis of cyclins and CDK1 and CDK2 expression in the oncogene-transformed MEFs grown in either adherent (A) or nonadherent (NA) conditions. (C) Western blot analysis of tumor suppressors p27, p21, and Rb in the oncogene-transformed MEFs grown in either adherent or nonadherent conditions. (D) Western blot analysis of cyclin expression following siRNA-mediated knockdown of individual cyclins in oncogene-transformed MEFs. The transformed cells (M, MYC; R, RAS; A, ABL) were first treated with the siRNAs against indicated cyclins alone for 48 h and then further cultured for an additional 48 h in the presence or absence of 1NM-PP1. (E) (Upper) Effect of cyclin knockdown on the growth of the oncogene-transformed MEFs. The cells were treated with the siRNAs against the indicated cyclins alone for 96 h, and cell counts were performed. The extent of growth inhibition caused by knockdown of each individual cyclin was normalized to the nontargeted control siRNA-treated cells. (Lower) Effects of individual cyclin knockdown on the sensitivity of the oncogene-transformed cells treated with 1NM-PP1. The transformed cells were first treated with siRNAs against individual cyclins or nontargeting (N.T) control siRNA for 48 h and then further cultured for additional 48 h in the presence of DMSO or 1NM-PP1 before cell counting was performed. The proliferation of 1NM-PP1–treated cells was normalized to that of DMSO-treated control cells. These experiments were independently repeated three times in triplicate. The error bars represent mean ± SEM. *Statistical significance as determined by a two-tailed t test.

Effect of small-molecule inhibition of CDK2 on cellular proliferation of MEF cells and anchorage-independent growth of human colon cancer cells. (A) (Upper) Model depicting the engineered CDK2^AS with the new binding pocket generated by the F80G mutation and the corresponding chemical inhibitor, 1NM-PP1, that selectively inhibits the kinase. The growth curve of MEFs carrying either the WT or analog-sensitive (AS) type of CDK2 treated with the specific inhibitor 1NM-PP1 or diluent DMSO (Lower Left) was compared with the growth curve of MEFs treated with either nontargeting control siRNA or CDK2-specific siRNA that is able to knock down CDK2 expression (Lower Right). (B) (Left) Tumorsphere formation of HCT116 cells seeded into ultra-low-attachment plates in media containing DMSO or 5 μM 1NM-PP1. (Magnification: 20×.) (Right) Luminescence-based assay was used to determine levels of ATP energy use as a measure of the relative number of live cells per plate.