Transient CDK4/6 inhibition protects hematopoietic stem cells from chemotherapy-induced exhaustion

Abstract

Conventional cytotoxic chemotherapy is highly effective in certain cancers but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise ("exhaustion"), which limits the use of chemotherapy and success of cancer therapy. We show that the coadministration of G1T28 (trilaciclib), which is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil treatment model. Consistent with a cell-intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity, and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer.

Fig. 1. G1T28 induces transient, reversible G₁ cell cycle arrest in both murine and human BM HSPCs

(A) Rate of murine BM HSC proliferation 12 hours after a single IP injection of the indicated doses of G1T28 (n = 5 mice/dose analyzed in 5 independent experiments, same for B and C). (B) Median effective dose (ED₅₀) of G1T28 to inhibit the in vivo proliferation of the indicated murine hematopoietic cell types with identification scheme shown in fig. S1 (LSK = Lin⁻Sca-1⁺c-Kit⁺ cells, MP = myeloid progenitors, E = erythroid, M = myeloid, B = B lymphocytes, T = T lymphocytes). (C) Relative proliferation rate of each murine hematopoietic cell type at maximum CDK4/6 inhibition, compared to untreated mice. (D) Rate of murine BM HSC proliferation at various time points after a single IP injection of 50 or 100 mg/kg of G1T28 (n = 3-6 mice/dose/time point). (E) Mean plasma concentration curve of G1T28 after single dose IV administration in healthy human volunteers. (F, G) Frequency of total human BM cells (F) and HSC-enriched CD45^dimCD34⁺CD38⁻ BM cells (G) in S/G₂/M phase of the cell cycle either before or at 24 and 32 hours after single dose of G1T28 treatment. Data were from Cohort 7 (BED) after single 192 mg/m² dose of G1T28. Error bars represent SD in A-D and SEM in E-G. The statistical significance of differences between vehicle and G1T28 treated groups in A and D and between indicated groups in B, C, and F was assessed using unpaired, two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 2. Transient G₁ arrest by G1T28 pretreatment accelerates hematologic recovery after single dose 5FU treatment

(A, B) Complete blood cell counts (A) and BM cellularity (B) at various time points after a single dose of 5FU with or without G1T28 pretreatment (n = 5-18 mice/treatment/time point). (C) Number of colony-forming cells (CFC) per femur 1 to 4 days after a single treatment with 5FU ± G1T28 (n = 4-11 mice/treatment/time point). Data represent mean ± SEM in A and mean ± SD in B and C. The statistical significance of differences between indicated groups was assessed using unpaired, two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3. G1T28 pretreatment protects mice from repeated doses of 5FU challenge

(A–D) Survival curves of mice that were treated with 5FU plus vehicle, G1T28, or pegfilgrastim at 7 (A) or 10 (B-D) day intervals. G1T28 was given 12 hours before 5FU in A and B, and either 30 minutes before, 24 hours before, or both in C. Pegfilgrastim was given 24 hours after 5FU in (D). Numbers in parenthesis indicate median survival (d = day), and animal number (n) in each treatment group. Significance for the pairwise comparison to 5FU + vehicle was calculated using the log-rank test in A, B, and D and Gehan-Breslow-Wilcoxon test in C (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant).

Fig. 4. G1T28-induced PQ protects HSCs from proliferative exhaustion

(A) Treatment and BM transplantation schematic. WBM, whole bone marrow cells (B-E) The percentage of donor-derived cells (CD45.1⁺) in total PB leukocytes (B), or myeloid cell (Mac1⁺, C), B lymphocyte (B220⁺, D), and T lymphocyte (CD3⁺, E) fractions 1 to 4 months after primary BM transplantation. 3-8 donor mice/treatment, 5 recipients/donor. Statistical significance of differences between treatment groups at 16 weeks after transplantation was assessed using one-way ANOVA with correction for multiple comparisons. (*, P < 0.05 compared to control; †, P < 0.05 compared to 5FU + vehicle, ‡, P < 0.05 compared to 5FU + G1T28). (F) The percentages of donor-derived cells in total BM cells, HSCs, and major BM cell lineages 32 weeks after BM transplantation. 5 donor mice/treatment, 5 recipients/donor. Statistical significance was assessed by two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Average donor cell reconstitution in B-F was calculated by first averaging the value of the 5 recipients from each donor, followed by calculating the mean of all donors. Error bars represent SEM.

Fig. 5. Transient CDK4/6 inhibition protects long-term HSC function against serial 5FU treatment by reducing overall proliferative burden

(A) The percentage of donor-derived cells (CD45.1⁺) in total PB leukocytes or myeloid cell (Mac1⁺), B lymphocyte (B220⁺), and T lymphocyte (CD3⁺) fractions 1 to 4 months after secondary BM transplantation. Data represent the mean ± SEM from 20-22 mice per group. (B, C) Cumulative HSC proliferation after a single dose of 5FU ± G1T28 treatment as determined by H2B-GFP label retention 4 weeks after 5FU administration. Representative H2B-GFP label dilution histograms (and curve fitting) are shown in (B), with quantification in (C). Proliferation index (PI) indicates the average number of daughter cells generated from a single, originally labeled HSC by the time of analysis, such that Log₂ (PI) equals the average number of HSC divisions. Data represent mean ± SD from 3-6 mice in 3 experiments. Statistical significance was assessed by unpaired, two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). div, division.