Myelofibrosis (myelofibrosis) is a deadly blood neoplasia with the worst prognosis among myeloproliferative neoplasms (MPN). The JAK2 inhibitors ruxolitinib and fedratinib have been approved for treatment of myelofibrosis, but they do not offer significant improvement of bone marrow fibrosis. CDK6 expression is significantly elevated in MPN/myelofibrosis hematopoietic progenitor cells. In this study, we investigated the efficacy of CDK4/6 inhibitor palbociclib alone or in combination with ruxolitinib in Jak2V617F and MPLW515L murine models of myelofibrosis. Treatment with palbociclib alone significantly reduced leukocytosis and splenomegaly and inhibited bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of myelofibrosis. Combined treatment of palbociclib and ruxolitinib resulted in normalization of peripheral blood leukocyte counts, marked reduction of spleen size, and abrogation of bone marrow fibrosis in murine models of myelofibrosis. Palbociclib treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, treatment with palbociclib or depletion of CDK6 inhibited Aurora kinase, NF-κB, and TGFβ signaling pathways in Jak2V617F mutant hematopoietic cells and attenuated expression of fibrotic markers in the bone marrow. Overall, these data suggest that palbociclib in combination with ruxolitinib may have therapeutic potential for treatment of myelofibrosis and support the clinical investigation of this drug combination in patients with myelofibrosis.

Significance:

These findings demonstrate that CDK6 inhibitor palbociclib in combination with ruxolitinib ameliorates myelofibrosis, suggesting this drug combination could be an effective therapeutic strategy against this devastating blood disorder.

Myelofibrosis (myelofibrosis) is the most aggressive form of myeloproliferative neoplasms (MPN), characterized by bone marrow (BM) fibrosis, leukocytosis, and extramedullary hematopoiesis. Patients with myelofibrosis with intermediate and high-risk disease have a median survival of 16 to 35 months (1). The oncogenic JAK2V617F mutation was detected in 50% to 60% patients with myelofibrosis (2, 3). Additional mutations in the thrombopoietin receptor (MPL) and calreticulin (CALR) were observed in myelofibrosis (4–7). JAK2, MPL, and CALR mutations are considered as MPN driver mutations and they lead to hyperactivation of the JAK/STAT signaling (3). The JAK1/2 inhibitor ruxolitinib is approved for treatment of myelofibrosis (8, 9). Although ruxolitinib treatment provides symptomatic relief, it does not cure or significantly improve BM fibrosis in patients with myelofibrosis (10, 11) Moreover, initial responses to ruxolitinib therapy are lost after prolonged treatment in many cases (12). Fedratinib, a new JAK2-selective inhibitor, has recently been approved for treatment of myelofibrosis (13, 14). Fedratinib therapy also reduces splenomegaly and constitutional symptoms but does not significantly improve BM fibrosis. Thus, there is a critical need to develop novel therapies for myelofibrosis that can effectively treat BM fibrosis.

Cyclin-dependent kinase 6 (CDK6) and its close homolog CDK4 are known to regulate G1 to S-phase cell-cycle progression via activation of the CDK4/6–cyclin D complex and subsequent phosphorylation of the retinoblastoma (Rb) protein, which drives E2F-dependent transcription (15, 16). Mice deficient in Cdk6 are viable and they exhibit only minor defects in erythrocyte and thymocyte development (17, 18). Expression of CDK6 is upregulated in various hematologic malignancies (16). CDK6 is required for leukemogenesis mediated by MLL- and NUP98-fusion oncoproteins (19, 20). CDK6 can also act as a transcriptional regulator (16). Both kinase-independent and kinase-dependent functions for CDK6 have been suggested (21). CDK6 has been shown to interact with NF-kB subunit p65 and serve as a transcriptional coregulator of NF-kB–dependent gene expression (22). Inhibitors targeting CDK4/6 have been developed and undergoing testing in various human cancers (23). Three CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, have been approved for treatment of hormone receptor (HR)–positive advanced breast cancer.

We have found that expression of CDK6 is significantly elevated in hematopoietic progenitors of Jak2V617F knock-in mice and patients with myelofibrosis. In this study, we investigated the efficacy of CDK4/6 inhibitor palbociclib alone and in combination with ruxolitinib in Jak2V617F and MPLW515L murine models of myelofibrosis. We show that palbociclib in combination with ruxolitinib normalizes blood leukocyte counts, reduces splenomegaly, and remarkably improves BM fibrosis in both Jak2V617F and MPLW515L mouse models of myelofibrosis.

Mice

Conditional Jak2V617F knock-in (24) and Mx1Cre (25) mice were previously described. Cre expression was induced by intraperitoneal injection of polyinosine-polycytosine (pI-pC) at 4 weeks after birth. To generate MPLW515L mouse model, 5-fluorouracil–primed BALB/c mice BM cells were transduced with MSCV-MPLW515L-IRES-GFP retroviruses and injected into lethally irradiated BALB/c (The Jackson Laboratory; stock # 000651) recipient mice. For competitive BM reconstitution assay, BM cells from Mx1Cre; Jak2VF/VF; GFP and wild-type C57BL/6 mice (The Jackson Laboratory; stock # 000664) were mixed at 1:1 ratio and injected into lethally irradiated C57BL/6 recipient mice. All animal studies were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of University of Virginia School of Medicine.

Patient samples

Peripheral blood and BM samples from patients with myelofibrosis were collected at University of Virginia Cancer Center (Charlottesville, VA). Informed written consent from the subjects was obtained for sample collection according to the protocols approved by the Institutional Review Board of the University of Virginia Health System and in accordance with the Declaration of Helsinki. Only adult patient samples were collected.

Cell cultures

Human HEL and SET-2 sells were from DSMZ. UKE-1 cell line was kindly provided by Dr. Ross Levine (MSKCC). Mouse parental BA/F3 cell line was obtained from Dr. James Griffin's Lab (Dana-Farber Cancer Institute, Boston, MA). These cell lines were obtained between 2006 and 2011. BA/F3-EpoR-JAK2V617F, BA/F3-MPLW515L, and BA/F3-MPL-CALR del52 cells were generated by Mohi laboratory and the cells were authenticated using PCR method. BA/F3-EpoR-JAK2V617F, BA/F3-MPLW515L, and BA/F3-MPL-CALR del52 cells and human HEL and SET-2 cells were maintained in RPMI1640 medium supplemented with 10% FBS and penicillin/streptomycin. UKE-1 cells were maintained in IMDM medium supplemented with 10% FBS, 10% DHS, 1 μmol/L hydrocortisone, and penicillin/streptomycin. Cells were monitored under microscope for contamination. However, they were not tested for Mycoplasma. Cells were cultured for less than one month before use.

Inhibitors

Palbociclib and ruxolitinib were purchased from Chemietek. To assess the in vivo efficacy of palbociclib/ruxolitinib in Jak2V617F model of myelofibrosis, BM cells from Mx1Cre; Jak2VF/VF mice were transplanted into lethally irradiated C57BL/6 recipient mice. Six weeks after transplantation, mice were randomized to receive treatment with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) plus ruxolitinib (60 mg/kg) by oral gavage once daily for 6 weeks. MPLW515 L BMT mice were treated with the drugs for 3 weeks.

Immunoblotting

SET-2 and HEL cells were washed in PBS following treatment with the inhibitors and lysed in RIPA lysis buffer containing protease inhibitors. Jak2V617F mouse BM cells and myelofibrosis patient BM and peripheral blood mononuclear cells (PBMC) were lysed directly by boiling in 2× sample buffer. Immunoblotting was performed using indicated phospho-specific or total antibodies. The following antibodies were used. Cell Signaling Technology: p-STAT5 (Tyr694) (#4322), STAT5 (#94205), p-RB (Ser795) (#9301), p-p65 (Ser536) (#3033), p-SMAD2 (Ser465/Ser467) (#18338), SMAD2 (#5339), Santa Cruz Biotechnology: p65 (#sc-372), CDK6 (#sc-177), RB (#sc-50). Sigma: β-Actin (#A5441), Abcam: HMGA2 (#ab202387), Abclonal: AURKA (#A2121), BD Biosciences: AURKB (#611082).

Immunostaining

Immunostaining for SNAIL and α-smooth muscle actin (αSMA) were performed as described below. Paraffin sections of BM were treated with 100% xylene for 5 minutes twice, 100% xylene, and 100/% ethanol 1:1 ratio for 3 minutes, 100% ethanol for 5 minutes twice, 95% ethanol for 5 minutes, 70% ethanol for 5 minutes, and 50% ethanol for 5 minutes and rinsed with cold water for 1 minute followed by antigen retrieval with sodium citrate buffer at 100°C for 20 minutes. Blocking was performed with 10% goat serum for 2 hours. Anti-SNAIL (Rabbit, 1:100, Abclonal) and anti-αSMA (rabbit, 1:300, Abclonal) were used for staining. Secondary staining was done using TRITC goat anti-rabbit antibody (1:200 dilution) (Jackson Immunoresearch) and mounted in Vectashield mounting medium with DAPI (H-1200, Vector Labs). Fluorescence was visualized using Zeiss LAM 710 confocal microscope. Data were analyzed using Image J software (Image J). Scale bars, 15 μm.

MSC culture and immunofluorescence staining

Mesenchymal stromal cells (MSC) were generated from mice BM as described previously (26). For immunofluorescence staining, MSCs were grown on coverslips and incubated with TGFβ1 (50 ng/mL) for 72 hours in the presence or absence of palbociclib (0.25 μmol/L). Collagen staining was performed with unconjugated antibodies against collagen I or collagen III (Abcam). Secondary staining was done using TRITC goat anti-rabbit antibody (Jackson Immunoresearch). Fluorescence was visualized using Zeiss LAM 710 Confocal microscope. Scale bars, 50 μm.

RNA sequencing

LSK (LinSca1+c-kit+) cells were sorted from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib plus ruxolitinib combination using a FACS Aria II (BD Biosciences). Total RNA was extracted from LSK cells using RNeasy Micro Kit (Qiagen). RNA sequencing (RNA-seq) was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) and Hiseq next-generation sequencing instrument (Illumina). Jak2V617F LT-HSC microarray data accession number: GSE79198. MPN patient data accession number: GSE54644. RNA-seq data generated in this study are deposited to NCBI GEO database (GSE173814).

Real-time quantitative PCR

Total RNA was extracted from LSK, MSC, or SET-2 cells with RNeasy Mini Kit (Qiagen), and cDNA samples were prepared by using QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative PCR (qRT-PCR) was performed in a Quantstudio 3 machine (Applied Biosystems) using SYBR Green PCR Master Mix (Quatabio). The data were normalized to GAPDH, 18S, or HPRT and fold changes in gene expression were determined by the ΔΔCt method. Sequences of the primers are available in the Supplementary Tables S1 and S2.

Statistical analysis

Results are expressed as mean ± SEM, and statistical significance was determined by one-way ANOVA or Student t test using Prism Version 8 software (GraphPad, Prism). P < 0.05 was considered statistically significant.

CDK6 expression is significantly upregulated in myelofibrosis

Using unbiased microarray gene expression analysis (GSE79198), we identified CDK6 as one of the significantly upregulated genes in long-term hematopoietic stem cells (LT-HSC) of Jak2V617F knock-in mice (24) compared with control LT-HSC (Fig. 1A). qRT-PCR further validated significantly increased expression of CDK6 in Jak2V617F LT-HSC as compared with wild-type LT-HSC (Fig. 1B). Analysis of MPN gene expression dataset (GSE54644) revealed that CDK6 expression is significantly increased in myelofibrosis patient's granulocytes compared with healthy controls (Fig. 1C). qRT-PCR further validated significantly elevated CDK6 mRNA expression in myelofibrosis CD34+ cells compared with healthy control CD34+ cells (Fig. 1D).

Figure 1.

CDK6 expression is significantly increased in myelofibrosis. A, Heatmap from microarray data analysis (GSE79198) showing significantly increased CDK6 expression in Jak2V617F mice LT-HSC (LinSca-1+c-kit+CD34CD135) compared with control mice LT-HSC. B, qRT-PCR analysis shows significant increase of CDK6 mRNA expression in Jak2V617F LT-HSC mice compared with control LT-HSC (n = 4). The mRNA expression was normalized with 18S. C, Analysis of MPN microarray gene expression data (GSE54644) shows that CDK6 expression is significantly increased in granulocytes of patients with myelofibrosis compared with healthy controls (controls = 11; myelofibrosis = 18). D, qRT-PCR analysis shows significant increase of CDK6 mRNA expression in myelofibrosis CD34+ cells compared with healthy control CD34+ cells (n = 4). The mRNA expression was normalized with 18S expression. E and F, Immunoblots showing elevated expression of CDK6 protein in the BM and PBMC of patients with myelofibrosis compared with healthy controls. β-Actin served as a loading control. G, Increased CDK6 protein expression in the BM of Jak2V617F heterozygous (Jak2VF/+) and homozygous (Jak2VF/VF) mice compared with wild-type control. β-Actin served as a loading control. H, Increased CDK6 protein expression in the BM of mice expressing MPLW515L compared with control mice. I, Elevated CDK6 protein expression in BA/F3-EpoR-JAK2V617F, BA/F3-MPLW515L, and BA/F3-MPL-CALR del52 cells compared with parental BA/F3 cells. *, P < 0.05; **, P < 0.005; Student t test.

Figure 1.

CDK6 expression is significantly increased in myelofibrosis. A, Heatmap from microarray data analysis (GSE79198) showing significantly increased CDK6 expression in Jak2V617F mice LT-HSC (LinSca-1+c-kit+CD34CD135) compared with control mice LT-HSC. B, qRT-PCR analysis shows significant increase of CDK6 mRNA expression in Jak2V617F LT-HSC mice compared with control LT-HSC (n = 4). The mRNA expression was normalized with 18S. C, Analysis of MPN microarray gene expression data (GSE54644) shows that CDK6 expression is significantly increased in granulocytes of patients with myelofibrosis compared with healthy controls (controls = 11; myelofibrosis = 18). D, qRT-PCR analysis shows significant increase of CDK6 mRNA expression in myelofibrosis CD34+ cells compared with healthy control CD34+ cells (n = 4). The mRNA expression was normalized with 18S expression. E and F, Immunoblots showing elevated expression of CDK6 protein in the BM and PBMC of patients with myelofibrosis compared with healthy controls. β-Actin served as a loading control. G, Increased CDK6 protein expression in the BM of Jak2V617F heterozygous (Jak2VF/+) and homozygous (Jak2VF/VF) mice compared with wild-type control. β-Actin served as a loading control. H, Increased CDK6 protein expression in the BM of mice expressing MPLW515L compared with control mice. I, Elevated CDK6 protein expression in BA/F3-EpoR-JAK2V617F, BA/F3-MPLW515L, and BA/F3-MPL-CALR del52 cells compared with parental BA/F3 cells. *, P < 0.05; **, P < 0.005; Student t test.

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Consistent with increased CDK6 mRNA expression, we observed elevated CDK6 protein levels in the BM and PBMCs of patients with myelofibrosis compared with healthy controls (Fig. 1E–F). We also observed elevated CDK6 protein levels in the BM of Jak2V617F mice (Fig. 1G) as well as in mice expressing MPLW515L (Fig. 1H). We next assessed the CDK6 protein levels in hematopoietic BA/F3 cells expressing MPN driver mutants JAK2V617F, MPLW515L, and CALRdel52. We found increased CDK6 protein levels in BA/F3 cells expressing JAK2V617F, MPLW515L, or CALRdel52 mutant compared with BA/F3 parent cells (Fig. 1I), suggesting that all three MPN driver mutants can induce CDK6 expression.

We tested whether inhibition of JAK2 can affect CDK6 expression. We observed dose-dependent reduction of STAT5 phosphorylation but no significant change in CDK6 expression by ruxolitinib treatment in HEL and myelofibrosis PBMC cells (Supplementary Fig. S1A and S1B). Similarly, in vivo treatment of ruxolitinib almost completely inhibited STAT5 phosphorylation but did not significantly affect CDK6 level or Rb phosphorylation in the BM of Jak2V617F knock-in (Jak2VF/VF) mice (Supplementary Fig. S1C and Fig. 6G), suggesting that ruxolitinib treatment does not inhibit CDK6 in MPN cells.

We next investigated the effects of CDK6 depletion on JAK2V617F-expressing hematopoietic cells. We observed that lentiviral shRNA-mediated knockdown of CDK6 significantly inhibited the proliferation of JAK2V617F-expressing murine BA/F3-EpoR-JAK2V617F cells and human SET-2 and HEL cells but not WT JAK2-expressing BA/F3-EpoR cells (Supplementary Fig. S2A–S2D). These suggest that CDK6 may play an important role in the growth/survival of MPN cells expressing oncogenic JAK2V617F mutant.

CDK6 inhibitor palbociclib alone or in combination with ruxolitinib significantly inhibits hematopoietic cells expressing MPN driver mutants

We investigated the effects of CDK6 inhibition by palbociclib on proliferation of hematopoietic cells expressing JAK2V617F and MPLW515L. We found that palbociclib treatment significantly reduced proliferation of murine BA/F3-EpoR-JAK2V617F and BAF3-MPLW515L cells, whereas WT JAK2-expressing BA/F3 cells were modestly affected by palbociclib treatment only at higher concentration (Fig. 2A–C). Palbociclib treatment also significantly inhibited proliferation of human JAK2V617F-positive HEL, SET-2, and UKE-1 cells (Fig. 2D–F). Combined treatment of palbociclib and ruxolitinib resulted in greater inhibition of growth in BA/F3-EpoR-JAK2V617F, HEL, and SET-2 cells (Fig. 2GI). We also tested the effects of palbociclib or palbociclib/ruxolitinib combination on apoptosis in BA/F3-EpoR-JAK2V617F cells. Treatment of palbociclib induced apoptosis in BA/F3-EpoR-JAK2V617F cells (Supplementary Fig. S3). Combined treatment of palbociclib and ruxolitinib caused marked increase in apoptosis of BA/F3-EpoR-JAK2V617F cells (Fig. 2J).

Figure 2.

Effects of palbociclib alone or in combination with ruxolitinib on hematopoietic cells and progenitors expressing MPN driver mutants. AC, BA/F3 (parent), BA/F3-EpoR-JAK2V617F, and BA/F3-MPLW515L cells were treated with vehicle (DMSO) or palbociclib, and cell proliferation was assessed by viable cell counts over 5 days. Palbociclib treatment significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and BA/F3-MPLW515L cells but exhibited modest inhibition of BA/F3 (parent) cells at higher concentration (0.5 μmol/L). DF, Palbociclib treatment showed significant reduction in cell proliferation of JAK2V617F-positive human HEL, SET-2, and UKE-1 cells. GI, Combined treatment palbociclib and ruxolitinib exhibited significantly greater inhibition of BA/F3-EpoR-JAK2V617F, HEL, and SET-2 cells compared with vehicle or single drug treatment. Values are expressed as percentages of controls (DMSO-treated). Data from three independent experiments are shown in bar graphs as mean ± SEM. J, Annexin V/propidium iodide staining followed by flow cytometry was performed to measure apoptosis in BA/F3-EpoR-JAK2V617F cells after treatment with palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination for 3 days. Representative dot plots of the percentage of apoptotic cells in BA/F3-EpoR-JAK2V617F cells treated with palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination (left). Bar graphs showing significant increase in apoptosis of BA/F3-EpoR-JAK2V617F cells by palbociclib/ruxolitinib combination treatment. K, Myelofibrosis CD34+ cells were plated in complete methylcellulose medium supplemented with cytokines in the presence of DMSO or palbociclib (0.06–0.5 μmol/L). Palbociclib treatment alone significantly inhibited hematopoietic progenitor colonies in myelofibrosis CD34+ progenitors (n = 7). L, Treatment of palbociclib in combination with ruxolitinib exhibited significantly greater inhibition of hematopoietic progenitor colonies in myelofibrosis CD34+ progenitors compared with single drug treatment (n = 7). Data are represented in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005; Student t test; NS, nonsignificant.

Figure 2.

Effects of palbociclib alone or in combination with ruxolitinib on hematopoietic cells and progenitors expressing MPN driver mutants. AC, BA/F3 (parent), BA/F3-EpoR-JAK2V617F, and BA/F3-MPLW515L cells were treated with vehicle (DMSO) or palbociclib, and cell proliferation was assessed by viable cell counts over 5 days. Palbociclib treatment significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and BA/F3-MPLW515L cells but exhibited modest inhibition of BA/F3 (parent) cells at higher concentration (0.5 μmol/L). DF, Palbociclib treatment showed significant reduction in cell proliferation of JAK2V617F-positive human HEL, SET-2, and UKE-1 cells. GI, Combined treatment palbociclib and ruxolitinib exhibited significantly greater inhibition of BA/F3-EpoR-JAK2V617F, HEL, and SET-2 cells compared with vehicle or single drug treatment. Values are expressed as percentages of controls (DMSO-treated). Data from three independent experiments are shown in bar graphs as mean ± SEM. J, Annexin V/propidium iodide staining followed by flow cytometry was performed to measure apoptosis in BA/F3-EpoR-JAK2V617F cells after treatment with palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination for 3 days. Representative dot plots of the percentage of apoptotic cells in BA/F3-EpoR-JAK2V617F cells treated with palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination (left). Bar graphs showing significant increase in apoptosis of BA/F3-EpoR-JAK2V617F cells by palbociclib/ruxolitinib combination treatment. K, Myelofibrosis CD34+ cells were plated in complete methylcellulose medium supplemented with cytokines in the presence of DMSO or palbociclib (0.06–0.5 μmol/L). Palbociclib treatment alone significantly inhibited hematopoietic progenitor colonies in myelofibrosis CD34+ progenitors (n = 7). L, Treatment of palbociclib in combination with ruxolitinib exhibited significantly greater inhibition of hematopoietic progenitor colonies in myelofibrosis CD34+ progenitors compared with single drug treatment (n = 7). Data are represented in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005; Student t test; NS, nonsignificant.

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We also investigated the effects of palbociclib/ruxolitinib treatment on myelofibrosis hematopoietic progenitors. CD34+ cells isolated from the peripheral blood of JAK2V617F-positive patients with myelofibrosis were plated in complete methylcellulose medium in the absence or presence of palbociclib or palbociclib/ruxolitinib and hematopoietic progenitor colonies were assessed. Palbociclib alone significantly reduced myeloid colony formation in myelofibrosis CD34+ progenitor cells (Fig. 2K). Combined treatment of palbociclib with ruxolitinib resulted in greater inhibition of colony formation in myelofibrosis CD34+ cells (Fig. 2L). Palbociclib/ruxolitinib treatment also caused reduction in myeloid colony formation in healthy control CD34+ cells (Supplementary Fig. S4A and S4B), although their effects were greater against myelofibrosis CD34+ cells than healthy controls.

Treatment of palbociclib alone or in combination with ruxolitinib markedly inhibits myelofibrosis in Jak2V617F mouse model of myelofibrosis

We previously reported the conditional Jak2V617F knock-in mice (24). Mice expressing homozygous Jak2V617F rapidly develop high-grade myelofibrosis (24, 27). So, we utilized the homozygous Jak2V617F knock-in mice in this study to test the in vivo efficacy of palbociclib alone or in combination with ruxolitinib. BM cells from the homozygous Jak2V617F knock-in mice (Mx1Cre; Jak2VF/VF) at 7 weeks after pI-pC induction were transplanted into lethally irradiated C57BL/6 recipient mice to generate a cohort of mice expressing Jak2VF/VF. The experimental approach is depicted in Fig. 3A. At six weeks after BMT, all mice showed elevated WBC and neutrophil counts (Fig. 3B), indicating MPN disease development in these animals. Mice were then randomized into four groups to receive treatment with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) plus ruxolitinib (60 mg/kg) by oral gavage once daily for a period of 6 weeks. Treatment of palbociclib alone significantly reduced WBC and neutrophil counts compared with vehicle treatment (Fig. 3B). Combined treatment of palbociclib and ruxolitinib normalized the WBC and neutrophil counts in these animals (Fig. 3B).

Figure 3.

In vivo administration of palbociclib alone or in combination with ruxolitinib ameliorates myelofibrosis in Jak2V617F mouse model of myelofibrosis. A, Experimental design to assess the in vivo effects of palbociclib/ruxolitinib in homozygous Jak2V617F (Jak2VF/VF) model of myelofibrosis. B, Peripheral blood WBC and neutrophil (NE) counts were assessed at 6 weeks after treatment (n = 8–10). C, Frequency of granulocyte/monocyte (Gr-1+/Mac-1+) precursors in the BM of palbociclib/ruxolitinib-treated mice is shown in bar graphs as mean ± SEM. D and E, Flow cytometric analysis of LSK (LinSca-1+c-kit+), LT-HSC (LinSca-1+c-kit+CD34CD135), ST-HSC (LinSca-1+c-kit+CD34+CD135), MPP (LinSca-1+c-kit+CD34+CD135+), CMP (LinSca-1c-kit+CD34+FcγRII/IIlow), GMP (LinSca-1c-kit+CD34+FcγRII/IIhigh), and MEP (LinSca-1c-kit+CD34FcγRII/III) in the BM is shown in bar graphs as mean ± SEM. F, BM cells (2 × 104) from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination were plated in methylcellulose medium (MethoCult 3434) with cytokines. CFU-GM colonies were scored 7 days after plating. G, Spleen size/weight in Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination (n = 10–12). *, P < 0.05; **, P < 0.005; ***, P < 0.0005; Student t test. H, Histopathologic analysis. Reticulin staining of the BM sections (magnification, ×500) from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination. Note that palbociclib treatment significantly reduced BM fibrosis while combined treatment of palbociclib/ruxolitinib almost completely ablated BM fibrosis in Jak2VF/VF mice.

Figure 3.

In vivo administration of palbociclib alone or in combination with ruxolitinib ameliorates myelofibrosis in Jak2V617F mouse model of myelofibrosis. A, Experimental design to assess the in vivo effects of palbociclib/ruxolitinib in homozygous Jak2V617F (Jak2VF/VF) model of myelofibrosis. B, Peripheral blood WBC and neutrophil (NE) counts were assessed at 6 weeks after treatment (n = 8–10). C, Frequency of granulocyte/monocyte (Gr-1+/Mac-1+) precursors in the BM of palbociclib/ruxolitinib-treated mice is shown in bar graphs as mean ± SEM. D and E, Flow cytometric analysis of LSK (LinSca-1+c-kit+), LT-HSC (LinSca-1+c-kit+CD34CD135), ST-HSC (LinSca-1+c-kit+CD34+CD135), MPP (LinSca-1+c-kit+CD34+CD135+), CMP (LinSca-1c-kit+CD34+FcγRII/IIlow), GMP (LinSca-1c-kit+CD34+FcγRII/IIhigh), and MEP (LinSca-1c-kit+CD34FcγRII/III) in the BM is shown in bar graphs as mean ± SEM. F, BM cells (2 × 104) from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination were plated in methylcellulose medium (MethoCult 3434) with cytokines. CFU-GM colonies were scored 7 days after plating. G, Spleen size/weight in Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination (n = 10–12). *, P < 0.05; **, P < 0.005; ***, P < 0.0005; Student t test. H, Histopathologic analysis. Reticulin staining of the BM sections (magnification, ×500) from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination. Note that palbociclib treatment significantly reduced BM fibrosis while combined treatment of palbociclib/ruxolitinib almost completely ablated BM fibrosis in Jak2VF/VF mice.

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Flow cytometric analysis showed significant reduction in myeloid precursors (Gr-1+/Mac-1+) in the BM of Jak2VF/VF mice treated with palbociclib or palbociclib/ruxolitinib combination (Fig. 3C). We also assessed the effects of palbociclib/ruxolitinib treatment on hematopoietic stem cells (HSC) and progenitors. We observed a significant reduction in LSK (LinSca1 +c-Kit+), short-term HSC (ST-HSC), and multipotent progenitors (MPP) in the BM of mice treated with palbociclib/ruxolitinib combination compared with vehicle treatment (Fig. 3D). In addition, we observed significant reduction of common myeloid progenitors (CMP), granulocyte–macrophage progenitors (GMP), and megakaryocyte–erythrocyte progenitors (MEP) in the BM of mice treated with palbociclib/ruxolitinib combination compared with vehicle treatment (Fig. 3E). Hematopoietic progenitor colony assays showed significant reduction of CFU-GM colonies in the BM of Jak2VF/VF mice treated with palbociclib or palbociclib/ruxolitinib combination compared with vehicle or ruxolitinib treatment (Fig. 3F). Spleen size/weight was significantly reduced in Jak2VF/VF mice upon palbociclib or ruxolitinib treatment alone (Fig. 3G). Combined treatment of palbociclib and ruxolitinib resulted in significantly greater reduction of spleen size/weight in Jak2VF/VF mice (Fig. 3G).

Histopathologic analyzes revealed extensive BM fibrosis in vehicle-treated Jak2VF/VF mice (Fig. 3H). Palbociclib treatment alone caused marked reduction of fibrosis in the BM of Jak2VF/VF mice, whereas ruxolitinib treatment did not significantly alter BM fibrosis (Fig. 3H). Combined treatment of palbociclib and ruxolitinib almost completely ablated fibrosis in the BM of Jak2VF/VF mice (Fig. 3H).

Palbociclib treatment preferentially inhibits Jak2V617F-mutant hematopoietic progenitors

To evaluate whether palbociclib can effectively inhibit disease causing Jak2V617F-mutant stem/progenitor cells, we generated Mx1Cre; Jak2VF/VF GFP+ mice and performed competitive BM transplantation assays followed by drug treatments. BM cells from Mx1Cre; Jak2VF/VF GFP+ mice (6 weeks after pI-pC induction) were mixed with WT C57BL/6 mice BM cells at a ratio of 1:1 and then transplanted into lethally irradiated C57BL/6 mice (outlined in Fig. 4A). At 6 weeks after BMT, mice were randomized to receive treatment with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) plus ruxolitinib (60 mg/kg) by oral gavage once daily for a period of 12 weeks. Treatment of palbociclib alone significantly reduced the WBC and neutrophil counts compared with vehicle treatment (Fig. 4B). Combined treatment of palbociclib and ruxolitinib resulted in normalization of WBC and neutrophil counts (Fig. 4B). Vehicle-treated chimeric mice exhibited high percentage (60%–70%) of GFP+ LSK and LK (Linc-Kit+) cells in their BM (Fig. 4C–D). Treatment of palbociclib or palbociclib/ruxolitinib combination significantly reduced the percentage of mutant GFP+ LSK and LK cells in the chimeric mice (Fig. 4C and D). Treatment of palbociclib or palbociclib/ruxolitinib combination also caused significant reduction in the percentage of mutant GFP+ myeloid (Gr-1+) and megakaryocytic (CD41+) cells in the BM of chimeric mice (Fig. 4E and F). Together, these results suggest that palbociclib treatment preferentially inhibits oncogenic Jak2V617F-mutant hematopoietic progenitors.

Figure 4.

Palbociclib treatment alone or in combination with ruxolitinib preferentially inhibits Jak2V617F-mutant hematopoietic progenitors. A, Scheme on competitive BM transplantation approach to assess the effects of palbociclib/ruxolitinib on Jak2V617F-mutant hematopoietic progenitors is depicted. BM cells (5 × 105) from Mx1Cre; Jak2VF/VF; GFP mice (6 weeks after pI-pC injection) were mixed with WT C57BL/6 mice BM (5 × 105) at a 1:1 ratio and were transplanted into lethally irradiated WT C57BL/6 recipient mice. Six weeks after BMT, recipient mice were randomized to receive treatment with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) and ruxolitinib (60 mg/kg). Drug was administered orally once daily for 12 weeks. B, Peripheral blood WBC and neutrophil (NE) counts were assessed at 12 weeks after treatment (n = 5). C–F, Percentages of Jak2V617F-mutant GFP+ LSK (LinSca-1+c-kit+), GFP+ LK (Linc-kit+), GFP+ Gr-1+, and GFP+CD41+ cells in the BM of chimeric animals at 12 weeks after treatment are shown; bar graphs, mean ± SEM (n = 5). G, Experimental design to assess the effects of drug withdrawal in secondary transplanted animals. After treatment of chimeric mice with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) and ruxolitinib (60 mg/kg) combination for 12 weeks, BM cells (1 × 106) were transplanted into WT C57BL/6 secondary recipient mice. No treatment was given to secondary recipient animals and they were analyzed at 16 weeks after transplantation. H–K, Percentages of Jak2V617F-mutant GFP+ LSK, GFP+ LK, GFP+ Gr-1+, and GFP+CD41+ cells in the BM of secondary recipient animals at 16 weeks after BMT are shown (n = 5 in each group). Data are represented in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005; Student t test.

Figure 4.

Palbociclib treatment alone or in combination with ruxolitinib preferentially inhibits Jak2V617F-mutant hematopoietic progenitors. A, Scheme on competitive BM transplantation approach to assess the effects of palbociclib/ruxolitinib on Jak2V617F-mutant hematopoietic progenitors is depicted. BM cells (5 × 105) from Mx1Cre; Jak2VF/VF; GFP mice (6 weeks after pI-pC injection) were mixed with WT C57BL/6 mice BM (5 × 105) at a 1:1 ratio and were transplanted into lethally irradiated WT C57BL/6 recipient mice. Six weeks after BMT, recipient mice were randomized to receive treatment with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) and ruxolitinib (60 mg/kg). Drug was administered orally once daily for 12 weeks. B, Peripheral blood WBC and neutrophil (NE) counts were assessed at 12 weeks after treatment (n = 5). C–F, Percentages of Jak2V617F-mutant GFP+ LSK (LinSca-1+c-kit+), GFP+ LK (Linc-kit+), GFP+ Gr-1+, and GFP+CD41+ cells in the BM of chimeric animals at 12 weeks after treatment are shown; bar graphs, mean ± SEM (n = 5). G, Experimental design to assess the effects of drug withdrawal in secondary transplanted animals. After treatment of chimeric mice with vehicle, palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) and ruxolitinib (60 mg/kg) combination for 12 weeks, BM cells (1 × 106) were transplanted into WT C57BL/6 secondary recipient mice. No treatment was given to secondary recipient animals and they were analyzed at 16 weeks after transplantation. H–K, Percentages of Jak2V617F-mutant GFP+ LSK, GFP+ LK, GFP+ Gr-1+, and GFP+CD41+ cells in the BM of secondary recipient animals at 16 weeks after BMT are shown (n = 5 in each group). Data are represented in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005; Student t test.

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To determine whether the effects of palbociclib or palbociclib/ruxolitinib treatment on mutant HSPC could sustain overtime after withdrawal of the treatment, we performed secondary transplantation experiment using BM from the primary transplanted chimeric mice after 12 weeks of treatment into lethally irradiated WT C57BL/6 recipient mice. The secondary transplanted mice were not given any treatment and they were analyzed at 16 weeks after transplantation (as outlined in Fig. 4G). We observed significantly reduced percentage of mutant GFP+ LSK and LK cells in the recipients of palbociclib-treated mice BM compared with recipients of vehicle-treated mice BM (Fig. 4H and I). Recipients of BM from Jak2VF/VF GFP+ chimeric mice that were initially treated with palbociclib/ruxolitinib combination exhibited greater reduction of GFP+ LSK and LK cells compared with recipients of single drug-treated mice BM (Fig. 4H and I). Recipients of palbociclib- or palbociclib/ruxolitinib-treated mice BM also exhibited significant reduction in the percentage of mutant GFP+ myeloid (Gr-1+) and megakaryocytic (CD41+) cells compared with recipients of vehicle or ruxolitinib-treated mice BM (Fig. 4J and K). Thus, decrease in mutant HSPC and myeloid/megakaryocytic precursors caused by the treatment of palbociclib or palbociclib/ruxolitinib was sustained overtime even after discontinuation of the treatment, that is, withdrawal of palbociclib or palbociclib/ruxolitinib combination treatment did not lead to recurrence of the disease in the secondary recipients.

Treatment of palbociclib or palbociclib/ruxolitinib combination significantly inhibits myelofibrosis in MPLW515L mouse model of myelofibrosis

We also tested the in vivo efficacy of palbociclib and palbociclib/ruxolitinib combination in MPLW515L mouse model of myelofibrosis (28). We generated a cohort of mice expressing MPLW515L by transduction of BALB/c mice BM cells with MSCV-MPLW515L retroviruses followed by transplantation into lethally irradiated syngeneic recipient mice. At three weeks after transplantation, mice were randomized to receive treatment with placebo (vehicle), palbociclib (50 mg/kg), ruxolitinib (60 mg/kg), or palbociclib (50 mg/kg) plus ruxolitinib (60 mg/kg; outlined in Fig. 5A). Whereas vehicle-treated MPLW515L mice exhibited markedly increased WBC, neutrophil, and platelet counts in their peripheral blood, palbociclib-treated animals exhibited significant decrease in peripheral blood WBC, neutrophil, and platelet counts (Fig. 5B). Ruxolitinib treatment also caused reduction in blood counts. However, combined treatment of palbociclib and ruxolitinib resulted in almost complete normalization of WBC, neutrophil, and platelet counts in these animals (Fig. 5B). Flow cytometric analysis revealed that palbociclib or palbociclib/ruxolitinib combination treatment significantly reduced myeloid precursors (Gr-1+/Mac-1+) and megakaryocytic precursors (CD61+/CD41+) in the BM and spleens of MPLW515L mice (Fig. 5C–F). Marked splenomegaly was observed in vehicle-treated MPLW515L mice (Fig. 5G). Palbociclib treatment alone significantly reduced splenomegaly in MPLW515L mice (Fig. 5G). However, combined treatment of palbociclib and ruxolitinib resulted in greater reduction of spleen size/weight than single drug treatment (Fig. 5G). Histopathologic analyzes showed that palbociclib treatment alone caused marked reduction of BM fibrosis whereas ruxolitinib treatment did not exhibit significant reduction of BM fibrosis in MPLW515L mice (Fig. 5H). Combined treatment of palbociclib with ruxolitinib ablated BM fibrosis in MPLW515L mice (Fig. 5H). Together, these data suggest that palbociclib treatment alone or in combination with ruxolitinib might be efficacious in the treatment of myelofibrosis.

Figure 5.

Palbociclib alone or in combination with ruxolitinib significantly reduces myelofibrosis in MPLW515L mouse model. A, Experimental design to test the efficacy of palbociclib/ruxolitinib in MPLW515L mouse model of myelofibrosis. B, Peripheral blood WBC, neutrophil (NE), and platelet (PLT) counts in the peripheral blood were assessed at 3 weeks after treatment (n = 5–9). Data are shown in bar graphs as mean ± SEM. C–F, Frequency of granulocyte/monocyte (Gr-1+/Mac-1+) precursors and megakaryocytic precursors (CD61+/CD41+) in the BM and spleens of MPLW515L mice treated with vehicle, palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination is shown in bar graphs as mean ± SEM (n = 5). G, Spleen size/weight in MPLW515L mice treated with vehicle, palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination (n = 5–9). Data are represented in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; Student t test. H, Histopathologic analysis. Reticulin staining showing extensive fibrosis (grade 2 and 3) in the BM of vehicle-treated or ruxolitinib-treated MPLW515L mice. Palbociclib-treated MPLW515L mice BM showed marked reduction in fibrosis, whereas combined treatment of palbociclib/ruxolitinib almost completely ablated fibrosis in the BM of MPLW515L mice.

Figure 5.

Palbociclib alone or in combination with ruxolitinib significantly reduces myelofibrosis in MPLW515L mouse model. A, Experimental design to test the efficacy of palbociclib/ruxolitinib in MPLW515L mouse model of myelofibrosis. B, Peripheral blood WBC, neutrophil (NE), and platelet (PLT) counts in the peripheral blood were assessed at 3 weeks after treatment (n = 5–9). Data are shown in bar graphs as mean ± SEM. C–F, Frequency of granulocyte/monocyte (Gr-1+/Mac-1+) precursors and megakaryocytic precursors (CD61+/CD41+) in the BM and spleens of MPLW515L mice treated with vehicle, palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination is shown in bar graphs as mean ± SEM (n = 5). G, Spleen size/weight in MPLW515L mice treated with vehicle, palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination (n = 5–9). Data are represented in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; Student t test. H, Histopathologic analysis. Reticulin staining showing extensive fibrosis (grade 2 and 3) in the BM of vehicle-treated or ruxolitinib-treated MPLW515L mice. Palbociclib-treated MPLW515L mice BM showed marked reduction in fibrosis, whereas combined treatment of palbociclib/ruxolitinib almost completely ablated fibrosis in the BM of MPLW515L mice.

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Inhibition of CDK6 by palbociclib alters gene expression and impairs cell signaling in hematopoietic progenitors expressing Jak2V617F

To gain insights into the mechanisms by which CDK6 inhibition ameliorates myelofibrosis, we performed RNA-seq on LSK cells from Jak2VF/VF mice treated with the inhibitors. Heatmap showed significantly downregulated genes in palbociclib- and palbociclib/ruxolitinib-treated LSK cells compared with vehicle-treated LSK cells (Fig. 6A). Gene set enrichment analysis (GSEA; ref. 29) of RNA-seq data revealed significant down regulation of genes related to cell cycle, stem cells, Aurora kinase (AURK), and NF-κB signaling pathways in palbociclib-treated LSK cells compared with vehicle-treated LSK cells (Fig. 6B). These pathways were also similarly affected in palbociclib/ruxolitinib–treated mice LSK cells compared with ruxolitinib-treated LSK cells (Supplementary Fig. S5A).

Figure 6.

Inhibition of CDK6 by palbociclib alters gene expression and cell signaling in hematopoietic progenitors expressing Jak2V617F. A, Heatmap showing top 100 significantly downregulated genes (P < 0.05, −1.5-fold) in palbociclib, ruxolitinib, and palbociclib/ruxolitinib–treated Jak2VF/VF mice LSK (LinSca-1+c-kit+) cells compared with vehicle-treated LSK cells. B, Gene set enrichment analyses of the RNA-seq data from palbociclib-treated Jak2VF/VF mice LSK cells compared with vehicle-treated Jak2VF/VF mice LSK. Enrichment plots of selected gene sets with normalized enrichment score (NES) and FDR are shown. C, Venn diagram showing the overlap between upregulated genes in patients with myelofibrosis and genes downregulated by palbociclib treatment in Jak2VF/VF mice LSK cells. The cutoffs were FDR-adjusted P < 0.05. D, Relative expression of AURKA, AURKB, and HMGA2 mRNA was determined by qRT-PCR in LSK cells obtained from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination. Data from four independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005. E, JAK2V617F-positive SET-2 cells were transduced with lentiviral CDK6 shRNA or control (scramble shRNA), and the infected cells were selected using puromycin. Relative expression of AURKA, AURKB, and HMGA2 mRNA was assessed by qRT-PCR and normalized with HPRT expression. Data from three independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005. F, PBMCs obtained from patients with myelofibrosis were treated with DMSO, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination at indicated concentrations for 6 hours. Immunoblotting was performed using phospho-specific or total antibodies as indicated. Palbociclib treatment reduced the phosphorylation of Rb and p65 subunit of NF-κB and decreased the expression of HMGA2, AURKA, and AURKB in myelofibrosis PBMCs. Combined treatment of palbociclib with ruxolitinib caused more pronounced inhibition of phosphorylation or expression of these target proteins. β-Actin was used as a loading control. G, BM cells obtained from Jak2VF/VF mice following in vivo treatment with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination were subjected to immunoblotting using phospho-specific or total antibodies as indicated. H, Immunoblot analysis showed reduced phosphorylation of Rb and p65 subunit of NF-κB and decreased expression of HMGA2, AURKA and AURKB upon CDK6 knockdown in SET-2 cells. β-Actin was used as a loading control.

Figure 6.

Inhibition of CDK6 by palbociclib alters gene expression and cell signaling in hematopoietic progenitors expressing Jak2V617F. A, Heatmap showing top 100 significantly downregulated genes (P < 0.05, −1.5-fold) in palbociclib, ruxolitinib, and palbociclib/ruxolitinib–treated Jak2VF/VF mice LSK (LinSca-1+c-kit+) cells compared with vehicle-treated LSK cells. B, Gene set enrichment analyses of the RNA-seq data from palbociclib-treated Jak2VF/VF mice LSK cells compared with vehicle-treated Jak2VF/VF mice LSK. Enrichment plots of selected gene sets with normalized enrichment score (NES) and FDR are shown. C, Venn diagram showing the overlap between upregulated genes in patients with myelofibrosis and genes downregulated by palbociclib treatment in Jak2VF/VF mice LSK cells. The cutoffs were FDR-adjusted P < 0.05. D, Relative expression of AURKA, AURKB, and HMGA2 mRNA was determined by qRT-PCR in LSK cells obtained from Jak2VF/VF mice treated with vehicle, palbociclib, ruxolitinib, and palbociclib/ruxolitinib combination. Data from four independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005. E, JAK2V617F-positive SET-2 cells were transduced with lentiviral CDK6 shRNA or control (scramble shRNA), and the infected cells were selected using puromycin. Relative expression of AURKA, AURKB, and HMGA2 mRNA was assessed by qRT-PCR and normalized with HPRT expression. Data from three independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005. F, PBMCs obtained from patients with myelofibrosis were treated with DMSO, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination at indicated concentrations for 6 hours. Immunoblotting was performed using phospho-specific or total antibodies as indicated. Palbociclib treatment reduced the phosphorylation of Rb and p65 subunit of NF-κB and decreased the expression of HMGA2, AURKA, and AURKB in myelofibrosis PBMCs. Combined treatment of palbociclib with ruxolitinib caused more pronounced inhibition of phosphorylation or expression of these target proteins. β-Actin was used as a loading control. G, BM cells obtained from Jak2VF/VF mice following in vivo treatment with vehicle, palbociclib, ruxolitinib, or palbociclib/ruxolitinib combination were subjected to immunoblotting using phospho-specific or total antibodies as indicated. H, Immunoblot analysis showed reduced phosphorylation of Rb and p65 subunit of NF-κB and decreased expression of HMGA2, AURKA and AURKB upon CDK6 knockdown in SET-2 cells. β-Actin was used as a loading control.

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We next compared genes that were significantly downregulated by palbociclib treatment with genes that were upregulated in myelofibrosis gene expression dataset (GSE54644). There was an overlap of 245 genes, which were upregulated in patients with myelofibrosis and significantly downregulated in palbociclib-treated Jak2VF/VF mice LSK cells (Fig. 6C). Molecular signature analysis of overlapping genes showed enrichment for cell cycle, Rb1 targets, cell proliferation, inflammatory response, and Aurora kinase pathway (Fig. 6C), indicating that these pathways were upregulated in myelofibrosis and downregulated by palbociclib treatment.

RNA-seq data analysis revealed that expression of AURKA, AURKB, and HMGA2 was significantly reduced in palbociclib-treated Jak2VF/VF mice LSK cells compared with vehicle-treated Jak2VF/VF mice LSK cells. qRT-PCR further validated that expression of AURKA, AURKB, and HMGA2 was significantly downregulated in palbociclib- and palbociclib/ruxolitinib–treated LSK cells compared with vehicle-treated LSK cells (Fig. 6D). Interestingly, expression of AURKA, AURKB, and HMGA2 is significantly elevated in myelofibrosis patient's hematopoietic cells as compared with healthy controls (Supplementary Fig. S5B). Lentiviral shRNA-mediated knockdown of CDK6 also significantly reduced the expression of AURKA, AURKB, and HMGA2 in SET-2 cells (Fig. 6E) similar to that observed with palbociclib treatment (Supplementary Fig. S5C), suggesting that they are bona fide targets of CDK6.

We next performed cell signaling studies in myelofibrosis patient PBMC and Jak2VF/VF mice BM following treatment with palbociclib or palbociclib/ruxolitinib combination. As expected, we observed decreased Rb phosphorylation upon palbociclib treatment in myelofibrosis PBMC and Jak2VF/VF BM cells (Fig. 6F–G). Ruxolitinib treatment, however, did not cause significant inhibition of Rb phosphorylation in these cells (Fig. 6F–G). We also observed reduced expression of HMGA2, AURKA, and AURKB proteins in myelofibrosis PBMC and Jak2VF/VF BM cells upon palbociclib or palbociclib/ruxolitinib treatment (Fig. 6F–G). Because CDK6 has been shown to regulate the NF-κB signaling pathway (22), we also assessed the effects of palbociclib on NF-κB signaling. Indeed, we observed reduced p65 phosphorylation in myelofibrosis PBMC and Jak2VF/VF mice BM treated with palbociclib or palbociclib/ruxolitinib (Fig. 6F–G). To further confirm that the signaling alterations caused by palbociclib treatment in myelofibrosis PBMC and Jak2VF/VF mice BM was due to inhibition of CDK6, we performed shRNA-mediated knockdown of CDK6 in SET-2 cells. We observed marked inhibition of Rb and p65 phosphorylation and decreased expression of HMGA2, AURKA, and AURKB proteins upon CDK6 knockdown in SET-2 cells (Fig. 6H) similar to that observed with palbociclib treatment (Supplementary Fig. S6).

Palbociclib treatment reduces the TGFβ1 level and attenuates expression of fibrotic markers

Analysis of the RNA-seq data also revealed that genes related to TGFβ signaling was significantly downregulated in palbociclib-treated Jak2VF/VF mice LSK cells (Fig. 7A). We showed previously that HMGA2 regulates TGFβ1 expression (26). Furthermore, increased levels of TGFβ1 have been found in patients with myelofibrosis (30), and TGFβ1 has been suggested to play a role in the pathogenesis of myelofibrosis (31, 32). We observed that palbociclib treatment significantly reduced serum TGFβ1 level in Jak2VF/VF mice (Fig. 7B). We also observed a marked decrease in SMAD2 phosphorylation, a downstream target of the TGFβ signaling, in myelofibrosis PBMC, Jak2VF/VF mice BM, and SET-2 cells upon palbociclib treatment (Fig. 7C–E). Combined treatment of palbociclib and ruxolitinib resulted in greater inhibition of SMAD2 phosphorylation in myelofibrosis PBMC and Jak2VF/VF mice BM (Fig. 7C and D). Knockdown of CDK6 also resulted in similar decrease in SMAD2 phosphorylation in SET-2 cells (Fig. 7F). Thus, suppression of CDK6 activity by gene depletion or palbociclib treatment can downmodulate the TGFβ signaling pathway. In addition, we observed that in vivo treatment of palbociclib alone or in combination with ruxolitinib significantly reduced serum levels of IL6 and IL1β in Jak2VF/VF mice (Supplementary Fig. S7A and S7B), suggesting that palbociclib/ruxolitinib combination treatment can alleviate inflammation.

Figure 7.

Inhibition of CDK6 by palbociclib reduces the expression of fibrotic markers. A, Analysis from the RNA-seq data shows downregulation of genes related to TGFβ pathway in palbociclib-treated Jak2VF/VF mice LSK cells compared with vehicle-treated Jak2VF/VF LSK cells. B, Serum TGFβ1 levels in Jak2VF/VF mice treated with vehicle and palbociclib were assessed by ELISA (n = 8–10; **, P < 0.005). C, Immunoblot showing decreased phosphorylation of SMAD2 in palbociclib-treated myelofibrosis PBMCs. Combined treatment of palbociclib and ruxolitinib resulted in greater inhibition of SMAD2 phosphorylation. The myelofibrosis PBMC samples used in Figs. 6F and 7C are from the same experiment and the same β-actin was used as a loading control. D, Immunoblot showing decreased phosphorylation of SMAD2 in palbociclib-treated Jak2VF/VF mice BM. Combined treatment of palbociclib and ruxolitinib resulted in greater inhibition of SMAD2 phosphorylation. The Jak2VF/VF mice BM samples used in Figs. 6G and 7D are from the same experiment and the same β-actin was used as a loading control. E, Immunoblot showing decreased phosphorylation of SMAD2 in palbociclib-treated SET-2 cells. F, Knockdown of CDK6 markedly reduced phosphorylation of SMAD2 in SET-2 cells. The SET-2 cell lysates used in Figs. 6H and 7F are from the same experiment and the same β-actin was used as a loading control. G, Stimulation with TGFβ1 (50 ng/mL) significantly increased collagen I and collagen III expression in BM MSCs. Treatment of palbociclib (0.25 μmol/L) in the presence of TGFβ1 (50 ng/mL) significantly reduced collagen (I and III) expression. The mRNA expression was assessed by qRT-PCR and normalized by Hprt. Data from four independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005. H, Immunofluorescence images showing increased expression of collagen I and collagen III in MSCs stimulated with TGFβ1 (50 ng/mL). Palbociclib (0.25 μmol/L) treatment significantly reduced TGFβ1 induced collagen I and III expression. Collagen I (red), collagen III (green), and DAPI (blue). I, Stimulation with TGFβ1 (50 ng/mL) significantly increases Snail1 and αSMA expression in BM MSCs. Treatment of palbociclib (0.25 μmol/L) significantly reduced Snail1 and αSMA expression in BM MSCs. The mRNA expression was assessed by qRT-PCR and normalized by Hprt. Data from four independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05. J, Representative immunofluorescence images showing decreased expression of Snail and αSMA in the BM sections of Jak2VF/VF mice treated with palbociclib alone or in combination with ruxolitinib. Shown are Snail (green), αSMA (yellow), and DAPI (blue).

Figure 7.

Inhibition of CDK6 by palbociclib reduces the expression of fibrotic markers. A, Analysis from the RNA-seq data shows downregulation of genes related to TGFβ pathway in palbociclib-treated Jak2VF/VF mice LSK cells compared with vehicle-treated Jak2VF/VF LSK cells. B, Serum TGFβ1 levels in Jak2VF/VF mice treated with vehicle and palbociclib were assessed by ELISA (n = 8–10; **, P < 0.005). C, Immunoblot showing decreased phosphorylation of SMAD2 in palbociclib-treated myelofibrosis PBMCs. Combined treatment of palbociclib and ruxolitinib resulted in greater inhibition of SMAD2 phosphorylation. The myelofibrosis PBMC samples used in Figs. 6F and 7C are from the same experiment and the same β-actin was used as a loading control. D, Immunoblot showing decreased phosphorylation of SMAD2 in palbociclib-treated Jak2VF/VF mice BM. Combined treatment of palbociclib and ruxolitinib resulted in greater inhibition of SMAD2 phosphorylation. The Jak2VF/VF mice BM samples used in Figs. 6G and 7D are from the same experiment and the same β-actin was used as a loading control. E, Immunoblot showing decreased phosphorylation of SMAD2 in palbociclib-treated SET-2 cells. F, Knockdown of CDK6 markedly reduced phosphorylation of SMAD2 in SET-2 cells. The SET-2 cell lysates used in Figs. 6H and 7F are from the same experiment and the same β-actin was used as a loading control. G, Stimulation with TGFβ1 (50 ng/mL) significantly increased collagen I and collagen III expression in BM MSCs. Treatment of palbociclib (0.25 μmol/L) in the presence of TGFβ1 (50 ng/mL) significantly reduced collagen (I and III) expression. The mRNA expression was assessed by qRT-PCR and normalized by Hprt. Data from four independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05; **, P < 0.005. H, Immunofluorescence images showing increased expression of collagen I and collagen III in MSCs stimulated with TGFβ1 (50 ng/mL). Palbociclib (0.25 μmol/L) treatment significantly reduced TGFβ1 induced collagen I and III expression. Collagen I (red), collagen III (green), and DAPI (blue). I, Stimulation with TGFβ1 (50 ng/mL) significantly increases Snail1 and αSMA expression in BM MSCs. Treatment of palbociclib (0.25 μmol/L) significantly reduced Snail1 and αSMA expression in BM MSCs. The mRNA expression was assessed by qRT-PCR and normalized by Hprt. Data from four independent experiments are shown in bar graphs as mean ± SEM. *, P < 0.05. J, Representative immunofluorescence images showing decreased expression of Snail and αSMA in the BM sections of Jak2VF/VF mice treated with palbociclib alone or in combination with ruxolitinib. Shown are Snail (green), αSMA (yellow), and DAPI (blue).

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Next, we assessed the levels of fibrotic markers in the BM following palbociclib treatment. Collagen deposition in the BM stromal cells is directly linked to myelofibrosis (30). We previously showed that TGFβ1 stimulation significantly increased collagen I and III expression in the BM MSCs (26). Because we observed that palbociclib treatment inhibits the TGFβ signaling pathway, we asked whether palbociclib treatment could inhibit TGFβ1-induced collagen expression in BM MSCs. Indeed, we observed that palbociclib significantly inhibited TGFβ1-induced collagen I and III expression in BM MSCs (Fig. 7G). Ruxolitinib treatment, however, did not significantly inhibit TGFβ1-induced collagen I and III expression in BM MSCs (Supplementary Fig. S8). Immunofluorescence staining also showed that palbociclib treatment markedly reduced TGFβ1-induced collagen I and III expression in the BM MSCs (Fig. 7H).

TGFβ1 can also induce Snail and αSMA, which promote epithelial–mesenchymal transition (EMT) and extracellular matrix (ECM) production in different types of tissue fibrosis and cancer (33, 34). We found that expression of Snail and αSMA was significantly elevated upon TGFβ1 stimulation, and treatment of palbociclib almost completely inhibited TGFβ1-induced Snail and αSMA expression in the BM MSCs (Fig. 7I). Immunohistochemistry analysis on the BM sections from vehicle-treated Jak2VF/VF mice showed high level expression of Snail and αSMA (Fig. 7J). Palbociclib treatment alone or in combination with ruxolitinib significantly reduced Snail and αSMA expression, whereas ruxolitinib treatment alone had little or no effect on their expression (Fig. 7J). These data strongly suggest that inhibition of CDK6 by palbociclib blocks TGFβ1-induced upregulation of fibrotic factors in the BM and thus prevent the development and progression of myelofibrosis.

Myelofibrosis is the most severe form of MPN with limited treatment options. Currently approved JAK2 inhibitors provide only symptomatic relief without offering significant reduction in BM fibrosis (12). Complete remissions similar to those observed in CML with ABL kinase inhibitors cannot be achieved with current JAK2 inhibitors in myelofibrosis. So, there is an unmet critical need for development of new therapeutic approaches for treatment of myelofibrosis.

CDK6 is known to regulate G1 to S-phase transition of the cell cycle (15, 16). However, CDK6 is not essential for mammalian cell cycle (15, 17) and hematopoiesis appears to be normal in CDK6-deficient mice under steady-state conditions (35). Dysregulated expression of CDK6 has been observed in a variety of malignancies (16). It has been shown recently that loss of CDK6 attenuates the development of PV-like MPN in mice, although CDK6 inhibition by palbociclib does not significantly improve PV disease in Jak2V617F/+ mice (36). The contribution of CDK6 in myelofibrosis pathogenesis and the effects of CDK6 inhibition against myelofibrosis has remained unknown.

We found significant upregulation of CDK6 expression in hematopoietic progenitors of Jak2V617F knock-in mice and patients with myelofibrosis. So, we evaluated the efficacy of CDK4/6 inhibitor palbociclib alone or in combination with JAK1/2 inhibitor ruxolitinib in hematopoietic cells expressing MPN driver mutants and murine models of myelofibrosis. We tested palbociclib because it is a well-tolerated FDA-approved drug for treatment of HR-positive advanced breast cancer (37). We demonstrate that palbociclib treatment significantly inhibits the growth/survival of hematopoietic cells expressing JAK2V617F and MPLW515L. Palbociclib treatment also caused pronounced inhibition of clonogenic growth of myelofibrosis patient CD34+ cells. More importantly, palbociclib treatment alone markedly reduced splenomegaly and BM fibrosis in Jak2VF/VF and MPLW515L mouse models of myelofibrosis. The effects of palbociclib in reducing splenomegaly and BM fibrosis were even more pronounced than those observed with currently approved ruxolitinib therapy. Combined treatment of palbociclib/ruxolitinib resulted in almost complete inhibition of BM fibrosis in Jak2VF/VF and MPLW515L mouse models. Thus, our data suggest that palbociclib alone or in combination with ruxolitinib may have therapeutic potential for treatment of myelofibrosis.

Previous studies have suggested that CDK6 can act as a transcriptional regulator (16, 21, 22). Using transcriptome analysis, we found that expression of AURKA, AURKB, and HMGA2 was significantly downregulated in Jak2VF/VF LSK by palbociclib or palbociclib/ruxolitinib treatment. AURKA and AURKB play an important role in cell-cycle progression during mitosis and cytokinesis, and their aberrant expression has been associated with various malignancies (38, 39). Previous studies also have shown that inhibition of AURKA/AURKB can reduce the growth and survival of JAK2V617F-positive MPN cells and attenuate the BM fibrosis in mouse models of MPN (40, 41). HMGA2 is a chromatin-binding protein that plays a role in the self-renewal of HSC (42). Overexpression of HMGA2 has been observed in myelofibrosis progenitors (43, 44). We showed previously that HMGA2 regulates TGFβ1 expression and promotes the development of myelofibrosis in Jak2V617F mice (26). Because Palbociclib can inhibit both CDK4 and CDK6, we performed CDK6 knockdown in JAK2V617F-positive SET-2 cells. We observed that knockdown of CDK6 significantly reduced expression of AURKA, AURKB, and HMGA2 similar to that observed with palbociclib treatment. Therefore, AURKA, AURKB, and HMGA2 are bona fide targets of CDK6, and inhibition of CDK6 by palbociclib may inhibit the MPN cells and ameliorates myelofibrosis by downregulation of these target genes. Future studies will determine how inhibition of CDK6 by palbociclib regulates the expression of these genes.

We observed downregulation of NF-κB pathway in Jak2VF/VF LSK cells and reduced phosphorylation of NF-κB p65 subunit in myelofibrosis primary cells and Jak2VF/VF mice BM by palbociclib treatment. NF-κB is known as a master regulator of inflammation, and constitutive activation of NF-κB has been observed in MPN/myelofibrosis (45). It has been shown that CDK6 interacts with p65 and regulates the expression of NF-κB target genes (22). A recent study also suggested that CDK6 deficiency downregulates the expression of NF-κB target genes in Jak2V617F mouse LSK cells (36). We also observed significant reduction of IL6 and IL1β levels in Jak2VF/VF mice treated with palbociclib/ruxolitinib combination. Thus, combined treatment of palbociclib and ruxolitinib might attenuate the myelofibrosis phenotype by inhibiting the inflammatory pathway.

Previous reports have suggested an important role for TGFβ signaling in various tissue fibrosis (33, 46). Increased levels of TGFβ1 also have been observed in patients with myelofibrosis as well as in mouse models of myelofibrosis (30–32, 47). We observed downregulation of genes related to TGFβ signaling in palbociclib-treated Jak2VF/VF mice LSK cells. We also observed decreased serum levels of TGFβ1 in Jak2VF/VF mice treated with palbociclib. In addition, we observed decreased phosphorylation of SMAD2 following treatment with palbociclib as well as with knockdown of CDK6. TGFβ1 can induce the expression of collagen, Snail, and αSMA, which have been implicated in tissue fibrosis and cancer metastasis (34, 48). Palbociclib treatment significantly reduced the TGFβ1 induced collagen (I and III), Snail, and αSMA expression in BM MSCs. We also observed that treatment of palbociclib significantly reduced Snail and αSMA expression in the BM of Jak2VF/VF mice. Thus, palbociclib treatment may prevent the development/progression of BM fibrosis by inhibiting the TGFβ signaling and reducing the expression of fibrotic markers.

In summary, we demonstrate that inhibition of CDK6 by palbociclib significantly inhibits MPN cells and progenitors, and ameliorates BM fibrosis in multiple murine models of myelofibrosis. Palbociclib treatment preferentially inhibits the mutant hematopoietic progenitors. We also show that dual targeting of CDK6 and JAK2 using palbociclib and ruxolitinib provides greater inhibition of MPN cells and more pronounced inhibition of BM fibrosis. Palbociclib treatment can induce myelosuppression in patients with breast cancer. A combinatorial therapeutic approach involving palbociclib and ruxolitinib will enable lowering the doses of each of the inhibitors and thus reducing toxicities while enhancing the therapeutic efficacy. Results from our study support the clinical investigation of palbociclib and ruxolitinib combination in patients with myelofibrosis.

G. Mohi reports grants from NIH during the conduct of the study and Tolero Pharmaceutical Inc. outside the submitted work. No disclosures were reported by the other authors.

A. Dutta: Data curation, formal analysis, investigation, writing–original draft. D. Nath: Formal analysis, Investigation. Y. Yang: Investigation. B.T. Le: Formal analysis. G. Mohi: Conceptualization, formal analysis, supervision, funding acquisition, writing–original draft.

The authors thank Matthew Stuver for assistance with CDK6 knockdown studies in cell lines. The authors also thank the Flow Cytometry and Microscopy Core Facilities and the Biorepository and Tissue Research Facility (BTRF) of the University of Virginia for assistance with FACS sorting, confocal microscopy and MPN specimen procurement and processing. Flow Cytometry and Microscopy Cores are supported by the UVA Cancer Center through P30CA044578 grant. This work was supported by grants from the National Institutes of Health (R01 HL095685, R01 HL149893, R21 CA235472) awarded to G. Mohi.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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