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GLI2 inhibition abrogates human leukemia stem cell dormancy.

Sadarangani A, Pineda G, Lennon KM, Chun HJ, Shih A, Schairer AE, Court AC, Goff DJ, Prashad SL, Geron I, Wall R, McPherson JD, Moore RA, Pu M, Bao L, Jackson-Fisher A, Munchhof M, VanArsdale T, Reya T, Morris SR, Minden MD, Messer K, Mikkola HK, Marra MA, Hudson TJ, Jamieson CH - J Transl Med (2015)

Bottom Line: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs.Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. asadarangani@ucsd.edu.

ABSTRACT

Background: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.

Methods: To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.

Results: Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.

Conclusion: In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

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PF-04449913 induced cell cycle activation enhances TKI sensitivity. a. Schematic of in vivo experiments. RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ BC CML cells within 48 hours of birth. Engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or the combination. b. Graph of myeloid sarcoma count in blast crisis CML engrafted mice in vehicle (n =13, blue), PF-04449913 (n=7, purple), dasatinib (n =6, red) and combination (n =3, black) treated mice +/− SEM; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis c. FACS analysis showing percentage of marrow engrafted blast crisis progenitor LSC (n = 3 patients) after 14-day treatment with vehicle (n =31, blue), PF-04449913 (n =25, purple), dasatinib (n =27, maroon) and combination (n=27, grey). *p < 0.05 by ANOVA and Tukey post-hoc analysis d. BCR-ABL transcripts in the blast crisis CML engrafted marrow mice after 14 days of treatment. Graph shows normalized BCR-ABL expression (HPRT) +/− SEM; *p < 0.05 by ANOVA and Tukey post-hoc analysis e. Hedgehog pathway gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n = 3, blue), PF-04449913 (n = 4, purple) dasatinib (n = 4, maroon), combination (n=3, dark grey). Expression levels of seven genes were significantly altered by synergistic effect of PF-04449913 and Dasatinib (NUMB, PRKACB, CTNNB1, FKBP8, CSNK1A1, CSNK1D and STK36), where five represent SHH regulatory genes (graphed). f. Mice serially transplanted with FACS purified human progenitors from LSC engrafted mice treated with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n = 8, maroon) or combination (n=7, grey) were examined for myeloid sarcomas; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis.
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Fig5: PF-04449913 induced cell cycle activation enhances TKI sensitivity. a. Schematic of in vivo experiments. RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ BC CML cells within 48 hours of birth. Engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or the combination. b. Graph of myeloid sarcoma count in blast crisis CML engrafted mice in vehicle (n =13, blue), PF-04449913 (n=7, purple), dasatinib (n =6, red) and combination (n =3, black) treated mice +/− SEM; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis c. FACS analysis showing percentage of marrow engrafted blast crisis progenitor LSC (n = 3 patients) after 14-day treatment with vehicle (n =31, blue), PF-04449913 (n =25, purple), dasatinib (n =27, maroon) and combination (n=27, grey). *p < 0.05 by ANOVA and Tukey post-hoc analysis d. BCR-ABL transcripts in the blast crisis CML engrafted marrow mice after 14 days of treatment. Graph shows normalized BCR-ABL expression (HPRT) +/− SEM; *p < 0.05 by ANOVA and Tukey post-hoc analysis e. Hedgehog pathway gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n = 3, blue), PF-04449913 (n = 4, purple) dasatinib (n = 4, maroon), combination (n=3, dark grey). Expression levels of seven genes were significantly altered by synergistic effect of PF-04449913 and Dasatinib (NUMB, PRKACB, CTNNB1, FKBP8, CSNK1A1, CSNK1D and STK36), where five represent SHH regulatory genes (graphed). f. Mice serially transplanted with FACS purified human progenitors from LSC engrafted mice treated with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n = 8, maroon) or combination (n=7, grey) were examined for myeloid sarcomas; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis.

Mentions: Having observed the reduction in dormancy following SMO inhibition, we investigated whether PF-04449913 sensitized BC LSC to dasatinib (Figure 5a) - a potent BCR-ABL TKI that targets dividing cells. Engrafted mice treated with a combination of PF-04449913 and dasatinib showed a significant reduction (p < 0.01, ANOVA) in myeloid sarcoma formation (Figure 5b) as well as in both bone marrow LSC burden (p < 0.05, ANOVA) (Figure 5c) and BCR-ABL1 (p < 0.05, ANOVA) expression (Figure 5d). Furthermore, quantitative RT-PCR Hh array analysis of FACS-purified progenitors revealed significantly (p = 0.05, Limma method) increased expression of negative Hh pathway regulators, including NUMB, PRKACB, FKBP8, CSNK1A1 and CSNK1D (Figure 5e) following combination therapy. Serial transplantation experiments demonstrated a significantly reduced capacity of LSC to form myeloid sarcomas (p < 0.01, ANOVA) following PF-04449913 and dasatinib therapy (Figure 5f). This study has provided the impetus for the on-going Phase 1b/ll combination SHH inhibitor and TKI inhibitor clinical trials for patients with advanced myeloid malignancies to both halt progression and prevent relapse.Figure 5


GLI2 inhibition abrogates human leukemia stem cell dormancy.

Sadarangani A, Pineda G, Lennon KM, Chun HJ, Shih A, Schairer AE, Court AC, Goff DJ, Prashad SL, Geron I, Wall R, McPherson JD, Moore RA, Pu M, Bao L, Jackson-Fisher A, Munchhof M, VanArsdale T, Reya T, Morris SR, Minden MD, Messer K, Mikkola HK, Marra MA, Hudson TJ, Jamieson CH - J Transl Med (2015)

PF-04449913 induced cell cycle activation enhances TKI sensitivity. a. Schematic of in vivo experiments. RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ BC CML cells within 48 hours of birth. Engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or the combination. b. Graph of myeloid sarcoma count in blast crisis CML engrafted mice in vehicle (n =13, blue), PF-04449913 (n=7, purple), dasatinib (n =6, red) and combination (n =3, black) treated mice +/− SEM; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis c. FACS analysis showing percentage of marrow engrafted blast crisis progenitor LSC (n = 3 patients) after 14-day treatment with vehicle (n =31, blue), PF-04449913 (n =25, purple), dasatinib (n =27, maroon) and combination (n=27, grey). *p < 0.05 by ANOVA and Tukey post-hoc analysis d. BCR-ABL transcripts in the blast crisis CML engrafted marrow mice after 14 days of treatment. Graph shows normalized BCR-ABL expression (HPRT) +/− SEM; *p < 0.05 by ANOVA and Tukey post-hoc analysis e. Hedgehog pathway gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n = 3, blue), PF-04449913 (n = 4, purple) dasatinib (n = 4, maroon), combination (n=3, dark grey). Expression levels of seven genes were significantly altered by synergistic effect of PF-04449913 and Dasatinib (NUMB, PRKACB, CTNNB1, FKBP8, CSNK1A1, CSNK1D and STK36), where five represent SHH regulatory genes (graphed). f. Mice serially transplanted with FACS purified human progenitors from LSC engrafted mice treated with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n = 8, maroon) or combination (n=7, grey) were examined for myeloid sarcomas; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis.
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Fig5: PF-04449913 induced cell cycle activation enhances TKI sensitivity. a. Schematic of in vivo experiments. RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ BC CML cells within 48 hours of birth. Engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or the combination. b. Graph of myeloid sarcoma count in blast crisis CML engrafted mice in vehicle (n =13, blue), PF-04449913 (n=7, purple), dasatinib (n =6, red) and combination (n =3, black) treated mice +/− SEM; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis c. FACS analysis showing percentage of marrow engrafted blast crisis progenitor LSC (n = 3 patients) after 14-day treatment with vehicle (n =31, blue), PF-04449913 (n =25, purple), dasatinib (n =27, maroon) and combination (n=27, grey). *p < 0.05 by ANOVA and Tukey post-hoc analysis d. BCR-ABL transcripts in the blast crisis CML engrafted marrow mice after 14 days of treatment. Graph shows normalized BCR-ABL expression (HPRT) +/− SEM; *p < 0.05 by ANOVA and Tukey post-hoc analysis e. Hedgehog pathway gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n = 3, blue), PF-04449913 (n = 4, purple) dasatinib (n = 4, maroon), combination (n=3, dark grey). Expression levels of seven genes were significantly altered by synergistic effect of PF-04449913 and Dasatinib (NUMB, PRKACB, CTNNB1, FKBP8, CSNK1A1, CSNK1D and STK36), where five represent SHH regulatory genes (graphed). f. Mice serially transplanted with FACS purified human progenitors from LSC engrafted mice treated with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n = 8, maroon) or combination (n=7, grey) were examined for myeloid sarcomas; *p < 0.05 and *p < 0.01 by ANOVA and Tukey post-hoc analysis.
Mentions: Having observed the reduction in dormancy following SMO inhibition, we investigated whether PF-04449913 sensitized BC LSC to dasatinib (Figure 5a) - a potent BCR-ABL TKI that targets dividing cells. Engrafted mice treated with a combination of PF-04449913 and dasatinib showed a significant reduction (p < 0.01, ANOVA) in myeloid sarcoma formation (Figure 5b) as well as in both bone marrow LSC burden (p < 0.05, ANOVA) (Figure 5c) and BCR-ABL1 (p < 0.05, ANOVA) expression (Figure 5d). Furthermore, quantitative RT-PCR Hh array analysis of FACS-purified progenitors revealed significantly (p = 0.05, Limma method) increased expression of negative Hh pathway regulators, including NUMB, PRKACB, FKBP8, CSNK1A1 and CSNK1D (Figure 5e) following combination therapy. Serial transplantation experiments demonstrated a significantly reduced capacity of LSC to form myeloid sarcomas (p < 0.01, ANOVA) following PF-04449913 and dasatinib therapy (Figure 5f). This study has provided the impetus for the on-going Phase 1b/ll combination SHH inhibitor and TKI inhibitor clinical trials for patients with advanced myeloid malignancies to both halt progression and prevent relapse.Figure 5

Bottom Line: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs.Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. asadarangani@ucsd.edu.

ABSTRACT

Background: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.

Methods: To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.

Results: Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.

Conclusion: In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

Show MeSH
Related in: MedlinePlus