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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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Selective shh inhibition reduces lsc burden in stromal co-cultures. a. Chemical structure of PF-04449913, a selective smoothened (SMO) antagonist. b. FACS analysis revealed a significant (Student’s t-test, *p = 0.047) reduction in blast crisis leukemic progenitor survival (n = 4 patient derived samples) following 7 days of PF-04449913 (1 μM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures. c. Cord blood (n = 3) CD34+ cells were plated on SL/M2 co-cultures and treated with vehicle (DMSO) or PF-04449913 (1uM) for 7 days. Colony forming unit (CFU) survival was determined and compared to vehicle treatment. d. Spleen weight in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n = 16, blue) or PF-04449913 (n = 12; 100 mg/kg daily, purple). A significant (Student t-test, *p = 0.006) reduction is observed after PF-044449913 treatment. e. Nanoproteomic (CB1000) traces of total GLI2 protein of CD34 + CD38+ FACS sorted derived from the spleen of mice (n = 5) after vehicle (blue) or PF-04449913 (green) treatment for 14 days with 100 mg/kg daily. f. GLI2 expression was determined after normalizing the area under the curve (AUC) to a β2-microglobulin (β2M) loading control (Student’s t-test *p = 0.001) g. Confocal fluorescence microscopic analysis of spleen sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913. Photomicrographs of sections stained with DAPI and antibodies specific for human CD45, human GLI2 and the merged image.
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Fig2: Selective shh inhibition reduces lsc burden in stromal co-cultures. a. Chemical structure of PF-04449913, a selective smoothened (SMO) antagonist. b. FACS analysis revealed a significant (Student’s t-test, *p = 0.047) reduction in blast crisis leukemic progenitor survival (n = 4 patient derived samples) following 7 days of PF-04449913 (1 μM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures. c. Cord blood (n = 3) CD34+ cells were plated on SL/M2 co-cultures and treated with vehicle (DMSO) or PF-04449913 (1uM) for 7 days. Colony forming unit (CFU) survival was determined and compared to vehicle treatment. d. Spleen weight in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n = 16, blue) or PF-04449913 (n = 12; 100 mg/kg daily, purple). A significant (Student t-test, *p = 0.006) reduction is observed after PF-044449913 treatment. e. Nanoproteomic (CB1000) traces of total GLI2 protein of CD34 + CD38+ FACS sorted derived from the spleen of mice (n = 5) after vehicle (blue) or PF-04449913 (green) treatment for 14 days with 100 mg/kg daily. f. GLI2 expression was determined after normalizing the area under the curve (AUC) to a β2-microglobulin (β2M) loading control (Student’s t-test *p = 0.001) g. Confocal fluorescence microscopic analysis of spleen sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913. Photomicrographs of sections stained with DAPI and antibodies specific for human CD45, human GLI2 and the merged image.

Mentions: A clinical small molecule SMO antagonist, PF-04449913 (Figure 2a), which was shown to compete for binding to human SMO (amino acids 181–787) with an IC50 of 4 nM (Additional file 1: Figure S1a), was utilized in this study, which is know to reduce GLI expression. PF-04449913 inhibited sonic hedgehog (Shh) stimulated luciferase expression in mouse embryonic fibroblasts with an IC50 of 6.8 nM (Additional file 1: Figure S1b, c); and significantly reduced medulloblastoma growth in a Ptch1+/−p53+/− allograft model (Additional file 1: Figure S1d, e) at doses that decreased murine Shh target gene expression (Additional file 1: Figure S1f-h). To recapitulate extrinsic growth regulatory cues provided by the LSC niche, human BC CML progenitors were co-cultured on human SCF, IL-3 and G-CSF secreting (SL/M2) stromal layers. In stromal co-culture experiments, FACS analysis demonstrated a significant reduction in BC LSC (Figure 2b) by PF-04449913 when compared with normal progenitors and (Figure 2c). Importantly, human BC LSC engrafted RAG2−/−γc−/− mice treated daily with PF-04449913 compared with vehicle treated controls had a significant spleen weight reduction (p = 0.006) (Figure 2d). This reduction in leukemic burden corresponded with decreased GLI2 protein expression, as determined by both nanoproteomic analysis of FACS purified human progenitors (Figure 2e, f) and GLI2 confocal fluorescence microscopic analysis of splenic sections (Figure 2 g).Figure 2


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)

Selective shh inhibition reduces lsc burden in stromal co-cultures. a. Chemical structure of PF-04449913, a selective smoothened (SMO) antagonist. b. FACS analysis revealed a significant (Student’s t-test, *p = 0.047) reduction in blast crisis leukemic progenitor survival (n = 4 patient derived samples) following 7 days of PF-04449913 (1 μM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures. c. Cord blood (n = 3) CD34+ cells were plated on SL/M2 co-cultures and treated with vehicle (DMSO) or PF-04449913 (1uM) for 7 days. Colony forming unit (CFU) survival was determined and compared to vehicle treatment. d. Spleen weight in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n = 16, blue) or PF-04449913 (n = 12; 100 mg/kg daily, purple). A significant (Student t-test, *p = 0.006) reduction is observed after PF-044449913 treatment. e. Nanoproteomic (CB1000) traces of total GLI2 protein of CD34 + CD38+ FACS sorted derived from the spleen of mice (n = 5) after vehicle (blue) or PF-04449913 (green) treatment for 14 days with 100 mg/kg daily. f. GLI2 expression was determined after normalizing the area under the curve (AUC) to a β2-microglobulin (β2M) loading control (Student’s t-test *p = 0.001) g. Confocal fluorescence microscopic analysis of spleen sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913. Photomicrographs of sections stained with DAPI and antibodies specific for human CD45, human GLI2 and the merged image.
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Related In: Results  -  Collection

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Fig2: Selective shh inhibition reduces lsc burden in stromal co-cultures. a. Chemical structure of PF-04449913, a selective smoothened (SMO) antagonist. b. FACS analysis revealed a significant (Student’s t-test, *p = 0.047) reduction in blast crisis leukemic progenitor survival (n = 4 patient derived samples) following 7 days of PF-04449913 (1 μM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures. c. Cord blood (n = 3) CD34+ cells were plated on SL/M2 co-cultures and treated with vehicle (DMSO) or PF-04449913 (1uM) for 7 days. Colony forming unit (CFU) survival was determined and compared to vehicle treatment. d. Spleen weight in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n = 16, blue) or PF-04449913 (n = 12; 100 mg/kg daily, purple). A significant (Student t-test, *p = 0.006) reduction is observed after PF-044449913 treatment. e. Nanoproteomic (CB1000) traces of total GLI2 protein of CD34 + CD38+ FACS sorted derived from the spleen of mice (n = 5) after vehicle (blue) or PF-04449913 (green) treatment for 14 days with 100 mg/kg daily. f. GLI2 expression was determined after normalizing the area under the curve (AUC) to a β2-microglobulin (β2M) loading control (Student’s t-test *p = 0.001) g. Confocal fluorescence microscopic analysis of spleen sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913. Photomicrographs of sections stained with DAPI and antibodies specific for human CD45, human GLI2 and the merged image.
Mentions: A clinical small molecule SMO antagonist, PF-04449913 (Figure 2a), which was shown to compete for binding to human SMO (amino acids 181–787) with an IC50 of 4 nM (Additional file 1: Figure S1a), was utilized in this study, which is know to reduce GLI expression. PF-04449913 inhibited sonic hedgehog (Shh) stimulated luciferase expression in mouse embryonic fibroblasts with an IC50 of 6.8 nM (Additional file 1: Figure S1b, c); and significantly reduced medulloblastoma growth in a Ptch1+/−p53+/− allograft model (Additional file 1: Figure S1d, e) at doses that decreased murine Shh target gene expression (Additional file 1: Figure S1f-h). To recapitulate extrinsic growth regulatory cues provided by the LSC niche, human BC CML progenitors were co-cultured on human SCF, IL-3 and G-CSF secreting (SL/M2) stromal layers. In stromal co-culture experiments, FACS analysis demonstrated a significant reduction in BC LSC (Figure 2b) by PF-04449913 when compared with normal progenitors and (Figure 2c). Importantly, human BC LSC engrafted RAG2−/−γc−/− mice treated daily with PF-04449913 compared with vehicle treated controls had a significant spleen weight reduction (p = 0.006) (Figure 2d). This reduction in leukemic burden corresponded with decreased GLI2 protein expression, as determined by both nanoproteomic analysis of FACS purified human progenitors (Figure 2e, f) and GLI2 confocal fluorescence microscopic analysis of splenic sections (Figure 2 g).Figure 2

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