Limits...
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.

Show MeSH

Related in: MedlinePlus

SHH pathway deregulation in chronic myeloid leukemia progression. a. Principal components plots derived from RNA-Seq data for 41 genes in the SHH pathway, from 7 chronic phase (CP; blue triangles) and 6 blast crisis (BC; red circles) untreated subjects, as well as 3 cord blood normal samples (CB; black diamonds) and 3 normal peripheral blood (NPB; black circles). b. Heatmap from unsupervised agglomerative hierarchical clustering of sonic hedgehog (SHH) pathway genes using RNA-Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients, 3 normal cord blood (CB) and 3 normal peripheral blood (NPB) sample. Samples labeled in bold correspond to patients that received clinical BCR-ABL inhibitor therapy. Red indicates over- and green, under-expression relative to the median RPKM (Log2 scale). Grey represent not expressed (RPKM = 0). c. Network analysis performed on differentially expressed genes between BC and CP revealed GLI2 as a key hub in the SHH pathway. d. Differentially expressed (DE) SHH genes at FDR 5% when comparing Blast crisis (BC) versus chronic phase (CP) or normal (CB and NPB). e. Box plots for GLI2 expression of 7 chronic phase (CP) and 6 blast crisis (BC) non-treated subjects, as well as 3 cord blood normal samples (CB) and 3 normal peripheral blood (NPB). Two-sided Jonckheere-Terpstra trend test: p = 0.014. f. GLI1 and GLI2 transcripts were compared using quantitative RT-PCR in FACS-purified human cord blood and normal peripheral blood CD34+CD38+Lin−PI− progenitor cells (n = 9, black), chronic phase CML (n = 7, blue) and in blast crisis CML (n = 10, red) patient derived samples. Values were normalized to RPL27 or HPRT housekeeping genes, and set to 1 for the normal progenitors. (Student’s t-test *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4414375&req=5

Fig1: SHH pathway deregulation in chronic myeloid leukemia progression. a. Principal components plots derived from RNA-Seq data for 41 genes in the SHH pathway, from 7 chronic phase (CP; blue triangles) and 6 blast crisis (BC; red circles) untreated subjects, as well as 3 cord blood normal samples (CB; black diamonds) and 3 normal peripheral blood (NPB; black circles). b. Heatmap from unsupervised agglomerative hierarchical clustering of sonic hedgehog (SHH) pathway genes using RNA-Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients, 3 normal cord blood (CB) and 3 normal peripheral blood (NPB) sample. Samples labeled in bold correspond to patients that received clinical BCR-ABL inhibitor therapy. Red indicates over- and green, under-expression relative to the median RPKM (Log2 scale). Grey represent not expressed (RPKM = 0). c. Network analysis performed on differentially expressed genes between BC and CP revealed GLI2 as a key hub in the SHH pathway. d. Differentially expressed (DE) SHH genes at FDR 5% when comparing Blast crisis (BC) versus chronic phase (CP) or normal (CB and NPB). e. Box plots for GLI2 expression of 7 chronic phase (CP) and 6 blast crisis (BC) non-treated subjects, as well as 3 cord blood normal samples (CB) and 3 normal peripheral blood (NPB). Two-sided Jonckheere-Terpstra trend test: p = 0.014. f. GLI1 and GLI2 transcripts were compared using quantitative RT-PCR in FACS-purified human cord blood and normal peripheral blood CD34+CD38+Lin−PI− progenitor cells (n = 9, black), chronic phase CML (n = 7, blue) and in blast crisis CML (n = 10, red) patient derived samples. Values were normalized to RPL27 or HPRT housekeeping genes, and set to 1 for the normal progenitors. (Student’s t-test *p < 0.05).

Mentions: To determine whether distinctive Hh pathway gene expression patterns predict CML progression, we utilized RNA-seq to compare expression levels of 41 a priori selected Hh pathway genes [19-21,45,46] in chronic phase (CP, n = 8), blast crisis (BC, n = 9), normal cord blood (CB, n = 3) and normal peripheral blood (NPB, n = 3) FACS-purified CD34+CD38+Lin− progenitors (Additional file 1: Table S1 and S2). Principal component analysis (PCA) and unsupervised hierarchical clustering revealed distinctive expression level differences among 41 Hh pathway genes that significantly (p = 0.047, permutation test) separated normal from untreated CP and BC progenitors and BCR-ABL inhibitor treated samples (Figure 1a, b). The expression levels of 20,613 genes above detectable expression levels (RPKM > 0.2 in over 75% of samples) were compared between untreated CP (n = 7) and BC (n = 6) progenitors. Among 1,495 differentially expressed (DE) genes (FDR < 0.05), key SHH genes were included (Figure 1d). Notably, a GLI2 network was enriched in BC LSC for upregulated DE genes using Ingenuity software (Figure 1c). Both RNA Seq (Figure 1e) and qRT PCR (Figure 1f) analyses confirmed significantly increased expression of the SHH pathway transcriptional activator, GLI2, during BC transformation. Together these data suggest that critical regulatory components of the Hh signaling pathway, such as GLI2, represent part of a core transcriptional program driving human myeloid LSC maintenance for BC CML, thereby providing the impetus for testing the capacity of a selective SMO antagonist to eradicate LSC.Figure 1


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)

SHH pathway deregulation in chronic myeloid leukemia progression. a. Principal components plots derived from RNA-Seq data for 41 genes in the SHH pathway, from 7 chronic phase (CP; blue triangles) and 6 blast crisis (BC; red circles) untreated subjects, as well as 3 cord blood normal samples (CB; black diamonds) and 3 normal peripheral blood (NPB; black circles). b. Heatmap from unsupervised agglomerative hierarchical clustering of sonic hedgehog (SHH) pathway genes using RNA-Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients, 3 normal cord blood (CB) and 3 normal peripheral blood (NPB) sample. Samples labeled in bold correspond to patients that received clinical BCR-ABL inhibitor therapy. Red indicates over- and green, under-expression relative to the median RPKM (Log2 scale). Grey represent not expressed (RPKM = 0). c. Network analysis performed on differentially expressed genes between BC and CP revealed GLI2 as a key hub in the SHH pathway. d. Differentially expressed (DE) SHH genes at FDR 5% when comparing Blast crisis (BC) versus chronic phase (CP) or normal (CB and NPB). e. Box plots for GLI2 expression of 7 chronic phase (CP) and 6 blast crisis (BC) non-treated subjects, as well as 3 cord blood normal samples (CB) and 3 normal peripheral blood (NPB). Two-sided Jonckheere-Terpstra trend test: p = 0.014. f. GLI1 and GLI2 transcripts were compared using quantitative RT-PCR in FACS-purified human cord blood and normal peripheral blood CD34+CD38+Lin−PI− progenitor cells (n = 9, black), chronic phase CML (n = 7, blue) and in blast crisis CML (n = 10, red) patient derived samples. Values were normalized to RPL27 or HPRT housekeeping genes, and set to 1 for the normal progenitors. (Student’s t-test *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4414375&req=5

Fig1: SHH pathway deregulation in chronic myeloid leukemia progression. a. Principal components plots derived from RNA-Seq data for 41 genes in the SHH pathway, from 7 chronic phase (CP; blue triangles) and 6 blast crisis (BC; red circles) untreated subjects, as well as 3 cord blood normal samples (CB; black diamonds) and 3 normal peripheral blood (NPB; black circles). b. Heatmap from unsupervised agglomerative hierarchical clustering of sonic hedgehog (SHH) pathway genes using RNA-Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients, 3 normal cord blood (CB) and 3 normal peripheral blood (NPB) sample. Samples labeled in bold correspond to patients that received clinical BCR-ABL inhibitor therapy. Red indicates over- and green, under-expression relative to the median RPKM (Log2 scale). Grey represent not expressed (RPKM = 0). c. Network analysis performed on differentially expressed genes between BC and CP revealed GLI2 as a key hub in the SHH pathway. d. Differentially expressed (DE) SHH genes at FDR 5% when comparing Blast crisis (BC) versus chronic phase (CP) or normal (CB and NPB). e. Box plots for GLI2 expression of 7 chronic phase (CP) and 6 blast crisis (BC) non-treated subjects, as well as 3 cord blood normal samples (CB) and 3 normal peripheral blood (NPB). Two-sided Jonckheere-Terpstra trend test: p = 0.014. f. GLI1 and GLI2 transcripts were compared using quantitative RT-PCR in FACS-purified human cord blood and normal peripheral blood CD34+CD38+Lin−PI− progenitor cells (n = 9, black), chronic phase CML (n = 7, blue) and in blast crisis CML (n = 10, red) patient derived samples. Values were normalized to RPL27 or HPRT housekeeping genes, and set to 1 for the normal progenitors. (Student’s t-test *p < 0.05).
Mentions: To determine whether distinctive Hh pathway gene expression patterns predict CML progression, we utilized RNA-seq to compare expression levels of 41 a priori selected Hh pathway genes [19-21,45,46] in chronic phase (CP, n = 8), blast crisis (BC, n = 9), normal cord blood (CB, n = 3) and normal peripheral blood (NPB, n = 3) FACS-purified CD34+CD38+Lin− progenitors (Additional file 1: Table S1 and S2). Principal component analysis (PCA) and unsupervised hierarchical clustering revealed distinctive expression level differences among 41 Hh pathway genes that significantly (p = 0.047, permutation test) separated normal from untreated CP and BC progenitors and BCR-ABL inhibitor treated samples (Figure 1a, b). The expression levels of 20,613 genes above detectable expression levels (RPKM > 0.2 in over 75% of samples) were compared between untreated CP (n = 7) and BC (n = 6) progenitors. Among 1,495 differentially expressed (DE) genes (FDR < 0.05), key SHH genes were included (Figure 1d). Notably, a GLI2 network was enriched in BC LSC for upregulated DE genes using Ingenuity software (Figure 1c). Both RNA Seq (Figure 1e) and qRT PCR (Figure 1f) analyses confirmed significantly increased expression of the SHH pathway transcriptional activator, GLI2, during BC transformation. Together these data suggest that critical regulatory components of the Hh signaling pathway, such as GLI2, represent part of a core transcriptional program driving human myeloid LSC maintenance for BC CML, thereby providing the impetus for testing the capacity of a selective SMO antagonist to eradicate LSC.Figure 1

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