GLI2 inhibition abrogates human leukemia stem cell dormancy

Anil Sadarangani,Gabriel Pineda,Ifat Geron,Minya Pu,Karen Messer,Amy Jackson-Fisher,Tannishtha Reya,Todd Vanarsdale,Russell Wall,Alice Shih,Lei Bao,Sacha Prashad ,Daniel Goff ,Sheldon R Morris ,Michael J Munchhof ,Kathleen M Lennon ,Angela C Court ,Marco A Marra ,Thomas J Hudson ,Hanna Mikkola ,Richard A Moore ,John Douglas Mcpherson ,Annelie Schairer ,Hye Jung Chun ,Mark D Minden ,Catriona Jamieson

doi:10.1186/s12967-015-0453-9

Abstract

BackgroundDormant 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.MethodsTo 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.ResultsNotably, 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.ConclusionIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0453-9) contains supplementary material, which is available to authorized users.

Highlights

Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs
Hh pathway deregulation signifies human leukemic progression and LSC generation To determine whether distinctive Hh pathway gene expression patterns predict Chronic myeloid leukemia (CML) progression, we utilized RNA sequencing (RNA-seq) to compare expression levels of 41 a priori selected Hh pathway genes [19,20,21,45,46] 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)
Both RNA Seq (Figure 1e) and qRT PCR (Figure 1f ) analyses confirmed significantly increased expression of the Sonic Hh (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 Spleen Weight (g) glioma associated (GLI) 2 Signal a d 0.8 0.6 0.4 0.2

Summary

Introduction

Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Chronic myeloid leukemia (CML) is a myeloproliferative disease arising from the acquisition of the Philadelphia chromosome (Ph) by hematopoietic stem cells (HSC) This is characterized by the reciprocal translocation between the long arms of chromosomes 9 and 22, t (9;22) (q34;q11) which in turn gives rise to a fusion of c-ABL and BCR to generate the BCR-ABL fusion gene that encodes the constitutively active tyrosine kinase BCR-ABL1 [1]. This results in the modulation of several key-signaling cascades leading to increased cell proliferation, induction of cellular transformation and blockage of apoptotic pathways. In advanced human myeloid malignancies that progress from antecedent hematologic malignancies, such as blast crisis (BC) CML, LSC have a proclivity for becoming dormant in protective niches [2,5,7,11,15,16,17,18]

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