icg-001 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Cancer stem cells (CSCs), including leukemia stem cells (LSCs), exhibit self-renewal capacity and differentiation potential and have the capacity to maintain or renew and propagate a tumor/leukemia. The initial isolation of CSCs/LSCs was in adult myelogenous leukemia, although more recently, the existence of CSCs in a wide variety of other cancers has been reported. CSCs, in general, and LSCs, specifically with respect to this review, are responsible for initiation of disease, therapeutic resistance and ultimately disease relapse. One key focus in cancer research over the past decade has been the development of therapies that safely eliminate the LSC/CSC population. One major obstacle to this goal is the identification of key mechanisms that distinguish LSCs from normal endogenous hematopoietic stem cells. An additional daunting feature that has recently come to light with advances in next-generation sequencing and single-cell sequencing is the heterogeneity within leukemias/tumors, with multiple combinations of mutations, gain and loss of function of genes, and so on being capable of driving disease, even within the CSC/LSC population. The focus of this review/perspective is on our work in identifying and validating, in both chronic myelogenous leukemia and acute lymphoblastic leukemia, a safe and efficacious mechanism to target an evolutionarily conserved signaling nexus, which constitutes a common "Achilles heel" for LSCs/CSCs, using small molecule-specific CBP/catenin antagonists.

Topics: beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cyclic AMP Response Element-Binding Protein; gamma Catenin; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Binding; Pyrimidinones

The development of the tyrosine kinase inhibitor Imatinib (IM) represents a milestone in CML (Chronic Myeloid Leukemia) treatment. However, it is not curative and patients develop IM resistance. IM resistance has been previously correlated with the emergence of drug-resistant LIC/LSC (Leukemia Initiating Cell/Leukemia Stem Cell) and increased nuclear catenin levels and enhanced Wnt signaling. It has been demonstrated previously that drug resistant CML LIC/LSC can be safely eliminated both in vitro and in vivo via disruption of the CBP/catenin interaction, utilizing the highly biochemically selective small molecule CBP/catenin antagonist ICG- 001.. Here, we utilized an in vitro IM selection of primary CML patients' samples to identify drug-resistant LIC/LSC populations. In this report, we characterized the drug-resistant CML LIC/LSC population using FACS, Smartchip qPCR and colony assays to analyze cell surface markers, transcriptomics and function.. As opposed to previous characterization of the CML leukemic stem cell population as being either CD34+CD38- or CD34+CD38+, the in vitro selected Imatinib resistant (IM-R) CML LSC population was consistently CD34-CD38-. In Long-Term Culture Initiating Cell assay (LTC-IC, a surrogate assay for long term repopulating stem cells), our results suggest that the CBP/catenin antagonist ICG- 001 sensitizes LIC/LSC to IM treatment by forced differentiative elimination of the CML LIC/LSC population.. In vitro selected IM resistant cells are negative for both CD34 and CD38 by FACS analysis. These cells acquire CD34/CD38 expression after co-culture with stromal cells. CBP/catenin antagonist ICG-001 facilitates IM function in eliminating these cells.

Topics: Antigens, CD; Biomarkers, Tumor; Bridged Bicyclo Compounds, Heterocyclic; Catenins; Cell Line, Tumor; CREB-Binding Protein; Drug Resistance, Neoplasm; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplasm Proteins; Neoplastic Stem Cells; Phenotype; Pyrimidinones; Tumor Stem Cell Assay

CREB-binding protein (CBP) and p300 are highly homologous transcriptional coactivators with unique, non-redundant roles that bind a wide array of proteins, including catenins-β and γ. ICG-001 is a small-molecule inhibitor that specifically inhibits the CBP/catenin interaction. Importantly, ICG-001 does not inhibit the p300/catenin interaction. We demonstrate that specifically inhibiting the interaction between CBP and catenin with ICG-001 results in the differentiation of quiescent drug-resistant chronic myelogenous leukemia-initiating cells (CML LICs), thereby sensitizing them to BCR-ABL tyrosine kinase inhibitors, for example, Imatinib. Using ICG-001 in a NOD/SCID/IL2Rγ(-/-) mouse model of engrafted human chronic myelogenous leukemia, we now demonstrate the complete elimination of engrafted leukemia after only one course of combined chemotherapy. Combination-treated animals live as long as their non-engrafted littermates. Results from these studies demonstrate that specifically antagonizing the CBP/catenin interaction with ICG-001 can eliminate drug-resistant CML LICs without deleterious effects to the normal endogenous hematopoietic stem cell population.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Catenins; Cell Line, Tumor; CREB-Binding Protein; Drug Resistance, Neoplasm; E1A-Associated p300 Protein; Female; Fusion Proteins, bcr-abl; Humans; Immunoblotting; Interleukin Receptor Common gamma Subunit; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Neoplastic Stem Cells; Protein Binding; Pyrimidinones; Reverse Transcriptase Polymerase Chain Reaction; Xenograft Model Antitumor Assays

Previously, we demonstrated that survivin expression is CBP/β-catenin/TCF-dependent. Now, using NCI-H28 cells, which harbor a homozygous deletion of β-catenin, we demonstrate that survivin transcription can similarly be mediated by nuclear γ-catenin. ICG-001, a specific inhibitor of binding to the N-terminus of CBP, effectively attenuates survivin expression. We demonstrate that γ-catenin by binding to TCF family members and specifically recruiting the coactivator CBP drives survivin transcription particularly in β-catenin-deficient cells. We also examined the relative expression of γ-catenin and β-catenin in 90 cases of chronic myeloid leukemia (CML) in a published gene expression microarray data base. A statistically significant negative correlation between γ-catenin and β-catenin was found in AP/BC cases (-0.389, P = 0.006). Furthermore, in subsequent independent validation studies by qPCR in 28 CP and BC patients increased γ-catenin expression predominated in BC cases and was associated with concomitantly increased survivin expression. Gene expression was 3- and 6-fold greater in BC patients as compared to CP patients, for γ-catenin and survivin, respectively. Consistent with this observation, nuclear γ-catenin accumulation was evident in this population consistent with a potential transcriptional role. Combined treatment with imatinib mesylate (IM) and ICG-001 significantly inhibited colony formation in sorted CD34(+) CML progenitors (survivin(+)/γ-catenin(high)/β-catenin(low)) isolated from one BC and one AP patient resistant to IM. Therefore, we believe that the ability of ICG-001 to block both the CBP/γ-catenin interaction and the CBP/β-catenin interaction may have clinical significance in cancers in which γ-catenin plays a significant transcriptional role.

Topics: Animals; Antigens, CD34; Antineoplastic Agents; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; CREB-Binding Protein; gamma Catenin; Hematopoietic Stem Cells; Humans; Inhibitor of Apoptosis Proteins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; p300-CBP Transcription Factors; Pyrimidinones; Recombinant Fusion Proteins; RNA, Messenger; RNA, Small Interfering; Survivin; TCF Transcription Factors; Transcription, Genetic