Rui-Yu Wang

Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions in lipid rafts

We and others have previously demonstrated that p210 Bcr-Abl tyrosine kinase inhibits stromal cell-derived factor-1α/CXCR4 chemokine receptor signaling, contributing to the deficient adhesion of chronic myeloid leukemia (CML) cells to bone marrow stroma. Conversely, exposure of CML cells to a tyrosine kinase inhibitor (TKI) enhances migration of CML cells towards stromal cell layers and promotes non-pharmacological resistance to imatinib. Src-related kinase Lyn is known to interact with CXCL12/CXCR4 signaling and is directly activated by p210 Bcr-Abl. In this study, we demonstrate that TKI treatment promoted CXCR4 redistribution into the lipid raft fraction, in which it co-localized with active phosphorylated form of Lyn (LynTyr396) in CML cells. Lyn inhibition or cholesterol depletion abrogated imatinib-induced migration, and dual Src/Abl kinase inhibitor dasatinib induced fewer CML cells to migrate to the stroma. These findings demonstrate the novel mechanism of microenvironment-mediated resistance through lipid raft modulation, which involves compartmental changes of the multivalent CXCR4 and Lyn complex. We propose that pharmacological targeting of lipid rafts may eliminate bone marrow-resident CML cells through interference with microenvironment-mediated resistance.

Activation of Apoptosis Signaling Eliminates CD34+ Progenitor Cells in Blast Crisis CML Independent of Response to Tyrosine Kinase Inhibitors

Despite being highly effective for newly diagnosed chronic myeloid leukemia (CML), imatinib not only is inactive against quiescent CML stem cells, but also has limited activity against blast crisis (BC) CML. The relative activity of Bcr-Abl and the expression levels of antiapoptotic proteins in proliferating and quiescent CD34+ BC CML progenitor cells and the effects of targeting antiapoptotic proteins in these cells are unknown. Here we report higher levels of p-CrkL in quiescent than in proliferating CD34+ progenitor cells and comparable expression levels of Bcl-2, Bcl-xL, Mcl-1 and XIAP in the two populations in BC CML. Inhibition of Bcl-2/Bcl-xL by ABT-737 in cells from patients with tyrosine kinase inhibitor (TKI)-resistant BC CML promoted apoptosis in quiescent CD34+ progenitor cells with an efficacy similar to that in proliferating cells. Combination of ABT-737 with imatinib (which decreases Mcl-1 levels) or triptolide (which decreases Mcl-1 and XIAP) synergistically induced death of both proliferating and quiescent CD34+ progenitor cells obtained from TKI-resistant BC CML patients. These results suggest that antiapoptotic proteins are critical targets in BC CML and that activation of apoptosis signaling can eliminate both proliferating and quiescent CD34+ progenitor cells in BC CML, independent of response to TKIs.

Leukemia cell mobilization with G-CSF plus plerixafor during busulfan-fludarabine conditioning for allogeneic stem cell transplantation.

We hypothesized that during conditioning chemotherapy for allogeneic stem cell transplant (allo-SCT), the disruption of stromal–leukemia interactions using G-CSF in combination with the CXCR4-specific inhibitor, plerixafor, may promote the release of leukemic cells from the niche and increase tumor elimination. In a phase 1/2 investigation, we treated 45 AML/myelodysplastic syndrome (MDS)/CML patients (34 AML, 7 MDS and 4 CML) with G-CSF (10 μg/kg daily for 6 days starting on day −9) plus plerixafor (doses of 0, 80, 160 or 240 μg/kg daily for 4 days starting on day −7) along with the busulfan–fludarabine (Bu–Flu) conditioning regimen. In the phase 1 part, we determined that G-CSF plus plerixafor is safe in this setting. We compared the clinical effects and outcomes of AML/MDS study patients (n=40) with 164 patients from a historical data set who received Bu–Flu alone before allo-SCT by stratifying on cytogenetics and disease status to correct for bias. Study patients had increased myeloid chimerism and lower rates of GvHD. There was no significant difference in relapse-free survival or overall survival. The G-CSF plus plerixafor combination increased circulating WBCs, CD34+ cells and CXCR4+ cells, and preferentially mobilized FISH+ leukemic cells.

Flow cytometry and fluorescence in situ hybridization to detect residual neuroblastoma cells in bone marrow

In patients with neuroblastoma morphological assessment of BM for residual NB cells is not precise, particularly when the number of tumor cells is small.

Mesenchymal stem cells as delivery systems for cancer and leukemia gene therapy

3194 Background: We previously demonstrated that intravenously (IV) injected bone marrow-derived mesenchymal stem cells (MSC) integrate into solid tumors as stromal fibroblasts and proliferate selectively in situ. We here propose a novel cancer therapy concept based on the intratumoral drug production by gene-modified MSC. METHODS MSC were transduced with adeno (AdV)- or adeno-associated (AAV) virus carrying marker- or therapeutic genes. Gene-modified MSC were injected IV into mice carrying cancer xenografts. RESULTS MSC were found only in tumors, where they proliferate not in normal tissues. MSC producing human interferon-beta (IFNβ-MSC) directly inhibited the growth of metastatic A375 melanoma and MDA 231 breast carcinoma following IV injection (p=0.0073), while recombinant IFNβ protein injected subcutaneously (SC) did not (p=0.14). IFNβ-MSC doubled the survival of tumor-bearing mice (p=0.001) while SC injected IFNβ was ineffective (p=0.021 vs p=0.4). Intraperitoneal (IP) injections of IFNβ-MSC into mice with ovarian carcinomas doubled their survival (SKOV-3) and cured 70% of OVAR-3 mice. IP administration of MSC carrying a replication competent oncolytic adenovirus prolonged survival. MSC injected into the carotid artery (IA) of mice with gliomas selectively proliferated in gliomas, not in normal brain tissues. Intratumoral injection of IFNβ-MSC in vivo significantly inhibited tumor growth. In a model of chronic myelogenous leukemia in blast crisis (KBM5), interferon α (IFNα) produced in vivo by MSC induced tumor regressions and doubled the survival of mice. The melanoma differentiation-associated gene 7 (MDA7) produced by MSC, preferentially inhibited Gleevec resistant KBM5 CML blast crisis cells. CONCLUSIONS Results suggest that gene-modified MSC selectively grow in tumors and inhibit the growth of leukemias, and metastatic cancers. Tumor inhibition required spatial proximity of MSC to the malignant cells. The findings suggest that therapeutic proteins or oncolytic replicating adenovirus have potent anti-tumor effects when produced in situ and suggest the use of MSC as tumor-selective gene therapy delivery systems. No significant financial relationships to disclose.

Abstract 1391: Single cell analysis of Lin-CD34-CD45- cells from primary AML samples reveals leukemia clones with stem cell-like properties distinct from CD34+CD38-CD123+ LSC

Phenotypically characterized Lin-CD34+CD38-CD123+ AML cells (leukemia stem cells, LSC) are believed to contain leukemia initiating cells (LIC). In the normal hematopoietic developmental hierarchy, CD34- cells are primitive hematopoietic stem cells that occupy the apex of the hematopoietic tree (Cell Stem Cell. 2013 Aug 1;13(2):161-74). A subpopulation of cells from normal individuals that lack cell surface markers (Lin-CD34-CD45- phenotype) were previously shown to have hematopoietic (and endothelial) potential (Blood. 2011 Aug 25;118(8):2105-15). Lin-CD34-CD45- cells from AML patient samples have also been shown to have LIC capacity in limited experiments (J Clin Invest. 2011 Jan;121(1):384-95). We investigated samples from 40 AML patients with active disease to determine the prevalence and frequency of Lin-CD34-CD45- cells, and determined that Lin-CD34-CD45- cells comprise 0.18 ± 0.07% of mononuclear cells in AML patients, comparable to the frequency found in normal samples (0.35 ± 0.20%, P = 0.32, n = 10). In 15 AML samples with informative cytogenetics, Lin-CD34-CD45- subpopulation contained high percentages of leukemia cells as determined by FISH (58.5 ± 8.5%). Relapsed patients with ≥ five prior different lines of therapy had significantly higher percentages of relative leukemic Lin-CD34-CD45- cells (84.6 ± 5.4% vs. 51.0 ± 11.3%, P = 0.015), and the number of prior therapies positively correlated with the fraction of leukemic Lin-CD34-CD45- cells (r = 0.55, P = 0.032). Using conventional flow cytometry and mass cytometry we sought to determine whether individual Lin-CD34-CD45- cells contained any identifying cell surface markers. The Lin-CD34-CD45- population was heterogeneous among and within patients, however some samples contained Lin-CD34-CD45- cells that expressed stem cell markers including CD38, CD47, CD99, CD117, CD146, or CD184. We further evaluated the Lin-CD34-CD45- population, LSC, and bulk AML by gene expression arrays using three primary AML samples. Gene set enrichment revealed that in comparison to LSC, Lin-CD34-CD45- cells have unique expression patterns similar to chemoresistant stem cells characterized by increased proliferation and oxidative phosphorylation. Single cell RNA-sequencing of Lin-CD34-CD45- cells, LSC, and bulk leukemia from one FLT3+ DNMT3A-mutated sample confirmed that individual Lin-CD34-CD45- cells had leukemia gene expression patterns. While some individual Lin-CD34-CD45- cells had gene expression similar to LSC, others possessed unique gene expression profiles. Results suggest that Lin-CD34-CD45- cells represent a putative stem cell state distinct from classically defined LSC. Investigations into alternate stem cell subpopulations that contribute to relapse and therapy resistance are warranted, and may reveal targets for eliminating LIC. Citation Format: Christopher B. Benton, Ahmed Al Rawi, Taejin Min, Rui-Yu Wang, Wendy Schober, Zhiqiang Wang, Zhihong Zeng, Jeffrey Hokanson, Zhu Zhu, Xiaoping Su, Xiaofeng Zheng, Karen Lu, Bing Carter, Richard E. Davis, Hagop Kantarjian, Marina Konopleva, Michael Andreeff. Single cell analysis of Lin-CD34-CD45- cells from primary AML samples reveals leukemia clones with stem cell-like properties distinct from CD34+CD38-CD123+ LSC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1391. doi:10.1158/1538-7445.AM2015-1391

Abstract LB-193: Ganglioside GD2 identifies cancer stem cells and inhibition of GD2 biosynthesis by targeting GD3 synthase exerts antitumor effects

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Currently, only very few markers either as single or in combination are available to identify cancer stem cells (CSCs). In this report, we identified that the ganglioside GD2, a marker known to express on mesenchymal stromal cells (MSCs) is expressed on a small fraction (5.5% ± 3.4%) of transformed human mammary epithelial cells (HMLER) and breast cancer patient tumors. FACS sorted GD2+ cells appear spindle shaped and proliferate 5 fold slower compared to GD2- cells in-vitro. Analysis of breast cancer cell lines (n=12) indicated that GD2 expression varies and that the basal breast cancer cell lines have a higher percentage of GD2+ cells (median 9%, range 1.2-17%, n=6), as compared to their luminal counterparts (median 0.2%, range 0-3%, n=6, p 10 fold. Moreover, we observed spontaneous generation of GD2+ from GD2- cells and vice versa in both in-vitro and in-vivo, suggesting a role of EMT in this process. Stable knock-down of GD3S in MDA-MB-231 cells using shRNA impaired in-vitro matrigel invasion by more than 10-fold and completely abolished tumor growth in-vivo. Importantly, Triptolide, an anti-inflammatory and anti-cancer drug, which was recently shown to inhibit GD3S expression in melanoma cells, also inhibited GD3S expression in MDA-MB-231 and SUM159 cells by >95% in a dose dependent manner and thereby inhibited growth of GD2+ cells in a time dependent manner. Intra-peritoneal administration of Triptolide (0.15mg/Kg/day) in NOD/SCID mice bearing MDA-MB-231 breast tumors completely eliminated tumors in 50% and reduced the tumor volume 7- to 8-fold in 25% of the mice. In conclusion, we identified GD2 as a new CSC specific cell surface marker and GD3 synthase as a potential therapeutic target for CSCs, with the potential of improving survival and cure rates of patients with breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-193. doi:1538-7445.AM2012-LB-193

Quantitative Determination of Bcl-2 Expression in AML Cell Lines and in Normal and Leukemic Progenitor Cell Compartments by Laser Scanning Cytometry: Comparison with Flow Cytometry and Western Blot

Laser Scanning Cytometry (LSC) is a newly developed microscope-based and computerized system that directly analyzes up to 3- color fluorescence simultaneously from single cells attached to slides. It permits correlations of quantitative cytometric data with morphology and with cytogenetic abnormalities determined by FISH. The data generated by LSC is equivalent to those obtained by flow cytometry in principle. In this study, Bcl-2 expression was measured in five leukemia cell lines by flow cytometry (FCM), LSC, and western blot. The order of Bcl-2 expression in cell lines from highest to lowest is HL60- DOX, OCI/AML3, HL60, MO7E, and TF-1. There was an excellent correlation of results of LSC vs. western blot, R2=0.97; FCM vs. western blot, R2=0.85; FCM vs. LSC, R2=0.83 (all p values <0.05). We then studied Bcl-2 expression in normal and AML progenitor cell populations. Bcl-2 expression was measured by LSC and western blot for CD34+/CD38+ and CD34+/CD38- cells after FACS-sorting. In normal bone marrow, Bcl-2 was significantly higher in CD34+/CD38+ than in CD34+/CD38- cells (p=0.009). Significant differences in Bcl-2 expression levels were noted in CD34+/CD38- cells between AML and normal bone marrow (NBM) populations (p=0.02), and AML-PB and NBM (p=0.005). All sorted populations contained 25% to 76.5% leukemia cells, based on FISH analysis. We conclude that LSC is a powerful technique for the analysis of gene expression in small numbers of FACS-sorted progenitor cells. It also allows correlations with morphology and cytogenetic abnormalities. The differential overexpression of Bcl-2 protein in CD34+/CD38- AML vs. normal bone marrow cells supports the concept of Bcl-2 as a novel target in the treatment of AML.