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γδ T cells play an important role in disease control in acute myeloid leukemia (AML) and have become an emerging area of therapeutic interest. These cells represent a minor population of T lymphocytes with intrinsic abilities to recognize antigens in a major histocompatibility complex-independent manner and functionally straddle the innate and adaptive immunity interface. AML shows high expression of phosphoantigens and UL-16 binding proteins that activate the Vδ2 and Vδ1 subtypes of γδ T cells, respectively, leading to γδ T cell-mediated cytotoxicity. Insights from murine models and clinical data in humans show improved overall survival, leukemia-free survival, reduced risk of relapse, enhanced graft-versus-leukemia effect, and decreased graft-versus-host disease in patients with AML who have higher reconstitution of γδ T cells following allogeneic hematopoietic stem cell transplantation. Clinical trials leveraging γδ T cell biology have used unmodified and modified allogeneic cells as well as bispecific engagers and monoclonal antibodies. In this review, we discuss γδ T cells' biology, roles in cancer and AML, and mechanisms of immune escape and antileukemia effect; we also discuss recent clinical advances related to γδ T cells in the field of AML therapeutics., Competing Interests: Competing interests: AR and AA: None. AM: Reports research funding to the institution from Chimeric Therapeutics, LinBio Sciences, Celgene, Sanofi-Aventis, CytoMed Therapeutics, BioSight, IGM Biosciences, Electra Therapeutics, Astex Pharmaceuticals. Other: Cero Therapeutics., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

We identified activin A receptor type I (ACVR1), a member of the TGF-β superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

MCL-1 is known to play a major role in resistance to BCL-2 inhibition, but the contribution of other BCL-2 family proteins has not been fully explored. We, here, demonstrate the ineffectiveness of MCL-1 inhibitor AMG176 in venetoclax-resistant, and conversely, of venetoclax in AMG176-resistant acute myelogenous leukemia (AML). Like cells with acquired resistance to venetoclax, cells with acquired resistance to AMG176 express increased MCL-1. Both cells with acquired resistance to venetoclax and to AMG176 express increased levels of BCL-2 and BCL-2A1, decreased BAX, and/or altered levels of other BCL-2 proteins. Cotargeting BCL-2 and MCL-1 was highly synergistic in AML cell lines with intrinsic or acquired resistance to BH3 mimetics or engineered to genetically overexpress BCL-2 or BCL-2A1 or downregulate BAX. The combination effectively eliminated primary AML blasts and stem/progenitor cells resistant to or relapsed after venetoclax-based therapy irrespective of mutations and cytogenetic abnormalities. Venetoclax and AMG176 combination markedly suppressed antiapoptotic BCL-2 proteins and AML stem/progenitor cells and dramatically extended mouse survival (median 336 vs. control 126 days; P < 0.0001) in a patient-derived xenograft (PDX) model developed from a venetoclax/hypomethylating agent therapy-resistant patient with AML. However, decreased BAX levels in the bone marrow residual leukemia cells after 4-week combination treatment may represent a resistance mechanism that contributed to their survival. Enhanced antileukemia activity was also observed in a PDX model of monocytic AML, known to be resistant to venetoclax therapy. Our results support codependence on multiple antiapoptotic BCL-2 proteins and suppression of BAX as mechanisms of AML resistance to individual BH3 mimetics. Cotargeting of MCL-1 and BCL-2 eliminates otherwise apoptosis-resistant cells., (©2022 American Association for Cancer Research.)

We observed that the immune checkpoint protein B7-H3 is overexpressed in acute myeloid leukemia (AML) patients with poor treatment outcomes. Inhibition of B7-H3 expression or blocking of its activity using a novel monoclonal antibody (T-1A5) in AML cells significantly enhanced natural killer (NK) cell-mediated cytotoxicity in AML cells in vitro and in vivo. Moreover, a human-mouse chimera of this antibody (ChT-1A5) induced antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells, but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping studies identified that both T-1A5 and ChT-1A5 antibodies bind to the FG-loop region of B7-H3, which is known to regulate the immunosuppressive function of B7-H3. Furthermore, treatment with ChT-1A5 in combination with human NK cells significantly prolonged survival in AML patient-derived xenograft (PDX) models. Our results suggest that the ChT-1A5 antibody can inhibit the immunosuppressive function of B7-H3 protein as well as induce ADCC in B7-H3+ AML., (© 2022 by The American Society of Hematology.)

Background: Metabolic stress resulting from nutrient deficiency is one of the hallmarks of a growing tumour. Here, we tested the hypothesis that metabolic stress induces breast cancer stem-like cell (BCSC) phenotype in triple-negative breast cancer (TNBC)., Methods: Flow cytometry for GD2 expression, mass spectrometry and Ingenuity Pathway Analysis for metabolomics, bioinformatics, in vitro tumorigenesis and in vivo models were used., Results: Serum/glucose deprivation not only increased stress markers but also enhanced GD2 + BCSC phenotype and function in TNBC cells. Global metabolomics profiling identified upregulation of glutathione biosynthesis in GD2 high cells, suggesting a role of glutamine in the BCSC phenotype. Cueing from the upregulation of the glutamine transporters in primary breast tumours, inhibition of glutamine uptake using small-molecule inhibitor V9302 reduced GD2 + cells by 70-80% and BCSC characteristics in TNBC cells. Mechanistic studies revealed inhibition of the mTOR pathway and induction of ferroptosis by V9302 in TNBC cells. Finally, inhibition of glutamine uptake significantly reduced in vivo tumour growth in a TNBC patient-derived xenograft model using NSG (non-obese diabetic/severe combined immunodeficiency with a complete null allele of the IL-2 receptor common gamma chain) mice., Conclusion: Here, we show metabolic stress results in GD2 + BCSC phenotype in TNBC and glutamine contributes to GD2 + phenotype, and targeting the glutamine transporters could complement conventional chemotherapy in TNBC., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways., (© 2022. The Author(s).)

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by lack of hormone receptor expression and is known for high rates of recurrence, distant metastases, and poor clinical outcomes. TNBC cells lack targetable receptors; hence, there is an urgent need for targetable markers for the disease. Breast cancer stem-like cells (BCSCs) are a fraction of cells in primary tumors that are associated with tumorigenesis, metastasis, and resistance to chemotherapy. Targeting BCSCs is thus an effective strategy for preventing cancer metastatic spread and sensitizing tumors to chemotherapy. The CD44 hi CD24 lo phenotype is a well-established phenotype for identification of BCSCs, but CD44 and CD24 are not targetable markers owing to their expression in normal tissues. The ganglioside GD2 has been shown to be upregulated in primary TNBC tumors compared with normal breast tissue and has been shown to identify BCSCs. In this review, we discuss GD2 as a BCSC- and tumor-specific marker in TNBC; epithelial-to-mesenchymal transition and the signaling pathways that are upstream and downstream of GD2 and the role of these pathways in tumorigenesis and metastasis in TNBC; direct and indirect approaches for targeting GD2; and ongoing clinical trials and treatments directed against GD2 as well as future directions for these strategies., (© 2021 New York Academy of Sciences.)

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood., Methods: We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis., Results: We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1., Conclusions: Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15's dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC., (© 2021. The Author(s).)

Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no effective standard therapy. Breast cancer stem-like cells (BCSCs) in primary TNBCs are reported to be responsible for metastatic spread of the disease and resistance to chemotherapy, but no available therapeutic tools target BCSCs. We previously reported that the ganglioside GD2 is highly expressed on BCSCs and that inhibition of its expression hampers TNBC growth. We therefore hypothesized that the anti-GD2 antibody dinutuximab (ch14.18) targets GD2 + BCSCs and inhibits TNBC growth., Method: To test our hypothesis, we first determined GD2 expression via immunohistochemistry in frozen primary tumor samples from patients with TNBC (n=89). Then, we examined the effects of dinutuximab on TNBC cell adhesion, migration, and mammosphere formation in vitro and on tumor growth in vivo using TNBC cell-line and patient-derived xenograft (PDX) models., Results: We found that GD2 was expressed in around 60% of primary TNBC tumors at variable levels and was associated with worse overall survival of patients with TNBC (p=0.002). GD2 was found to be expressed in tumors and stroma, but normal ducts and lobules in adjacent tissues have shown low or no GD2 staining, indicating that GD2 is potentially a novel biomarker for tumor and its microenvironment. Treatment with dinutuximab significantly decreased adhesion and migration of MDA-MB-231 and SUM159 TNBC cells. Moreover, dinutuximab treatment inhibited mTOR signaling, which has been shown to be regulated by GD2 in BCSCs. Dinutuximab also reduced tumor growth in nude mice bearing TNBC cell-line xenografts. Finally, dinutuximab in combination with activated natural killer cells inhibited tumor growth in a TNBC PDX model and improved overall survival of tumor-bearing mice., Conclusions: Dinutuximab successfully eliminated GD2 + cells and reduced tumor growth in both in vivo models. Our data provide proof-of-concept for the criticality of GD2 in BCSCs and demonstrate the potential of dinutuximab as a novel therapeutic approach for TNBC., Competing Interests: Competing interests: MA and VLB were awarded a patent for therapeutic targeting of GD2 in cancer (US9846160B2)., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) consists of various cell types that support the growth of AML cells and protect them from chemotherapy. Mesenchymal stromal cells (MSCs) in the BME have been shown to contribute immensely to leukemogenesis and chemotherapy resistance in AML cells. However, the mechanism of stroma-induced chemotherapy resistance is not known. Here, we hypothesized that stromal cells promote a stem-like phenotype in AML cells, thereby inducing tumorigenecity and therapy resistance. To test our hypothesis, we co-cultured AML cell lines and patient samples with BM-derived MSCs and determined aldehyde dehydrogenase (ALDH) activity and performed gene expression profiling by RNA sequencing. We found that the percentage of ALDH+ cells increased dramatically when AML cells were co-cultured with MSCs. However, among the 19 ALDH isoforms, ALDH2 and ALDH1L2 were the only two that were significantly upregulated in AML cells co-cultured with stromal cells compared to cells cultured alone. Mechanistic studies revealed that the transforming growth factor-β1 (TGF-β1)-regulated gene signature is activated in AML cells co-cultured with MSCs. Knockdown of TGF-β1 in BM-MSCs inhibited stroma-induced ALDH activity and ALDH2 expression in AML cells, whereas treatment with recombinant TGF-β1 induced the ALDH+ phenotype in AML cells. We also found that TGF-β1-induced ALDH2 expression in AML cells is mediated by the non-canonical pathway through the activation of p38. Interestingly, inhibition of ALDH2 with diadzin and CVT-10216 significantly inhibited MSC-induced ALDH activity in AML cells and sensitized them to chemotherapy, even in the presence of MSCs. Collectively, BM stroma induces ALDH2 activity in AML cells through the non-canonical TGF-β pathway. Inhibition of ALDH2 sensitizes AML cells to chemotherapy., Competing Interests: The authors have declared that no competing interests exist.

Given the proven importance of the CXCL12/CXCR4 axis in the stroma-acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-b signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-b in stromal cells or TGF-b-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-b signaling.

The bone marrow microenvironment, also known as the bone marrow niche, is a complex network of cell types and acellular factors that supports normal hematopoiesis. For many years, leukemia was believed to be caused by a series of genetic hits to hematopoietic stem and progenitor cells, which transform them to preleukemic, and eventually to leukemic, cells. Recent discoveries suggest that genetic alterations in bone marrow niche cells, particularly in osteogenic cells, may also cause myeloid leukemia in mouse models. The osteogenic niche, which consists of osteoprogenitors, preosteoblasts, mature osteoblasts, osteocytes and osteoclasts, has been shown to play a critical role in the maintenance and expansion of hematopoietic stem and progenitor cells as well as in their oncogenic transformation into leukemia stem/initiating cells. We have recently shown that acute myeloid leukemia cells induce osteogenic differentiation in mesenchymal stromal cells to gain a growth advantage. In this review, we discuss the role of the osteogenic niche in the maintenance of hematopoietic stem and progenitor cells, as well as in their transformation into leukemia cells. We also discuss the signaling pathways that regulate osteogenic niche-hematopoietic stem and progenitor cells or osteogenic niche-leukemic stem/initiating cell interactions in the bone marrow, together with novel approaches for therapeutically targeting these interactions., (Copyright© 2018 Ferrata Storti Foundation.)

Breast cancer stem-like cells (BCSC) are implicated in cancer recurrence and metastasis of triple-negative breast cancer (TNBC). We have recently discovered that ganglioside GD2 expression defines BCSCs and that ST8SIA1 regulates GD2 expression and BCSC function. In this report, we show that ST8SIA1 is highly expressed in primary TNBC; its expression is positively correlated with the expression of several BCSC-associated genes such as BCL11A, FOXC1, CXCR4, PDGFRβ, SOX2, and mutations in p53. CRISPR knockout of ST8SIA1 completely inhibited BCSC functions, including in vitro tumorigenesis and mammosphere formation. Mechanistic studies discovered activation of the FAK-AKT-mTOR signaling pathway in GD2 + BCSCs, and its tight regulation by ST8SIA1. Finally, knockout of ST8SIA1 completely blocked in vivo tumor growth and metastasis by TNBC cells. In summary, these data demonstrate the mechanism by which ST8SIA1 regulates tumor growth and metastasis in TNBC and identifies it as a novel therapeutic target., (©2018 American Association for Cancer Research.)

Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high β-galactosidase staining. In addition, overexpression of BCL-X L in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response., (Copyright © 2018 Ferrata Storti Foundation.)

High-grade Bladder Cancer (BLCA) represents the most aggressive and treatment-resistant cancer that renders the patients with poor survival. However, only a few biomarkers have been identified for the detection and treatment of BLCA. Recent studies show that ganglioside GD2 can be used as cancer biomarker and/or therapeutic target for various cancers. Despite its potential relevance in cancer diagnosis and therapeutics, the role of GD2 is unknown in BLCA. Here, we report for the first time that high-grade BLCA tissues and cell lines have higher expression of GD2 compared to low-grade by high-resolution Mass Spectrometry. The muscle invasive UMUC3 cell line showed high GD2, mesenchymal phenotype, and cell proliferation. Besides, we have shown the cancer stem cells (CSC) property (CD44hiCD24lo) of GD2+ UMUC3 and J82 cells. Also, the evaluation of lipid metabolism in GD2+ BLCA cell lines revealed higher levels of Phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin (CL) and lower levels of Phosphatidylserine (PS), plasmenyl-phosphatidylethanolamines (pPE), plasmenyl-phosphocholines (pPC), sphingomyelins (SM), triglycerides (TGs) and N-Acetylneuraminic acid. These findings are significantly correlated with the tissues of BLCA patients. Based on this evidence, we propose that GD2 may be used as an effective diagnostic and therapeutic target for aggressive BLCA., Competing Interests: CONFLICTS OF INTEREST The authors do not have any conflicts of interest.

Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

We have identified that the ganglioside GD2 is a marker for breast cancer stem cells (BCSCs), and that targeting the enzyme GD3 synthase (GD3S, which regulates GD2 biosynthesis) reduces breast tumorigenesis. The pathways regulating GD2 expression, and their anomalous functions in BCSC, are unclear. Proteomic analysis of GD2+ and GD2- cells from breast cancer cell lines revealed the activation of NFκB signaling in GD2+ cells. Dose- and time-dependent suppression of NFκB signaling by the small molecule inhibitor BMS-345541 reduced GD2+ cells by > 90%. Likewise, BMS-345541 inhibited BCSC GD3S expression, mammosphere formation, and cell migration/invasion in vitro. Breast tumor-bearing mice treated with BMS-345541 showed a statistically significant decrease in tumor volume and exhibited prolonged survival compared to control mice, with a median survival of 78 d for the BMS-345541-treated group vs. 58 d for the controls. Moreover, in an experimental metastases model, treatment with BMS-345541 reduced the lung metastases by > 5-fold. These data suggest that GD2 expression and function,and NFκB signaling, are related, and they control BCSCs tumorigenic characteristics. Thus, the suppression of NFκB signaling by BMS-345541 is a potentially important advance in controlling breast cancer growth and metastases.

The epithelial-mesenchymal transition (EMT) bestows cancer cells with increased stem cell properties and metastatic potential. To date, multiple extracellular stimuli and transcription factors have been shown to regulate EMT. Many of them are not druggable and therefore it is necessary to identify targets, which can be inhibited using small molecules to prevent metastasis. Recently, we identified the ganglioside GD2 as a novel breast cancer stem cell marker. Moreover, we found that GD3 synthase (GD3S)--an enzyme involved in GD2 biosynthesis--is critical for GD2 production and could serve as a potential druggable target for inhibiting tumor initiation and metastasis. Indeed, there is a small molecule known as triptolide that has been shown to inhibit GD3S function. Accordingly, in this manuscript, we demonstrate that the inhibition of GD3S using small hairpin RNA or triptolide compromises the initiation and maintenance of EMT instigated by various signaling pathways, including Snail, Twist and transforming growth factor-β1 as well as the mesenchymal characteristics of claudin-low breast cancer cell lines (SUM159 and MDA-MB-231). Moreover, GD3S is necessary for wound healing, migration, invasion and stem cell properties in vitro. Most importantly, inhibition of GD3S in vivo prevents metastasis in experimental as well as in spontaneous syngeneic wild-type mouse models. We also demonstrate that the transcription factor FOXC2, a central downstream effector of several EMT pathways, directly regulates GD3S expression by binding to its promoter. In clinical specimens, the expression of GD3S correlates with poor prognosis in triple-negative human breast tumors. Moreover, GD3S expression correlates with activation of the c-Met signaling pathway leading to increased stem cell properties and metastatic competence. Collectively, these findings suggest that the GD3S-c-Met axis could serve as an effective target for the treatment of metastatic breast cancers.

Our recent study suggests that targeting GD3 synthase (also known as ST8SIA1)-the rate-limiting enzyme in biosynthesis of the breast cancer stem cell marker GD2-abrogates metastasis and depletes the cancer stem cell populations within a tumor, thus providing an effective therapeutic strategy against metastatic breast cancers.

Leukemia cells are protected from chemotherapy-induced apoptosis by their interactions with bone marrow mesenchymal stromal cells (BM-MSCs). Yet the underlying mechanisms associated with this protective effect remain unclear. Genome-wide gene expression profiling of BM-MSCs revealed that coculture with leukemia cells upregulated the transcription of genes associated with nuclear factor (NF)-κB signaling. Moreover, primary BM-MSCs from leukemia patients expressed NF-κB target genes at higher levels than their normal BM-MSC counterparts. The blockade of NF-κB activation via chemical agents or the overexpression of the mutant form of inhibitor κB-α (IκBα) in BM-MSCs markedly reduced the stromal-mediated drug resistance in leukemia cells in vitro and in vivo. In particular, our unique in vivo model of human leukemia BM microenvironment illustrated a direct link between NF-κB activation and stromal-associated chemoprotection. Mechanistic in vitro studies revealed that the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen-4 (VLA-4) played an integral role in the activation of NF-κB in the stromal and tumor cell compartments. Together, these results suggest that reciprocal NF-κB activation in BM-MSCs and leukemia cells is essential for promoting chemoresistance in the transformed cells, and targeting NF-κB or VLA-4/VCAM-1 signaling could be a clinically relevant mechanism to overcome stroma-mediated chemoresistance in BM-resident leukemia cells.

Connective tissue growth factor (CTGF/CCN2) is involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in precursor B-acute lymphoblastic leukemia (ALL) and that increased expression of CTGF is associated with inferior outcome in B-ALL. In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knockdown CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA. Silencing of CTGF resulted in significant suppression of leukemia cell growth compared to control vector, which was associated with AKT/mTOR inactivation and increased levels of cyclin-dependent kinase inhibitor p27. CTGF knockdown sensitized ALL cells to vincristine and methotrexate. Treatment with an anti-CTGF monoclonal antibody, FG-3019, significantly prolonged survival of mice injected with primary xenograft B-ALL cells when co-treated with conventional chemotherapy (vincristine, L-asparaginase and dexamethasone). Data suggest that CTGF represents a targetable molecular aberration in B-ALL, and blocking CTGF signaling in conjunction with administration of chemotherapy may represent a novel therapeutic approach for ALL patients.

Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment. Connective tissue growth factor (CTGF) is highly expressed in MSCs, but its role in the BM stroma is unknown. Therefore, we knocked down (KD) CTGF expression in human BM-derived MSCs by CTGF short hairpin RNA. CTGF KD MSCs exhibited fivefold lower proliferation compared with control MSCs and had markedly fewer S-phase cells. CTGF KD MSCs differentiated into adipocytes at a sixfold higher rate than controls in vitro and in vivo. To study the effect of CTGF on engraftment of leukemia cells into BM, an in vivo model of humanized extramedullary BM (EXM-BM) was developed in NOD/SCID/IL-2rg(null) mice. Transplanted Nalm-6 or Molm-13 human leukemia cells engrafted at a threefold higher rate in adipocyte-rich CTGF KD MSC-derived EXM-BM than in control EXM-BM. Leptin was found to be highly expressed in CTGF KD EXM-BM and in BM samples of patients with acute myeloid and acute lymphoblastic leukemia, whereas it was not expressed in normal controls. Given the established role of the leptin receptor in leukemia cells, the data suggest an important role of CTGF in MSC differentiation into adipocytes and of leptin in homing and progression of leukemia.

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial-mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-β to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.

Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have increased resistance to conventional therapies and are capable of establishing metastasis. However, only a few biomarkers of CSCs have been identified. Here, we report that ganglioside GD2 (a glycosphingolipid) identifies a small fraction of cells in human breast cancer cell lines and patient samples that are capable of forming mammospheres and initiating tumors with as few as 10 GD2+ cells. In addition, the majority of GD2+ cells are also CD44hiCD24lo, the previously established CSC-associated cell surface phenotype. Gene expression analysis revealed that GD3 synthase (GD3S) is highly expressed in GD2+ as well as in CD44hiCD24lo cells and that interference with GD3S expression, either by shRNA or using a pharmacological inhibitor, reduced the CSC population and CSC-associated properties. GD3S knockdown completely abrogated tumor formation in vivo. Also, induction of epithelial-mesenchymal transition (EMT) in transformed human mammary epithelial cells (HMLER cells) dramatically increased GD2 as well as GD3S expression in these cells, suggesting a role of EMT in the origin of GD2+ breast CSCs. In summary, we identified GD2 as a new CSC-specific cell surface marker and GD3S as a potential therapeutic target for CSCs, with the possibility of improving survival and cure rates in patients with breast cancer.

The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.

The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-beta in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44(+), CD24(-), and CD45(-). Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-beta), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.

Multipotent mesenchymal stromal/stem cells (MSC) have shown potential clinical utility. However, previous assessments of MSC behavior in recipients have relied on visual detection in host tissue following sacrifice, failing to monitor in vivo MSC dispersion in a single animal and limiting the number of variables that can be observed concurrently. In this study, we used noninvasive, in vivo bioluminescent imaging to determine conditions under which MSC selectively engraft in sites of inflammation. MSC modified to express firefly luciferase (ffLuc-MSC) were injected into healthy mice or mice bearing inflammatory insults, and MSC localization was followed with bioluminescent imaging. The inflammatory insults investigated included cutaneous needle-stick and surgical incision wounds, as well as xenogeneic and syngeneic tumors. We also compared tumor models in which MSC were i.v. or i.p. delivered. Our results demonstrate that ffLuc-expressing human MSC (hMSC) systemically delivered to nontumor-bearing animals initially reside in the lungs, then egress to the liver and spleen, and decrease in signal over time. However, hMSC in wounded mice engraft and remain detectable only at injured sites. Similarly, in syngeneic and xenogeneic breast carcinoma-bearing mice, bioluminescent detection of systemically delivered MSC revealed persistent, specific colocalization with sites of tumor development. This pattern of tropism was also observed in an ovarian tumor model in which MSC were i.p. injected. In this study, we identified conditions under which MSC tropism and selective engraftment in sites of inflammation can be monitored by bioluminescent imaging over time. Importantly, these consistent findings were independent of tumor type, immunocompetence, and route of MSC delivery.

Background: Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells., Design and Methods: Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining., Results: Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were approximately 90-fold enriched in the MSCA-1(+)CD56(-) fraction and approximately 180-fold in the MSCA-1(+)CD56(+) fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1(+)CD56(-) mesenchymal stem cells subset and CD166 to MSCA-1(+)CD56(+/-) mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1(+)CD56(+/-) cells whereas adipocytes emerged exclusively from MSCA-1(+)CD56(-) cells. The culture of single sorted MSCA-1(+)CD56(+) cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities., Conclusions: Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1(+)CD56(+) subset is an attractive starting population for autologous chondrocyte transplantation.

We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9(+) cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9(+) PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9(+) but not in the FZD9(-) fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9(+)CD10(+)CD26(+) fraction but were absent in the FZD9(+)CD10(-)CD26(-) population. Cultured FZD9(+) cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL.

The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)-derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271(+) population. The recognized CD271(+) populations were fractionated by fluorescence-activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU-F. The results showed that only the CD271(bright) but not the CD271(dim) population contained CFU-F. Two-color flow cytometry analysis revealed that only the CD271(bright) population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271(bright) population but no other BM cells. The new MSC-specific molecules included the platelet-derived growth factor receptor-beta (CD140b), HER-2/erbB2 (CD340), frizzled-9 (CD349), the recently described W8B2 antigen, as well as cell-surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9-3C2F1, and HEK-3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM-MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.