BackgroundTriple-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.MethodTo 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.ResultsWe 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.ConclusionsDinutuximab 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.
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.
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 NK cell-mediated cytotoxicity in AML cells in vitro and in vivo. Moreover, 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 models. Our results suggest that ChT-1A5 antibody can inhibit the immunosuppressive function of B7-H3 protein as well as induce ADCC in B7-H3+ AML.
Breast cancer stem cells (BSCs) constitute a fraction of primary tumor cells that exhibit drug resistance and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Nutrient deprivation-associated metabolic stress observed during tumor progression is reportedly associated with the cancer stem cell phenotype, and we have shown that oxidative stress caused by serum deprivation induces GD2 expression in vitro and in vivo. To identify metabolic signatures associated with GD2+ cells and their potential adaptive mechanisms to oxidative stress, global metabolic profiling was performed using a mass spectroscopy-based approach. We found that metabolites associated with amino acid metabolism, particularly glutathione metabolism, to be most highly upregulated in GD2+ compared to GD2− cells. In addition, glutathione biosynthesis and oxidation to glutathione disulfide was also increased in cells undergoing oxidative stress. These data suggest that the glutathione-mediated detoxification pathways play a key role in GD2+ or basal type breast cancer cells. As glutamine is a major precursor molecule for glutathione biosynthesis, we hypothesized that glutamine, at least in part, regulates cellular redox tone and the GD2+ phenotype in breast cancer cells. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 in media containing 0%, 0.5%, 1%, 2%, or 4% of L-glutamine for 3 days. Flow cytometry analysis revealed that the percentage of GD2+ increased from 6% to 12% and from 12% to 22% in MDA-MB-231 and SUM159 cells, respectively when the cells were cultured at 0 or 4% glutamine. Glutaminase, an enzyme that converts glutamine to glutamate, which further leads to glutathione biosynthesis, has been shown to be overexpressed in basal-type breast cancer cells. To investigate the role of glutaminase on stem cell redox homeostasis and phenotype, we treated TNBC cell lines MDA-MB-231 and SUM159 cells with glutaminase inhibitors CB-839 or 968 compound for 3 days. Flow analysis revealed 70-80% reduction in GD2 expression after treatment in a dose dependent manner. In addition, treatment with CB-839 inhibited cell proliferation by over 80% in a dose-dependent manner in MDA-MB-231 cells and SUM159 cells (p<0.001). Next, to determine the effect of glutaminase inhibition on BCSC function, we cultured MDA-MB-231 and SUM-159 cells in low adherent cell culture dishes with or without glutaminase inhibitors 968 or CB-839 for 7 days. Interestingly, we observed a 40-50% reduction in mammosphere formation in both treated cell lines compared to the untreated controls, suggesting that inhibition of glutaminase is critical for BCSC function. In conclusion, the anti-oxidant glutathione is highly upregulated in GD2+ BCSCs, and this appears to be associated with the concentration of glutamine, and appears to regulate, perhaps via the regulation of redox homeostaisis, GD2 expression in BCSCs. Targeting glutaminase and redox homeostasis using CB-839 or CB968 inhibits BCSC as measured by GD2 positivity and mammosphere formation. Glutaminase inhibition in combination with chemotherapy could be a valuable future therapeutic strategy for targeting BCSCs in TNBC. Citation Format: Stanley Ly, Khoa Nguyen, Michael Andreeff, Venkata Lokesh Battula. Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-05.
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