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.

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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.

Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancy that originates in the bone marrow (BM). Despite advances in the molecular characterization of AML, factors regulating its progression are still not known. Among several BM niches that support AML growth in the BM, the osteogenic niche has gained attention in recent years owing to its potential role in leukemogenesis. Genetic alterations in osteoprogenitor cells have been shown to induce myeloid leukemia in mouse models. We reported recently that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSCs) in the BM to facilitate faster AML engraftment in mice (Battula et al., JCI Insight, 2017). However specifics of this osteogenic niche generated by AML are not known. Here we hypothesize that AML expands osteo-progenitor rich niche in the BM, but that the mature bone is reduced. To determine the type of AML-induced osteo-lineage differentiation in the BM, we generated transgenic reporter mice by crossing Osx-CreERt2 mice with Ocn-GFP; ROSA-tdTomato mice. The resulting triple transgenic mice has the genotype of Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. In these mice the tdTomato (red) positive cells represents osteo-lineage cells that originate from Osterix expressing (Osx+) cells, whereas a GFP+ cell represents an osteocalcin-expressing (Ocn+) mature osteoblast. Seven day old triple transgenic mice were injected with tamoxifen to activate Osx-CreERT2 to mark the Osx+ cells with tomato reporter. To investigate the osteogenic cell type that is induced by AML cells in the bone marrow, we implanted murine AML cells with MLL-ENL fusion proteins into Osx-CreERt2;Ocn-GFP;ROSA-tdTomato mice. Three weeks after implantation of AML cells, the femurs and tibia of these mice were dissected and subjected to histological evaluation using fluorescence microscopy. In control BM without AML, the GFP+ (Ocn+) cells were found in the trabecular bone surface as well as the periosteum of the bone, whereas the tdTomato+ (Osx+)cells were found in the marrow and the bone matrix; this suggests that some of the osteocytes originated from tamoxifen-induced Osx+ osteoprogenitor cells. Interestigly, in mice implanted with AML cells, we found a 3-4 fold increase in Osx+ cells in the marrow compared to normal BM (Fig 1A). However, the number of GFP+ cells on the endosteum and trabecular bone surface was reduced, suggesting that AML cells might expand osteoprogenitor cells but not fully differentiated mature osteoblasts. Next, to investigate whether AML cells affect the mature bone, AML PDX cells developed in our laboratory were implanted into NSG mice. The PDX models usually take 12-14 weeks to achieve >90% engraftment in the peripheral blood which provides ample time to observe alterations in bone composition. At this stage, the mice were subjected to computed tomography imaging to measure bone architecture, volume (BV), mineral density (BMD) and bone volume fraction (BVF). Interestingly, we observed large bone cavities close to epiphysis and metaphysis areas in the femur and tibia of mice with AML (Fig 1B). In addtion, BMD and BVF in these mice were reduced by 20-30% compared to control mice without leukemia. To validate the bone resorption in these mice, bone histomorphometric analysis was performed on femurs and tibias from mice with and without AML. Masson-Goldner's Trichrome staining revealed a 5- to 10-fold decrease in the trabecular and cortical bone thickness in AML femurs compared to normal femurs. Moreover, measurements of osteoclast activation by tartrate-resistant acidic phosphatase (TRAP) revealed positive staining for osteoclasts on the endosteal surface and massive bone resorption in AML bone compared to normal bone. Mechanistic studies showed that AML cells inhibit osteoprotegerin (OPG) ~10 fold in MSCs, a factor that inhibits the RNAK ligand which in turn activates osteoclasts that breakdown the bone. In conclusion, our data suggest that bone homeostasis is dysregulated in AML by induction of osteogenic and osteolytic activities simultaneously. AML cells induce an osteoprogenitor niche but also activate osteoclasts resulting in osteopenia/osteoporosis in mouse models. In-depth analysis of bone remodeling in AML patients could result in new insights into the pathobiology of the disease and provide therapeutic avenues for AML. Disclosures Andreeff: Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Celgene: Consultancy; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership. Battula:United Therapeutics Inc.: Patents & Royalties, Research Funding.

Acute myeloid leukemia (AML) is the most common and aggressive acute leukemia found in adults. Immune checkpoint inhibition has led to important clinical advances in cancer therapy in recent years due to superior cure rates compared with standard therapy. We hypothesize that B7-H3 (CD276) an immune checkpoint protein is overexpressed in AML cells and targeting B7-H3 activates immune cells against AML cells. We analyzed B7-H3 expression in peripheral blood (PB) and bone marrow (BM) mononuclear cells from AML patients (n=65) and healthy donors (n=10) at MD Anderson Cancer Center. Cell surface expression analysis by flow cytometry revealed that the cells of ~60% of the patients were positive for B7-H3 and its expression was 2- to 3-fold higher in AML cells than in healthy donor cells. B7-H3 expression is relatively higher in CD34+ AML cells than in CD34- AML cells (p<0.01). In contrast, no difference was observed between CD34+ and CD34- cells from healthy donors. The Cancer Genome Atlas RNA sequencing data revealed that patients with high B7-H3 expression had significantly lower overall and disease-free survival durations than did patients with low B7-H3 expression (p=0.024). To investigate the role of B7-H3 in immunomodulation, we stably knocked down B7-H3 in OCI-AML3 and co-cultured them with or without human PB-derived NK cells at a 2:1 ratio and measured apoptosis induction in AML cells by annexin-v binding approach. We found that knockdown of B7-H3 induced NK cell-mediated apoptosis in AML cells 3-fold compared to control AML cells. These data indicate that inhibition of B7-H3 in AML cells enhances NK cell-mediated apoptosis in AML cells. To target B7-H3, we have generated four monoclonal antibodies: B1, B2, B3 and B4 (codenamed to protect IP). To investigate whether these novel anti-B7-H3 monoclonal antibodies are able to block B7-H3 immunomodulatory function and activate NK cells, we performed a co-culture experiment with GFP-expressing OCI-AML3 cells and PB-derived NK cells in the presence or absence of anti-B7-H3 antibodies. Apoptosis induction was measured by real-time annexin-v binding using IncuCyte live cell imaging system. The addition of anti-B7-H3 monoclonal antibodies, B1, B2, B3 and B4 at 25μg/ml enhanced NK cell-induced apoptosis 3-fold in OCI-AML3 cells. These data indicate that anti-B7-H3 antibodies block the immunomodulatory function of B7-H3 and induce NK cell-mediated apoptosis in AML cells. In vivo testing of these antibodies against AML-PDX models is currently ongoing. In conclusion, we found that B7-H3 is overexpressed in AML cells and its expression is associated with bad prognosis in AML patients. Knockdown or antibody-mediated blocking of B7-H3 enhanced NK cell-induced apoptosis in AML cells. These data indicated that B7-H3 is a novel immune-checkpoint protein in AML and patients could potentially benefit from anti-B7-H3 therapies. Citation Format: Stanley Ly, Bin Yuan, Sabrina Grimm, Michael Andreeff, Hans-Jörg Bühring, Venkata Lokesh Battula. B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3248.

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