Human Triple-negative Breast Cancer Tumors Research Articles

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Abstract 2912: SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment

Abstract Introduction: A promising target for breast cancer is secreted frizzled-related protein 2 (SFRP2), and a humanized monoclonal antibody to SFRP2 (hSFRP2 mAb) inhibits primary triple negative breast cancer (TNBC) in vivo. CD38 is a known cause of PD-1 inhibitor resistance in some tumors, and in metastatic osteosarcoma hSFRP2 mAb overcomes PD-1 mAb resistance by reducing CD38 in tumor infiltrating lymphocytes (TILs). Could this phenomenon be replicated in TNBC? This is unknown as the expression of CD38 in TNBC has not been studied. This study will establish the presence of SFRP2 and CD38 in the TNBC microenvironment. Methods: Immunohistochemistry (IHC) was performed on an 88 core human TNBC tissue microarray (TMA) with antibodies to SFRP2 and CD38. Spatial analysis software was used to determine percent positivity. Each core was categorized using the scoring system for the estrogen receptor in breast cancer (<1% negative, 1-10% low positive, and >10% positive). Multiplex IHC was performed on 4 human triple TNBCs sections. Tissues were stained with antibodies to CD38, CD68 (macrophage), CD3 (T-cell), CD19 (B-cell), CytoKeratin (tumor), and SFRP2. Percent positivity of each marker was analyzed with a software package. For flow cytometry of tumor associated macrophages (TAMs), 1 million EO771 cells were injected into the mammary fat pad of female C57/BL6 mice. After 4 weeks tumors were extracted and a cell suspension was incubated in a mastermix with antibodies to SFRP2, CD38, PD1, CD11B and F480. Cells were fixed and conjugated to a fluorochrome secondary antibody. TAM gated cells were analyzed for the presence of SFRP2 and CD38. Results: For the TNBC TMA, 83 cores were positive and 4 cores were low positive for SFRP2, 1 not evaluable; 87 cores were positive and 1 low positive for CD38. For multiplex IHC there was a total of 1,753,544 individual cells in all 4 human TNBC tumors that were stained. 94% of cells stained positive for SFRP2, and 65% stained positive for CD38. Of the cells that were positive for SFRP2, 70% were also positive for CD38, 15% were positive for CD68, 1% were positive for CD19, 77% were positive for CytoKeratin, and 10% were positive for CD3. Of the cells that were CD38 positive, 99% were also positive for SFRP2, 15% were positive for CD68, 1% were positive for CD19, 81% were positive for CytoKeratin, and 9% were positive for CD3. Flow cytometry analysis of TAMs confirmed the presence of SFRP2, CD38 on TAMs. TAMs that were negative for PD1 and CD38 (pro-inflammatory) had a 56% reduction in SFRP2 compared to TAMs that were double positive for PD1 and CD38 (anti-inflammatory). Conclusion: SFRP2 and CD38 are prevalent in human TNBC and colocalize in the TNBC microenvironment in tumor cells, TAMs and TILs, but not B-cells. The presence and co-localization of SFRP2 and CD38 in TAMs was confirmed with flow cytometry. This confirms that SFRP2 is a therapeutic target in TNBC and provides a mechanistic rational for combination therapy with PD-1 inhibitor. Citation Format: Lillian Hsu, Patrick Nasarre, Rupak Mukherjee, Eleanor Hilliard, Martin Romeo, Elizabeth O'Quinn, Whitney Christians, Nancy Klauber DeMore. SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2912.

The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non–small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

Transcriptome reprogramming through alternative splicing triggered by apigenin drives cell death in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by its aggressiveness and resistance to cancer-specific transcriptome alterations. Alternative splicing (AS) is a major contributor to the diversification of cancer-specific transcriptomes. The TNBC transcriptome landscape is characterized by aberrantly spliced isoforms that promote tumor growth and resistance, underscoring the need to identify approaches that reprogram AS circuitry towards transcriptomes, favoring a delay in tumorigenesis or responsiveness to therapy. We have previously shown that flavonoid apigenin is associated with splicing factors, including heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2). Here, we showed that apigenin reprograms TNBC-associated AS transcriptome-wide. The AS events affected by apigenin were statistically enriched in hnRNPA2 substrates. Comparative transcriptomic analyses of human TNBC tumors and non-tumor tissues showed that apigenin can switch cancer-associated alternative spliced isoforms (ASI) to those found in non-tumor tissues. Apigenin preferentially affects the splicing of anti-apoptotic and proliferation factors, which are uniquely observed in cancer cells, but not in non-tumor cells. Apigenin switches cancer-associated aberrant ASI in vivo in TNBC xenograft mice by diminishing proliferation and increasing pro-apoptotic ASI. In accordance with these findings, apigenin increased apoptosis and reduced tumor proliferation, thereby halting TNBC growth in vivo. Our results revealed that apigenin reprograms transcriptome-wide TNBC-specific AS, thereby inducing apoptosis and hindering tumor growth. These findings underscore the impactful effects of nutraceuticals in altering cancer transcriptomes, offering new options to influence outcomes in TNBC treatments.

Adjuvant Auger Electron-Emitting Radioimmunotherapy with [111In]In-DOTA-Panitumumab in a Mouse Model of Local Recurrence and Metastatic Progression of Human Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a high risk for recurrence and metastasis. We studied the effectiveness of Auger electron (AE) radioimmunotherapy (RIT) with antiepidermal growth factor receptor (EGFR) panitumumab conjugated with DOTA complexed to 111In ([111In]In-DOTA-panitumumab) for preventing metastatic progression after local treatment of 231/LM2-4 Luc+ human TNBC tumors in the mammary fat pad of NRG mice. Prior to RIT, the primary tumor was resected, and tumor margins were treated with X-irradiation (XRT; 5 days × 6 Gy/d). RIT was administered 1 day post-XRT by intravenous injection of 26 MBq (15 μg) or 2 × 10 MBq (15 μg each) separated by 7 d. These treatments were compared to tumor resection with or without XRT combined with DOTA-panitumumab (15 μg) or irrelevant [111In]In-DOTA-IgG2 (24 MBq; 15 μg), and efficacy was evaluated by Kaplan-Meier survival curves. The effect of [111In]In-DOTA-panitumumab (23 MBq; 15 μg) after tumor resection without local XRT was also studied. Tumor resection followed by XRT and RIT with 26 MBq [111In]In-DOTA-panitumumab significantly increased the median survival to 35 d compared to tumor resection with or without XRT (23-24 d; P < 0.0001). Local treatment with tumor resection and XRT followed by 2 × 10 MBq of [111In]In-DOTA-panitumumab, DOTA-panitumumab, or [111In]In-DOTA-IgG2 did not significantly improve median survival (26 days for all treatments). RIT alone with [111In]In-DOTA-panitumumab postresection of the tumor without XRT increased median survival to 29 days, though this was not significant. Despite significantly improved survival in mice treated with tumor resection, XRT, and RIT with [111In]In-DOTA-panitumumab, all mice eventually succumbed to advanced metastatic disease by 45 d post-tumor resection. SPECT/CT with [111In]In-DOTA-panitumumab, PET/MRI with [64Cu]Cu-DOTA-panitumumab F(ab')2, and PET/CT with [18F]FDG were used to detect recurrent and metastatic disease. Uptake of [111In]In-DOTA-panitumumab at 4 d p.i. in the MFP tumor was 26.8 ± 9.7% ID/g and in metastatic lymph nodes (LN), lungs, and liver was 34.2 ± 26.9% ID/g, 17.5 ± 6.0% ID/g, and 9.4 ± 2.4%ID/g, respectively, while uptake in the lungs (6.0 ± 0.9% ID/g) and liver (5.2 ± 2.9% ID/g) of non-tumor-bearing NRG was significantly lower (P < 0.05). Radiation-absorbed doses in metastatic LN, lungs, and liver were 9.7 ± 6.1, 6.4 ± 2.1, and 10.9 ± 2.7 Gy, respectively. In conclusion, we demonstrated that RIT with [111In]In-DOTA-panitumumab combined with tumor resection and XRT significantly improved the survival of mice with recurrent TNBC. However, the aggressive nature of 231/LM2-4 Luc+ tumors in NRG mice may have contributed to the tumor recurrence and progression observed.

Schlafen 12 Slows TNBC Tumor Growth, Induces Luminal Markers, and Predicts Favorable Survival.

The Schlafen 12 (SLFN12) protein regulates triple-negative breast cancer (TNBC) growth, differentiation, and proliferation. SLFN12 mRNA expression strongly correlates with TNBC patient survival. We sought to explore SLFN12 overexpression effects on in vivo human TNBC tumor xenograft growth and performed RNA-seq on xenografts to investigate related SLFN12 pathways. Stable SLFN12 overexpression reduced tumorigenesis, increased tumor latency, and reduced tumor volume. RNA-seq showed that SLFN12 overexpressing xenografts had higher luminal markers levels, suggesting that TNBC cells switched from an undifferentiated basal phenotype to a more differentiated, less aggressive luminal phenotype. SLFN12-overexpressing xenografts increased less aggressive BC markers, HER2 receptors ERBB2 and EGFR expression, which are not detectable by immunostaining in TNBC. Two cancer progression pathways, the NAD signaling pathway and the superpathway of cholesterol biosynthesis, were downregulated with SLFN12 overexpression. RNA-seq identified gene signatures associated with SLFN12 overexpression. Higher gene signature levels indicated good survival when tested on four independent BC datasets. These signatures behaved differently in African Americans than in Caucasian Americans, indicating a possible biological difference between these races that could contribute to the worse survival observed in African Americans with BC. These results suggest an increased SLFN12 expression modulates TNBC aggressiveness through a gene signature that could offer new treatment targets.

The deubiquitinating enzyme STAMBP is a newly discovered driver of triple-negative breast cancer progression that maintains RAI14 protein stability

Triple-negative breast cancer (TNBC) is a heterogeneous malignancy in women. It is associated with poor prognosis, aggressive malignant behavior, and limited treatment options. In the ubiquitin‒proteasome system (UPS), deubiquitinases (DUBs) are potential therapeutic targets for various tumors. In this study, by performing unbiased siRNA screening, we identified STAMBP, a JAMM metalloprotease in the DUB family, as a driver of human TNBC tumor growth. Functionally, the knockdown of STAMBP inhibited the proliferation, migration, and invasion of multiple TNBC cell lines. Immunoprecipitation–mass spectrometry combined with functional and morphological analysis verified the interaction between STAMBP and the actin-binding protein RAI14. Mechanistically, STAMBP stabilized the RAI14 protein by suppressing the K48-linked ubiquitination of RAI14 and thus prevented its proteasomal degradation. Therefore, knocking down STAMBP resulted in the reduction in RAI14 protein levels and suppression of tumor growth in vitro and in vivo. Importantly, high levels of STAMBP were correlated with poor prognosis in TNBC patients. In summary, we reveal a previously unrecognized DUB pathway that promotes TNBC progression and provides a rationale for potential therapeutic interventions for the treatment of TNBC.

Abstract A39: A therapeutic strategy to inhibit Wnt signaling also reprograms breast tumor-immune cell interactions: Perspectives for conferring immune checkpoint inhibitor susceptibility

Abstract Excessive Wnt signaling is associated with 1) poor prognosis in triple-negative breast cancer (TNBC) and other cancers and 2) immune checkpoint inhibitor resistance, thus limiting its therapeutic application. From our previous work and Wnt signaling principles, we devised CHA1 as an inhibitor of Wnt signaling. CHA1 combines the green tea catechin EGCG (Epigallocatechin-3-gallate) and the DNA methyltransferase inhibitor decitabine, which have been used in numerous clinical trials and/or FDA approved for other cancers, respectively. Our investigations showed that CHA1 treatment (but not EGCG or decitabine alone) reduced primary tumors and metastases in TNBC xenograft and decreased Wnt signaling. Unexpectedly, CHA1 reprogrammed intrinsic tumor properties for antigen presentation and immune cell infiltration. The implications governing tumor-immune cell interactions are discussed in the context of increasing checkpoint inhibitor susceptibility. Both Immune-compromised and immune-competent TNBC preclinical models were used for mechanistic elaboration. In CHA1-treated human TNBC tumors in immune-compromised mice, biochemical and RNA seq analyses show that Wnt signaling was decreased due to induction of Wnt pathway inhibitors (e.g., SFRP1, DKK1, HBP1). The RNA seq analysis also revealed induction of &amp;gt;100 genes for antigen presentation and associated processes. MHC staining of CHA1-treated tumors verified the genotypic changes, which were also accompanied by robust gamma interferon signaling. All results were recapitulated in the immune-competent 4T1 syngeneic TNBC model. We observed large increases in tumor-infiltrating CD8+ T cells with CHA1 treatment. Lastly, recent reports collated the molecularly disparate properties of a “cold-to-hot” transition, in which “hot” tumors have increased immune cell infiltration and sensitivity to checkpoint inhibitors. Remarkably, the TNBC tumors (typically “cold”) that were treated with CHA1 now exhibited the unrelated list of “hot” tumor properties: 1) epigenetic reprogramming, 2) suppressed Wnt signaling, 3) E-cadherin and epithelial marker re-expression, 4) CD8+ T-cell enrichment, 5) increased tumor antigen presentation properties, and 6) increased tumor PD-L1 expression. The CHA1 mechanism is consistent with a global reprogramming of intrinsic tumor properties, triggered by differential Wnt and interferon signaling. CHA1 engages the fundamental processes that regulate tumor-immune cell dynamics, which, in turn, govern immune cell infiltration and determine checkpoint inhibitor sensitivity. Our work additionally establishes a molecular framework for assessing compounds that engage a fundamental “cold-to-hot” tumor reprogramming and that may predict new immune checkpoint inhibitor sensitivity. Thus, CHA1 treatment may reprogram tumor-immune cell dynamics to significantly expand the spectrum of TNBC and other tumors that can be efficaciously treated with immune checkpoint inhibitors (e.g., anti-PDL1 and/or anti-PD1). Citation Format: Mariam Alamoudi, Mollie Chipman, Francesca Deleso-Frechette, Eileen Liu, Rui Zhang, Zixu Wang, K. Eric Paulsopn, Amy S. Yee. A therapeutic strategy to inhibit Wnt signaling also reprograms breast tumor-immune cell interactions: Perspectives for conferring immune checkpoint inhibitor susceptibility [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A39.

Effectiveness and normal tissue toxicity of Auger electron (AE) radioimmunotherapy (RIT) with [111In]In-Bn-DTPA-nimotuzumab in mice with triple-negative or trastuzumab-resistant human breast cancer xenografts that overexpress EGFR

IntroductionOur objective was to evaluate the effectiveness and normal tissue toxicity of nimotuzumab labeled with the Auger electron (AE)-emitter, 111In ([111In]In-Bn-DTPA-nimotuzumab) for radioimmunotherapy (RIT) of human triple-negative breast cancer (TNBC) or trastuzumab-resistant HER2-positive BC tumors overexpressing epidermal growth factor receptors (EGFR) in athymic mice. MethodsNormal tissue toxicity was studied in non-tumor-bearing Balb/c mice i.v. administered 9.0 or 28.6 MBq (3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab, unlabeled nimotuzumab (3 mg/kg) or normal saline. A complete blood cell count (CBC) and serum alanine aminotransferase (ALT) and creatinine (Cr) were measured at 14 days. Body weight was monitored. RIT studies were performed in CD-1 athymic mice engrafted s.c. with MDA-MB-468 human TNBC tumors or TrR1 HER2-positive but trastuzumab-resistant BC tumors. Mice were i.v. administered two amounts (15.5 MBq; 3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab separated by 14 days. Control mice received unlabeled Bn-DTPA-nimotuzumab (3 mg/kg) or anti-HER2 [111In]In-Bn-DTPA-trastuzumab or normal saline. Tumor growth and body weight were measured for 6 weeks. A tumor growth index (TGI) and body weight index (BWI) were calculated to compare the tumor size and body weight post-treatment with the pre-treatment values. A tumor doubling ratio (TDR) was calculated for each treatment group compared to control mice receiving normal saline. ResultsThere was no loss of body weight or decreased red blood cells (RBC) or platelets (PLT) or increased serum ALT or Cr in Balb/c mice administered 9.0 or 28.6 MBq (3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab compared to mice treated with unlabeled Bn-DTPA-nimotuzumab (3 mg/kg) or normal saline. There was a significant decrease in white blood cell (WBC) counts in Balb/c mice receiving 28.6 MBq but not 9.0 MBq of [111In]In-Bn-DTPA-nimotuzumab. Based on these results, an administered amount of 15.5 MBq (3 mg/kg) was selected for RIT studies. Administration of two amounts (15.5 MBq; 3 mg/kg) separated by 14 days to CD-1 athymic mice with s.c. MDA-MB-468 xenografts strongly inhibited tumor growth. The TDR for mice treated with [111In]In-Bn-DTPA-nimotuzumab was 2.15 compared to control mice receiving normal saline. In contrast, treatment with unlabeled Bn-DTPA-nimotuzumab or [111In]In-Bn-DTPA-trastuzumab had no significant effect on tumor growth (TDR = 0.96 and 1.08, respectively). RIT with [111In]In-Bn-DTPA-nimotuzumab also strongly inhibited the growth of TrR1 tumors in athymic mice (TDR = 2.13) compared to unlabeled Bn-DTPA-nimotuzumab (TDR = 0.91). There were no losses in body weight over 6 weeks in tumor bearing mice receiving [111In]In-Bn-DTPA-nimotuzumab, unlabeled Bn-DTPA-nimotuzumab, [111In]In-Bn-DTPA-trastuzumab or normal saline. Conclusions[111In]In-Bn-DTPA-nimotuzumab was effective for treatment of TNBC or trastuzumab-resistant HER2-positive human BC tumors in mice that overexpress EGFR at administered amounts that caused no decrease in body weight or normal tissue toxicity in non-tumor-bearing Balb/c mice. Advances in knowledge and implications for patient careOur results suggest that Auger electron RIT with [111In]In-Bn-DTPA-nimotuzumab may provide a novel therapeutic option for patients with TNBC or trastuzumab-resistant HER2-positive BC that overexpresses EGFR. The low normal tissue toxicity of this approach may allow combination with other targeted therapies such as antibody-drug conjugates (ADCs).

4T1 Mammary Carcinoma Colonization of Metastatic Niches Is Accelerated by Obesity.

Breast cancer (BC) remains the leading cause of cancer-related deaths among women, and the chances to develop it are duplicated by obesity. Still, the impact of obesity during BC progression remains less understood. We investigated the role of obesity in tumor progression using the murine model of 4T1 mammary carcinoma in BALB/c female mice, previously high-fat-diet (HFD) fed. HFD induced obesity, metabolic impairment, and high serum and fat leptin levels. After injection of 4T1-cells, HFD-mice accelerated tumor progression and metastasis. 4T1-cells found within HFD-mice metastatic niches presented higher clonogenic potential. 4T1-cells treated in vitro with fat-conditioned medium derived from HFD-mice, increased migration capacity through CXCL12 and CCL25 gradients. In HFD-mice, the infiltration and activation of immune cells into tumor-sentinel lymph nodes was overall reduced, except for activated CD4+ T cells expressing low CD25 levels. Within the bone marrow, the levels of haematopoiesis-related IL-6 and TNF-α decreased after 4T1-cells injection in HFD-mice whereas increased in the controls, suggesting that upregulation of both cytokines, regardless of the tumor, is disrupted by obesity. Finally, the expression of genes for leptin, CXCR4, and CCR9 (receptors of CXCL12 and CCL25, respectively) was negatively correlated with the infiltration of CD8 T cells in human triple-negative BC tumors from obese patients compared to non-obese. Together, our data present early evidence of systemic networks triggered by obesity that promote BC progression to the metastatic niches. Targeting these pathways might be useful to prevent the rapid BC progression observed among obese patients.

Profile of Alexander Y. Rudensky, winner of the 2018 Vilcek Prize in Biomedical Science

In October 2017, the Chicago-based MacArthur Foundation announced the names of the latest winners of the renowned “genius” grants: fellowships awarded for “originality, insight, and potential,” to outstanding scientists, writers, visual artists, and members of other professions. Of the 24 fellows selected, at least one-third are immigrants to the United States, working in fields as varied as immunology, anthropology, psychology, social work, and creative writing. Foreign-born scientists in the United States are routinely well-represented among recipients of prestigious honors, including the Nobel Prizes and election to the National Academy of Sciences, to name but a couple of high honors bestowed on scientists. Alexander Y. Rudensky. Image courtesy of The Vilcek Foundation. T regs in the T cell zone of a lymph node. Foxp3 is shown in blue, CD4 in green, T-bet in red, and CD44 in white. Image courtesy of Alejandra Mendoza (Memorial Sloan Kettering Cancer Center, New York City). Immunohistochemical staining of a human triple-negative breast cancer tumor with anti-Foxp3 antibody (brown) and hematoxylin. Image courtesy of Hannah Y. Wen (Memorial Sloan Kettering Cancer Center, New York City). In 2005, the New York City-based Vilcek Foundation began an annual prize program to recognize exceptionally accomplished foreign-born scientists working in the United States. The program’s mission is to raise public awareness of the role of immigrant talent in sustaining the nation’s global leadership in science, technology, and the arts. The Vilcek Foundation’s prize winners are chosen by accomplished peers and receive a cash award of $100,000. Since 2009, the Vilcek Foundation has also recognized promising young scientists who have carved a niche for themselves early in their careers through pathbreaking work that has caught the attention of the scientific community; each winner of the Creative Promise Prize currently receives a $50,000 cash award* (concomitantly, the Vilcek Foundation recognizes foreign-born … [↵][1]1To whom correspondence should be addressed. Email: jan.vilcek{at}nyumc.org. [1]: #xref-corresp-1-1

Synthetic Lethality Exploitation by an Anti-Trop-2-SN-38 Antibody-Drug Conjugate, IMMU-132, Plus PARP Inhibitors in BRCA1/2-wild-type Triple-Negative Breast Cancer.

Purpose: Both PARP inhibitors (PARPi) and sacituzumab govitecan (IMMU-132) are currently under clinical evaluation in triple-negative breast cancer (TNBC). We sought to investigate the combined DNA-damaging effects of the topoisomerase I (Topo I)-inhibitory activity of IMMU-132 with PARPi disruption of DNA repair in TNBC.Experimental Design:In vitro, human TNBC cell lines were incubated with IMMU-132 and various PARPi (olaparib, rucaparib, or talazoparib) to determine the effect on growth, double-stranded DNA (dsDNA) breaks, and cell-cycle arrest. Mice bearing BRCA1/2-mutated or -wild-type human TNBC tumor xenografts were treated with the combination of IMMU-132 and PARPi (olaparib or talazoparib). Study survival endpoint was tumor progression to >1.0 cm3 and tolerability assessed by hematologic changes.Results: Combining IMMU-132 in TNBC with all three different PARPi results in synergistic growth inhibition, increased dsDNA breaks, and accumulation of cells in the S-phase of the cell cycle, regardless of BRCA1/2 status. A combination of IMMU-132 plus olaparib or talazoparib produces significantly improved antitumor effects and delay in time-to-tumor progression compared with monotherapy in mice bearing BRCA1/2-mutated HCC1806 TNBC tumors. Furthermore, in mice bearing BRCA1/2-wild-type tumors (MDA-MB-468 or MDA-MB-231), the combination of IMMU-132 plus olaparib imparts a significant antitumor effect and survival benefit above that achieved with monotherapy. Most importantly, this combination was well tolerated, with no substantial changes in hematologic parameters.Conclusions: These data demonstrate the added benefit of combining Topo I inhibition mediated by IMMU-132 with synthetic lethality provided by PARPi in TNBC, regardless of BRCA1/2 status, thus supporting the rationale for such a combination clinically. Clin Cancer Res; 23(13); 3405-15. ©2017 AACR.

Targeting synthetic lethality between the SRC kinase and the EPHB6 receptor may benefit cancer treatment.

Application of tumor genome sequencing has identified numerous loss-of-function alterations in cancer cells. While these alterations are difficult to target using direct interventions, they may be attacked with the help of the synthetic lethality (SL) approach. In this approach, inhibition of one gene causes lethality only when another gene is also completely or partially inactivated. The EPHB6 receptor tyrosine kinase has been shown to have anti-malignant properties and to be downregulated in multiple cancers, which makes it a very attractive target for SL applications. In our work, we used a genome-wide SL screen combined with expression and interaction network analyses, and identified the SRC kinase as a SL partner of EPHB6 in triple-negative breast cancer (TNBC) cells. Our experiments also reveal that this SL interaction can be targeted by small molecule SRC inhibitors, SU6656 and KX2-391, and can be used to improve elimination of human TNBC tumors in a xenograft model. Our observations are of potential practical importance, since TNBC is an aggressive heterogeneous malignancy with a very high rate of patient mortality due to the lack of targeted therapies, and our work indicates that FDA-approved SRC inhibitors may potentially be used in a personalized manner for treating patients with EPHB6-deficient TNBC. Our findings are also of a general interest, as EPHB6 is downregulated in multiple malignancies and our data serve as a proof of principle that EPHB6 deficiency may be targeted by small molecule inhibitors in the SL approach.

The TRAIL receptor agonist drozitumab targets basal B triple-negative breast cancer cells that express vimentin and Axl.

Previously, we found that GST-tagged tumor necrosis factor-related apoptosis inducing ligand preferentially killed triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype by activating death receptor 5 (DR5). The purpose of this study was to explore the sensitivity of breast cancer cell lines to drozitumab, a clinically tested DR5-specific agonist; identify potential biomarkers of drozitumab-sensitive breast cancer cells; and determine if those biomarkers were present in tumors from patients with TNBC. We evaluated viability, caspase activity, and sub-G1 DNA content in drozitumab-treated breast cancer cell lines and we characterized expression of potential biomarkers by immunoblot. Expression levels of vimentin and Axl were then explored in 177 TNBC samples from a publically available cDNA microarray dataset and by immunohistochemistry (IHC) in tumor tissue samples obtained from 53 African-American women with TNBC. Drozitumab-induced apoptosis in mesenchymal TNBC cell lines but not in cell lines from other breast cancer subtypes. The drozitumab-sensitive TNBC cell lines expressed the mesenchymal markers vimentin and Axl. Vimentin and Axl mRNA and protein were expressed in a subset of human TNBC tumors. By IHC, ~15 % of TNBC tumors had vimentin and Axl expression in the top quartile for both. These findings indicate that drozitumab-sensitive mesenchymal TNBC cells express vimentin and Axl, which can be identified in a subset of human TNBC tumors. Thus, vimentin and Axl may be useful to identify TNBC patients who would be most likely to benefit from a DR5 agonist.

Abstract P5-04-05: Co-stimulation through 4-1BB/CD137 improves expansion and function of tumor-infiltrating T lymphocytes from primary and metastatic triple-negative breast cancer and inflammatory breast cancer

Abstract Background: Increased CD8+ tumor-infiltrating lymphocytes (TIL) is associated with improved prognosis in triple-negative breast cancer (TNBC) suggesting that T-cell responses at the tumor site can be harnessed for autologous T-cell therapy using TIL expanded ex vivo. Although TIL therapy has been developed for solid tumors such as melanoma, cervical, and ovarian cancer. Moreover, methods facilitating CD8+ TIL expansion from TNBC are desirable given their cytotoxic potential against tumor cells. One approach to address this need is to provide agonist signals through the 4-1BB/CD137 pathway during TIL expansion selectively costimulating CD8+ T-cell activation. In this study, we established a method of expanding TIL from surgical specimens and core biopsies from primary TNBC patients and compared the phenotype and function of these TIL to lymphocytes from peripheral blood. Patients and methods: Eight primary human TNBC tumor samples were obtained by surgical resection or core biopsy after neo-adjuvant chemotherapy. Small (4-6 mm2) tumor fragments were cultured for 28 days in 24-well plates in medium containing 3000 IU/ml IL-2 alone or in combination with 10 µg/ml agonistic anti-4-1BB IgG4 (BMS663513) added at the start of culture. Viable cell numbers and the expression of CD3, CD8, CD4, CD27, CD28, CD56, CD16, Granzyme B, and Perforin were determined by flow cytometry on day 28 after culture. Cytotoxic function of the TIL was evaluated by measuring Caspase 3 cleavage in target cells. Blood samples collected at surgery were immunophenotyped for subsets of T cells and NK cells, and assessed for ability of T cells to synthesize Th1/Th2 cytokines following activation through the T-cell receptor (TCR), and potential of NK cells to kill K562 targets. Results: TIL were successfully expanded from tumor fragments in 6/8 of the cases, with addition of anti-4-1BB greatly increasing the percentage and yield of CD8+CD3+ T cells. CD8+ TIL isolated from cultures receiving 4-1BB costimulation however had decreased CD27 and CD28 expression together with increased cytotoxic T-cell activity. Gene expression analysis also found that TIL from these 4-1BB costimulated cultures had a more differentiated CD8+ T-cell gene profile. Peripheral blood CD3+, CD8+, and CD4+ T cells were lower than those of healthy controls, but TCR-activated cytokine synthesis was not significantly different. Peripheral blood NK cells expressed normal Granzyme A/B and Perforin levels and exhibited normal IFN-? secretion and CD107a-release following exposure to IL-12/IL-18 or with K562 targets. Conclusions: TIL can be reproducibly expanded from TNBC tumors with IL-2 after neo-adjuvant therapy, with CD8+ TIL outgrowth and effector activity increased by provision of 4-1BB/CD137 costimulatory signals during culture initiation. These CD8+ TIL however had a more differentiated phenotype with higher cytotoxic activity. Peripheral blood T-cell and NK cell function was comparable to those of healthy donors. Our results support further development of an autologous TIL expansion protocol after neo-adjuvant therapy for use in an adoptive cell therapy approach to treat TNBC recurrence or metastasis. Citation Format: Michiko Harao, Hui Gao, Jie Qing Chen, Elizabeth A Mittendorf, Gildy V Babiera, Sarah M DeSnyder, Korrene F Rockwood, Savitri Krishnamurthy, Huiming Sun, Jie S Willey, Naoto T Ueno, James M Reuben, Laszlo G Radvanyi. Co-stimulation through 4-1BB/CD137 improves expansion and function of tumor-infiltrating T lymphocytes from primary and metastatic triple-negative breast cancer and inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-05.

Abstract LB-263: Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with few treatment options. Recently, it was shown that infiltration of TNBC with CD8+ tumor-infiltrating lymphocytes (TIL) is associated with improved prognosis, suggesting that T-cell responses at the tumor site can be harnessed as an autologous T-cell therapy for TNBC. Although TIL therapy has been developed for solid tumors such as melanoma, cervical, and ovarian cancer, whether TIL, especially CD8+ cells, with effector-memory properties can be expanded with reasonable yields from primary TNBC is not known. Given the high rates of relapse and refractory nature of recurrent TNBC disease, autologous TIL therapy may offer a life-saving option for these patients. Moreover, methods to facilitate CD8+ TIL expansion from TNBC are also desirable given the cytotoxic potential of these cells. One approach to address this need is to manipulate TNF-R family member signaling, such as 41BB/CD137, to provide potent costimulatory signals for CD8+ T-cell activation and division. In this study, we established a method of isolating and generating TIL from patients with primary TNBC surgical specimens and core biopsies using an agonistic 41BB/CD137 antibody. Patients and methods: Four primary human TNBC tumor samples were obtained by surgical resection or core biopsy after neoadjuvant chemotherapy. Small tumor fragments were cultured in 24-well plates in medium containing 3000 IU/ml IL-2, or 3000 IU/ml IL-2 plus 10 μg/ml added agonistic anti-41BB IgG4 (BMS663513). Viable cell numbers and the expression of CD3, CD8, CD4, CD27, CD28, CCR7, CD45RA, CD56, CD16, Granzyme B, and Perforin was determined by flow cytometry on days 7, 14, 21, 28, and 35 after culture set-up. Cytotoxic function of the TIL was evaluated by measuring Caspase 3 cleavage in target cells. Results: Although TIL were successfully expanded from the tumor fragments in 4/4 tumor samples, IL-2 alone yielded fewer cells than IL-2 together with 41BB costimulation provided early during the culture. Anti-41BB markedly increased the percentage and yield of CD8+CD3+ T cells at all times (6.34x106 vs , 202x106, P&amp;lt;0.01). However, the resulting CD8+ cells had decreased CD27 expression coincident with increased CD70 when anti-41BB was added. 41BB costimulation also increased the cytotoxic T-cell activity of the expanded TIL. The expanded TIL could also be cryopreserved and later expanded using anti-CD3 and IL-2. Conclusions: We have established a method to expand CD8+ TIL with cytotoxic activity with high yield from small samples of remaining primary tumor after neoadjuvant chemotherapy using IL-2. Provision of 41BB costimulation immediately upon tumor fragment culture initiation was critical in enhancing the rate and yield of CD8+ T cells with increased effector function; these could be cryopreserved and later thawed with high viability for secondary expansion. Our results support further development of an autologous TIL expansion protocol after neoadjuvant therapy for use in an adoptive cell therapy approach to treat TNBC recurrence or metastasis. Citation Format: Michiko Harao, Elizabeth A. Mittendorf, Gildy V. Babiera, Naoto T. Ueno, Laszlo G. Radvanyi. Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2014-LB-263