Triple-negative Breast Cancer Mouse Research Articles

Effect of doxorubicin on efficacy of immune checkpoint inhibitors through increasing infiltration of CD8-positive T cells and activating STAT1-IRF1-CXCL10 axis.

e13095 Background: Immune checkpoint inhibitors (ICIs) generate an enduring clinical remission in metastatic cancers. However, the efficacy of ICIs monotherapy for triple negative breast cancer (TNBC) is limited. Strategies are urgently needed to enhance anticancer immune responses in TNBC to prolong survival of patients. Methods: BALB/c mice subcutaneous tumor experiments were done to evaluate the efficacy of doxorubicin combined with PD-1 inhibitor, compared with other chemotherapy including cisplatin and paclitaxel. RNA-seq and single-sample gene set enrichment analysis (ssGSEA) were done with our collected mice tumor tissue to observe changes of tumor immune environment in TNBC after chemotherapy combined with immunotherapy. Then, we performed coculture, Transwell assay and FACs in TNBC cell lines, including MDA-MB-231, BT549 and 4T1, and primary CD8+T cells, to assess effects of doxorubicin on CD8+T cell migration in vitro. Gene Ontology (GO) and Kyoto Encyclopedia of Genes, Genomes (KEGG) analysis were enriched from RNA-seq data to seek for a pathway. We analyzed the difference of CD8+T cell infiltration related gene expression, including CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11. Western blot, qPCR and ELISA were utilized to explore possible mechanism in stronger migration of CD8+T cells by doxorubicin. Rescue assays were used to confirm the involved mechanisms. Results: Subcutaneous tumor experiment showed that doxorubicin could enhance the efficacy of immune checkpoint inhibitors (p＜0.05),andthe immune microenvironment in TNBC transferred from "anti-inflammatory" to "proinflammatory" status after doxorubicin combined with immunotherapy, compared with other chemotherapy drugs combined with immunotherapy. Higher CD8+T cells infiltration occurred in TNBC treated with doxorubicin in vivo (p＜0.05). The CD8+T cells migration assay demonstrated a higher CD8+T cell migration with doxorubicin in vitro (p＜0.05). KEGG analysis revealed that the chemokine-related pathway was significantly activated after doxorubicin treatment, in which CXCL10 chemokine was most likely to be responsible for CD8+T cell infiltration based on its expression and secretion (both p<0.01). Further experiments demonstrated that the expression of the whole STAT1-IRF1-CXCL10 axis was up-regulated in TNBC mice treated with doxorubicin (p＜0.01), and the STAT1-IRF1-CXCL10 axis was also activated in doxorubicin-treated TNBC cell lines (p＜0.0001). Conclusions: This research demonstrated that doxorubicin can enhance efficacy of immune checkpoint inhibitors through increasing infiltration of CD8+T cells in triple-negative breast cancer via activating STAT1-IRF1-CXCL10 axis. These results provide rationales of doxorubicin combined with PD-1 inhibitor for extending survival of TNBC patients.

High-dose paclitaxel and its combination with CSF1R inhibitor in polymeric micelles for chemoimmunotherapy of triple negative breast cancer

The presence of immunosuppressive immune cells in tumors is a significant barrier to the generation of therapeutic immune responses. Similarly, in vivo triple-negative breast cancer (TNBC) models often contain prevalent, immunosuppressive tumor-associated macrophages in the tumor microenvironment (TME), resulting in breast cancer initiation, invasion, and metastasis. Here, we test systemic chemoimmunotherapy using small-molecule agents, paclitaxel (PTX), and colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397, to enhance the adaptive T cell immunity against TNBCs in immunocompetent mouse TNBC models. We use high-capacity poly(2-oxazoline) (POx)-based polymeric micelles to greatly improve the solubility of insoluble PTX and PLX3397 and widen the therapeutic index of such drugs. The results demonstrate that high-dose PTX in POx, even as a single agent, exerts strong effects on TME and induces long-term immune memory. In addition, we demonstrate that the PTX and PLX3397 combination provides consistent therapeutic improvement across several TNBC models, resulting from the repolarization of the immunosuppressive TME and enhanced T cell immune response that suppress both the primary tumor growth and metastasis. Overall, the work emphasizes the benefit of drug reformulation and outlines potential translational path for both PTX and PTX with PLX3397 combination therapy using POx polymeric micelles for the treatment of TNBC.

Dual-drug loaded ultrasound-responsive nanodroplets for on-demand combination chemotherapy

Phase-changing nanodroplets are nanometric sized constructs that can be vaporized via external stimuli, such as focused ultrasound, to generate gaseous bubbles that are visible in ultrasound. Their activation can also be leveraged to release their payload, creating a method for ultrasound-modulated localized drug delivery. Here, we develop a perfluoropentane core nanodroplet that can simultaneously load paclitaxel and doxorubicin, and release them in response to an acoustic trigger. A double emulsion method is used to incorporate the two drugs with different physio-chemical properties, which allows for a combinatorial chemotherapy regimen to be used. Their loading, release, and biological effects on a triple negative breast cancer mouse model are investigated. We show that activation enhances the drug-delivery effect and delays the tumor growth rate in vivo. Overall, the phase-changing nanodroplets are a useful platform to allow on-demand delivery of combinations of drugs.

OTUD1 chemosensitizes triple-negative breast cancer to doxorubicin by modulating P16 expression

Chemotherapy remains a critical component of triple-negative breast cancer (TNBC) treatment; however, patients often develop resistance to chemotherapeutic agents. Accumulating evidence indicates that deubiquitylases (DUBs) play pivotal roles in regulating cell proliferation, differentiation, apoptosis, and tumorigenesis. Deubiquitylase OTUD1 is considered a tumor suppressor in various cancers, yet its role in doxorubicin sensitivity in breast cancer patients remains inadequately understood. In this study, we investigated the expression levels and prognostic role of OTUD1 in breast cancer. Our findings demonstrated that OTUD1 was downregulated in TNBC, and lower OTUD1 expression levels were correlated with poor prognosis. We utilized the CCK-8 cell viability assay, flow cytometric analysis, and a TNBC mouse xenograft model to examine the influence of OTUD1 on doxorubicin (DOX) chemotherapy sensitivity in vitro and in vivo. Western blot and immunohistochemistry were employed to explore the correlation between OTUD1 and P16. Our results indicated that upregulation of OTUD1 expression inhibits TNBC cell proliferation and enhances its sensitivity to doxorubicin. Additionally, rescue experiments confirmed that the chemosensitizing effect of OTUD1 overexpression could be reversed by the inhibition of P16. Therefore, our findings reveal that OTUD1 sensitizes TNBC cells to DOX by upregulating P16 expression, suggesting a potential new diagnostic biomarker and therapeutic target for the future treatment of TNBC.

Ultrasound-Responsive Oxygen-Carrying Pollen for Enhancing Chemo-Sonodynamic Therapy of Breast Cancer.

The tumor-suppressing efficacy of either chemotherapeutics or gaseous drugs has been confirmed in treating the triple negative breast cancer (TNBC), while the efficacy of single treatment is usually dissatisfactory. Herein, a novel ultrasound responsive natural pollen delivery system is presented to simultaneously load chemotherapeutics and gaseous drugs for synergistic treatment of TNBC. The hollow structure of pollen grains carries oxygen-enriched perfluorocarbon (PFC), and the porous spinous process structure adsorbs the chemotherapeutic drug doxorubicin (DOX) (PO/D-PGs). Ultrasound can trigger the oxygen release from PFC and excite DOX, which is not only a chemotherapeutic but also a sonosensitizer, to realize chemo-sonodynamic therapy. The PO/D-PGs are demonstrated to effectively enhance oxygen concentration and increase the production of reactive oxygen species in the presence of low-intensity ultrasound, synergistically enhancing the tumor killing ability. Thus, the synergistic therapy based on ultrasound-facilitated PO/D-PGs significantly enhances the antitumor effect in the mouse TNBC model. It is believed that the proposed natural pollen cross-state microcarrier can be used as an effective strategy to enhance chemo-sonodynamic therapy for TNBC.

Development of effective immunotherapy for triple negative breast cancer by WEE1 kinase inhibition

Abstract Treatment options for triple negative breast cancer (TNBC) are limited because they are usually insensitive to most available hormonal or targeted therapeutic agents owing to their triple negative nature. Exciting clinical results using immune checkpoint inhibitors (ICI) directed against PD-1/PD-L1 have been reported. However, only a subset of patients (~20%) respond to ICI. Therefore, identifying rational combinations is needed to enhance the efficacy of immunotherapy in TNBC. We demonstrated there that inhibition of WEE1, a G2/M cell cycle checkpoint regulator induced by DNA damage, increased expression levels of MHC-I, and pro-inflammatory chemokines (CXCL9 and CXCL10) together with PD-L1 in human and mouse TNBC cells. Consequently, WEE1 inhibition enhanced cytotoxic T-lymphocyte (CTL)-mediated killing of tumor cells, and CTL infiltration into the tumor, making tumors susceptible to anti-PD-1 mAb immune checkpoint blockade in mouse TNBC models in a CD8+ T cell–dependent manner. Mechanistically, WEE1 inhibition triggered the cellular damage stress and interferon response through the double-stranded DNA/RNA (dsDNA/RNA) by activating the TLR9/IRF7 pathway. Furthermore, combining WEE1 inhibition and TLR9 agonist CpG DNA resulted in tumor regression, facilitating an effective antitumor T cell response. Our findings have rapid translational implications for the exploration of WEE1 inhibitors alongside agents that stimulate CD8+ T cell activity, including ICI and TLR9 agonists, which are both clinically available to treat TNBC patients. We acknowledge the funding support from NCI R01CA222963 and DoD W81XWH-19-1-0106.

PRR15 deficiency facilitates malignant progression by mediating PI3K/Akt signaling and predicts clinical prognosis in triple-negative rather than non-triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast neoplasms with a higher risk of recurrence and metastasis than non-TNBC. Nevertheless, the factors responsible for the differences in the malignant behavior between TNBC and non-TNBC are not fully explored. Proline rich 15 (PRR15) is a protein involved in the progression of several tumor types, but its mechanisms are still controversial. Therefore, this study aimed to investigate the biological role and clinical applications of PRR15 on TNBC. PRR15 gene was differentially expressed between TNBC and non-TNBC patients, previously described as an oncogenic factor in breast cancer. However, our results showed a decreased expression of PRR15 that portended a favorable prognosis in TNBC rather than non-TNBC. PRR15 knockdown facilitated the proliferation, migration, and invasive ability of TNBC cells in vitro and in vivo, which was abolished by PRR15 restoration, without remarkable effects on non-TNBC. High-throughput drug sensitivity revealed that PI3K/Akt signaling was involved in the aggressive properties of PRR15 silencing, which was confirmed by the PI3K/Akt signaling activation in the tumors of PRR15Low patients, and PI3K inhibitor reversed the metastatic capacity of TNBC in mice. The reduced PRR15 expression in TNBC patients was positively correlated with more aggressive clinicopathological characteristics, enhanced metastasis, and poor disease-free survival. Collectively, PRR15 down-regulation promotes malignant progression through the PI3K/Akt signaling in TNBC rather than in non-TNBC, affects the response of TNBC cells to antitumor agents, and is a promising indicator of disease outcomes in TNBC.

C-Myc-Targeting PROTAC Based on a TNA-DNA Bivalent Binder for Combination Therapy of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is highly aggressive with a poor clinical prognosis and no targeted therapy. The c-Myc protein is a master transcription factor and a potential therapeutic target for TNBC. In this study, we develop a PROTAC (PROteolysis TArgeting Chimera) based on TNA (threose nucleic acid) and DNA that effectively targets and degrades c-Myc. The TNA aptamer is selected in vitro to bind the c-Myc/Max heterodimer and appended to the E-box DNA sequence to create a high-affinity, biologically stable bivalent binder. The TNA-E box-pomalidomide (TEP) conjugate specifically degrades endogenous c-Myc/Max, inhibits TNBC cell proliferation, and sensitizes TNBC cells to the cyclin-dependent kinase inhibitor palbociclib in vitro. In a mouse TNBC model, combination therapy with TEP and palbociclib potently suppresses tumor growth. This study offers a promising nucleic acid-based PROTAC modality for both chemical biology studies and therapeutic interventions of TNBC.

Abstract 2480: Chemotherapy increases invasive cancer stem cells in breast cancer

Abstract Background: Metastasis is the leading cause of breast cancer (BC) death. To successfully metastasize, tumor cells must have both invasive and stem-like phenotypes. BC cells that express MenaINV, an invasive isoform of the actin regulatory protein Mena, have an enhanced ability to migrate and intravasate within the primary tumor, and extravasate at secondary sites. BC cells with a stem-like phenotype can withstand anoikis during hematogenous dissemination and initiate metastatic growth. Therefore, MenaINVHigh cancer stem cells (CSCs) have high metastatic potential and can be considered the most dangerous cells in the tumor. We recently showed that MenaINV and the CSC program can be induced independently in BC cells via direct contact with tumor associated macrophages (TAMs). Though chemotherapy improves survival, recent evidence shows that for some BC patients, chemotherapy may increase metastatic dissemination, though the reasons for this are unclear. Chemotherapy causes extensive cell death and a proinflammatory cytokine surge that recruits macrophages to the primary tumor and enhances the assembly of the tumor cell dissemination portals TMEM doorways. Given that TAM-tumor cell contact induces MenaINV and the CSC program in BC cells, we hypothesized that chemotherapy promotes dissemination by increasing TAM-tumor cell contact, leading to the induction of MenaINVHigh cells, CSCs, and “double positive” MenaINVHigh CSCs. Methods: We used two models of BC (MMTV-PyMT mice bearing spontaneous mammary tumors, and HT17 patient-derived xenografts) which recapitulate metastatic triple-negative BC in mice. Mice were treated with paclitaxel (10 mg/kg, IV) every 5 days for a total of 3 treatments. Mouse subgroups received an additional treatment of liposomes with the macrophage-depleting agent clodronate (200 µL, 18.4 µM concentration, IP), or PBS control liposomes, every 2 days. Mammary tumors were evaluated for: (1) TAM density, (2) number of “single positive” cells - defined as those expressing high levels of Sox9 (CSC marker) or MenaINV (invasion marker) - and (3) the number of “double positive” MenaINVHigh CSCs (Sox9HighMenaINVHigh). Results: We found that chemotherapy increased TAM density, and this corresponded with an increase in “double positive” MenaINVHigh CSCs. Upon macrophage depletion, MenaINVHigh CSCs fell to below baseline levels regardless of chemotherapy treatment. Chemotherapy did not increase “single positive” cells. Conclusion: The induction of prometastatic MenaINVHigh CSCs during chemotherapy is macrophage dependent. The finding that chemotherapy only affected double positive cells suggests that MenaINV and the CSC program are induced together, rather than independently, by TAMs. Further work is required to determine the mechanism of this co-induction. These results lay the groundwork for novel microenvironment-based therapies to alleviate the prometastatic effects of chemotherapy in BC. Citation Format: Madeline Friedman, Camille L. Duran, Ved P. Sharma, John S. Condeelis, Maja H. Oktay, George S. Karagiannis, David Entenberg. Chemotherapy increases invasive cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2480.

Abstract 5811: Epigenetic regulation of chromosomal instability in triple-negative breast cancer

Abstract Metastasis is the leading cause of cancer-related death among women with breast cancer. Chromosomal instability (CIN) has emerged as a hallmark of triple-negative breast cancer (TNBC) as it has recently been shown to promote metastasis. However, the underlying molecular mechanisms by which CIN drives metastasis are not completely understood. We have identified a discrete population of highly metastatic SOX2/OCT4+ cells expressing elevated levels of epigenetic regulator EZH2 associated with increased CIN in both human and mouse TNBC. EZH2 histone methyl transferase (HMT) is the catalytic subunit of the Polycomb repressive complex 2 (PRC2), represses target genes through trimethylation of Histone 3 at lysine 27 (H3K27me3). Importantly, genetic and pharmacologic inhibition of EZH2 lead to reduction of CIN and impaired metastasis. These findings led to the hypothesis that EZH2-mediated CIN constitutes a novel mechanism of metastasis regulation. To directly demonstrate epigenetic regulation of CIN, we used genome wide-Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in parallel with RNA-seq. Gene set enrichment of EZH2-repressed target genes directly implicated the spindle formation pathway network. The central core of this network comprised of tankyrase (TNKS), a multifunctional poly (ADP-ribose) polymerase (PARP) previously implicated in DNA repair, telomere function and centrosome maturation. ChIP-PCR confirmed that EZH2 directly binds to the TNKS promoter, and CRISPR knockout of TNKS abrogated the ability of EZH2 inhibition in suppressing CIN, consistent with pharmacological inhibition of TNKS. More specifically, dysregulation of TNKS by EZH2 in OCT4/SOX2+ cells lead to increased numbers of centrosomes and multipolar mitosis. To directly demonstrate the role of aberrant H3K27me3 in regulating chromosomal segregation during mitosis, we used dCas9-EZH2 or dCas9-EZH2 catalytically dead mutant together with chromosome-specific CRISPR guides to ectopically enhance H3K27 trimethylation at the pericentromeric region of specific chromosomes. Ectopic deposition of EZH2 at pericentromeric regions lead to increased CIN. Conceptually, our work provides an unappreciated link between epigenetic regulation and CIN which have been hitherto studied in isolation. From a clinical perspective, demonstrating epigenetic regulation of CIN has opened the possibility for the development of first CIN suppressive therapeutic strategies targeting TNBC metastasis. Citation Format: Yang Bai, Albert Agustinus, Cem Meydan, Dylan R. McNally, Shira Yomtoubian, Liron Yoffe, Ari M. Melnick, Samuel Bakhoum, Vivek Mittal. Epigenetic regulation of chromosomal instability in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5811.

Abstract 3991: NOS inhibition enhances docetaxel-mediated anti-tumor effect in obese mice with triple negative breast cancer

Abstract Purpose of the study: To evaluate the role of obesity in tumor progression and tumor microenvironment (TME) dynamic after the inhibition of nitric oxide synthase (NOS) in syngeneic murine models engrafted with triple negative breast cancer (TNBC) tumors. For this study, we wanted to deepen the results of our recently completed phase I/II clinical trial, where treatment with NG-methyl-L-arginine (L-NMMA, a pan-NOS inhibitor) increased the response rate to chemotherapy (docetaxel) in obese metastatic TNBC (ER-/PR-/HER2-) patients to 86% vs. 60% in normal weight individuals. TME analysis from responders revealed a neutrophil phenotype shift from protumor N2 to antitumor N1. We thus hypothesize that combined treatment with L-NMMA and docetaxel enhances antitumor effect by modulating TME in obese mice with TNBC. Experimental procedures: 3-week old female C57BL/6 mice were fed for 10 weeks either with high fat diet (HFD) or normal diet (ND). At week 13, TNBC mouse E0771 tumor cells were injected into the mice mammary fat pad. Once tumors reached 100 mm3, the animals were randomized to four groups: vehicle; docetaxel; L-NMMA; and docetaxel plus L-NMMA treatment. Tumor volume was measured throughout the experiment and growth rate was compared between groups at the end of treatment. Blood, tumor and perigonadal white adipose tissue (pgWAT) were collected. Cytokines and nitrite/nitrate levels (NO2-/NO3-, a NO indicator) were measured from blood and conditioned media (CM) from pgWAT using appropriate kits. Tumor immune cell infiltration was evaluated by immunohistochemistry. GraphPad software was used for statistical analysis and P-values <0.05 were considered to describe statistically significant differences between groups. Results: Tumor growth rate was significantly higher in HFD mice compared to ND mice. Treatment with the docetaxel/L-NMMA combination significantly slowed tumor growth in HFD mice (p=0.015), and showed a similar trend in ND mice (p=0.92) vs. the corresponding vehicle-treated groups. The reduction of tumor growth was significantly higher in HFD mice vs. ND mice (median of differences -2.0, p=0.031). HFD mice presented higher levels of pro-inflammatory cytokines, [IL-1b (p=0.011), IL-6 (p<0.0001) and TNFa (p<0.0001)] vs ND mice in pgWAT CM, as well as increased levels of NO2-/NO3- and expression of iNOS in tumors and in perigonadal adipose tissue. Conclusion: HFD mice had a pro-inflammatory profile with a significantly faster tumor growth and higher expression of iNOS in tumor and adipose tissues. Treatment with the docetaxel/L-NMMA combination resulted in a more significant anti-tumor effect in HFD mice, likely through remodeling of TME. Spatial analysis, including CODEX and immunophenotyping are being conducted to determine the TME influence in obesity associated TNBC and the effect of NOS inhibition. Citation Format: Ivonne Uzair, Kai Sun, Ann Anselme, Wei Qian, Jianying Zhou, Roberto Rosato, Jenny Chang. NOS inhibition enhances docetaxel-mediated anti-tumor effect in obese mice with triple negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3991.

Abstract 484: VIP236: A small molecule drug conjugate with an optimized camptothecin payload has significant activity in patient-derived and metastatic cancer models

Abstract Introduction: We previously presented the design and preliminary characterization of VIP236—an SMDC consisting of an αvβ3 integrin binder linked to an optimized camptothecin topoisomerase I (TOP1) inhibitor payload released by neutrophil elastase in the tumor microenvironment (Lerchen et al, Cancers 2022). Herein we evaluate VIP236 in patient-derived and metastatic models of cancer. Methods: Subcutaneous patient-derived xenograft mouse models in non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), colorectal cancer (CRC), and triple negative breast cancer (TNBC) were treated with VIP236. Metastasis formation was measured by human DNA PCR in an orthotopic TNBC mouse model. Gastric cancer models included patient-derived and cell line xenograft mouse models. Tumor/control (T/C) ratios were calculated, and body weight was measured. Pharmacodynamic (PD) effect of VIP236 in a gastric cancer SNU16 xenograft mouse model was investigated. Tumor samples from vehicle- and VIP236-treated groups collected up to 144 hours postdose were analyzed for % positive γH2AX cells by a fit-for-purpose qualified immunohistochemistry assay. Results: VIP236 is efficacious in vivo patient-derived models of NSCLC, RCC, CRC and TNBC with T/C ratios of 0.0, 8.9,14.5 and 5.6 compared with vehicle control (p<0.0001, CRC p<0.01). VIP236 showed statistically improved in vivo efficacy in gastric cancer models versus trastuzumab deruxtecan (a commercial antibody-drug conjugate [ADC] with a camptothecin payload) in NCIH87 (HER2high, p<0.05), SNU16 (HER2neg, p<0.001), and GXA3040 (patient-derived, HER2low, p<0.001) xenografts. VIP236 induced statistically significant tumor growth inhibition in a patient-derived CRC liver metastasis model (p<0.01) and statistically significant reduction in lung (p<0.001) and brain (p<0.01) metastasis from an orthotopic TNBC model was observed. Across all in vivo models no clinically significant weight loss or treatment-related mortality were observed. Furthermore, a time-dependent increase of γH2AX % positive cells in SNU16 in vivo VIP236-treated tumors confirms that VIP236 drives robust TOP1 inhibition and subsequent DNA damage. Conclusions: In summary, VIP236 monotherapy efficacy in NSCLC, gastric, TNBC, RCC, CRC, and metastatic TNBC in vivo cancer models provides a rationale for clinical investigation in advanced metastatic solid tumors. In gastric cancer, VIP236 showed improved efficacy compared with the approved ADC, trastuzumab deruxtecan, regardless of HER2 expression. These results warrant further evaluation in clinical trials. Citation Format: Beatrix Stelte-Ludwig, Melanie M. Frigault, Hans-Georg Lerchen, Tibor Schomber, Raquel Izumi, Amy J. Johnson, Ahmed Hamdy. VIP236: A small molecule drug conjugate with an optimized camptothecin payload has significant activity in patient-derived and metastatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 484.

Abstract 3608: MLL1 regulates cytokine-driven cell migration and metastasis

Abstract Cell migration, particularly in three-dimensional (3D) environments, is a critical requirement for metastasis - the spread of cancer cells from a primary tumor to distant sites. Despite causing the vast majority of cancer-related deaths, targeting metastasis remains challenging clinically. Cancer cell migration can be triggered and maintained by elevated levels of cytokines, which are presumed to be predominantly secreted by immune cells in the tumor microenvironment. However, recent work has highlighted the importance and impact of cytokine production by cancer cells themselves. While the signaling cascades that regulate cell migration have been studied extensively, the epigenetic regulation of cell migration is still poorly understood. MLL1 is a histone methyltransferase, which along with its scaffold protein Menin, is integral to methylation at the histone 3 lysine 4 (H3K4) site. Here, we show that the MLL1-Menin interaction is essential for the migration of cancer cells and that the disruption of this interaction impairs cell migration and metastasis. MLL1 depletion reduces 3D cell migration in vitro and led to lesser metastatic burden and prolonged survival in vivo. Reduced lung metastatic burden was observed even after accounting for the difference in the growth rate of primary tumors. Mechanistically, MLL1-Menin interaction controls actin filament assembly via the IL-6/pSTAT3/Arp3 axis and acto-myosin contractility via the TGF-β1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1-Menin inhibition also decreases cell proliferation by inhibiting mitosis and cell cycle-related pathways. MLL1 depletion decreases primary tumor growth, metastatic spread, and metastatic outgrowth in mouse models of triple-negative breast cancer (TNBC), thus improving survival. When comparing metastatic burden at a threshold tumor size, rather than at a timepoint, MLL1 depletion still lowered metastatic burden. Combining an MLL1-Menin inhibitor with Paclitaxel, a standard TNBC chemotherapeutic, abrogated tumor growth and metastasis in a syngeneic TNBC mouse model. Overall, our studies show that MLL1 is an epigenetic regulator of 3D cancer cell migration, which exerts its effects by controlling the production of the key cytokines IL-6 and TGF-β1. Our in vivo studies demonstrate the potential of targeting the MLL1-Menin interaction in metastasis prevention and its potential to be combined with currently administered chemotherapeutics. Citation Format: Praful R. Nair, Ludmila Danilova, Estibaliz Gómez-de-Mariscal, Dongjoo Kim, Rong Fan, Arrate Muñoz-Barrutia, Elana Fertig, Denis Wirtz. MLL1 regulates cytokine-driven cell migration and metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3608.

Abstract 2507: Chronic stress promotes tumor progression and metastasis through the tumor microenvironment in triple-negative breast cancer

Abstract Background: Breast cancer is the most common cancer diagnosed in American women. Triple-negative breast cancer (TNBC) is three times more likely to end in death than other breast cancer subtypes and disproportionately affects young African American women. Chronic stress as a risk factor promotes tumor progression, but its role in TNBC progression and metastasis remains elusive. Methods: In this study, mice were implanted with 4T1-luciferase cells in the tibia or the fourth mammary pad of BALB/c mice and NSG mice. After four weeks of restraining inducing chronic stress and (or) β-adrenergic receptor inhibitor treatment, the impact of chronic stress on the progression and metastasis of tumor, tumor microenvironment was assessed. Results: Our results showed that chronic stress facilitated tumor progression and metastasis in both BALB/c mice and NSG TNBC mice models. In contrast, chronic stress reduced the number and function of tumor-infiltrating TCD8+ cells but increased the expression of the β2-adrenergic receptor of TCD8+ cells in the TNBC tumor microenvironment (TME) of BALB/c mice. Interestingly, chronic stress increased the density of neuron fiber in the TME of both BALB/c and NSG TNBC mice. Notably, the treatment of Propranolol (Prop, a β1- and β2-adrenergic receptor inhibitor) significantly decreased the tumor burden and metastasis of both BALB/c mice and NSG TNBC mice model. Accompany by recovering the function and number of TCD8+ cells in the TME of BALB/c mice. Conclusion: Our study indicates that chronic stress promotes TNBC progression and metastasis by regulating the tumor-immune network in the TME. Citation Format: Chao Zhang, Lizhong Wang, Runhua Liu. Chronic stress promotes tumor progression and metastasis through the tumor microenvironment in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2507.

Abstract 689: The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor models

Abstract Multiple oncolytic viruses have been shown to induce various beneficial changes in the tumor microenvironment (TME), which ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are confirming the mechanisms of action of the virus in combination with immune checkpoint inhibition in murine and human preclinical models. Efficacy after intratumoral injection of 108 PFU CodaLytic in combination with αPD-1 and/or αCTLA-4 checkpoint blockade was determined in αPD-1-resistant B16-F10 melanoma and 4T1 triple-negative breast cancer mouse models. Changes in the tumor immune infiltrate after treatment were characterized using flow cytometry. Primary human tumoroids were incubated with CodaLytic +/- 10 μg/ml pembrolizumab using the 3D-Explore platform by Nilogen Oncosystems. Tumor cell killing (TCK) was detected by high-content imaging at 72h and effects on the TME were characterized by cytokine release and RNA sequencing at 24h and/or 48h. In B16-F10, addition of CodaLytic to αPD-1 blockade rescued non-significant tumor growth inhibition (TGI) with αPD-1 alone, achieving 86% TGI (p < 0.01), 40% complete regressions and 60% survival beyond 45 days (69% TGI, 20% regressions and 30% long-term survival after CodaLytic monotherapy). Mechanistically, CodaLytic-containing regimens increased tumor immune infiltration, in particular with CD8+ T cells and cross-presenting dendritic cells (DCs). 4T1 tumors did not significantly respond to αPD-1, αCTLA-4 or CodaLytic monotherapies, but achieved 86% TGI in combination with 50% long-term survival past 55 days. CTLA-4 blockade emerged as the initial driver of slowed tumor growth and αPD-1 drove long-term survival, which was further augmented by CodaLytic addition. Infiltration with CD45+ leukocytes and cross-presenting DCs correlated with tumor volumes. The benefit of CodaLytic combination treatment was also demonstrated ex vivo in human tumoroid cultures. Meaningful TCK was observed in 50% of specimens vs 17% with pembrolizumab and 33% with CodaLytic alone. In this system without immune cell recruitment from a systemic reservoir, sustained release of CXCL9 and CXCL10 and temporal decreases of MIP-1β and CCL2 associated with response. Additional transcriptional analyses are ongoing. Taken together, this and additional preclinical data confirms the emergence of CodaLytic as a potent immunostimulatory agent capable of restoring immune cell infiltration and anti-tumor efficacy when used in combination with checkpoint inhibition in PD-1 resistant models. This data supports future development of CodaLytic for immuno-virotherapy of tumor types in which checkpoint therapy is less established. Citation Format: Yiwen Zhao, Nusrat Jahan, Marcin Stawowczyk, Katie Pfeffer, Juliana Tafrova, James Rodriguez, Chen Yang, Sybil A. Tasker, Steffen Mueller, J. Robert Coleman, Johanna K. Kaufmann. The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 689.

Abstract 2799: Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology

Abstract Transforming growth factor-beta (TGF-ß) is a master regulatory cytokine with pleotropic effects important in tumor development. TGF-ß1 binds TGF-ß receptor 2, which recruits and phosphorylates TGF-ß receptor 1 (TGFBR1). TGFBR1 in turn phosphorylates SMAD2 and SMAD3. Phosphorylated SMAD2/3 (pSMAD2/3) complexes with SMAD4 to translocate to the nucleus and enact transcriptional programs. In late-stage tumors, TGF-ß signaling is oncogenic, promoting growth, invasion, immune suppression, tissue remodeling, and epithelial to mesenchymal transition (EMT). In an effort to counter the oncogenic effects of TGF-ß signaling, Sumitomo Pharma Oncology, Inc. is developing TP-6379, an investigational, orally available, small molecule, selective TGFBR1 inhibitor. TP-6379 has a biochemical IC50 of 1.07 nM and has been shown to reduce pSMAD2 in Rhabdomyosarcoma cells with an IC50 of 108 nM. A dose dependent increase of TP-6379 was observed in plasma and tumors of A549 tumor bearing athymic nude mice (10, 50, 75, and 100 mg/kg PO). In another study using the same mouse model, pSMAD2 has shown an inverse correlation with pharmacokinetics showing > 90% inhibition for up to 4 hours (75 mg/kg PO). Pharmacodynamic (PD) biomarkers are critical for determining biological activity, but procuring serial tumor biopsies for PD analysis is challenging for many tumor types. We hypothesize that pSMAD2 can be measured in non-tumor tissues as a surrogate for activity within the tumor. Surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin punches, and even circulating tumor cells (CTCs) are much less invasive and can be sampled over multiple timepoints. We observed a measurable decrease of PD biomarkers pSMAD2/3 in PBMCs and skin after treatment with TP-6379 using a bead-based immunoassay (Luminex). Healthy human donor PBMCs treated ex vivo for 2 hours with up to 1 µM TP-6379 showed a 96% reduction in pSMAD2/3 signal. Although TP-6379 was still active in reducing pSMAD2 in PBMCs when treated in whole blood for 2 hours, it required around 30-fold more drug to achieve a similar effect, possibly due to serum protein binding. Skin samples from in vivo dosed tumor bearing mice also show significant inhibition of pSMAD2/3, which correlated with significant pSMAD2/3 inhibition in tumors from both EMT6 and 4T1 syngeneic triple-negative breast cancer mouse tumor models. We confirmed that the Luminex assay is sensitive enough to reliably measure pSMAD2/3 in healthy human doner skin punches down to 3 mm in diameter. Lastly, CTCs from breast cancer patients’ whole blood treated ex vivo for 24 hours with as low as 1 µM TP-6379 showed a decrease in pSMAD2 and EMT marker SNAI1 by as much as 96% and 86%, respectively, when measured by immunofluorescence. In summary, our research has shown that pSMAD2/3 and EMT marker SNAI1 are valid PD biomarkers for TP-6379 therapy. We propose that these biomarkers would be best measured clinically using PBMCs, CTCs and/or skin punches. Citation Format: Richard E. Heinz, Curtis A. Allred, David A. Kircher, Yuta Matsumura, Bettina Franz, Zhizhou Ye, Jinny Lee, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui, Steven L. Warner. Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2799.

DIF-1 exhibits anticancer activity in breast cancer via inhibition of CXCLs/CXCR2 axis-mediated communication between cancer-associated fibroblasts and cancer cells.

The tumor microenvironment (TME), largely composed of tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), plays a key role in cancer progression. A small molecule, differentiation-inducing factor-1 (DIF-1) secreted by Dictyostelium discoideum, is known to exhibit anticancer activity; however, its effect on the TME remains unknown. In this study, we investigated the effect of DIF-1 on the TME using mouse triple-negative breast cancer 4T1-GFP cells, mouse macrophage RAW 264.7 cells, and mouse primary dermal fibroblasts (DFBs). Polarization of 4T1 cell-conditioned medium-induced macrophage into TAMs was not affected by DIF-1. In contrast, DIF-1 decreased 4T1 cell co-culturing-induced C-X-C motif chemokine ligand 1 (CXCL1), CXCL5, and CXCL7 expression in DFBs and suppressed DFB differentiation into CAF-like cells. Additionally, DIF-1 inhibited C-X-C motif chemokine receptor 2 (CXCR2) expression in 4T1 cells. Immunohistochemical analyses of tumor tissue samples excised from breast cancer-bearing mice showed that DIF-1 did not affect the number of CD206-positive TAMs; however, it decreased the number of α-smooth muscle actin-positive CAFs and CXCR2 expression. These results indicated that the anticancer effect of DIF-1 was partially attributed to the inhibition of CXCLs/CXCR2 axis-mediated communication between breast cancer cells and CAFs.

Nanosecond Pulsed Electric Field Induces an Antitumor Effect in Triple-Negative Breast Cancer via CXCL9 Axis Dependence in Mice

Triple-negative breast cancer (TNBC) is a refractory tumor, and therapeutic options are very limited. Local ablation has been applied recently. Chemokines play a critical role in the recruitment of immune cells into ablative tumors. Nanosecond pulsed electric field (nsPEF) shows potential anti-tumor efficacy, but the mechanism for maintaining the immune effect is not very clear. Here, we applied nsPEF for treating 4T1 breast cancer cells in vitro. RNA sequencing (RNA-seq) was applied. Anti-CXCL9 was used alone or combined with nsPEF to treat triple-negative breast cancer in mice. We demonstrated that nsPEF effectively induced cell apoptosis and inhibited the growth and metastasis of triple-negative breast cancer. An immune effect, especially chemotaxis, was activated by nsPEF. The number of infiltrated CD8+ T cells was increased significantly. We found that the inhibition of residual breast cancer growth by nsPEF was dependent on the CXCL9 axis. In conclusion, our work demonstrated that nsPEF effectively ablated the tumor, aroused an immune response, and inhibited residual breast cancer growth via CXCL9 axis dependence in mice.

Multi-Targeted and On-Demand Non-Coding RNA Regulation Nanoplatform against Metastasis and Recurrence of Triple-Negative Breast Cancer.

Dysregulation of microRNAs (miRs) is the hallmark of triple-negative breast cancer (TNBC), which is closely involved with itsgrowth, metastasis, and recurrence. Dysregulated miRs are promising targets for TNBC therapy, however, targeted and accurate regulation of multiple disordered miRs in tumors is still a great challenge. Here, a multi-targeting and on-demand non-coding RNA regulation nanoplatform (MTOR) is reported to precisely regulate disordered miRs, leading to dramatical suppression of TNBC growth, metastasis, and recurrence. With the assistance of long blood circulation, ligands of urokinase-type plasminogen activator peptide and hyaluronan located in multi-functional shells enable MTOR to actively target TNBC cells and breast cancer stem cell-like cells (BrCSCs). After entering TNBC cells and BrCSCs, MTOR is subjected to lysosomal hyaluronidase-induced shell detachment, leading to an explosion of the TAT-enriched core, thereby enhancing nuclear targeting. Subsequently, MTOR could precisely and simultaneously downregulate microRNA-21 expression and upregulate microRNA-205 expression in TNBC. In subcutaneous xenograft, orthotopic xenograft, pulmonary metastasis, and recurrence TNBC mouse models, MTOR shows remarkably synergetic effects on the inhibition of tumor growth, metastasis, and recurrence due to its on-demand regulation of disordered miRs. This MTOR system opens a new avenue for on-demand regulation of disordered miRs against growth, metastasis, and recurrence of TNBC.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming

BackgroundChemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized.MethodsThe differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model.ResultsWe provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model.ConclusionsOur in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.