Triple-negative Cells Research Articles

Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression

BackgroundThe glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer.ResultsHere we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins.ConclusionsThe results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase’s role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

CCNE1 amplification as marker of poor prognosis and novel therapeutic target in advanced breast cancer.

1040 Background: Cyclin E1 (CCNE1) overexpression or gene amplification is linked to dismal outcomes in various tumors, including breast cancer (BC). Recent studies suggest that CCNE1 amplification is a potential target for new synthetic lethality-based treatments, including CDK2-selective and PKMYT1 inhibition. This study explores the clinical and genomic characteristics of CCNE1-amplified BCs. Methods: We analyzed genomic and clinical data from consecutive BCs that underwent clinical tumor-normal targeted panel sequencing (MSK-IMPACT) between April 2014 and December 2021. Allele-specific copy number, including CCNE1 amplification, and fraction genome altered (FGA) were calculated by FACETS. Mutual exclusivity and co-occurrence analyses were performed using CoMEt. Benjamini–Hochberg method for multiple testing correction (q) was applied. Real-world progression-free survival (rwPFS) was assessed by the Kaplan Meier method and Cox models, focusing on patients with available pre-treatment samples. Results: Out of 3,753 BCs, 125 (3.3%) had CCNE1 amplification. A higher occurrence of CCNE1 amplification was noted in post-treatment (n=2,368) compared to treatment-naïve tumors (n=1,385; 4% vs 2.4%, p=0.007). CCNE1 amplification was less common in hormone receptor (HR)+/HER2- BCs (2%) compared to HER2+ (7.6%) and triple-negative (7.2%) tumors (p<0.001). BCs with CCNE1 amplification showed higher median FGA, indicating increased genomic instability. In primary BC, TP53 alterations were more frequent in CCNE1-amplified tumors (q<0.001), while CDH1 alterations were mutually exclusive (q<0.001) suggesting that lobular BCs rarely harbor CCNE1 amplification. Similar trends were seen in post-treatment samples. CCNE1 amplification detected at baseline was linked to shorter median rwPFS in HR+/HER2- metastatic BCs treated with CDK4/6 inhibitors plus endocrine therapy (8.8 vs 15.2 months in CCNE1-amplified [n=9] vs CCNE1-non amplified [n=402]; hazard ratio [HR] 2.82, 95% CI 1.38-5.75), and in HER2+ metastatic BCs treated with first-line THP regimen (7.3 vs 20.8 months in CCNE1-amplified [n=5] vs CCNE1-non amplified [n=106]; HR 3.1, 95% CI 1.24-7.87). In a CCNE1-amplified triple-negative cell model (HCC1569), treatment with the PKMYT1 inhibitor RP-6306 significantly reduced tumor growth. Conclusions: CCNE1 amplification in BCs is associated with greater genomic instability and characterizes a group of metastatic BCs with poor response to standard of care therapies. Novel therapies targeting CCNE1-amplified tumors are under evaluation in preclinical and clinical studies.

Slow Sulfide Donor GYY4137 Increased the Sensitivity of Two Breast Cancer Cell Lines to Paclitaxel by Different Mechanisms.

Paclitaxel (PTX) is a chemotherapeutic agent affecting microtubule polymerization. The efficacy of PTX depends on the type of tumor, and its improvement would be beneficial in patients' treatment. Therefore, we tested the effect of slow sulfide donor GYY4137 on paclitaxel sensitivity in two different breast cancer cell lines, MDA-MB-231, derived from a triple negative cell line, and JIMT1, which overexpresses HER2 and is resistant to trastuzumab. In JIMT1 and MDA-MB-231 cells, we compared IC50 and some metabolic (apoptosis induction, lactate/pyruvate conversion, production of reactive oxygen species, etc.), morphologic (changes in cytoskeleton), and functional (migration, angiogenesis) parameters for PTX and PTX/GYY4137, aiming to determine the mechanism of the sensitization of PTX. We observed improved sensitivity to paclitaxel in the presence of GYY4137 in both cell lines, but also some differences in apoptosis induction and pyruvate/lactate conversion between these cells. In MDA-MB-231 cells, GYY4137 increased apoptosis without affecting the IP3R1 protein, changing the morphology of the cytoskeleton. A mechanism of PTX sensitization by GYY4137 in JIMT1 cells is distinct from MDA-MB-231, and remains to be further elucidated. We suggest different mechanisms of action for H2S on the paclitaxel treatment of MDA-MB-231 and JIMT1 breast cancer cell lines.

All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells.MethodsMCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(β) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student’s t-test or one-way ANOVA was used to analyze data from replicates.ResultsOur results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(β) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(β) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression.ConclusionsTo the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

A recombinant adeno-associated virus expressing secretory TGF-β type Ⅱ receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice

To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-β (TGF-β) type Ⅱ receptor (sTβRⅡ) extracellular domain-IgG2a Fc fusion protein (sTβRⅡ-Fc) on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice. The pAAV-sTβRⅡ-Fc vector expressing sTβRⅡ-Fc fusion protein constructed by molecular cloning, the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTβRⅡ virus, which was purified by density gradient centrifugation with iodixanol. Western blotting was used to examine the effects of AAV-sTβRⅡ virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin, vimentin and p-Smad2/3 in 4T1 cell xenografts in mice. BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTβRⅡ virus, AAV-GFP virus or PBS (n=6) through the tail vein, and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging. Ki67 expression in the tumor tissues and sTβRⅡ protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining, and tumor metastases in the vital organs were examined with HE staining. The recombinant pAAV-sTβRⅡ-Fc vector successfully expressed sTβRⅡ in HEK 293T cells. Infection with AAV2-sTβRⅡ virus significantly reduced TGF-β1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice (P<0.05). In the tumor-bearing mice, intravenous injection of AAV-sTβRⅡ virus significantly increased E-cadherin expression, reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues (P<0.05 or 0.01), and induced liver-specific sTβRⅡ expression without causing body weight loss or heart, liver, spleen or kidney pathologies. The recombinant AVV2 vector encoding sTβRⅡ extracellular domain is capable of blocking the TGF-β signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.

Enhancing therapeutic efficacy: sustained delivery of 5-fluorouracil (5-FU) via thiolated chitosan nanoparticles targeting CD44 in triple-negative breast cancer

Our current study reports the successful synthesis of thiolated chitosan-based nanoparticles for targeted drug delivery of 5-Fluorouracil. This process was achieved through the ionic gelation technique, aiming to improve the efficacy of the chemotherapeutic moiety by modifying the surface of the nanoparticles (NPs) with a ligand. We coated these NPs with hyaluronic acid (HA) to actively target the CD44 receptor, which is frequently overexpressed in various solid malignancies, including breast cancer. XRD, FTIR, SEM, and TEM were used for the physicochemical analysis of the NPs. These 5-Fluorouracil (5-FU) loaded NPs were evaluated on MDA-MB-231 (a triple-negative breast cell line) and MCF-10A (normal epithelial breast cells) to determine their in vitro efficacy. The developed 5-FU-loaded NPs exhibited a particle size within a favorable range (< 300 nm). The positive zeta potential of these nanoparticles facilitated their uptake by negatively charged cancer cells. Moreover, they demonstrated robust stability and achieved high encapsulation efficiency. These nanoparticles exhibited significant cytotoxicity compared to the crude drug (p < 0.05) and displayed a promising release pattern consistent with the basic diffusion model. These traits improve the pharmacokinetic profile, efficacy, and ability to precisely target these nanoparticles, offering a potentially successful anticancer treatment for breast cancer. However, additional in vivo assessments of these formulations are obligatory to confirm these findings.

Prostate Specific Membrane Antigen Expression in a Syngeneic Breast Cancer Mouse Model

PurposeProstate specific membrane antigen (PSMA) has been studied in human breast cancer (BCa) biopsies, however, lack of data on PSMA expression in mouse models impedes development of PSMA-targeted therapies, particularly in improving breast conserving surgery (BCS) margins. This study aimed to validate and characterize the expression of PSMA in murine BCa models, demonstrating that PSMA can be utilized to improve therapies and imaging techniques.MethodsMurine triple negative breast cancer 4T1 cells, and human cell lines, MDA-MB-231, MDA-MB-468, implanted into the mammary fat pads of BALB/c mice, were imaged by our PSMA targeted theranostic agent, PSMA-1-Pc413, and tumor to background ratios (TBR) were calculated to validate selective uptake. Immunohistochemistry was used to correlate PSMA expression in relation to CD31, an endothelial cell biomarker highlighting neovasculature. PSMA expression was also quantified by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).ResultsAccumulation of PSMA-1-Pc413 was observed in 4T1 primary tumors and associated metastases. Average TBR of 4T1 tumors were calculated to be greater than 1.5—ratio at which tumor tissues can be distinguished from normal structures—at peak accumulation with the signal intensity in 4T1 tumors comparable to that in high PSMA expressing PC3-pip tumors. Extraction of 4T1 tumors and lung metastases followed by RT-PCR analysis and PSMA-CD31 co-staining shows that PSMA is consistently localized on tumor neovasculature with no expression in tumor cells and surrounding normal tissues.ConclusionThe selective uptake of PSMA-1-Pc413 in these cancer tissues as well as the characterization and validation of PSMA expression on neovasculature in this syngeneic 4T1 model emphasizes their potential for advancements in targeted therapies and imaging techniques for BCa. PSMA holds great promise as an oncogenic target for BCa and its associated metastases.

Abstract PO1-20-08: Primary breast implant-associated squamous cell carcinoma with subsequent TEMPUS testing and failure of immunotherapy

Abstract Primary squamous cell carcinoma of the breast is a rare entity, and there are a limited number of cases in the literature describing squamous cell carcinoma arising from breast implants. It has been hypothesized that these implant-associated cancers may arise from epithelialization of the implant capsule followed by chronic inflammation. None of the cases in the literature were treated with immunotherapy. We present a case of an aggressive, primary sarcomatoid squamous cell carcinoma of the breast arising from a breast implant that was treated unsuccessfully with simultaneous chemotherapy and immunotherapy following TEMPUS testing. Introduction: Breast augmentation is the most common plastic surgery procedure performed worldwide, with more than 300,000 done in the United States every year. While breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is extensively discussed in recent literature, other breast implant-associated cancers are rarer and less studied. Breast implant-associated squamous cell carcinoma (BIA-SCC) specifically is an extremely rare complication of breast augmentation and no guidelines for treatment exist. The American Society of Plastic Surgeons (ASPS) released a statement on BIA-SCC on 9/2022 following a safety communication from the FDA, and it is recommended health care providers submit case reports of BIA-SCC to a patient registry. Case study: We present the case of a female patient who underwent bilateral breast augmentation in 2006 with Mcghan textured, pre-pectoral, areolar saline implants. In 2022, she presented with swelling and a lump in her breast and underwent bilateral breast implant removal with capsulectomy. Pathology showed triple negative sarcomatoid squamous cell carcinoma of the right breast capsule. TEMPUS testing on the breast biopsy revealed a PIK3CA mutation and PD-L1 negative status. Her case was discussed with a multi-disciplinary team including plastic surgery, multiple oncologists, and radiation oncology and her clinical picture was determined to be most consistent with BIA-SCC. Based on the KEYNOTE-522 study, she was determined to be a candidate for neoadjuvant therapy with pembrolizumab and chemotherapy as she was newly diagnosed, previously untreated, nonmetastatic, and had a triple negative tumor. She received two cycles of carboplatin, paclitaxel, and pembrolizumab but unfortunately rapidly progressed with chest wall extension, multiple new pulmonary nodules, worsening lymphadenopathy, right ventricular tumor thrombus, and new malignant pleural effusion and was transitioned to hospice. Discussion: Seven cases of BIA-SCC, one case of squamous cell carcinoma (SCC) after liquid silicone injection augmentation, one case of squamous metaplasia on the breast implant capsule, and one case of epithelization of the breast implant capsule have been previously described in the literature (Table 1). None of the available cases of BIA-SCC report TEMPUS testing, and none of the patients were treated with immunotherapy. The origin of BIA-SCC is unclear, but several theories have been described including chronic inflammation leading to conversion of epithelial tissue on the capsule to SCC. It is unknown how the breast implant capsule might become epithelialized to begin with – introduction of epithelium intraoperatively during augmentation, rupture of epidermal cysts, existence of epithelium in the area from a childhood injury or from birth, or epithelization from chronic shear forces from the augmentation or leakage of the implant are all possibilities. With the ongoing data gathering through the FDA patient registry, we will continue to learn more about these rare cancers and potentially get further insight into their origins. Table 1 An overview of cases of BIA-SCC in the literature Citation Format: Emily Podany, Katherine Clifton. Primary breast implant-associated squamous cell carcinoma with subsequent TEMPUS testing and failure of immunotherapy [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 PO1-20-08.

Abstract PO2-15-11: Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models

Abstract Background: Glutamine is an important metabolic substrate in many aggressive tumors, with comparable importance to glucose metabolism. Utilizing human breast cancer mouse xenograft models, we studied the kinetics of the PET imaging agent, [5-11C] glutamine, a biochemical authentic substrate for glutamine metabolism, to further characterize the metabolism of glutamine and downstream labeled metabolites. Studies were performed with and without inhibition of the enzyme, glutaminase (GLS), the first step in glutamine catabolism that generates glutamate, and key target for therapy directed to glutamine-metabolizing cancers. Methods: The study used xenograft mouse models for two breast cancer cell lines, HCC1806, a highly glutaminolytic triple-negative cell line, and MCF-7, a hormone receptor positive line with only low levels of glutaminolysis. Mice were injected with 5-[11C]L-glutamine, and the contributions of individual metabolites to the total [11C] signal in blood and tumor tissue were estimated at 10, 20, and 30 minutes after injection by assaying the fractional contributions of [5-11C] glutamine and labeled metabolites, specifically [5-11C] glutamate, [11C] CO2 and all other metabolites with [11C], in both blood and tumor tissue. This generated estimated time activity curves for [5-11C] glutamine and downstream metabolites in both HCC1806 and MCF-7 mouse models with and without CB-839 treatment, an inhibitor of GLS. We also investigated a compartment model that describes these data, including model simulations that fit the model to match the measured curves. Results: We found that, out to 30 minutes post-injection, the vast majority of tumor radioactivity was in the form of either 5-11C] glutamine or [5-11C] glutamate, with smaller amounts of radioactivity in the form of more downstream metabolites, including [11C]CO2. Systemic administration of the glutaminase inhibitor alters the arterial input curve by reducing the systemic rate of conversion of [5-11C] glutamine to [5-11C] glutamate. However, in GLS active tumors, [5-11C] glutamate retained in the large cellular glutamate pool represents a greater fraction of total radioactivity than in the blood and leads to a total tumor time-activity curve that is only marginally different than the total tumor time-activity curve after GLS inhibition. Data from blood and tissue analysis were used to test a hypothetical kinetic model for [5-11C] glutamine, which provided estimates of glutamate and glutamate tumor tissue concentrations and tumor GLS activity that agree with direct measurements in the animal models. Conclusion: The study of [ 5-11C] glutamine in breast cancer models leads to kinetic insight that are in line with biochemical studies and direct measurement taken for tumor tissue obtained from the animal models. Retention of metabolized [5-11C] glutamine as [5-11C] glutamate in a large tissue pool in tumors with high GLS activity makes non-invasive inference of GLS activity challenging without additional ex vivo analysis, supporting the use of non-metabolized glutamine analogs such as such as [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) to infer in vivo glutamine metabolism. Citation Format: Christopher Hensley, Prashanth Padakanti, Hoon Choi, Christina Dulal, Hsiaojiu Lee, Austin Pantel, Rong Zhou, David Mankoff. Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models [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 PO2-15-11.

Anticancer Activity of Encapsulated Pearl Millet Polyphenol-Rich Extract against Proliferating and Non-Proliferating Breast Cancer Cells In Vitro.

Plant-derived polyphenols are bioactive compounds with potential health-promoting properties including antioxidant, anti-inflammatory, and anticancer activity. However, their beneficial effects and biomedical applications may be limited due to their low bioavailability. In the present study, we have considered a microencapsulation-based drug delivery system to investigate the anticancer effects of polyphenol-rich (apigenin, caffeic acid, and luteolin) fractions, extracted from a cereal crop pearl millet (Pennisetum glaucum), using three phenotypically different cellular models of breast cancer in vitro, namely triple negative HCC1806, ER-positive HCC1428, and HER2-positive AU565 cells. Encapsulated polyphenolic extract induced apoptotic cell death in breast cancer cells with different receptor status, whereas it was ineffective against non-tumorigenic MCF10F cells. Encapsulated polyphenolic extract was also found to be cytotoxic against drug-resistant doxorubicin-induced senescent breast cancer cells that were accompanied by increased levels of apoptotic and necrotic markers, cell cycle inhibitor p21 and proinflammatory cytokine IL8. Furthermore, diverse responses to the stimulation with encapsulated polyphenolic extract in senescent breast cancer cells were observed, as in the encapsulated polyphenolic extract-treated non-proliferating AU565 cells, the autophagic pathway, here cytotoxic autophagy, was also induced, as judged by elevated levels of beclin-1 and LC3b. We show for the first time the anti-breast cancer activity of encapsulated polyphenolic extract of pearl millet and postulate that microencapsulation may be a useful approach for potentiating the anticancer effects of phytochemicals with limited bioavailability.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome

BackgroundAcute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated.MethodsC57BL/6J mice (9–14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45−, TER-119−, CD31−) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment.ResultsAt homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs.ConclusionsTPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Selective Ru(II)/benzoate complexes against triple-negative breast tumor cells and their interactions with DNA and BSA

New ruthenium(II) complexes bearing benzoate (AB) ligand with the general formula [Ru(AB)(bipy)(PP)]PF6, where 2,2′-bipyridine (bipy) and different diphosphine ligands, (PP) = 1,2′-bis(diphenylphosphine)ethane (dppe, 1), 1,3′-bis(diphenylphosphine)propane (dppp, 2) and 1,2′-bis(diphenylphosphine)ferrocene (dppf, 3), were synthesized. The compounds were characterized by molar conductivity, elemental analysis, cyclic voltammetry, infrared and UV–Vis spectroscopies, NMR, and by single-crystal X-ray diffraction for complexes 1 and 2. The complexes showed a weak interaction to CT-DNA through DNA minor groove, with Kb values at around 103-104 M−1. CT-DNA interaction assays by viscosity and circular dichroism (CD) suggested that the compounds do not significantly alter the secondary DNA structure. The complexes are cytotoxic against MDA-MB-231, MCF-7 (breast) and A549 (lung) tumor cell lines, with IC50 values in the range of 1 to 17 µM. The compounds 1-3 showed high selectivity against triple-negative breast tumor cells. Remarkably, complexes 1 and 3 show greater cytotoxic activity against cells than cisplatin, being promising agents for tumor treatment.

Liposome-encapsulated progesterone efficiently suppresses B-lineage cell proliferation

Progesterone suppresses several ancient pathways in a concentration-dependent manner. Based on these characteristics, progesterone is considered a candidate anticancer drug. However, the concentration of progesterone used for therapy should be higher than the physiological concentration, which makes it difficult to develop progesterone-based anticancer drugs. We previously developed liposome-encapsulated progesterone (Lipo-P4) with enhanced anticancer effects, which strongly suppressed triple-negative breast cancer cell proliferation in humanized mice. In this study, we aimed to clarify whether Lipo-P4 effectively suppresses the proliferation of B-lineage cancer cells. We selected six B-cell lymphoma and two myeloma cell lines, and analyzed their surface markers using flow cytometry. Next, we prepared liposome-encapsulated progesterone and examined its effect on cell proliferation in these B-lineage cancer cells, three ovarian clear cell carcinoma cell lines, two prostate carcinoma cell lines, and one triple-negative breast cancer adenocarcinoma cell line. Lipo-P4 suppressed the proliferation of all cancer cell lines. All B-lineage cell lines, except for the HT line, were more susceptible than the other cell types, regardless of the expression of differentiation markers. Empty liposomes did not suppress cell proliferation. These results suggest that progesterone encapsulated in liposomes efficiently inhibits the proliferation of B-lineage cells and may become an anticancer drug candidate for B-lineage cancers.

Transcriptomics Studies Reveal Functions of Transglutaminase 2 in Breast Cancer Cells Using Membrane Permeable and Impermeable Inhibitors

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer.The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

PFDN4 as a Prognostic Marker Was Associated with Chemotherapy Resistance through CREBP1/AURKA Pathway in Triple-Negative Breast Cancer.

Breast cancer is the most common malignancy and its incidence is increasing. It is currently mainly treated by clinical chemotherapy, but chemoresistance remains poorly understood. Prefolded proteins 4 (PFDN4) are molecular chaperone complexes that bind to newly synthesized polypeptides and allow them to fold correctly to stabilize protein formation. This study aimed to investigate the role of PFDN4 in chemotherapy resistance in breast cancer. Our study found that PFDN4 was highly expressed in breast cancer compared to normal tissues and was statistically significantly associated with stage, nodal status, subclasses (luminal, HER2 positive and triple negative), triple-negative subtype and disease-specific survival by TCGA database analysis. CRISPR knockout of PFDN4 inhibited the growth of 89% of breast cancer cell lines, and the triple-negative cell line exhibited a stronger inhibitory effect than the non-triple-negative cell line. High PFDN4 expression was associated with poor overall survival in chemotherapy and resistance to doxorubicin and paclitaxel through the CREBP1/AURKA pathway in the triple-negative MDAMB231 cell line. This study provides insightful evidence for the value of PFDN4 in poor prognosis and chemotherapy resistance in breast cancer patients.

Abstract 2790: Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study

Abstract Triple-negative breast cancers are the most challenging to treat with targeted therapy because of their non-expression of cell surface receptors, unlike other types of breast cancer. However, conventional therapies such as chemotherapy, radiotherapy, and surgery are standard treatment modalities. Moreover, drug encapsulation strategies to deliver drugs at the targeted site have been sought-after alternatives. Hematopoietic Stem Cells (HSCs) have unique features to migrate towards inflammatory sites and other body organs during any injuries and release various cytokines, which lead to interaction with molecular pathways and results in reduced inflammation and tissue injuries. From this characteristic, we have hypothesized that hematopoietic stem cells can show tumor tropism and modulate the metabolic alteration in cancer and cancer stem cells, mainly focusing on Triple Negative Breast Cancer Stem Cells (TNBC CSCs). In this study, we have sorted both cancer stem cells and hematopoietic stem cells from TNBC and hematological cell lines, respectively. Sorted HSCs were cultured in specific conditions to prepare conditioned media. Then, both cell’ types were used for co-cultured assay and conditioned media cultured with TNBC-CSCs were performed. Metabolomics studies were performed to decipher the molecular interactions and TNBC-CSCs metabolic alterations. The result indicates that the HSCs are showing positive tropism towards TNBC stem cells. Furthermore, HSCs-derived conditioned media (CM) interact with the cell cycle pathway by downregulating the CDK genes and causing DNA breakage in the TNBC-CSCs. Moreover, the Metabolomics studies further confirmed that HSCs CM-derived cytokines interact with various pathways, dysregulating the TNBC-CSCs proliferation and altering the metabolic activities. Citation Format: Sumit Mallick, Sudheer Shenoy P, Bipasha Bose. Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study [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 2790.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [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 1530.

Abstract 1779: De novo purine pathway enzyme ATIC promotes tumor progression and modulates metabolic reprogramming in breast cancer

Abstract Breast cancer (BC), a complex and heterogeneous disease, poses a significant global health challenge with a pressing need for enhanced prognostic markers and therapeutic targets. This study focuses on 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), an enzyme essential in de novo purine biosynthesis and previously implicated in myeloma and hepatocellular carcinoma progression. Leveraging publicly available datasets, including TCGA, METABRIC, and GENT2, we identified elevated ATIC expression in BC tissues compared to normal tissues. Notably, patients with elevated ATIC expression displayed significantly diminished overall survival rates, underscoring ATIC's potential as a prognostic marker. Functional assays involving ATIC downregulation in BC cell lines revealed suppressed cell growth, migration, and increased sensitivity to chemotherapeutic drug, Doxorubicin, implicating ATIC in BC progression. Further, RNA-sequencing analysis of ATIC knockdown (KD) BC cell lines revealed a positive correlation between ATIC expression and the activation of MYC and E2F-regulated pathways. The marked attenuation in cell growth and various tumor properties upon ATIC KD provides mechanistic insights into the role of ATIC in fueling BC progression, potentially through the modulation of MYC and E2F target gene expression. Gene Set Enrichment Analysis (GSEA) on differentially expressed genes identified between tumors with high ATIC expression and low ATIC expression in BC patients from the TCGA cohort revealed a notable enrichment of genes associated with MYC and E2F targets, providing additional support for the observed molecular associations in our experimental model. Intriguingly, ATIC KD in Triple Negative BC cell line unexpectedly led to an increase in fatty acid β-oxidation (FAO), uncovering a metabolic vulnerability in these cells. In conclusion, this study unravels the multifaceted role of ATIC in BC, demonstrating its influence on BC tumor progression and metabolism. The observed increase in FAO upon ATIC KD presents a novel metabolic vulnerability that could be explored for targeted therapeutic interventions in BC. This comprehensive analysis contributes valuable insights toward understanding ATIC as a potential therapeutic target and prognostic indicator in BC. Citation Format: Divya Murthy, Kuldeep S. Attri, Jun Hyoung Park, Benny A. Kaipparettu. De novo purine pathway enzyme ATIC promotes tumor progression and modulates metabolic reprogramming in breast cancer [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 1779.

Abstract 5956: FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients

Abstract Backgroud: The prognosis of B cell ALL patients (pts) relapsing after Hematopoietic Transplant is poor and new drugs are needed. In acute myeloid leukemia, FLT3-inhibitors (FLT3i) are showing promising results in FLT3-mutated (mut) cases. Their potential use in FLT3-mut B-ALL has been poorly investigated. Aim: To assess if FLT3 could be a valid targetable marker in B-ALL. Methods: We sequenced with a capture based large RNA-seq panel (1385genes) 183 adult B-ALL [n=52 Pos for Ph or t(4;11) or t(1;19) and n=131 Triple-Negative (TN) pts that are negative for Ph (n=43), t(4;11) and t(1;19) translocations] and 15 donors. To confirm FLT3 muts, 13 available samples were further sequenced with Extended Myeloid Solution (98 genes; SOPHiA) where FLT3 gene is fully covered. In vitro studies using 5 FLT3i [Gilteritinib (Gil), Midostaurin, Crenolanib, Sorafenib and Quizartibin] and Venetoclax (Ven) were conducted on pt primary cells and on TN and other B-ALL cell lines (MUTZ5, MHH-CALL4 Ph-like; NALM6;RS4;11, 697, REH) and 2 AML cell lines (OCI-AML3 FLT3;MV-4-11 FLT3-ITD). Results: We found 15 FLT3 mut in 14/131 TN B-ALL and in 1/43 Ph+ cases: 43.8% were TKD mut, 3 ITD mut, 3 splicing site mut,1 N-terminal, 1 juxta-membrane domain and 1 Ig-like site (Fig. 1A). After pathogenicity interpretation we excluded 3 variants and one mutation was not confirmed at DNA level. Overall 7.6% of TN were mut (Ph+ 2.3%). 81.8% of FLT3mut pts potentially druggable with FLT3i, mainly TKD (63.3%). Targeted NGS revealed the co-occurrence with other mutations (range 2-15), and in particular with CSMD1 (n=4), KDM6A, KMT2D and CREBBP (n=3) alterations. Moreover, 11/13 samples carried copy number alteration (mean 12), mostly amplifications. CDKN2A &amp; IKZF1 were the most frequently altered genes (n=5;4 CN-loss). FLT3 expression was increased in 11/15 cases compared to donors and wt pts. As reported, the expression was higher in ZNF384 or MLLr B-ALL subtypes.To evaluate the effect of FLT3is in ALL, we treated ex vivo primary leukemic cells of 6 adult ALL patients (FLT3-mut n=4; FLT3-wt n=2) with increasing concentrations of 5 FLT3i for 24, 48 and 72h. We observed a trend towards greater response of FLT3-mut ALL cells compared to wt ones. The average IC50 values in response to the five FLT3i was 1.6 uM and 24.5 uM after 72h of treatment in FLT3-mut and FLT3-wt B-ALL, respectively (Fig. 1B). We than evaluated the sensitivity to the 5 Inhs in all cell lines. Interestingly MHH-CALL4 wt were more sensitive to Gil compared to OCI AML3 wt, and showed the same effect compare to MV-4-11 AML mut (F.1C). Given the effect of Gil in single agent, we combined it with Ven in wt B-ALL cells at increasing concentrations. Interestingly We noticed a strong additive effect with the higher Gil concentration and all Ven doses(F.1D). Conclusions: FLT3 alterations identify a novel subgroup of TN B-ALL with therapeutic potential also in combination regimens. Ricerca Corrente Italian MoH L3P1946. Citation Format: Anna Ferrari, Andrea Ghelli Luserna di Rorà, Federico Lo Schiavo, Eugenio Fonzi, Giorgia Capirossi, Chiara Salvesi, Maria Teresa Bochicchio, Lorenzo Ledda, Chiara Servilli, Matteo Paganelli, Giovanni Marconi, Cristina Papayannidis, Chiara Sartor, Michela Rondoni, Anna Maria Mianulli, Barbara Giannini, Crescenza Pasciolla, Fabio Giglio, Sara Galimberti, Monica Fumagalli, Prassede Salutari, Valentina Gaidano, Giovanni Martinelli, Giorgia Simonetti. FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients [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 5956.

Abstract 6951: The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive type of breast cancer in which the lymphatic vessels become blocked by cancer cells in the breast tissue. The five-year survival rate for this type of cancer is around 40%, one of the worst among breast cancer. Most IBCs are estrogen receptor (ER) and progesterone receptor (PR) negative and HER2 positive, with some presenting an amplification of HER2 while others are ER, PR, and HER2 negative, formally known as the triple-negative (TNBC) subtype. However, it has been reported that treatment with 17β-Estradiol (E2) of ER-negative IBC and non-IBC cells can exert rapid non-genomic signaling mediated by ERα-36, GPR30, and/or estrogen receptor beta, activating MAPK pathways involved in oncogenic phenotypes. Previous data generated in our lab showed that when cells were treated with estradiol it led to increased phosphorylation levels of p38. However, the specific role p38 MAPK signaling takes on in IBC is unknown. Thus, this study centers on examining the role of estradiol and p38 regulation in the triple-negative IBC cell line model, SUM149PT. We hypothesize that upon estradiol treatment p38 signaling pathway is activated resulting in the over-expression of pro-inflammatory cytokines which regulate pro-tumorigenic phenotypes including the growth and survival of IBC cells. Inflammatory cytokines can promote pro-oncogenic features by promoting cell proliferation, angiogenesis, and immune suppression. We treated SUM149 IBC cells with 10nM of estradiol and measured the levels of human cytokines using a Human Cytokine Antibody Array. In addition, treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines in a dose-dependent manner. Ongoing studies include assessing the effects of p38 inhibition proliferation, invasion, and migration. In summary, a better characterization of the effect of E2 and p38 activation in ER-negative cell lines may provide great insight into novel targeted therapies for IBC. Citation Format: Keyla V. Ramirez, Adrian D. Bonilla-González, Jeisac Guzman-Rivera, Esther A. Peterson-Peguero. The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer [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 6951.