Treatment Of Triple-negative Breast Cancer Research Articles

Abstract PO1-03-11: Camrelizumab in Combination with Nab-Paclitaxel and Carboplatin versus Nab-Paclitaxel and Carboplatin as Neoadjuvant Therapy for Triple Negative Breast Cancer: A Multicenter, Open-Label, Randomized Controlled Study

Abstract Background: Triple-negative breast cancer (TNBC) is recognized for its aggressive nature and unfavorable prognosis. Immune checkpoint inhibitors have demonstrated promising antitumor activity in both neoadjuvant and metastatic settings. This study aimed to assess the efficacy and safety of camrelizumab, an anti-PD-1 antibody, in conjunction with chemotherapy as a neoadjuvant treatment for early-stage TNBC. Methods: This two-phase prospective, multicenter, randomized controlled study was initiated with the enrollment of previously untreated stage II-III TNBC patients in the first phase. They received neoadjuvant therapy comprising six 3-week cycles of camrelizumab (200 mg, q3w), along with nab-paclitaxel (220 mg/m2, q3w) and carboplatin (AUC 5, q3w). In the second phase, patients were enrolled and randomized 1:1 to either the camrelizumab plus nab-paclitaxel and carboplatin group or the nab-paclitaxel plus carboplatin group. The present report covers the data from the first phase of the study. The primary endpoint was the total pathological complete response (tpCR, ypT0/is ypN0) rate. Secondary endpoints included breast pathological complete response (bpCR, ypT0/is) rate, objective response rate (ORR), event-free survival (EFS), residual cancer burden (RCB), and safety. This study is registered with ClinicalTrials.gov, under the identifier NCT04907344. Results: Between September 2021 and March 2023, 39 patients were enrolled in the first phase. A total of 35 (89.7%) were node-positive. Among the 32 patients with evaluable efficacy, the tpCR rate was 59.4% (19/32), and the bpCR rate was 68.8% (22/32). The ORR at the end of neoadjuvant treatment stood at 87.2% (34/39). Among the 39 patients, 11 (28.2%) experienced grade 3 or higher treatment-related adverse events, the most common of which were thrombocytopenia (6 [15.4%]), leucopenia (5 [12.8%]), neutropenia (5 [12.8%]), alanine aminotransferase increased (2 [5.1%]), aspartate aminotransferase increased (1 [2.6%]), and hypothyroidism (1 [2.6%]). Adverse events leading to discontinuation of any agent occurred in 5 (12.8%) patients, while adverse events leading to delay in treatment occurred in 7 (17.9%) patients. Serious treatment-related adverse events were reported by 4 (10.4%) patients. Conclusions: In patients with early TNBC, the neoadjuvant treatment of camrelizumab in combination with nab-paclitaxel and carboplatin demonstrated a high pCR rate with an acceptable safety profile. The second phase of the study is currently in progress to validate these findings. Citation Format: Weipeng Zhao, Xin Wang, Bin Zhang, Yehui Shi, Chen Wang, Xu Wang, Shufen Li, Chunfang Hao, Ning Lu, Yongsheng Jia, Li Zhang, Lan Zhang, Weixin Sun, Xuchen Cao, Zhongsheng Tong. Camrelizumab in Combination with Nab-Paclitaxel and Carboplatin versus Nab-Paclitaxel and Carboplatin as Neoadjuvant Therapy for Triple Negative Breast Cancer: A Multicenter, Open-Label, Randomized Controlled Study [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 PO1-03-11.

Abstract PO1-18-07: Phase I trial of combination pembrolizumab and ruxolitinib in metastatic triple negative breast cancer

Abstract BACKGROUND: The amplification of genes PDL-1, PDL-2, and JAK2 on chromosome 9p24, known as the PDJ amplicon, has been identified in 15% of triple negative breast cancer (TNBC) and is associated with tumor infiltrating lymphocytes and elevated JAK2 mRNA, suggesting a coordinated inflammatory response in TNBC. Adding pembrolizumab to chemotherapy in KEYNOTE trials 355 and 522 has validated the use of PD-1 blockade in TNBC and redefined the standard of care. Despite preclinical data suggesting a role for targeting the IL- 6/JAK2/STAT3 pathway, the JAK1/JAK2 inhibitor ruxolitinib has not shown efficacy as a single Agent in breast cancer. This is the first phase I clinical trial evaluating the combination of pembrolizumab and ruxolitinib in the treatment of metastatic TNBC. METHODS: We conducted a phase I clinical trial with accrual from 2018 to 2023 at Mayo Clinic Arizona. Patients (n=12) received 200 mg of IV pembrolizumab per cycle (every 3 weeks) with 3+3 dose escalation of oral ruxolitinib twice a day (dose level 0: 10 mg PO BID, level 1: 15 mg PO BID, level 2: 20 mg PO BID). The primary endpoint was to determine the maximum tolerated dose (MTD) of ruxolitinib and secondary endpoints were treatment safety and efficacy. Clinical response was assessed by computed tomography of the chest, abdomen, and pelvis every 3 cycles. PD-L1 expression in tumor was measured by immunohistochemistry (IHC). The effect of the combined therapies on T and B cell populations were assessed by flow cytometry and antibody profiling of 695 tumor antigens and 8 viral antigens by nucleic acid protein programmable array. RESULTS: Twelve female patients with chemotherapy refractory metastatic TNBC were treated on study. All received 200 mg IV pembrolizumab every cycle. Due to rapid disease progression of three patients early after treatment initiation, eligibility was restricted to prohibit patients with extensive pulmonary disease or elevated LDH. Those patients were excluded from efficacy or correlative analysis. The most common toxicities were transaminitis (n=12), anemia/leukopenia (n=12), and fatigue (n=9). The majority of adverse events were grade 1 and occurred at dose level 2. There were 5 adverse events grade 3 or higher: respiratory or thoracic complications (n=3), hypertension (n=1), and abdominal pain (n=1), but MTD was not established. The median number of cycles was 4 for each dose level (range n=2.0-9.0) and the median time to progression was 3 months (range n=1.4-6.3). No patient had tumor response by RECIST v1.1 criteria and there was no association of disease stability with PD-L1 tumor expression by IHC. Circulating antibodies to tumor antigens remained stable, and viral-specific antibodies mildly decreased by month 2 in all patients at dose levels 0 and 1. Of the 7 patients reactive to TRIM21 autoantibodies, 2 patients had a transient increase at 2 months. CONCLUSIONS: The combination of pembrolizumab with ruxolitinib was well tolerated at all dose levels, and the number but not the severity of adverse events increased with higher ruxolitinib dose. Two of six patients in dose level 0 and 1 had stable disease for six months on treatment, suggesting that the lower dose levels of ruxolitinib (i.e. 10 mg PO BID) may have more activity in combination with pembrolizumab. Citation Format: Anelia Kassi, Jacqueline Carmona, Sarah M. Green, Heidi Kosiorek, Michael T. Barrett, Barbara A. Pockaj, Donald Northfelt, Karen S. Anderson. Phase I trial of combination pembrolizumab and ruxolitinib in metastatic triple negative breast cancer [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 PO1-18-07.

Abstract PO2-14-10: A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model

Abstract Background Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses but up to 30% of breast cancer-related deaths. Similarly, Inflammatory breast cancer (IBC) accounts for 2-4% of breast cancer diagnosis but causes 8%–10% of all breast cancer-related deaths. The myeloid cell leukemia-1 (MCL-1) is a member of the BCL-2 family of proteins, which is highly amplified in numerous human cancers and associated with cell immortalization, transformation, and chemoresistance. In fact, MCL-1 is amplified in 55% of TNBC after preoperative chemotherapy. Amongst the MCL-1 inhibitors that have been developed, AZD5991 showed high selectivity and affinity for MCL-1. In myeloma and acute myeloid leukemia cells AZD5991 induces apoptosis and showed anti-tumor effects in in vivo models. In the present study, we evaluated the biological role of the MCL-1 inhibitor AZD5991 in IBC and TNBC in vitro and in vivo models. Materials and methods The expression of the MCL-1 gene was analyzed in the World Consortium IBC patient dataset. Next, we screened a panel of TNBC and IBC cell lines for MCL-1 expression by western blot analysis. The effect of AZD5991 on TNBC and IBC cells was analyzed via cell titer-blue proliferation, clonogenic assays and soft agar assay. To further validate these findings, we used siRNA to knockdown MCL-1 and performed cell proliferation, clonogenic and migration assays. To identify signaling pathways involved in MCL-1 Inhibitor’s effects we performed a proteome profiler human apoptosis array in TNBC cells in the presence of AZD5991. The anti-tumor effect of the MCL-1 inhibitor was evaluated using a SUM-149 (TN-IBC) orthotopic xenograft mouse model. The mice were treated with AZD5991 at doses of 15 and 30mg/kg once weekly via tail vein injection for a duration of 4 weeks. Results The mean expression level of MCL-1 was higher in patients with TNBC than in those with non-TNBC. Further, compared to the patients with ER positive, HER-2 negative tumors those with TNBC showed an increased in the mean expression of MCL-1 gene (p value= 0.05). We screened a panel of TNBC and IBC cell lines in which most of the cell lines showed high levels of MCL-1. The cell proliferation assay showed IC50 ranging from 0.27 – 19.6 μM. The SUM149, SUM190, FCIBC02, MDA-IBC3 and MDA-MB-468 cells showed high sensitivity and showed significant decrease in the proliferation rate while BCX010, KPL4, MDA-MB-231, 4T1.2 and E0771 cells as well as the normal breast cells MCF10A exhibited resistance with no significant decrease in proliferation. The clonogenic and soft agar assays with these sensitive and resistant cell lines showed similar results. Further, these findings were validated via genetic knockdown of MCL-1 which resulted in a significant decrease in cell proliferation, colony formation and migration capability of the TNBC cells. To identify signaling pathways involved in MCL-1 Inhibitor’s phenotypic changes, we performed a proteome profiler human apoptosis array which showed a decreased in BCLx expression, and an increase in expression of BAX and phospho-Rad17 proteins in the AZD5991 treated TN-IBC cells. Finally, in a SUM-149 (TN-IBC) orthotopic xenograft mouse model, mice injected with 30mg/kg showed a significant tumor regression compared to the vehicle-treated mice (vehicle vs 30 mg/kg, p value=0.0006). Conclusions and Future directions Our data demonstrates that AZD5991 inhibits tumorigenesis in an inflammatory breast cancer xenograft model. We are currently validating several significant targets to understand the biological mechanisms related to our candidate drug. Our long-term goal is to develop MCL-1 targeted therapy in combination with standard-of-care treatment for inflammatory and triple-negative breast cancers. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Michael White, Bisrat Debeb, Emilly Villodre, Xiaoding Hu, Debu Tripathy, Wendy Woodward, Rachel Layman, Geoffrey Bartholomeusz, Chandra Bartholomeusz. A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model [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 PO2-14-10.

Abstract PO5-21-09: Clinical outcomes by age subgroups in the phase 3 TROPiCS-02 study of sacituzumab govitecan vs treatment of physician’s choice in HR+/HER2‒ metastatic breast cancer

Abstract Background Sacituzumab govitecan (SG) is a Trop-2directed antibody-drug conjugate approved for second-line or later (2L+) treatment of metastatic triple-negative breast cancer in multiple countries and for 2L+ treatment of HR+/HER2‒ (IHC 0, 1+, or 2+/ISH) metastatic breast cancer (mBC) in the US. SG significantly improved progression-free survival (PFS) and overall survival (OS) vs treatment of physician’s choice (TPC) in patients (pts) with pretreated, endocrine-resistant HR+/HER2‒ mBC in the TROPiCS-02 study. The incidence of HR+/HER2‒ mBC increases with age, especially after 65, and older pts often have poorer prognosis due to comorbidities, increased toxicity, and reduced efficacy of chemotherapy. In TROPiCS-02, pts aged ≥ 65 years (yr) derived consistent PFS benefit and clinically meaningful improved OS with SG vs TPC. In this analysis, we present additional efficacy and safety outcomes by age subgroup. Methods Pts with HR+/HER2‒ mBC who had received at least 1 endocrine therapy, taxane, and CDK4/6 inhibitor, and 2 to 4 prior lines of chemotherapy for mBC, were randomized to either SG (10 mg/kg IV days 1 and 8, Q3W) or TPC. The primary end point was PFS, and secondary end points included OS, objective response rate (ORR), and safety. A post hoc analysis was conducted by age subgroups (< 65 vs ≥ 65, < 75 vs ≥ 75). Results In total, 543 pts were randomized to the SG (n = 272; 73 [27%] ≥ 65, 16 [6%] ≥ 75) and TPC (n = 271; 67 [25%] ≥ 65, 8 [3%] ≥ 75) groups. Baseline characteristics were generally similar across treatment arms in each age subgroup, but a higher proportion of pts had performance status 1 vs 0 in the ≥ 65 vs < 65 and ≥ 75 vs < 75 subgroups, respectively. The data cutoff date was July 1, 2022. SG demonstrated longer median PFS and OS vs TPC across age subgroups. Hazard ratios (HR) for PFS with SG vs TPC were 0.69, 0.59, 0.70, and 0.30 for the < 65, ≥ 65, < 75, and ≥ 75 subgroups, respectively (Table). The HR for OS were 0.81, 0.80, 0.82, and 0.56 in these subgroups, respectively (Table). Median duration of response (DoR) was also longer with SG vs TPC across age subgroups, although median DoR was not estimable in the ≥ 75 subgroup (Table). ORR was higher with SG vs TPC, except in the ≥ 75 subgroup (Table). Safety was generally similar with SG vs TPC across subgroups with the following differences. Grade ≥ 3 treatment-emergent adverse events were more common in the ≥ 75 subgroup than the other subgroups (SG, 81% in ≥ 75 vs 68%-73% for all others; TPC, 71% in ≥ 75 vs 60%-61% for all others). TEAEs leading to dose reduction were also more common in pts treated with SG in the ≥ 75 subgroup, but were consistent across other subgroups (Table). However, sample sizes in the ≥ 75 subgroup were small, which limits interpretation (Table). TEAEs leading to treatment discontinuation were more common in older pts, irrespective of treatment group (Table). Conclusions SG showed benefit in PFS and OS vs TPC in pts with HR+/HER2‒ mBC across age subgroups, consistent with the intent to treat population. SG was associated with a slight increase in toxicity for elderly pts, as expected. SG demonstrated a favorable benefit/risk profile, with efficacy benefit and acceptable toxicity, for elderly pts. Table. Citation Format: Aditya Bardia, Peter Schmid, Sara Tolaney, Frederik Marmé, Javier Cortés, Theresa Valdez, Hao Wang, Wendy Verret, Hope Rugo. Clinical outcomes by age subgroups in the phase 3 TROPiCS-02 study of sacituzumab govitecan vs treatment of physician’s choice in HR+/HER2‒ metastatic breast cancer [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 PO5-21-09.

Abstract PO3-06-11: Undated analyses from a Phase Ib open-label study of Pucotenlimab (HX008) in combination with gemcitabine and cisplatin as first-line treatment of metastatic triple-negative breast cancer

Abstract Background: Gemcitabine plus cisplatin (GP) therapy war preferred in China as the first-line therapy for metastatic triple-negative breast cancer (mTNBC), with a median progression-free survival (PFS) of 7.73 months, and a median overall survival (OS) of 19.37 months [1]. Pucotenlimab (HX008) is a novel humanized IgG4 anti-PD-1 monoclonal antibody with an engineered Fc domain. Preliminary results from the phase 1b trial [2] have shown that HX008 combined with GP has a manageable safety profile and demonstrates promising antitumor activity in patients (pts) with mTNBC. Here, we present the updated results from the long-term survival analysis. Methods: Eligible subjects were those who were treatment-naïve mTNBC patients and had measurable lesion. Participants received HX008 at a dose of 3 mg/kg every 3 weeks in combination with chemotherapy (gemcitabine + cisplatin) for 6 cycles and maintained with HX008 until disease progression or unacceptable toxicity occurred. The primary endpoints included overall response rate (ORR) and safety, and the secondary endpoints included duration of response (DoR), PFS, and OS. Results: Between July 2019 and March 2020, a total of 31 pts were enrolled in this study. The median age of patients was 50 years (range 29–69). By the cut-off date (April 24th, 2023), the median follow-up period was 20.7 m (IQR: 12.2, 25.6). The confirmed ORR was consistent with the previously reported data and remains at 74.2% (95% CI: 55.4%, 88.1%). The median PFS was 9.0 months (95% CI, 7.3, 9.3). The median DoR was 7.3 months (95% CI, 5.0, 9.8). The OS was immature, with the result of 23.1 months (95% CI, 13.4, NE), while the 12-m, 24-m and 36-m OS rate was 77.4% (95%CI: 64.0%, 93.6%), 42.7% (95%CI: 28.0%, 65.3%), and 24.4% (95%CI: 11.3%, 52.7%), respectively. The most common grade 3 or 4 treatment-related adverse events included neutropenia (71.0%), leukopenia (45.2%), anemia (38.7%), and thrombocytopenia (32.3%). There was no treatment-related death and no new safety signals were identified. Conclusion: The combination therapy of HX008 plus GP demonstrates favorable clinical efficacy with a manageable safety profile in pts with mTNBC. The combination therapy led to a longer PFS and OS than GP therapy reported previously, regardless of PD-L1 status. The safety profile was consistent with the known toxic effects of each agent. In this updated analysis, no new safety signals were identified. Clinical trial information: NCT04750382

Abstract PO4-03-12: De-escalating treatment with neoadjuvant chemotherapy in early triple-negative breast cancer

Abstract Neoadjuvant chemotherapy (NAT) is a standard treatment in early triple-negative breast cancer, but the regimen and duration of this treatment are controversial. We analyze a cohort of early triple negative breast cancer treated with NAT with different schemas to determine its efficacy. 272 patients treated with NAT since 2001 to 2020 were selected. The median age of the group was 57 years (27-85), initial size 30 mm (9-122) and Ki67 expression was 60 (7-100). 107 patients (39.3%) had histologically confirmed initial axillary involvement and the regimens of chemotherapy used were carboplatin AUC 5 + weekly paclitaxel (80 mg/m2) x 4 cycles in 56 cases (21%) (SCHEME A), same scheme followed by 4 cycles of adriamycin 50 mg/m2 + cyclophosphamide 500 mg/m2 in 85 cases (31%) (SCHEME B), Adriamycin 50 mg/m2 + cyclophosphamide 500 mg/m2 × 4 cicles in 44 (16%) (SCHEME C) and Adriamycin 50 mg/m2 + cyclophosphamide 500 mg/m2 × 4 cicles followed by docetaxel 100 mg/m2 × 4 cicles in 87 (32%) (SCHEME D). We achieved a 51% of pathologic complete response (pCR),and a total of 24% of recurrences. The proportion of PCR and recurrence according to the different schemes is represented in the following table. The clinical factors associated with recurrence were the initial size (OR 8.6), the initial axillary involvement (OR 4.21) and the absence of pCR (OR 8.14), and for patients with pCR it was only the initial axillary involvement (OR 3.4). Patients that received schema A in NAT only presented a total of 5% of recurrence (3/56), only in patients with initial axillary involvement. The NAT schema with carboplatin AUC 5 + weekly paclitaxel (80 mg/m2) x 4 cycles is safe in patients without initial nodal involvement with high pathologic complete response (62%) and lower recurrence rate (9% in nonPCR and 3% in pCR). Therefore, it could be considered as a standard scheme of neoadjuvant chemotherapy for patients without axillary involvement. Table. SCHEMA A (20%) B (31%) C (16%) D (32%) Pcr 62% 63% 41% 37% Recurrence in pCR 3% 7% 16% 9% Recurrence in nonpCR 9% 16% 50% 63% Citation Format: Serafin Morales Murillo, Ariadna Gasol Cudós, Noemí Tuset Der-Abrain. De-escalating treatment with neoadjuvant chemotherapy in early triple-negative breast cancer [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 PO4-03-12.

Abstract PO4-28-07: SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism

Abstract Background: The disturbance of lipid metabolism in the microenvironment plays an important role in the occurrence and development of triple-negative breast cancer(TNBC). SLC26A9, a member of the SLC26A family of anion transporters, is involved in breast oncogenesis. However, the effect of SLC26A9 in triple-negative breast cancer on lipid metabolism in the tumor microenvironment remains unclear. Methods: Bioinformatics, tissue microarrays and TNBC cell lines were used to detect the expression of SLC26A9 and its clinical significance. By altering the expression of SLC26A9 gene in TNBC cells, the effects of SLC26A9 on the regulation of lipid metabolism, cell biological behavior and its related molecular mechanisms in the TNBC microenvironment were explored using adoptive staining techniques, flow cytometric analysis and animal models. Results: We found by tissue microarray (TNBC=105 cases, Nor=67 cases) that the expression of SLC26A9 was significantly up-regulated in BC compared to paracancerous tissues, where SLC26A9 expression was higher in TNBC, and the same results were obtained in BC cell lines. Secondly, we found that SCL26A9 was positively correlated with lipid metabolism status in TNBC microenvironment and possessed a close association with P53 mutation by analyzing GEO and TCGA databases. Therefore, we used two cell lines (MDA-MB-231, MDA-MB-468) in which SCL26A9 was highly expressed in TNBC cell lines. It was demonstrated by oil red O staining and lipid fluorescence staining that knockdown/silencing of SLC26A9 diminished the ability of MDA-MB-231 and MDA-MB-468 cells to uptake and utilize lipids, especially for metabolites such as neutral lipids: triglycerides, lipid droplets, and steroidal substances. Then we demonstrated by using fatty acid flow cytometric analysis that knockdown/silencing of SLC26A9 significantly reduced the utilization and production of fatty acids in MDA-MB-231 and MDA-MB-468 cells, which are the key metabolic indicators of cancer cells as well as important energy-providing substances for cancer progression. Therefore, we further verified functionally that cell proliferation, migration, invasion and anti-apoptosis were significantly inhibited in in vitro after knockdown/silencing of SLC26A9, and in vivo tumorigenesis was significantly inhibited in BALB/c nude mice in in vivo. In addition, the expression of markers of lipid metabolic activity (FASN, ACLY) was reduced after knockdown/silencing of SLC26A9 in MDA-MB-231 cells by Western Blot, and the reduced expression of FASN and ACLY mainly inhibited the ab initio synthesis, uptake and utilization of fatty acids in TNBC cells, which in turn regulated the proliferation and invasion of TNBC. Mechanistically, SLC26A9 silencing/knockdown inhibited the synthesis, uptake, and utilization of multiple metabolites of lipids in TNBC, which combined with the TCGA database results revealed the activation of the key signaling pathway, the JAK-STAT3 signaling pathway, to promote the progression of TNBC. Conclusion(s): SLC26A9 is involved in the disturbance of lipid metabolism in the TNBC microenvironment and regulates the progression of TNBC. The search for new SLC26A9 inhibitors is expected to provide a new direction for the treatment of TNBC, but the exact molecular mechanisms need to be further explored. SLC26A9 upregulation in TNBC leads to a poor prognosis by referencing lipid metabolism in the TNBC microenvironment Wench. SLC26A9 is involved in lipid metabolism disorders and cell biological functions in the TNBC microenvironment in vivo. SLC26A9 is involved in disordered lipid metabolism in the TNBC microenvironment. Citation Format: Yingming Zhou, Hu Wang, Zhengxing Zhou, Xuemei Liu, Biguang Tuo, Taolang Li. SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism [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 PO4-28-07.

Abstract PO1-18-11: Imaging of tumour microvasculature using high-resolution contrast enhanced ultrasound together with markers of proliferation/angiogenesis/vascular mimicry to characterise response to NACT in TNBC

Abstract Background: In the UK, triple-negative breast cancer (TNBC) comprises 10-15% of breast cancer diagnoses annually. TNBC represents a clinical challenge due to a lack of expression of three treatable drug targets, namely oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER-2). The mainstay of treatment involves the use of neoadjuvant chemotherapy (NACT) followed by surgical excision where up to 50% of patients will achieve a pathological complete response (pCR). The addition of neo-adjuvant immunotherapy may increase the pCR rate to 60%. The presence of lymphatics and blood vessels within/around malignant tumours plays a role in cancer progression as does the formation of new blood vessels (angiogenesis). The vascular endothelial growth factor (VEGF) family and their receptors (VEGFRs) play an important role in angiogenesis as well as promoting the growth and survival of cancer cells. VEGFR is frequently overexpressed in TNBC and promotes changes in vascular endothelial cells, the basement membrane and the surrounding extracellular matrix. Co-expression of epidermal growth factor receptor (EGFR) and VEGFR enhances tumour growth and angiogenesis in an autocrine and paracrine manner. An alternate mechanism for tumour microcirculation in tumours is vasculogenic mimicry (VM). This is distinct from classical tumour angiogenesis. The presence of VM is associated with poor overall survival in breast cancer patients and anti-angiogenic treatment of TNBC may even promote tumour growth, proliferation and metastasis by stimulating VM. Conventional imaging techniques such as B-mode ultrasound and MRI are commonly used to monitor disease response in the breast but have limited accuracy in predicting pCR or residual disease. High-resolution contrast-enhanced ultrasound (HRCEUS) may offer a more accurate test to monitor TNBC response to NACT. This method utilises intravenously injected microbubbles, which consist of a gas core with an outer shell of lipid/albumin to image tumour microvasculature as well as gross tumour morphology. Trial Design: Single-centre study to evaluate HRCEUS to image the microvasculature of TNBC tumours in patients undergoing (NACT) and correlate imaging results with established markers of angiogenesis, proliferation and vasculogenic mimicry. Eligibility Criteria: Female, 18 to 60 years, histologically confirmed invasive TNBC with planned NACT, able to consent and in the Investigator’s opinion, adhering to the trial recommendations and governance. Aims: To investigate if imaging changes in the tumour microvasculature indicate a response to NACT and are reflected in the expression of proliferative markers. To determine whether changes in the tumour microvasculature and disease response are driven by angiogenesis +/- VM. To investigate whether overall disease response and changes in the microvasculature are influenced by the basal type phenotype or germline mutations in BRCA 1/2. Investigate patient satisfaction with the contrast ultrasound test. Statistical Methods: Quantify HRCEUS imaging characteristics of microvasculature at 3 treatment points. Compare this with the results of conventional imaging and the histopathological results of surgical excision at the end of NACT. Quantify immunohistochemical markers of angiogenesis, proliferation and VM at three treatment points and compare this with HRCEUS. Determine phenotype of all cases using immunohistochemistry to identify the basal Perform subtype analysis to assess if the basal phenotype is associated with imaging and immunohistochemical changes in the microvasculature during NACT. Test patients for germline BRCA1/2 gene mutations. Perform subtype analysis to assess if germline BRCA1/2 mutations are associated with imaging and immunohistochemical changes in the microvasculature during NACT. Present accrual (2 patients) and target accrual (5 patients) Contact details: j.rait@nhs.net Citation Format: Jaideep Rait, Michelle Garrett, Catherine Harper-Wynne, Sonia Saw, Mengxing Tang, Priya Palanisamy, Matthieu TOULEMONDE, Karina Cox. Imaging of tumour microvasculature using high-resolution contrast enhanced ultrasound together with markers of proliferation/angiogenesis/vascular mimicry to characterise response to NACT in TNBC [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 PO1-18-11.

Abstract PO3-13-07: MUC1-C INTEGRATES CHRONIC ACTIVATION OF INTERFERON PATHWAYS WITH CHROMATIN REMODELING IN TREATMENT RESISTANCE OF TRIPLE-NEGATIVE BREAST CANCER

Abstract Chronic inflammation is intimately linked to cancer progression and resistance to treatment. Triple-negative breast cancer (TNBC) is a recalcitrant malignancy largely unresponsive to cytotoxic, targeted and immunotherapeutic agents. The Mucin1 (MUC1) transmembrane heterodimeric protein, which is aberrantly expressed in TNBCs, evolved in mammals to provide protection of epithelia from the external environment. With loss of homeostasis, the (i) MUC1 N-terminal (MUC1-N) subunit is shed and released into serum as detected by the CA15-3 assay, and (ii) MUC1 C-terminal (MUC1-C) subunit is activated and thereby induces inflammatory, proliferative and remodeling signaling pathways associated with wound healing. However, prolonged MUC1-C activation in settings of chronic inflammation promotes cancer progression and treatment resistance. The SWI/SNF PBAF chromatin remodeling complex includes the polybromo-1 (PBRM1) subunit and drives DNA damage resistance and immune evasion in certain cancer cells through mechanisms that remain unclear. Recent studies in TNBCs have demonstrated that MUC1-C drives intrinsic activation of type II interferon (IFN) pathway that is linked to chronic inflammation and immune evasion. In extending this work, we have found that MUC1-C activates the type I IFN pathway, which contributes to genomic instability and DNA damage tolerance in TNBC cells. Importantly, our results further demonstrate that MUC1-C interacts with interferon regulatory factor 1 (IRF1) as a key regulatory node in integrating activation of the type I and II IFN pathways. Here, we report that MUC1-C is necessary for PBRM1 expression and that it forms a nuclear complex with PBRM1 in TNBC cells. Analysis of global transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) profiles further demonstrated that MUC1-C and PBRM1 drive STAT1 and IRF1 expression by increasing chromatin accessibility of promoter-like signatures (PLS) on their respective genes. We also found that MUC1-C, PBRM1, and IRF1 increase the expression and chromatin accessibility on PLSs of the (i) type II IFN pathway IDO1 and WARS genes and (ii) type I IFN pathway RIG-I, MDA5, cGAS, STING and ISG15 genes that collectively contribute to DNA damage resistance and immune evasion. In support of these results, targeting MUC1-C in wild-type BRCA1/2 TNBC cells enhanced carboplatin-induced DNA damage and loss of self- renewal capacity. In addition, MUC1-C was necessary for DNA damage resistance, self-renewal and tumorigenicity of olaparib-resistant BRCA1-mutant TNBC cells. Analysis of TNBC clinical samples corroborated that (i) MUC1 and PBRM1 are associated with decreased responsiveness to chemotherapy and (ii) MUC1-C expression is associated with the depletion of tumor-infiltrating lymphocytes. These findings demonstrate that MUC1-C activates PBRM1, and thereby chromatin remodeling of IFN-stimulated genes that promote chronic inflammation, DNA damage resistance, and immune evasion. These findings have identified MUC1-C as a potential target for the treatment of TNBC that have limited therapeutic options. To that end, the generation of MAb 3D1 against the MUC1-C extracellular domain provided an opportunity for the development of agents that target MUC1-C on the cancer cell surface. As one example, an allogeneic anti-MUC1-C CAR T cell using MAb 3D1 sequences is undergoing Phase I evaluation for the treatment of MUC1-C-expressing cancers (NCT05239143: P-MUC1C-ALLO1 Allogeneic CAR-T Cells in the Treatment of Subjects with Advanced or Metastatic Solid Tumors). In addition, anti-MUC1-C huMAb3D1-MMAE ADCs are under development by the NCI NExT Program for IND-enabling studies and performing early phase clinical trials in patients with TNBC. Citation Format: Nami Yamashita, Yoshihiro Morimoto, Atsushi Fushimi, Rehan Ahmad, Atrayee Bhattacharya, Yuka Inoue, Henry Withers, Geoffrey Shapiro, Tomoharu Yoshizumi, Takayuki Ueno, Shinji Ohno, Mark Long, Donald Kufe. MUC1-C INTEGRATES CHRONIC ACTIVATION OF INTERFERON PATHWAYS WITH CHROMATIN REMODELING IN TREATMENT RESISTANCE OF TRIPLE-NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-07.

Abstract PO1-03-08: Association of patient-reported triple-negative breast cancer (TNBC) knowledge with clinical trial participation

Abstract Background: Patients with TNBC are highly encouraged to participate in clinical trials given the lack of targeted treatment options for this breast cancer subtype. Yet little is known regarding barriers to trial participation in this population, including the impact of patients’ understanding about TNBC biology. We examined whether disease knowledge and other patient-level factors were associated with TNBC patients’ participation in trials. Methods: From a multicenter, prospective cohort study of patients enrolled from 2019-present with newly diagnosed TNBC (ER/PR ≤10% also eligible), we identified those with stage I-III tumors who completed at least one survey. Surveys assessed demographics, TNBC knowledge, risk perceptions, and participation in clinical trials. Patients were assigned to Cohort A if surgery was the first intervention and Cohort B if neoadjuvant therapy was given. Medical records were reviewed to verify the accuracy of tumor characteristics reported by patients. Data was summarized with descriptive statistics and logistic regression was used to identify factors associated with trial participation. Results: Of the 137 patients included, median age was 56 years (IQR 46-63), 104 (82%) were non-Hispanic White, 10 (8%) Black, 5 (4%) Hispanic. and 110 (86%) had at least some college education. Overall, 55 (41%) had stage I tumors, 65 (48%) stage II, and 14 (10%) stage III. Among 116 patients with available clinical trial participation data, 55 (47%) reported trial participation, 59 (51%) reported no trial participation, and 2 (2%) were unsure. The majority (36/55, 65%) of trial participants were in Cohort B. Among 129 patients with TNBC knowledge data, 82 (64%) and 73 (57%) correctly reported their tumor stage and grade, respectively. Most (104/129, 81%) knew TNBC was defined as ER/PR/HER2-negative, but only 16% (21/128) knew the ER/PR-negative cut off was ≤1%. Of 98 patients with risk perception data, 30 (31%) estimated their likelihood of recurrence within the next 5 years as 0%, 60 (61%) between 10-50%, and 8 (8%) between 60-100%. When asked if TNBC patients who remain cancer-free after 3 years will most likely not experience recurrence, 44 (45%) said “True,” 13 (13%) said “False,” and 40 (41%) were unsure. On univariate analysis, trial participation was less likely in Cohort A patients (OR 0.42, 95% CI 0.20 – 0.89, P=0.02) and more likely in stage II patients (vs. stage I, OR 2.43, 95% CI 1.09 – 5.39, P=0.03). On multivariable analysis, patients with at least some college education had higher odds of trial participation compared to patients with a college degree or higher (OR 3.21, 95% CI 1.11 – 9.28, P=0.03). No associations were found for other factors, including TNBC knowledge. Conclusions: About half of TNBC patients surveyed reported participating in clinical trials. Most of these patients were undergoing neoadjuvant therapy, likely reflective of greater study availability in this setting. While TNBC knowledge was not significantly associated with trial participation in this small sample, a considerable number of patients reported their tumor characteristics incorrectly and/or appeared to underestimate their recurrence risks. This highlights the need for enhanced informational support for TNBC patients during treatment decision-making. More research is needed to understand barriers to trial participation in this population to leverage patient engagement and accelerate progress towards more effective treatments for TNBC. Citation Format: Megan Tesch, Noah Graham, Sean Ryan, Melissa Hughes, Eileen Wrabel, Nadine Tung, Steve Lo, Thomas Fynan, Meredith Faggen, Natalie Sinclair, Maria Constantinou, Sarah Sinclair, Nabihah Tayob, Nancy Lin, Ann Partridge, Eric Winer, Sara Tolaney, Ana Garrido-Castro. Association of patient-reported triple-negative breast cancer (TNBC) knowledge with clinical trial participation [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 PO1-03-08.

Abstract PO3-25-11: Unprecedented responses to neoadjuvant sequential administration of Tocilizumab followed by cisplatin/docetaxel for locally advanced triple negative breast cancer patients

Abstract Background: Triple negative breast cancer (TNBC) remains a challenge for clinicians due to its aggressive behavior and scarcity of effective treatment. Interleukin 6 (IL-6) is well known to contribute to poor therapeutic gain, tumor relapse and aggressive tumor growth. Here we have explored targeting the IL-6 signaling pathway by Tocilizumab (Actemra) as a new potential therapeutic approach for the treatment of TNBC. Methods: The study population includes thirty (30) patients with (T2-T4, N0-3) stage (IIB-III) TNBC treated uniformly with Tocilizumab followed with cisplatin/docetaxel according to a traditional 3+3 design using a sentinel patient at each cohort. Eligible patients received Tocilizumab on day 1 followed by cisplatin/docetaxel on day 2 every 4 weeks for a total of six cycles. The primary end points were pathological complete response in breast and axilla (pCR) (ypT0/ypN0) and assessment for tolerability/toxicities profiles. Additionally, the status of the BRCA1 and BRCA2 genes was determined in all patients. Results: Eligible patients who were enrolled in this phase I/II trial had a median age of 42 years, with 63% premenopausal and 67% had nodal involvement at base line. The median tumor size was 45 mm (range 25-150 mm), stage III A/B 26% and stage IIIC 50%. Germline BRCA1/2 mutations represented 27%. The overall pCR and (RCB class 0+1) were 66% and 69%, respectively. Interestingly, pCR reached 81% in axilla, and 100% in patients with tumors bearing BRCA1/2 mutations. Interestingly, tocilizumab affected the blood level of several pro-carcinogenic cytokines. Conclusions: To our knowledge this novel therapeutic strategy is the first to report the inhibition of a cytokine signaling (a new class of biological therapy) in combination with chemotherapy for the intricate and hard-to-treat TNBC patients with unprecedented responses and acceptable tolerability/toxicities, which merits further investigation. Pending Patents: US Patent Application No. 18/122,839 European Patent Application No. 23 162 694.6 Citation Format: Taher Al-Tweigeri, Noura Alraouji, Asma Tulbah, Syed Akhtar, Meshari Alzahrani, Saleh Najjar, Dahish Ajarim, Kauser Suleman, Adher Al Sayed, Abdelilah Aboussekhra. Unprecedented responses to neoadjuvant sequential administration of Tocilizumab followed by cisplatin/docetaxel for locally advanced triple negative breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-11.

Abstract PO3-29-04: Patient selection for high RSK2 expression is key for achieving improved PFS in metastatic breast cancer in the PMD-026 Phase 1/1b study

Abstract Background: PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK and PDK-1 pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Both pathways are implicated in HR+ and triple negative breast cancer (TNBC). RSK2 can modulate the growth of breast cancer (BC) by promoting cell cycle progression through G2/M, specifically through pYB-1S102 which nucleates spindle pole formation in M-phase of cancer cells. This mechanism of action (MOA) for PMD-026 can be monitored by measuring the loss of pYB-1S102 and pCDK2Y15, a marker for G2/M arrest. Therefore, PMD-026 presents a novel mechanism to inhibit resistance at G1/S which is common for CDK4/6 inhibitors as well as ADCs such as those that contain Topoisomerase 1 conjugates. Here in, we show for the first time that PMD-026 inhibits P-YB-1S102 and pCDK2y15 in a dose-dependent manner in TNBC cells and address whether this could be translated as a pharmacodynamic marker of PMD-026 activity in patients. To guide patient selection, we are co-developing a unique and simple IHC based companion diagnostic (CDx) with Roche based on the principle that activated RSK2 translocates from the cytoplasm into the nucleus. In mBC, RSK2 was found to be highly expressed in ~70% of patients using a staining algorithm. An H-score was developed and ultimately H-score >180 (75% nuclear staining with staining intensity of 2+ or 3+ by IHC) was used as the cut-off in our Phase 1/1b trial retrospective study. This IHC assay will be used to select patients in the upcoming Phase II, as PFS in the PMD-026 Phase I/1b study showed remarkable differences when comparing patients with high and low RSK2. Results: In this Phase 1/1b study, PMD-026 was evaluated in an ethnically diverse population of women across all breast cancer subtypes. Patients had a median of 5 prior lines of therapy and PMD-026 achieved stable disease in 44% (11/25) of the subjects. Consistent with the MOA, PMD-026 inhibited pCDK2Y15 in PBMCs from patients at C1D15. Corresponding plasma concentrations were ~ 1µM, which correlates with the IC90 of PMD-026 in preclinical models. To guide patient selection in the future, the RSK2 IHC with an H:Score >180 was used to evaluate patient outcomes. Using this criterion, PFS was significantly longer in RSK2 high vs low patients (3.6 months vs 1.3 month, HR = 0.27, p = 0.031, N=20 patients representing de novo TNBC and HR+/CDK4/6 refractory subtypes). Upon further analysis, patients who had 5 lines of therapy or less benefited the most. PFS in these less heavily pretreated RSK2 high patients was 4.8 months vs 1.3 months for RSK2 low patients (HR = 0.07, p = 0.001 N=14 representing the same subtypes above). It was clear that the time on study was longer for patients with high RSK2 vs low, and this was specifically noted in de novo TNBC and HR+ patients that previously failed CDK4/6 inhibitors as well as endocrine therapies. A key observation in this study was that the PFS benefit was based on the RSK2 levels and not on any pharmacokinetic differences between patients, as the AUC (p=0.53), Cmax (p=0.73) and plasma levels (p=0.76) did not differ between RSK2 high and RSK2 low groups. These data strongly support the activity of this first in class RSK inhibitor, PMD-026, in RSK2 high breast cancers. Our integrated IHC based CDx strategy plans to prospectively enroll RSK2 high patients for our Phase 2 trials will be described. Conclusions/Translational Significance: CDx guided breast cancer therapeutics are often lacking for the treatment of TNBC, as well as HR+ breast cancers that have failed CDK4/6 inhibitors. The ability to identify patients who could potentially benefit most from RSK-targeted agents such as PMD-026 is an important step forward in delivering practice changing therapies for patients. Citation Format: Sandra Dunn, Judy Wang, Hyo Han, Robert Wesolowski, Amita Patnaik, Shakeela Bahadur, Nicolaos Palaskas, My-my Huynh, Aarthi Jayanthan, Gerrit Los, Andrew Dorr. Patient selection for high RSK2 expression is key for achieving improved PFS in metastatic breast cancer in the PMD-026 Phase 1/1b study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-29-04.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [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 PO1-06-11.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-10.

A holistic review of recent advances in nano-based drug delivery systems for the treatment of triple-negative breast cancer (TNBC)

A holistic review of recent advances in nano-based drug delivery systems for the treatment of triple-negative breast cancer (TNBC)

Quercetin exerts anti-tumor immune mechanism by regulating IL-6/JAK2/STAT3 signaling pathway to deplete Treg cells

Breast cancer is still the leading cause of death among women worldwide. Due to the lack of effective drug targets, triple-negative breast cancer has a worse prognosis and higher mortality compared with other types of breast cancer, and chemotherapy is still the main treatment for triple-negative breast cancer at present. Quercetin (QUE) is a flavonoid compound found in a variety of fruits and vegetables. The mechanism of QUE has been extensively studied, such as prostate cancer, colon cancer, ovarian cancer, etc. However, the anti-tumor immune mechanism of QUE in triple-negative breast cancer remains unclear. Therefore, we assessed the anti-tumor immune effects of QUE on triple-negative breast cancer using both 4T1 cells and a xenograft mouse model of 4T1 cells. In vitro, we examined the inhibitory effects of QUE on 4T1 cells and its molecular mechanisms through MTT, Transwell, ELISA, and Western blotting. In vivo, by establishing a xenograft mouse model, we utilized flow cytometry, immunohistochemistry, ELISA, and Western blotting to evaluate the anti-tumor immune effects of QUE on triple-negative breast cancer. The results indicate that QUE inhibits the proliferation, migration, and invasion of 4T1 cells, concurrently significantly suppressing the IL-6/JAK2/STAT3 signaling pathway. Furthermore, it depletes Treg cell content in 4T1 xenograft mice, thereby improving the tumor immune microenvironment and promoting the cytotoxicity of relevant tumor immune cells. These findings suggest that QUE may serve as a potential adjuvant for immune therapy in triple-negative breast cancer.

Biotin-stapled mixed micelles of lapatinib: Unleashing apoptosis power in triple-negative breast cancer

The treatment options currently offered for triple-negative breast cancer (TNBC) encounter numerous obstacles, including disappointing overall survival rates, unavoidable side effects, and the development of drug resistance. Additionally, there is a growing need for nutrients, like vitamins (e.g., biotin), to sustain the unregulated growth of TNBC cells. Hence, targeted therapy (LPT B-MM) for enhancing the treatment of TNBC was explored which encompassed hydrophobized [using α-TOS (α-Tocopheryl succinate] Pluronic-based mixed micelles having inherent sodium-dependent multivitamin transporter (SMVT) targeting (biotin-stapled) ability. LPT B-MM had optimal size (210.72± 25.88 nm), PDI (0.272± 0.023), and enhanced drug loading capacity (∼13.31± 2.12%). Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT B-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. This study highlights the considerable potential of LPT B-MM to induce apoptosis, showcasing its promise in enhancing payload capabilities and enabling SMVT specific targeting within the context of TNBC. LPT B-MM demonstrated the synergistic effects of LPT and α-TOS in inducing apoptosis in TNBC cells, leading to enhanced therapeutic efficacy. This novel combination therapy offers a promising strategy for improving treatment outcomes in TNBC patients and represents a significant advancement in the field of TNBC therapy.

Dual nanoparticle immunostimulant hydrogel for synergistic combating “Cold” tumor

The immunosuppressive tumor microenvironment (TME) in “cold” triple-negative breast cancer (TNBC) leads to resistance to current immunotherapies. Here, we designed an in-situ hydrogel reservoir to deliver two types of nanoparticles (NPs) targeting 4T1 tumor cells and tumor-associated macrophages (TAMs) for cancer cell elimination and reversal of the immunosuppressive TME. The 4T1 tumor cell-targeted NPs could specifically deliver the mitochondria-targeted photosensitizer and catalase into 4T1 tumor cells to execute an enhanced photodynamic therapy and induce immunogenic cell death. The TAMs-targeted NPs delivered the stimulator of interferon genes (STING) agonist into macrophages to activate the STING pathway, triggering a type I interferon-driven innate immune response that induces a shift to a “hot” T cell-inflamed TME. This local synergistic therapy could effectively stimulate dendritic cell activation and the continuous T-cell infiltration, thereby reprograming the immunosuppressive TME, reinvigorating a powerful anti-tumor immune response and inhibiting tumor growth and metastasis in TNBC tumor model. In addition, this study combined with immune checkpoint blockade further achieves reinforced therapeutic outcome against “cold” tumor, providing a promising hydrogel-mediated platform for treatment of TNBC.

Chlorin Conjugates in Photodynamic Chemotherapy for Triple-Negative Breast Cancer.

Breast cancer (BC) is the most common type of cancer in women and the number of new cases in the US is still increasing each year. Triple-negative breast cancer (TNBC), which comprises 15-20% of all breast cancer, is a heterogeneous disease and is considered the most aggressive type of breast cancer due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expressions for treatments. Traditional chemotherapy is the standard protocol for the treatment of TNBC. Toxicity and multidrug resistance are major drawbacks to chemotherapy. The lack of molecular targets and poor prognosis for TNBC prompts an urgent need to discover novel therapeutic strategies to improve clinical outcomes and quality of life for patients. Photodynamic therapy (PDT) or light treatment is a binary anti-cancer procedure that uses a photosensitizer (PS) that, upon light activation, produces cytotoxic oxygen species, destroying tumor cells. PDT is minimally invasive and can be repeated a few times without accumulating significant toxicity in the surrounding tissues. The primary goal of this study was to investigate in vitro photodynamic chemotherapy as a ternary combination therapy using our synthesized photosensitizers (chlorin-vitamin conjugates and their corresponding indium complexes) co-treated with known chemotherapeutic agents (taxol, doxorubicin, cisplatin, fluorouracil, or methotrexate) in the presence of light and determine the optimum conditions as a pre-clinical study of an enhanced tumoricidal effect against TNBC. Our results indicated that the best combination for an effective chemophotodynamic effect involves a ternary treatment of the indium complex of the chlorin-lipoic acid conjugate (InCLA) co-treated with taxol, which exhibited strong synergism at the nanomolar concentration when combined in the presence of visible light irradiation. Other ternary combinations containing taxol with a synergistic anti-tumor effect against TNBC include chlorin-pantothenic acid (CPA) and chlorin-biotin (CBTN) conjugates. Several other ternary combinations containing InCLA, CBTN, and CPA with either cisplatin, fluorouracil, or methotrexate were identified to generate a synergistic or additive effect. The light dosage remained constant, but the dosages of photosensitizers and chemotherapy drugs were varied to obtain the lowest possible concentration for the desired effect. The synergistic, additive or antagonistic effects of the drug combinations were determined based on the Chou-Talalay method, with InCLA-taxol having the lowest combination index (CI) of 0.25. Fluorescence and transmission electron microscopy (TEM) images provided evidence of apoptosis as the preferred mode of cell death. Our study demonstrated the combination of PDT and chemotherapy as a potential treatment option for TNBC patients.

Pharmacological Mechanism of Mume Fructus in the Treatment of Triple-Negative Breast Cancer Based on Network Pharmacology.

Our study aims to find the relevant mechanism of Mume Fructus in the treatment of triple-negative breast cancer (TNBC) by network pharmacology analysis and experimental validation. The effective compounds of Mume Fructus and TNBC-related target genes were imported into Cytoscape to construct a Mume Fructus-effective compounds-disease target network. The common targets of Mume Fructus and TNBC were determined by drawing Venn diagrams. Then, the intersection targets were transferred to the STRING database to construct a protein-protein interaction (PPI) network. To investigate the mechanism of Mume Fructus in treatment of TNBC, breast cancer cell (MDA-MB-231) was treated with Mume Fructus and/or transfected with small interference RNA-PKM2(siPKM2).CCK-8 assay, cell clonal formation assay, transwell, flow cytometry, qRT-PCR, and western blotting were performed. Eight effective compounds and 145 target genes were obtained, and the Mume Fructus- effective compounds-disease target network was constructed. Then through the analysis of the PPI network, we obtained 10 hub genes including JUN, MAPK1, RELA, AKT1, FOS, ESR1, IL6, MAPK8, RXRA, and MYC. KEGG enrichment analysis showed that JUN, MAPK1, RELA, FOS, ESR1, IL6, MAPK8, and RXRA were enriched in the Th17 cell differentiation signaling pathway. Loss of PKM2 and Mume Fructus both inhibited the malignant phenotype of MDA-MB-231 cells. And siPKM2 further aggravated the Mume Fructus inhibition of malignancy of breast cancer cells. Network pharmacology analysis suggests that Mume Fructus has multiple therapeutic targets for TNBC and may play a therapeutic role by modulating the immune microenvironment of breast cancer.