Resistant Triple Negative Breast Cancer Research Articles

Discovery of novel small molecules targeting the USP21/JAK2/STAT3 axis for the treatment of triple-negative breast cancer

The deficiency in available targeted agents and frequency of chemoresistance are primary challenges in clinical management of triple-negative breast cancer (TNBC). The aberrant expression of USP21 and JAK2 represents a characterized mechanism of TNBC progression and resistance to paclitaxel (PTX). Despite its clear that high expression of USP21-mediated de-ubiquitination leads to increased levels of JAK2 protein, we lack regulator molecules to dissect the mechanisms that the interaction between USP21 and JAK2 contributes to the phenotype and resistance of TNBC. Here, we report a USP21/JAK2/STAT3 axis-targeting regulator 13c featuring a N-anthraniloyl tryptamine scaffold that showed excellent anti-TNBC potency and promising safety profile. Importantly, the therapeutic potential of using 13c in combination with PTX in PTX-resistant TNBC was demonstrated. This study showcases N-anthraniloyl tryptamine derivatives as a novel anti-TNBC chemotype with a pharmacological mode of action targeting the USP21/JAK2/STAT3 axis and provides a potential therapeutic target for the treatment of TNBC.

Novel markers of MCL1 inhibitor sensitivity in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is an aggressive breast cancer sub-type with limited treatment options and poor prognosis. Currently, standard treatments for TNBC includes surgery, chemotherapy, and anti-PDL1 therapy. These therapies have limited efficacy in advanced stages. Myeloid-cell leukemia 1 (MCL1) is an anti-apoptotic BCL2 family protein. High expression of MCL1 contributes to chemotherapy resistance and is associated with worse prognosis in TNBC. MCL1 inhibitors are in clinical trials for TNBC, but response rates to these inhibitors can vary and predictive markers are lacking. Currently we identified a 4-member (AXL, ETS1, IL6, EFEMP1) gene signature (GS) that predicts MCL1 inhibitor sensitivity in TNBC cells. Factors encoded by these genes regulate signaling pathways to promote MCL1 inhibitor resistance. Small molecule inhibitors of the GS factors can overcome resistance and sensitize otherwise resistant TNBC cells to MCL1 inhibitor treatment. These findings offer insights into potential therapeutic strategies and tumor stratification for MCL1 inhibitor use in TNBC.

Evaluation of copanlisib in combination with eribulin in triple negative breast cancer patient-derived xenograft models.

The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.

Abstract PO3-15-01: Overexpression of N-glycosylation related gene OST4 promotes chemotherapy resistance in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with high malignancy and poor outcome. Resistance to chemotherapy leads to treatment failure. How to overcome drug resistance is the key point in TNBC treatment. Previously, we initiated a prospective multicenter single-arm NeoPath clinical study (ClinicalTrial.gov identifier: NCT03907800) to assess the efficacy and safety of nab-paclitaxel combined with carboplatin in the neoadjuvant therapy of triple negative and HER2-positive early breast cancer. A total of 99 TNBC patients were enrolled and the pCR rate in TNBC was 33.78%. Samples before and after treatment were collected. RNA-sequencing results showed that OST4 was of high-expression in drug-resistant TNBC tumor tissues compared with chemo-sensitive samples. To verify the clinical significance of OST4 expression levels in TNBC, we tested the expression of OST4 by immunohistochemical technique on a tissue microarray including 87 TNBC cases treated with chemotherapy. We found that high expression of OST4 was associated with poor tumor differentiation, and patients with high OST4 expression had more relapse after chemotherapy. Ex-vivo proliferation assay and foci formation assay further confirmed that over-expression of OST4 promoted chemo-resistance, whereas down-regulating OST4 could reverse resistance in TNBC cell lines. As an oligosaccharyltransferase, it is reported that OST4 played parts in N-glycosylation. So we try to elucidate the relationship between N-glycosylation and drug resistance. Glycoproteomics results verified the existence of differential N-glycosylation atlas in drug-resistant tumor tissues compared with drug sensitive samples. NGS1 was identified as a potential target of OST4 by co-immunoprecipitation and mass spectrometry. In conclusion, high expression of OST4 could promote chemotherapy resistance in TNBC trough N-glycosylation regulation. Targeting OST4 may help reverse drug-resistance and improve therapeutic effect in TNBC. Citation Format: Wei Wang, Deyue Liu, Shuning Ding, Yan Fang, Jiayi Wu, Li Zhu. Overexpression of N-glycosylation related gene OST4 promotes chemotherapy resistance in 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 PO3-15-01.

Abstract PO4-13-11: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results in early TNBC, many patients do not respond, and chemotherapy remains the main treatment. High recurrence rates in TNBC are in part due to its inherent molecular heterogeneity and sub-clonal diversity, whereby cells present in minority sub-populations escape therapeutic pressure. Recurrent genomic alterations are not selected for upon therapy resistance in neoadjuvant chemotherapy (NAC) resistant TNBC, however sub-clonal transcriptionally defined cells are “primed” to drive resistance and are pre-existing, suggesting phenotypic heterogeneity is governed by inheritable activation of epigenetically defined regulatory regions. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Driver DNA mutations and copy number alterations (CNA’s) were identified using a clinically validated targeted capture panel. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine meta-clusters across the dataset. A distinct immune-like MC was contributed by epithelial G1 cells, suggesting a cross-talk of epithelial cells with spatially proximal immune cells. Two MCs were dominated by cells from ChemoR patients and were defined by pathways related to hypoxia, EMT, and extra-cellular matrix signalling. Using scATAC profiles, we identified that the sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of distal enhancer chromatin modification in regulating MC transcription. None of the meta-clusters were dominated by tumours with specific genetic mutations or CNA’s. Spatial ATAC and RNA profiling of a subset of tumours identified spatial epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and associated with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommati, Shefali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in 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 PO4-13-11.

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory 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 PO1-29-01.

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 PO2-04-14: Dual Inhibition of WEE1 and PKMYT1 Synergistically Overcomes CDK4/6 Inhibitor Resistance in Breast Cancer

Abstract Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are approved for the treatment of advanced estrogen receptor-positive, HER2-negative (ER+ve) breast cancer when combined with endocrine therapy. However, the emergence of resistance to CDK4/6 inhibitors poses a significant clinical challenge. This study investigates the mechanisms underlying CDK4/6 inhibitor resistance and identifies a common occurrence of high replication stress in triple-negative breast cancer (TNBC) and CDK4/6 inhibitor-resistant ER+ve breast cancers. While ER+ve breast cancer patients have benefited from targeted therapy, TNBC patients, particularly those without germline BRCA1/2 or PALB2 mutations, lack effective treatment options. TNBC and CDK4/6 inhibitor-resistant ER+ve breast cancer cells exhibit an increased dependence on cell cycle arrest to repair replication stress-induced DNA damage. Failure to exit the cell cycle results in excessive DNA damage, thereby limiting their proliferative potential. Based on these findings, we hypothesize that dual inhibition of WEE1 and PKMYT1, two kinases involved in G2/M cell cycle transition and crucial for cell cycle arrest and DNA damage repair, could synergistically inhibit tumor growth. Although WEE1 and PKMYT1 share a common function in promoting G2/M cell cycle transition through CDK1 phosphorylation, their functions are not entirely redundant, suggesting that targeting both kinases may elicit a synergistic response. Recent clinical trials have shown resistance to WEE1-targeted monotherapies, emphasizing the need for combining WEE1 inhibitors with other agents, such as PKMYT1 inhibitors. We demonstrate the synergistic effects of WEE1 inhibitor (MK1775/AZD1775) and PKMYT1 inhibitor (RP6306) in a panel of TNBC and ER+ve breast cancer cells with acquired palbociclib resistance (PalboR). Combination treatment with both inhibitors leads to excessive unrepaired DNA damage, as indicated by increased γH2AX foci following 48 hours of combination treatment, whereas single-agent treatments induce significantly lower levels of DNA damage. Furthermore, we observed the AZD1775/RP6306 combination decreases the expression of the DNA repair protein Rad51 in PalboR cells. To evaluate the clinical potential of dual WEE1 and PKMYT1 inhibition, we generated organoids from metastatic breast cancer lesions of patients who had progressed on CDK4/6 inhibitors for ex vivo studies. Our results demonstrate that dual inhibition of WEE1 and PKMYT1 is more effective than single-agent treatments in eliminating CDK4/6 inhibitor-resistant organoids. Additionally, using patient-derived xenograft mouse models from patients who had progressed on palbociclib and endocrine treatment, we show that the AZD1775/RP6306 combination exhibits superior tumor suppression effects compared to single agents. Overall, this study highlights the potential therapeutic benefits of dual inhibition of WEE1 and PKMYT1 in overcoming CDK4/6 inhibitor resistance in TNBC and ER+ve breast cancer patients. These findings provide a rationale for future clinical trials aimed at exploring the clinical utility of this combination therapy. Citation Format: Mei-Kuang Chen, Grace DeAlessandro, Tuyen Bui, Khandan Keyomarsi. Dual Inhibition of WEE1 and PKMYT1 Synergistically Overcomes CDK4/6 Inhibitor Resistance in 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 PO2-04-14.

Abstract PO1-06-14: Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC)

Abstract Background: Aberrant PI3K pathway signaling is frequently observed in TNBC. Increasing evidence indicate that PI3K pathway activation maintains the stemness and chemoresistance of breast cancer stem cells (CSCs). PI3K inhibition sensitizes CSCs to chemotherapy. Eribulin (E), a non-taxane microtubule dynamics inhibitor, showed overall survival benefit in metastatic HER2 negative BC. Preclinically, the addition of copanlisib (C), a potent pan-class I PI3K inhibitor that is highly selective against α and δ PI3K isoforms, improved anti-tumor effect in both E-sensitive and resistant TNBC patient-derived xenograft models, irrespective of PIK3CA/PTEN alteration status. Here we report the preliminary data of the phase I dose escalation trial of E+C in patients with metastatic TNBC. Methods: This trial includes a phase I study to determine the dose limiting toxicity (DLT) and recommended phase 2 dose (RP2D) of E+C, followed by a randomized phase II study of E+C (at RP2D) versus E (stratified by tumor PIK3CA/PTEN mutation status) with progression-free survival (PFS) as the primary endpoint. Key eligibility included patients with: metastatic TNBC who progressed on ≤5 lines of chemotherapy, prior anthracycline/taxane chemotherapy (unless contraindicated), ECOG 0-1, adequate organ function and known tumor PIK3CA/PTEN/AKT mutation status. Key exclusions include: prior receipt of E or any PI3K/mTOR/AKT inhibitor, grade ≥2 neuropathy, presence of tumor AKT mutation, and uncontrolled diabetes, hypertension or congenital QT prolongation. A 3+3 design was used to determine the DLT and RP2D. Dose level 1 (DL1) started with E at 1.1 mg/m2 mg IV and C at 45 mg IV on days (D) 1/8 of 21-D cycle, with the potential to escalate to 2 additional dose levels – DL2: E 1.4 mg/m2 and C 45 mg on D 1/8; DL3: E 1.4 mg/m2 and C 60 mg on D 1/8. Results: Eight patients were enrolled in the phase I study and received at least 1 dose of E+C. The median age was 48.5 (range, 26-67) years. The majority of patients identified as White (additionally 2 Hispanic, 1 Black) and had an ECOG performance status of 1 (n=6, 75.0%). Tumor PIK3CA and PTEN alterations were each observed in 1 patient. Median number of cycles received were 2 (range, 1-5). All 8 patients had at least 1 treatment-related adverse event (TRAE). The most common TRAEs across all dose levels were: lymphopenia (n=5, 62.5%), neutropenia (n=5, 62.5%), hyperglycemia (n=4, 50.0%), anemia (n=4, 50.0%) and fatigue (n=4, 50.0%); most toxicities were grade (G) 1-2. G3 hyperglycemia and hypertension were observed in 1 patient each, were medically managed and did not lead to treatment interruption. 1 patient was diagnosed with pulmonary embolism, possibly attributed to study therapy and breast cancer. DLTs were reported in 2 of 2 patients treated at DL2 - 1 patient developed G3 rash responsive to oral corticosteroids, and 1 patient developed G3 acute kidney injury in the setting of hypovolemia and discontinued study therapy. No additional patients were enrolled in DL2. No DLTs were observed in all 6 patients treated at DL1. 1 patient treated at DL1 had possible G1 non-infectious pneumonitis that resolved spontaneously on short interval imaging. Stable disease was observed in 2 of 6 response-evaluable patients (33.3%). 1 patient achieved regression of cutaneous disease following 1 cycle of therapy as assessed by the treating investigator. DL1, E at 1.1 mg/m2 plus C at 45 mg IV, was determined to be the RP2D. Tumor tissue biomarker analysis is underway. Conclusion: These data indicate that E+C at DL1 is a well-tolerated regimen warranting further investigation. The study is actively enrolling patients to the phase II study. Clinical trial information: NCT04345913. Funding Source: National Cancer Institute. Citation Format: Nusayba Bagegni, Brittney Haas, Leslie Nehring, Jingqin Luo, Laura Kennedy, Meghna Trivedi, Manali Bhave, Rabih Said, Cynthia Ma. Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (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 PO1-06-14.

Ganoderic acid D attenuates gemcitabine resistance of triple-negative breast cancer cells by inhibiting glycolysis via HIF-1α destabilization

BackgroundGemcitabine (GEM) resistance is the primary reason why combination chemotherapy is limited in triple-negative breast cancer (TNBC). Ganoderic acid D (GAD), a natural triterpenoid compound obtained from Ganoderma lucidum, has been shown to have antitumor activities. However, whether GAD can reverse GEM resistance in TNBC requires further investigation. PurposeThis study investigated whether and how GAD could reverse GEM resistance in TNBC as an antitumor adjuvant. MethodsThe effects of GAD on cell proliferation, cell cycle, and glycolysis were studied in vitro using a GEM-resistant (GEM-R) TNBC cell model. We enriched key pathways affected by GAD using proteomics techniques. Western blotting and qPCR were used to detect the expression of glycolysis-related genes after GAD treatment. A mouse resistance model was established using GEM-R TNBC cells, and hematoxylin-eosin staining and immunohistochemistry were used to assess the role of GAD in reversing resistance in vivo. ResultsCellular functional assays showed that GAD significantly inhibited proliferation and glucose uptake in GEM-R TNBC cells. GAD reduces HIF-1α accumulation in TNBC cells under hypoxic conditions through the ubiquitinated protease degradation pathway. Mechanistically, GAD activates the p53/MDM2 pathway, promoting HIF-1α ubiquitination and proteasomal degradation and downregulating HIF-1α-dependent glycolysis genes like GLUT1, HK2, and PKM2. Notably, GAD combined with gemcitabine significantly reduced the growth of GEM-R TNBC cells in a subcutaneous tumor model. ConclusionsThis study reveals a novel antitumor function of GAD, which inhibits glycolysis by promoting HIF-1α degradation in GEM-R TNBC cells, offering a promising therapeutic strategy for TNBC patients with GEM resistance.

PYCR3 modulates mtDNA copy number to drive proliferation and doxorubicin resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) poses significant challenges in treatment due to its aggressive nature and limited therapeutic targets. Understanding the underlying molecular mechanisms driving TNBC progression and chemotherapy resistance is imperative for developing effective therapeutic strategies. Thus, in this study, we aimed to elucidate the role of pyrroline-5-carboxylate reductase 3 (PYCR3) in TNBC pathogenesis and therapeutic response. We observed that PYCR3 is significantly upregulated in TNBC specimens compared to normal breast tissues, correlating with a poorer prognosis in TNBC patients. Knockdown of PYCR3 not only suppresses TNBC cell proliferation but also reverses acquired resistance of TNBC cells to doxorubicin, a commonly used chemotherapeutic agent. Mechanistically, we identified the mitochondrial localization of PYCR3 in TNBC cells and demonstrated its impact on TNBC cell proliferation and sensitivity to doxorubicin through the regulation of mtDNA copy number and mitochondrial respiration. Importantly, Selective reduction of mtDNA copy number using the mtDNA replication inhibitor 2′, 3′-dideoxycytidine effectively recapitulates the phenotypic effects observed in PYCR3 knockout, resulting in decreased TNBC cell proliferation and the reversal of doxorubicin resistance through apoptosis induction. Thus, our study underscores the clinical relevance of PYCR3 and highlight its potential as a therapeutic target in TNBC management. By elucidating the functional significance of PYCR3 in TNBC, our findings contribute to a deeper understanding of TNBC biology and provide a foundation for developing novel therapeutic strategies aimed at improving patient outcomes.

PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib via the EGFR pathway

Epidermal growth factor receptor (EGFR)-targeted drugs (erlotinib, etc.) are used to treat multiple types of tumours. EGFR is highly expressed in most triple-negative breast cancer (TNBC) patients. However, only a small proportion of TNBC patients benefit from EGFR-targeted drugs in clinical trials, and the resistance mechanism is unclear. Here, we found that PDZ domain containing 1 (PDZK1) is downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation is related to erlotinib resistance in TNBC. PDZK1 binds to EGFR. Through this interaction, PDZK1 promotes EGFR degradation by enhancing the binding of EGFR to c-Cbl and inhibits EGFR phosphorylation by hindering EGFR dimerisation. We also found that PDZK1 is specifically downregulated in TNBC tissues and correlated with a poor prognosis in TNBC patients. In vitro and in vivo functional assays showed that PDZK1 suppressed TNBC development. Restoration of EGFR expression or kinase inhibitor treatment reversed the degree of cell malignancy induced by PDZK1 overexpression or knockdown, respectively. PDZK1 overexpression sensitised TNBC cells to erlotinib both in vitro and in vivo. In conclusion, PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells.

SF3A2 promotes progression and cisplatin resistance in triple-negative breast cancer via alternative splicing of MKRN1.

Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer owing to the lack of effective therapeutic targets. Splicing factor 3a subunit 2 (SF3A2), a poorly defined splicing factor, was notably elevated in TNBC tissues and promoted TNBC progression, as confirmed by cell proliferation, colony formation, transwell migration, and invasion assays. Mechanistic investigations revealed that E3 ubiquitin-protein ligase UBR5 promoted the ubiquitination-dependent degradation of SF3A2, which in turn regulated UBR5, thus forming a feedback loop to balance these two oncoproteins. Moreover, SF3A2 accelerated TNBC progression by, at least in part, specifically regulating the alternative splicing of makorin ring finger protein 1 (MKRN1) and promoting the expression of the dominant and oncogenic isoform, MKRN1-T1. Furthermore, SF3A2 participated in the regulation of both extrinsic and intrinsic apoptosis, leading to cisplatin resistance in TNBC cells. Collectively, these findings reveal a previously unknown role of SF3A2 in TNBC progression and cisplatin resistance, highlighting SF3A2 as a potential therapeutic target for patients with TNBC.

Abstract CT128: Phase I/II clinical trials of LAE005, afuresertib plus nab-paclitaxel in patients with advanced solid tumors, primarily in patients with triple-negative breast cancer (TNBC)

Abstract Background: AKT inhibitor was reported to be a potential treatment for drug-resistant tumors by reducing AKT activity, and a synergistic agent of ICIs. Afuresertib is an oral pan-AKT inhibitor that has shown clinical efficacy in multiple tumors. The phase I part of this study assessed the safety, tolerability, and anti-tumor activity of the combination therapy of LAE005 (PD-L1 antibody) plus afuresertib plus nab-paclitaxel in advanced solid tumors, primarily in TNBC. Methods: This is a multi-center, open-label, dose-escalation and proof-of-concept phase I/II study (NCT05390710). In phase I part, patients with advanced solid tumors (TNBC preferred) who have failed 0 to 3 lines of standard treatment are eligible. ECOG score 0-1. Efficacy evaluation is based on RECIST 1.1. Dose escalation uses Bayesian optimal interval (BOIN) design. Results: As of December 11, 2023, 22 subjects were enrolled and dosed. The median prior line of systematic anti-tumor therapy was 1 (0-3), including chemotherapy, PAPR inhibitor or ICIs. Six subjects (3 TNBC, 2 HR+ BC, 1 lung adenocarcinoma) were enrolled in cohort 1 (afuresertib 100 mg PO QD + LAE005 1200 mg IV Q3W + nab-paclitaxel 125 mg/m2 IV D1, D8, Q3W). Six TNBC subjects were enrolled in cohort 2 (afuresertib 100 mg PO QD + LAE005 1200 mg IV Q3W + nab-paclitaxel 100 mg/m2 IV D1, D8, Q3W). 10 TNBC subjects were enrolled in cohort 3 (afuresertib 125 mg PO QD + LAE005 1200 mg IV Q3W + nab-paclitaxel 100 mg/m2 IV D1, D8, Q3W). 2, 0, 2 dose-limiting toxicity (DLT) cases were reported out of 6, 6, 7 DLT evaluable subjects in cohort 1, 2, 3, respectively. DLTs were grade 3 rash (including rash, rash maculopapular, and erythema multiforme) and fever. The most common treatment-emergent AEs were rash (90.9%), white blood cell count decreased (81.8%), and neutrophil count decreased (77.3%). The most common grade 3 or above AEs were neutrophil count decreased (40.9%), white blood cell count decreased (36.4%), rash (18.2%) and lymphocyte count decreased (18.2%). Most of the AEs are manageable, reversible, and recovered after routine treatments. The median follow-up time for 19 TNBC subjects was 16.0 months (min-max: 8.7-28.9 months). The median PFS of these TNBC subjects was 5.4 months (95% CI: 1.4-14.0 months). Among 14 TNBC patients who completed at least 2 cycles of treatment, 5 cases had confirmed PR (ORR 35.7%), 4 cases (28.6%) had SD as the best response. DCR was 64.3%, ORR in three cohorts was 1/2, 2/5, 2/7, respectively. The median DOR was 9.26 months (95%CI: 2.3-NE months).5 TNBC subjects were treated for more than 32 weeks, and the longest duration of treatment was 73 weeks. Conclusions: The triplet regimen shows a manageable safety profile and encouraging preliminary activity in the drug resistant TNBC patients. This triplet regimen warrants further investigation. Citation Format: Binghe Xu, Pin Zhang, Ying Wang, Zhongsheng Tong, Tingjing Yao, Xian Wang, Zijia Wang, Pengfei Guo, Wenyue Ma, Yong YUE. Phase I/II clinical trials of LAE005, afuresertib plus nab-paclitaxel in patients with advanced solid tumors, primarily in patients with triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT128.

SOD1-high fibroblasts derived exosomal miR-3960 promotes cisplatin resistance in triple-negative breast cancer by suppressing BRSK2-mediated phosphorylation of PIMREG

Platinum-based neoadjuvant therapy represented by cisplatin is widely employed in treating Triple-Negative Breast Cancer (TNBC), a particularly aggressive subtype of breast cancer. Nevertheless, the emergence of cisplatin resistance presents a formidable challenge to clinical chemotherapy efficacy. Herein, we revealed the critical role of tumor microenvironment (TME) derived exosomal miR-3960 and phosphorylation at the S16 site of PIMREG in activating NF-κB signaling pathway and promoting cisplatin resistance of TNBC. Detailed regulatory mechanisms revealed that SOD1-upregulated fibroblasts secrete miR-3960 and are then transported into TNBC cells via exosomes. Within TNBC cells, miR-3960 targets and inhibits the expression of BRSK2, an AMPK protein kinase family member. Furthermore, we emphasized that BRSK2 contributes to ubiquitination degradation of PIMREG and modulates subsequent activation of the NF-κB signaling pathway by mediating PIMREG phosphorylation at the S16 site, ultimately affects the cisplatin resistance of TNBC. In conclusion, our research demonstrated the crucial role of SOD1high fibroblast, exosomal miR-3960 and S16 site phosphorylated PIMREG in regulating the NF-κB signaling pathway and cisplatin resistance of TNBC. These findings provided significant potential as biomarkers for accurately diagnosing cisplatin-resistant TNBC patients and guiding chemotherapy strategy selection.

Oncogenic miR-106b-5p promotes cisplatin resistance in triple-negative breast cancer by targeting GDF11.

Cytoplatin (CDDP) is a standard treatment for triple-negative breast cancer (TNB), but patient resistance to CDDP limits its efficacy. A growing study confirms that microRNAs (miRNAs) are significantly important in breast cancer, especially TNBC. This research was carried out to examine the function of miR-106b-5p in CDDP resistance of TNBC as well as the downstream mechanism. The miR-106b-5p and growth-differentiation factor 11 (GDF11) expressions in the tissues from TNBC patients and CDDP-treated TNBC cell lines were measured by RT-qPCR. Thereafter, cell proliferation and migration in the presence of CDDP treatment were evaluated via CCK-8 and Transwell assays in the TNBC cells. A xenograft mice model was also established to verify the miR-106b-5p silencing effect on the growth of CDDP resistance TNBC cells in vivo. Luciferase reporter experiments were performed to predict the relationship between miR-106b-5p and GDF11 expression. The results showed that miR-106b-5p was upregulated in the TNBC tumor cells and TNBC cells treated with CDDP and knockdown of this caused inhibition of the TNBC cell lines' proliferation, migration and suppressed the growth of the TNBC xenografted tumors, in the presence of CDDP treatment. In addition, it was observed that miR-106b-5p can bind to GDF11; as a result in the TNBC tissues and CDDP-treated TNBC cell lines the down-regulation of GDF11 was observed. Moreover, GDF11 silencing promoted CDDP-treated TNBC cell lines' proliferation and migration and reversed the interference effect of miR-106b-5p. MiR-106b-5p was upregulated in TNBC and this upregulation may promote CDDP resistance of the TNBC cells by targeting GDF11 and inhibiting its expression.

CircRNA (circ)_0007823 Contributes to Triple-Negative Breast Cancer Progression and Cisplatin Resistance via the miR-182-5p/FOXO1 Pathway.

Cisplatin (DDP) is used for the clinical management of triple-negative breast cancer (TNBC). However, the development of drug resistance limits its therapeutic efficacy. Circular RNAs (circRNAs) are known to be involved in tumor DDP resistance. In our previous study, we reported that circ_0007823 expression is downregulated and correlated with adverse prognosis in TNBC. However, its association with DDP resistance remains unclear. This study aimed to determine the role of circ_0007823 and miR-182-5p in DDP-resistant TNBC and explore the underlying mechanisms. First, expression profiles circ_0007823, microRNA (miR)-182-5p, and forkhead box O1 (FOXO1) in TNBC cells were determined. Additionally, biological characteristics of cells, including apoptosis, cell cycle, proliferation, and migration, were analyzed using various assays. Luciferase reporter and rescue assays were used to determine the correlations among circ_0007823, miR-182-5p, and FOXO1 expression. MiR-182-5p was overexpressed in DDP-resistant TNBC cells. MiR-182-5p knockdown suppressed the invasiveness and increased the apoptosis of drug-resistant cells, contributing to G1 arrest and S phase reduction. Mechanistically, circ_0007823 targeted miR-182-5p, and its overexpression drastically reduced the promotional effects of the miR-182-5p mimic on the aggression and transfer ability of drug-resistant cells. Furthermore, FOXO1 overexpression increased the sensitivity of cells to DDP and reduced their malignant progression. Therefore, FOXO1 was established as the downstream target of miR-182-5p that may be used to treat DDP-resistant TNBC. In summary, circ_0007823 overexpression attenuated DDP resistance in TNBC via the miR-182-5p-FOXO1 axis, indicating the therapeutic potential of circ_0007823 DDP-resistant TNBC treatment.

Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells.

Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, the SPAG5 knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines. The knockout of SPAG5 was confirmed on both gene and protein levels using genomic PCR, DNA sequencing and western blotting. The functional loss of SPAG5 was determined by colony-formation assay. SPAG5-regulated doxorubicin- and docetaxel-resistance was assessed by MTT and apoptosis assays. The results indicated that all the SPAG5 knockout MDA-MB-231 and BT549 clones were biallelic, where one allele was replaced by the donor template, and the other allele had the same "T" insertion (indel) adjacent to the cutting sites of gRNAs at the exon 1 boundary, irrespective of the gRNAs and cell lines. The locus of indel interrupted the SPAG5 transcription by damaging the GT-AG mRNA processing rule. Deletion of SPAG5 decreased clonogenicity in both MDA-MB-231 and BT549 cells. SPAG5 was able to regulate the resistance and the drug-induced apoptosis of both doxorubicin and docetaxel. In conclusion, recombinant plasmid-based CRISPR-Cas9 technology can be used to delete the SPAG5 gene in the TNBC cell lines. SPAG5 has an important role in regulating cell proliferation and doxorubicin- and docetaxel-resistance in MDA-MB-231 and BT549 cells.

Abstract 179: Identification of a novel crosstalk between MDSC and NKregs in immunotherapy resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer and lacks standard treatment options. Although immunotherapy shows some response in these patients along with chemotherapy, heterogeneity of tumor cells and immune cells results in significant low response to immunotherapy treatment. Based on single cell sequencing which enables identity of heterogenous population at single cell level and functional studies involving preclinical mouse models, our laboratory recently identified distinct CD56brightSOCS3highCD11b−CD27− immature NK cells subpopulations, with diminished cytotoxic granzyme signatures that promote TNBC tumor progression. However, the precise characterization and functionality of these rogue tumor promoting NK cells remains unclear in clinical samples. Here, we aimed to illustrate mechanistic insight of these novel immature NK subpopulation in context of immunotherapy resistant TNBC patient samples. The scRNA seq analysis of TNBC patient samples identified four specific subclusters of NK cells in TNBC. We further found that NK2 and NK3 subclusters are CD56dim Granzymehigh cytotoxic NK cells and the other two subclusters (NK0 and NK1) are CD56bright immature NKs. Interestingly, the NK0 and NK1 subcluster specifically exhibit distinctive characteristics and functions. Notably, CD56bright immature NK1 cells, identified as tissue-resident NK cells, were engaged in direct communication with myeloid-derived suppressor cells (MDSCs). The CD56bright NK0 possesses moderate to low SOCS3 expression and moderate to high granzyme expression with elevated early NK activation markers and inhibitory receptor expressions, suggesting its regulatory property (NKregs). scRNA seq and flow cytometry analysis showed higher NKregs and immunosuppressive MDSC population, specifically with high monocytic-MDSCs (mMDSCs) in non-responder TNBC (to adjuvant combinational treatment of chemo and immunotherapy) patient samples rather than responder patients’ tissue. Based on in silico analysis, we further found a potential crosstalk between these mMDSC & NKreg within the tumors of non-responsiveness of TNBC patients. Overall, our studies show a connection of MDSCs and NKregs in TNBC patients, who do not respond to immunotherapy, highlighting a possible mechanism for immunotherapy resistance, which could be targeted in near future for better treatment options in TNBC. Citation Format: Megha Das, Samantha Henry, Shailesh Singh, Camila Dos Santos, Rumela Chakrabarti. Identification of a novel crosstalk between MDSC and NKregs in immunotherapy resistant triple negative 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 179.

Abstract 7205: PLK1 inhibition overcomes PARP inhibitor resistance in triple-negative breast cancer

Abstract Patients with aggressive breast cancer (BC) subtypes usually do not have favorable prognosis despite improvements in treatment modalities. These cancers remain a major cause of morbidity and mortality in women globally. This has fostered a major effort to discover actionable molecular targets to treat these patients. Polo-like Kinase (PLK1) is one of these molecular targets that are under comprehensive investigation for the treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not been explored. Therefore, we examined the expression of PLK1 protein in a large cohort of more than 1500 Middle Eastern ethnicity BC by immunohistochemistry. Correlation with clinico-pathological parameters and the prognosis was performed. PLK1 overexpression was observed in 27.4% of all BC and was significantly associated with aggressive clinico-pathological markers. The antitumor effect of PLK1 and PARP inhibitors either alone or in combination was tested in two BRCA mutated, and one BRCA proficient TNBC cell lines. We showed that combined inhibition significantly reduced cell survival and induced apoptosis in TNBC cell lines. Furthermore, our data suggest that PLK1 inhibition overcomes PARP inhibitor resistance in TNBC cell lines. These findings would pave the way to explore the future use of PLK1 inhibitors in the clinical setting of PARPi-resistant patients. Citation Format: Abdul K. Siraj, Pratheesh Kumar Poyil, Divya Padmaja, Sandeep Kumar Parvathareddy, Khadija Alobaisi, Rafia Begum, Osama Almalik, Fouad Al-Dayel, Khawla S. Al-Kuraya. PLK1 inhibition overcomes PARP inhibitor resistance in triple-negative 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 7205.