Chemotherapy-resistant Triple-negative Breast Cancer Research Articles

EMP2 Serves as a Functional Biomarker for Chemotherapy-Resistant Triple-Negative Breast Cancer.

Breast cancer (BC) remains among the most commonly diagnosed cancers in women worldwide. Triple-negative BC (TNBC) is a subset of BC characterized by aggressive behavior, a high risk of distant recurrence, and poor overall survival rates. Chemotherapy is the backbone for treatment in patients with TNBC, but outcomes remain poor compared to other BC subtypes, in part due to the lack of recognized functional targets. In this study, the expression of the tetraspan protein epithelial membrane protein 2 (EMP2) was explored as a predictor of TNBC response to standard chemotherapy. We demonstrate that EMP2 functions as a prognostic biomarker for patients treated with taxane-based chemotherapy, with high expression at both transcriptomic and protein levels following treatment correlating with poor overall survival. Moreover, we show that targeting EMP2 in combination with docetaxel reduces tumor load in syngeneic and xenograft models of TNBC. These results provide support for the prognostic and therapeutic potential of this tetraspan protein, suggesting that anti-EMP2 therapy may be beneficial for the treatment of select chemotherapy-resistant TNBC tumors.

ΔNp63 regulates MDSC survival and metabolism in triple-negative breast cancer

Triple-negative breast cancer (TNBC) contributes greatly to mortality of breast cancer, demanding new targetable options. We have shown that TNBC patients have high ΔNp63 expression in tumors. However, the function of ΔNp63 in established TNBC is yet to be explored. In current studies, targeting ΔNp63 with inducible CRISPR knockout and Histone deacetylase inhibitor Quisinostat showed that ΔNp63 is important for tumor progression and metastasis in established tumors by promoting myeloid-derived suppressor cell (MDSC) survival through tumor necrosis factor alpha. Decreasing ΔNp63 levels are associated with decreased CD4+ and FOXP3+ T-cells but increased CD8+ T-cells. RNA sequencing analysis indicates that loss of ΔNp63 alters multiple MDSC properties such as lipid metabolism, chemotaxis, migration, and neutrophil degranulation besides survival. We further demonstrated that targeting ΔNp63 sensitizes chemotherapy. Overall, we showed that ΔNp63 reprograms the MDSC-mediated immunosuppressive functions in TNBC, highlighting the benefit of targeting ΔNp63 in chemotherapy-resistant TNBC.

Diverging prognostic effects of CD155 and CD73 expressions in locally advanced triple-negative breast cancer.

Immune checkpoint inhibition, combined with novel biomarkers, may provide alternative pathways for treating chemotherapy-resistant triple-negative breast cancer (TNBC). This study investigates the expression of new immune checkpoint receptors, including CD155 and CD73, which play a role in T and natural killer (NK) cell activities, in patients with residual TNBC after neoadjuvant chemotherapy (NAC). The expression of biomarkers was immunohistochemically examined by staining archival tissue from surgical specimens (n = 53) using specific monoclonal antibodies for PD-L1, CD155, and CD73. Of those, 59.2% (29/49) were found to be positive (>1%) for PD-L1 on the tumour and tumour-infiltrating lymphocytes (TILs), while CD155 (30/53, 56.6%) and CD73 (24/53, 45.3%) were detected on tumours. Tumour expressions of CD155 and CD73 significantly correlated with PD-L1 expression on the tumour (p = 0.004 for CD155, p = 0.001 for CD73). Patients with CD155 positivity ≥10% were more likely to have a poor chemotherapy response, as evidenced by higher MDACC Residual Cancer Burden Index scores and Class II/III than those without CD155 expression (100% vs 82.6%, p = 0.03). At a median follow-up time of 80 months (range, 24-239), patients with high CD73 expression showed improved 10-year disease-free survival (DFS) and disease-specific survival (DSS) rates compared to those with low CD73 expression. In contrast, patients with CD155 (≥10%) expression exhibited a decreasing trend in 10-year DFS and DSS compared to cases with lower expression, although statistical significance was not reached. However, patients with coexpression of CD155 (≥10%) and low CD73 were significantly more likely to have decreased 10-year DFS and DSS rates compared to others (p = 0.005). These results demonstrate high expression of CD73 and CD155 in patients with residual tumours following NAC. CD155 expression was associated with a poor response to NAC and poor prognosis in this chemotherapy-resistant TNBC cohort, supporting the use of additional immune checkpoint receptor inhibitor therapy. Interestingly, the interaction between CD155 and CD73 at lower levels resulted in a worse outcome than either marker alone, which calls for further investigation in future studies.

Abstract 2774: Moesin-induced adriamycin resistance through IL-6/MSN/STAT3 axis and therapeutic efficacy of atovaquone in chemotherapy resistant triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high frequency of chemotherapy resistance. Moesin (MSN) is a cytoskeleton linker discovered as a novel oncogene in our previous proteomics analysis. Here, we evaluated the molecular mechanisms of MSN and how it functions in response to chemotherapy, which remains unclear in TNBC. Method: The 178 in-house TNBC patient cohort and METABRIC data set were enrolled for clinicopathologic analysis. The multiple TNBC four-cell lines were used to establish MSN knock-down and over-expressed in vitro models using a lentiviral system. RNA sequencing was analyzed with MDA-MB-231 parent cell and MDA-MB-231/ADR12 (adriamycin resistance cell line). Invasion and migration assay was done. The drug response based on MSN gene expression was observed by cell viability assay, western blot, and AV/PI flow cytometry. Drug resistance concerning cancer stemness was confirmed with sphere formation and ALDH activity assay. Immunofluorescence and immunoprecipitation were performed. The combination index(CI), which is to define the synergism of combined treatment was calculated with the Compusyn program. Results: The cohort with METABRIC and our in-house clinical data showed concordant findings that overexpressed MSN mRNA and protein expressions have a poor prognosis in TNBC patients. The MSN protein expression in TNBC was increased compared to the protein level in both the normal human tissues and non-TNBC cell lines. Overexpressed MSN induced high cell proliferation activity, invasion, and migration ability. The apoptosis rate (AV+/PI+) along with cleaved PARP and caspase-3 expression was increased in TNBC cell lines with MSN downregulation. Further tests to determine the role of MSN in adriamycin resistance showed an increased number and size of spheres in the MSN overexpressed cell lines. Moreover, the protein and mRNA levels of MSN expression in MDA-MB-231/ADR12 were significantly higher than the level in the matched parental cell line. MSN and phospho-STAT3(T705) were increased by treatment of IL-6 in TNBC cell lines. The direct binding between MSN and STAT3, and the co-localization of MSN and pSTAT3 were consistently confirmed by co-immunoprecipitation and immunofluorescence, which were all activated by IL-6. Blocking the STAT3 pathway with atovaquone restored ADR response in MSN overexpressed tumor cells. CI value on the combined treatment of ADR and atovaquone in the MDA-MB-231/ADR12 cell line showed higher synergetic efficacy than the parental cell line. Conclusion: This research demonstrates the involvement of the MSN in doxorubicin resistance through IL-6/MSN/STAT3 pathway and provides a novel therapeutic candidate for TNBC patients. Citation Format: Cheng Hyun Lee, Da Sol Kim, Soo Young Park, Han Suk Ryu. Moesin-induced adriamycin resistance through IL-6/MSN/STAT3 axis and therapeutic efficacy of atovaquone in chemotherapy resistant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2774.

Identification of Src Family Kinases as Potential Therapeutic Targets for Chemotherapy-Resistant Triple Negative Breast Cancer.

Simple SummaryBreast cancer remains the leading cause of cancer-related deaths in women worldwide. One particular subtype, triple negative breast cancer (TNBC), is known for being highly aggressive and metastatic, with a high prevalence of resistance to chemotherapy. Patients with chemotherapy-resistant TNBC tumors have virtually no treatment options. Here, we studied tyrosine phosphorylation-mediated signaling networks in chemotherapy-resistant TNBC xenografts, to identify potential therapeutic targets. We identified Src family kinases (SFK) as potential drug targets. Inhibition of SFKs radically reduced tumor growth in xenografts that had a signature of high SFK-activity. We assessed a small number of human tumor tissue specimens and found a low prevalence of SFK-driven tumors. These data may explain the previous unsuccessful clinical trials targeting SFKs in TNBC and warrant further studies with a higher number of patient samples. These results emphasize the importance of the characterization of phosphotyrosine signaling profiles for better patient stratification, in addition to gaining novel therapeutic insights.Neoadjuvant chemotherapy (NAC) remains the cornerstone of the treatment for triple negative breast cancer (TNBC), with the goal of complete eradication of disease. However, for patients with residual disease after NAC, recurrence and mortality rates are high and the identification of novel therapeutic targets is urgently needed. We quantified tyrosine phosphorylation (pTyr)-mediated signaling networks in chemotherapy sensitive (CS) and resistant (CR) TNBC patient-derived xenografts (PDX), to gain novel therapeutic insights. The antitumor activity of SFK inhibition was examined in vivo. Treated tumors were further subjected to phosphoproteomic and RNAseq analysis, to identify the mechanism of actions of the drug. We identified Src Family Kinases (SFKs) as potential therapeutic targets in CR TNBC PDXs. Treatment with dasatinib, an FDA approved SFK inhibitor, led to inhibition of tumor growth in vivo. Further analysis of post-treatment PDXs revealed multiple mechanisms of actions of the drug, confirming the multi-target inhibition of dasatinib. Analysis of pTyr in tumor specimens suggested a low prevalence of SFK-driven tumors, which may provide insight into prior clinical trial results demonstrating a lack of dasatinib antitumor activity in unselected breast cancer patients. Taken together, these results underscore the importance of pTyr characterization of tumors, in identifying new targets, as well as stratifying patients based on their activated signaling networks for therapeutic options. Our data provide a strong rationale for studying SFK inhibitors in biomarker-selected SFK-driven TNBC.

Oxidative Phosphorylation Is a Metabolic Vulnerability in Chemotherapy-Resistant Triple-Negative Breast Cancer.

Oxidative phosphorylation (OXPHOS) is an active metabolic pathway in many cancers. RNA from pretreatment biopsies from patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy demonstrated that the top canonical pathway associated with worse outcome was higher expression of OXPHOS signature. IACS-10759, a novel inhibitor of OXPHOS, stabilized growth in multiple TNBC patient-derived xenografts (PDX). On gene expression profiling, all of the sensitive models displayed a basal-like 1 TNBC subtype. Expression of mitochondrial genes was significantly higher in sensitive PDXs. An in vivo functional genomics screen to identify synthetic lethal targets in tumors treated with IACS-10759 found several potential targets, including CDK4. We validated the antitumor efficacy of the combination of palbociclib, a CDK4/6 inhibitor, and IACS-10759 in vitro and in vivo. In addition, the combination of IACS-10759 and multikinase inhibitor cabozantinib had improved antitumor efficacy. Taken together, our data suggest that OXPHOS is a metabolic vulnerability in TNBC that may be leveraged with novel therapeutics in combination regimens. SIGNIFICANCE: These findings suggest that triple-negative breast cancer is highly reliant on OXPHOS and that inhibiting OXPHOS may be a novel approach to enhance efficacy of several targeted therapies.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

Development of targeted therapy therapeutics to sensitize triple-negative breast cancer chemosensitivity utilizing bacteriophage phi29 derived packaging RNA

BackgroundTo date, triple-negative breast cancer (TNBC) treatment options are limited because of the loss of target receptors and, as a result, are only managed with chemotherapy. What is worse is that TNBC is frequently developing resistance to chemotherapy. By using small interfering RNA (siRNA)-based therapeutics, our recent work demonstrated X-box-binding protein 1 (XBP1) was linked to human epidermal growth factor receptor 2 positive (HER2+) breast cancer development and chemoresistance. Given the instability, off-target effects, net negative charge, and hydrophobicity of siRNA in vivo utilization and clinical transformation, its use in treatment is hampered. Thus, the development of a siRNA-based drug delivery system (DDS) with ultra-stability and specificity is necessary to address the predicament of siRNA delivery.ResultsHere, we assembled RNase resistant RNA nanoparticles (NPs) based on the 3WJ structure from Phi29 DNA packaging motor. To improved targeted therapy and sensitize TNBC to chemotherapy, the RNA NPs were equipped with an epidermal growth factor receptor (EGFR) targeting aptamer and XBP1 siRNA. We found our RNA NPs could deplete XBP1 expression and suppress tumor growth after intravenous administration. Meanwhile, RNA NPs treatment could promote sensitization to chemotherapy and impede angiogenesis in vivo.ConclusionsThe results further demonstrate that our RNA NPs could serve as an effective and promising platform not only for siRNA delivery but also for chemotherapy-resistant TNBC therapy.

Discovery of tumoricidal DNA oligonucleotides by response-directed in\xa0vitro evolution

Drug discovery is challenged by ineffectiveness of drugs against variable and evolving diseases, and adverse effects due to poor selectivity. We describe a robust platform which potentially addresses these limitations. The platform enables rapid discovery of DNA oligonucleotides evolved in vitro for exerting specific and selective biological responses in target cells. The process operates without a priori target knowledge (mutations, biomarkers, etc). We report the discovery of oligonucleotides with direct, selective cytotoxicity towards cell lines, as well as patient-derived solid and hematological tumors. A specific oligonucleotide termed E8, induced selective apoptosis in triple-negative breast cancer (TNBC) cells. Polyethylene glycol-modified E8 exhibited favorable biodistribution in animals, persisting in tumors up to 48-hours after injection. E8 inhibited tumors by 50% within 10 days of treatment in patient-derived xenograft mice, and was effective in ex vivo organ cultures from chemotherapy-resistant TNBC patients. These findings highlight a drug discovery model which is target-tailored and on-demand.

Abstract P5-03-10: Circulating exosomal microRNA profiling to depict mechanisms of chemotherapy resistance among triple negative breast cancer

Abstract Background: Chemotherapeutic resistance leads to high mortality among triple negative breast cancer (TNBC) and underlying mechanisms are poorly understood. Exosomes have been new member of liquid biophsy. MicroRNAs (miRNAs), as the most important inclusions in exosomes, making them ideal candidate biomarkers and therapeutical targets. Methods: We isolated exosomes and analyzed exosome-carried miRNAs signatures in several TNBC cells lines sensitive or resistant to adariamycin, docetaxel or cisplatin. The resistance transfer capacity was determined by flow cytometry after sensitive cells incubated 48 hours with exosomes from drug resistant cells. Locked nucleic acid probes and enzyme-labeled fluorescence (LNA-ELF-FISH) was performed to detect exosomal miRNA molecules transfer. Animal mode was constructed to evaluate treatment feasibility using miRNA modified exosomes. Serum exosomes from 40 TNBC stage IV patients underwent chemotherapy before or after progress disease (PD) status were isolated to analyze miRNA profiling for potential biomarkers identification. Results: We successfully isolated and identified exosomes from several drug sensitive and resistant TNBC cell lines and patients. Exosomal miR-222, miR-4443, miR-100, miR-17, miR-210 were found significantly upregulated from chemotherapy resistance cells. Incubation of exosomes from the resistant cells with the sensitive cells resulted in increasing resistant capacity among sensitive cells. Exosomal miRNA molecules transfer were detected using LNA-ELF-FISH. Transfection of synthesized miRNAs competitors into exosomes increased drug sensitivity in vivo. Exosomal miR-222, miR-4443, miR-100, miR-17, miR-210 were also found upregulated significantly from serums of patients after PD status. These five miRNAs were able to differentiate patients with PD status from those with CR or PR status with at least 89% accuracy. Conclusion: Exosomes from chemotherapy resistant TNBC cells could transfer drug resistance to sensitive cells via exosomal miRNAs. A circulating exosomal microRNA profiling was estabilished for potential biomarkers and therapeutical targets identification. Citation Format: He Y, Tang J. Circulating exosomal microRNA profiling to depict mechanisms of chemotherapy resistance among triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-10.

Abstract 840: ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC)

Abstract Purpose: TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. Patients with residual TNBC after neoadjuvant chemotherapy (NAC) have a high risk of locoregional recurrence despite aggressive subsequent local therapies, including RT. Thus, new radiosensitizers that address chemoresistant TNBC (i.e residual disease after NAC) are needed. ATR plays a key role in the cellular response to replication stress and DNA damage. TNBC is characterized by G1 checkpoint loss, homologous recombination deficiency (HRD), and other features which increase dependence on ATR pathway signaling. We hypothesized that VX-970, a selective inhibitor of ATR, would effectively radiosensitize TNBC. Methods: Cell lines representing varying subtypes of TNBC (MDA-MB-231, HCC1806, and BT-549) and the normal breast epithelial cell line MCF10a were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, Mayo Clinic (MC)TNBC1 and MCTNBC2, patient derived xenograft models (PDXs) generated prospectively from baseline and chemoresistant residual disease surgical specimens after NAC, respectively, of unique patients in the Breast Cancer Genome Guided Therapy Study were analyzed. Tumors were injected into hind-legs of athymic nude mice and animals were randomized to: 1) Vehicle 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX-970 was administered daily 1 hour prior to RT at a dose of 60 mg/kg. Exome sequencing was assessed for germ-line and/or somatic alterations in HR genes and an ex-vivo RAD51 foci formation assay was applied to confirm the functional status of HR in each PDX model. Results: In vitro, VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the RT-induced G2/M checkpoint, delayed resolution of γH2AX and 53BP1 foci and reduced colony formation after RT in MDA-MB-231, HCC1806, and BT-549 relative to MCF10a. In vivo, VX-970 did not exhibit single agent activity at the dose administered but markedly sensitized both MCTNBC1 and MCTNBC2 to RT, suggesting that VX-970 could overcome chemoresistant TNBC biology. For MCTNBC1 and MCTNBC2, the median time to tumor tripling was 26 vs 49 days (p=0.002) and 25 vs 43 days (p=0.006) for the RT and RT + VX-970 groups, respectively. Ex-vivo RAD51 foci formation was robust in MCTNBC1, a PDX without HR gene alterations. In contrast, there was no RT-induced RAD51 foci formation in MCTNBC2, suggesting a BRCA1 mutation in the corresponding human tumor resulted in loss of HR function in that model. Conclusion: VX-970 + RT effectively radiosensitized TNBC PDXs established from both pre-treatment biopsy specimens and chemoresistant residual tumor samples and in both HR deficient and HR proficient settings. ATR inhibition should be investigated in the clinic in combination with RT for patients with residual TNBC after NAC. Citation Format: Robert W. Mutter, Mohamed M. Kahila, Xinyi Tu, Qin Zhou, Jia Yu, Krishna R. Kalari, Liewei Wang, Judy C. Boughey, Matthew P. Goetz, Jann N. Sarkaria, Zhenkun Lou. ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 840.

Abstract 144: Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells

Abstract Many patients with triple negative breast cancer (TNBC) exhibit poor prognosis and are at high risk of tumor relapse and metastasis due to the resistance to conventional chemotherapy. These tumor recurrences have been attributed to the presence of breast cancer stem cells (BCSCs), a subpopulation of breast cancer cells that possesses stem-like properties and displays resistance to chemotherapy. Therefore, targeting BCSCs is a priority to overcoming chemotherapy failure in TNBCs. We generated chemotherapy-resistant TNBC cells through cyclic treatments with paclitaxel (pac). The pac-resistant cells displayed increased self-renewal potential compared to the parental TNBC cells, higher percentage BCSCs and increased phosphorylation of Src kinase at Tyr416. After performing a kinase library screening on BCSCs in chemotherapy-resistant cells, we identified dasatinib, a Src kinase family inhibitor, potently reduced BCSC self-renewal and two BCSC populations (ALDH+ and CD24lowCD44high) as well as phosphor-Src. We also demonstrated dasatinib to block pac-induced BCSC enrichment and Src activation in the parental TNBC cells. Interestingly, we found that dasatinib induced an epithelial differentiation of pac-resistant cells, resulting in their enhanced sensitivity to paclitaxel. The combination treatment of dasatinib and paclitaxel not only decreased the proportion of BCSCs and their self-renewal capacity but also synergistically reduced cell viability of pac-resistant cells. In vivo studies further demonstrated the effectiveness of dasatinib/paclitaxel combination treatment in inhibiting breast tumor growth. All these data suggest that dasatinib is a promising anti-BCSC agent and can be used in combination with paclitaxel to overcome chemotherapy resistance in TNBCs. Citation Format: Jun Tian, Chieh Lo, Fatmah Al Raffa, Meiou Dai, Jean-Jacques Lebrun. Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 144.

Abstract 956: Systematic identification of the actionable kinase dependencies of chemotherapy-resistant triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for approximately 15% of all breast cancer cases, with over 35,000 newly diagnosed women per year in the USA. TNBC patients are at high risk of recurrence, and neoadjuvant standard chemotherapy leads to pathologic complete response in only about 30% of patients. No targeted therapy has yet been conclusively established to improve outcome. The management of TNBC will significantly improve once mechanisms responsible for TNBC resistance to chemotherapies are identified. Here, we applied a new functional proteomic strategy to reveal which (dys)regulated phospho-signaling circuits are the effective dependencies of chemotherapy-resistant TNBC cells. The high-throughput kinase activity-mapping (HT-KAM) assay is our new screening technology to assess the catalytic activity of many kinases in parallel. HT-KAM relies on collections of biologic peptide probes that are computationally derived from PhosphoAtlas (Olow and Chen et al., Cancer Research 2016) and are physically used as combinatorial sensors to measure the activity of kinase enzymes in biologic extracts. The HT-KAM system provides access to a vast, untapped resource of meaningful measurements, whether readouts are interpreted irrespective of which enzymes phosphorylate which probes, or analyzed to convert global phospho-signatures into functional profiles of kinase activities. Kinome maps reveal how signaling networks are rewired by drug interventions in the context of different cellular backgrounds or exogenously mutated proteins/pathways, and provide insight into potentially targetable pathways. We previously successfully showed that the HT-KAM platform identifies new, actionable kinases responsible for intrinsic or acquired targeted-therapy resistance in BRAFV600E colorectal cancer cells and melanoma tumors from patients. To study TNBC, we used a 615-peptide sensor library that supports the mapping of >100 kinases and represents >900 functional kinase-substrate interactions relevant to tumor biology, and matches hundreds of druggable components of signaling circuits. We characterized the phospho-catalytic signatures of 10 TNBC cell lines, either untreated or treated with chemotherapeutic drugs (5-FU, carboplatin, doxorubicin) at IC50 concentrations. We also included a panel of 8 luminal BC cells. Based on our advanced preliminary data, we anticipate showing a comprehensive map of the oncogenic kinome of TNBC, and how it rewires its signaling network in response to chemotherapies. The most hyperactive and conserved pathways establish a priority map of strategic kinase hot spots to explore as therapeutic candidates. We find that chemotherapy-induced kinases such as SRC, AKT, CHEK, or regulating NFkB or PARP pathways can be inhibited to improve therapeutic sensitivity in TNBC. Citation Format: Jean-Philippe F. Coppe, Pan Bo, Carolien van der Borden, Nina Koemans, Changjun Wang, Denise Wolf, Christina Yau, Sietske Bakker, Marij Hartog, Miki Mori, Ana Ruiz-Saenz, Zhongzhong Chen, Aleksandra Olow, Pei Rong Evelyn Lee, Laura van 't Veer. Systematic identification of the actionable kinase dependencies of chemotherapy-resistant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 956.

Abstract P2-10-05: The impact of TP53 mutation and pathological response to neoadjuvant chemotherapy on triple negative breast cancer outcomes

Abstract Background: Triple Negative Breast Cancer (TNBC) accounts for 15-20% of breast cancers, and has a poor prognosis compared with hormone receptor-positive or HER2-amplified disease. There is currently no targeted therapy to salvage chemotherapy resistant TNBC confirming a need for predictive biomarkers of sensitivity to therapy. This study aimed to evaluate the impact of TP53 mutations, common in TNBC, on sensitivity to therapy and patient outcomes. Methods: TNBC samples (pretreatment biopsies and surgical specimens, n=106) had annotated response to standardized anthracycline/taxane based neoadjuvant chemotherapy (NACT) and 3-year survival data, by which time most recurrences occur in TNBC. RNAseq was performed using Illumina HiSeq with mRNA library preparation protocol to identify TP53 mutations, which were then scored using the Evolutionary Action Score (EAS), a novel computational model shown to be successful at identifying deleterious mutations. This work was supported by an ASCO Conquer Cancer Foundation Young Investigator Award grant. Results: 106 TNBC patients were enrolled on this study, 14 had stage I, 65 stage II, and 24 had stage III disease. 64 patient samples have been analyzed to date, of which 39 (61%) harbored a TP53 mutation, and 24 (38%) were TP53 wildtype. Pathologic complete response (pCR) and survival Of 47 patients who received NACT, 14 (30%) achieved a pCR, and 33 (70%) had residual disease (RD). Patients who achieved a pCR had a significantly better 3-year recurrence free survival (RFS) (100% vs 59%, p=0.011) and overall survival (OS) (100% vs 62%, p-0.01) compared to patients who had residual disease. TP53 status and pCR The rate of pCR was significantly higher in the TP53 mutant group (11/29, 38%) compared to TP53 wildtype (2/17, 12%) TNBC (OR = 4.6, (0.9, 24), p = 0.046). Patients with wild-type and mutant TP53 TNBC who achieved a pCR had excellent 3-year RFS and OS at 100%. Among patients with RD, TP53 wild-type TNBC exhibited a non-significant trend towards higher 3-year RFS (72% vs 49%,HR = 2.5 (0.8, 8.0) p = 0.12) and OS (79% vs 48%,HR = 2.6 (0.8, 8.4) p = 0.11) compared to TP53 mutant TNBC. TP53 status and survival The 3-year OS for TP53 mutant TNBC was 72%, compared to 86% for TP53 wildtype TNBC (HR = 1.5 (0.5, 4.3), p = 0.48). There was no difference in 3-year RFS between the two groups. TP53 EAS and survival Among 39 non-silent TP53 mutant cases, EAS ranged from 42.3 to 100 with a median score of 87.6. The 3-year OS for patients with EAS ≥86.7 was 73%, compared to 74% for patients with EAS <86.7 (HR 0.8 (0.2, 3.0), p=0.78). When separated into tertiles, the 3-year OS rates were 75%, 68%, and 77% for the lower, middle, and upper thirds respectively. The 3-year OS rates for TNBC with EAS = 0 (TP53 wildtype or silent TP53 mutation) was 87%. Conclusion: TP53 mutations were associated with a higher chance of achieving a pCR; NACT Patients who achieved a pCR to NACT had excellent outcomes, regardless of TP53 mutation status. In patients with RD following NACT, TP53 mutant TNBC had a trend towards worse outcomes compared to TP53 wild-type TNBC. Larger cohorts are required to further evaluate the impact of TP53 mutations and TP53 EAS on survival outcomes in TNBC. Citation Format: Mitri Z, Lin C-H, Hess KR, Thompson A, Lichtarge O, Hubbard R, Fu C, Symmans WF, Moulder-Thompson SL. The impact of TP53 mutation and pathological response to neoadjuvant chemotherapy on triple negative breast cancer outcomes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-05.

Preliminary results from a single-arm, phase II study assessing the efficacy of pembrolizumab plus radiotherapy in metastatic triple negative breast cancer.

95 Background: Two trials have demonstrated overall response rates of 19% with immune checkpoint inhibitors alone in chemotherapy-resistant triple negative breast cancer (TNBC). Radiation therapy (RT) is frequently used to enhance local control in TNBC and has been reported to induce distant (abscopal) tumor responses when combined with immunotherapy. We evaluate the safety and efficacy of the combination of RT and pembrolizumab, a programmed cell death protein 1 (PD-1) inhibitor, in a single-arm, two-stage, phase II study in metastatic TNBC. Results from the first stage are reported. Methods: Eligibility criteria includes women with biopsy-proven TNBC, ECOG performance status 0-2, ≥ 2 measurable sites of metastatic disease with at least one site requiring RT. A total RT dose of 3000 cGy is delivered in one week in 5 fractions. Pembrolizumab is given intravenously at 200 mg/kg D1+/- 3 days and then every 3 weeks until disease progression as defined by RECIST v 1.1. The primary endpoint is overall response rate at week 13 in the non-targeted, non-irradiated lesions. Secondary endpoints include safety and overall survival. Tumor biopsies are obtained at baseline and at week 7. PD-L1 expression was not required for study entry. Results: Nine patients were enrolled in the first phase and assessed for antitumor activity and safety. Median age was 52 years (range 37-73). Three patients died secondary to disease-related complications (at weeks 2, 6 and 8) and 1 came off study due to disease progression prior to week 13. Of the 5 patients evaluable at week 13, 2 had a partial response, 1 had stable disease and 2 had disease progression. Common toxicities were mild and included fatigue, myalgia and nausea. One grade 3 event was reported - hyperbilirubinemia not attributable to study therapy after one dose of pembrolizumab. Conclusions: The combination of pembrolizumab and RT was well-tolerated in the first stage of a phase II study. Responses were observed outside of the RT fields in 2 of 5 patients who were evaluable at 13 weeks. The contribution of RT to pembrolizumab is unknown and will be investigated in the second stage of the study, which is ongoing. Correlative studies are planned. Clinical trial information: NCT02730130.

Abstract 738: Correlation between tumor infiltrating lymphocytes and CCL5 gene expression in chemotherapy-resistant triple negative breast cancer

Abstract Background: The presence of tumor infiltrating lymphocytes (TILs) have been associated with a better outcome in triple negative breast cancer (TNBC). In the other hand, we have found that CCL5, a chemokine involved in the recruitment of immune cells to the tumor microenvironment is an independent prognostic marker for distant-recurrence free survival (DFRS) in TNBC. The aim of this work was to evaluate the relationship between these two immunological prognostic markers in chemotherapy-resistant TNBC. Material and Methods: We evaluated a retrospective cohort (n = 74) of TNBC patients with residual disease after neoadjuvante chemotherapy. Residual tumors were profiled with Nanostring for genes previously related to TNBC aggressiveness. Gene expression values for CCL5 were log2 transformed and median centered. TIL count was performed according to the methods published by Salgado et al (2015). Values of TILs count were log2 transformed (values with 0% were removed from the cohort; n = 1). Spearman's rank correlation analysis and proportional hazards regression was used to investigate the relationship and impact in the outcome between these two markers. Results: The median TIL count was 10% (interquartile range: 21.25). A total of 59.5% of cases had TILs <15%. There was a significant correlation between TILs and CCL5 (ñ = 0.345, P = 0.032). In univariate analysis (as continuous variable), TILs (HR = 0.968 per unit of change; 95%IC: 0.946-0.990; P = 0.006) and CCL5 (HR = 0.95 per unit of change; 95%IC: 0.635-0.996; P = 0.046) were both associated with outcome. After adjusting to CCL5 expression, TIL's count, was the only significant marker with a P-value = 0.007 (HR = 0.702 per unit of change; 95%IC: 0.54-0.909), in contrast to CCL5 (HR = 0.925, CI95%:0.717-1,194; P-value = 0.550). Conclusions: TILs and CCL5 gene expression are significantly correlated in chemotherapy-resistant TNBC. However, although CCL5 expression is strongly associated with the progression of BC, particularly TNBC, TIL assessment remains the stronger and more significant prognostic immunological marker. Citation Format: Joseph A. Pinto, Zaida D. Morante, Roberto Salgado, Nadezhda Cardenas, Jhajaira Araujo, Jaime Ponce, Franco Doimi, Justin Balko, Henry Gomez. Correlation between tumor infiltrating lymphocytes and CCL5 gene expression in chemotherapy-resistant triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 738.

Abstract 178: PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer

Abstract Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene. This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an “un-targetable” oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis. We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs. Citation Format: Fara Braso-Maristany, Simone Filosto, Steven Catchpole, Rebecca Marlow, Jelmar Quist, Erika Francesch Domenech, Gianmaria Liccardi, Leila Zakka, Violeta Serra, Albert Gris, Maggie Cheang, Nirmesh Patel, Anna Perdrix Rosell, Patrycja Gazinska, Elodie Noel, Jonathan Watkins, Pierfrancesco Marra, Anita Grigoriadis, Andrew Tutt. PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 178.

Abstract P2-06-07: PRKCQ regulates taxol sensitivity of triple negative breast cancer cells via IL-6/Stat3 signaling

Abstract Background/Rationale. While some patients with triple negative breast cancer achieve long-term remission with chemotherapy, many have cancers that are chemotherapy resistant. The lack of targeted therapies for this subtype also makes some triple negative cancers difficult to treat and control. PRKCQ, a member of the novel protein kinase C family, is preferentially expressed in triple negative breast cancers compared to ER+/Luminal cancers. We previously reported that PRKCQ expression drives growth-factor independent growth, anoikis resistance and migration of breast epithelial cells. In addition, PRKCQ is required for in vivo growth of triple negative breast cancers tumor xenografts. We sought to determine if PRKCQ expression modulates sensitivity of triple negative breast cancer cells to standard of care chemotherapy and whether PRKCQ inhibition could be a strategy to induce death of chemotherapy-resistant triple negative breast cancer cells. Methods. We determined the effects of modulating PRKCQ expression, using PRKCQ cDNA or shRNA vectors, on Doxorubicin or Taxol treatment-induced effects on triple negative breast cancer cells, including those that are relatively chemotherapy resistant at baseline. We determined the mechanisms by which PRKCQ expression regulates sensitivity to Taxol. Results. Increased PRKCQ expression in MCF-10A breast epithelial cells suppresses the apoptosis-inducing effects of Doxorubicin or Taxol treatment. PRKCQ-induced Taxol resistance is dependent on PRKCQ kinase activity. PRKCQ-expressing MCF-10A cells secrete enhanced levels of IL-6, leading to the autocrine activation of Stat3; IL-6/Stat3 activation is necessary for PRKCQ-induced resistance to Taxol. Finally, downregulation of PRKCQ sensitized MDA-231-Luc cells to Taxol treatment and induced apoptosis of these cells which are relatively resistant to Taxol at baseline. Conclusions. PRKCQ regulates sensitivity to standard of care chemotherapies used in the treatment of triple negative breast cancer. IL-6/Stat3 signaling induced by PRKCQ kinase activity is responsible for resistance to the effects of Taxol treatment. Targeting PRKCQ therefore could be an attractive strategy to overcome chemotherapy resistance of a subset of triple negative breast cancers. Citation Format: Irie HY, Byerly J. PRKCQ regulates taxol sensitivity of triple negative breast cancer cells via IL-6/Stat3 signaling. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-07.

The sialyl-glycolipid SSEA4 marks a subpopulation of chemotherapy resistant breast cancer cells with mesenchymal features

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with high risk of early relapse and metastasis [1]. At the moment chemotherapy remains the main option for systemic therapy of TNBC patients but complete remission occurs only in 20% of the patients [2]. In order to identify biomarker for chemotherapy-resistant TNBC cells, we performed a cell surface marker screen in 4 TNBC patient-derived xenograft (PDX) models that respond well to adriamycin/cyclophosphamide-based (A/C) chemotherapy but fail to reach complete pathological response. We used multi-parameter flow cytometry to screen the expression of 45 cell surface markers during the course of chemotherapy. We identified the sialyl-glycolipid SSEA4 as a marker of chemotherapy-resistant cancer cells in all four models. In addition, 3 out of 4 TNBC PDXs showed higher percentage of SSEA4-positive cells compared to all A/C-sensitive TNBC PDXs analysed. Gene expression comparison between SSEA4-positive and SSEA4-negative tumor cells from 3 TNBC PDXs highlighted an overexpression of mesenchymal-associated genes and a deregulation of drug resistance pathway-associated genes and miRNAs in SSEA4+ breast cancer cells. In addition, high expression of ST3 beta-galactoside alpha-2,3-sialyltransferase 2 (ST3Gal2), the enzyme catalyzing the last step of SSEA4 synthesis, was found associated with poor outcome in ER-, PR- breast cancer patients treated with chemotherapy (p < 0.01, HR 3.08). Thus, we propose SSEA4 as a novel marker of mesenchymal and chemoresistant breast cancer cells, and ST3GAL2 expression as a predictive marker for chemoresistance associated with poor outcome in breast cancer patients.

Glucocorticoid receptor antagonism as a novel therapy for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 10% to 20% of newly diagnosed invasive breast cancer. Finding effective targets for chemotherapy-resistant TNBC has proven difficult in part because of TNBC's molecular heterogeneity. We have previously reported that likely because of the antiapoptotic activity of glucocorticoid receptor (GR) in estrogen receptor (ER)-negative breast epithelial and cancer cells, high GR expression/activity in early-stage TNBC significantly correlates with chemotherapy resistance and increased recurrence. We hypothesized that pretreatment with mifepristone, a GR antagonist, would potentiate the efficacy of chemotherapy in GR+ TNBCs by inhibiting the antiapoptotic signaling pathways of GR and increasing the cytotoxic efficiency of chemotherapy. TNBC cell apoptosis was examined in the context of physiologic glucocorticoid concentrations, chemotherapy, and/or pharmacologic concentrations of mifepristone. We used high-throughput live microscopy with continuous recording to measure apoptotic cells stained with a fluorescent dye and Western blot analysis to detect caspase-3 and PARP cleavage. The effect of mifepristone on GR-mediated gene expression was also measured. TNBC xenograft studies were performed in female severe combined immunodeficient (SCID) mice and tumors were measured following treatment with vehicle, paclitaxel, or mifepristone/paclitaxel. We found that although mifepristone treatment alone had no significant effect on TNBC cell viability or clonogenicity in the absence of chemotherapy, the addition of mifepristone to dexamethasone/paclitaxel treatment significantly increased cytotoxicity and caspase-3/PARP cleavage. Mifepristone also antagonized GR-induced SGK1 and MKP1/DUSP1 gene expression while significantly augmenting paclitaxel-induced GR+ MDA-MB-231 xenograft tumor shrinkage in vivo. These results suggest that mifepristone pretreatment could be a useful strategy for increasing tumor cell apoptosis in chemotherapy-resistant GR+ TNBC.