Triple-negative Breast Cancer Patient-derived Xenografts Research Articles

Carbonic Anhydrase-IX Guided Albumin Nanoparticles for Hypoxia-mediated Triple-Negative Breast Cancer Cell Killing and Imaging of Patient-derived Tumor.

Triple-Negative Breast Cancer (TNBC) is considered as the most onerous cancer subtype, lacking the estrogen, progesterone, and HER2 receptors. Evaluating new markers is an unmet need for improving targeted therapy against TNBC. TNBC depends on several factors, including hypoxia development, which contributes to therapy resistance, immune evasion, and tumor stroma formation. In this study, we studied the curcumin analogue (3,4-Difluorobenzylidene Curcumin; CDF) encapsulated bovine serum albumin (BSA) nanoparticle for tumor targeting. For tumor targeting, we conjugated Acetazolamide (ATZ) with CDF and encapsulated it in the BSA to form a nanoparticle (namely BSA-CDF-ATZ). The in vitro cytotoxicity study suggested that BSA-CDF-ATZ is more efficient when compared to free CDF. The BSA-CDF-ATZ nanoparticles showed significantly higher cell killing in hypoxic conditions compared to normoxic conditions, suggesting better internalization of the nanoparticles into cancer cells under hypoxia. Fluorescent-dye labeled BSA-CDF-ATZ revealed higher cell uptake of the nanoparticle compared to free dye indicative of better delivery, substantiated by a high rate of apoptosis-mediated cell death compared to free CDF. The significantly higher tumor accumulation and low liver and spleen uptake in TNBC patient-derived tumor xenograft models confirm the significant potential of BSA-CDF-ATZ for targeted TNBC imaging and therapy.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs

BackgroundThe human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs).MethodsFifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100–200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (IP) injection of either formulation buffer (vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for Western blot, RNA, and immunohistochemistry analyses.ResultsAll 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation.ConclusionsThe marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

Targeting triple-negative breast cancers with the Smac-mimetic birinapant

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.

Patient-Derived Xenografts as an Innovative Surrogate Tumor Model for the Investigation of Health Disparities in Triple Negative Breast Cancer.

Despite a decline in overall incidence rates for cancer in the past decade, due in part to impressive advancements in both diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer-related deaths in women. BC alone accounts for ∼30% of all new cancer diagnoses in women worldwide. Triple-negative BC (TNBC), defined as having no expression of the estrogen or progesterone receptors and no amplification of the HER2 receptor, is a subtype of BC that does not benefit from the use of estrogen receptor-targeting or HER2-targeting therapies. Differences in socioeconomic factors and cell intrinsic and extrinsic characteristics have been demonstrated in Black and White TNBC patient tumors. The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with TNBC has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established TNBC PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. The primary aim of this review is to emphasize the importance of using clinically relevant translatable tumor models that reflect TNBC human tumor biology and heterogeneity in high-risk patient populations. The focus is to highlight the complexity of BC as it specifically relates to the management of TNBC in Black women. We discuss the importance of utilizing PDX models to study the extracellular matrix (ECM), and the distinct differences in ECM composition and biophysical properties in Black and White women. Finally, we demonstrate the crucial importance of PDX models toward novel drug discovery in this patient population.

Wee1 Inhibition Enhances the Anti-Tumor Effects of Capecitabine in Preclinical Models of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. Adavosertib (AZD1775) is a potent, small-molecule, ATP-competitive inhibitor of the Wee1 kinase that potentiates the activity of many DNA-damaging chemotherapeutics and is currently in clinical development for multiple indications. The purpose of this study was to investigate the combination of AZD1775 and capecitabine/5FU in preclinical TNBC models. TNBC cell lines were treated with AZD1775 and 5FU and cellular proliferation was assessed in real-time using IncuCyte® Live Cell Analysis. Apoptosis was assessed via the Caspase-Glo 3/7 assay system. Western blotting was used to assess changes in expression of downstream effectors. TNBC patient-derived xenograft (PDX) models were treated with AZD1775, capecitabine, or the combination and assessed for tumor growth inhibition. From the initial PDX screen, two of the four TNBC PDX models demonstrated a better response in the combination treatment than either of the single agents. As confirmation, two PDX models were expanded for statistical comparison. Both PDX models demonstrated a significant growth inhibition in the combination versus either of the single agents. (TNBC012, p < 0.05 combo vs. adavosertib or capecitabine, TNBC013, p < 0.01 combo vs. adavosertib or capecitabine.) An enhanced anti-proliferative effect was observed in the adavosertib/5FU combination treatment as measured by live cell analysis. An increase in apoptosis was observed in two of the four cell lines in the combination when compared to single-agent treatment. Treatment with adavosertib as a single agent resulted in a decrease in p-CDC2 in a dose-dependent manner that was also observed in the combination treatment. An increase in γH2AX in two of the four cell lines tested was also observed. No significant changes were observed in Bcl-xL following treatment in any of the cell lines. The combination of adavosertib and capecitabine/5FU demonstrated enhanced combination effects both in vitro and in vivo in preclinical models of TNBC. These results support the clinical investigation of this combination in patients with TNBC, including those with brain metastasis given the CNS penetration of both agents.

Abstract P3-02-04: Combined aurora kinase A and mTORC 1/2 inhibition results in disruption of tumor cell energetics in triple negative breast cancer

Abstract Background: We have previously demonstrated the efficacy of pharmacologic targeting of Aurora Kinase A (AurA) in cell line and patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC). We observed that inherent or acquired resistance to AurA inhibitors was associated with a senescence phenotype and up-regulation of the PI3K/Akt/mTOR pathway. This provided the rationale to assess whether this resistance could be overcome with the combination of the AurA inhibitor alisertib with the TORC1/2 inhibitor TAK-228. In select TNBC PDX models, we found that there were highly significant anti-tumor effects of the combination compared to single agents. The purpose of the current study was to evaluate the potential role for alterations in tumor metabolic profiles in anti-tumor activity after dual inhibition of AurA and TORC1/2, as both pathways are known to mediate cellular metabolism and energy utilization in normal and cancer cells. Methods: Three TNBC PDX models were used for these studies. Tumor sections were implanted subcutaneously on bilateral flanks of athymic nude mice. When tumors reached ~150 mm3 mice were randomized into four treatment arms: vehicle; alisertib (30 mg/kg); TAK-228 (0.75 mg/kg); or the combination and dosed daily by oral gavage for 30 days (n=10 tumors per arm). At end of study, tumors were harvested and polar metabolites extracted for global ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics with a focus on central carbon and nitrogen metabolism. Metabolites were separated using a high throughput gradient-based C18 method and identified and validated manually against an in-house library of 800+ compounds. Results: Global metabolomics analysis of PDX tumors revealed a significant impact of TAK-228 treatment on fatty acid oxidation, resulting in increased levels of saturated fatty acids (C8, C10, C12) and palmitoleic acid, an increase in arginine, proline, metabolism and a decrease in glutamine metabolism. Alisertib treatment impacted tryptophan metabolism; initial results suggest a disruption of metabolite balance downstream of tryptophan. These disruptions in nitrogen and carbon metabolism occurred in the combination treatment arm as well. Of note, glucose utilization via glycolysis and the pentose phosphate pathway (PPP) were decreased with treatments, with the most profound decrease in the combination arm. Conclusions: In addition to increased anti-tumor growth activity, TNBC PDX tumor metabolic profiles were altered in mice treated alisertib, TAK-228 and the combination. Global metabolomics analysis revealed that several key metabolic pathways, including fatty acid oxidation, amino acid metabolism, glycolysis and PPP activity were significantly dysregulated, likely contributing to the observed tumor growth inhibition. Importantly, the effects on several pathways by the single agents persisted in the combination, while others, including the decrease in glycolysis and PPP activity, were more pronounced in the combination arm. These results support the hypothesis that dual targeting of AurA and TORC1/2 may exert potent anti-tumor activity in TNBC through several mechanisms, including the disruption of tumor cell energetics. This combination is currently being evaluated in a Phase I clinical trial with planned expansion cohorts with serial tumor biopsies (NCT02719691). These clinical samples may provide a means for validation of these preclinical observations and allow continued elucidation of the potential antitumor metabolic reprogramming resulting from this dual targeting strategy and the clinical setting most likely to benefit from this combination. Citation Format: John J Tentler, Angelo D'Allesandro, Julie A Reisz, Kyrie L Dailey, Anna Capasso, Anastasia Ionkina, Stacey M Bagby, Todd M Pitts, Jennifer R Diamond. Combined aurora kinase A and mTORC 1/2 inhibition results in disruption of tumor cell energetics in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-04.

Abstract PD8-05: Single-cell analysis of breast cancer metastasis using patient-derived xenograft model reveals clonal relationship between primary tumor and its metastases

Abstract Introduction Breast cancer is the most common cancer in female and triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer which shows high rate of recurrence and metastasis. Malignant cells that comprise primary tumor are heterogeneous and during disease progression selection of tumor cells occur as a mean to adapt and survive. Tumor heterogeneity can be studied by grouping cells as clones which refer to a group of cells related to each other by descent from a unitary origin. Understanding the mechanism of clonal dynamics and selection during cancer evolution is important to develop new therapeutic strategies for cancer metastasis. Moreover, it is imperative to be able to detect rare subclones that are responsible for metastasis. Thus, single-cell analysis is critical to identify cellular heterogeneity of cancer and molecular basis of metastatic phenotype in depth. Measuring genome and transcriptome in single-cell level will enable us to discover clonal dynamics during cancer metastasis and infer molecular determinants of metastasis fitness. Here, we propose to investigate the clonal dynamics, genomic, and transcriptomic profiles of human breast tumor metastasis using TNBC patient-derived xenograft (PDX) model to understand the mechanism of breast cancer progression and metastasis. Methods Tumor cells from previously established PDXs were transplanted into mammary fat pad of mice. Tumors were removed when it reached maximum allowed endpoint size (1,000mm3) and mice were monitored and allowed to grow metastasis. PDXs that developed primary tumor and metastasis were selected for subsequent single cell analysis. Single-cell whole-genome sequencing (scWGS) was performed using direct library preparation (DLP+) method which is preamplification-free single-cell genome sequencing approach. scWGS data was used to call cell-specific copy number events, which allows us to cluster cells and identify clones. Hierarchical clustering was used to identify clonal structure of each sample. Phylogenetic tree was computed using copy number data of samples from each PDX. Results Nine different TNBC PDX lines were tested and 6 PDX lines developed metastasis. We focused on 2 PDX lines of which metastatic tissues were available for single cell sequencing. SA919 developed metastatic mass near cervical or lumbar spine area. SA535 developed metastasis to lung, axillary or inguinal area and tumor recurrence at mammary fat pad site. scWGS for SA919 and SA535 was carried out and we generated libraries from single cells of primary tumor and its metastases. Hierarchical clustering using copy number data revealed clonal structure of primary tumor and metastases. SA919 showed oligoclonal primary tumor and metastasis whereas SA535 showed polyclonal metastasis from polyclonal primary tumor. scWGS data of primary tumor and its metastases in each PDX were merged to compute phylogenetic trees. Phylogenetic analysis revealed that not all clones of primary tumor contributed to metastasis in both PDXs. Different metastatic lesions of SA535 harbored different clones from primary tumor. For example, clones consisting left axillary metastasis and right axillary metastasis in SA535 were different from each other, however they both originated from primary tumor clones. Metastatic lesions from SA919 and SA535 also had newly appearing clones that were not present in primary tumor inferring clonal evolution during metastasis. Conclusion We were able to capture different patterns of metastasis in several PDXs. Phylogenetic analysis using scWGS data revealed clonal relationship between primary tumor and metastases. Further analysis of single cell genomic and transcriptomic profiles will provide deeper understanding of metastasis in breast cancer. Citation Format: Hakwoo Lee, Farhia Kabeer, Ciara O'Flanagan, Jazmine Brimhall, Justina Biele, Biexi Wang, Teresa Ruiz Algara, So Ra Lee, Daniel Lai, Michael Yuen, Simong Song, Patricia Ye, Jenifer Pham, Richard Moore, Andy J Mungall, Sohrab P Shah, Samuel Aparicio. Single-cell analysis of breast cancer metastasis using patient-derived xenograft model reveals clonal relationship between primary tumor and its metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-05.

Abstract P3-11-08: Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease. TNBC lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elucidate feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. Here, we examined whether the novel LIFR inhibitor, EC359, has the ability to counteract negative effects of LIFR signaling to enhance HDACIs therapeutic efficacy in the treatment of TNBC. Methods: We tested multiple HDACIs currently in clinical trials including vorinostat, panobinostat, romidepsin, and givinostat using multiple TNBC models. The effect of combination therapy of HDACIs and EC359 on TNBC cell viability and invasion was examined using MTT assays and matrigel invasion assays respectively. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and Caspase 3/7 assays, respectively. Mechanistic studies were performed using Western blotting, qRT-PCR, and reporter gene assays. The efficacy of combination therapy in vivo was examined using Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models. Results: We demonstrated that the treatment of TNBC models with HDACIs increased the expression of LIFR. Immunohistochemistry analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC samples. Knockdown of LIFR or treatment with a small molecule inhibitor of LIFR (EC359) significantly enhanced the efficacy of HDACIs in reducing cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy of HDACIs or EC359 in TNBC cell lines. Mechanistic studies, reporter gene assays and biochemical studies using multiple TNBC models exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359+HDACI combination therapy. Treatment of human breast tumors utilizing PDEX assays showed that EC359 enhanced the ability of HDACIs to decrease the proliferation (Ki-67 positivity) compared to monotherapy. Furthermore, using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides greater therapeutic efficacy than monotherapy. In addition, treatment with EC359 can overcome the feedback activation of LIFR currently observed in the treatment of TNBC with HDACIs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P Pratap, Yiliao Luo, Junhao Liu, Kristin A Altwegg, Xiaonan Li, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R Sareddy, Rajeshwar R Tekmal, Hareesh B Nair, Klaus J Nickisch, Ratna K Vadlamudi. Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-08.

Abstract P3-13-01: SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates strong inhibition of the Wnt signaling pathway and antitumor effects as monotherapy and in combination with chemotherapy in triple-negative breast cancer (TNBC) models

Abstract Aberrant activation of the Wnt signaling pathway is associated with tumorigenesis, relapse/chemoresistance, and distance metastasis in TNBC. SM08502 is a novel, oral, small-molecule pan-CLK inhibitor that has been shown to potently inhibit the Wnt signaling pathway in several preclinical cancer models. The purpose of these studies was to examine the antitumor activity of SM08502 as monotherapy and in combination with standard chemotherapy in preclinical models of TNBC. The effect of SM08502 on cell proliferation was tested 13 BC cell lines, 7 of which were TNBC derived. Cell proliferation was strongly impaired by SM08502 across all lines (average EC50=0.170 µM [0.055-0.510]). SM08502 demonstrated similar potency between HR+ and TNBC cell lines with average EC50 values of 0.202 µM (0.058-0.510) and 0.142 µM (0.055-0.240), respectively. Relative to DMSO, SM08502 (1 μM) potently inhibited Wnt pathway-related gene and protein expression (TCF7, DVL2, LRP5, and ERBB2) in TNBC cell lines. Notably, SM08502 showed little inhibitory effect on cell proliferation in normal breast cells (Hs578Bst) compared with their paired TNBC cells (Hs578T) with an EC50 of 1.517 μM and 0.080 μM, respectively. Wnt pathway-related proteins were also overexpressed in Hs578T cells relative to Hs578Bst, and SM08502 (1 µM) strongly reduced their expression. In vivo antitumor effects and tolerability of oral SM08502 (25 mg/kg QD for 20 days) were assessed in mice bearing orthotopically implanted, luciferase-expressing, TNBC (MDA-MB231)-derived xenografts (n=5 mice per group). Significant tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with SM08502 (80%, p&amp;lt;0.01). Metastasis was assessed by ex vivo imaging utilizing luciferase activity in bilateral lungs collected at study end. Luminescence was observed in 9/10 lungs from the vehicle-treated mice. In the SM08502-treated mice, only 3/10 lungs had measurable luminescence, suggesting that SM08502 reduced lung metastasis of TNBC tumors in this model. Additionally, SM08502 (12.5 and 25 mg/kg QD), gemcitabine (G)/Nab-paclitaxel (Nab-P) (75/30 mg/kg Q7D i.p.), and SM08502 combined with G/Nab-P were tested in MDA-MB231 xenografts. SM08502 (12.5 and 25 mg/kg) and G/Nab-P alone induced strong TGI vs. vehicle (81, 86, and 91%, respectively; p&amp;lt;0.0001), although no tumor regressions occurred. SM08502 (12.5 and 25 mg/kg) in combination with G/Nab-P achieved improved TGI (93% and 95%, respectively; p&amp;lt;0.0001 vs. vehicle) and induced tumor regression in 30% (3/10) and 60% (6/10) of mice, respectively. SM08502 (25 mg/kg QD) was also assessed in 4 patient-derived xenograft (PDX) models of TNBC (Crown Biosciences). Inhibition of tumor growth was observed in all tested PDX models (average TGI=69% [60-81], p&amp;lt;0.01 vs. vehicle). SM08502 was well tolerated in all tested xenograft models based on body weight measurements. In summary, SM08502 potently inhibited cell proliferation and expression of Wnt pathway-related genes in TNBC cell lines. SM08502 demonstrated strong in vivo antitumor effects in TNBC xenografts and PDX models and appeared to suppress lung metastasis. Additionally, SM08502 induced tumor regression in combination with G/Nab-P in TNBC xenografts, whereas G/Nab-P alone did not. These data suggest that SM08502 as a single agent or combined with standard chemotherapy has the potential to provide clinical benefit in TNBC. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM08502 in subjects with advanced solid tumors is ongoing (NCT03355066). Citation Format: Heekyung Chung, Lauren Sitts, Emily Creger, John Duc Nguyen, Brian Eastman, Chi-Ching Mak, Sunil KC, Betty Tam, Carine Bossard, Timothy Phalen, Steven Cha. SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates strong inhibition of the Wnt signaling pathway and antitumor effects as monotherapy and in combination with chemotherapy in triple-negative breast cancer (TNBC) models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-13-01.

Abstract P5-05-04: A novel inhibitor of cancer stem cells overcomes chemotherapy resistance of triple negative breast cancers

Abstract Background/Rationale: Sensitivity to chemotherapy is a strong, consistent predictor of survival for patients diagnosed with triple negative breast cancer (TNBC) and chemotherapy resistance remains a critical problem for a subset of patients. Cancer cells with a stem cell-like profile are thought to represent a relatively chemotherapy resistant subpopulation. A screen for novel inhibitors of these cancer stem cell-like cells identified the multi-kinase inhibitor ON108600 that inhibits Casein kinase 2 (CK2), TNIK and Dyrk kinases. We sought to determine whether treatment with ON108600 enhances chemotherapy sensitivity of aggressive, multi-drug resistant TNBC, thereby offering a novel therapeutic option for patients with high risk TNBC. Methods: Patient-derived xenograft (PDX) models derived from patients with chemotherapy-resistant primary or metastatic TNBC were screened for expression of ON108600 targets prior to selection for in vivo studies. Studies to confirm target engagement in vivo were performed to optimize dosing schedules. Selected PDX models were expanded and treated with vehicle control, ON108600, Paclitaxel, or ON108600/Paclitaxel combination. Tumor growth was assessed. Tumors were harvested at end point for histological analysis. Results: Chemotherapy- resistant TNBC PDX models were screened for protein expression of CK2, Dyrk and TNIK kinases. Two models with the highest level of these kinases were selected and propagated for in vivo studies; one model is derived from a primary TNBC with intrinsic resistance to chemotherapy while the second is derived from a heavily pre-treated metastatic TNBC. Target engagement was confirmed by assessing levels of CK2 substrates in tumors treated with different doses of ON108600. Expanded cohorts of these PDX models were treated with ON108600, Paclitaxel or combined ON108600/Paclitaxel. While Paclitaxel chemotherapy had minimal effect on tumor growth in these PDX models, combined ON108600/Paclitaxel treatment significantly suppressed growth and/or caused regression of these aggressive tumors. Importantly, combination treatment was associated with minimal toxicity. RNASeq profiling of PDX tumors treated with ON108600 or vehicle control was performed, revealing suppression of multiple oncogenic pathways by ON108600 which are currently being validated. Conclusions: ON108600, identified as an inhibitor of a cancer stem cell-like population, overcomes Paclitaxel resistance to suppress growth of aggressive TNBC for which treatment options remain limited. These results support further study and clinical translation of ON108600. Citation Format: Katsutoshi Sato, Amol Padgaonkar, Stephen Cosenza, Stacey Baker, Ramana Reddy, E Premkumar Reddy, Hanna Y. Irie. A novel inhibitor of cancer stem cells overcomes chemotherapy resistance of triple negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-04.

Abstract P4-08-01: Tumor suppressor PLK2 may serve as a biomarker in triple negative breast cancer for combinational therapy of PLK1 inhibitors with the standard-of-care chemotherapy

Abstract Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Surprisingly, PLK2 is frequently deleted in human breast cancer, preferentially in triple negative breast cancer (TNBC). Pilot studies using an RNAi screen and colony formation assays suggested that PLK2 might act as a tumor suppressor in breast cancer. This paradox stimulated us to investigate the functions of PLK1 and PLK2 in preclinical models and to understand their clinical implications. In preliminary experiments, we found that knockdown of PLK1 mitigated PLK2-deletion induced phenotypes including inhibiting colony formation, and rescued Plk2 null mammary epithelial cell phenotypes in vivo, including gland hyperbranching, epithelial cell hyperproliferation, and abnormal spindle orientation. Furthermore, we demonstrated that PLK2 interacted with PLK1 using a bimolecular fluorescence complementation assay and by co-immunoprecipitation, and using a proximity ligation assay this interaction was shown to occur in prometaphase. Due to the lack of targeted therapy, chemotherapy, such as carboplatin usually in combination with Taxol, is still one of the only systematic treatments for TNBC. Although PLK2 is frequently deleted in TNBC, there is currently no viable strategy to target this loss of function. The relationship between PLK2 and PLK1 suggests targeting PLK1 might provide a potential target to treat TNBC patients with loss of PLK2. In order to test this hypothesis, we used p53 null mammary epithelial cells as a sensitized background to generate a cohort of p53/Plk2 double null TNBC tumors. RNA-seq analysis showed that these tumors can be classified into three distinct molecular TNBC sublines: luminal-like, basal-like and claudin-low. Combinational treatment with the PLK1 inhibitor Volasertib and carboplatin showed a strong synergistic effect on tumor growth and led to regression of the Plk2/p53 null claudin-low tumors, but not control Plk2+/p53 null claudin-low tumors. The effect of combinational treatment of PLK1 inhibitor and carboplatin on the other two sublines as well as PLK2-low TNBC patient-derived xenograft (PDX) models is currently under investigation. We hope that these preclinical experiments will provide a rationale for the application PLK1 inhibitors in combination with chemotherapy in a subset of TNBC patients with deletion of PLK2. [Supported by Susan G. Komen Foundation grant SAC110031] Citation Format: Yang Gao, Elena Kabotyanski, Deanna Acosta, Elizabeth Villegas, Thomas F Westbrook, Charles M Perou, Jeffrey M Rosen. Tumor suppressor PLK2 may serve as a biomarker in triple negative breast cancer for combinational therapy of PLK1 inhibitors with the standard-of-care chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-01.

Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically.

AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro, and using a lung xenograft model, we show that systemic delivery of AZD0156 enhances the tumor growth inhibitory effects of radiation treatment in vivo Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition.

Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required.Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo.Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity.Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.

Abstract C016: Oxidative phosphorylation is a metabolic vulnerability in chemotherapy resistant triple negative breast cancer

Abstract There is a pressing need to identify improved therapies for triple negative breast cancers (TNBC) resistant to standard chemotherapy. To identify potential molecular targets, we performed RNA sequencing of pre-treatment biopsies from 43 patients with operable TNBC who received neoadjuvant anthracycline and taxane-based chemotherapy. Ingenuity pathway analysis demonstrated that the top canonical pathway associated with higher likelihood of recurrence was higher expression of oxidative phosphorylation (OXPHOS) signature. We therefore sought to determine the efficacy of IACS-10759, a potent inhibitor of OXPHOS, in 10 TNBC patient-derived xenografts (PDX), 8 generated from chemotherapy-resistant tumors. Partial response was observed in one PDX model and prolonged disease stabilization in 5 of 10 PDXs. PDXs with higher expression of protein coding mitochondrial genes were more sensitive to IACS-10759. AXL overexpression was associated with intrinsic and acquired IACS-10759 resistance. The combination of cabozantinib, a multi-kinase inhibitor targeting AXL, with IACS-10759 significantly improved responses in TNBC PDXs. In contrast, selective AXL inhibitor BGB324 or knockdown of AXL did not enhance IACS-10759 sensitivity. In addition, an in vivo synthetic lethality screen identified CDK4, PARP1 and PARP2 as potential combination targets for IACS-10759. Palbociclib as well as talazoparib enhanced growth inhibitory effect of OXPHOS inhibition in vitro and in vivo. Our data suggests that OXPHOS is a promising target in chemoresistant TNBC. IACS-10759 is currently in Phase 1 testing, including TNBC. Further work is needed to determine the optimal biomarker-driven combination partners. Citation Format: Kurt Evans, Stacy Moulder, Erkan Yuca, Stephen Scott, Natalia Paez Arango, Maryam Shariati, Christopher P Vellano, Turcin Saridogan, Xiaofeng Zheng, Ana Maria Gonzalez-Angulo, Ming Zhao, Xiaoping Su, Coya Tapia, Ken Chen, Argun Akcakanat, Charles M Perou, Bora Lim, Debu Tripathy, Timothy A Yap, Maria E Di Francesco, Giulio Draetta, Philip Jones, Joe Marszalek, Funda Meric-Bernstam. Oxidative phosphorylation is a metabolic vulnerability in chemotherapy resistant triple negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C016. doi:10.1158/1535-7163.TARG-19-C016

Abstract C091: Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications

Abstract Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional control (e.g. MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g. RB1, CCNE1, CDKN2A alterations). SY-5609 is an oral, potent, and highly selective CDK7 inhibitor that is advancing through IND-enabling studies to support initiation of a planned Phase 1 oncology trial in early 2020. Here we report on the relationship between pharmacokinetics (PK), pharmacodynamics (PD), and tumor growth inhibition (TGI) in xenograft models of tumor types with transcriptional and/or cell cycle aberrations including high grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and estrogen receptor positive breast cancer (ER+BC). Methods: The relationship between SY-5609 PK, PD, and TGI was evaluated in xenograft models of HGSOC (OVCAR3) and TNBC (HCC70) using once daily (QD) or twice daily (BID) dosing via oral gavage. SY-5609 TGI was evaluated as a single agent (SA) QD in patient-derived xenograft (PDX) models of HGSOC (n=3), SCLC (n=4), TNBC (n=4), and in combination with once weekly fulvestrant in ER+BC PDX models selected in vivo for resistance to the CDK4/6 inhibitor palbociclib (n=1) or resistance to both palbociclib and fulvestrant (n=1). Results: SY-5609 plasma exposure was dose proportional and did not accumulate after repeated therapeutic doses. Dose-dependent transcriptional responses in xenograft tissue were observed within 4 hours of SY-5609 dosing and were sustained for 24 hours. TGI was dose-dependent, with tumor regressions observed at doses significantly below the maximum tolerated dose (MTD). Similar TGI was seen when the same daily dose was administered either QD or BID suggesting that the effect was driven by overall exposure or minimum concentration. In HGSOC, SCLC, and TNBC PDX models, SA SY-5609 induced &amp;gt;50% TGI in all models tested (11/11), with 7/11 (64%) demonstrating robust anti-tumor activity (≥90% TGI or regression): 3/3 HGSOC, 2/4 SCLC, and 2/4 TNBC. In a palbociclib-resistant ER+BC PDX model, the combination of SY-5609 and fulvestrant induced significant TGI (89%), with no evident tumor regrowth up to 21 days after dosing cessation, distinguishing the observed effects from SY-5609 SA or fulvestrant SA. In a palbociclib and fulvestrant double-resistant ER+ BC PDX model, SY-5609 SA resulted in 33% TGI and fulvestrant SA had no activity. In contrast, the combination of SY-5609 and fulvestrant demonstrated significant TGI (68%; p&amp;lt;0.0001 vs fulvestrant SA), suggesting re-sensitization to fulvestrant. Conclusions: We have characterized PK, PD, and anti-tumor activity of the CDK7 inhibitor SY-5609 in a series of xenograft models. Exposure in plasma is dose-proportional and does not accumulate at therapeutic dose levels. SY-5609 induces dose-dependent transcriptional responses in tumor xenograft tissue and shows robust TGI, including regressions, in PDX models derived from multiple solid tumor indications. These results highlight the broad potential for SY-5609 across a variety of solid tumor types, including treatment resistant ER+BC, and support the development of SY-5609 in early phase clinical trials. Citation Format: Liv H Johannessen, Shanhu Hu, Nan Ke, Anthony D'Ippolito, Nisha Rajagopal, Jason Marineau, Anneli Savinainen, William Zamboni, Graeme Hodgson. Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C091. doi:10.1158/1535-7163.TARG-19-C091

Preclinical PERCIST and 25% of SUVmax Threshold: Precision Imaging of Response to Therapy in Co-clinical 18F-FDG PET Imaging of Triple-Negative Breast Cancer Patient-Derived Tumor Xenografts.

Numerous recent works highlight the limited utility of established tumor cell lines in recapitulating the heterogeneity of tumors in patients. More realistic preclinical cancer models are thought to be provided by transplantable, patient-derived xenografts (PDXs). The inter- and intratumor heterogeneity of PDXs, however, presents several challenges in developing optimal quantitative pipelines to assess response to therapy. The objective of this work was to develop and optimize image metrics for 18F-FDG PET to assess response to combination docetaxel and carboplatin therapy in a co-clinical trial involving triple-negative breast cancer PDXs. We characterized the reproducibility of standardized uptake value (SUV) metrics to assess response to therapy, and we optimized a preclinical PERCIST paradigm to complement clinical standards. Considerations in this effort included variability in tumor growth rate and tumor size, solid tumors versus tumor heterogeneity and a necrotic phenotype, and optimal selection of tumor slices versus whole tumor. Methods: A test-retest protocol was implemented to optimize the reproducibility of 18F-FDG PET SUV thresholds, SUVpeak metrics, and preclinical PERCIST parameters. In assessing response to therapy, 18F-FDG PET imaging was performed at baseline and 4 d after therapy. The reproducibility, accuracy, variability, and performance of imaging metrics to assess response to therapy were determined. We defined an index called the Quantitative Response Assessment Score to integrate parameters of prediction and precision and thus aid in selecting the optimal image metric to assess response to therapy. Results: Our data suggest that a threshold of 25% of SUVmax (SUV25) was highly reproducible (<9% variability). The concordance and reproducibility of preclinical PERCIST were maximized at α = 0.7 and β = 2.8 and exhibited a high correlation with SUV25 measures of tumor uptake, which in turn correlated with the SUV of metabolic tumor. Conclusion: The Quantitative Response Assessment Score favors SUV25 followed by SUVpeak for a sphere with a volume of 14 mm3 (SUVP14) as optimal metrics of response to therapy. Additional studies are warranted to fully characterize the utility of SUV25 and preclinical PERCIST SUVP14 as image metrics for response to therapy across a wide range of therapeutic regimens and PDX models.

QPI Allows in vitro Drug Screening of Triple Negative Breast Cancer PDX Tumors and Fine Needle Biopsies

The development of resistance to initially successful cancer therapies is a major cause of the morbidity and mortality associated with cancer. Identifying evolving resistance at an early stage could inform clinical decision making to adapt therapies before resistant cancer cell phenotypes have become clonally dominant or metastasized. This goal of early detection has prompted heavy investments in liquid biopsy, organoid, and high-throughput screening methodologies. Recently, High-Speed Live-Cell Interferometry (HSLCI), a quantitative phase imaging (QPI) methodology, was shown to predict triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) sensitivity to carboplatin only 40 hours after tumor removal from a mouse. Before HSLCI can be tested in the clinic, it must be adapted for minimally invasive sample acquisition techniques, throughput must be increased to enhance drug screening capacity, and, like any screening method, protocols must be adjusted to reflect drug-specific effects. To overcome these barriers, the system’s hardware was redesigned to increase throughput six-fold and enable the simultaneous screening of multiple therapeutics, and experiments were expanded to include several new classes of drugs. Additionally, the updated system was then incorporated into fine needle biopsy-compatible protocols that were developed for HSLCI and yielded data concordant with recently-published in vivo screens.

The Altered Transcriptome and DNA Methylation Profiles of Docetaxel Resistance in Breast Cancer PDX Models

Taxanes are standard therapy in clinical practice for metastatic breast cancer; however, primary or acquired chemoresistance are a common cause of mortality. Breast cancer patient-derived xenografts (PDX) are powerful tools for the study of cancer biology and drug treatment response. Specific DNA methylation patterns have been associated to different breast cancer subtypes but its association with chemoresistance remains unstudied. Aiming to elucidate docetaxel resistance mechanisms, we performed genome-wide DNA methylation in breast cancer PDX models, including luminal and triple-negative breast cancer (TNBC) models sensitive to docetaxel, their matched models after emergence of chemoresistance and residual disease after short-term docetaxel treatment. We found that DNA methylation profiles from breast cancer PDX models maintain the subtype-specific methylation patterns of clinical samples. Two main DNA methylation clusters were found in TNBC PDX and remain stable during the emergence of docetaxel resistance; however, some genes/pathways were differentially methylated according to docetaxel response. A DNA methylation signature of resistance able to segregate TNBC based on chemotherapy response was identified. Transcriptomic profiling of selected sensitive/resistant pairs and integrative analysis with methylation data demonstrated correlation between some differentially methylated and expressed genes in docetaxel-resistant TNBC PDX models. Multiple gene expression changes were found after the emergence of docetaxel resistance in TNBC. DNA methylation and transcriptional changes identified between docetaxel-sensitive and -resistant TNBC PDX models or residual disease may have predictive value for chemotherapy response in TNBC. IMPLICATIONS: Subtype-specific DNA methylation patterns are maintained in breast cancer PDX models. While no global methylation changes were found, we uncovered differentially DNA methylated and expressed genes/pathways associated with the emergence of docetaxel resistance in TNBC.

6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase-2 Regulates TP53-Dependent Paclitaxel Sensitivity in Ovarian and Breast Cancers.

Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases.Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo. Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma. PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53.

Test-Retest Performance of a 1-Hour Multiparametric MR Image Acquisition Pipeline With Orthotopic Triple-Negative Breast Cancer Patient-Derived Tumor Xenografts.

Preclinical imaging is critical in the development of translational strategies to detect diseases and monitor response to therapy. The National Cancer Institute Co-Clinical Imaging Resource Program was launched, in part, to develop best practices in preclinical imaging. In this context, the objective of this work was to develop a 1-hour, multiparametric magnetic resonance image-acquisition pipeline with triple-negative breast cancer patient-derived xenografts (PDXs). The 1-hour, image-acquisition pipeline includes T1- and T2-weighted scans, quantitative T1, T2, and apparent diffusion coefficient (ADC) parameter maps, and dynamic contrast-enhanced (DCE) time-course images. Quality-control measures used phantoms. The triple-negative breast cancer PDXs used for this study averaged 174 ± 73 μL in volume, with region of interest–averaged T1, T2, and ADC values of 1.9 ± 0.2 seconds, 62 ± 3 milliseconds, and 0.71 ± 0.06 μm2/ms (mean ± SD), respectively. Specific focus was on assessing the within-subject test–retest coefficient-of-variation (CVWS) for each of the magnetic resonance imaging metrics. Determination of PDX volume via manually drawn regions of interest is highly robust, with ∼1% CVWS. Determination of T2 is also robust with a ∼3% CVWS. Measurements of T1 and ADC are less robust with CVWS values in the 6%–11% range. Preliminary DCE test–retest time-course determinations, as quantified by area under the curve and Ktrans from 2-compartment exchange (extended Tofts) modeling, suggest that DCE is the least robust protocol, with ∼30%–40% CVWS.