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List of genes differentially regulated between resistant and responder PDX.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX.Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605–15. ©2018 AACR.

Boxplot of the expression of genes encoding proteins involved in capecitabine metabolism (TYMP, TYMS, TK1, DPYD) and cell-cycle control (RB1, CDKN2A, CCND1).

Supplemental data include a Supplemental Table-1, Supplemental Figure 1, Supplemental Figure 2, Supplemental Figure 3, Supplemental Figure 4, Supplemental Figure 5 and Supplemental Figure 6. Supplemental Table-1 is related to Figure 1 Supplemental Figure 1 is related to Figure 2 Supplemental Figure 2 is related to Figure 5 Supplemental Figures 3-5 are related to Figure 6 Supplemental Figure 6 is related to Figure 1 and Figure 5

Purpose:Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB).Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments.Results:EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone.Conclusions:We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Breast cancer is the most common cancer type worldwide accounting for more than 2 million newly diagnosed cases in 2020. Approximately 20% of all breast tumors in the USA is the highly metastatic subtype triple-negative breast cancer (TNBC). Despite recent development of new therapies, disease-free survival rates of TNBC patients are still relatively low. In this study we describe the role of histone modifier lysine methyltransferase 2C and 2D (KMT2C and KMT2D) in metastasis. KMT2C and KMT2D are among the most commonly mutated genes across all cancer types including TNBC. To examine the functional relevance of Kmt2c and Kmt2d mutations in a murine TNBC mouse model, we mutated Kmt2c and Kmt2d using CRISPR/Cas9. In order to minimize confounder effects of constitutive CRISPR/Cas9 expression we utilized a unique strategy of consecutive transient transfections. Mutation of Kmt2c or Kmt2d did not impact tumor growth in vivo, however, Kmt2c and Kmt2d mutant tumors were highly metastatic to multiple organs while no metastases were found in mice with wildtype tumors. To unravel the underlying mechanisms, we first performed histone mass spectrometry. As expected, direct histone modification target of Kmt2cd and Kmt2d, H3K4me1 levels were reduced in mutant cells. Interestingly, we also identified decreased levels of H3K27me3 and increased levels of H3K27ac. To further delineate the impact of these changes we performed ChIP-seq for these three histone modifications. In accordance with the histone mass spectrometry data peak intensities for H3K4me1, H3K27me3, and H3K27ac were increased or decreased, respectively, in mutant cells. Additionally, to examine effects of these changes on chromatin remodeling we performed ATAC sequencing. We identified differencent chromatin structures between wildtype and mutant cells, validating the impact of Kmt2c and Kmt2d perturbation on histone modification and chromatin remodeling. Lastly, we performed RNA-seq and PRO-seq analyses to identify genes and pathways that could explain the increased metastatic capacity of the mutant cells. Gene set enrichment analyses revealed differential regulation of pro-inflammatory pathways in Kmt2c mutant cells correlating with increased number of T-cells in Kmt2c mutant tumors. Kmt2d mutant cells showed alterations in metabolic pathways which we also identified by unbiased metabolomics showing significant changes. This study revealed that mutations in Kmt2c and Kmt2d are sufficient to induce distant metastases and we deciphered underlying mechanism that could be explored as novel strategies to treat or prevent metastasis. Citation Format: Marco Seehawer, Zheqi Li, Jun Nishida, Paloma Cejas, Andrew Reiter, Seth Goldman, Pierre Foidart, Ernesto Rojas, Malvina Papanastasiou, Karen Adelman, Henry Long, Kornelia Polyak. Histone modification by Kmt2c and Kmt2d regulate metastasis in triple-negative breast cancer in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A020.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Background: Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC. Methods: We conducted a retrospective immunohistochemical analysis on human TNBC samples (n=81) and validated our findings in in vitro and in vivo assays. Results: Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours. Conclusions: We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs.

Purpose: Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB). Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments. Results: EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone. Conclusions: We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments. Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX. Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment. Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605–15. ©2018 AACR.