Targeting Transcriptional Regulation with a CDK9 Inhibitor Suppresses Growth of Endocrine- and Palbociclib-Resistant ER+ Breast Cancers.

The combination of endocrine therapy and CDK4/6 inhibitors such as palbociclib is an effective and well-tolerated treatment for estrogen receptor-positive (ER+) breast cancer, yet many patients relapse with therapy-resistant disease. Determining the mechanisms underlying endocrine therapy resistance is limited by the lack of ability to fully recapitulate inter- and intratumor heterogeneity in vitro and of availability of tumor samples from women with disease progression or relapse. In this study, multiple cell line models of resistant disease were used for both two-dimensional (2D)- and three-dimensional (3D)-based inhibitor screening. The screens confirmed the previously reported role of pro-proliferative pathways, such as PI3K-AKT-mTOR, in endocrine therapy resistance and additionally identified the transcription-associated cyclin-dependent kinase CDK9 as a common hit in ER+ cell lines and patient-derived organoids modeling endocrine therapy-resistant disease in both the palbociclib-sensitive and palbociclib-resistant settings. The CDK9 inhibitor, AZD4573, currently in clinical trials for hematologic malignancies, acted synergistically with palbociclib in these ER+in vitro 2D and 3D models. In addition, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell-cycle regulators differentially downregulated only in combination-treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease. SIGNIFICANCE: Targeting transcription-associated CDK9 synergizes with CDK4/6 inhibitor to drive tumor regression in multiple models of endocrine- and palbociclib-resistant ER+ breast cancer, which could address the challenge of overcoming resistance in patients.

Oxidative phosphorylation is a metabolic vulnerability of endocrine therapy and palbociclib resistant metastatic breast cancers.

Resistance to endocrine treatments and CDK4/6 inhibitors is considered a near-inevitability in most patients with estrogen receptor positive breast cancers (ER + BC). By genomic and metabolomics analyses of patients' tumours, metastasis-derived patient-derived xenografts (PDX) and isogenic cell lines we demonstrate that a fraction of metastatic ER + BC is highly reliant on oxidative phosphorylation (OXPHOS). Treatment by the OXPHOS inhibitor IACS-010759 strongly inhibits tumour growth in multiple endocrine and palbociclib resistant PDX. Mutations in the PIK3CA/AKT1 genes are significantly associated with response to IACS-010759. At the metabolic level, in vivo response to IACS-010759 is associated with decreased levels of metabolites of the glutathione, glycogen and pentose phosphate pathways in treated tumours. In vitro, endocrine and palbociclib resistant cells show increased OXPHOS dependency and increased ROS levels upon IACS-010759 treatment. Finally, in ER + BC patients, high expression of OXPHOS associated genes predict poor prognosis. In conclusion, these results identify OXPHOS as a promising target for treatment resistant ER + BC patients.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

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.

Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.