Up to date clinical tests for predicting cancer chemotherapy response are not available and individual markers have shown little predictive value. We hypothesized that gene expression patterns ...attributable to chemotherapy-resistant cells can predict response and cancer prognosis. We contrasted the expression profiles of 13 different human tumor cell lines of gastric (EPG85-257), pancreatic (EPP85-181), colon (HT29) and breast (MCF7 and MDA-MB-231) origin and their counterparts resistant to the topoisomerase inhibitors daunorubicin, doxorubicin or mitoxantrone. We interrogated cDNA arrays with 43 000 cDNA clones ( approximately 30 000 unique genes) to study the expression pattern of these cell lines. We divided gene expression profiles into two sets: we compared the expression patterns of the daunorubicin/doxorubicin-resistant cell lines and the mitoxantrone-resistant cell lines independently to the parental cell lines. For identifying predictive genes, the Prediction Analysis for Mircorarrays algorithm was used. The analysis revealed 79 genes best correlated with doxorubicin resistance and 70 genes with mitoxantrone resistance. In an independent classification experiment, we applied our model of resistance for predicting the sensitivity of 44 previously characterized breast cancer samples. The patient group characterized by the gene expression profile similar to those of doxorubicin-sensitive cell lines exhibited longer survival (49.7+/-26.1 months, n=21, P=0.034) than the resistant group (32.9+/-18.7 months, n=23). The application of gene expression signatures derived from doxorubicin-resistant and -sensitive cell lines allowed to predict effectively clinical survival after doxorubicin monotherapy. Our approach demonstrates the significance of in vitro experiments in the development of new strategies for cancer response prediction.
Activation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and ...phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone.
► Inhibition of PI3K-AKT signaling in tumors activates receptor tyrosine kinases ► Induction is due to relief of feedback inhibition of FOXO-dependent RNA expression ► AKT activates feedback via FOXO- and mTORC1-dependent pathways ► Inhibition of both AKT and induced RTKs is a promising therapeutic strategy
BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors ...(PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity.
We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi.
RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor.
Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.
The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients ...with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure.
In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples.
The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders OR 55.6%, 95% confidence interval (CI) 33.7-75.4 contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P = 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P = 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P = 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P = 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PD-L1 expression all correlated to olaparib response.
Olaparib yielded a high clinical response rate in treatment-naïve TNBCs revealing HR deficiency, beyond germline HR mutations.
ClinicalTrials.gov identifier: NCT02624973.
•We observed an objective response to olaparib in 18 out of 32 (56%) of unselected, primary triple negative breast cancers.•Homologous recombination deficiency (HRD) was determined by targeted DNA sequencing and BRCA1 methylation.•HRD was predictive of response to olaparib and present in 16 out of 18 responders.•HRD was also predictive of response to olaparib beyond germline BRCA1/2 and gPALB2 mutations; 12 out of 14 responders.•Olaparib was associated with minor side-effects and did not influence subsequent tolerance to chemotherapy.
Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including ...the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.
The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts ...(PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.
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•We developed a biobank of breast cancer patient-derived tumor xenografts (PDTXs)•PDTXs represent diverse molecular subtypes and retain intra-tumor heterogeneity•PDTX-derived tumor cells (PDTCs) were used for high-throughput drug testing•PDTXs/PDTCs are a robust platform for pre-clinical pharmacogenomic studies
Development and analysis of a collection of breast-cancer-patient-derived xenografts indicate that the xenografts and cell cultures derived from them preserve the heterogeneity of the original tumors and can be used for drug screening.
Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of ...patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.
Abstract
A major obstacle in the fight against cancer is the development of drug resistance in response to therapy. Acquired resistance can be mediated by a small population of drug-tolerant ...persister cancer cells (DTPs), which are characterized by an increased capacity of adaptation to various stresses. DTPs are a small slow-cycling population of cells that tolerate cancer drugs, and most likely are responsible for generating a stable resistance. Growth differentiation factor 15 (GDF15) is a well-established marker of cellular stress. It is a member of the TGF-β superfamily, and while its physiological expression is barely detectable in most human somatic tissues, it is prominently induced under stress conditions to maintain cell and tissue homeostasis. Evidence suggests that GDF15 can be secreted in different cancer types. Nevertheless, GDF15 can have opposite effects depending on cellular context and disease stage, and further studies are needed to confirm its biological role in cancer. The working hypothesis of this study is that GDF15 plays a functional role in mediating the eribulin tolerance in DTP cells. MDA-MB-231 and MCF7 cell lines were treated for a month with an IC80 of eribulin. Initially, cells were sensitive to eribulin and approx. 85 % of the cells died after 7 days of treatment, then for about three weeks the surviving cells remained in a quiescent-DTP state. After approx. 3 weeks of treatment with a high dose of eribulin, cells started to proliferate in the presence of the drug, and we assume that at this point a stable mechanism of resistance is established. Interestingly, the analysis of the GDF15 levels during the chronic treatment showed that GDF15 expression/secretion was only detectable in the DTP state, suggesting a role of GDF15 in the maintenance of DTPs. To demonstrate the GDF15 protecting role against eribulin, we conducted loss of GDF15 function experiments, targeting both GDF15 and its receptor GFRAL. siGDF15 cells showed higher sensitivity to eribulin treatment than the siCTRL cells. We confirmed the same results targeting the GFRAL receptor, confirming an autocrine role of the secreted GDF15 in the cells that survive eribulin treatment. These results open the possibility for combining eribulin and a GDF15 antibody to specifically target DTP cells, and overcome eribulin resistance. Moreover, we envision that the circulating levels of GDF15 in metastatic breast cancer patients could be used as a pharmacodynamic biomarker during eribulin treatment. Furthermore, we are confident that GDF15 could help to better understand the onset of acquired resistance to eribulin in metastatic breast cancer patients.
Citation Format: Chiara Bellio, Marta Emperador, Esther Zamora, Violeta Serra, Cristina Saura, Bruce A. Littlefield, Josep Villanueva. GDF15 contributes to the maintenance of the drug-tolerant persister state in cells responding to eribulin abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P069.
Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution ...during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.
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