Search

PDF file - 58K

PDF file - 301K, Combined PIK3CA siRNA and PARP suppression in TNBC cell lines

PDF file - 3.4MB, Concordance in ER, PR, HER2, and PTEN expression between original patient tumors and the derived tumor grafts

PDF file - 195K, PI3K regulates BRCA expression

PDF file - 208K, PI3K blockade suppresses BRCA1/2 transcription

PDF file - 162K, Western blot of cell lysates from MDA-MB-468 or PDC44 cells treated with 750nM and 500nM BKM120 respectively for 4 days using the indicated antibodies

Supplementary Table 1 - XLSX 39K, Exome sequencing results. The table contains the raw data for Figure 2B as well as the mutation allele frequency, the prediction of protein functionality, the inclusion in the Cancer Gene Census and the protein function. Sample legend: B, DCIS right; C, DCIS left; D, NSCLC left; E, NSCLC right; F, N2(4R)

Supplementary Figures - PDF file 585K, Supplementary Figure 1. Fluorescence in situ hybridization (FISH) of HER2. Supplementary Figure 2. EGFR and HER2 mutations identified. Supplementary Figure 3. Comparisons of allele frequencies. Supplementary Figure 4. HER2-V659E response to erlotinib and neratinib in MCF10A cells

PDF file - 190K, ERK and ETS1 downregulate BRCA1/2 expression

PDF file - 139K, Common BRCA1 and BRCA2 promoter putative transcription factors analysis

Supplementary Figure from Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance

Supplementary Data from Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance

Purpose:PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance.Experimental Design:We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models.Results:Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress.Conclusions:Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.

Background: HER3 is overexpressed in 30-50% of breast cancers and has been associated with poor prognosis. Patritumab deruxtecan (HER3-DXd; U3-1402) is a HER3-directed ADC with a potent topoisomerase I (TOP1) inhibitor payload. A phase 1/2 study of HER3-DXd (NCT02980341) demonstrated promising antitumor activity in hormone receptor positive (HR+) metastatic BC patients with clinical activity observed across baseline levels of HER3 protein or mRNA expression. A window of opportunity clinical trial is currently ongoing to evaluate the biological activity of HER3-DXd in patients with treatment naïve BC according to HER3 mRNA/protein expression levels (NCT04610528). Here, we aimed to describe the activity of HER3-DXd in PDX models to identify robust biomarkers of response. Methods: The antitumor activity of HER3-DXd was assessed in 21 BC PDX models (14 HR+ and 7 triple negative). HER3-DXd sensitivity was established as a complete response that lasted longer than 120 days, following 4 weekly doses of 10 mg/kg. HER3-DXd antitumor activity was compared to the antitumor activity of irinotecan (50 mg/kg dosed once weekly), which was evaluated according to modified RECIST criteria in a subset of 14 BC PDX models. PDX models of acquired resistance to HER3-DXd were generated by repeated treatment cycles. Pharmacodynamic (PD) experiments were conducted by collecting tumor samples from PDXs after a single dose of HER3-DXd or irinotecan. Baseline HER3 expression was assessed by immunohistochemistry (IHC) and Western blot. mRNA expression of 72 genes including ERBB3 and genes from the PAM50 signature were measured using the nCounter platform. Two-class unpaired significance analysis of microarrays (SAM), using a false-discovery rate Citation Format: Andreu Òdena, Laia Monserrat, Fara Brasó-Maristany, Marta Guzmán, Judit Grueso, Olga Rodríguez, Maurizio Scaltriti, Sarat Chandarlapaty, Yang Qiu, Kumiko Koyama, Mafalda Oliveira, Aleix Prat, Violeta Serra. Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-14.

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Response to AZD5363 in PDX models recapitulates patients' response

Supplementary Figure 1. FGFR1-4 DNA CN, mRNA levels and ROC curves analysis. A) Waterfall plot representing the best antitumor response of FGFRi in 24 patients from 8 different cancer types treated at VHIO. FGFR1/2 amplification status and high FGFR1-4 mRNA levels are indicated in the underneath panel. B) Copy number (CN) values of FGFR1-4 by MSK-IMPACT in the FGFR/FGFamp PDX collection and categorized analysis in PDXs harboring CN amplification (CN>4), according to the response to rogaratinib (responders (SD and PR) and non-responders (PD)). C) Evaluation of FGFR1/4 CN ratio by FISH ratio (the dashed line indicates the CN ratio>2.2 that is considered amplified) and categorized analysis according to the response to rogaratinib. D) Correlation analysis between the CN values (X-axis) and mRNA levels (Y-axis) of FGFR1-4 obtained from MSK-IMPACT or nCounter, respectively. Pearson correlation coefficient (r2) is shown. E) Categorized analysis of FGFR1-4 mRNA composite biomarker according to the response to rogaratinib, obtained from HTG or qPCR data. Statistical test: Mann-Whitney U test. ROC curve analysis the FGFR1-4 mRNA composite biomarker, obtained from HTG or qPCR (AUC=0.89, both).

Supplementary Figure 2. FGFR1-3 protein expression in BC PDXs models. A) Western blot (WB) analysis of FGFR1 and FGFR3 protein expression in PDX models; CAL120 cells were used as positive control for FGFR1 expression. B) Clinical evolution of a refractory metastatic triple negative breast cancer (mTNBC) patient -Pt306-, whose primary tumor harbored a high-level FGFR2amp (CN 21.8) and co-amplification of 11q13 (CN 7.0), assessed by FISH. Pt achieved disease stabilization (SD) lasting 8 months: reduction in lung target lesions (TL, red arrow) and overall decrease in other lung non-target lesions (NTL, red circle). Primary tumor biopsy was available for further molecular characterization with IHC FGFR2. C) IHC images of FGFR2 in PDX models and the primary tumor Bx from Pt306, according to the model/patient response to FGFRi (PD or SD). D) Correlation analysis of FGFR1/2/3 mRNA levels vs. protein levels by WB for FGFR1/3 or IHC for FGFR2. FGFR1/3 were quantified with ImageJ (normalized by tubulin levels) and FGFR2 with QuPath and ImageJ. Statistical test: Pearson correlation coefficient (r2) is shown.

PDF file, 523KB, Cell cycle analysis of trastuzumab/lapatinib resistant cells.

PARP inhibitors (PARPi) are approved drugs for platinum-sensitive, high-grade serous ovarian cancer (HGSOC) and for breast, prostate, and pancreatic cancers (PaC) harboring genetic alterations impairing homologous recombination repair (HRR). Detection of nuclear RAD51 foci in tumor cells is a marker of HRR functionality, and we previously established a test to detect RAD51 nuclear foci. Here, we aimed to validate the RAD51 score cut off and compare the performance of this test to other HRR deficiency (HRD) detection methods. Laboratory models from BRCA1/BRCA2-associated breast cancer, HGSOC, and PaC were developed and evaluated for their response to PARPi and cisplatin. HRD in these models and patient samples was evaluated by DNA sequencing of HRR genes, genomic HRD tests, and RAD51 foci detection. We established patient-derived xenograft models from breast cancer (n = 103), HGSOC (n = 4), and PaC (n = 2) that recapitulated patient HRD status and treatment response. The RAD51 test showed higher accuracy than HRR gene mutations and genomic HRD analysis for predicting PARPi response (95%, 67%, and 71%, respectively). RAD51 detection captured dynamic changes in HRR status upon acquisition of PARPi resistance. The accuracy of the RAD51 test was similar to HRR gene mutations for predicting platinum response. The predefined RAD51 score cut off was validated, and the high predictive value of the RAD51 test in preclinical models was confirmed. These results collectively support pursuing clinical assessment of the RAD51 test in patient samples from randomized trials testing PARPi or platinum-based therapies.Significance:This work demonstrates the high accuracy of a histopathology-based test based on the detection of RAD51 nuclear foci in predicting response to PARPi and cisplatin.

PDF file, 85KB.

RPPA data analysis

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Purpose:AZD5363/capivasertib is a pan-AKT catalytic inhibitor with promising activity in combination with paclitaxel in triple-negative metastatic breast cancer harboring PI3K/AKT-pathway alterations and in estrogen receptor–positive breast cancer in combination with fulvestrant. Here, we aimed to identify response biomarkers and uncover mechanisms of resistance to AZD5363 and its combination with paclitaxel.Experimental Design:Genetic and proteomic markers were analyzed in 28 HER2-negative patient-derived xenografts (PDXs) and in patient samples, and correlated to AZD5363 sensitivity as single agent and in combination with paclitaxel.Results:Four PDX were derived from patients receiving AZD5363 in the clinic which exhibited concordant treatment response. Mutations in PIK3CA/AKT1 and absence of mTOR complex 1 (mTORC1)-activating alterations, for example, in MTOR or TSC1, were associated with sensitivity to AZD5363 monotherapy. Interestingly, excluding PTEN from the composite biomarker increased its accuracy from 64% to 89%. Moreover, resistant PDXs exhibited low baseline pAKT S473 and residual pS6 S235 upon treatment, suggesting that parallel pathways bypass AKT/S6K1 signaling in these models. We identified two mechanisms of acquired resistance to AZD5363: cyclin D1 overexpression and loss of AKT1 p.E17K.Conclusions:This study provides insight into putative predictive biomarkers of response and acquired resistance to AZD5363 in HER2-negative metastatic breast cancer.

Purpose: PI3K pathway activation occurs in concomitance with RAS/BRAF mutations in colorectal cancer, limiting the sensitivity to targeted therapies. Several clinical studies are being conducted to test the tolerability and clinical activity of dual MEK and PI3K pathway blockade in solid tumors.Experimental Design: In the present study, we explored the efficacy of dual pathway blockade in colorectal cancer preclinical models harboring concomitant activation of the ERK and PI3K pathways. Moreover, we investigated if TP53 mutation affects the response to this therapy.Results: Dual MEK and mTORC1/2 blockade resulted in synergistic antiproliferative effects in cell lines bearing alterations in KRAS/BRAF and PIK3CA/PTEN. Although the on-treatment cell-cycle effects were not affected by the TP53 status, a marked proapoptotic response to therapy was observed exclusively in wild-type TP53 colorectal cancer models. We further interrogated two independent panels of KRAS/BRAF- and PIK3CA/PTEN-altered cell line– and patient-derived tumor xenografts for the antitumor response toward this combination of agents. A combination response that resulted in substantial antitumor activity was exclusively observed among the wild-type TP53 models (two out of five, 40%), but there was no such response across the eight mutant TP53 models (0%). Interestingly, within a cohort of 14 patients with colorectal cancer treated with these agents for their metastatic disease, two patients with long-lasting responses (32 weeks) had TP53 wild-type tumors.Conclusions: Our data support that, in wild-type TP53 colorectal cancer cells with ERK and PI3K pathway alterations, MEK blockade results in potent p21 induction, preventing apoptosis to occur. In turn, mTORC1/2 inhibition blocks MEK inhibitor–mediated p21 induction, unleashing apoptosis. Clin Cancer Res; 21(24); 5499–510. ©2015 AACR.

Supplementary Table from Preclinical In Vivo Validation of the RAD51 Test for Identification of Homologous Recombination-Deficient Tumors and Patient Stratification

Supplementary Data from Preclinical In Vivo Validation of the RAD51 Test for Identification of Homologous Recombination-Deficient Tumors and Patient Stratification

Table S1. MiSeq 57-gene panel list

Supplementary Table S2 HTG OBP panel raw data

PDF file, 233KB, In vitro cell death induced by lapatinib and INK-128 of trastuzumab/lapatinib resistant cell lines.

Low pAkt S473 is associated with resistance to AZD5363 monotherapy

PDF file, 281KB, Trastuzumab treatment in trastuzumab-resistant patient derived-xenografts.

PIK3R1 mutation in head and neck squamous cell carcinoma (HNSCC) PDX and activation of AKT/mTORC1-pathway in two AKT1 p.E17K-mutant PDXs determine sensitivity to AZD5363

PDF file, 593KB, In vitro cell death induced by lapatinib and INK-128 of trastuzumab/lapatinib resistant cell lines.

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2–mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1–S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1–CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301–13. ©2016 AACR.

Analysis of relevant signaling pathways, proliferation, apoptosis and S-phase entry in relation with AZD5363 resistance

Table S3. Molecular characteristics of colorectal cancer cell lines and PDX (VHIO panel)

Supplementary Figure and Table Legends. The text describes the panels and tables included as Supplementary Figure and Table Legends

Purpose: The PI3K/Akt/mTOR pathway is an attractive target in HER2-positive breast cancer that is refractory to anti-HER2 therapy. The hypothesis is that the suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance.Experimental Design: We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR, and extracellular signal-regulated kinase (ERK) signaling pathways was evaluated by Western blotting. Tumor growth inhibition after treatment with lapatinib, INK-128, or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed on trastuzumab-based therapy.Results: The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade produced synergistic induction of cell death in five different HER2-positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line–based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, which resulted in durable tumor shrinkage and exhibited no signs of toxicity in these models.Conclusions: The simultaneous blockade of both PI3K/Akt/mTOR and ERK pathways obtained by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer models refractory to anti-HER2 therapy. Clin Cancer Res; 18(9); 2603–12. ©2012 AACR.

Table S4. Molecular characteristics of colorectal cancer PDX (IRCC panel)

Supplementary Materials and Methods

Figure S2. Immunohistochemistry of p53 in tumors from CRC patients treated with MEK- plus PI3K-inhibitors

Supplementary Figure Legends

Figure S3. p53 proficiency is required to induce apoptosis in CRC cell lines and tumors upon anti MEK- and mTOR-inhibition

PDF file, 714KB, Combination index (CI) of the combination of lapatinib and INK-128 in resistant cell lines.

Supplementary Figure 5. Relative expression levels of lucitanib-specific RTK targets and ligands in PDX and patient tumors. A) mRNA expression levels of 2544 genes detected by HTG in Pt325 samples (Pre-tt versus On-tt). Highlighted are some differentially expressed genes between both samples, including FGFR1. Data is log-2 transformed. B) and C) Relative mRNA levels of lucitanib-specific RTK targets or cognate ligands in PDX and patient tumors from the MTKI/FGFRi-treated patient cohort from Figure 3D, respectively.

Table S2. Primer pairs for the relevant actionable genes in this study

Supplementary Table S1 PDXs subtype, genotype and drug efficacy data

Supplementary Figure Legends

PDF file, 760KB, Biochemical analyses of trastuzumab/lapatinib sensitive cells treated with lapatinib.