Targeting mTOR to overcome resistance to hormone and CDK4/6 inhibitors in ER-positive breast cancer models

Cancer; Cell biologyCàncer; Biologia cel·lularCáncer; Biología celularResistance to therapy remains a major obstacle in cancer management. Although treatment with hormone and CDK4/6 inhibitors is successful in luminal breast cancer, resistance to these treatments is frequent, highlighting the need for novel therapeutic strategies to delay disease progression and improve patient survival. Here, we assessed the mechanisms of acquired resistance using T47D and MCF-7 tamoxifen- and palbociclib-resistant cell-line variants in culture and as xenografts, and patient-derived cells (PDCs) obtained from sensitive or resistant patient-derived xenografts (PDXs). In these models, we analyzed the effect of specific kinase inhibitors on survival, signaling and cellular aggressiveness. Our results revealed that mTOR inhibition is more effective than PI3K inhibition in overcoming resistance, irrespective of PIK3CA mutation status, by decreasing cell proliferation and tumor growth, as well as reducing cell migration and stemness. Moreover, a combination of mTOR and CDK4/6 inhibitors may prevent pathway reactivation downstream of PI3K, interfering with the survival of resistant cells and consequent tumor escape. In conclusion, we highlight the benefits of incorporating mTOR inhibitors into the current therapy in ER + breast cancer. This alternative therapeutic strategy not only enhances the antitumor response but may also delay the emergence of resistance and tumor recurrence.This work was supported by CONICET, ANPCYT (Grants PICT2509 & PICT0345), Instituto Nacional del Cáncer (Grants 2016 & 2018), Fundación Williams, Fundación Bunge & Born (Oster Grant) (Argentina); Instituto de Salud Carlos III (Grants PI20/00892 & CPII19/0033), Ministerio de Economía y Competitividad (FJCI-2015-25412) (Spain)

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors

Proteïnes supressores de tumors; Inhibidors de la quinasaProteínas supresoras de tumores; Inhibidores de la quinasaTumor suppressor proteins; Kinase inhibitorsCyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C–cyclin D–CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. Significance: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6–INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.The Chandarlapaty lab has received generous funding support for this research from the Cancer Couch Foundation, the Shen Family Fund, the Smith Fund for Cancer Research, the Breast Cancer Research Foundation, an NIH Cancer Center Support Grant (P30 CA008748), and NIH R01234361. Q. Li has received support from Translational Research Oncology Training Fellowship (MSKCC) made possible by the generous contribution of First Eagle Investment Management. V. Serra reports grants from the Susan G. Komen Foundation (CCR15330331) and Instituto de Salud Carlos III (CPII19/00033) during the conduct of the study and grants from Novartis, Genentech, and AstraZeneca outside the submitted work. The Chodera laboratory receives or has received funding from multiple sources, including the NIH and an NIH Cancer Center Support Grant (P30 CA008748), the National Science Foundation, the Parker Institute for Cancer Immunotherapy, Relay Therapeutics, Entasis Therapeutics, Silicon Therapeutics, EMD Serono (Merck KGaA), AstraZeneca, Vir Biotechnology, Bayer, XtalPi, Foresite Laboratories, the Molecular Sciences Software Institute, the Starr Cancer Consortium, the Open Force Field Consortium, Cycle for Survival, a Louis V. Gerstner Young Investigator Award, and the Sloan Kettering Institute. J. Guo acknowledges support from NIH grant R01 GM121505. J.D. Chodera acknowledges support from NIH grant P30 CA008748, NIH grant R01 GM121505, and NIH grant R01 GM132386. A complete funding history for the Chodera lab can be found at http://choderalab.org/funding, including complete funding information and grant numbers. The authors thank Dr. Marie Will and Madeline Dorso for helpful comments on the manuscript and Dr. Zhan Yao for helpful advice on the kinase assays

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

Cáncer de mama y de ovario; Inhibición WEE1Càncer de mama i d'ovari; Inhibició WEE1Breast and ovarian cancer; WEE1 inhibitionPurpose: 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.This work was supported by the Spanish Instituto de Salud Carlos III (ISCIII), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds (FIS PI17/01080 to V. Serra, PI12/02606 to J. Balmaña); European Research Area-NET, Transcan-2 (AC15/00063), Asociación Española contra el Cáncer (AECC; LABAE16020PORTT), the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR; 2017 SGR 540), La Marató TV3 (654/C/2019), and ERAPERMED2019–215 to V. Serra. We also acknowledge the GHD-Pink program, the FERO Foundation, and the Orozco Family for supporting this study (to V. Serra). V. Serra was supported by the Miguel Servet Program (ISCIII; CPII19/00033); M. Castroviejo-Bermejo and C. Cruz (AIOC15152806CRUZ) by AECC; A. Herencia-Ropero by Generalitat de Catalunya-PERIS (SLT017/20/000081); M. Palafox by Juan de la Cierva (FJCI-2015–25412); A. Lau by AECC and Generalitat de Catalunya-PERIS (INVES20095LLOP, SLT002/16/00477); A. Gris-Oliver by FI-AGAUR (2015 FI_B 01075). This work was supported by Breast Cancer Research Foundation (BCRF-19–08), Instituto de Salud Carlos III Project Reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (to J. Arribas). The xenograft program in the Caldas laboratory was supported by Cancer Research UK and also received funding from an EU H2020 Network of Excellence (EuroCAN). The RPPA facility is funded by NCI #CA16672

High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER+ breast cancer

Breast cancer; Cancer models; Predictive markersCáncer de mama; Modelos de cáncer; Marcadores predictivosCàncer de pulmó; Models de càncer; Marcadors predictiusCDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies

Resistance to taxanes in triple negative breast cancer associates with the dynamics of a CD49f+ tumor initiating population

Taxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer

Estudio de la vía de señalización de RANK y RANKL en células de mama humanas y generación de ortoxenopacientes de càncer de mama

[spa] Este proyecto de tesis doctoral se ha centrado en tres objetivos principales: el primero es el estudio de la función de la vía de señalización de RANK-RANKL-OPG en el desarrollo y progresión del cáncer de mama en humanos. El segundo es el análisis de la expresión de RANK en muestras de tumores humanos y análisis modificaciones en el gen que afecten en la incidencia de cáncer de mama en humanos. El tercero es la generación de modelos ortotópicos de ratón de cáncer de mama humano que sirvan como herramienta para la caracterización de nuevas dianas terapéuticas así como para testar la eficacia de nuevas drogas dirigidas a pacientes de cáncer de mama resistente a los tratamientos ya existentes. Respecto al primero objetivo, los resultados obtenidos indican que la sobre-expresión de RANK en células epiteliales de mama humanas no transformadas MCF10A induce la adquisición de un fenotipo asociado al proceso de transición epitelio-mesenquina (EMT) así como la adquisición de un fenotipo característico de células madre. El mecanismo por el cual la sobre-expresión de RANK induce EMT está relacionado con el aumento de la producción del factor de crecimiento transformate beta (TGF-beta) mediante la activación de las vías de señalización del factor nuclear potenciador de las cadenas ligeras kappa de las células B activadas (NF-κB) y la proteína quinasa activada por mitógenos p38. La inducción del fenotipo de célula madre está asociado a la activación de vía de señalización de las quinasas reguladas por señales extracelulares 1 y 2 (ERK 1/2). Los resultados referentes al segundo objetivo indican que en muestras de pacientes humanos, los niveles de expresión de RANK son más elevados en tumores con un peor pronóstico clínico (fenotipo triple negativo, alto índice de proliferación y algo grado histológico) y mediante la expresión de los niveles de mARN de RANK y RANKL se puede diferenciar entre tumores metastáticos y no metastáticos a nódulo linfático. Además, se ha descrito la existencia de un polimorfismo de nucleótido simple (SNP) presente en la región 5´del gen de RANK que está asociado con una menor probabilidad de padecer cáncer de mama en pacientes que poseen mutaciones en el gen BRCA2 (“breast cancer 2”). Finalmente, en este trabajo se describe la generación de cinco modelos ortotópicos de cáncer de mama a partir de muestras clínicas de pacientes. Estos modelos mantienen las características histológicas de los tumores humanos de origen así como los patrones de metástasis de los pacientes de los que proceden. De los cinco modelos generados, dos tienen unas características histológicas relativas a tumores triples negativos (TNBC), dos a tumores luminales (positivos para la expresión del receptor de estrógenos y progesterona) y uno a tumor Her2+. Los tumores Luminales y Her2+ manifiestan diferentes comportamientos de crecimiento en respuesta a hormonal, siendo un reflejo de los distintos tipos de comportamientos que existen en clínica. Por último, se ha testado la sensibilidad de estos modelos a taxanos (Docetaxel), una de las drogas quimioterapéuticas mas empleadas en la actualidad. Los dos modelos TNBC muestran más sensibilidad a la droga mientras que los modelos hormono-dependientes son resistentes. A partir de un modelo sensible TNBC se ha desarrollado un segundo modelo de ratones resistente a Docetaxel mediante la administración continuada del fármaco. Lo que se pretende en futuros proyectos es emplear estos modelos generados en el estudio de nuevas dianas terapéuticas en cáncer de mama así como testar nuevos fármacos en su fase pre-clínica de desarrollo.[eng] This study has been focused in three main goals: study of RANK and RANKL pathway in the development and progression of human breast cancer; analysis of RANK expression in clinical human samples and analysis in RANK gene modifications implicated in human breast cancer; generation of orthoxenopatients of human breast cancer. The results of first objective showed RANK over-expression in human mammary cells MCF10A induces epithelial-mesenchymal transition (EMT) and Stemness phenotype. The molecular mechanism though RANK induces these effects implicates the increment of transforming growth factor beta (TGF-beta) ligands expression thorugh activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen activated kinase protein p38. On the other hand, the induction of Stemnes phenotype implicates the activation of Extracellular signal-regulated kinases 1/2 (ERK 1/2). The analysis of RANK expression in clinical human samples showed that tumor with a poor prognosis (such as triple negative tumors, tumors with high proliferative index and high histological grade) has higher levels of RANK expression. Moreover, through analysis of RANK and RANKL expression is able discriminate metastatic and non metastatic tumors. In addition, a lower probability to undergo breast cancer in patients with mutations in BRCA2 gene has been correlated with a SNP in the 5´region of RANK gene. Finally, five models of ortothopic mouse models of human breast cancer have been generated. The models obtained were two triple negative, two luminal and one Her2+. These models resemble de histological characteristics of original tumors and the metastasis patterns of the patients. Also the growth rate, latency and dependence to hormones were evaluated in these models. The sensitivity of these tumors was tested to Docetaxel. The triple negative tumors were sensitive whereas Luminal and Her2+ tumors were resistant to Docetaxel. Moreover, resistant models to Docetaxel were generated from sensitive models by continued Docetaxel administration. Samples were collected during the process in order to investigate the mechanisms implicated in resistance acquisition to Decetaxel in the future

Dual Fatty Acid Synthase and HER2 Signaling Blockade Shows Marked Antitumor Activity against Breast Cancer Models Resistant to Anti-HER2 Drugs

Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti- HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockad

High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER+ breast cancer.

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies

Resistance to taxanes in triple negative breast cancer associates with the dynamics of a CD49f+ tumor initiating population

Taxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer

Resistance to taxanes in triple negative breast cancer associates with the dynamics of a CD49f+ tumor initiating population

Taxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer