ER+ metastatic breast cancer targeted therapy: biomarkers of response and mechanisms of resistance to PI3K and FGFR inhibitors

La teràpia endocrina és un dels majors avenços en el tractament de càncer de mama (CM) dels últims 30 anys. Tot i això, les pacients acaben recaient degut a mecanismes de resistència, com ara les alteracions de la via de PI3K/mTOR i de FGFR. La via de PI3K està freqüentment alterada en CM, especialment en el conjunt luminal on el 30-40% de les pacients tenen mutacions al gen PIK3CA. S’han desenvolupat inhibidors específics d’mTOR i de PI3K (PI3Kinh), i alguns han sigut aprovats per la FDA per a CM ER+ metastàtic. Tanmateix, mecanismes de resistència han estat limitant la resposta. En aquest sentit, alteracions posteriors o en paral·lel a PI3K, com la amplificació de la regió 11q13, són recurrents en CM metastàtic i és important esbrinar si aquestes confereixen resistència a PI3Kinh. L’amplificació de FGFR1 es troba en 10% dels casos de CM metastàtic. Inhibidors específics contra FGFR1/2/3 (FGFRinh) han mostrat una eficàcia limitada en el conjunt d’amplificats de FGFR1. D’altra banda, els inhibidors multi-tirosina quinasa (MTKI, específics per a les famílies de FGFR, VEGFR i PDFGR) han mostrat una millor eficàcia però amb més toxicitat. En aquest estudi, hem investigat mecanismes de resistència a PI3Kinh i biomarcadors de resposta a FGFRinh. En la primera part, hem determinat l’activitat antitumoral d’alpelisib, un PI3Kinh específic per a la isoforma α, en una cohort de 24 xenògrafts derivats de tumor de pacient (PDX). Hem detectat una agregació d’alteracions posteriors a PI3K i amplificacions en l’11q13 entre els models resistents i les hem validat en models in vitro, incloent la sobreexpressió de ciclina D1 i FGFs. A més, 2 de 3 models PDX amb alteracions possiblement activadores d’mTORC1 han mostrat una eficàcia superior amb l’inhibidor d’mTORC1/2 (mTORC1/2inh) en comparació amb l’PI3Kinh. En la segona part d’aquesta tesi hem analitzat l’activitat antitumoral d’un FGFRinh, rogaratinib, en una cohort de 17 PDXs que contenen amplificacions en FGFR1/4 i/o FGF3/4/19 (l’amplicó 11q13). D’entre els potencials biomarcadors de resposta analitzats (nombre de còpies de gen de FGFR1-4, expressió gènica de FGFR1-4, i co-amplificació i expressió de lligands FGF), hem identificat que alts nivells de mRNA de FGFR1-4 prediuen la resposta a FGFRinh. Hem comparat l’eficàcia de rogaratinib amb un MTKI, lucitanib, on hem observat una millor eficàcia. A més, hem observat un elevat bloqueig de proliferació i vascularització amb lucitanib. Testant teràpia antiangiogènica en dos models resistents a rogaratinib, hem observat nivells similars d’eficàcia al lucitanib, suggerint que la resposta a lucitanib és deguda al bloqueig vascular en ambdós models. En conclusió, hem determinat que models que contenen alteracions activadores d’mTORC1 (e.g. TSC1del) són resistents a PI3Kinh però sensibilitzen a mTORC1/2inh i que la sobreexpressió de ciclina D1, FGF i ambdues alteracions generen resistència a PI3Kinh. Tanmateix, hem determinat que nivells alts d’mRNA de FGFR1-4 prediuen la resposta a FGFRinh i que els MTKI presenten millor eficàcia degut a un elevat bloqueig de la proliferació i la vasculatura. Creiem que aquests resultats ajudaran a millorar la selecció de pacients en futurs assajos clínics de teràpia dirigida a CM ER+.Endocrine therapy has been one of the major advances in the treatment of breast cancer (BC) for the past 30 years. However, patients eventually relapse due to mechanisms of resistance. These involve several pathways related to proliferation and growth, including the PI3K/mTOR and FGFR pathways. The PI3K pathway is frequently altered in BC, especially in the luminal setting where 30-40% of patients harbor PIK3CA mutations. Specific inhibitors targeting key nodes in the PI3K pathway have been developed, such as mTOR and PI3K inhibitors (PI3Kinh). Clinical trials combining these inhibitors with endocrine therapy showed improved responses, leading to FDA approval of some of these inhibitors for metastatic ER+ BC. However, mechanisms of resistance are a major concern. In this sense, alterations downstream PI3K or in parallel pathways are recurrent in the metastatic disease, and it is important to understand if they confer resistance to PI3K inhibitors. Another common alteration in luminal BC is the amplification of the 11q13 amplicon, containing important genes for proliferation such as CCND1, FGF3/4/19, PAK1 and RPS6KB2, whose impact on PI3K sensitivity is unknown. FGFR1 amplification is found in 10% of metastatic luminal BC. Specific inhibitors against FGFR1/2/3 (FGFRinh) have shown limited efficacy in the FGFR1amp BC. This is in contrast with the clinical efficacy observed previously with Multi-targeted Tyrosine Kinase Inhibitors (MTKI, targeting FGFR, VEGFR and PDGFR families), albeit these agents showed severe adverse effects. In this study, we investigated mechanisms of resistance to PI3Kinh and biomarkers of response to FGFRinh. In the first part, we determined the antitumoral activity of alpelisib, an α-specific PI3Kinh, in a cohort of 24 genetically annotated Patient-Derived Xenografts (PDX). We observed an aggregation of genetic alterations downstream PI3K and 11q13 amplifications among the resistant models and validated these with in vitro models as mechanisms of resistance to alpelisib, including overexpression of cyclin D1 and high FGF-signaling. In addition, in 2 out of 3 PDX models with putative mTORC1 activating alterations, we observed higher efficacy of an mTORC1/2 inhibitor (mTORC1/2inh) compared with a PI3Kinh. In the second part of this thesis we analyzed the antitumor activity of an FGFRinh, rogaratinib, in a cohort of 17 PDX harboring amplifications in FGFR1/4 and/or FGF3/4/19 (11q13 amplicon). Amongst the potential biomarkers of response tested (FGFR1-4 gene copy number, FGFR1-4 gene expression, and FGF ligand co-amplification and expression), we identified that high mRNA of FGFR1-4 could predict for FGFRinh response. We compared rogaratinib efficacy with a MTKI, lucitanib, and we observed higher efficacy of lucitanib across 7 models. We further analyzed the mode of action of rogaratinib vs. lucitanib and we observed an increased blockade of proliferation and vascularization under lucitanib treatment. However, when testing an antiangiogenic therapy in two rogaratinib-resistant models, similar levels of efficacy as with lucitanib monotherapy were achieved, suggesting that lucitanib efficacy was due to the blockade of the vascularization in both models. In conclusion, we established that PDX models harboring genetic alterations consistent with mTORC1 activation (e.g. TSC1del) are resistant to PI3Kinh but sensitive to an mTORC1/2inh and that cyclin D1 overexpression, high FGF and both alterations together result in resistance to PI3Kinh. We further determined that high mRNA of FGFR1-4 predicts for FGFRinh response and that a MTKI exhibits higher efficacy than specific FGFRinh due to a higher blockade of proliferation and vascularization. These results will help for a better patient selection in future clinical trials for ER+ BC targeted therapy

The impact and function of C-terminal degrons and protein misfolding in the degradation of truncated proteins

Curs 2019-2020Premature Termination Codons (PTCs) are mainly generated after nonsense and frameshift alterations. The Nonsense Mediated Decay (NMD) system detects and degrades mRNAs containing PTCs. However, the efficiency of this surveillance mechanism is limited and some PTC-containing mRNAs can escape from degradation, thus potentially generating truncated protein forms. The Ubiquitin Proteasome System (UPS) is involved in the degradation of proteins either by detecting specific amino acid sequence motifs (degrons) or by detecting misfolding features of the protein. Degrons located at the c-terminal region of proteins (c-degrons) potentiate a rapid degradation of proteins, however, their impact on the degradation of proteins with truncated c-terminus remains unknown. In this study, we employed two cancer datasets as natural experiments to study the degradation of truncated proteins: a dataset of more than 600 primary tumor biopsies from the Clinical Proteomics Tumor Analysis Consortium (CPTAC), and a dataset with more than 300 cancer-derived cell lines from CCLE with integrated genome, transcriptome and proteome information. In order to select those mutations producing truncated proteins, we annotated the NMD efficiency probability of all the PTCs in both datasets and classified them between NMD-skipping or NMD-triggering. To our surprise, not only NMD-skipping but also NMD-triggering showed decreased protein stability. We annotated all c-degrons instances in the predicted truncated proteins and analysed the changes in protein stability, but no significant differences nor tendencies were observed. We further explored whether the length of the truncated protein could impact protein stability and yet that was the case for NMD-skipping protein products but not for NMD-triggering, where protein stability decrease was independent of the mutation localization in the protein. In conclusion, c-degrons failed to explain the overall protein stability decrease of truncated proteins. However, major protein sequence loss increases destabilisation but only for proteins from NMD-skipping PTCs. Results presented in this study suggest that truncated proteins could follow different degradation pathways depending on NMD efficiency.Director/a: Serrat Jurado, Josep Mari

ER+ metastatic breast cancer targeted therapy: biomarkers of response and mechanisms of resistance to PI3K and FGFR inhibitors /

Departament responsable de la tesi: Departament de Bioquímica i Biologia Molecular.La teràpia endocrina és un dels majors avenços en el tractament de càncer de mama (CM) dels últims 30 anys. Tot i això, les pacients acaben recaient degut a mecanismes de resistència, com ara les alteracions de la via de PI3K/mTOR i de FGFR.La via de PI3K està freqüentment alterada en CM, especialment en el conjunt luminal on el 30-40% de les pacients tenen mutacions al gen PIK3CA. S'han desenvolupat inhibidors específics d'mTOR i de PI3K (PI3Kinh), i alguns han sigut aprovats per la FDA per a CM ER+ metastàtic. Tanmateix, mecanismes de resistència han estat limitant la resposta. En aquest sentit, alteracions posteriors o en paral·lel a PI3K, com la amplificació de la regió 11q13, són recurrents en CM metastàtic i és important esbrinar si aquestes confereixen resistència a PI3Kinh.L'amplificació de FGFR1 es troba en 10% dels casos de CM metastàtic. Inhibidors específics contra FGFR1/2/3 (FGFRinh) han mostrat una eficàcia limitada en el conjunt d'amplificats de FGFR1. D'altra banda, els inhibidors multi-tirosina quinasa (MTKI, específics per a les famílies de FGFR, VEGFR i PDFGR) han mostrat una millor eficàcia però amb més toxicitat.En aquest estudi, hem investigat mecanismes de resistència a PI3Kinh i biomarcadors de resposta a FGFRinh. En la primera part, hem determinat l'activitat antitumoral d'alpelisib, un PI3Kinh específic per a la isoforma α, en una cohort de 24 xenògrafts derivats de tumor de pacient (PDX). Hem detectat una agregació d'alteracions posteriors a PI3K i amplificacions en l'11q13 entre els models resistents i les hem validat en models in vitro, incloent la sobreexpressió de ciclina D1 i FGFs. A més, 2 de 3 models PDX amb alteracions possiblement activadores d'mTORC1 han mostrat una eficàcia superior amb l'inhibidor d'mTORC1/2 (mTORC1/2inh) en comparació amb l'PI3Kinh.En la segona part d'aquesta tesi hem analitzat l'activitat antitumoral d'un FGFRinh, rogaratinib, en una cohort de 17 PDXs que contenen amplificacions en FGFR1/4 i/o FGF3/4/19 (l'amplicó 11q13). D'entre els potencials biomarcadors de resposta analitzats (nombre de còpies de gen de FGFR1-4, expressió gènica de FGFR1-4, i co-amplificació i expressió de lligands FGF), hem identificat que alts nivells de mRNA de FGFR1-4 prediuen la resposta a FGFRinh. Hem comparat l'eficàcia de rogaratinib amb un MTKI, lucitanib, onhem observat una millor eficàcia. A més, hem observat un elevat bloqueig de proliferació i vascularització amb lucitanib. Testant teràpia antiangiogènica en dos models resistents a rogaratinib, hem observat nivells similars d'eficàcia al lucitanib, suggerint que la resposta a lucitanib és deguda al bloqueig vascular en ambdós models.En conclusió, hem determinat que models que contenen alteracions activadores d'mTORC1 (e.g. TSC1del) són resistents a PI3Kinh però sensibilitzen a mTORC1/2inh i que la sobreexpressió de ciclina D1, FGF i ambdues alteracions generen resistència a PI3Kinh. Tanmateix, hem determinat que nivells alts d'mRNA de FGFR1-4 prediuen la resposta a FGFRinh i que els MTKI presenten millor eficàcia degut a un elevat bloqueig de la proliferació i la vasculatura. Creiem que aquests resultats ajudaran a millorar la selecció de pacients en futurs assajos clínics de teràpia dirigida a CM ER+.Endocrine therapy has been one of the major advances in the treatment of breast cancer (BC) for the past 30 years. However, patients eventually relapse due to mechanisms of resistance. These involve several pathways related to proliferation and growth, including the PI3K/mTOR and FGFR pathways. The PI3K pathway is frequently altered in BC, especially in the luminal setting where 30-40% of patients harbor PIK3CA mutations. Specific inhibitors targeting key nodes in the PI3K pathway have been developed, such as mTOR and PI3K inhibitors (PI3Kinh). Clinical trials combining these inhibitors with endocrine therapy showed improved responses, leading to FDA approval of some of these inhibitors for metastatic ER+ BC. However, mechanisms of resistance are a major concern. In this sense, alterations downstream PI3K or in parallel pathways are recurrent in the metastatic disease, and it is important to understand if they confer resistance to PI3K inhibitors. Another common alteration in luminal BC is the amplification of the 11q13 amplicon, containing important genes for proliferation such as CCND1, FGF3/4/19, PAK1 and RPS6KB2, whose impact on PI3K sensitivity is unknown. FGFR1 amplification is found in 10% of metastatic luminal BC. Specific inhibitors against FGFR1/2/3 (FGFRinh) have shown limited efficacy in the FGFR1amp BC. This is in contrast with the clinical efficacy observed previously with Multi-targeted Tyrosine Kinase Inhibitors (MTKI, targeting FGFR, VEGFR and PDGFR families), albeit these agents showed severe adverse effects. In this study, we investigated mechanisms of resistance to PI3Kinh and biomarkers of response to FGFRinh. In the first part, we determined the antitumoral activity of alpelisib, an α-specific PI3Kinh, in a cohort of 24 genetically annotated Patient-Derived Xenografts (PDX). We observed an aggregation of genetic alterations downstream PI3K and 11q13 amplifications among the resistant models and validated these with in vitro models as mechanisms of resistance to alpelisib, including overexpression of cyclin D1 and high FGF-signaling. In addition, in 2 out of 3 PDX models with putative mTORC1 activating alterations, we observed higher efficacy of an mTORC1/2 inhibitor (mTORC1/2inh) compared with a PI3Kinh. In the second part of this thesis we analyzed the antitumor activity of an FGFRinh, rogaratinib, in a cohort of 17 PDX harboring amplifications in FGFR1/4 and/or FGF3/4/19 (11q13 amplicon). Amongst the potential biomarkers of response tested (FGFR1-4 gene copy number, FGFR1-4 gene expression, and FGF ligand co-amplification and expression), we identified that high mRNA of FGFR1-4 could predict for FGFRinh response. We compared rogaratinib efficacy with a MTKI, lucitanib, and we observed higher efficacy of lucitanib across 7 models. We further analyzed the mode of action of rogaratinib vs. lucitanib and we observed an increased blockade of proliferation and vascularization under lucitanib treatment. However, when testing an antiangiogenic therapy in two rogaratinib-resistant models, similar levels of efficacy as with lucitanib monotherapy were achieved, suggesting that lucitanib efficacy was due to the blockade of the vascularization in both models. In conclusion, we established that PDX models harboring genetic alterations consistent with mTORC1 activation (e.g. TSC1del) are resistant to PI3Kinh but sensitive to an mTORC1/2inh and that cyclin D1 overexpression, high FGF and both alterations together result in resistance to PI3Kinh. We further determined that high mRNA of FGFR1-4 predicts for FGFRinh response and that a MTKI exhibits higher efficacy than specific FGFRinh due to a higher blockade of proliferation and vascularization. These results will help for a better patient selection in future clinical trials for ER+ BC targeted therapy

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