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Background: Novel effective and safe therapies are required for advanced TNBC after progression to standard-of-care first line treatment with anti-PD-1/L1 + chemotherapy. We have found that the most aggressive TNBC variants are driven by a heterogeneous set of genetic aberrations that converge in the increased activity of 6 kinases: KIT, PNKP, PRKCE, P70S6K, ERK and CDK6 (Nat Commun; 9:3501-18). Although the inhibition of each kinase in monotherapy yielded little efficacy in preclinical models, the combinations targeting pairs of the former kinases led to therapeutic synergy in most cases. The combined inhibition of CDK6 and ERK led to 5-fold increase in overall survival in preclinical TNBC models (PDXs and spontaneous murine cancer models). The greatest activity was observed in models with increased activity of either CDK6 or ERK. Thus, we aimed to test the safety and preliminary efficacy of combined ERK and CDK6 inhibition with binimetinib and palbociclib in women with advanced TNBC with hyperactivation of ERK and/or CDK6. The combination has been preliminary tested in lung cancer, where a phase I dose-escalation trial established the RP2D in binimetinib 45 mg/BID plus Palbociclib 125 mg daily 21/7. Trial design: Single-arm, prospective, multicentric, open-label, phase IB/II trial with intra-patient dose-escalation. Patients candidate for pre-screening will have determined the activity of ERK and CDK6 with an in-house developed assay and acquisition algorithm at the central lab. Those testing positive for either marker will undergo full screening. Patients will start treatment with continuous oral binimetinib at 45 mg/BID and palbociclib 100 mg/day from days 1 to 21, in 28-day cycles. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity will be allowed to escalate to palbociclib to 125 in cycle 2. Fresh biopsies will be harvested at baseline and disease progression, in order to establish patient-derived organoids (PDOs) and perform WES. PBMCs will be harvested at baseline, +24 hours, and at the beginning of cycle 2, to study changes in the immunophenotype. RECIST 1.1 and NCI CTC AE V 5.0 criteria will be used for assessing disease control (q8 weeks) and toxicity. Eligibility criteria: Pre-screening: Women >18 year old diagnosed of advanced, non-curable TNBC; 2) who have received a minimum of 1 treatment line including immunotherapy; 3) no more than 2 treatment lines for advanced disease; 4) Adequate organ function and recovery from previous toxicity to < tolerable G2. Full screening: 5) Demonstration of hyper-activation of CDK6 and/or ERK in the tumor sample; 6) PD to the previous treatment regimen within the last 28 days. Specific aims: Primary: 1) To determine the progression-free survival time of the combination of binimetinib and palbociclib in TNBC patients with hyperactivation of ERK and/or CDK6 in second/third line 2) To assess the safety and tolerability of the former combination Secondary: 1) 6-month PFS rate, response rate, and overall survival of the combination 2) To study biomarkers of sensitivity and primary and acquired resistance to the combination taking advantage of PDOs 3) To determine the immunodynamics of the combination. Statistical methods: The null hypothesis is that the median PFS time for second/third line TNBC with best physician’s choice is 1.7 months (NEJM; 384:1529-41, 2021). With alpha and beta errors of 0.05 and 0.2, the minimum number of patients to demonstrate a 30% improvement in PFS to 2.5 months is 25. Preliminary data suggest that approximately 40% of the patients show hyper-activation of CDK6 and/or ERK; thus, and assuming a methodological failure of 10%, the minimum number of patients to screen is 69. Present accrual/target accrual: 27 screened of 69 planed; 11 accrued of 25 planned Citation Format: Luis Manso, Alfonso Cortes, Juan M Cejalvo, Serafin Morales, Jose A García Saenz, Ramon Colomer, Rodrigo Sanchez-Bayona, Jorge Silva, Juan A Guerra, Diego Malon, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Phase IB/II trial of palbociclib and binimetinib in advanced triple-negative breast cancer with hyperactivation of ERK and/or CDK4/6 [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 OT2-19-08.

Background: Novel therapies are needed upon progression to first-line CDK4/6 inhibitor (CDKi) plus endocrine therapy in advanced HR+ breast cancer. One frequent alteration driving resistance to CDKi plus hormones is FGFR1/2 amplification/overexpression. We have demonstrated that a combined assay of RNAScope and FISH, detecting RNA overexpression and genomic amplification of FGFR1/2, captures more FGFR-aberrant cases than either test alone. When these alterations exist, the triple estrogen receptor (ER), CDK4/6 and FGFR1/2 blockade is able to revert acquired resistance to CDKi (Breast Cancer Res; 23:21-37; 2021). Rogaratinib is a selective FGFR1-4 kinase inhibitor with significant activity in FGFR-aberrant malignancies. In this trial we will test the safety of the triple blockade combining fulvestrant, palbociclib and rogaratinib. We will also study its role as a resistant-reversion combo for FGFR1/2-positive, HR+ patients progressing to a CDK4/6 inhibitor plus aromatase inhibitor (AI) in the first-line metastatic setting. Trial design: Single-arm, prospective, multicentric, open-label, phase I dose-escalation trial. Patients will be pre-screened for amplification and/or overexpression of FGFR1/2 by FISH and RNAScope, during their first-line treatment. Positive patients will start treatment: on day 1, patients will receive rogaratinib q12 hours. On day 2, patients will continue on rogaratinib, receive fulvestrant (500 ug IM) and start on palbociclib (100 mg/day on days 1-21). A second fulvestrant dose (500 ug) will be administered in day +15. The first cycle will be 29 days, followed by 28-day cycles (continuous rogaratinib q12 hours days 1-28, fulvestrant 500 ug day 1, and palbociclib 100 mg/day days 1-21). Rogaratinib will be escalated, starting on 400 mg/bid in 200 mg/bid increments, following a classic 3+3 schedule. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity will be allowed to escalate palbociclib to 125 in cycle 2. A full pharmacokinetic profile and pharmacodynamics (plasma FGF23 and phosphate levels) will be performed on days 1 and 15 of cycle 1. The impact of the triple combination in immunodynamics will be assessed on PBMCs on days 2 and 15. Ten additional HR+, FGFR1/2 + patients will be accrued at the RP2D in an expansion cohort to study efficacy. RECIST 1.1 and NCI CTC AE V 5.0 criteria will be used for assessing disease control (q8 weeks) and toxicity. Eligibility criteria: Pre-screening: Women >18 year old diagnosed with advanced, non-curable HR+ breast cancer; 2) who are receiving first-line treatment with an AI and any oral CDKi; 3) Adequate organ function. Full screening: 4) Positivity for amplification (FISH ratio>2.2) and/or overexpression (RNAScope score of 3+ or 4+) of either FGFR1/2. 5) PD to first-line AI + CDKi within the last 28 days. 6) Recovery from previous toxicity to < tolerable G2. Specific aims: Primary: 1) To determine RP2D of the triple combination of fulvestrant, palbociclib and rogaratinib. 2) To assess the safety and toxicity of the former combination 3) To study the pharmacokinetics, pharmacodynamics and immunodynamics of the triplet. Secondary: 1) To determine the PFS time and 6-month PFS rate of the triplet in FGFR1/2 amplified/overexpressed patients AI + CDKi doublet. Statistical methods: We will follow a 3+3 escalation schedule; thus, the minimum and maximum number of patients for the dose-escalation phase will range between 6 and 18 (no escalation planned beyond 800 mg/bid rogaratinib). Ten additional patients will be accrued in the expansion cohort (N=28). The anticipated rate of positive screening is 40%; thus, we will screen approximately 70 patients. . Present accrual/target accrual: 55/70 screened; 16 FGFR1/2-positive (of 28 planned); 2 accrued and treated. Citation Format: Sonia Pernas, Cristina Hernando, Begoña Bermejo, Noelia Martinez-Jañez, Jose A García Saenz, Serafin Morales, Luis Manso, Jorge Silva, Juan A Guerra, Diego Malon, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Rogaratinib, palbociclib and fulvestrant in advanced hormone receptor-positive (HR+), FGFR1/2-positive breast cancer: Phase I trial plus an expansion cohort [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 OT2-20-01.

Biomarker; Hormone receptor-positive; Breast cancer Biomarcador; Cáncer de mama; Receptor hormonal positivo Biomarcador; Càncer de mama; Receptor hormonal positiu CDK4/6 inhibitors benefit a minority of patients who receive them in the breast cancer adjuvant setting. p27Kip1 is a protein that inhibits CDK/Cyclin complexes. We hypothesized that single-nucleotide polymorphisms that impaired p27Kip1 function could render patients refractory to endocrine therapy but responsive to CDK4/6 inhibitors, narrowing the patient subpopulation that requires CDK4/6 inhibitors. We found that the p27Kip1 V109G single-nucleotide polymorphism is homozygous in approximately 15% of hormone-positive breast cancer patients. Polymorphic patients experience rapid failure in response to endocrine monotherapy compared with wild-type or heterozygous patients in the first-line metastatic setting (progression-free survival: 92 vs 485 days, P

Supplementary material

Supplementary Figure 1: Microvessel confocal staining broken down by fluorescence channel

Purpose:We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Vascular normalization can be tracked with 2[18F]fluoro-2-deoxy-d-glucose (FDG)-PET. We tested the efficacy of the mitochondrial inhibitor ME-344 or placebo added to bevacizumab in early breast cancer.Patients and Methods:Treatment-naïve HER2-negative patients with T > 1 cm (any N) underwent a breast-centered 18F-fluorodeoxyglucose (FDG)-PET (day 1) and received a single dose of bevacizumab (15 mg/kg), followed by a second FDG-PET (day 8). Patients were then randomized (1:1) to Arm A (ME-344 10 mg/kg intravenous on days 8, 15, and 21) or Arm B (placebo). Tumors were biopsied on days 0 and 29. Succinate dehydrogenase enzyme histochemistry (SDH-EHC), confocal microscopy of vessel architecture, and HIF1α staining were performed in pre- and posttreatment biopsies to assess the pharmacodynamics, vessel normalization, and tissue re-oxygenation by bevacizumab, respectively.Results:ME-344 displayed significant biological activity versus placebo: compared with a 186% increase in Arm B, Ki67 decreased by 23.4% from days 0 to 28 in Arm A (P < 0.001) (N = 42 patients). FDG-PET predicted vascular normalization in about one-third of the patients in each arm, which was confirmed using confocal microscopy and HIF1α staining. In the subgroup with vascular normalization, ME-344 induced a Ki67 decrease of 33.4% (placebo: 11.8 increase). SDH-EHC suggested on-target effects of ME-344.Conclusions:ME-344 has significant biological antitumor activity in HER2-negative breast cancer, particularly after induction of vascular normalization and tissue reoxygenation with bevacizumab.

Among others, expression levels of programmed cell death 1 ligand 1 (PD‐L1) have been explored as biomarkers of the response to immune checkpoint inhibitors in cancer therapy. Here, we present the results of a chemical screen that interrogated how medically approved drugs influence PD‐L1 expression. As expected, corticosteroids and inhibitors of Janus kinases were among the top PD‐L1 downregulators. In addition, we identified that PD‐L1 expression is induced by antiestrogenic compounds. Transcriptomic analyses indicate that chronic estrogen receptor alpha (ERα) inhibition triggers a broad immunosuppressive program in ER‐positive breast cancer cells, which is subsequent to their growth arrest and involves the activation of multiple immune checkpoints together with the silencing of the antigen‐presenting machinery. Accordingly, estrogen‐deprived MCF7 cells are resistant to T‐cell‐mediated cell killing, in a manner that is independent of PD‐L1, but which is reverted by estradiol. Our study reveals that while antiestrogen therapies efficiently limit the growth of ER‐positive breast cancer cells, they concomitantly trigger a transcriptional program that favors their immune evasion., Under prolonged hormone therapy, ER+ breast cancer cells activate an inflammatory transcriptional program, which includes a generalized upregulation of immune checkpoint mediators together with the downregulation of the antigen‐presenting machinery. These findings reveal that, while hormone therapies efficiently arrest the growth of ER+ breast cancer cells, they also promote a phenotype switch that favors their immune evasion.

Background: CDK4/6 inhibitors benefit a limited percentage of hormone receptor-positive breast cancer (HRPBC) patients in the adjuvant setting: according to the MonarchE study, from all patients treated with the endocrine plus CDK4/6 inhibitor combination, 84% were adequately treated with endocrine therapy alone, ~5% experienced benefit from the combination, and 11% were not rescued from relapse by abemaciclib. Given the side effects and the cost, biomarkers to guide treatment decisions in this setting are appealing. We found that the p27Kip1 V109G SNP was enriched in HRPBC patients experiencing relapse despite endocrine treatment. p27Kip1 binds to cyclins and CDKs, restraining cells from cycling by inhibiting the formation of CDK/cyclin complexes and their kinase activity, resulting in less phosphorylation of Rb. A functionally impaired p27Kip1 could render tumor cells insensitive to endocrine therapy, while being rescued by CDK4/6 inhibitors. Thus, this SNP could narrow down the patient population that requires adjuvant CDK4/6 inhibitors. Methods: Isogenic HRPBC cell lines, wild-type or polymorphic homozygous for the p27Kip1 V109G SNP were generated with CRISPR-Cas9. Cell cycle and cell viability were assessed with BRDU incorporation and colony assays. Immunoprecipitation coupled with western blot (WB) was used to measure the formation of CDK/Cyclin complexes; Rb phosphorylation was assessed by WB. An in vitro kinase assay was set up to measure the CDK4 activity of p27Kip1/CDK/Cyclin complexes. Patients (n=115) with metastatic, HRPBC receiving endocrine monotherapy or in combination with CDK4/6 inhibitors were genotyped for the p27Kip1 V109G SNP, and PFS by genotype and therapy compared with the Kaplan-Meier method. All statistical tests were two-sided. Results: three isogenic polymorphic clones were generated from the wild-type T47-D hormone-positive cell line. The three clones were resistant to hormonal deprivation compared to wild-type cells. The relative plating efficiency (RPE) in the colony assays of the polymorphic clones exposed to hormonal deprivation compared to that of deprived T47-D cells was 550% (clone C1), 165% (clone E1) and 100% (Clone F5); P< 0.005. The three clones were also resistant to fulvestrant (Fulv) (300%, 170% and 180%, respectively); P< 0.005. Cell cycle (positive BRDU cells) decreased ~3 fold in wild type cells (18% to 6.5%) when exposed to hormonal deprivation or Fulv, but remained unaltered in the polymorphic clones. However, when palbociclib was added to hormonal deprivation or Fulv, the effects in RPE increased and were similar in polymorphic clones and parental cells (>5% RPE compared to vehicle, both in polymorphic and wild-type cells). The p27Kip1 V109G SNP was found in homozygosity in ~15% of metastatic HRPBC patients. When patients received endocrine monotherapy in the first-line setting, polymorphic patients experience rapid failure (N=51) compared to wild-type/heterozygous patients (4.3 vs. 21.1 months; P < 0.0001). However, when patients received hormonal plus CDK4/6 inhibitors, the differences disappeared (18.3 vs. 24.3 months; P=0.85). Mechanistically, we observed that the formation of CDK2/CyclinA, CDK2/CyclinE and CDK4/Cyclin D1 complexes was >200% higher in polymorphic than in wild-type cells (P< 0.05). Regarding CDK4 kinase activity of p27Kip1/CDK/Cyclin complexes, as opposed to wild-type p27Kip1, p27Kip1 V109G was unable to suppress the kinase activity of CDK4 in presence of Fulv or hormonal deprivation. However, palbociclib was able to fully suppress CDK4 kinase activity regardless of the p27Kip1 genotype. Conclusion: Germline p27Kip1 genotyping can constitute a tool for treatment selection: whereas wild-type patients are adequately treated with endocrine monotherapy, polymorphic patients are inherently resistant, but are rescued with CDK4/6 inhibitors. Thus, hormonal+CDK4/6 inhibitor combos could be reserved for the polymorphic patients. Citation Format: Miguel Quintela-Fandino, Silvana Mouron, Maria J. Bueno, Manuel Muñoz, Raul Torres, Sandra Rodriguez, Rodrigo Sánchez-Bayona, Luis Manso, Jorge Silva, Marcos Malumbres. p27Kip1 V109G single-nucleotide polymorphism (SNP): pinpointing the hormone-receptor positive breast cancer subpopulation that requires CDK4/6 inhibitors in addition to endocrine therapy. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-28.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor denosumab in BC patients, beyond its bone-related effects, is unclear. Here, we investigated the prognostic value of RANK expression and its functionality in human BC. We analyzed RANK and RANKL expression in more than 1500 BC cases (777 being estrogen receptor-negative (ER-)) from four independent cohorts. We confirmed that RANK is more frequently expressed in ER- tumors, but it is also found in a subset of ER+ tumors. In ER- BC, RANK expression was independently associated with poor outcome, especially in postmenopausal patients and those who received adjuvant chemotherapy. Gene expression analyses unraveled distinct biology associated with RANK in relation to ER expression and menopause, and evidenced enhanced RANK activation in ER- postmenopausal tumors, together with regulation of metabolic pathways. Functional studies and transcriptomic analyses in ER- RANK+ patients-derived orthoxenografts demonstrated that activation of RANK signaling pathway promotes tumor cell proliferation and stemness, and regulates multiple biological processes including tumor immune surveillance and metabolism. Our results demonstrate that RANK expression is an independent poor prognosis biomarker in postmenopausal ER- BC patients and support the rational of using RANK pathway inhibitors in combination with chemotherapy in ER- BC.

Purpose: We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Vascular normalization can be tracked with 2[18F]fluoro-2-deoxy-d-glucose (FDG)-PET. We tested the efficacy of the mitochondrial inhibitor ME-344 or placebo added to bevacizumab in early breast cancer. Patients and Methods: Treatment-naïve HER2-negative patients with T > 1 cm (any N) underwent a breast-centered 18F-fluorodeoxyglucose (FDG)-PET (day 1) and received a single dose of bevacizumab (15 mg/kg), followed by a second FDG-PET (day 8). Patients were then randomized (1:1) to Arm A (ME-344 10 mg/kg intravenous on days 8, 15, and 21) or Arm B (placebo). Tumors were biopsied on days 0 and 29. Succinate dehydrogenase enzyme histochemistry (SDH-EHC), confocal microscopy of vessel architecture, and HIF1α staining were performed in pre- and posttreatment biopsies to assess the pharmacodynamics, vessel normalization, and tissue re-oxygenation by bevacizumab, respectively. Results: ME-344 displayed significant biological activity versus placebo: compared with a 186% increase in Arm B, Ki67 decreased by 23.4% from days 0 to 28 in Arm A (P < 0.001) (N = 42 patients). FDG-PET predicted vascular normalization in about one-third of the patients in each arm, which was confirmed using confocal microscopy and HIF1α staining. In the subgroup with vascular normalization, ME-344 induced a Ki67 decrease of 33.4% (placebo: 11.8 increase). SDH-EHC suggested on-target effects of ME-344. Conclusions: ME-344 has significant biological antitumor activity in HER2-negative breast cancer, particularly after induction of vascular normalization and tissue reoxygenation with bevacizumab.

Simple Summary Triple-negative breast cancers (TNBC) represent approximately 15% of all breast cancers and lack the expression of a defined molecular target. This absence makes this subtype of cancer difficult to treat and control. Current chemotherapy drugs cause various side effects and toxicities that can jeopardize the quality of life of patients with TNBC cancer. Therefore, this research focuses on a new semi-synthetic compound derived from [6]-gingerol, where we demonstrate that it does not cause significant toxic effects in vivo and, more importantly, we demonstrate its antitumor and antimetastatic effects using preclinical xenograft models simulating two clinical scenarios of a woman with breast cancer. Abstract Breast cancer metastasis is the most common cause of cancer death in women worldwide. Triple-negative breast cancers (TNBC) form a heterogeneous group of tumors that have higher relapse rates and poorer survival compared to other breast cancer subtypes. Thus, this work reports the antitumor and antimetastatic activities of a [6]-gingerol-derived semi-synthetic compound named SSi6 on MDA-MB-231 TNBC cells using xenograft models. SSi6 did not cause toxic effects in vivo as demonstrated by body weight and hematological and histological evaluations. From the orthotopic xenograft model, we demonstrated that SSi6 slows and inhibits the growth of the primary tumor, as well as prevents metastatic spontaneous progression from lymph nodes to the lungs. Moreover, a second xenograft model with resection of the primary tumor showed that SSi6 also blocks the progression of metastases from the lymph nodes to other visceral organs. Taken together, our results demonstrate that SSi6 is a promising compound to be investigated in other preclinical and clinical models to be applied as a complementary therapy for TNBC.

Preclinical evidence indicates the potential of targeting mitochondrial respiration as a therapeutic strategy. We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Using molecular imaging, we showed how the same antiangiogenic agent may display different normalizing properties in patients with the same tumor type. This is of key importance, since patients experiencing normalization seem to get more benefit from standard chemotherapy combinations, and also could be eligible for combination with antimitochondrial agents. This scenario emphasizes the need for monitoring vascular normalization in order to optimize the use of antiangiogenics. We have also proposed a method to evaluate anti-mitochondrial agents' pharmacodynamics; despite promising accuracy in animal studies the clinical results were inconclusive, highlighting the need for research in this field. Regarding patients that respond to antiangiogenics increasing vessel abnormality, in this case an immunosuppressive tumor microenvironment is generated. Whether anti-mitochondrial agents can positively modulate the activity of T effector cell subpopulations remains an area of active research. Our research sheds light on the importance of refining the use of antiangiogenics, highlighting the relevance of tracing vascular normalization as a potential biomarker for antiangiogenics to assist patient-tailored medicine and exploring the role of mitochondrial inhibitors in the context of vascular normalization and correction of hypoxia. Sí

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations, Background FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear. Methods FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied. Results FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P, MQF is a recipient of the following grants: AES - PI16/00354 and AES – PI19/00454 funded by the ISCIII (Instituto de Salud Carlos III, Ministry of Science and Innovation, Spain) and co-funded by the European Regional Development Fund (ERDF); 2017/BMD3733 - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. RC and MJB are co-recipients of an AES – PI17/01865 grant funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF). RC is a recipient of funding PIE15/00068, PI20/01458, and PT17/0017/0007 by the ISCIII. This study was partially funded by Bayer Inc. CRIS Contra el Cancer Foundation contributed with a generous donation to this study

© The Author(s)., [Background]: Preclinical research suggests that the efficacy of immune checkpoint inhibitors in breast cancer can be enhanced by combining them with antiangiogenics, particularly in a sequential fashion. We sought to explore the efficacy and biomarkers of combining the anti-PD-L1 durvalumab plus the antiangiogenic bevacizumab after bevacizumab monotherapy for advanced HER2-negative breast cancer. [Methods]: Patients had advanced HER2-negative disease that progressed while receiving single-agent bevacizumab maintenance as a part of a previous chemotherapy plus bevacizumab regimen. Treatment consisted of bi-weekly durvalumab plus bevacizumab (10 mg/kg each i.v.). Peripheral-blood mononuclear cells (PBMCs) were obtained before the first durvalumab dose and every 4 weeks and immunophenotyped by flow-cytometry. A fresh pre-durvalumab tumor biopsy was obtained; gene-expression studies and immunohistochemical staining to assess vascular normalization and characterize the immune infiltrate were conducted. Patients were classified as “non-progressors” if they had clinical benefit (SD/PR/CR) at 4 months. The co-primary endpoints were the changes in the percentage T cell subpopulations in PBMCs in progressors versus non-progressors, and PFS/OS time. [Results]: Twenty-six patients were accrued. Median PFS and OS were 3.5 and 11 months; a trend for a longer OS was detected for the hormone-positive subset (19.8 versus 7.4 months in triple-negatives; P = 0.11). Clinical benefit rate at 2 and 4 months was 60% and 44%, respectively, without significant differences between hormone-positive and triple-negative (P = 0.73). Non-progressors’ tumors displayed vascular normalization features as a result of previous bevacizumab, compared with generally abnormal patterns observed in progressors. Non-progressors also showed increased T-effector and T-memory signatures and decreased TREG signatures in gene expression studies in baseline—post-bevacizumab—tumors compared with progressors. Notably, analysis of PBMC populations before durvalumab treatment was concordant with the findings in tumor samples and showed a decreased percentage of circulating TREGs in non-progressors. [Conclusions]: This study reporting on sequential bevacizumab+durvalumab in breast cancer showed encouraging activity in a heavily pre-treated cohort. The correlative studies agree with the preclinical rationale supporting an immunopriming effect exerted by antiangiogenic treatment, probably by reducing TREGs cells both systemically and in tumor tissue. The magnitude of this benefit should be addressed in a randomized setting., MQF is a recipient of the following grants: AES - PI16/00354 funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) and B2017/BMD3733 (Immunothercan-CM) - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. SM is a recipient of the following grants: Spanish Ministerio de Economía y Competitividad (MINECO) (SAF2017-83732-R; AEI/FEDER, EU) and co-funded by Comunidad de Madrid (B2017/BMD3733; Immunothercan-CM). RC is a recipient RC is a recipient of the ISCIII grants PIE15/00068 and PI17/01865.

Estrogen receptor (ER)-positive breast tumors are routinely treated with estrogen-depriving therapies. Despite their effectiveness, patients often progress into a more aggressive form of the disease. Through a chemical screen oriented to identify chemicals capable of inducing the expression of the immune-checkpoint ligand PD-L1, we found antiestrogens as hits. Subsequent validations confirmed that estrogen deprivation or ERα depletion induces PD-L1 expression in ER-positive breast cancer cells, both in vitro and in vivo. Likewise, PD-L1 expression is increased in metastasis arising from breast cancer patients receiving adjuvant hormonal therapy for their local disease. Transcriptome analyses indicate that estrogen deprivation triggers a broad immunosuppressive program, not restricted to PD-L1. Accordingly, estrogen deprived MCF7 cells are resistant to T-cell mediated cell killing, in a manner that can be reverted by estradiol. Our study reveals that while antiestrogen therapies effectively limit tumor growth in ER-positive breast cancers, they also trigger a transcriptional program that favors immune evasion.

Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability. M.Q.-F. is a recipient of the following grants: FIS PI10/0288 and FIS PI13/00430 from the Ministry of Health (Spain), 2010-BECA-RETORNO from theAECC Scientific Foundation, and donations from the Rosae Foundation andAvon Espan ̃a S.A.U. I.Z. is a recipient of La Caixa-CNIO PhD 2011 fellowship.M.Q.-F. received research funds from Boehringer-Ingelheim and Novartis. Sí

The combined use of a FGFR1 blocker and aromatase inhibitors is appealing for treating breast cancer patients with FGFR1 amplification. However, no pharmacodynamic studies have addressed the effects of this combined target modulation. We conducted a phase 0/I clinical trial in an adjuvant setting, with the goal of obtaining pharmacodynamic proof of the effects of combined aromatase and FGFR1 inhibition and to establish the RP2D for nintedanib combined with letrozole. Patients and methods: Women with early-stage luminal breast cancer were eligible for enrollment in the study. Dose level 1 was nintedanib (150 mg/bid) plus letrozole (2.5 mg/day) administered for a single 28-day cycle (DLT assessment period), followed by a classic 3 + 3 schedule. FGF23 and 17-B-estradiol levels were determined on days 0 and 15; pharmacokinetic parameters were assessed on days 1 and 28. Patients were allowed to continue treatment for 6 cycles. The primary study endpoint was a demonstration of FGFR1 modulation (defined as a 25% increase in the plasma FGF23 level). Results: A total of 19 patients were enrolled in the study (10 in the expansion cohort following dose escalation). At the RP2D (nintedanib 200 mg/bid plus letrozole 2.5 mg/day), we observed a 55% mean increase in the plasma FGF23 level, and 81.2% of the patients had no detectable level of 17-B-estradiol in their plasma (87.5% of the patients treated with letrozole alone). Nintedanib and letrozole displayed a pharmacokinetic interaction that led to three- and twofold increases in their respective plasma concentrations. Most G3 toxic events (5 out of 6: 2 diarrhea and 3 hypertransaminasemia) occurred subsequent to the DLT assessment period. Conclusion: Combined treatment with nintedanib (200 mg/bid) plus letrozole (2.5 mg/day) effectively suppressed FGFR1 and aromatase activity, and these respective doses can be used as starting doses in any subsequent trials. However, drug-drug interactions may produce tolerability issues when these drugs are co-administered for an extended time period (e.g., 6 months). Patients enrolled in future trials with these drugs should be carefully monitored for their FGF23 levels and signs of toxicity, and those findings should guide individualized treatment decisions, MQF is a recipient of the following grants: AES - PI16/00354 funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) and B2017/BMD3733 - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. RC is a recipient of the following grants: AES PI17/01865 and PIE15/00068 by the ISCIII and co-funded by the European Regional Development Fund (ERDF). This study was partially funded by Boehringer-Ingelheim. CRIS Contra el Cancer Foundation contributed with a generous donation to this study

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair. The DNA Replication Group is part of the BFU2016-81796-REDC network of excellence. We thank all members of the group for discussions and Dr. Oscar Fernández-Capetillo and Dr. Ana Losada for useful comments on the manuscript Sí

// Miguel Quintela-Fandino 1 , Nora Soberon 2 , Ana Lluch 3 , Luis Manso 4 , Isabel Calvo 5 , Javier Cortes 6, 7 , Fernando Moreno-Anton 8 , Miguel Gil-Gil 9 , Noelia Martinez-Janez 7 , Antonio Gonzalez-Martin 10 , Encarna Adrover 11 , Raquel de Andres 12 , Gemma Vinas 13 , Antonio Llombart-Cussac 14 , Emilio Alba 15 , Silvana Mouron 1 , Juan Guerra 16 , Begona Bermejo 3 , Esther Zamora 6 , Jose Angel Garcia-Saenz 8 , Sonia Pernas Simon 9 , Eva Carrasco 17 , Maria Jose Escudero 17 , Ruth Campo 17 , Ramon Colomer 18 , Maria A. Blasco 2 1 Breast Cancer Clinical Research Unit, CNIO-Spanish National Cancer Research Center, Madrid, Spain 2 Telomeres and Telomerase Group, Molecular Oncology Programme, CNIO-Spanish National Cancer Research Center, Madrid, Spain 3 Medical Oncology Department, Hospital Clinico Universitario, Valencia, Spain 4 Medical Oncology Department, Hospital 12 de Octubre, Madrid, Spain 5 Medical Oncology Department, Hospital de Monteprincipe, Madrid, Spain 6 Medical Oncology Department, Hospital Vall d´Hebron, Barcelona, Spain 7 Medical Oncology Department, Hospital Ramon y Cajal, Madrid, Spain 8 Medical Oncology Department, Hospital Clinico San Carlos, Madrid, Spain 9 Medical Oncology Department, Institut Catala d'Oncologia-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain 10 Medical Oncology Department, MD Anderson Cancer Center, Madrid, Spain 11 Medical Oncology Department, Hospital General de Albacete, Albacete, Spain 12 Medical Oncology Department, Hospital Lozano Blesa, Zaragoza, Spain 13 Medical Oncology Department, Institut Catala d'Oncologia-Hospital Dr. Josep Trueta, Girona, Spain 14 Medical Oncology Department, Hospital Arnau de Vilanova, Valencia, Spain 15 Medical Oncology Department, Hospital Clinico Universitario Virgen de la Victoria, Malaga, Spain 16 Medical Oncology Department, Hospital Universitario de Fuenlabrada, Fuenlabrada, Spain 17 GEICAM, Madrid, Spain 18 Medical Oncology Department, Hospital Universitario La Princesa, Madrid, Spain Correspondence to: Miguel Quintela-Fandino, email: mquintela@cnio.es Maria A. Blasco, email: mblasco@cnio.es Keywords: telomere length, critically short telomeres, breast cancer, weekly paclitaxel, toxicity Received: October 25, 2016 Accepted: January 27, 2017 Published: February 21, 2017 ABSTRACT Cumulative toxicity from weekly paclitaxel (myalgia, peripheral neuropathy, fatigue) compromises long-term administration. Preclinical data suggest that the burden of critically short telomeres ( 21.9% CSTs) had 2-fold higher number of neuropathy ( P = 0.04) or fatigue ( P = 0.019) episodes and >3-fold higher number of myalgia episodes ( P = 0.005). The average telomere length was unrelated to the incidence of side effects. The percentage of CSTs, but not the average telomere size, is associated with weekly paclitaxel-derived toxicity.

Background: Rationalization of antiangiogenics requires biomarkers. Vascular re‐normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with 18F‐misonidazole ([18F]‐FMISO, a probe that detects hypoxia) PET, in response to window‐of‐opportunity (WoO) treatment with the antiangiogenic dovitinib. Methods: Two patient‐derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV‐PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [18F]‐FMISO‐PET, [18F]‐FDG‐PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase‐3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. Results: [18F]‐FMISO SUV did not change after dovitinib‐WoO treatment compared to vehicle‐WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P, Highlights We describe a new technique to monitor the efficacy of antiangiogenics.This drug class currently lacks biomarkers.Antiangiogenics exert their positive effect by inducing stromal normalization.18F‐fluoromisonidazole PET is able to monitor stromal normalization.Tumors showing stromal normalization by PET experience benefit from antiangiogenics.

Cell-cycle transitions are controlled by CDKs (cyclin-dependent kinases), whose activation is usually associated with the binding of cyclins. RINGO/Speedy proteins can also bind to and activate CDKs, although they do not have amino acid sequence homology with cyclins. The RINGO/Speedy family members studied so far positively regulate cell-cycle progression. In the present paper, we report the biochemical and functional characterization of RINGO/Speedy E. We show that RINGO/Speedy E is a functionally distant member of this protein family that negatively affects cell-cycle progression. RINGO/Speedy E overexpression inhibits the meiotic progression in Xenopus oocytes as well as the proliferation of mammalian cells. RINGO/Speedy E can bind to endogenous CDK1 and CDK2 in both cellular systems. However, the RINGO/Speedy E-activated CDKs have different substrate specificity than the CDKs activated by other RINGO/Speedy proteins, which may account for their different effects on the cell cycle. Our results indicate that, although all RINGO/Speedy family members can activate CDKs, they may differently regulate cell-cycle progression.

Summary We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication., Graphical Abstract, Highlights • PrimPol is a second human primase that is able to start DNA chains with dNTPs • Identification of PrimPol as a DNA polymerase that tolerates AP sites and 8oxoG lesions • PrimPol is located at both DNA compartments, mitochondria, and the nucleus • PRIMPOL gene silencing affects mtDNA replication

DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy. © 2013 Nature America, Inc. All rights reserved., Comunidad de Madrid (S2011/BMD-236); Spanish Ministry of Economy and Competitiveness (BFU2010-21467, BFU2012-37969,Consolider CSD2007-00015)

In the Acknowledgments of our report we unintentionally omitted to include a sentence stating that J.G. holds a postdoctoral fellowship (JAE-Doc) from the Program “Junta para la Ampliacion de Estudios” co-funded by the Fondo Social Europeo (FSE). The authors regret this omission.

Criado-García, Olga et al., Lafora disease (LD), a fatal neurodegenerative disorder characterized by the presence of intracellular inclusions called Lafora bodies (LBs), is caused by loss-of-function mutations in laforin or malin. Previous studies suggested a role of these proteins in the regulation of glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of the intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. We have generated a malin-deficient (Epm2b-/-) mouse with a phenotype similar to that of LD patients. By 3-6 months of age, Epm2b-/- mice present neurological and behavioral abnormalities that correlate with a massive presence of LBs in the cortex, hippocampus and cerebellum. Sixteen-day-old Epm2b-/- mice, without detectable LBs, show an impairment of macroautophagy (hereafter called autophagy), which remains compromised in adult animals. These data demonstrate similarities between the Epm2a-/- and Epm2b-/- mice that provide further insights into LD pathogenesis. They illustrate that the dysfunction of autophagy is a consequence of the lack of laforin-malin complexes and a common feature of both mouse models of LD. Because this dysfunction precedes other pathological manifestations, we propose that decreased autophagy plays a primary role in the formation of LBs and it is critical in LD pathogenesis. © The Author 2011. Published by Oxford University Press. All rights reserved., This work was funded by the Spanish Ministerio de Ciencia eInnovacio ́ n (SAF2008-00226 to S.R.C.; BFU2007-61774 andBFU2010-16031toP.B.;BFU2008-00186toE.K.;SAF2008-01907 to P.S.; and SAF2010-18586 to J.M.S.), theRed de Biobancos del FIS (RD09/0076/00011 to C.N.), theFondo de Investigacio ́ n Sanitaria (PI10/02628 to C.N.;PI070023 to M.S.), the Fundacio ́ n Marato TV3 (Ref. 100130to P.S., E.K. and S.R.C.) and the Centro de Investigacio ́ n Bio-me ́dica en Red de Enfermedades Raras (CIBERER).A.M.G.-C. holds a fellowship from the Fundacio ́ n ConchitaRa ́bago

Knecht, Erwin et al., Lafora disease (LD), a fatal neurodegenerative disorder characterized by intracellular inclusions called Lafora bodies (LBs), is caused by recessive loss-of-function mutations in the genes encoding either laforin or malin. Previous studies suggested a role of these proteins in regulating glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. Here we review our recent finding that dysfunction of autophagy is a common feature of both laforin- and malin-deficient mice, preceding other pathological manifestations. We propose that autophagy plays a primary role in LD pathogenesis and is a potential target for its treatment. © 2012 Landes Bioscience., Spanish Ministry of Science and Innovation (SAF2008-00226 to SRdeC, BFU2010-16031 to PB; BFU2008-00186, SAF2008-01907, SAF2010-18586); the Red de Biobancos del FIS (RD09/0076/00011); the Fondo de Investigación Sanitaria (PI10/02628, PI070023); the Fundación Marato TV3 (Ref.100130); the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); Fundación Conchita Rábago

RINGO/Speedy proteins are direct activators of Cdk1 and Cdk2 that have no sequence homology to cyclins. We have characterized the role in cell-cycle progression of a new human member of this protein family referred to as RINGO C. We show that siRNA-mediated knockdown of RINGO C results in premature mitotic exit with misaligned chromosomes, even in the presence of microtubule poisons. Time-lapse-microscopy experiments suggest that RINGO C is involved in the spindle-assembly checkpoint (SAC). Consistent with this idea, RINGO-C-depleted cells show impaired recruitment of the SAC components Mad2, Bub1 and BubR1. As the checkpoint is overridden, cells display defective chromosome segregation, which leads to an increased number of micronuclei and binucleated structures. Intriguingly, we found that RINGO C can associate with the mitotic kinase Aurora B, and downregulation of RINGO C produces mislocalization of the active form of Aurora B in prometaphase. Taken together, our results indicate a role for RINGO C in the mitotic checkpoint, which might be mediated by defective recruitment of SAC components and deregulation of the activity of Aurora kinase B.

Purpose Triple-negative breast cancer (TNBC) is characterized by large heterogeneity and relative lack of available targeted therapies. To find therapeutic strategies for distinct patients with TNBC, several approaches have been used for TNBC clustering, including recently immune and phosphoproteomic patterns. Based on 70-kDa ribosomal protein S6 kinase (P70S6K)-TNBC clustering, the current study explores the immune profiling in TNBC tumors. Methods Stromal tumor-infiltrating lymphocytes (sTILs) were evaluated in human TNBC tumor samples. Furthermore, immunohistochemistry staining for CD8, CD4, Foxp3, and CD20 was performed in tissue microarrays (TMA) sections. Results Histological analysis showed decreased sTILs, CD20+ cells, and CD8+/CD4+ ratio in high phosphorylated P70S6K (p-P70S6K) tumors. Moreover, p-P70S6K score was directly correlated with CD4+ and Foxp3+ T cells, while it was inversely correlated with CD8+/CD4+ and CD8+/Foxp3+ ratios. Conclusion sTIL infiltration and lymphocyte profiling vary in the context of hyperactivation of P70S6K in TNBC tumors.

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p