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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. This study has been supported by the Susan G. Komen Foundation (CCR15330331) and by the Catalan Agency AGAUR (2017 SGR 540) [to V.S.]. V.S. received funds from the Instituto de Salud Carlos III: grants PI13/01714, CP14/00228, MV15/00041, CPII19/00033 and PI20/00892. M.P. received a Juan de la Cierva Grant from the Ministerio de Economía y Competitividad (FJCI-2015-25412), L.Mo. a grant from FI-AGAUR (2019 FI_B 01199), F.B-M. a grant from the Fundación Científica Asociación Española Contra el Cáncer (AECC_Postdoctoral17-1062) and M.S-G, a Marie Slodowska-Curie Innovative Training Networks PhD fellowship (H2020-MSCA-ITN-2015_675392). 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.A.). R.B.C. laboratory is supported by Breast Cancer Now (grant numbers: MAN-Q1 and MAN-Q2), NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20007) and EdiREX Horizon 2020 grant No.731105. The xenograft program in the C.C. laboratory is supported by Cancer Research UK and also received funding from an EU H2020 Network of Excellence (EuroCAN). This work has been supported by NIH grants P30 CA008748 and RO1CA190642-01, the CDMRP grant BC171535P1, and the Breast Cancer Research Foundation [to M.S.]. A.P. received funds from Instituto de Salud Carlos III—PI16/00904 and PI19/01846, Breast Cancer Now—2018NOVPCC1294, Breast Cancer Research Foundation-AACR Career Development Awards for Translational Breast Cancer Research 19-20-26-PRAT, Fundació La Marató TV3 201935-30, the European Union’s Horizon 2020 research and innovation program H2020-SC1-BHC-2018-2020. IRB Barcelona is a recipient of a Severo Ochoa Centre of Excellence Award from the Spanish Ministry of Economy and Competitiveness (MINECO; Government of Spain) and is supported by CERCA (Generalitat de Catalunya). C. S. Verma reports grants from MSD International and grants from Ipsen outside the submitted work.

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

Proteïnes supressores de tumors; Inhibidors de la quinasa Proteínas supresoras de tumores; Inhibidores de la quinasa Tumor suppressor proteins; Kinase inhibitors Cyclin-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.

Molecular profiling of personal cancer genomes, and the identification of actionable vulnerabilities and drug-response biomarkers, are the basis of precision oncology. Tumors often present several driver alterations that might be connected by cross-talk and feedback mechanisms, making it difficult to mark single oncogenic variations as reliable predictors of therapeutic outcome. In the current work, we uncover and exploit driver alteration co-occurrence patterns from a recently published in vivo screening in patient-derived xenografts (PDXs), including 187 tumors and 53 drugs. For each treatment, we compare the mutational profiles of sensitive and resistant PDXs to statistically define Driver Co-Occurrence (DCO) networks, which capture both genomic structure and putative oncogenic synergy. We then use the DCO networks to train classifiers that can prioritize, among the available options, the best possible treatment for each tumor based on its oncogenomic profile. In a cross-validation setting, our drug-response models are able to correctly predict 66% of sensitive and 77% of resistant drug-tumor pairs, based on tumor growth variation. Perhaps more interesting, our models are applicable to several tumor types and drug classes for which no biomarker has yet been described. Additionally, we experimentally validated the performance of our models on 15 new tumor samples engrafted in mice, achieving an overall accuracy of 75%. Finally, we adapted our strategy to derive drug-response models from continuous clinical outcome measures, such as progression free survival, which better represent the data acquired during routine clinical practice and in clinical trials. We believe that the computational framework presented here could be incorporated into the design of adaptive clinical trials, revealing unexpected connections between oncogenic alterations and increasing the clinical impact of genomic profiling.