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Supplemental File 1. Excel file with additional details on the mathematical model.

Supplemental Tables 1-4. Table S1. Sequences of sgRNAs and oligonucleotides; Table S2. GSEA pathway enrichment analysis of enriched and dropout genes in MCF7 and T47D cells; Table S3. The top 2.5% (3745) enriched sgRNAs of whole-genome CRISPR/Cas9 sgRNA knockout screen in MCF7 cells; Table S4. The top 2.5% (3745) enriched sgRNAs of whole-genome CRISPR/Cas9 sgRNA knockout screen in T47D cells.

Supplemental File 2. Excel file with the cbioportal data the genomic data analysis is based on.

Word file containing the Supplemental Text, Supplemental Figures S1-S6, and Supplemental Tables S1-S2.

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this. Cancer Res; 75(4); 732–41. ©2014 AACR.

Previous studies have suggested that PTEN loss is associated with p110β signaling dependency, leading to the clinical development of p110β-selective inhibitors. Here we use a panel pre-clinical models to reveal that PI3K isoform dependency is not governed by loss of PTEN and is impacted by feedback inhibition and concurrent PIK3CA/PIK3CB alterations. Furthermore, while pan-PI3K inhibition in PTEN-deficient tumors is efficacious, upregulation of Insulin Like Growth Factor 1 Receptor (IGF1R) promotes resistance. Importantly, we show that this resistance can be overcome through targeting AKT and we find that AKT inhibitors are superior to pan-PI3K inhibition in the context of PTEN loss. However, in the presence of wild-type PTEN and PIK3CA-activating mutations, p110α-dependent signaling is dominant and selectively inhibiting p110α is therapeutically superior to AKT inhibition. These discoveries reveal a more nuanced understanding of PI3K isoform dependency and unveil novel strategies to selectively target PI3K signaling nodes in a context-specific manner., Understanding the mechanisms driving PI3K isoform dependency in prostate cancer can help the design of future clinical trials. Here, the authors show that gain-of-function mutations in PIK3CA or PIK3CB can confer PI3K p110 isoform dependency and that the direct inhibition of AKT may be superior to PI3K inhibition in PTEN-deficient prostate cancers.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Kα inhibitor alpelisib in estrogen receptor positive (ER+) PIK3CA-mutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, for example, MCL1 inhibitors, was experimentally validated in ER+ breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line–specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL2 family members were highly expressed. On the basis of these results, newly developed cell line–specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance. Significance: Network-based mathematical models of oncogenic signaling and experimental validation of its predictions can identify resistance mechanisms for targeted therapies, as this study demonstrates for PI3Kα-specific inhibitors in breast cancer.

This paper brings forth a kind of automatic pressurizing device which can resolve the problem of poor hydraulic support setting load directly without modifying the original hydraulic system excessively. On the basis of the control loop that is designed according to working requirements and conditions to realize automatic pressurization of stand column system, it is divided into pressurizing structure and auxiliary structure. Reserved liquid opening and unit design are used to design the automatic pressurizing device from the principle perspective, and it is also made associated with all units reasonably by improving the details. The automatic pressurizing device combines machinery and hydraulic pressure so that all structural units can finish the expected action and coordinate mutually under the effect of emulsion in order to finally realize the overall function of automatic and fast pressurization. On the basis of the simulated analysis of AMESim hydraulic simulation platform on the control loop and lifting support system loop of support stand column, the working performance of automatic pressurizing device and dynamic characteristics of control loop are simulated. The result after comparing and analyzing the simulation curve and site installation test of underground ZY12000/28/63D support finally shows that the device is able to enhance the setting load to above 30 MPa, which is basically the same with simulation result. It demonstrates that the design of automatic pressurizing device conforms to the expected pressurization requirements and the pressurization effect is significant.

By use of the concept of impedance, this paper establishes a random energy harvester output model of double-ended fixed beam piezoelectric energy harvester under random White Gaussian noise excitation, which is based on the discussion of the output characteristics influenced by changing vibration beam shape and piezoelectric sheet length under random excitation, while improving piezoelectric structure of double-ended fixed beam piezoelectric energy harvester. It also deduces the output voltage, current and power of energy harvester with Fokker–Planck equation, which are verified by Monte Carlo simulation and experimental testing. It can be found that improving vibration beam shape can reduce resonant frequency and increase average output power of the system. The optimal value of piezoelectric sheet length can be different corresponding to different structures, which proves that double-ended fixed beam piezoelectric energy harvester under random White Gaussian noise excitation can achieve maximum output with optimal piezoelectric sheet length. In addition, in order to further improve the output performance of piezoelectric energy harvester under random excitation, it is necessary to increase the acceleration spectral density under random excitation and set an optimal value of the piezoelectric loading resistance, which is more suitable for energy harvester applied under random low-frequency environment.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER(+)) breast cancer. We identify that ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER(+) breast cancer. An epigenome CRISPR-CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the eR degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARIDIA-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER-FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER(+) breast cancer.

Seeing double can be a good thingMany human breast cancers harbor activating mutations inPIK3CA, the gene coding for the catalytic subunit of phosphoinositide 3-kinase (PI3K). Clinical trials are underway to evaluate the efficacy of PI3K inhibitors in cancer patients. Vasanet al.found unexpectedly that a subset of breast cancers harbor not one—but two—PIK3CAmutations, and the mutations occur on the same allele (see the Perspective by Toker). In model systems, the double mutations hyperactivate PI3K signaling and enhance tumor growth. Preliminary analysis of clinical trial data suggests that breast cancers with double mutations are more responsive to PI3K inhibitors than those with a single mutation.PIK3CAmutational status could help identify the breast cancer patients most likely to benefit from these drugs.Science, this issue p.714; see also p.685

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Kα inhibitor alpelisib in ER+ PIK3CA mutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, e.g., MCL1 inhibitors, was experimentally validated in ER+ breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line-specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL-2 family members were highly expressed. Based on these results, newly developed cell line-specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance.

Significance We report on X-ray and cryo-EM structural studies on the assembly of the human Shieldin complex composed of SHLD2, SHLD3, and REV7, as well as its complex with bound TRIP13 toward understanding the principles underlying TRIP13-mediated disassembly of Shieldin. Our studies identify a conformational heterodimeric alignment of open (O) and closed (C) conformers of REV7 when bound to a fused SHLD2–SHLD3 construct. The AAA+ ATPase TRIP13 captures the N terminus of C-REV7 (REVNT) within its central hexameric channel, with the rotatory motion associated with sequential ATP hydrolysis within individual TRIP13 subunits. This facilitates the stepwise pulling of the REV7NT through the central channel, resulting in initial disassembly of C-REV7 followed by dissociation of the Shieldin complex., The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA+ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3–REV7 binary and fused SHLD2–SHLD3–REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2–SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2–SHLD3–REV7–TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.

Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer. Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance. RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented. Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth. Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) breast cancer patients. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2 and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacological inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. A promising approach to tackle the cancer drug resistance problem is to build mechanistic mathematical models of the signaling network of cancer cells, and explicitly model the dynamics of information flow through this network under distinct genetic conditions and in response to perturbations.In this work, we used a network-based mathematical model to identify sensitivity factors and drug combinations for the PI3Kα inhibitor alpelisib, which was recently approved for ER+PIK3CAmutant breast cancer. We experimentally validated the model-predicted efficacious combination of alpelisib and BH3 mimetics (e.g. MCL1 inhibitors) in ER+ breast cancer cell lines. We also experimentally validated the reduced sensitivity to alpelisib caused by FOXO3 knockdown, which is a novel potential resistance mechanism. Our experimental results showed cell line-specific sensitivity to the combination of alpelisib and BH3 mimetics, which was driven by the choice of BH3 mimetics. We find that cell lines were sensitive to the addition of either MCL1 inhibitor s63845 alone or in combination with BCL-XL/BCL-2 inhibitor navitoclax, and that the need for the combination of both BH3 mimetics was predicted by the expression of BCL-XL. Based on these results, we developed cell line-specific network models that are able to recapitulate the observed differential response to alpelisib and BH3 mimetics, and also incorporate the most recent knowledge on resistance and response to PI3Kα inhibitors.Overall, we present an approach for the development, experimental testing, and refining of mathematical models, which we apply to the context of PI3Kα inhibitor drug resistance in breast cancer. Our approach predicted and validated PI3Kα inhibitor sensitivity factors (FOXO3 knockdown) and drug combinations (BH3 mimetics), and illustrates that network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance.

(ER)-positive tumors, also known as luminal breast cancers represent over 70% of breast cancers and they are treated primarily with endocrine therapy. Despite the success of endocrine therapies, resistance to these agents is a major clinical problem, and it eventually develops in all patients with metastatic disease. Therefore, the identification and understanding of the mechanisms of resistance to endocrine therapy is a major research priority. Important insights into the mechanisms of endocrine resistance have been gained with the identification of recurrent, activating mutations in ESR1 (the gene coding ER) in approximately 18 % of the tumors with acquired resistance to aromatase inhibitors. However, the mechanisms of resistance in the remaining 82 % of the cases largely unknown. We have recently genomically characterized the largest cohort of endocrine resistant metastatic breast tumors (n=1918 tumors). In addition to ESR1 mutations, we identified the presence of inactivating mutations in the ARID1A gene of the SWI/SNF complex in metastatic endocrine-resistant breast tumors. In parallel, as part of our efforts to elucidate mechanisms of resistance to endocrine therapy, we performed an epigenome CRISPR/CAS9 knockout screen that identified ARID1A as the top candidate whose loss determined resistance to the ER degrader in the clinic fulvestrant. We validated these findings showing that ARID1A knockout cells are resistant to endocrine therapy in vitro and in vivo. Hence, from both clinical observations and our unbiased CRISPR screen from our laboratory, we identified ARID1A loss as a candidate of endocrine resistance. We next performed for the first time the mechanistic characterization of ARID1A loss in ER-positive breast cancer. We determined that ARID1A loss remodels the chromatin landscape of breast cancer on a genome-wide scale, converging to a basal-like gene expression program resulting from downregulation of master transcription factors (TFs) that are required for luminal (ER+) identity. Transcriptome profiling of ARID1A knockout cell lines and patient samples with loss of ARID1A were consistent with our initial findings showing an induction of basal-like gene expression programs. Thus, we elucidated a new mechanism of endocrine resistance through lineage switching mediated by the inactivation of ARID1A. Mechanistically, we showed that ARID1A-dependent SWI/SNF complex binding was impaired at the genomic sites of the major luminal-lineage determining TFs including ER, FOXA1, and GATA3. We also determined that ARID1A regulates ER-chromatin interactions and ER-dependent transcription, the defining driver in ER+ breast cancer. Hence, tumors cells with loss of ARID1A shift from ER-dependent luminal cells to ER-independent basal-like cells and become less responsive to luminal specific anti-ER therapy. Altogether, we uncovered a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER+ breast cancer. This work which I am the last and corresponding author of was recently published to Nature Genetics (Xu et al, Nature Genetics 2020).Interestingly, further analyses of Arid1a knockout in the mammary gland and breast organoids validated a key role of ARID1A in mammary gland morphogenesis and luminal cell differentiation. Moreover, single cell RNA-seq analyses demonstrated that ARID1A knockout breast cells did not differentiate to mature luminal cells. In the cohort of breast cancer tumors (n=1918), we also identified the presence of hotspot missense mutations in the pioneer transcription factor FOXA1 to be enriched in metastatic endocrine-resistant breast tumors. Mutations in the pioneer transcription factor FOXA1 are a hallmark of ER-positive lobular breast cancers. However, the functional consequences of FOXA1 mutations and whether they affect the therapeutic response to endocrine therapy are currently unknown. We undertook a comprehensive approach to investigate the structural consequences, including the genome-wide chromatin recruitment, chromatin accessibility, and transcriptional network in breast cancer models harboring recurrent hotspot and breast cancer specific FOXA1 mutations. By examining the landscape of FOXA1 mutations in a larger cohort (4,952 breast cancer patients), we identified several hotspot mutations in the Wing2 region, and a breast-cancer specific mutation located in the third beta strand, namely SY242CS. Interestingly, FOXA1 hotspot missense mutations were found to be more common in metastatic samples compared to primary tumors, and mutually exclusive with ESR1 mutations, suggesting a positive correlation between presence of FOXA1 mutations and resistance to endocrine therapy. Indeed, FOXA1 mutations were associated with a lower response to aromatase inhibitors in breast patient samples. Mechanistically, we uncovered that FOXA1 Wing2 mutations display increased chromatin binding affinity at ER sites upon estrogen stimulation, and enhanced ER-mediated transcription. Accordingly, 3D structural molecular simulations determined a highly organized 3D conformation conferred by FOXA1 mutations in the Wing2 loop, which may drive augmented ER recruitment or potentially allow more stable/durable interactions with ER and other cooperating factors at the chromatin. By contrast, FOXA1 SY242CS displayed a novel pioneering function over distinct genomic regions that were enriched by the presence of a new FOXA1-binding motif. This alternative binding motif was FOXA1 SY242CS-specific and common at both unique accessible and chromatin bound sites, leading to novel open chromatin regions and the induction of an alternative transcriptome regardless of estrogen stimulation, granting this variant a cellular growth advantage. Thus mechanistically, two phenotypic groups of FOXA1 mutants were established: hypermorphic Wing2 mutants that augment estrogen response, and a neomorphic SY242CS mutant that promotes an alternative pioneering, and cistromic and transcriptomic function leading to endocrine therapy resistance. This work which I am a corresponding author of was recently published to Cancer Cell Arruabarrena-Aristorena et al, Cancer Cell 2020). In summary, our data provide insights into how mutations in transcriptional regulators perturb their function to dictate breast cancer progression and therapeutic response. Our work highlights the power of examining recurrent cancer-associated mutations to advance our understanding of transcriptional regulators in breast cancer. These results also provide mechanistic insights into how FOXA1 and ARID1A mutations are associated with worse outcome to endocrine therapy and position these genetic alterations as potential biomarkers of endocrine therapy response for the treatment of metastatic ER+ breast cancer. Citation Format: Eneda Toska, Guotai Xu, Amaia Arruabarrena-Aristorena, Maurizio Scaltriti. Epigenetic mechanisms of endocrine therapy response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG05.

Summary: The PI3K pathway integrates extracellular stimuli to phosphorylate effectors such as AKT and serum-and-glucocorticoid-regulated kinase (SGK1). We have previously reported that the PI3K pathway regulates estrogen receptor (ER)-dependent transcription in breast cancer through the phosphorylation of the lysine methyltransferase KMT2D by AKT. Here, we show that PI3Kα inhibition, via a negative-feedback loop, activates SGK1 to promote chromatin-based regulation of ER-dependent transcription. PI3K/AKT inhibitors activate ER, which promotes SGK1 transcription through direct binding to its promoter. Elevated SGK1, in turn, phosphorylates KMT2D, suppressing its function, leading to a loss of methylation of lysine 4 on histone H3 (H3K4) and a repressive chromatin state at ER loci to attenuate ER activity. Thus, SGK1 regulates the chromatin landscape and ER-dependent transcription via the direct phosphorylation of KMT2D. These findings reveal an ER-SGK1-KMT2D signaling circuit aimed to attenuate ER response through a role for SGK1 to program chromatin and ER transcriptional output. : Toska, Castel, et al. show that the PI3K pathway propagates its effects to control chromatin and estrogen receptor (ER) function through SGK1, a PI3K effector. PI3K inhibitors, via a negative-feedback loop, activate SGK1, which phosphorylates the histone lysine methyltransferase KMT2D to attenuate its activity and regulate ER response. Keywords: SGK1, KMT2D, PI3K pathway, estrogen receptor, breast cancer, chromatin regulation, AKT, PI3K inhibitors

Activating mutations in

PIK3CA mutations represent the most frequent oncogenic driver lesion found across all human cancers. Given that single PIK3CA mutations are a predictive biomarker for response to PI3K inhibition, and that these clinical benefits are small, we hypothesized that additional genomic factors may cooperate with single PIK3CA mutations in oncogenesis and response to PI3K inhibitors. We analyzed several large pan-cancer datasets and have discovered a phenomenon of dual PIK3CA mutations in 15% of all PIK3CA-mutant cancers, including breast cancer. Dual PIK3CA mutations are clonal, are found in both primary untreated and metastatic tumors, and are enriched at conserved amino acid positions. We have proved that the majority of dual PIK3CA mutations—including the most frequent dual PIK3CA mutant combinations in breast cancer—are in cis compound mutations (i.e., on the same allele, resulting in a protein molecule with two mutations) in patient samples and cell lines, as shown through multiple sequencing techniques including long-range single-molecule real-time sequencing (SMRT-seq). Compound PIK3CA mutations increase PI3K pathway signaling and growth proliferation in nontransformed cells and cancer cells in vitro and in vivo. Biochemical experiments using recombinant PI3K complexes reveal a mechanism where compound mutants increase PI3K complex thermal instability, resulting in increased kinase activity and liposome binding as compared to single mutants. Compound PIK3CA mutations render cells more sensitive to the PI3Kα inhibitors BYL719 and GDC-0077 than single PIK3CA mutations. These data translate to the clinic, where patients with dual PIK3CA-mutant metastatic breast cancer exhibit more durable responses to PI3K inhibition versus patients with single PIK3CA-mutant breast tumors. Our data support a novel mutational dose-response model for oncogene activation, where compound mutations amplify PI3K signaling and growth to a greater degree than single mutants, resulting in increased sensitivity to PI3Kα inhibitor targeted therapy. These findings also have therapeutic relevance, where compound PIK3CA mutations may be a superior predictive biomarker for response to PI3Kα inhibitors than single PIK3CA mutations, across PIK3CA-mutant cancers. Citation Format: Neil Vasan, Jared Johnson, Hong Shao, Pedram Razavi, Alexander Gorelick, Erik Ladewig, Alesia Antoine, Hardik Shah, Eneda Toska, Guotai Xu, Abiha Kazmi, Barry Taylor, Komal Jhaveri, Maura Dickler, Elisa de Stanchina, Eduard Reznik, Raul Rabadan, Melissa Smith, Robert Sebra, Lewis Cantley, Maurizio Scaltriti, Jose Baselga. Compound PIK3CA mutations support a mutational dose-response model for oncogene activation and response to PI3K inhibitor targeted therapy in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A21.

PIK3CA is the most frequently mutated oncogene across all human cancers, and codes for p110α, the catalytic subunit of the PI3K complex. PI3K catalyzes the phosphorylation of the lipid PIP2 to PIP3, which initiates a downstream signaling cascade involving the activation of AKT and mTOR. Hotspot E545K and H1047R mutations constitutively activate PI3K and are oncogenic in multiple cancer histologies including breast cancer, where PIK3CA mutations are present in 40% of tumors and are a target for cancer therapy. Recently, the PI3Kα inhibitor alpelisib has demonstrated improved PFS in patients with ER+ PIK3CA mutant metastatic breast cancer on a phase 3 randomized clinical trial, resulting in its FDA approval. In early clinical trials, we observed a population of patients with displayed prolonged clinical benefit to alpelisib, with double PIK3CA mutant tumors. This prompted us to undertake a comprehensive analysis of the prevalence of multiple PIK3CA mutations and to investigate their potential biological relevance and correlation with sensitivity to PI3Kα inhibitors.We analyzed multiple independent primary and metastatic tumor cohorts and found that 10-15% of all PIK3CA mutant tumors across all cancer histologies contain multiple PIK3CA mutations, the vast majority of which carry exactly two mutations. Double mutations occur at specific amino acid positions by codon enrichment analysis, where the most frequent combinations in breast cancer are comprised of a canonical “major mutant” hotspot (involving either E542, E545, or H1047) combined with a second “minor mutant” site (involving either E453, E726, or M1043). Double PIK3CA mutations are enriched in ER+/HER2- breast cancers and occur at similar frequencies in therapy-naïve primary tumors and metastatic tumors. To study the allelic configuration of double mutations, we leveraged single molecule real time sequencing (SMRT-seq) on fresh breast tumor samples from patients known to carry two PIK3CA mutations in their tumors by NGS. Six tumors representative of the most frequent double mutants in breast cancer were obtained from patients, and were analyzed by SMRT-seq. All contained double mutations in cis. We overexpressed double cis mutants and constituent single mutants in nontransformed and ER+ breast cancer cells. Double PIK3CA mutations in cis increased downstream PI3K pathway signaling and cell/tumor proliferation in vitro and in vivo, when compared to single mutations. In contrast, mutations in trans do not increase cell signaling or growth proliferation more than single mutations.The prevailing model of PI3K activation occurs through the engagement of the p85α regulatory subunit with phosphotyrosines on RTK signaling complexes, which relieves catalytic inhibition. Single oncogenic mutations recapitulate these events by weakening the interactions between p110α and p85α (“disrupters”), or by promoting binding to membrane (“binders”). We purified recombinant full length PI3Kα complexes containing single and double cis p110α mutations to dissect the biochemical mechanisms by which these double PIK3CA mutations in cis modulate kinase activity. We demonstrate that cis mutants have increased kinase activity compared to single mutants and that this activation is due to both increased p85α disruption and increased lipid binding, compared to single mutants.We tested whether cis mutant cells exhibit differential sensitivity to PI3Kα inhibitors. While in the absence of treatment, cis mutant signaling is increased compared to single mutants, treatment with the PI3Kα inhibitors alpelisib or GDC-0077 results in a similar PI3K pathway inhibition in nontransformed and ER+ breast cancer cells. With respect to cell viability, E545K and H1047R major hotspot mutants are more sensitive to alpelisib and GDC-0077 compared to minor mutants and WT. In turn, all cis mutants are synergistically more sensitive to alpelisib and GDC-0077 compared to single major and minor hotspots.We analyzed response data from SANDPIPER, a randomized phase 3 clinical trial that tested the efficacy of the ER degrader fulvestrant with or without the PI3Kα/γ/δ inhibitor taselisib in metastatic ER+ PIK3CA mutant breast cancer. We used ctDNA to detect the presence of PIK3CA mutations. PIK3CA mutant patients on the taselisib arm had an objective response rate (ORR) of 20.3% vs 9.7% compared to the placebo arm, which was statistically significant, confirming that PIK3CA mutation predicts response to PI3Kα inhibition. We then compared responses of patients with single vs multiple mutations. Single mutant patients on the taselisib arm had an ORR of 18.1% vs 10.0% compared to the placebo arm, which was not statistically significant. On the contrary, multiple mutant patients on the taselisib arm achieved an ORR of 30.2% vs 8.7% compared to the placebo arm, which was statistically significant. These findings confirm that breast cancer patients with multiple mutant tumors achieve higher clinical benefit to PI3Kα inhibition compared to single mutant tumors.In this work, we have identified double mutations in cis as a novel, and relatively frequent, genomic alteration in PIK3CA, which translates into a clinically meaningful number of patients who may derive additional benefit from targeted therapy. Our results implicate a model of oncogene addiction to double mutant PIK3CA in breast cancer, with graded levels of activation and inhibition for single vs double mutants. PI3Kα inhibitors are now a standard of care in PIK3CA mutant ER+ metastatic breast cancer and are being explored in other PIK3CA mutant tumor histologies. Our findings provide a rationale for testing whether patients with multiple PIK3CA mutant tumors are exquisitely sensitive to PI3Kα inhibitors. Citation Format: Neil Vasan, Pedram Razavi, Jared L. Johnson, Hong Shao, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Ting-Yu Lin, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T. Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Sarat Chandarlapaty, Raul Rabadan, Ed Reznik, Melissa L. Smith, Robert Sebra, Frauke Schimmoller, Timothy R. Wilson, Lori S. Friedman, Lewis C. Cantley, Maurizio Scaltriti, Jose Baselga. Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG16.

Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide-3-kinase (PI3K), are the most frequent oncogenic alterations in estrogen receptor-positive (ER+) breast cancer and are also prevalent in other tumor types. PI3Kα inhibitors including alpelisib have recently been shown to be clinically active in ER+ PIK3CA mutant breast cancer. To characterize determinants of sensitivity to these agents, we undertook a comprehensive analysis of PIK3CA mutant cancer genomes and observed the presence of double PIK3CA mutations in 12-15% of breast cancer and other tumor types. These double PIK3CA mutations are clonal, located in cis on the same allele, and are composed of a single hotspot mutation combined with a recurrent second-site mutation. Double PIK3CA mutations in cis result in increased PI3K activity and downstream signaling together with enhanced cell proliferation and tumor growth compared to single hotspot mutations. The biochemical mechanisms underlying this increased oncogenicity include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased membrane lipid binding. Finally, these double PIK3CA mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations (e.g. E545K or H1047R) in experimental models and in patients with ER+ PIK3CA mutant metastatic breast cancer from the SANDPIPER randomized phase III clinical trial. These findings implicate double PIK3CA mutations in cis as a novel mechanism of oncogene addiction relative to single hotspot mutations, providing a rationale to develop PI3Kα inhibitors for the therapy of double PIK3CA mutant cancers. Citation Format: Neil Vasan, Pedram Razavi, Jared L Johnson, Hong Shao, Timothy Wilson, Frauke Schimmoller, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Ting-Yu Lin, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Sarat Chandarlapaty, Raul Rabadan, Ed Reznik, Melissa L Smith, Robert Sebra, Lori Friedman, Lewis C Cantley, Maurizio Scaltriti, José Baselga. Double PIK3CA mutations in cis drive oncogene addiction and enhance sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-01.

Although platinum‐based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume‐regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8‐dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug‐induced apoptosis independently from drug uptake, possibly by impairing VRAC‐dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D‐containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common somatic alteration of the SWI/SNF complex across all cancers including estrogen receptor positive (ER)+ breast cancer. We have recently reported that ARID1A inactivating mutations are present at a high frequency in advanced endocrine resistant ER+ breast cancer. However, the mechanisms whereby disruption of ARID1A influences breast cancer progression and/or endocrine therapy resistance are unknown. In order to elucidate mechanisms of resistance to endocrine therapy, we performed an epigenome CRISPR/CAS9 knockout screen that identified ARID1A as the top candidate whose loss determines resistance to the ER degrader, fulvestrant. ARID1A knockout cells were found to be less responsive to endocrine therapy compared to intact ARID1A cells in vitro and in vivo. These observations led us to undertake a comprehensive chromatin-based mechanistic characterization of ARID1A loss in ER+ breast cancer and its role in endocrine therapy response. ARID1A disruption in ER+ breast cancer cells led to widespread changes in chromatin accessibility converging on the master transcription factors (TFs) that regulate gene expression programs critical for luminal (ER+) lineage identity. Global transcriptome profiling of ARID1A knockout cell lines and patient samples harboring ARID1A inactivating mutations revealed an enrichment for basal-like (ER-) gene expression signatures. The state of increased cellular plasticity of luminal cells that acquire a basal-like phenotype upon ARID1A inactivation is enabled by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the major luminal-lineage determining transcription factors including ER, FOXA1, and GATA3. Thus, through widespread chromatin reprograming and functional regulation of mater luminal TFs, tumor cells alter lineage fidelity and become less responsive to luminal-specific anti-ER therapy. We also show that ARID1A regulates genome-wide ER-chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in the determination of breast luminal cell identity and endocrine therapeutic response in breast cancer. Citation Format: Eneda Toska, Guotai Xu, Sagar Chhangawala, Emiliano Cocco, Pedram Razavi, Jordan Otto, Yanyan Cai, Carmen Chan, Drew R. D' Avino, Clayton Collings, Ross L. Levine, Maurizo Scaltriti, Jorge S. Reis-Filho, Cigall Kadoch, Christina Leslie, Jose Baselga. ARID1A is a critical regulator of luminal identity and therapeutic response in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 949.

Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations. Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case. In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest. Citation Format: Jorge Gómez Tejeda Zañudo, Pingping Mao, Joan Montero, Guotai Xu, Kailey J. Kowalski, Gabriela N. Johnson, José Baselga, Maurizio Scaltriti, Anthony G. Letai, Nikhil Wagle, Reka Albert. Network modeling of drug resistance mechanisms and drug combinations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 675.

Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide 3-kinase (PI3K), are the most frequent oncogenic alterations in all cancers, including estrogen receptor-positive (ER+) breast cancer. There are many distinct oncogenic PIK3CA mutations including major hotspot mutations (e.g. E542K, E545K, H1047R) which are predictive of response to PI3Kα inhibitors. While responses to PI3Kα inhibitors can be variable, some patients with single hotspot mutant tumors derive a deep and prolonged clinical benefit. In a comprehensive analysis of the genomic data available in breast cancer, we observed double PIK3CA mutations and investigated their potential biological relevance and potential correlation with sensitivity to PI3Kα inhibitors. We detected double PIK3CA mutations in 10-15% of all PIK3CA-mutant cancers including breast cancer. Double PIK3CA mutations in breast cancer are defined by a pattern of co-expression of a major hotspot mutation associated with a recurrent second-site PIK3CA mutation (‘minor mutation’). We have found that these double PIK3CA mutations are compound, that is in cis on the same allele, using single molecule real time sequencing (SMRT-seq) of fresh breast tumor samples. Double compound PIK3CA mutations result in increased PI3K activity and downstream signaling compared to single hotspot mutants in nontransformed cells and in ER+ breast cancer cells. Double compound mutations also increase cell proliferation and xenograft growth compared to single hotspot mutants. Biochemical experiments using recombinant PI3K compound mutant protein complexes reveal a combination mechanism of action, with simultaneous PI3K destabilization and increased membrane binding compared to single hotspot mutants, leading to increased kinase activity. Importantly, these compound mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutants in both preclinical models and also in patients with breast cancer. Together, our data support that double compound PIK3CA mutations enhance the oncogenicity of single hotspot PIK3CA mutations and this increased dependency results in increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations. We propose that double compound activating mutations are an additional mechanism of oncogenic transformation in addition to other known mechanisms such as gene amplification, point mutation, or gene translocation. We speculate that double compound mutant PIK3CA may function as a clinical biomarker of increased sensitivity to PI3K-directed targeted therapies and may improve the therapeutic window of PI3K inhibitors in ER+ breast cancer and other PIK3CA-mutant tumor histologies. Citation Format: Neil Vasan, Pedram Razavi, Jared Johnson, Hong Shao, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T. Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Raul Rabadan, Ed Reznik, Melissa L. Smith, Robert Sebra, Lewis C. Cantley, Maurizio Scaltriti, Jose Baselga. Double PIK3CA mutations in cis enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3917.

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

To investigate the application value of three-dimensional reconstruction and virtual preoperative planning for Pilon fractures.Between July 2010 and June 2014, 16 patients with closed Pilon fracture were treated, including 12 males and 4 females with an average age of 36.5 years (range, 22-53 years) and a mean disease duration of 10.2 days (range, 6-14 days). According to AO/Orthopaedic Trauma Association (AO/OTA) typing, 2 cases were rated as 43.B2 type, 3 cases as 43.B3 type, 3 cases as 43.C1 type, 2 cases as 43.C2 type, and 6 cases as 43.C3 type. The preoperative CT data from 16 patients were imported into Mimics10.01 software to establish the detailed fracture three-dimensional digital models. Virtual operation of fracture reduction and implanting internal fixation was performed on the models, and the optional surgical planning was made. Based on the virtual preoperative planning, operations were performed.Established detailed three-dimensional Pilon fracture digital models could perfectly reflect the fracture characteristics, could be observed at any direction, and aided for fracture classification accurately. Virtual fracture operations of reduction, internal fixation and other could be performed to simulate the clinical operation, which could assist the surgeon better preoperative planning in achieving visual presentation and improving the communication. The operation time was 70-130 minutes (mean, 87.8 minutes); intraoperative blood loss volume was 30-150 mL (mean, 71.9 mL). The wounds healed by first intension in all patients. The mean follow-up time was 11.6 months (range, 8-18 months). Postoperative radiological results at 3 groups showed good anatomic reduction according to the Burwell-Charnley criteria, and the fracture healing time was 3-6 months (mean, 3.7 months). There was no complication of internal fixation loosening or breakage during follow-up. The American Orthopedic Foot and Ankle Society (AOFAS) score was 71-100 (mean, 92.3); the results were excellent in 10 cases, good in 5 cases, and fair in 1 case, with an excellent and good rate of 93.8% at last follow-up. No loss of fracture reduction was observed on the X-ray film.The clinical feasibility of virtual reconstruction preoperative planning is good in the treatment of Pilon fractures, which helps surgeons better understanding Pilon fracure and making the surgical planning.

We integrated the genomic sequencing of 1,918 breast cancers, including 1,501 hormone receptor-positive tumors, with detailed clinical information and treatment outcomes. In 692 tumors previously exposed to hormonal therapy, we identified an increased number of alterations in genes involved in the mitogen-activated protein kinase (MAPK) pathway and in the estrogen receptor transcriptional machinery. Activating ERBB2 mutations and NF1 loss-of-function mutations were more than twice as common in endocrine resistant tumors. Alterations in other MAPK pathway genes (EGFR, KRAS, among others) and estrogen receptor transcriptional regulators (MYC, CTCF, FOXA1, and TBX3) were also enriched. Altogether, these alterations were present in 22% of tumors, mutually exclusive with ESR1 mutations, and associated with a shorter duration of response to subsequent hormonal therapies.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

Nearly 10 years ago the usefulness of poly(ADP-ribose) polymerase (PARP) inhibitors to kill BRCA1 or BRCA2-deficient cells was reported, and this finding has served as a prime example of the concept of synthetic lethality in the context of anticancer therapy. The clinical translation of this finding has undergone several ups and downs, however. Despite spectacular responses seen in some patients with BRCA-deficient breast or ovarian cancers, other patients did not show the expected benefit from PARP inhibitor therapy. Thus, like for all novel tailored anti-cancer drugs, upfront and secondary resistance remain major hurdles in the implementation of the initial preclinical finding. We know at least one clinically relevant mechanism of PARP inhibitor resistance: the reversion of BRCA function by secondary mutations. Nevertheless, it is also clear that this mechanism does not explain all cases of resistance. At the moment, we only have a poor understanding of BRCA reversion-independent resistance mechanisms. Preclinical data have pointed in several directions, e.g. increased drug efflux, reduced drug target levels, or alternative DNA repair. Here, we discuss these mechanisms with a focus on potential DNA repair adaptations.

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting ‘off-target’ sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0617-1) contains supplementary material, which is available to authorized users.

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this. Cancer Res; 75(4); 732–41. ©2014 AACR.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in diverse human cancers, including human lung cancer. In this study, STAT3β, a dominant negative (DN) form of STAT3, was used to block activated STAT3 in human lung cancer cells and to confirm the effects of DN STAT3 on lung cancer cell proliferation and invasion in?vitro. The results showed that the pIRES-STAT3β plasmid is efficiently transfected into and overexpressed in human lung cancer cells. Overexpressed STAT3β specifically blocked STAT3 transcriptional activation, inhibiting the proliferation and augmenting the apoptosis of human lung cancer cells. This was associated with the down-regulation of the anti-apoptotic gene bcl-xl and the cell cycle gene cyclin D1. Additionally, the invasive activity of A549 and PG cells was significantly inhibited by overexpressed STAT3β, and was accompanied by a decrease in matrix metalloproteinase-2 activation. These findings suggest that interfering with the DN STAT3, STAT3β, may induce potent antitumor activity. STAT3? is therefore a potential candidate for the treatment of human lung cancers with high metastasic ability.

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway(1). In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers(2,3). Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration(4). Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases(5). In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent endresection of DSBs in BRCA1 deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance(6). Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

We have previously shown that mammary tumors spontaneously arising in a genetically engineered mouse model for human BRCA1-associated breast cancer (K14Cre;Brca1F/F;p53F/F) are highly sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib due to their deficiency in homology-directed DNA repair (Rottenberg et al. PNAS 2008;105:17079-84). Despite the initial sensitivity, tumors are not eradicated and eventually develop drug resistance. In this project we aim to identify mechanisms that explain this resistance. In particular we are interested in alterations that result in restoration of DNA repair by homologous recombination (HR). For this purpose we performed a loss-of-function shRNA screen using Brca1; p53-deficient cell lines derived from a mouse mammary tumor. The CGH profile of these cell lines is highly similar to that of the original tumor. Moreover, these cells have successfully been transplanted orthotopically into syngeneic mice and they recapitulate the morphology and olaparib response of the donor tumor. In our screen we included 2000 hairpins from the Sigma Mission library which target 409 mouse genes involved in the DNA damage response. As a result we found enrichments for hairpins targeting Rev7 in olaparib-surviving colonies. This finding was validated with 3 individual hairpins which caused substantially decreased Rev7 mRNA levels. Moreover, Rev7-deficient Brca1−/−;p53−/− tumor cells are less sensitive to doxorubicin, carboplatin and cisplatin. Loss of Rev7 rescued mES cells from Brca1 deficiency and also correlated with the formation of Rad51 foci in both Brca1-deficient mES cells and Brca1−/−;p53−/− tumor cells after irradiation. We therefore think that homology-directed DNA repair is restored in Rev7-depleted Brca1−/−;p53−/− cells. We are currently confirming this finding using HR reporter assays and we are investigating whether the loss of Rev7 causes olaparib resistance in mouse tumors. Our data suggest that absence of REV7 function, like lack of 53BP1, contributes to PARP inhibitor resistance in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-392. doi:1538-7445.AM2012-LB-392

Many breast cancers respond to chemotherapy or hormonal therapy, however, the lack of tumor eradication is a central clinical problem preceding the development of drug resistant tumors. We have previously studied responses of mammary tumors generated in the K14cre;Brca1F5-13/F5-13;p53F2-10/F2-10 mouse model for hereditary breast cancer to clinically relevant anti-cancer drugs such as doxorubicin, topotecan, cisplatin and the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib (1-4). The BRCA1- and p53-deficient tumors generated in this model are hypersensitive to these drugs and never become resistant to cisplatin due to the large, irreversible deletion in Brca1 (5). We show here that even dose-dense treatment with a maximum tolerable dose of cisplatin does not result in tumor eradication. This is not due to the trivial possibility that the drug-resistant remnants are poorly accessible to drug, since we found a homogeneous distribution of the platinum-DNA adducts throughout the tumor. To explain this result we have addressed the hypothesis that the lack of eradication of drug-sensitive tumors is due to increased in vivo chemotherapy resistance of tumor-initiating cells (TICs). Using the CD24 and CD49f cell surface markers which detect normal mouse mammary stem cells, we have identified TICs in BRCA1- and p53-deficient tumors. In addition to the Lin-/CD24+/CD49f+ subpopulation, we show that a larger population of Lin-/CD24+/CD49f-cells also has tumor-initiating capability in at least two serial orthotopic transplantations, suggesting that these are not more differentiated transit-amplifying cells. However, we did not find an enrichment of TICs in cisplatin-treated tumor remnants. We conclude that in this model the resistance of cisplatin-surviving cells cannot be attributed to special biochemical defense mechanisms of TICs. Instead we will present data supporting the hypothesis that surviving TICs arrest in their cell cycle and ‘hibernate’ until the drug is gone. Using Brca1−/−;p53−/− cell lines derived from the mouse tumors we found that only Brca1−/−;p53−/− cells that were in the G0/G1 phase of the cell cycle 24h after cisplatin treatment were capable of forming new colonies, whereas cells in G2 or M were not. Hence, a p53-independent arrest in G0/G1 appears to underlie the lack of eradication of residual Brca1−/−;p53−/− cells. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A14