Phase 2 study of neoadjuvant enzalutamide and paclitaxel for luminal androgen receptor-enriched TNBC: Trial results and insights into "ARness".

Luminal androgen receptor (LAR)-enriched triple-negative breast cancer (TNBC) is a distinct subtype. The efficacy of AR inhibitors and the relevant biomarkers in neoadjuvant therapy (NAT) are yet to be determined. We tested the combination of the AR inhibitor enzalutamide (120 mg daily by mouth) and paclitaxel (80 mg/m2 weekly intravenously) (ZT) for 12 weeks as NAT for LAR-enriched TNBC. Eligibility criteria included a percentage of cells expressing nuclear AR by immunohistochemistry (iAR) of at least 10% and a reduction in sonographic volume of less than 70% after four cycles of doxorubicin and cyclophosphamide. Twenty-four patients were enrolled. Ten achieved a pathologic complete response or residual cancer burden-I. ZT was safe, with no unexpected side effects. An iAR of at least 70% had a positive predictive value of 0.92 and a negative predictive value of 0.97 in predicting LAR-enriched TNBC according to RNA-based assays. Our data support future trials of AR blockade in early-stage LAR-enriched TNBC.

Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer.

Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

Phylogenetic inference from single-cell RNA-seq data.

Tumors are comprised of subpopulations of cancer cells that harbor distinct genetic profiles and phenotypes that evolve over time and during treatment. By reconstructing the course of cancer evolution, we can understand the acquisition of the malignant properties that drive tumor progression. Unfortunately, recovering the evolutionary relationships of individual cancer cells linked to their phenotypes remains a difficult challenge. To address this need, we have developed PhylinSic, a method that reconstructs the phylogenetic relationships among cells linked to their gene expression profiles from single cell RNA-sequencing (scRNA-Seq) data. This method calls nucleotide bases using a probabilistic smoothing approach and then estimates a phylogenetic tree using a Bayesian modeling algorithm. We showed that PhylinSic identified evolutionary relationships underpinning drug selection and metastasis and was sensitive enough to identify subclones from genetic drift. We found that breast cancer tumors resistant to chemotherapies harbored multiple genetic lineages that independently acquired high K-Ras and ß-catenin, suggesting that therapeutic strategies may need to control multiple lineages to be durable. These results demonstrated that PhylinSic can reconstruct evolution and link the genotypes and phenotypes of cells across monophyletic tumors using scRNA-Seq.

PTEN in triple-negative breast carcinoma: protein expression and genomic alteration in pretreatment and posttreatment specimens.

Background: Recent advances have been made in targeting the phosphoinositide 3-kinase pathway in breast cancer. Phosphatase and tensin homolog (PTEN) is a key component of that pathway. Objective: To understand the changes in PTEN expression over the course of the disease in patients with triple-negative breast cancer (TNBC) and whether PTEN copy number variation (CNV) by next-generation sequencing (NGS) can serve as an alternative to immunohistochemistry (IHC) to identify PTEN loss. Methods: We compared PTEN expression by IHC between pretreatment tumors and residual tumors in the breast and lymph nodes after neoadjuvant chemotherapy in 96 patients enrolled in a TNBC clinical trial. A correlative analysis between PTEN protein expression and PTEN CNV by NGS was also performed. Results: With a stringent cutoff for PTEN IHC scoring, PTEN expression was discordant between pretreatment and posttreatment primary tumors in 5% of patients (n = 96) and between posttreatment primary tumors and lymph node metastases in 9% (n = 33). A less stringent cutoff yielded similar discordance rates. Intratumoral heterogeneity for PTEN loss was observed in 7% of the patients. Among pretreatment tumors, PTEN copy numbers by whole exome sequencing (n = 72) were significantly higher in the PTEN-positive tumors by IHC compared with the IHC PTEN-loss tumors (p < 0.0001). However, PTEN-positive and PTEN-loss tumors by IHC overlapped in copy numbers: 14 of 60 PTEN-positive samples showed decreased copy numbers in the range of those of the PTEN-loss tumors. Conclusion: Testing various specimens by IHC may generate different PTEN results in a small proportion of patients with TNBC; therefore, the decision of testing one versus multiple specimens in a clinical trial should be defined in the patient inclusion criteria. Although a distinct cutoff by which CNV differentiated PTEN-positive tumors from those with PTEN loss was not identified, higher copy number of PTEN may confer positive PTEN, whereas lower copy number of PTEN would necessitate additional testing by IHC to assess PTEN loss. Trial registration: NCT02276443.

Simple combination of multiple somatic variant callers to increase accuracy.

Publications comparing variant caller algorithms present discordant results with contradictory rankings. Caller performances are inconsistent and wide ranging, and dependent upon input data, application, parameter settings, and evaluation metric. With no single variant caller emerging as a superior standard, combinations or ensembles of variant callers have appeared in the literature. In this study, a whole genome somatic reference standard was used to derive principles to guide strategies for combining variant calls. Then, manually annotated variants called from the whole exome sequencing of a tumor were used to corroborate these general principles. Finally, we examined the ability of these principles to reduce noise in targeted sequencing.

Multiomic analysis of homologous recombination-deficient end-stage high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC) is frequently characterized by homologous recombination (HR) DNA repair deficiency and, while most such tumors are sensitive to initial treatment, acquired resistance is common. We undertook a multiomics approach to interrogate molecular diversity in end-stage disease, using multiple autopsy samples collected from 15 women with HR-deficient HGSC. Patients had polyclonal disease, and several resistance mechanisms were identified within most patients, including reversion mutations and HR restoration by other means. We also observed frequent whole-genome duplication and global changes in immune composition with evidence of immune escape. This analysis highlights diverse evolutionary changes within HGSC that evade therapy and ultimately overwhelm individual patients.

Glycolysis regulates KRAS plasma membrane localization and function through defined glycosphingolipids.

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.

Predictors of success in establishing orthotopic patient-derived xenograft models of triple negative breast cancer.

Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient's diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient's tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.

Predicting clinical response to everolimus in ER+ breast cancers using machine-learning.

Endocrine therapy remains the primary treatment choice for ER+ breast cancers. However, most advanced ER+ breast cancers ultimately develop resistance to endocrine. This acquired resistance to endocrine therapy is often driven by the activation of the PI3K/AKT/mTOR signaling pathway. Everolimus, a drug that targets and inhibits the mTOR complex has been shown to improve clinical outcomes in metastatic ER+ breast cancers. However, there are no biomarkers currently available to guide the use of everolimus in the clinic for progressive patients, where multiple therapeutic options are available. Here, we utilized gene expression signatures from 9 ER+ breast cancer cell lines and 23 patients treated with everolimus to develop and validate an integrative machine learning biomarker of mTOR inhibitor response. Our results show that the machine learning biomarker can successfully distinguish responders from non-responders and can be applied to identify patients that will most likely benefit from everolimus treatment.

Human NOP2/NSUN1 regulates ribosome biogenesis through non-catalytic complex formation with box C/D snoRNPs.

5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5'ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.

Tumor Immune Microenvironment Changes by Multiplex Immunofluorescence Staining in a Pilot Study of Neoadjuvant Talazoparib for Early-Stage Breast Cancer Patients with a Hereditary BRCA Mutation.

PURPOSE: The immunological profile of early-stage breast cancer treated with neoadjuvant PARP inhibitors has not been described. The aim of this study was to delineate the changes in the tumor immune microenvironment (TiME) induced by talazoparib. PATIENTS AND METHODS: Patients with operable germline BRCA1/2 pathogenic variant (gBRCA1/2+) breast cancer were enrolled in a feasibility study of neoadjuvant talazoparib. Thirteen patients who received 8 weeks of neoadjuvant talazoparib were available for analysis, including 11 paired pre- and post-talazoparib core biopsies. Treatment-related changes in tumor-infiltrating lymphocytes were examined and immune cell phenotypes and their spatial distribution in the TiME were identified and quantified by multiplex immunofluorescence using a panel of 6 biomarkers (CD3, CD8, CD68, PD-1, PD-L1, and CK). RESULTS: Neoadjuvant talazoparib significantly increased infiltrating intratumoral and stromal T-cell and cytotoxic T-cell density. There was no difference in PD-1 or PD-L1 immune cell phenotypes in the pre- and post-talazoparib specimens and PD-L1 expression in tumor cells was rare in this cohort. Spatial analysis demonstrated that pre-talazoparib interactions between macrophages and T cells may correlate with pathologic complete response. CONCLUSIONS: This is the first study with phenotyping to characterize the immune response to neoadjuvant talazoparib in patients with gBRCA1/2+ breast cancer. These findings support an emerging role for PARP inhibitors in enhancing tumor immunogenicity. Further investigation of combinatorial strategies is warranted with agents that exploit the immunomodulatory effects of PARP inhibitors on the TiME.

Identification of biomarkers of response to preoperative talazoparib monotherapy in treatment naïve gBRCA+ breast cancers.

Germline mutations in BRCA1 or BRCA2 exist in ~2-7% of breast cancer patients, which has led to the approval of PARP inhibitors in the advanced setting. We have previously reported a phase II neoadjuvant trial of single agent talazoparib for patients with germline BRCA pathogenic variants with a pathologic complete response (pCR) rate of 53%. As nearly half of the patients treated did not have pCR, better strategies are needed to overcome treatment resistance. To this end, we conducted multi-omic analysis of 13 treatment naïve breast cancer tumors from patients that went on to receive single-agent neoadjuvant talazoparib. We looked for biomarkers that were predictive of response (assessed by residual cancer burden) after 6 months of therapy. We found that all resistant tumors exhibited either the loss of SHLD2, expression of a hypoxia signature, or expression of a stem cell signature. These results indicate that the deep analysis of pre-treatment tumors can identify biomarkers that are predictive of response to talazoparib and potentially other PARP inhibitors, and provides a framework that will allow for better selection of patients for treatment, as well as a roadmap for the development of novel combination therapies to prevent emergence of resistance.

Molecular Characterization and Prospective Evaluation of Pathologic Response and Outcomes with Neoadjuvant Therapy in Metaplastic Triple-Negative Breast Cancer.

PURPOSE: Metaplastic breast cancer (MpBC) is a rare subtype of breast cancer that is commonly triple-negative and poorly responsive to neoadjuvant therapy in retrospective studies. EXPERIMENTAL DESIGN: To better define clinical outcomes and correlates of response, we analyzed the rate of pathologic complete response (pCR) to neoadjuvant therapy, survival outcomes, and genomic and transcriptomic profiles of the pretreatment tumors in a prospective clinical trial (NCT02276443). A total of 211 patients with triple-negative breast cancer (TNBC), including 39 with MpBC, received doxorubicin-cyclophosphamide-based neoadjuvant therapy. RESULTS: Although not meeting the threshold for statistical significance, patients with MpBCs were less likely to experience a pCR (23% vs. 40%; P = 0.07), had shorter event-free survival (29.4 vs. 32.2 months, P = 0.15), metastasis-free survival (30.3 vs. 32.4 months, P = 0.22); and overall survival (32.6 vs. 34.3 months, P = 0.21). This heterogeneity is mirrored in the molecular profiling. Mutations in PI3KCA (23% vs. 9%, P = 0.07) and its pathway (41% vs. 18%, P = 0.02) were frequently observed and enriched in MpBCs. The gene expression profiles of each histologically defined subtype were distinguishable and characterized by distinctive gene signatures. Among nonmetaplastic (non-Mp) TNBCs, 10% possessed a metaplastic-like gene expression signature and had pCR rates and survival outcomes similar to MpBC. CONCLUSIONS: Further investigations will determine if metaplastic-like tumors should be treated more similarly to MpBC in the clinic. The 23% pCR rate in this study suggests that patients with MpBC should be considered for NAT. To improve this rate, a pathway analysis predicted enrichment of histone deacetylase (HDAC) and RTK/MAPK pathways in MpBC, which may serve as new targetable vulnerabilities.

Chemotherapy Coupled to Macrophage Inhibition Induces T-cell and B-cell Infiltration and Durable Regression in Triple-Negative Breast Cancer.

Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful antitumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small-molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single-cell RNA sequencing characterized tumor-infiltrating lymphocytes including Th cells and antigen-presenting B cells that were highly enriched in responders to combination therapy. In one model that exhibited long-term posttreatment tumor regression, high-dimensional imaging techniques identified the close spatial localization of B220+/CD86+-activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks posttreatment. The transcriptional and metabolic heterogeneity of TAMs was also characterized in two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model was highly conserved in human claudin-low breast cancers, and high expression of the TAM signature correlated with reduced overall survival in patients with breast cancer. This TAM signature may help identify human patients with claudin-low breast cancer that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rational immunotherapy combinations. SIGNIFICANCE: Immunostimulatory chemotherapy combined with pharmacologic inhibition of TAMs results in durable treatment responses elicited by Th cells and B cells in claudin-low TNBC models.

Prognostic Impact of High Baseline Stromal Tumor-Infiltrating Lymphocytes in the Absence of Pathologic Complete Response in Early-Stage Triple-Negative Breast Cancer.

High stromal tumor-infiltrating lymphocytes (sTILs) are associated with an improved pathologic complete response (pCR) and survival in triple-negative breast cancer (TNBC). We hypothesized that high baseline sTILs would have a favorable prognostic impact in TNBC patients without a pCR after neoadjuvant chemotherapy (NACT). In this prospective NACT study, pretreatment biopsies from 318 patients with early-stage TNBC were evaluated for sTILs. Recursive partitioning analysis (RPA) was applied to search for the sTIL cutoff best associated with a pCR. With ≥20% sTILs identified as the optimal cutoff, 33% patients had high sTILs (pCR rate 64%) and 67% had low sTILs (pCR rate 29%). Patients were stratified according to the sTIL cutoff (low vs. high) and response to NACT (pCR vs. residual disease (RD)). The primary endpoint was event-free survival (EFS), with hazard ratios calculated using the Cox proportional hazards regression model and the 3-year restricted mean survival time (RMST) as primary measures. Within the high-sTIL group, EFS was better in patients with a pCR compared with those with RD (HR 0.05; 95% CI 0.01-0.39; p = 0.004). The difference in the 3-year RMST for EFS between the two groups was 5.6 months (95% CI 2.3-8.8; p = 0.001). However, among patients with RD, EFS was not significantly different between those with high sTILs and those with low sTILs (p = 0.7). RNA-seq analysis predicted more CD8+ T cells in the high-sTIL group with favorable EFS compared with the high-sTIL group with unfavorable EFS. This study did not demonstrate that high baseline sTILs confer a benefit in EFS in the absence of a pCR.

ABCA1 Expression Is Upregulated in an EMT in Breast Cancer Cell Lines via MYC-Mediated De-Repression of Its Proximal Ebox Element.

The ATP-Binding Cassette transporter A1 (ABCA1) reverse cholesterol transport channel has been associated with a number of phenotypes in breast cancer, including reduced proliferation and increased metastatic capacity. It is induced in an epithelial-mesenchymal transition (EMT), but little is known about how this occurs, and whether it is sufficient to promote metastatic phenotypes. To address these questions, we have deciphered the transcriptional regulation of ABCA1 across EMT states and found that it is repressed by MYC via an E-box element in its P1 alternative promoter. De-repression of the promoter by MYC knockdown leads to induction of ABCA1 expression. This indicates that ABCA1 expression is regulated in an EMT, revealing another link between ABCA1 and malignant phenotypes.

A combinatorial oligogenic basis for the phenotypic plasticity between late-onset dilated and arrhythmogenic cardiomyopathy in a single family.

INTRODUCTION: Primary dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) are the two common and distinct forms of hereditary cardiomyopathies caused by defined pathogenic variants (PVs) typically in different sets of genes. DCM is characterized by left ventricular dilatation, dysfunction, and failure, whereas ARVC classically involves the right ventricle and is characterized by fibrofatty infiltration of the myocardium. DCM is caused primarily by the PVs in genes encoding sarcomere and cytoskeletal protein, while ARVC is mainly a disease of the desmosome proteins. DCM and ARVC exhibit partial phenotypic and genetic overlaps. AIM: To analyze the genetic basis of the phenotypic heterogeneity of cardiomyopathy in members of a single family. METHODS AND RESULTS: We recruited, clinically characterized, and performed whole-exome sequencing in five affected, three probably affected, and two clinically unaffected members of a single family. The family members mainly exhibited late-onset DCM associated with conduction defects and arrhythmias. One family member who died suddenly was diagnosed with the classic ARVC at autopsy and another presented with isolated ventricular tachycardia. A novel splicing (truncating) and a rare missense variant in the TTN gene, likely in cis, co-segregated with the phenotype in all affected and probably affected family members and were likely the causal variants. Several PVs and LPVs in other genes involved in cardiomyopathies and arrhythmias were also identified that seem to modify the expression of the phenotype. Notably, LPVs in the DSP and PKP2 genes, which are known genes for ARVC, were identified in the family member who also carried the TTN variants but developed the classic ARVC. CONCLUSION: The findings indicate the causal role of the TTN variants, exhibiting an age-dependent penetrance in late-onset DCM, and highlight the potential modifying role of the concomitant LPVs in additional genes on the expression of the phenotype, including a phenotypic switch from the anticipated DCM to ARVC. The findings support an oligogenic basis of the cardiac phenotype in hereditary cardiomyopathies. A comprehensive genetic analysis involving all PVs and LPVs along with detailed phenotypic characterization is necessary to gain insights into the molecular pathogenesis of hereditary cardiomyopathies.

Simplifying the development of portable, scalable, and reproducible workflows.

Command-line software plays a critical role in biology research. However, processes for installing and executing software differ widely. The Common Workflow Language (CWL) is a community standard that addresses this problem. Using CWL, tool developers can formally describe a tool's inputs, outputs, and other execution details. CWL documents can include instructions for executing tools inside software containers. Accordingly, CWL tools are portable-they can be executed on diverse computers-including personal workstations, high-performance clusters, or the cloud. CWL also supports workflows, which describe dependencies among tools and using outputs from one tool as inputs to others. To date, CWL has been used primarily for batch processing of large datasets, especially in genomics. But it can also be used for analytical steps of a study. This article explains key concepts about CWL and software containers and provides examples for using CWL in biology research. CWL documents are text-based, so they can be created manually, without computer programming. However, ensuring that these documents conform to the CWL specification may prevent some users from adopting it. To address this gap, we created ToolJig, a Web application that enables researchers to create CWL documents interactively. ToolJig validates information provided by the user to ensure it is complete and valid. After creating a CWL tool or workflow, the user can create 'input-object' files, which store values for a particular invocation of a tool or workflow. In addition, ToolJig provides examples of how to execute the tool or workflow via a workflow engine. ToolJig and our examples are available at https://github.com/srp33/ToolJig.

Serial single-cell genomics reveals convergent subclonal evolution of resistance as early-stage breast cancer patients progress on endocrine plus CDK4/6 therapy.

Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+) breast cancer patients but its value in earlier stage patients is unclear. We examined evolutionary trajectories of early-stage breast cancer tumors, using single cell RNA sequencing (scRNAseq) of serial biopsies from the FELINE clinical trial (#NCT02712723) of endocrine therapy (letrozole) alone or combined with the CDK inhibitor ribociclib. Despite differences in subclonal diversity evolution across patients and treatments, common resistance phenotypes emerged. Resistant tumors treated with combination therapy showed accelerated loss of estrogen signaling with convergent up-regulation of JNK signaling through growth factor receptors. In contrast, cancer cells maintaining estrogen signaling during mono- or combination therapy showed potentiation of CDK4/6 activation and ERK upregulation through ERBB4 signaling. These results indicate that combination therapy in early-stage ER+ breast cancer leads to emergence of resistance through a shift from estrogen to alternative growth signal-mediated proliferation.