Modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) promotes tumor cell survival, proliferation, epigenetic changes, angiogenesis, invasion, and metastasis. Here we demonstrate that in conditions of elevated glucose, there is increased expression of key drug resistance proteins (ABCB1, ABCG2, ERCC1, and XRCC1), all of which are regulated by the Hedgehog pathway. In elevated glucose conditions, we determined that the Hedgehog pathway transcription factors, GLI1 and GLI2, are modified by O-GlcNAcylation. This modification functionally enhanced their transcriptional activity. The activity of GLI was enhanced when O-GlcNAcase was inhibited, while inhibiting O-GlcNAc transferase caused a decrease in GLI activity. The metabolic impact of hyperglycemic conditions impinges on maintaining PKM2 in the less active state that facilitates the availability of glycolytic intermediates for biosynthetic pathways. Interestingly, under elevated glucose conditions, PKM2 directly influenced GLI activity. Specifically, abrogating PKM2 expression caused a significant decline in GLI activity and expression of drug resistance proteins. Cumulatively, our results suggest that elevated glucose conditions upregulate chemoresistance through elevated transcriptional activity of the Hedgehog/GLI pathway. Interfering in O-GlcNAcylation of the GLI transcription factors may be a novel target in controlling cancer progression and drug resistance of breast cancer.

HER2-targeted therapy dramatically improves outcomes in early breast cancer. Here we report the results of two HER2-targeted combinations in the neoadjuvant I-SPY2 phase 2 adaptive platform trial for early breast cancer at high risk of recurrence: ado-trastuzumab emtansine plus pertuzumab (T-DM1/P) and paclitaxel, trastuzumab and pertuzumab (THP). Eligible women have >2.5 cm clinical stage II/III HER2+ breast cancer, adaptively randomized to T-DM1/P, THP, or a common control arm of paclitaxel/trastuzumab (TH), followed by doxorubicin/cyclophosphamide, then surgery. Both T-DM1/P and THP arms 'graduate' in all subtypes: predicted pCR rates are 63%, 72% and 33% for T-DM1/P (n = 52), THP (n = 45) and TH (n = 31) respectively. Toxicity burden is similar between arms. Degree of HER2 pathway signaling and phosphorylation in pretreatment biopsy specimens are associated with response to both T-DM1/P and THP and can further identify highly responsive HER2+ tumors to HER2-directed therapy. This may help identify patients who can safely de-escalate cytotoxic chemotherapy without compromising excellent outcome.

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

The METRIC study (NCT#0199733) explored a novel antibody-drug conjugate, glembatumumab vedotin (GV), targeting gpNMB that is overexpressed in ~40% of patients with triple-negative breast cancer (TNBC) and associated with poor prognosis. The study was a randomized, open-label, phase 2b study that evaluated progression-free survival (PFS) of GV compared with capecitabine in gpNMB-overexpressing TNBC. Patients who had previously received anthracycline and taxane-based therapy were randomized 2:1 to receive, GV (1.88 mg/kg IV q21 days) or capecitabine (2500 mg/m2 PO daily d1-14 q21 days). The primary endpoint was RECIST 1.1 PFS per independent, blinded central review. In all, 327 patients were randomized to GV (213 treated) or capecitabine (92 treated). Median PFS was 2.9 months for GV vs. 2.8 months for capecitabine. The most common grade ≥3 toxicities for GV were neutropenia, rash, and leukopenia, and for capecitabine were fatigue, diarrhea, and palmar-plantar erythrodysesthesia. The study did not meet the primary endpoint of improved PFS over capecitabine or demonstrate a relative risk/benefit improvement over capecitabine.

The identification of prognostic markers in patients receiving neoadjuvant therapy is crucial for treatment optimization in HER2-positive breast cancer, with the immune microenvironment being a key factor. Here, we investigate the complexity of B and T cell receptor (BCR and TCR) repertoires in the context of two phase III trials, NeoALTTO and CALGB 40601, evaluating neoadjuvant paclitaxel with trastuzumab and/or lapatinib in women with HER2-positive breast cancer. BCR features, particularly the number of reads and clones, evenness and Gini index, are heterogeneous according to hormone receptor status and PAM50 subtypes. Moreover, BCR measures describing clonal expansion, namely evenness and Gini index, are independent prognostic factors. We present a model developed in NeoALTTO and validated in CALGB 40601 that can predict event-free survival (EFS) by integrating hormone receptor and clinical nodal status, breast pathological complete response (pCR), stromal tumor-infiltrating lymphocyte levels (%) and BCR repertoire evenness. A prognostic score derived from the model and including those variables, HER2-EveNT, allows the identification of patients with 5-year EFS > 90%, and, in those not achieving pCR, of a subgroup of immune-enriched tumors with an excellent outcome despite residual disease.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is a particularly aggressive form of the disease, and ultimately progresses in patients with metastases on standard therapies. Anthracyclines, such as doxorubicin, are an effective treatment for HER2-positive breast cancer, particularly when administered in combination with trastuzumab - however, doxorubicin-related cardiotoxicity has limited its use. Many patients are therefore never treated with anthracyclines, even upon disease progression, despite the potential for benefit. MM-302 is a novel, HER2-targeted antibody-liposomal doxorubicin conjugate that specifically targets HER2-overexpressing cells. Preclinical and Phase 1 data suggest that MM-302, as a monotherapy or in combination with trastuzumab, could be effective for managing previously treated, anthracycline-naïve, HER2-positive breast cancer, without the cardiotoxicity observed with free doxorubicin formulations.

MET expression and activation appear to be important for initiation and progression of triple-negative breast cancer. Tivantinib (ARQ 197) is an orally administered agent that targets MET, although recent preclinical data suggests the agent may have mechanisms of action that are independent of MET signaling. We conducted a phase 2 study of tivantinib monotherapy in patients with metastatic triple-negative breast cancer.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate comprising trastuzumab and DM1, a microtubule polymerization inhibitor, covalently bound via a stable thioether linker. To characterize the pharmacokinetics (PK) of T-DM1 in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer, data from four studies (TDM3569g, TDM4258g, TDM4374g, and TDM4688g) of single-agent T-DM1 administered at 3.6 mg/kg every 3 weeks (q3w) were assessed in aggregate.

Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated.

Treatment options for triple-negative breast cancer remain limited. Activation of the PI3K pathway via loss of PTEN and/or INPP4B is common. Buparlisib is an orally bioavailable, pan-class I PI3K inhibitor. We evaluated the safety and efficacy of buparlisib in patients with metastatic triple-negative breast cancer.

Human epidermal growth factor receptor 3 (HER3) is broadly expressed in breast cancer; high expression is associated with an adverse prognosis. Patritumab deruxtecan (HER3-DXd) is an investigational HER3-targeted antibody-drug conjugate that is being evaluated as a novel treatment in HER3-expressing advanced breast cancer in the U31402-A-J101 study.

Brentuximab vedotin is a CD30-directed antibody-drug conjugate. Retreatment with brentuximab vedotin monotherapy was investigated in patients with CD30-positive Hodgkin lymphoma (HL) or systemic anaplastic large cell lymphoma (ALCL) who relapsed after achieving complete or partial remission (CR or PR) with initial brentuximab vedotin therapy in a previous study (ClinicalTrials.gov NCT00947856).

We aimed to improve efficacy and reduce toxicity of high-risk human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC) treatment by replacing taxanes and trastuzumab with trastuzumab emtansine (T-DM1).

Despite the availability of multiple human epidermal growth factor receptor 2-targeted (HER2-targeted) treatments, therapeutic resistance in HER2+ breast cancer remains a clinical challenge. Intratumor heterogeneity for HER2 and resistance-conferring mutations in the PIK3CA gene (encoding PI3K catalytic subunit α) have been investigated in response and resistance to HER2-targeting agents, while the role of divergent cellular phenotypes and tumor epithelial-stromal cell interactions is less well understood. Here, we assessed the effect of intratumor cellular genetic heterogeneity for ERBB2 (encoding HER2) copy number and PIK3CA mutation on different types of neoadjuvant HER2-targeting therapies and clinical outcome in HER2+ breast cancer. We found that the frequency of cells lacking HER2 was a better predictor of response to HER2-targeted treatment than intratumor heterogeneity. We also compared the efficacy of different therapies in the same tumor using patient-derived xenograft models of heterogeneous HER2+ breast cancer and single-cell approaches. Stromal determinants were better predictors of response than tumor epithelial cells, and we identified alveolar epithelial and fibroblastic reticular cells as well as lymphatic vessel endothelial hyaluronan receptor 1-positive (Lyve1+) macrophages as putative drivers of therapeutic resistance. Our results demonstrate that both preexisting and acquired resistance to HER2-targeting agents involve multiple mechanisms including the tumor microenvironment. Furthermore, our data suggest that intratumor heterogeneity for HER2 should be incorporated into treatment design.

In preclinical studies, the expression of vascular endothelial growth factor (VEGF) in hormone receptor-positive breast cancer is associated with estrogen-independent tumor growth and resistance to endocrine therapies. This study investigated whether the addition of bevacizumab, a monoclonal antibody against VEGF, to letrozole enhanced the antitumor activity of the letrozole in the preoperative setting.

Poly (ADP-ribose)-polymerase inhibitors (PARPi) have been approved for cancer patients with germline BRCA1/2 (gBRCA1/2) mutations, and efforts to expand the utility of PARPi beyond BRCA1/2 are ongoing. In preclinical models of triple-negative breast cancer (TNBC) with intact DNA repair, we have previously shown an induced synthetic lethality with combined EGFR inhibition and PARPi. Here, we report the safety and clinical activity of lapatinib and veliparib in patients with metastatic TNBC.

Assaying for large numbers of low-frequency mutations requires sequencing at extremely high depth and accuracy. Increasing sequencing depth aids the detection of low-frequency mutations yet limits the number of loci that can be simultaneously probed. Here we report a method for the accurate tracking of thousands of distinct mutations that requires substantially fewer reads per locus than conventional hybrid-capture duplex sequencing. The method, which we named MAESTRO (for minor-allele-enriched sequencing through recognition oligonucleotides), combines massively parallel mutation enrichment with duplex sequencing to track up to 10,000 low-frequency mutations, with up to 100-fold fewer reads per locus. We show that MAESTRO can be used to test for chimaerism by tracking donor-exclusive single-nucleotide polymorphisms in sheared genomic DNA from human cell lines, to validate whole-exome sequencing and whole-genome sequencing for the detection of mutations in breast-tumour samples from 16 patients, and to monitor the patients for minimal residual disease via the analysis of cell-free DNA from liquid biopsies. MAESTRO improves the breadth, depth, accuracy and efficiency of mutation testing by sequencing.

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

Brain metastases represent the greatest clinical challenge in treating HER2-positive breast cancer. We report the development of orthotopic patient-derived xenografts (PDXs) of HER2-expressing breast cancer brain metastases (BCBM), and their use for the identification of targeted combination therapies. Combined inhibition of PI3K and mTOR resulted in durable tumor regressions in three of five PDXs, and therapeutic response was correlated with a reduction in the phosphorylation of 4EBP1, an mTORC1 effector. The two nonresponding PDXs showed hypermutated genomes with enrichment of mutations in DNA-repair genes, which suggests an association of genomic instability with therapeutic resistance. These findings suggest that a biomarker-driven clinical trial of PI3K inhibitor in combination with an mTOR inhibitor should be conducted for patients with HER2-positive BCBM.