Ganetespib is a small molecule, nongeldanamycin HSP90 inhibitor with potent inhibitory effects on HSP90-dependent oncoproteins of relevance to breast cancer pathogenesis. We therefore tested ganetespib in an unselected cohort of patients with MBC.

Trastuzumab is effective in human epidermal growth factor receptor 2 (HER2)-over-expressing breast and gastric cancers. However, patients may develop resistance through downstream signaling via the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. This phase 1 trial was conducted to determine the safety and tolerability of the investigational AKT inhibitor MK-2206, to prepare for future studies to determine whether the combination with trastuzumab could inhibit compensatory signaling.

Prostate cancer is characterized by its dependence on androgen receptor (AR) and frequent activation of PI3K signaling. We find that AR transcriptional output is decreased in human and murine tumors with PTEN deletion and that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates AKT signaling by reducing levels of the AKT phosphatase PHLPP. Thus, these two oncogenic pathways cross-regulate each other by reciprocal feedback. Inhibition of one activates the other, thereby maintaining tumor cell survival. However, combined pharmacologic inhibition of PI3K and AR signaling caused near-complete prostate cancer regressions in a Pten-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival.

T-cell-based therapies have emerged as one of the most clinically effective ways to target solid and non-solid tumors. HER2 is responsible for the oncogenesis and treatment resistance of several human solid tumors. As a member of the HER family of tyrosine kinase receptors, its over-activity confers unfavorable clinical outcome. Targeted therapies directed at this receptor have achieved responses, although development of resistance is common. We explored a novel HER2/CD3 bispecific antibody (HER2-BsAb) platform that while preserving the anti-proliferative effects of trastuzumab, it recruits and activates non-specific circulating T-cells, promoting T cell tumor infiltration and ablating HER2(+) tumors, even when these are resistant to standard HER2-targeted therapies. Its in vitro tumor cytotoxicity, when expressed as EC50, correlated with the surface HER2 expression in a large panel of human tumor cell lines, irrespective of lineage or tumor type. HER2-BsAb-mediated cytotoxicity was relatively insensitive to PD-1/PD-L1 immune checkpoint inhibition. In four separate humanized mouse models of human breast cancer and ovarian cancer cell line xenografts, as well as human breast cancer and gastric cancer patient-derived xenografts (PDXs), HER2-BsAb was highly effective in promoting T cell infiltration and suppressing tumor growth when used in the presence of human peripheral blood mononuclear cells (PBMC) or activated T cells (ATC). The in vivo and in vitro antitumor properties of this BsAb support its further clinical development as a cancer immunotherapeutic.

Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.

Five to ten percent of ER+ metastatic breast cancer (MBC) tumors harbor somatic PTEN mutations. Loss of function of this tumor-suppressor gene defines a highly aggressive, treatment-refractory disease for which new therapies are urgently needed. This Phase I multipart expansion study assessed oral capivasertib with fulvestrant in patients with PTEN-mutant ER+ MBC. Safety and tolerability were assessed by standard methods. Plasma and tumor were collected for NGS and immunohistochemistry analyses of PTEN protein expression. In 31 eligible patients (12 fulvestrant naive; 19 fulvestrant pretreated), the 24-week clinical benefit rate was 17% in fulvestrant-naive and 42% in fulvestrant-pretreated patients, with objective response rate of 8% and 21%, respectively. Non-functional PTEN was centrally confirmed in all cases by NGS or immunohistochemistry. Co-mutations occurred in PIK3CA (32%), with less ESR1 (10% vs 72%) and more TP53 (40% vs 28%) alterations in fulvestrant-naive versus fulvestrant-pretreated patients, respectively. PTEN was clonally dominant in most patients. Treatment-related grade ≥3 adverse events occurred in 32% of patients, most frequently diarrhea and maculopapular rash (both n = 2). In this clinical study, which selectively targeted the aggressive PTEN-mutant ER+ MBC, capivasertib plus fulvestrant was tolerable and clinically active. Phenotypic and genomic differences were apparent between fulvestrant-naive and -pretreated patients.Trial registration number for the study is NCT01226316.

Although most primary estrogen receptor (ER)-positive breast cancers respond well to endocrine therapies, many relapse later as metastatic disease due to endocrine therapy resistance. Over one third of these are associated with mutations in the ligand-binding domain (LBD) that activate the receptor independent of ligand. We have used an array of advanced computational techniques rooted in molecular dynamics simulations, in concert with and validated by experiments, to characterize the molecular mechanisms by which specific acquired somatic point mutations give rise to ER constitutive activation. By comparing structural and energetic features of constitutively active mutants and ligand-bound forms of ER-LBD with unliganded wild-type (WT) ER, we characterize a spring force originating from strain in the Helix 11-12 loop of WT-ER, opposing folding of Helix 12 into the active conformation and keeping WT-ER off and disordered, with the ligand-binding pocket open for rapid ligand binding. We quantify ways in which this spring force is abrogated by activating mutations that latch (Y537S) or relax (D538G) the folded form of the loop, enabling formation of the active conformation without ligand binding. We also identify a new ligand-mediated hydrogen-bonding network that stabilizes the active, ligand-bound conformation of WT-ER LBD, and similarly stabilizes the active conformation of the ER mutants in the hormone-free state. IMPLICATIONS: Our investigations provide deep insight into the energetic basis for the structural mechanisms of receptor activation through mutation, exemplified here with ER in endocrine-resistant metastatic breast cancers, with potential application to other dysregulated receptor signaling due to driver mutations.

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are key therapeutic agents in the management of metastatic hormone-receptor-positive breast cancer. However, the emergence of drug resistance limits their long-term efficacy. Here, we show that breast cancer cells develop CDK4/6i resistance via a sequential two-step process of E2F activation. This process entails retinoblastoma (Rb)-protein degradation, followed by c-Myc-mediated amplification of E2F transcriptional activity. CDK4/6i treatment halts cell proliferation in an Rb-dependent manner but dramatically reduces Rb-protein levels. However, this reduction in Rb levels insufficiently induces E2F activity. To develop CDK4/6i resistance, upregulation or activating mutations in mitogenic or hormone signaling are required to stabilize c-Myc levels, thereby augmenting E2F activity. Our analysis of pre-treatment tumor samples reveals a strong correlation between c-Myc levels, rather than Rb levels, and poor therapeutic outcomes after CDK4/6i treatment. Moreover, we propose that proteasome inhibitors can potentially reverse CDK4/6i resistance by restoring Rb levels.

Physiologic activation of estrogen receptor α (ERα) is mediated by estradiol (E2) binding in the ligand-binding pocket of the receptor, repositioning helix 12 (H12) to facilitate binding of coactivator proteins in the unoccupied coactivator binding groove. In breast cancer, activation of ERα is often observed through point mutations that lead to the same H12 repositioning in the absence of E2. Through expanded genetic sequencing of breast cancer patients, we identified a collection of mutations located far from H12 but nonetheless capable of promoting E2-independent transcription and breast cancer cell growth. Using machine learning and computational structure analyses, this set of mutants was inferred to act distinctly from the H12-repositioning mutants and instead was associated with conformational changes across the ERα dimer interface. Through both in vitro and in-cell assays of full-length ERα protein and isolated ligand-binding domain, we found that these mutants promoted ERα dimerization, stability, and nuclear localization. Point mutations that selectively disrupted dimerization abrogated E2-independent transcriptional activity of these dimer-promoting mutants. The results reveal a distinct mechanism for activation of ERα function through enforced receptor dimerization and suggest dimer disruption as a potential therapeutic strategy to treat ER-dependent cancers.

Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.

Cellular proteins continuously undergo non-enzymatic covalent modifications (NECMs) that accumulate under normal physiological conditions and are stimulated by changes in the cellular microenvironment. Glycation, the hallmark of diabetes, is a prevalent NECM associated with an array of pathologies. Histone proteins are particularly susceptible to NECMs due to their long half-lives and nucleophilic disordered tails that undergo extensive regulatory modifications; however, histone NECMs remain poorly understood. Here we perform a detailed analysis of histone glycation in vitro and in vivo and find it has global ramifications on histone enzymatic PTMs, the assembly and stability of nucleosomes, and chromatin architecture. Importantly, we identify a physiologic regulation mechanism, the enzyme DJ-1, which functions as a potent histone deglycase. Finally, we detect intense histone glycation and DJ-1 overexpression in breast cancer tumors. Collectively, our results suggest an additional mechanism for cellular metabolic damage through epigenetic perturbation, with implications in pathogenesis.

PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells.

Cyclin dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) are effective in breast cancer; however, drug resistance is frequently encountered and poorly understood. We conducted a genomic analysis of 348 estrogen receptor-positive (ER+) breast cancers treated with CDK4/6i and identified loss-of-function mutations affecting FAT1 and RB1 linked to drug resistance. FAT1 loss led to marked elevations in CDK6, the suppression of which restored sensitivity to CDK4/6i. The induction of CDK6 was mediated by the Hippo pathway with accumulation of YAP and TAZ transcription factors on the CDK6 promoter. Genomic alterations in other Hippo pathway components were also found to promote CDK4/6i resistance. These findings uncover a tumor suppressor function of Hippo signaling in ER+ breast cancer and establish FAT1 loss as a mechanism of resistance to CDK4/6i.

Identification of actionable genomic vulnerabilities is key to precision oncology. Utilizing a large-scale drug screening in patient-derived xenografts, we uncover driver gene alteration connections, derive driver co-occurrence (DCO) networks, and relate these to drug sensitivity. Our collection of 53 drug-response predictors attains an average balanced accuracy of 58% in a cross-validation setting, rising to 66% for a subset of high-confidence predictions. We experimentally validated 12 out of 14 predictions in mice and adapted our strategy to obtain drug-response models from patients' progression-free survival data. Our strategy reveals links between oncogenic alterations, increasing the clinical impact of genomic profiling.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

Driver mutations in oncogenes encode proteins with gain-of-function properties that enhance fitness. Heterozygous mutations are thus viewed as sufficient for tumorigenesis. We describe widespread oncogenic mutant allele imbalance in 13,448 prospectively characterized cancers. Imbalance was selected for through modest dosage increases of gain-of-fitness mutations. Negative selection targeted haplo-essential effectors of the spliceosome. Loss of the normal allele comprised a distinct class of imbalance driven by competitive fitness, which correlated with enhanced response to targeted therapies. In many cancers, an antecedent oncogenic mutation drove evolutionarily dependent allele-specific imbalance. In other instances, oncogenic mutations co-opted independent copy-number changes via the evolutionary process of exaptation. Oncogenic allele imbalance is a pervasive evolutionary innovation that enhances fitness and modulates sensitivity to targeted therapy.

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.

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA's selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR-FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell-based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.