PIK3CA inhibition in models of proliferative glomerulonephritis and lupus nephritis.

Proliferative glomerulonephritis is a severe condition often leading to kidney failure. There is a significant lack of effective treatment for these disorders. Here, following the identification of a somatic PIK3CA gain-of-function mutation in podocytes of a patient, we demonstrate using multiple genetically engineered mouse models, single-cell RNA sequencing and spatial transcriptomics the crucial role played by this pathway for proliferative glomerulonephritis development by promoting podocyte proliferation, dedifferentiation and inflammation. Additionally, we show that alpelisib, a PI3Kα inhibitor, improves glomerular lesions and kidney function in different mouse models of proliferative glomerulonephritis and lupus nephritis by targeting podocytes. Surprisingly, we determined that pharmacological inhibition of PI3Kα affects B and T lymphocyte population in lupus nephritis mouse models with decrease in the production of proinflammatory cytokines, autoantibodies and glomerular complement deposition, which are all characteristic features of PI3K delta (PI3Kδ) inhibition, the primary PI3K isoform expressed in lymphocytes. Importantly, PI3Kα inhibition does not impact lymphocyte function under normal conditions. These findings were then confirmed in human lymphocytes isolated from patients with active lupus nephritis. In conclusion, we demonstrate the major role played by PI3Kα in proliferative glomerulonephritis and show that in this condition, alpelisib acts on both podocytes and the immune system.

Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3.

KMT2C and KMT2D, encoding histone H3 lysine 4 methyltransferases, are among the most commonly mutated genes in triple-negative breast cancer (TNBC). However, how these mutations may shape epigenomic and transcriptomic landscapes to promote tumorigenesis is largely unknown. Here we describe that deletion of Kmt2c or Kmt2d in non-metastatic murine models of TNBC drives metastasis, especially to the brain. Global chromatin profiling and chromatin immunoprecipitation followed by sequencing revealed altered H3K4me1, H3K27ac and H3K27me3 chromatin marks in knockout cells and demonstrated enhanced binding of the H3K27me3 lysine demethylase KDM6A, which significantly correlated with gene expression. We identified Mmp3 as being commonly upregulated via epigenetic mechanisms in both knockout models. Consistent with these findings, samples from patients with KMT2C-mutant TNBC have higher MMP3 levels. Downregulation or pharmacological inhibition of KDM6A diminished Mmp3 upregulation induced by the loss of histone-lysine N-methyltransferase 2 (KMT2) and prevented brain metastasis similar to direct downregulation of Mmp3. Taken together, we identified the KDM6A-matrix metalloproteinase 3 axis as a key mediator of KMT2C/D loss-driven metastasis in TNBC.

Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

HER2 heterogeneity and treatment response-associated profiles in HER2-positive breast cancer in the NCT02326974 clinical trial.

BACKGROUNDHER2-targeting therapies have great efficacy in HER2-positive breast cancer, but resistance, in part due to HER2 heterogeneity (HET), is a significant clinical challenge. We previously described that in a phase II neoadjuvant trastuzumab emtansine (T-DM1) and pertuzumab (P) clinical trial in early-stage HER2-positive breast cancer, none of the patients with HER2-HET tumors had pathologic complete response (pCR).METHODSTo investigate cellular and molecular differences among tumors according to HER2 heterogeneity and pCR, we performed RNA sequencing and ERBB2 FISH of 285 pretreatment and posttreatment tumors from 129 patients in this T-DM1+P neoadjuvant trial. A subset of cases was also subject to NanoString spatial digital profiling.RESULTSPretreatment tumors from patients with pCR had the highest level of ERBB2 mRNA and ERBB signaling. HER2 heterogeneity was associated with no pCR, basal-like features, and low ERBB2 expression yet high ERBB signaling sustained by activation of downstream pathway components. Residual tumors showed decreased HER2 protein levels and ERBB2 copy number heterogeneity and increased PI3K pathway enrichment and luminal features. HET tumors showed minimal treatment-induced transcriptomic changes compared with non-HET tumors. Immune infiltration correlated with pCR and HER2-HET status.CONCLUSIONResistance mechanisms in HET and non-HET tumors are distinct. HER2-targeting antibodies have limited efficacy in HET tumors. Our results support the stratification of patients based on HET status and the use of agents that target downstream components of the ERBB signaling pathway in patients with HET tumors.TRIAL REGISTRATIONClinicalTrials.gov NCT02326974.FUNDINGThis study was funded by Roche and the National Cancer Institute.

TBCRC 039: a phase II study of preoperative ruxolitinib with or without paclitaxel for triple-negative inflammatory breast cancer.

BACKGROUND: Patients with inflammatory breast cancer (IBC) have overall poor clinical outcomes, with triple-negative IBC (TN-IBC) being associated with the worst survival, warranting the investigation of novel therapies. Preclinical studies implied that ruxolitinib (RUX), a JAK1/2 inhibitor, may be an effective therapy for TN-IBC. METHODS: We conducted a randomized phase II study with nested window-of-opportunity in TN-IBC. Treatment-naïve patients received a 7-day run-in of RUX alone or RUX plus paclitaxel (PAC). After the run-in, those who received RUX alone proceeded to neoadjuvant therapy with either RUX + PAC or PAC alone for 12 weeks; those who had received RUX + PAC continued treatment for 12 weeks. All patients subsequently received 4 cycles of doxorubicin plus cyclophosphamide prior to surgery. Research tumor biopsies were performed at baseline (pre-run-in) and after run-in therapy. Tumors were evaluated for phosphorylated STAT3 (pSTAT3) by immunostaining, and a subset was also analyzed by RNA-seq. The primary endpoint was the percent of pSTAT3-positive pre-run-in tumors that became pSTAT3-negative. Secondary endpoints included pathologic complete response (pCR). RESULTS: Overall, 23 patients were enrolled, of whom 21 completed preoperative therapy. Two patients achieved pCR (8.7%). pSTAT3 and IL-6/JAK/STAT3 signaling decreased in post-run-in biopsies of RUX-treated samples, while sustained treatment with RUX + PAC upregulated IL-6/JAK/STAT3 signaling compared to RUX alone. Both treatments decreased GZMB+ T cells implying immune suppression. RUX alone effectively inhibited JAK/STAT3 signaling but its combination with PAC led to incomplete inhibition. The immune suppressive effects of RUX alone and in combination may negate its growth inhibitory effects on cancer cells. CONCLUSION: In summary, the use of RUX in TN-IBC was associated with a decrease in pSTAT3 levels despite lack of clinical benefit. Cancer cell-specific-targeting of JAK2/STAT3 or combinations with immunotherapy may be required for further evaluation of JAK2/STAT3 signaling as a cancer therapeutic target. TRIAL REGISTRATION: www. CLINICALTRIALS: gov , NCT02876302. Registered 23 August 2016.

Heterogeneity and transcriptional drivers of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with limited treatment options. To characterize TNBC heterogeneity, we defined transcriptional, epigenetic, and metabolic subtypes and subtype-driving super-enhancers and transcription factors by combining functional and molecular profiling with computational analyses. Single-cell RNA sequencing revealed relative homogeneity of the major transcriptional subtypes (luminal, basal, and mesenchymal) within samples. We found that mesenchymal TNBCs share features with mesenchymal neuroblastoma and rhabdoid tumors and that the PRRX1 transcription factor is a key driver of these tumors. PRRX1 is sufficient for inducing mesenchymal features in basal but not in luminal TNBC cells via reprogramming super-enhancer landscapes, but it is not required for mesenchymal state maintenance or for cellular viability. Our comprehensive, large-scale, multiplatform, multiomics study of both experimental and clinical TNBC is an important resource for the scientific and clinical research communities and opens venues for future investigation.

Peripheral blood TCR clonotype diversity as an age-associated marker of breast cancer progression.

Immune escape is a prerequisite for tumor growth. We previously described a decline in intratumor activated cytotoxic T cells and T cell receptor (TCR) clonotype diversity in invasive breast carcinomas compared to ductal carcinoma in situ (DCIS), implying a central role of decreasing T cell responses in tumor progression. To determine potential associations between peripheral immunity and breast tumor progression, here, we assessed the peripheral blood TCR clonotype of 485 breast cancer patients diagnosed with either DCIS or de novo stage IV disease at younger (<45) or older (≥45) age. TCR clonotype diversity was significantly lower in older compared to younger breast cancer patients regardless of tumor stage at diagnosis. In the younger age group, TCR-α clonotype diversity was lower in patients diagnosed with de novo stage IV breast cancer compared to those diagnosed with DCIS. In the older age group, DCIS patients with higher TCR-α clonotype diversity were more likely to have a recurrence compared to those with lower diversity. Whole blood transcriptome profiles were distinct depending on the TCR-α Chao1 diversity score. There were more CD8+ T cells and a more active immune environment in DCIS tumors of young patients with higher peripheral blood TCR-α Chao1 diversity than in those with lower diversity. These results provide insights into the role that host immunity plays in breast cancer development across different age groups.

HATS off to KAT6A/B inhibitors: A new way to target estrogen-receptor-positive breast cancer.

Inhibitors for the KAT6 family of histone acetyltransferases (HATs) have been actively pursued due to the oncogenic roles of KAT6A in human cancer. CTx-648 is a novel KAT6A/B inhibitor with excellent pharmacokinetic properties and in vivo efficacy that represents a promising new treatment strategy for estrogen-receptor-positive breast cancer.

Combination Therapies to Improve the Efficacy of Immunotherapy in Triple-negative Breast Cancer.

Immune checkpoint inhibition combined with chemotherapy is currently approved as first-line treatment for patients with advanced PD-L1-positive triple-negative breast cancer (TNBC). However, a significant proportion of metastatic TNBC is PD-L1-negative and, in this population, chemotherapy alone largely remains the standard-of-care and novel therapeutic strategies are needed to improve clinical outcomes. Here, we describe a triple combination of anti-PD-L1 immune checkpoint blockade, epigenetic modulation thorough bromodomain and extra-terminal (BET) bromodomain inhibition (BBDI), and chemotherapy with paclitaxel that effectively inhibits both primary and metastatic tumor growth in two different syngeneic murine models of TNBC. Detailed cellular and molecular profiling of tumors from single and combination treatment arms revealed increased T- and B-cell infiltration and macrophage reprogramming from MHCIIlow to a MHCIIhigh phenotype in mice treated with triple combination. Triple combination also had a major impact on gene expression and chromatin profiles shifting cells to a more immunogenic and senescent state. Our results provide strong preclinical evidence to justify clinical testing of BBDI, paclitaxel, and immune checkpoint blockade combination.

A convolutional neural network STIFMap reveals associations between stromal stiffness and EMT in breast cancer.

Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. We registered high-elasticity regions within human breast tumors colocalizing with markers of mechanical activation and an epithelial-to-mesenchymal transition (EMT). The findings highlight the utility of STIFMap to assess mechanical heterogeneity of human tumors across length scales from single cells to whole tissues and implicates stromal stiffness in tumor cell heterogeneity.

Clinical and translational relevance of intratumor heterogeneity.

Intratumor heterogeneity (ITH) is a driver of tumor evolution and a main cause of therapeutic resistance. Despite its importance, measures of ITH are still not incorporated into clinical practice. Consequently, standard treatment is frequently ineffective for patients with heterogeneous tumors as changes to treatment regimens are made only after recurrence and disease progression. More effective combination therapies require a mechanistic understanding of ITH and ways to assess it in clinical samples. The growth of technologies enabling the spatially intact analysis of tumors at the single-cell level and the development of sophisticated preclinical models give us hope that ITH will not simply be used as a predictor of a poor outcome but will guide treatment decisions from diagnosis through treatment.

Copy Number Variation in Inflammatory Breast Cancer.

Identification of a unique genomic biomarker in de novo inflammatory breast cancer (IBC) may provide an insight into the biology of this aggressive disease. The goal of our study was to elucidate biomarkers associated with IBC. We examined breast biopsies collected from Dana-Farber Cancer Institute patients with IBC prior to initiating preoperative systemic treatment (30 samples were examined, of which 14 were eligible). Patients without available biopsies (n = 1), with insufficient tumor epithelial cells (n = 10), or insufficient DNA yield (n = 5) were excluded from the analysis. Molecular subtype and tumor grade were abstracted from a medical records' review. Ten IBC tumors were estrogen-receptor-positive (ER+) and human epidermal growth factor receptor 2 (HER2)-negative (n = 10 out of 14). Sufficient RNA and DNA were simultaneously extracted from 14 biopsy specimens using the Qiagen AllPrep Kit. RNA was amplified using the Sensation kit and profiled using the Affymetrix Human Transcriptome Array 2.0. DNA was profiled for genome-wide copy number variation (CNV) using the Affymetrix OncoScan Array and analyzed using the Nexus Chromosome Analysis Suite. Among the 14 eligible samples, we first confirmed biological concordance and quality control metrics using replicates and gene expression data. Second, we examined CNVs and gene expression change by IBC subtype. We identified significant CNVs in IBC patients after adjusting for multiple comparisons. Next, to assess whether the CNVs were unique to IBC, we compared the IBC CNV data to fresh-frozen non-IBC CNV data from The Cancer Genome Atlas (n = 388). On chromosome 7p11.2, we identified significant CN gain located at position 58,019,983-58,025,423 in 8 ER+ IBC samples compared to 338 non-IBC ER+ samples (region length: 5440 bp gain and 69,039 bp, False Discovery Rate (FDR) p-value = 3.12 × 10-10) and at position 57,950,944-58,025,423 in 3 TN-IBC samples compared to 50 non-IBC TN samples (74,479 base pair, gain, FDR p-value = 4.27 × 10-5; near the EGFR gene). We also observed significant CN loss on chromosome 21, located at position 9,648,315-9,764,385 (p-value = 4.27 × 10-5). Secondarily, differential gene expression in IBC patients with 7p11.2 CN gain compared to SUM149 were explored after FDR correction for multiple testing (p-value = 0.0016), but the results should be interpreted with caution due to the small sample size. Finally, the data presented are hypothesis-generating. Validation of CNVs that contribute to the unique presentation and biological features associated with IBC in larger datasets may lead to the optimization of treatment strategies.

Challenges and opportunities for modeling aging and cancer.

Age is among the main risk factors for cancer, and any cancer study in adults is faced with an aging tissue and organism. Yet, pre-clinical studies are carried out using young mice and are not able to address the impact of aging and associated comorbidities on disease biology and treatment outcomes. Here, we discuss the limitations of current mouse cancer models and suggest strategies for developing novel models to address these major gaps in knowledge and experimental approaches.

JAK-STAT Signaling in Inflammatory Breast Cancer Enables Chemotherapy-Resistant Cell States.

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.

Breast cancer prevention by short-term inhibition of TGFß signaling.

Cancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFß signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFß in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy.

Molecular classification and biomarkers of clinical outcome in breast ductal carcinoma in situ: Analysis of TBCRC 038 and RAHBT cohorts.

Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We perform multiscale, integrated molecular profiling of DCIS with clinical outcomes by analyzing 774 DCIS samples from 542 patients with 7.3 years median follow-up from the Translational Breast Cancer Research Consortium 038 study and the Resource of Archival Breast Tissue cohorts. We identify 812 genes associated with ipsilateral recurrence within 5 years from treatment and develop a classifier that predicts DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions are identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

Untangling the web of intratumour heterogeneity.

Intratumour heterogeneity (ITH) is a hallmark of cancer that drives tumour evolution and disease progression. Technological and computational advances have enabled us to assess ITH at unprecedented depths, yet this accumulating knowledge has not had a substantial clinical impact. This is in part due to a limited understanding of the functional relevance of ITH and the inadequacy of preclinical experimental models to reproduce it. Here, we discuss progress made in these areas and illuminate future directions.

Bacterial Darth Vader: May the force be with you.

The microbiome has a major impact on tumor progression, yet the mechanisms are poorly defined. Fu et al. report in Cell that intracellular bacteria in a breast cancer model promote metastasis via reorganizing the cytoskeleton to enhance resistance to mechanical stress, thereby facilitating survival in the circulation during dissemination.