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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.

Intratumoral T-cell dysfunction is a hallmark of pancreatic tumors, and efforts to improve dendritic cell (DC)-mediated T-cell activation may be critical in treating these immune therapy unresponsive tumors. Recent evidence indicates that mechanisms that induce dysfunction of type 1 conventional DCs (cDC1) in pancreatic adenocarcinomas (PDACs) are drivers of the lack of responsiveness to checkpoint immunotherapy. However, the impact of PDAC on systemic type 2 conventional DC (cDC2) development and function has not been well studied. Herein, we report the analysis of 3 cohorts, totaling 106 samples, of human blood and bone marrow (BM) from PDAC patients for changes in cDCs. We found circulating cDC2s and their progenitors were significantly decreased in the blood of PDAC patients, and repressed numbers of cDC2s were associated with poor prognosis. Serum cytokine analyses identified IL6 as significantly elevated in PDAC patents and negatively correlated with cDC numbers. In vitro, IL6 impaired the differentiation of cDC1s and cDC2s from BM progenitors. Single-cell RNA sequencing analysis of human cDC progenitors in the BM and blood of PDAC patients showed an upregulation of the IL6/STAT3 pathway and a corresponding impairment of antigen processing and presentation. These results suggested that cDC2s were systemically suppressed by inflammatory cytokines, which was linked to impaired antitumor immunity.

The effects of radiotherapy (RT) on tumor immunity in pancreatic ductal adenocarcinoma (PDAC) are not well understood. To better understand if RT can prime antigen-specific T-cell responses, we analyzed human PDAC tissues and mouse models. In both settings, there was little evidence of RT-induced T-cell priming. Using in vitro systems, we found that tumor–stromal components, including fibroblasts and collagen, cooperate to blunt RT efficacy and impair RT-induced interferon signaling. Focal adhesion kinase (FAK) inhibition rescued RT efficacy in vitro and in vivo, leading to tumor regression, T-cell priming, and enhanced long-term survival in PDAC mouse models. Based on these data, we initiated a clinical trial of defactinib in combination with stereotactic body RT in patients with PDAC (NCT04331041). Analysis of PDAC tissues from these patients showed stromal reprogramming mirroring our findings in genetically engineered mouse models. Finally, the addition of checkpoint immunotherapy to RT and FAK inhibition in animal models led to complete tumor regression and long-term survival.Significance:Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy.This article is highlighted in the In This Issue feature, p. 2711

Supplementary Table from Stromal Reprogramming by FAK Inhibition Overcomes Radiation Resistance to Allow for Immune Priming and Response to Checkpoint Blockade

Supplementary Figure from Stromal Reprogramming by FAK Inhibition Overcomes Radiation Resistance to Allow for Immune Priming and Response to Checkpoint Blockade

Supplementary Table S4

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.

Supplementary Figures

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

The effects of radiotherapy (RT) on tumor immunity in pancreatic ductal adenocarcinoma (PDAC) are not well understood. To better understand if RT can prime antigen-specific T-cell responses, we analyzed human PDAC tissues and mouse models. In both settings, there was little evidence of RT-induced T-cell priming. Using in vitro systems, we found that tumor–stromal components, including fibroblasts and collagen, cooperate to blunt RT efficacy and impair RT-induced interferon signaling. Focal adhesion kinase (FAK) inhibition rescued RT efficacy in vitro and in vivo, leading to tumor regression, T-cell priming, and enhanced long-term survival in PDAC mouse models. Based on these data, we initiated a clinical trial of defactinib in combination with stereotactic body RT in patients with PDAC (NCT04331041). Analysis of PDAC tissues from these patients showed stromal reprogramming mirroring our findings in genetically engineered mouse models. Finally, the addition of checkpoint immunotherapy to RT and FAK inhibition in animal models led to complete tumor regression and long-term survival. Significance: Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy. This article is highlighted in the In This Issue feature, p. 2711

Severe congenital neutropenia is an inborn disorder of granulopoiesis. Approximately one third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 severe congenital neutropenia cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase that is implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the adenosine triphosphate-binding pocket. Both genetic loss of CLPB and expression of CLPB variants result in impaired granulocytic differentiation of human hematopoietic progenitor cells and increased apoptosis. These CLPB variants associate with wild-type CLPB and inhibit its adenosine triphosphatase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.

Tumor-associated macrophages (TAMs) are involved in many aspects of cancer progression and correlate with poor clinical outcomes in many cancer types, including pancreatic ductal adenocarcinomas (PDACs). Previous studies have shown that TAMs can populate PDAC tumors not only by monocyte recruitment but also by local proliferation. However, the impact local proliferation might have on macrophage phenotype and cancer progression is unknown. Here, we utilized genetically engineered cancer models, single-cell RNA-sequencing data, andin vitrosystems to show that proliferation of TAMs was driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. The p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppressionin vivodrove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression, also drive response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.SummaryTAMs are indicative of poor clinical outcomes and in PDAC their number is sustained in part by local proliferation. This study shows that stromal desmoplasia drives local proliferation of TAMs, and induces their immunosuppressive ability through altering cell cycle machinery, including p21 expression. Serendipitously, these changes in p21 in TAMs also potentially render tumors more sensitive to CD40 agonist therapy.

Most human tumours are heterogeneous, composed of cellular clones with different properties present at variable frequencies. Highly heterogeneous tumours have poor clinical outcomes, yet the underlying mechanism remains poorly understood. Here, we show that minor subclones of breast cancer cells expressing IL11 and FIGF (VEGFD) cooperate to promote metastatic progression and generate polyclonal metastases composed of driver and neutral subclones. Expression profiling of the epithelial and stromal compartments of monoclonal and polyclonal primary and metastatic lesions revealed that this cooperation is indirect, mediated through the local and systemic microenvironments. We identified neutrophils as a leukocyte population stimulated by the IL11-expressing minor subclone and showed that the depletion of neutrophils prevents metastatic outgrowth. Single-cell RNA-seq of CD45+ cell populations from primary tumours, blood and lungs demonstrated that IL11 acts on bone-marrow-derived mesenchymal stromal cells, which induce pro-tumorigenic and pro-metastatic neutrophils. Our results indicate key roles for non-cell-autonomous drivers and minor subclones in metastasis.

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.

A key step in the biosynthesis of the polyene polyketide ECO-0501 by Amycolatopsis orientalis ATCC 43491 is thought to involve oxidative decarboxylation of arginine or Nδ-methylarginine to the corresponding primary amide. This reaction is the centerpiece of a recently identified biosynthetic cassette that generates 4-guanidinobutyryl thioesters to serve as starter units for polyketide synthesis. We examined the reaction of ORF7, the predicted ECO-0501 biosynthetic decarboxylase, with arginine, and saw no evidence of decarboxylation. Instead, we observed exclusive amine oxidation to generate 2-oxoarginine, with a kcat/KM,Arg of 5.6 × 106 M− 1 s− 1, typical of values measured for physiological amino acid decarboxylases. In contrast, when ORF7 was incubated with Nδ-methylarginine, we observed exclusive decarboxylation to generate 4-(N1-methylguanidino)butyramide. These differing reactive pathways provide insight into the biosyntheses of guanidinobutyryl-derived polyketides and demonstrate the biosynthetic versatility of arginine-processing decarboxylases. In addition, it suggests that ORF7 may be an incisive model system for dissecting the determinants of flavoprotein-catalyzed oxidase and monooxygenase modes of reactivity.

A key step in the biosynthesis of the polyene polyketide ECO-0501 by Amycolatopsis orientalis ATCC 43491 is thought to involve oxidative decarboxylation of arginine or N

Most breast tumors display a high degree of intratumor heterogeneity, with many distinct subpopulations of cancer cells present. Elevated diversity within a tumor increases the chance for cellular adaptation, as individual clones may react differently to changes in the tumor microenvironment. Thus, treatment of heterogeneous tumors my lead to selection of a resistant clone, its expansion, and tumor progression. However, the fitness of cancer cells depends not only on their intrinsic properties, but could also be affected through interactions between different subpopulations. These interactions could be the reason for maintenance of minor clones along the major population. Therefore, intratumor heterogeneity may have functional relevance in tumor progression and colonization of metastatic sites. To emulate clonal interactions, we used the previously developed polyclonal breast cancer model of MDA-MB-468 cell line expressing soluble factors, IL-11 and FIGF. The IL-11 and FIGF clones, when present as minor population, support the growth of other clones in vivo. Moreover, polyclonal tumors with minor driving clone population are highly metastatic. Thus, we hypothesized that clonal interactions could not only drive tumor growth, but could also play an important role in metastasis. We have found that polyclonal tumors lead to polyclonal metastases, composed of mixture of neutral and driver clones. To investigate the mechanisms of clonal interactions driving polyclonal metastasis, we performed RNA profiling of subpopulations and stroma from polyclonal tumors. Our results suggest that this cooperation is indirect and that driver clones promote metastasis by altering the tumor microenvironment. We have also found that minor driver clones affect the immune cells within primary tumor, circulating blood, and metastatic site. These systemic changes significantly influence the metastatic progression. We are currently testing whether a treatment targeting this indirect clonal interaction mechanism could prevent polyclonal metastatic spread. Our study shows that the interaction between minor clones and other cancer cells could drive tumor growth and metastasis. Moreover, our results suggest that clonal cooperation in metastatic progression may be indirect and involve modulation of immune microenvironment of the primary tumor and distant organs. This abstract is also being presented as Poster B03. Citation Format: Michalina Janiszewska, Doris P. Tabassum, Andriy Marusyk, Muhammad B. Ekram, Zafira Castaño, Natalie L. Kingston, Yuanbo Qin, Tyler Laszewski, Minsuk Kwak, Kimiyo Nakamura, Rong Fan, Franziska Michor, Sandra S. McAllister, Kornelia Polyak. Minor clones can drive polyclonal metastasis by affecting immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr PR02.