Anti-CD73 antibody activates human B cells, enhances humoral responses and induces redistribution of B cells in patients with cancer.

BACKGROUND: CD73 is widely expressed on immune cells playing a critical role in immunomodulatory functions including cell adhesion and migration, as a costimulatory molecule for T cells and in production of adenosine. The function of CD73 expressed on B cells has not been fully characterized. Mupadolimab is an anti-human CD73 antibody that activates B cells. We evaluated the characteristics of this antibody and its effects on immune cells in vitro and in vivo. METHODS: Mupadolimab binding to CD73, inhibition of CD73 enzymatic activity, and effects on lymphocyte activation were evaluated in vitro by measuring changes in immunophenotype by flow cytometry. Cryogenic-transmission electron microscopy was used to determine epitope binding. Effects on human B cells in vivo were evaluated in immunodeficient NSG-SGM3 mice immunized with SARS-CoV-2 and influenza viral antigens. Safety and immune effects were evaluated in the completed dose escalation portion of a phase 1 trial conducted in patients with cancer. RESULTS: Mupadolimab binds to a unique epitope on CD73POS B cells resulting in their activation and differentiation through B cell receptor signaling pathways. Mupadolimab induces expression of CD69, CD83, CD86 and MHC class II on B cells along with morphological transformation into plasmablasts and expression of CD27, CD38 and CD138. These effects are independent of adenosine. Mupadolimab binds to the N-terminal of CD73 in the closed position and competitively inhibits substrate binding. Mupadolimab enhanced antigen specific antibody response to SARS-CoV-2 spike protein and influenza hemagglutinin in humanized mouse models. Mupadolimab was evaluated as a monotherapy in a phase 1 trial (NCT03454451) in 34 patients with advanced cancer and demonstrated binding to CD73POS circulating cells and transient reduction in the number of B cells, with return of CD73NEG B cells with memory phenotype. No dose-limiting toxicities or changes in serum immunoglobulins were seen. CONCLUSIONS: Mupadolimab activates B cells and stimulates the production of antigen specific antibodies. The effects in patients with cancer suggest that activated, CD69POS B cells redistribute to lymphoid tissues. Minor tumor regression was observed in several patients. These results support further investigation of mupadolimab as an immunotherapy for cancer and its potential use as a vaccine adjuvant. TRIAL REGISTRATION NUMBER: NCT03454451.

Combining CD47 blockade with trastuzumab eliminates HER2-positive breast cancer cells and overcomes trastuzumab tolerance.

Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-α (SIRPα) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.

Therapeutic blockade of CXCR2 rapidly clears inflammation in arthritis and atopic dermatitis models: demonstration with surrogate and humanized antibodies.

Neutrophils are the most abundant effector cells of the innate immune system and represent the first line of defense against infection. However, in many common pathologies, including autoimmune diseases, excessive recruitment and activation of neutrophils can drive a chronic inflammatory response leading to unwanted tissue destruction. Several strategies have been investigated to tackle pathologic neutrophil biology, and thus provide a novel therapy for chronic inflammatory diseases. The chemokine receptor CXCR2 plays a crucial role in regulating neutrophil homeostasis and is a promising pharmaceutical target. In this study, we report the discovery and validation of a humanized anti-human CXCR2 monoclonal antibody. To enable in vivo studies, we developed a surrogate anti-mouse CXCR2 antibody, as well as a human knock-in CXCR2 mouse. When administered in models of atopic dermatitis (AD) and rheumatoid arthritis (RA), the antibodies rapidly clear inflammation. Our findings support further developments of anti-CXCR2 mAb approaches not only for RA and AD, but also for other neutrophil-mediated inflammatory conditions where neutrophils are pathogenic and medical needs are unmet.

Targeting the A2AR in cancer; early lessons from the clinic.

The immunosuppressive tumor microenvironment (TME) represents a challenge that all immunotherapies must overcome to enable a robust and durable anti-tumor response. One of the dominant mechanisms of immunosuppression in the TME is hypoxia and the generation of extracellular adenosine [1]. Pioneering work from Drs Ohta and Sitkovsky demonstrating that adenosine signaling through the adenosine 2A receptor (A2AR) inhibits T cells has led to the development of several agents designed to inhibit the production or downstream signaling of adenosine [2••,3••]. This review will focus on the safety, efficacy, and biomarkers associated with A2AR antagonists in clinical development.

Adenosine 2A Receptor Blockade as an Immunotherapy for Treatment-Refractory Renal Cell Cancer.

Adenosine mediates immunosuppression within the tumor microenvironment through triggering adenosine 2A receptors (A2AR) on immune cells. To determine whether this pathway could be targeted as an immunotherapy, we performed a phase I clinical trial with a small-molecule A2AR antagonist. We find that this molecule can safely block adenosine signaling in vivo. In a cohort of 68 patients with renal cell cancer (RCC), we also observe clinical responses alone and in combination with an anti-PD-L1 antibody, including subjects who had progressed on PD-1/PD-L1 inhibitors. Durable clinical benefit is associated with increased recruitment of CD8+ T cells into the tumor. Treatment can also broaden the circulating T-cell repertoire. Clinical responses are associated with an adenosine-regulated gene-expression signature in pretreatment tumor biopsies. A2AR signaling, therefore, represents a targetable immune checkpoint distinct from PD-1/PD-L1 that restricts antitumor immunity. SIGNIFICANCE: This first-in-human study of an A2AR antagonist for cancer treatment establishes the safety and feasibility of targeting this pathway by demonstrating antitumor activity with single-agent and anti-PD-L1 combination therapy in patients with refractory RCC. Responding patients possess an adenosine-regulated gene-expression signature in pretreatment tumor biopsies.See related commentary by Sitkovsky, p. 16.This article is highlighted in the In This Issue feature, p. 1.

Dysregulated integrin αVß3 and CD47 signaling promotes joint inflammation, cartilage breakdown, and progression of osteoarthritis.

Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVß3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVß3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVß3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVß3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVß3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVß3 and CD47 signaling in OA pathogenesis and suggest that activation of αVß3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVß3, CD47, and their signaling pathways as a disease-modifying therapy.

A2AR Antagonism with CPI-444 Induces Antitumor Responses and Augments Efficacy to Anti-PD-(L)1 and Anti-CTLA-4 in Preclinical Models.

Adenosine signaling through A2A receptors (A2AR) expressed on immune cells suppresses antitumor immunity. CPI-444 is a potent, selective, oral A2AR antagonist. Blockade of A2AR with CPI-444 restored T-cell signaling, IL2, and IFNγ production that were suppressed by adenosine analogues in vitro CPI-444 treatment led to dose-dependent inhibition of tumor growth in multiple syngeneic mouse tumor models. Concentrations of extracellular adenosine in the tumor microenvironment, measured using microdialysis, were approximately 100-150 nmol/L and were higher than corresponding subcutaneous tissue. Combining CPI-444 with anti-PD-L1 or anti-CTLA-4 treatment eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that incompletely responded to anti-PD-L1 or anti-CTLA-4 monotherapy. Tumor growth was fully inhibited when mice with cleared tumors were later rechallenged, indicating that CPI-444 induced systemic antitumor immune memory. CD8+ T-cell depletion abrogated the efficacy of CPI-444 with and without anti-PD-L1 treatment, demonstrating a role for CD8+ T cells in mediating primary and secondary immune responses. The antitumor efficacy of CPI-444 with and without anti-PD-L1 was associated with increased T-cell activation, a compensatory increase in CD73 expression, and induction of a Th1 gene expression signature consistent with immune activation. These results suggest a broad role for adenosine-mediated immunosuppression in tumors and justify the further evaluation of CPI-444 as a therapeutic agent in patients with solid tumors. Cancer Immunol Res; 6(10); 1136-49. ©2018 AACR.

A CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer.

CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant 'don't eat me' signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47-associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance.

Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors.

Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential.

CD47 is a widely expressed cell surface protein that functions as a regulator of phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin's lymphoma (NHL), and many solid tumors in xenograft models. Here, we report the development of a humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for and has been entered into clinical trials in patients with AML and solid tumors (ClinicalTrials.gov identifier: NCT02216409).

Learning from host-defense peptides: cationic, amphipathic peptoids with potent anticancer activity.

Cationic, amphipathic host defense peptides represent a promising group of agents to be developed for anticancer applications. Poly-N-substituted glycines, or peptoids, are a class of biostable, peptidomimetic scaffold that can display a great diversity of side chains in highly tunable sequences via facile solid-phase synthesis. Herein, we present a library of anti-proliferative peptoids that mimics the cationic, amphipathic structural feature of the host defense peptides and explore the relationships between the structure, anticancer activity and selectivity of these peptoids. Several peptoids are found to be potent against a broad range of cancer cell lines at low-micromolar concentrations including cancer cells with multidrug resistance (MDR), causing cytotoxicity in a concentration-dependent manner. They can penetrate into cells, but their cytotoxicity primarily involves plasma membrane perturbations. Furthermore, peptoid 1, the most potent peptoid synthesized, significantly inhibited tumor growth in a human breast cancer xenotransplantation model without any noticeable acute adverse effects in mice. Taken together, our work provided important structural information for designing host defense peptides or their mimics for anticancer applications. Several cationic, amphipathic peptoids are very attractive for further development due to their high solubility, stability against protease degradation, their broad, potent cytotoxicity against cancer cells and their ability to overcome multidrug resistance.

Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips.

Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of "liquid biopsies" from cancer patients.

Use of a KIT-specific monoclonal antibody to bypass imatinib resistance in gastrointestinal stromal tumors.

Acquired resistance to imatinib is a significant problem for the clinical management of gastrointestinal stromal tumor (GIST) patients, and second-line small molecules have shown limited efficacy in this setting. We have recently demonstrated that a monoclonal antibody targeting KIT could potentially bypass imatinib resistance in preclinical models of GIST.

Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.

Anti-KIT monoclonal antibody inhibits imatinib-resistant gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.

The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma.

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

Three differentiation states risk-stratify bladder cancer into distinct subtypes.

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

Cutting edge: NLRC5-dependent activation of the inflammasome.

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1ß and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1ß, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1ß, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.