S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

BACKGROUND: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21.

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.

Homeostatic IL-23 receptor signaling limits Th17 response through IL-22-mediated containment of commensal microbiota.

Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. In mice, the presence of segmented filamentous bacteria (SFB) promotes Th17 differentiation and the development of autoimmune disease. Here, we demonstrate that the IL-23 pathway dynamically regulates the abundance of SFB as well as mucosal barrier function in the adult animal. Genetic or pharmacological inactivation of the pathway selectively perturbs the abundance of a small group of commensals, including SFB, and results in an impaired mucosal barrier. Defective barrier function leads to systemic dissemination of microbial products, provoking induction of the IL-23 pathway with dual consequences: IL-23 drives IL-22 production to reinforce mucosal barrier function and elicit antimicrobial activities, and it also drives the differentiation of Th17 cells in an attempt to combat escaped microbes in the lamina propria and in distal tissues. Thus, barrier defects generate a systemic environment that facilitates Th17 development.

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.

A requirement for wild-type Ras isoforms in mutant KRas-driven signalling and transformation.

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.

Disruption of stem cell niche-confined R-spondin 3 expression leads to impaired hematopoiesis.

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine.

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors.

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations.

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

Stem cell plasticity enables hair regeneration following Lgr5+ cell loss.

Under injury conditions, dedicated stem cell populations govern tissue regeneration. However, the molecular mechanisms that induce stem cell regeneration and enable plasticity are poorly understood. Here, we investigate stem cell recovery in the context of the hair follicle to understand how two molecularly distinct stem cell populations are integrated. Utilizing diphtheria-toxin-mediated cell ablation of Lgr5+ (leucine-rich repeat-containing G-protein-coupled receptor 5) stem cells, we show that killing of Lgr5+ cells in mice abrogates hair regeneration but this is reversible. During recovery, CD34+ (CD34 antigen) stem cells activate inflammatory response programs and start dividing. Pharmacological attenuation of inflammation inhibits CD34+ cell proliferation. Subsequently, the Wnt pathway controls the recovery of Lgr5+ cells and inhibition of Wnt signalling prevents Lgr5+ cell and hair germ recovery. Thus, our study uncovers a compensatory relationship between two stem cell populations and the underlying molecular mechanisms that enable hair follicle regeneration.

Requirement for BUB1B/BUBR1 in tumor progression of lung adenocarcinoma.

Lung adenocarcinoma is often discovered as metastatic disease with very poor prognosis. However, much remains unknown about the mechanisms of lung adenocarcinoma tumor progression. In this study we showed that knockdown of BUB1B/BUBR1, a critical mitotic checkpoint protein, significantly inhibited anchorage-independent growth of lung adenocarcinoma cell lines. In allograft and tail vein mouse model studies, BUB1B suppression inhibited primary tumor growth and reduced metastasis to the lung and lymph nodes, resulting in prolonged survival in both tumor prevention and tumor intervention settings. Mechanistic studies revealed that BUB1B knockdown sensitized cells to anoikis. The N-terminal region and GLEBS domain of BUB1B were required for its functions in both anchorage-independent growth and anoikis resistance, whereas the kinase domain was less critical. Overexpression of BUB1B is associated with disease progression and poor survival in human lung adenocarcinoma patients. Collectively, these data reveal a novel function for BUB1B in mediating anchorage-independent survival and growth, thereby facilitating lung adenocarcinoma dissemination during metastasis. Thus, targeting BUB1B could provide potential therapeutic benefit in suppressing metastasis and prolonging survival in lung adenocarcinoma patients.

Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration.

The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Lgr5 marks mitotically active intestinal stem cells (ISCs). Importantly, intestinal homeostasis can be maintained after depletion of Lgr5(+) cells due to the activation of Lgr5(-) reserve ISCs. The Lgr5(-) ISC populations are thought to play a similar role during intestinal regeneration following radiation-induced damage. We tested this regeneration hypothesis by combining depletion of Lgr5(+) ISCs with radiation exposure. In contrast to the negligible effect of Lgr5(+) ISC loss during homeostasis, depletion of Lgr5(+) cells during radiation-induced damage and subsequent repair caused catastrophic crypt loss and deterioration of crypt-villus architecture. Interestingly though, we found that crypts deficient for Lgr5(+) cells are competent to undergo hyperplasia upon loss of Apc. These data argue that Lgr5(-) reserve stem cells are radiosensitive and that Lgr5(+) cells are crucial for robust intestinal regeneration following radiation exposure but are dispensable for premalignant hyperproliferation.

Comparative oncogenomics identifies PSMB4 and SHMT2 as potential cancer driver genes.

Cancer genomes maintain a complex array of somatic alterations required for maintenance and progression of the disease, posing a challenge to identify driver genes among this genetic disorder. Toward this end, we mapped regions of recurrent amplification in a large collection (n=392) of primary human cancers and selected 620 genes whose expression is elevated in tumors. An RNAi loss-of-function screen targeting these genes across a panel of 32 cancer cell lines identified potential driver genes. Subsequent functional assays identified SHMT2, a key enzyme in the serine/glycine synthesis pathway, as necessary for tumor cell survival but insufficient for transformation. The 26S proteasomal subunit, PSMB4, was identified as the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo. Elevated expression of SHMT2 and PSMB4 was found to be associated with poor prognosis in human cancer, supporting the development of molecular therapies targeting these genes or components of their pathways.

Oncogenic ERBB3 mutations in human cancers.

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

IL-27 promotes T cell-dependent colitis through multiple mechanisms.

Interleukin-27 (IL-27) is a cytokine known to have both proinflammatory and immunoregulatory functions. The latter appear to dominate in vivo, where IL-27 suppresses TH17 responses and promotes the differentiation of Tr1 cells expressing interferon-γ and IL-10 and lacking forkhead box P3 (Foxp3). Accordingly, IL-27 receptor α (Il27ra)-deficient mice suffer from exacerbated immune pathology when infected with various parasites or challenged with autoantigens. Because the role of IL-27 in human and experimental mouse colitis is controversial, we studied the consequences of Il27ra deletion in the mouse T cell transfer model of colitis and unexpectedly discovered a proinflammatory role of IL-27. Absence of Il27ra on transferred T cells resulted in diminished weight loss and reduced colonic inflammation. A greater fraction of transferred T cells assumed a Foxp3(+) phenotype in the absence of Il27ra, suggesting that IL-27 functions to restrain regulatory T cell (T(reg)) development. Indeed, IL-27 suppressed Foxp3 induction in vitro and in an ovalbumin-dependent tolerization model in vivo. Furthermore, effector cell proliferation and IFN-γ production were reduced in the absence of Il27ra. Collectively, we describe a proinflammatory role of IL-27 in T cell-dependent intestinal inflammation and provide a rationale for targeting this cytokine in pathological situations that result from a breakdown in peripheral immune tolerance.

IL-27 supports germinal center function by enhancing IL-21 production and the function of T follicular helper cells.

Maturation and selection of high-affinity B cell clones in the germinal center (GC) relies on support from T follicular helper (T(FH)) cells. T(FH) cells are characterized by their localization to the B cell follicle and their high expression of the costimulatory molecules ICOS and PD1 and the cytokine IL-21, which promotes immunoglobulin (Ig) class switching and production by B cells. We show that the heterodimeric cytokine IL-27 is critical for the function of T(FH) cells and for normal and pathogenic GC responses. IL-27 signaling to T cells results in the production of IL-21, a known autocrine factor for the maintenance of T(FH) cells, in a STAT3-dependent manner. IL-27 also enhances the survival of activated CD4(+) T cells and the expression of T(FH) cell phenotypic markers. In vivo, expression of the IL-27Rα chain is required to support IL-21 production and T(FH) cell survival in a T cell-intrinsic manner. The production of high-affinity antibodies is reduced, and pristane-elicited autoantibodies and glomerulonephritis are significantly diminished, in Il27ra(-/-) mice. Together, our data show a nonredundant role for IL-27 in the development of T cell-dependent antibody responses.

Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

BACKGROUND: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy. CONCLUSION/SIGNIFICANCE: Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

Somatic mutations in p85alpha promote tumorigenesis through class IA PI3K activation.

Members of the mammalian phosphoinositide-3-OH kinase (PI3K) family of proteins are critical regulators of various cellular process including cell survival, growth, proliferation, and motility. Oncogenic activating mutations in the p110alpha catalytic subunit of the heterodimeric p110/p85 PI3K enzyme are frequent in human cancers. Here we show the presence of frequent mutations in p85alpha in colon cancer, a majority of which occurs in the inter-Src homology-2 (iSH2) domain. These mutations uncouple and retain p85alpha's p110-stabilizing activity, while abrogating its p110-inhibitory activity. The p85alpha mutants promote cell survival, AKT activation, anchorage-independent cell growth, and oncogenesis in a p110-dependent manner.

Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection.

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Cutting edge: IL-27 is a potent inducer of IL-10 but not FoxP3 in murine T cells.

The cytokine IL-27 is important for restricting inflammation in response to a wide variety of immune challenges. In this study, we demonstrate that IL-27 induces expression of the anti-inflammatory cytokine IL-10 by CD4+ and CD8+ T cells. IL-27 relied upon the Th1 transcription factor STAT1 to induce IL-10+IFN-gamma+FoxP3- Th1 cells, which were recently shown to be key negative regulators during certain infections. Il27ra-/- mice generated fewer IL-10+ T cells during both Listeria monocytogenes infection and experimental autoimmune encephalomyelitis. The data presented here indicate a novel mechanism for the induction of IL-10 expression by T cells and provide a mechanistic basis for the suppressive effects of IL-27.