The genomes of medicinal skullcaps reveal the polyphyletic origins of clerodane diterpene biosynthesis in the family Lamiaceae.

The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.

Deciphering and reprogramming the cyclization regioselectivity in bifurcation of indole alkaloid biosynthesis.

The metabolism of monoterpene indole alkaloids (MIAs) is an outstanding example of how plants shape chemical diversity from a single precursor. Here we report the discovery of novel enzymes from the Alstonia scholaris tree, a cytochrome P450, an NADPH dependent oxidoreductase and a BAHD acyltransferase that together synthesize the indole alkaloid akuammiline with a unique methanoquinolizidine cage structure. The two paralogous cytochrome P450 enzymes rhazimal synthase (AsRHS) and geissoschizine oxidase (AsGO) catalyse the cyclization of the common precursor geissoschizine and they direct the MIA metabolism towards to the two structurally distinct and medicinally important MIA classes of akuammilan and strychnos alkaloids, respectively. To understand the pathway divergence, we investigated the catalytic mechanism of the two P450 enzymes by homology modelling and reciprocal mutations. Upon conducting mutant enzyme assays, we identified a single amino acid residue that mediates the space in active sites, switches the enzymatic reaction outcome and impacts the cyclization regioselectivity. Our results represent a significant advance in MIA metabolism, paving the way for discovery of downstream genes in akuammilan alkaloid biosynthesis and facilitating future synthetic biology applications. We anticipate that our work presents, for the first time, insights at the molecular level for plant P450 catalytic activity with a significant key role in the diversification of alkaloid metabolism, and provides the basis for designing new drugs.

Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling.

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-ß signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.

Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models.

Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.

Neutralization of PD-L2 is Essential for Overcoming Immune Checkpoint Blockade Resistance in Ovarian Cancer.

PURPOSE: Ovarian cancer represents a major clinical hurdle for immune checkpoint blockade (ICB), with reported low patient response rates. We found that the immune checkpoint ligand PD-L2 is robustly expressed in patient samples of ovarian cancers and other malignancies exhibiting suboptimal response to ICB but not in cancers that are ICB sensitive. Therefore, we hypothesize that PD-L2 can facilitate immune escape from ICB through incomplete blockade of the PD-1 signaling pathway. EXPERIMENTAL DESIGN: We engineered a soluble form of the PD-1 receptor (sPD-1) capable of binding and neutralizing both PD-L2 and PD-L1 with ×200 and ×10,000 folds improvement in binding affinity over wild-type PD-1 leading to superior inhibition of ligand-mediated PD-1 activities. RESULTS: Both in vitro and in vivo analyses performed in this study demonstrated that the high-affinity sPD-1 molecule is superior at blocking both PD-L1- and PD-L2-mediated immune evasion and reducing tumor growth in immune-competent murine models of ovarian cancer. CONCLUSIONS: The data presented in this study provide justification for using a dual targeting, high-affinity sPD-1 receptor as an alternative to PD-1 or PD-L1 therapeutic antibodies for achieving superior therapeutic efficacy in cancers expressing both PD-L2 and PD-L1.

The m6A RNA demethylase FTO is a HIF-independent synthetic lethal partner with the VHL tumor suppressor.

Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism.

Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.

S100A10 Is a Critical Mediator of GAS6/AXL-Induced Angiogenesis in Renal Cell Carcinoma.

Angiogenesis is a hallmark of cancer that promotes tumor progression and metastasis. However, antiangiogenic agents have limited efficacy in cancer therapy due to the development of resistance. In clear cell renal cell carcinoma (ccRCC), AXL expression is associated with antiangiogenic resistance and poor survival. Here, we establish a role for GAS6/AXL signaling in promoting the angiogenic potential of ccRCC cells through the regulation of the plasminogen receptor S100A10. Genetic and therapeutic inhibition of AXL signaling in ccRCC tumor xenografts reduced tumor vessel density and growth under the renal capsule. GAS6/AXL signaling activated the expression of S100A10 through SRC to promote plasmin production, endothelial cell invasion, and angiogenesis. Importantly, treatment with the small molecule AXL inhibitor cabozantinib or an ultra-high affinity soluble AXL Fc fusion decoy receptor (sAXL) reduced the growth of a pazopanib-resistant ccRCC patient-derived xenograft. Moreover, the combination of sAXL synergized with pazopanib and axitinib to reduce ccRCC patient-derived xenograft growth and vessel density. These findings highlight a role for AXL/S100A10 signaling in mediating the angiogenic potential of ccRCC cells and support the combination of AXL inhibitors with antiangiogenic agents for advanced ccRCC. SIGNIFICANCE: These findings show that angiogenesis in renal cell carcinoma (RCC) is regulated through AXL/S100A10 signaling and support the combination of AXL inhibitors with antiangiogenic agents for the treatment of RCC.

Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1/PD-1 targeted immunotherapy.

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg-/- (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10-14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.

CD215+ Myeloid Cells Respond to Interleukin 15 Stimulation and Promote Tumor Progression.

Interleukin 15 (IL-15) regulates the development, survival, and functions of multiple innate and adaptive immune cells and plays a dual role in promoting both tumor cell growth and antitumor immunity. Here, we demonstrated that the in vivo injection of recombinant human IL-15 (200 µg/kg) or murine IL-15 (3 µg/kg) to tumor-bearing NOD-SCID-IL2Rg-/- (NSI) mice resulted in increased tumor progression and CD45+ CD11b+ Gr-1+ CD215+ cell expansion in the tumors and spleen. In B16F10-bearing C57BL/6 mice model, we found that murine IL-15 has antitumoral effect since the activation and expansion of CD8+ T cells with murine IL-15 treatment. But no enhanced or reduced tumor growth was observed in mice when human IL-15 was used. However, both murine and human IL-15 promote CD45+ CD11b+ Gr-1+ CD215+ cells expansion. In xenograft tumor models, CD215+ myeloid cells, but not CD215- cells, responded to human IL-15 stimulation and promoted tumor growth. Furthermore, we found that human IL-15 mediated insulin-like growth factor-1 production in CD215+ myeloid cells and blocking IGF-1 reduced the tumor-promoting effect of IL-15. Finally, we observed that higher IGF-1 expression is an indicator of poor prognosis among lung adenocarcinoma patients. These findings provide evidence that IL-15 may promote tumor cell progression via CD215+ myeloid cells, and IGF-1 may be an important candidate that IL-15 facilitates tumor growth.

GZD824 suppresses the growth of human B cell precursor acute lymphoblastic leukemia cells by inhibiting the SRC kinase and PI3K/AKT pathways.

Available therapeutic options for advanced B cell precursor acute lymphoblastic leukemia (pre-B ALL) are limited. Many lead to neutropenia, leaving patients at risk of life-threatening infections and result in bad outcomes. New treatment options are needed to improve overall survival. We previously showed that GZD824, a novel BCR-ABL tyrosine kinase inhibitor, has anti-tumor activity in Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia cells and tumor models. Here, we show that GZD824 decreases cell viability, induces cell-cycle arrest, and causes apoptosis in pre-B ALL cells. Furthermore, Ph- pre-B ALL cells were more sensitive to GZD824 than Ph+ pre-B ALL cells. GZD824 consistently reduced tumor loads in Ph- pre-B ALL xenografts but failed to suppress Ph+ pre-B ALL xenografts. GZD824 decreased phosphorylation of SRC kinase, STAT3, RB and C-myc. It also downregulated the expression of BCL-XL, CCND1 and CDK4 and upregulated expression of CCKN1A. Expression of IRS1 was decreased in GZD824-treated pre-B ALL cells, blocking the PI3K/AKT pathway. These data demonstrate that GZD824 suppresses pre-B ALL cells through inhibition of the SRC kinase and PI3K/AKT pathways and may be a potential therapeutic agent for the management of pre-B ALL.

Heterogeneity of CD34 and CD38 expression in acute B lymphoblastic leukemia cells is reversible and not hierarchically organized.

The existence and identification of leukemia-initiating cells in adult acute B lymphoblastic leukemia (B-ALL) remain controversial. We examined whether adult B-ALL is hierarchically organized into phenotypically distinct subpopulations of leukemogenic and non-leukemogenic cells or whether most B-ALL cells retain leukemogenic capacity, irrespective of their immunophenotype profiles. Our results suggest that adult B-ALL follows the stochastic stem cell model and that the expression of CD34 and CD38 in B-ALL is reversibly and not hierarchically organized.

Repulsive guidance molecule B inhibits metastasis and is associated with decreased mortality in non-small cell lung cancer.

Repulsive guidance molecules (RGMs) are co-receptors of bone morphogenetic proteins (BMPs) and programmed death ligand 2 (PD-L2), and might be involved in lung and other cancers. We evaluated repulsive guidance molecule B (RGMB) expression in 165 non-small cell lung cancer (NSCLC) tumors and 22 normal lung tissue samples, and validated the results in an independent series of 131 samples. RGMB was downregulated in NSCLC (P ≤ 0.001), possibly through promoter hypermethylation. Reduced RGMB expression was observed in advanced-stage tumors (P = 0.017) and in tumors with vascular invasion (P < 0.01), and was significantly associated with poor overall survival (39 vs. 62 months, P < 0.001) and with disease-associated patient mortality (P = 0.015). RGMB knockdown promoted cell adhesion, invasion and migration, in both NSCLC cell lines and an in vivo mouse model, which enhanced metastatic potential. Conversely, RGMB overexpression and secretion suppressed cancer progression. The tumor-suppressing effect of RGMB was exerted through inhibition of the Smad1/5/8 pathway. Our results demonstrate that RGMB is an important inhibitor of NSCLC metastasis and that low RGMB expression is a novel predictor or a poor prognosis.

A New Strategy to Target Acute Myeloid Leukemia Stem and Progenitor Cells Using Chidamide, a Histone Deacetylase Inhibitor.

Leukemia stem cells (LSCs) are responsible for treatment failure and relapse in acute myeloid leukemia (AML). Therefore, development of novel LSCs-targeting therapeutic strategies is of crucial clinical importance to improve the treatment outcomes of AML. Histone deacetylase (HDAC) inhibitors have shown potent and specific anticancer stem cell activities in preclinical studies. Chidamide, a novel benzamide-type selectively HDAC inhibitor, has been reported to induce G1 arrest and apoptosis in the relatively mature progenitor population, whereas its effect on primitive LSCs has not been clarified. In this study, we demonstrated that chidamide specifically induces apoptosis in LSC-like cells and primary AML CD34(+) cells in a concentration- and time-dependent manner. Our further molecular mechanistic study uncovered that chidamide induces LSCs death by activation of reactive oxygen species (ROS). It compromises the mitochondria membrane potential, modulates antiapoptotic and pro-apoptotic proteins in BCL2 family and activates caspase-3 leading to PARP degradation. Meanwhile, chidamide activates CD40 and modulates its downstream signaling pathways, JNK and NFκB. The results of this study suggest that chidamide may be a novel LSC-targeting agent for AML therapeutics.

Quantitative evaluation of the immunodeficiency of a mouse strain by tumor engraftments.

BACKGROUND: The mouse is an organism that is widely used as a mammalian model for studying human physiology or disease, and the development of immunodeficient mice has provided a valuable tool for basic and applied human disease research. Following the development of large-scale mouse knockout programs and genome-editing tools, it has become increasingly efficient to generate genetically modified mouse strains with immunodeficiency. However, due to the lack of a standardized system for evaluating the immuno-capacity that prevents tumor progression in mice, an objective choice of the appropriate immunodeficient mouse strains to be used for tumor engrafting experiments is difficult. METHODS: In this study, we developed a tumor engraftment index (TEI) to quantify the immunodeficiency response to hematologic malignant cells and solid tumor cells of six immunodeficient mouse strains and C57BL/6 wild-type mouse (WT). RESULTS: Mice with a more severely impaired immune system attained a higher TEI score. We then validated that the NOD-scid-IL2Rg-/- (NSI) mice, which had the highest TEI score, were more suitable for xenograft and allograft experiments using multiple functional assays. CONCLUSIONS: The TEI score was effectively able to reflect the immunodeficiency of a mouse strain.

High expression of TACC3 in esophageal squamous cell carcinoma correlates with poor prognosis.

To analyze the expression of the transforming acidic coiled-coil protein 3 (TACC3) in esophageal squamous cell carcinoma (ESCC) samples, and to identify whether TACC3 can serve as a biomarker for the diagnosis and prognosis of ESCC, qPCR, western blotting and immunohistochemistry staining (IHC) were utilized to detect the expression of TACC3. Furthermore, cell growth, colony formation, migration ability and the epithelial-mesenchymal transition markers of ESCC cells in which TACC3 were knocked-down were measured. The mRNA and protein levels of TACC3 were higher in ESCC specimens compared to non-tumorous esophageal epithelial tissues. IHC results revealed TACC3 expression was significantly correlated to differentiation (p = 0.017) and lymphoid nodal status (p = 0.028). The patients with high-expression of TACC3 had a significantly poor prognosis compared to those of low-expression (p = 0.017), especially in the patients at stages I-II (p = 0.028). Multivariate analysis indicated that TACC3 expression was an independent prognostic factor for ESCC patients (p = 0.025). Knockdown of TACC3 inhibited the ability of cell proliferation, colony formation and migration. This study first identifies TACC3 not only as a useful biomarker for diagnose and prognosis of ESCC, but also as a potential therapeutic target for patients with ESCC.

Loss of Angiopoietin-like 7 diminishes the regeneration capacity of hematopoietic stem and progenitor cells.

Successful expansion of hematopoietic stem cells (HSCs) would benefit the use of HSC transplants in the clinic. Angiopoietin-like 7 promotes the expansion of hematopoietic stem and progenitor cells (HSPC) in vitro and ex vivo. However, the impact of loss of Angptl7 on HSPCs in vivo has not been characterized. Here, we generated Angptl7-deficient mice by TALEN-mediated gene targeting and found that HSC compartments in Angptl7-null mice were compromised. In addition, wild type (WT) HSPCs failed to repopulate in the BM of Angptl7-null mice after serial transplantations while the engraftment of Angptl7-deficient HSPCs in WT mice was not impaired. These results suggest that Angptl7 is required for HSPCs repopulation in a non-cell autonomous manner.

ANGPTL7 regulates the expansion and repopulation of human hematopoietic stem and progenitor cells.

Successful expansion of hematopoietic stem cells would benefit the use of hematopoietic stem cell transplants in the clinic. Several angiopoietin-like proteins, including angiopoietin-like 7, can support the activity of hematopoietic stem cells. However, effects of ANGPTL7 on human hematopoietic stem cells and the downstream signaling cascade activated by ANGPTL7 are poorly understood. Here, we established a human hematopoietic stem and progenitor cell-supportive mouse fetal liver cell line that specifically expressed the Angptl7 protein. Furthermore, we found ANGPTL7 is capable of stimulating human hematopoietic stem and progenitor cell expansion and increasing the repopulation activities of human hematopoietic progenitors in xenografts. RNA-sequencing analysis showed that ANGPTL7 activated the expression of CXCR4, HOXB4 and Wnt downstream targets in human hematopoietic progenitors. In addition, chemical manipulation of Wnt signaling diminished the effects of ANGPTL7 on human hematopoietic stem and progenitor cells in culture. In summary, we identify the secreted growth factor ANGPTL7 as a regulator of both human hematopoietic stem and progenitor cell expansion and regeneration.

Mechanisms determining the fate of hematopoietic stem cells.

Successful in vitro expansion of hematopoietic stem cells (HSCs) will facilitate the application of HSC transplantation for the treatment of various diseases, including hematological malignancies. To achieve this expansion, the molecular mechanisms that control the fate of HSCs must be deciphered. Leukemia-initiating cells (LICs) or leukemia stem cells (LSCs) may originate from normal HSCs, which suggest that the dysregulation of the mechanisms that regulate the cell fate of HSCs may underlie leukemogenesis. Here we review the recent progress in the application of HSCs, the regulatory mechanisms of the fate of HSCs, and the origins of leukemia.

Using mouse models to study function of transcriptional factors in T cell development.

Laboratory mice have widely been used as tools for basic biological research and models for studying human diseases. With the advances of genetic engineering and conditional knockout (CKO) mice, we now understand hematopoiesis is a dynamic stepwise process starting from hematopoietic stem cells (HSCs) which are responsible for replenishing all blood cells. Transcriptional factors play important role in hematopoiesis. In this review we compile several studies on using genetic modified mice and humanized mice to study function of transcriptional factors in lymphopoiesis, including T lymphocyte and Natural killer (NK) cell development. Finally, we focused on the key transcriptional factor Bcl11b and its function in regulating T cell specification and commitment.