Dissecting the molecular determinants of clinical PARP1 inhibitor selectivity for tankyrase1.

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/ß-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/ß-catenin pathway-related cancers, such as colorectal cancers.

Herbal medicine derived carbon dots: synthesis and applications in therapeutics, bioimaging and sensing.

Since the number of raw material selections for the synthesis of carbon dots (CDs) has grown extensively, herbal medicine as a precursor receives an increasing amount of attention. Compared with other biomass precursors, CDs derived from herbal medicine (HM-CDs) have become the most recent incomer in the family of CDs. In recent ten years, a great many studies have revealed that HM-CDs tend to be good at theranostics without drug loading. However, the relevant development and research results are not systematically reviewed. Herein, the origin and history of HM-CDs are outlined, especially their functional performances in medical diagnosis and treatment. Besides, we sort out the herbal medicine precursors, and analyze the primary synthetic methods and the key characteristics. In terms of the applications of HM-CDs, medical therapeutics, ion and molecular detection, bioimaging, as well as pH sensing are summarized. Finally, we discuss the crucial challenges and future prospects.

Helicity of perfluoroalkyl chains controlled by the self-assembly of the Ala-Ala dipeptides.

Four Ala-Ala dipeptides with a perfluoroalkyl chain at the N-terminal were synthesized. They were able to self-assemble into helical nanofibers and/or twisted nanobelts in a mixture of DMSO/H2 O. The handedness of nanofibers and nanobelts was controlled by the chirality of the alanine at the N-terminal. The stacking handedness of the phenylene groups and the helicity of the perfluoroalkyl chain were studied using circular dichroism spectroscopy and vibrational circular dichroism, respectively. The chirality of the alanine at N-terminal controlled the stacking handedness of the neighboring phenylene groups. Moreover, due to the low potential barrier between M- and P-helices of the perfluorocarbon chain, the handedness of the organic self-assemblies eventually controlled the helicity of the perfluorocarbon chain. X-ray diffraction indicated that a lamellar structure was formed by the dimers of the dipeptides.

Alignment of twisted nanoribbons formed by C17H35CO-Val-Ala sodium salts.

C17H35CO-l-Val-l-Ala and C17H35CO-d-Val-d-Ala sodium salts can form physical gels in water, and self-assemble into right- and left-handed twisted nanoribbons, respectively. FT-IR and 1H NMR spectra indicate that the H-bonding between the neighboring valine residues and electrostatic interactions of carboxylate groups play important roles in the formation of helical nanoribbons. Circular dichroism characterization and theoretical chemical calculations indicate that the dipeptide segments pack in a helical manner. X-ray diffraction patterns and theoretical chemical simulations indicate an interdigitated bilayer structure. The hydrogels exhibit a thixotropic behavior. The twisted nanoribbons are able to align under directional force.

Aggregation-Induced Chirality: Twist and Stacking Handedness of the Biphenylene Groups of n-C12H25O-BP-CO-Ala-Ala Dipeptides.

In mixtures of water and dimethyl sulfoxide, 4'-(n-dodecyloxy)-1,1'-biphenyl-4-carbonyl Ala-Ala dipeptides can self-assemble into tubular structures that are formed by coiled nanoribbons. The twist and stacking handedness of biphenylene groups were studied using circular dichroism and confirmed by theoretical chemical calculations. The handedness of the coiled nanoribbons and the stacking handedness of biphenylene groups are controlled by the chirality of alanine at the C-terminus, whereas the twist handedness of biphenylene groups is determined by the chirality of alanine at the N-terminus. 1H NMR spectra indicated that the hydrogen bond formed by the N-H group of alanine at the N-terminus plays an important role in the formation of organic self-assemblies. On the basis of small-angle X-ray scattering characterization, a dimer structure was proposed to form through the terminal COOH groups.

How does virtual water influence the water stress pattern in Africa? A research perspective from the perspectives of production and trade.

Northern Africa has become the first region in the world to exhaust its water resources, with a 40 % decrease in per capita water availability south of the Sahara over the past decade. While adjusting production structures and consumption can regulate the supply-demand dynamics of water resources, the extent of the impact of virtual water-induced pressure on both the regional and national levels in Africa remains largely understudied. Applying the standard Penman formula, this research calculates the water footprint of eight cereal crops in 54 African countries from 1990 to 2021. By integrating corresponding data on cereal trade, the study analyzes changes in virtual water stress. The findings indicate a decline in the per-unit production and consumption water footprints for African cereals. However, the continuous expansion of cultivation areas contributes to a rising water stress. In comparison to 1990, the water stress for soybeans, sorghum, rice, maize, and cassava increased by 149.72 %, 146.88 %, 133.89 %, 123.30 %, and 90.8 %, respectively, in 2021. Only barley showed a reduction in water stress by 23.22 %. The study underscores the growing interconnectedness of virtual water trade (VWT) among African nations from 1990 to 2021, leading to a more balanced trade distribution. VWT has reduced water stress by 7.65 %, 2.08 %, and 1.8 % in Western, Central, and Northern Africa, respectively, while increasing pressure in Southern and Eastern Africa by 10.51 % and 1.01 %. The flow of virtual water in Africa is most influenced by spatial proximity, primarily occurring between adjacent countries or regions. Forecasts for water stress under the five scenarios of SSPs-RCP8.5 have been conducted, revealing a continuous increase in water stress across Africa. Furthermore, analysis of the SSP2-RCP8.5 scenario indicates that by 2030 and 2040, African cereal crops are projected to face virtual water resource stress increases of 7 % and 18.76 %, respectively, compared to 2020 levels. During the same period, Sierra Leone is anticipated to experience a growth rate in virtual water stress of approximately 1903.38 %. Consequently, altering crop cultivation structures and enhancing VWT are poised to alleviate water resource pressure, promoting the scientific management of agricultural water resources in Africa.

Natural Products from Herbal Medicine Self-Assemble into Advanced Bioactive Materials.

Novel biomaterials are becoming more crucial in treating human diseases. However, many materials require complex artificial modifications and synthesis, leading to potential difficulties in preparation, side effects, and clinical translation. Recently, significant progress has been achieved in terms of direct self-assembly of natural products from herbal medicine (NPHM), an important source for novel medications, resulting in a wide range of bioactive supramolecular materials including gels, and nanoparticles. The NPHM-based supramolecular bioactive materials are produced from renewable resources, are simple to prepare, and have demonstrated multi-functionality including slow-release, smart-responsive release, and especially possess powerful biological effects to treat various diseases. In this review, NPHM-based supramolecular bioactive materials have been revealed as an emerging, revolutionary, and promising strategy. The development, advantages, and limitations of NPHM, as well as the advantageous position of NPHM-based materials, are first reviewed. Subsequently, a systematic and comprehensive analysis of the self-assembly strategies specific to seven major classes of NPHM is highlighted. Insights into the influence of NPHM structural features on the formation of supramolecular materials are also provided. Finally, the drivers and preparations are summarized, emphasizing the biomedical applications, future scientific challenges, and opportunities, with the hope of igniting inspiration for future research and applications.

Progressive vascular changes in a transgenic mouse model of squamous cell carcinoma.

Phage display was used to identify homing peptides for blood vessels in a mouse model of HPV16-induced epidermal carcinogenesis. One peptide, CSRPRRSEC, recognized the neovasculature in dysplastic skin but not in carcinomas. Two other peptides, with the sequences CGKRK and CDTRL, preferentially homed to neovasculature in tumors and, to a lesser extent, premalignant dysplasias. The peptides did not home to vessels in normal skin, other normal organs, or the stages in pancreatic islet carcinogenesis in another mouse model. The CGKRK peptide may recognize heparan sulfates in tumor vessels. The dysplasia-homing peptide is identical to a loop in kallikrein-9 and may bind a kallikrein inhibitor or substrate. Thus, characteristics of the angiogenic vasculature distinguish premalignant and malignant stages of skin tumorigenesis.

A two-step mechanism governing PARP1-DNA retention by PARP inhibitors.

PARP inhibitors (PARPi) have emerged as promising cancer therapeutics capable of targeting specific DNA repair pathways, but their mechanism of action with respect to PARP1-DNA retention remains unclear. Here, we developed single-molecule assays to directly monitor the retention of PARP1 on DNA lesions in real time. Our study reveals a two-step mechanism by which PARPi modulate the retention of PARP1 on DNA lesions, consisting of a primary step of catalytic inhibition via binding competition with NAD+ followed by an allosteric modulation of bound PARPi. While clinically relevant PARPi exhibit distinct allosteric modulation activities that can either increase retention of PARP1 on DNA or induce its release, their retention potencies are predominantly determined by their ability to outcompete NAD+ binding. These findings provide a mechanistic basis for improved PARPi selection according to their characteristic activities and enable further development of more potent inhibitors.

PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib.

The synthetic lethal association between BRCA deficiency and poly (ADP-ribose) polymerase (PARP) inhibition supports PARP inhibitor (PARPi) clinical efficacy in BRCA-mutated tumors. PARPis also demonstrate activity in non-BRCA mutated tumors presumably through induction of PARP1-DNA trapping. Despite pronounced clinical response, therapeutic resistance to PARPis inevitably develops. An abundance of knowledge has been built around resistance mechanisms in BRCA-mutated tumors, however, parallel understanding in non-BRCA mutated settings remains insufficient. In this study, we find a strong correlation between the epithelial-mesenchymal transition (EMT) signature and resistance to a clinical PARPi, Talazoparib, in non-BRCA mutated tumor cells. Genetic profiling demonstrates that SNAI2, a master EMT transcription factor, is transcriptionally induced by Talazoparib treatment or PARP1 depletion and this induction is partially responsible for the emerging resistance. Mechanistically, we find that the PARP1 protein directly binds to SNAI2 gene promoter and suppresses its transcription. Talazoparib treatment or PARP1 depletion lifts PARP1-mediated suppression and increases chromatin accessibility around SNAI2 promoters, thus driving SNAI2 transcription and drug resistance. We also find that depletion of the chromatin remodeler CHD1L suppresses SNAI2 expression and reverts acquired resistance to Talazoparib. The PARP1/CHD1L/SNAI2 transcription axis might be therapeutically targeted to re-sensitize Talazoparib in non-BRCA mutated tumors.

Systematic surface engineering of magnetic nanoworms for in vivo tumor targeting.

In the design of nanoparticles that can target disease tissue in vivo, parameters such as targeting ligand density, type of target receptor, and nanoparticle shape can play an important role in determining the extent of accumulation. Herein, a systematic study of these parameters for the targeting of mouse xenograft tumors is performed using superparamagnetic iron oxide as a model nanoparticle system. The type of targeting peptide (recognizing cell surface versus extracellular matrix), the surface coverage of the peptide, its attachment chemistry, and the shape of the nanomaterial [elongated (nanoworm, NW) versus spherical (nanosphere, NS)] are varied. Nanoparticle circulation times and in vivo tumor-targeting efficiencies are quantified in two xenograft models of human tumors (MDA-MB-435 human carcinoma and HT1080 human fibrosarcoma). It is found that the in vivo tumor-targeting ability of the NW is superior to that of the NS, that the smaller, neutral CREKA targeting group is more effective than the larger, positively charged F3 molecule, that a maximum in tumor-targeting efficiency and blood half-life is observed with approximately 60 CREKA peptides per NW for either the HT1080 or the MDA-MB-435 tumor types, and that incorporation of a 5-kDa polyethylene glycol linker improves targeting to both tumor types relative to a short linker. It is concluded that the blood half-life of a targeting molecule-nanomaterial ensemble is a key consideration when selecting the appropriate ligand and nanoparticle chemistry for tumor targeting.

Circularly polarized luminescence of single-handed helical tetraphenylethylene-silica nanotubes.

Two single-handed helical tetraphenylethylene-silica nanotubes with circularly polarized luminescence (CPL) properties and enhanced fluorescence efficiency were fabricated through a supramolecular templating approach using the self-assemblies of chiral gelators as templates. This work provides a facile strategy for constructing CPL-active organic-inorganic hybrid nanomaterials with single-handed helical morphologies.

Peptide targeting of tumor lymph vessels.

In addition to having a blood vasculature, most normal organs and pathologic conditions involve a second vascular system, the lymphatic vasculature, and many tumors induce the growth of new lymphatic vessels. However, compared to the blood vasculature, very little is known about the lymphatic vessels in tumors. We have used the in vivo phage display technology to map tumor-specific differences in the lymphatic vasculature, and identified peptides that specifically home to tumor lymphatics. Each of these peptides recognizes lymphatic vessels in a different set of tumors, and some of them also recognize tumor cells. Furthermore, these peptides can differentiate lymphatic vasculature of a premalignant lesion from that of a full-blown tumor, indicating tumor stage-specific differences in the lymphatic vessels. None of the lymphatic homing peptides recognizes blood endothelial cells, nor do they home to any normal organ. Of interest, some of our homing peptides are able to penetrate the target cells in a cell type-specific manner. These peptides appear to be able to concentrate in the target tissue, making them particularly efficient delivery vectors for the targeting of therapeutic moieties and imaging agents. Conjugation of the lymphatic homing peptides to drugs provides an opportunity to specifically deliver therapeutic agents into tumors using a route not previously exploited. The surprising degree of selectivity of these lymphatic homing peptides suggests extensive molecular specialization of tumor lymphatic vessels, positing the existence of a molecular lymphatic "zip code" system, as has been previously demonstrated for the tumor blood vasculature.

Lymphatic zip codes in premalignant lesions and tumors.

Blood vessels in tumors are morphologically and functionally distinct from normal resting blood vessels. We probed lymphatic vessels in premalignant lesions and tumors by in vivo screening of phage-displayed peptide libraries, asking whether they too have distinctive signatures. The resulting peptides begin to define such signatures. One peptide identified the lymphatics in a human melanoma xenograft. Another recognized the lymphatics in prostate cancers but not in premalignant prostate lesions; this peptide similarly identifies human prostate cancer lymphatics. A third was selective for the lymphatics in the premalignant prostate lesions. A fourth identified the lymphatics in dysplasias and squamous carcinomas of the cervix and skin. None recognize lymphatics in normal tissues. Thus, tumor development is associated with organ- and stage-specific changes in lymphatics. Systemic treatment of mice with fusions of a lymphatic homing peptide and a proapoptotic motif reduced the number of tumor lymphatics in prostate tumor and melanoma, forecasting future lymphatic targeting agents for detection and therapeutic intervention.

Processing of PatS, a morphogen precursor, in cell extracts of Anabaena sp. PCC 7120.

Upon N-stepdown, Anabaena sp. PCC 7120 differentiates heterocysts along filaments in a semiregular pattern. A 17-amino acid peptide called PatS is a morphogen precursor for pattern formation. The principal PatS derivative involved in heterocyst patterning has been proposed to be the C-terminal peptide PatS-5 (RGSGR), PatS-6 (ERGSGR), or PatS-8 (CDERGSGR). We present the first evidence for processing of PatS in cell extracts of this cyanobacterium. PatS is probably cleaved between the C-terminal 7th and 8th amino acid residues, producing PatS-7 (DERGSGR), then converted into PatS-6 and PatS-5. The processing site could be changed by a substitution at the C-terminal 8th residue.

Molecular profiling of heart endothelial cells.

BACKGROUND: Endothelial cells that line the vascular lumen can express cell-surface proteins that are specific to the endothelium of a particular tissue. In this study, we probed the heart vasculature for heart-specific endothelial markers by phage display. METHODS AND RESULTS: We used a novel combination of in vivo phage selection and a bacterial 2-hybridization scheme against a heart cDNA library, which allows simultaneous identification of peptides that specifically bind to the target endothelium, as well as the endothelial molecules (receptors) recognized by the peptides. We found 5 heart-targeting peptides and their receptors. We confirmed and quantified the selective expression of 4 of the proteins in heart endothelial cells by independent methods. The heart specificity of phages was as high as 300-fold greater than that of nonrecombinant control phages. The proteins selectively expressed by the heart endothelium were in most cases also expressed by cardiomyocytes and, at lower levels, in some other tissues. CONCLUSIONS: These findings provide new markers for the endothelium of heart vessels and reveal a commonality between parenchymal and endothelial gene expression in the heart. The heart-homing peptides provide a means of targeting diagnostic and therapeutic agents to the heart, and their receptors are potential drug discovery targets.

Combined anti-fetal liver kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits angiogenesis and growth of human soft tissue sarcoma xenografts by induction of endothelial cell apoptosis.

Vascular endothelial growth factor (VEGF) and VEGF receptor 2 [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] have been shown to play a major role in tumor angiogenesis. In this study, we investigated whether anti-Flk-1 monoclonal antibody DC101 could therapeutically inhibit growth and angiogenesis of human soft tissue sarcoma, and we explored its capacity to enhance the tumoricidal effects of doxorubicin. Treatment of well-established leiomyosarcoma SKLMS-1 and rhabdomyosarcoma RD xenografts in severe combined immunodeficient mice with DC101 resulted in significant antitumor activity. In a parallel study, we compared tumor inhibition with continuous low-dose "antiangiogenic" schedule versus once-every-2-weeks high-dose standard schedule of doxorubicin. We found that continuous low-dose treatment inhibited the tumor growth of RD xenografts about 46.5% of that with standard-schedule treatment, but that continuous low-dose treatment did not inhibit the tumor growth of SKLMS-1 xenografts. Notably, combined DC101 and continuous low-dose doxorubicin resulted in more effective growth inhibition of SKLMS-1 and RD xenografts than has been observed with any agent alone in a long-term s.c. tumor xenograft model. The combination therapy was associated with no additional toxicity to the host animal compared with low-dose doxorubicin alone. Histological examination of xenografts showed significantly reduced microvessel counts in the tumors given combined therapy compared with the tumors given either agent alone. These results are consistent with an enhanced inhibition of angiogenesis in vivo by combined DC101 and doxorubicin using Matrigel plug assay. Additionally, DC101 plus doxorubicin directly exerted enhanced inhibitory effects on endothelial cell migration, proliferation, and tube-like formation in vitro. Furthermore, the combination induced an enhanced apoptosis of endothelial cells that was associated with an increase of capase-3 activity. Thus, the inhibition of angiogenesis and induction of endothelial cell apoptosis are likely important mechanisms for the antitumor activity of combined DC101 and doxorubicin. Collectively, our data suggested that anti-VEGF receptor 2 in combination with continuous low-dose doxorubicin may provide a new therapeutic approach for human soft tissue sarcoma in the clinic.

A first-in-human study of the anti-α5ß1 integrin monoclonal antibody PF-04605412 administered intravenously to patients with advanced solid tumors.

PURPOSE: A first-in-human clinical trial of a fully human, Fc-engineered IgG1 monoclonal antibody targeting integrin α5ß1 was conducted to evaluate tolerability, maximum tolerated dose, pharmacokinetics, pharmacodynamics and preliminary anti-tumor activity. METHODS: Escalating doses of PF-04605412 were given IV on day 1, 28 and every 2 weeks thereafter to patients with advanced solid tumors until disease progression or unacceptable toxicity. Sequential dose cohorts were evaluated based on a modified 3 + 3 dose-escalation design. The starting dose was 7.5 mg based on preclinical data. RESULTS: Thirty-three patients were enrolled to six dose levels (7.5, 11.25, 16.9, 34, 68 and 136 mg). Twenty-three patients were evaluable for the primary endpoint (determination of the maximum tolerated dose). Five patients required permanent drug discontinuation due to acute infusion-related reactions, which occurred as grade 3 events in two patients. PK analysis indicated that the targeted drug exposure based on preclinical models was not achieved by the tolerated doses and PK modeling suggesting that doses at least fivefold higher would be necessary. No anti-tumor activity was observed. CONCLUSION: Based on the safety data, the risks associated with the likelihood of significant cytokine-mediated infusion reactions at higher doses, the projected high dose necessary to affect on the biological target and the lack of anti-tumor activity at the doses explored, the trial was prematurely terminated without determining a formal maximum tolerated dose. Further clinical development of PF-04605412 has been discontinued.

Targeting activin receptor-like kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

Dual functional monoclonal antibody PF-04605412 targets integrin alpha5beta1 and elicits potent antibody-dependent cellular cytotoxicity.

Integrin α5ß1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5ß1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies.