Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

Transient compartmentalization of simian immunodeficiency virus variants in the breast milk of african green monkeys.

Natural hosts of simian immunodeficiency virus (SIV), African green monkeys (AGMs), rarely transmit SIV via breast-feeding. In order to examine the genetic diversity of breast milk SIV variants in this limited-transmission setting, we performed phylogenetic analysis on envelope sequences of milk and plasma SIV variants of AGMs. Low-diversity milk virus populations were compartmentalized from that in plasma. However, this compartmentalization was transient, as the milk virus lineages did not persist longitudinally.

Lack of B cell dysfunction is associated with functional, gp120-dominant antibody responses in breast milk of simian immunodeficiency virus-infected African green monkeys.

The design of an effective vaccine to reduce the incidence of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) via breastfeeding will require identification of protective immune responses that block postnatal virus acquisition. Natural hosts of simian immunodeficiency virus (SIV) sustain nonpathogenic infection and rarely transmit the virus to their infants despite high milk virus RNA loads. This is in contrast to HIV-infected women and SIV-infected rhesus macaques (RhMs), nonnatural hosts which exhibit higher rates of postnatal virus transmission. In this study, we compared the systemic and mucosal B cell responses of lactating, SIV-infected African green monkeys (AGMs), a natural host species, to that of SIV-infected RhMs and HIV-infected women. AGMs did not demonstrate hypergammaglobulinemia or accumulate circulating memory B cells during chronic SIV infection. Moreover, the milk of SIV-infected AGMs contained higher proportions of naive B cells than RhMs. Interestingly, AGMs exhibited robust milk and plasma Env binding antibody responses that were one to two logs higher than those in RhMs and humans and demonstrated autologous neutralizing responses in milk at 1 year postinfection. Furthermore, the plasma and milk Env gp120-binding antibody responses were equivalent to or predominant over Env gp140-binding antibody responses in AGMs, in contrast to that in RhMs and humans. The strong gp120-specific, functional antibody responses in the milk of SIV-infected AGMs may contribute to the rarity of postnatal transmission observed in natural SIV hosts.

Mucosal immunization of lactating female rhesus monkeys with a transmitted/founder HIV-1 envelope induces strong Env-specific IgA antibody responses in breast milk.

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.

Memory CD4(+) T lymphocytes in the gastrointestinal tract are a major source of cell-associated simian immunodeficiency virus in chronic nonpathogenic infection of African green monkeys.

Simian immunodeficiency virus (SIV) infection of natural hosts is characterized by nonpathogenic chronic viremia, maintenance of gastrointestinal epithelial barrier integrity, and low numbers of target cells. Assessment of cell-associated virus load in T cell subsets in multiple anatomic compartments of chronically SIV-infected sabeus African green monkeys (AGMs) revealed that gastrointestinal memory CD4(+) T lymphocytes are a major source of cell-associated virus and a significant contributor to SIV viremia in AGMs.

Phase I, pharmacokinetic and pharmacodynamic evaluation of CYT997, an orally-bioavailable cytotoxic and vascular-disrupting agent.

PURPOSE: CYT997 is a novel microtubule inhibitor and vascular disrupting agent. This phase I trial examined the safety, tolerability, pharmacokinetics and vascular-disrupting effects of orally-administered CYT997. EXPERIMENTAL DESIGN: We performed a phase I accelerated dose-escalation study of CYT997 given orally once every 2 to 3 weeks in patients with advanced solid tumours. Vascular disruption was assessed by measurement of plasma von Willebrand factor (vWF) levels and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). RESULTS: A total of 56 doses were administered to 21 patients over 8 dose levels (15-164 mg/m(2)). Grade 3 fatigue and grade 3 hypoxia were dose limiting. Oral bioavailability was observed with approximate linear pharmacokinetics over the 11-fold dose range. At doses of 84 mg/m(2) and above, plasma vWF levels increased above baseline and DCE-MRI scans showed reductions in tumour K(trans) in some patients. CONCLUSIONS: CYT997 is orally bioavailable. The 118 mg/m(2) dose level should be used to guide dosing in future studies.

Harnessing autologous immune effector mechanisms in acute myeloid leukemia: 2023 update of trials and tribulations.

Numerous recent advances have been made in therapeutic approaches toward acute myeloid leukemia (AML). Since 2017, we have seen eleven novel Food & Drug Administration (FDA)-approved medications for AML, all of which extend beyond the classical cytarabine-based cytostatic chemotherapy. In the recent two decades, the role of immune surveillance in AML has been intensively investigated. The power of one's own innate and adaptive immunity has been harnessed pharmacologically toward the goal of clearance of AML cells. Specifically, pre-clinical studies have shown great promise for antibodies that disinhibit T cells and macrophages by blocking checkpoint receptors within the immunologic synapse, thereby resulting in the elimination of AML cells. Anti-CD33 CAR-T therapies and anti-CD3/CD123 bispecific antibodies have also exhibited encouraging results in pre-clinical and early clinical studies. However, despite these translational efforts, we currently have no immune-based therapies for AML on the market, with the exception of gemtuzumab ozogamicin. In this focused review, we discuss molecular target validation and the most relevant clinical updates for immune-based experimental therapeutics including anti-CD47 monoclonal antibodies, CAR-T therapies, and bispecific T cell engagers. We highlight barriers to the clinical translation of these therapies in AML, and we propose solutions to optimize the manufacturing and delivery of the most novel immune-based therapies in the pipeline.

High cell-free virus load and robust autologous humoral immune responses in breast milk of simian immunodeficiency virus-infected african green monkeys.

The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4(+) T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.

HIV-specific functional antibody responses in breast milk mirror those in plasma and are primarily mediated by IgG antibodies.

Despite months of mucosal virus exposure, the majority of breastfed infants born to HIV-infected mothers do not become infected, raising the possibility that immune factors in milk inhibit mucosal transmission of HIV. HIV Envelope (Env)-specific antibodies are present in the milk of HIV-infected mothers, but little is known about their virus-specific functions. In this study, HIV Env-specific antibody binding, autologous and heterologous virus neutralization, and antibody-dependent cell cytotoxicity (ADCC) responses were measured in the milk and plasma of 41 HIV-infected lactating women. Although IgA is the predominant antibody isotype in milk, HIV Env-specific IgG responses were higher in magnitude than HIV Env-specific IgA responses in milk. The concentrations of anti-HIV gp120 IgG in milk and plasma were directly correlated (r = 0.75; P < 0.0001), yet the response in milk was 2 logarithm units lower than in plasma. Similarly, heterologous virus neutralization (r = 0.39; P = 0.010) and ADCC activity (r = 0.64; P < 0.0001) in milk were directly correlated with that in the systemic compartment but were 2 log units lower in magnitude. Autologous neutralization was rarely detected in milk. Milk heterologous virus neutralization titers correlated with HIV gp120 Env-binding IgG responses but not with IgA responses (r = 0.71 and P < 0.0001, and r = 0.17 and P = 0.30). Moreover, IgGs purified from milk and plasma had equal neutralizing potencies against a tier 1 virus (r = 0.65; P < 0.0001), whereas only 1 out of 35 tested non-IgG milk fractions had detectable neutralization. These results suggest that plasma-derived IgG antibodies mediate the majority of the low-level HIV neutralization and ADCC activity in breast milk.

Origin and evolution of HIV-1 in breast milk determined by single-genome amplification and sequencing.

HIV transmission via breastfeeding accounts for a considerable proportion of infant HIV acquisition. However, the origin and evolution of the virus population in breast milk, the likely reservoir of transmitted virus variants, are not well characterized. In this study, HIV envelope (env) genes were sequenced from virus variants amplified by single-genome amplification from plasmas and milk of 12 chronically HIV-infected, lactating Malawian women. Maximum likelihood trees and statistical tests of compartmentalization revealed interspersion of plasma and milk HIV env sequences in the majority of subjects, indicating limited or no compartmentalization of milk virus variants. However, phylogenetic tree analysis further revealed monotypic virus variants that were significantly more frequent in milk (median proportion of identical viruses, 29.5%; range, 0 to 61%) than in plasma (median proportion of identical viruses, 0%; range, 0 to 26%) (P = 0.002), suggesting local virus replication in the breast milk compartment. Moreover, clonally amplified virus env genes in milk produced functional virus Envs that were all CCR5 tropic. Milk and plasma virus Envs had similar predicted phenotypes and neutralization sensitivities to broadly neutralizing antibodies in both transmitting and nontransmitting mothers. Finally, phylogenetic comparison of longitudinal milk and plasma virus env sequences revealed synchronous virus evolution and new clonal amplification of evolved virus env genes in milk. The limited compartmentalization and the clonal amplification of evolving, functional viruses in milk indicate continual seeding of the mammary gland by blood virus variants, followed by transient local replication of these variants in the breast milk compartment.

Robust vaccine-elicited cellular immune responses in breast milk following systemic simian immunodeficiency virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Limited contribution of mucosal IgA to Simian immunodeficiency virus (SIV)-specific neutralizing antibody response and virus envelope evolution in breast milk of SIV-infected, lactating rhesus monkeys.

Breast milk transmission of human immunodeficiency virus (HIV) remains an important mode of infant HIV acquisition. Interestingly, the majority of infants remain uninfected during prolonged virus exposure via breastfeeding, raising the possibility that immune components in milk prevent mucosal virus transmission. HIV-specific antibody responses are detectable in the milk of HIV-infected women and simian immunodeficiency virus (SIV)-infected monkeys; however, the role of these humoral responses in virus neutralization and local virus quasispecies evolution has not been characterized. In this study, four lactating rhesus monkeys were inoculated with SIVmac251 and monitored for SIV envelope-specific humoral responses and virus evolution in milk and plasma throughout infection. While the kinetics and breadth of the SIV-specific IgG and IgA responses in milk were similar to those in plasma, the magnitude of the milk responses was considerably lower than that of the plasma responses. Furthermore, a neutralizing antibody response against the inoculation virus was not detected in milk samples at 1 year after infection, despite a measurable autologous neutralizing antibody response in plasma samples obtained from three of four monkeys. Interestingly, while IgA is the predominant immunoglobulin in milk, the milk SIV envelope-specific IgA response was lower in magnitude and demonstrated more limited neutralizing capacity against a T-cell line-adapted SIV compared to those of the milk IgG response. Finally, amino acid mutations in the envelope gene product of SIV variants in milk and plasma samples occurred in similar numbers and at similar positions, indicating that the humoral immune pressure in milk does not drive distinct virus evolution in the breast milk compartment.

Preservation of memory CD4(+) T lymphocytes in breast milk of lactating rhesus monkeys during acute simian immunodeficiency virus infection.

Acute human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection is associated with a massive depletion of memory CD4(+) T lymphocytes in the gastrointestinal tract. To define the dynamics of the CD4(+) T lymphocyte subpopulations in breast milk during acute HIV or SIV infection, lymphocyte populations were monitored in blood and milk of 4 Mamu-A*01(+) rhesus monkeys after SIVmac251 inoculation. Strikingly, although the CD4(+) T lymphocytes in blood were depleted during the peak of viremia, the milk CD4(+) T lymphocyte counts remained unchanged, despite active virus replication in the breast milk compartment. Moreover, CD4(+) memory T lymphocytes were preserved in breast milk during acute infection. CD4(+) T lymphocytes in breast milk and other mucosal compartments of uninfected monkeys were similar in their memory phenotype, activation status, and chemokine (C-C motif) receptor 5 expression. Interestingly, the number and proportion of effector CD8(+) T lymphocytes in milk were increased during acute SIV infection, suggesting effective control of virus-mediated CD4(+) T lymphocyte destruction in the breast milk compartment.

Local replication of simian immunodeficiency virus in the breast milk compartment of chronically-infected, lactating rhesus monkeys.

Breast milk transmission remains a major mode of infant HIV acquisition, yet anatomic and immunologic forces shaping virus quasispecies in milk are not well characterized. In this study, phylogenic analysis of envelope sequences of milk SIV variants revealed groups of nearly identical viruses, indicating local virus production. However, comparison of the patterns and rates of CTL escape of blood and milk virus demonstrated only subtle differences between the compartments. These findings suggest that a substantial fraction of milk viruses are produced by locally-infected cells, but are shaped by cellular immune pressures similar to that in the blood.

Colony-stimulating factor-1 (CSF-1) delivers a proatherogenic signal to human macrophages.

M-CSF/CSF-1 supports the proliferation and differentiation of monocytes and macrophages. In mice, CSF-1 also promotes proinflammatory responses in vivo by regulating mature macrophage functions, but little is known about the acute effects of this growth factor on mature human macrophages. Here, we show that in contrast to its effects on mouse bone marrow-derived macrophages, CSF-1 did not induce expression of urokinase plasminogen activator mRNA, repress expression of apolipoprotein E mRNA, or prime LPS-induced TNF and IL-6 secretion in human monocyte-derived macrophages (HMDM) from several independent donors. Instead, we show by expression profiling that CSF-1 modulates the HMDM transcriptome to favor a proatherogenic environment. CSF-1 induced expression of the proatherogenic chemokines CXCL10/IFN-inducible protein 10, CCL2, and CCL7 but repressed expression of the antiatherogenic chemokine receptor CXCR4. CSF-1 also up-regulated genes encoding enzymes of the cholesterol biosynthetic pathway (HMGCR, MVD, IDI1, FDPS, SQLE, CYP51A1, EBP, NSDHL, DHCR7, and DHCR24), and expression of ABCG1, encoding a cholesterol efflux transporter, was repressed. Consistent with these effects, CSF-1 increased levels of free cholesterol in HMDM, and the selective CSF-1R kinase inhibitor GW2580 ablated this response. These data demonstrate that CSF-1 represents a further link between inflammation and cardiovascular disease and suggest two distinct mechanisms by which CSF-1, which is known to be present in atherosclerotic lesions, may contribute to plaque progression.

Discovery of CYT997: a structurally novel orally active microtubule targeting agent.

CYT997 was discovered as a potent tubulin polymerization inhibitor possessing potent cytotoxic activity against a range of cancer cells. Details of SAR studies, pharmacokinetic investigations and synthesis of compounds leading to the discovery of CYT997 are reported.

Discovery of 2-(alpha-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs).

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

The 2.7 A crystal structure of the autoinhibited human c-Fms kinase domain.

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.