Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Inhibition of de novo lipogenesis targets androgen receptor signaling in castration-resistant prostate cancer.

A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119-mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7-driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.

CDKI-73: an orally bioavailable and highly efficacious CDK9 inhibitor against acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia with dismal long-term prognosis with age. The most aggressive subtype of AML is MLL-AML that is characterized by translocations of the mixed-lineage leukemia gene (MLL) and resistance to conventional chemotherapy. Cyclin dependent kinase 9 (CDK9) plays a crucial role in the MLL-driven oncogenic transcription, and hence, inhibiting activity of CDK9 has been proposed as a promising strategy for treatment of AML. We investigated the therapeutic potential of CDKI-73, one of the most potent CDK9 inhibitors, against a panel of AML cell lines and samples derived from 97 patients. CDKI-73 induced cancer cells undergoing apoptosis through transcriptional downregulation of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP by majorly targeting CDK9. Contrastively, it was relatively low toxic to the bone marrow cells of healthy donors. In MV4-11 xenograft mouse models, oral administration of CDKI-73 resulted in a marked inhibition of tumor growth (p < 0.0001) and prolongation of animal life span (P < 0.001) without causing body weight loss and other overt toxicities. The study suggests that CDKI-73 can be developed as a highly efficacious and orally deliverable therapeutic agent for treatment of AML.

Ex uno plures: clonal reinforcement drives evolution of a simple microbial community.

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community's underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103's phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an "engine" generating diversity by creating new metabolic niches, but not the occupants themselves.

A Central Bioactive Region of LTBP-2 Stimulates the Expression of TGF-ß1 in Fibroblasts via Akt and p38 Signalling Pathways.

Latent transforming growth factor-ß-1 binding protein-2 (LTBP-2) belongs to the LTBP-fibrillin superfamily of extracellular proteins. Unlike other LTBPs, LTBP-2 does not covalently bind transforming growth factor-ß1 (TGF-ß1) but appears to be implicated in the regulation of TGF-ß1 bioactivity, although the mechanisms are largely unknown. In experiments originally designed to study the displacement of latent TGF-ß1 complexes from matrix storage, we found that the addition of exogenous LTBP-2 to cultured human MSU-1.1 fibroblasts caused an increase in TGF-ß1 levels in the medium. However, the TGF-ß1 increase was due to an upregulation of TGF-ß1 expression and secretion rather than a displacement of matrix-stored TGF-ß1. The secreted TGF-ß1 was mainly in an inactive form, and its concentration peaked around 15 h after addition of LTBP-2. Using a series of recombinant LTBP-2 fragments, the bioactivity was identified to a small region of LTBP-2 consisting of an 8-Cys motif flanked by four epidermal growth factor (EGF)-like repeats. The LTBP-2 stimulation of TGF-ß expression involved the phosphorylation of both Akt and p38 mitogen-activated protein kinase (MAPK) signalling proteins, and specific inactivation of each protein individually blocked TGF-ß1 increase. The search for the cell surface receptor mediating this LTBP-2 activity proved inconclusive. Inhibitory antibodies to integrins ß1 and αVß5 showed no reduction of LTBP-2 stimulation of TGF-ß1. However, TGF-ß1 upregulation was partially inhibited by anti-αVß3 integrin antibodies, suggestive of a direct or indirect role for this integrin. Overall, the study indicates that LTBP-2 can directly upregulate cellular TGF-ß1 expression and secretion by interaction with cells via a short central bioactive region. This may be significant in connective tissue disorders involving aberrant TGF-ß1 signalling.

Experimental microbial evolution: history and conceptual underpinnings.

We chronicle and dissect the history of the field of Experimental Microbial Evolution, beginning with work by Monod. Early research was largely carried out by microbiologists and biochemists, who used experimental evolutionary change as a tool to understand structure-function relationships. These studies attracted the interest of evolutionary biologists who recognized the power of the approach to address issues such as the tempo of adaptive change, the costs and benefits of sex, parallelism, and the role which contingency plays in the evolutionary process. In the 1980s and 1990s, an ever-expanding body of microbial, physiological and biochemical data, together with new technologies for manipulating microbial genomes, allowed such questions to be addressed in ever-increasing detail. Since then, technological advances leading to low-cost, high-throughput DNA sequencing have made it possible for these and other fundamental questions in evolutionary biology to be addressed at the molecular level.

A multifaceted kinase axis regulates plant organ abscission through conserved signaling mechanisms.

Plants have evolved mechanisms to abscise organs as they develop or when exposed to unfavorable conditions.1 Uncontrolled abscission of petals, fruits, or leaves can impair agricultural productivity.2,3,4,5 Despite its importance for abscission progression, our understanding of the IDA signaling pathway and its regulation remains incomplete. IDA is secreted to the apoplast, where it is perceived by the receptors HAESA (HAE) and HAESA-LIKE2 (HSL2) and somatic embryogenesis receptor kinase (SERK) co-receptors.6,7,8,9 These plasma membrane receptors activate an intracellular cascade of mitogen-activated protein kinases (MAPKs) by an unknown mechanism.10,11,12 Here, we characterize brassinosteroid signaling kinases (BSKs) as regulators of floral organ abscission in Arabidopsis. BSK1 localizes to the plasma membrane of abscission zone cells, where it interacts with HAESA receptors to regulate abscission. Furthermore, we demonstrate that YODA (YDA) has a leading role among other MAPKKKs in controlling abscission downstream of the HAESA/BSK complex. This kinase axis, comprising a leucine-rich repeat receptor kinase, a BSK, and an MAPKKK, is known to regulate stomatal patterning, early embryo development, and immunity.10,13,14,15,16 How specific cellular responses are obtained despite signaling through common effectors is not well understood. We show that the identified abscission-promoting allele of BSK1 also enhances receptor signaling in other BSK-mediated pathways, suggesting conservation of signaling mechanisms. Furthermore, we provide genetic evidence supporting independence of BSK1 function from its kinase activity in several developmental processes. Together, our findings suggest that BSK1 facilitates signaling between plasma membrane receptor kinases and MAPKKKs via conserved mechanisms across multiple facets of plant development.

The pre-clinical absorption, distribution, metabolism and excretion properties of IPI-926, an orally bioavailable antagonist of the hedgehog signal transduction pathway.

1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h(-1)), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties.

Candidate genes affecting stomatal density in rice (Oryza sativa L.) identified by genome-wide association.

Stomata regulate photosynthesis and water loss. They have been an active subject of research for centuries, but our knowledge of the genetic components that regulate stomatal development in crops remains very limited in comparison to the model plant Arabidopsis thaliana. Leaf stomatal density was found to vary by over 2.5-fold across a panel of 235 rice accessions. Using GWAS, we successfully identified five different QTLs associated with stomatal density on chromosomes 2, 3, 9, and 12. Forty-two genes were identified within the haplotype blocks corresponding to these QTLs. Of these, nine genes contained haplotypes that were associated with different stomatal densities. These include a gene encoding a trehalose-6-phosphate synthase, an enzyme that has previously been associated with altered stomatal density in Arabidopsis, and genes encoding a B-BOX zinc finger family protein, a leucine-rich repeat family protein, and the 40 S ribosomal protein S3a, none of which have previously been linked to stomatal traits. We investigated further and show that a closely related B-BOX protein regulates stomatal development in Arabidopsis. The results of this study provide information on genetic associations with stomatal density in rice. The QTLs and candidate genes may be useful in future breeding programs for low or high stomatal density and, consequently, improved photosynthetic capacity, water use efficiency, or drought tolerance.

The development of proteasome inhibitors as anticancer drugs.

The ubiquitin-proteasome pathway plays a central role in the targeted destruction of cellular proteins, including cell cycle regulatory proteins. Because these pathways are critical for the proliferation and survival of all cells, and in particular cancerous cells, proteasome inhibition is a potentially attractive anticancer therapy. Based on encouraging cytotoxic activity, bortezomib was the first proteasome inhibitor to be evaluated in clinical trials. Efficacy and safety results from a phase 2 clinical trial contributed to approval of bortezomib for use in patients with relapsed and refractory multiple myeloma who have received at least 2 prior therapies and have demonstrated disease progression on their last therapy.

Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo.

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer.

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Regiospecific and stereoselective syntheses of (+/-) morphine, codeine, and thebaine via a highly stereocontrolled intramolecular 4 + 2 cycloaddition leading to a phenanthrofuran system.

Total syntheses of the morphine alkaloids are described that use a direct stereoselective formation of the phenanthrofuran system via an intramolecular 4 + 2 cycloaddition of a diene tethered to the 4-position of a 7-methoxybenzofuran-3-carboxylic acid ester.

Targeting CDK9 for treatment of colorectal cancer.

Colorectal cancer (CRC) remains one of the most lethal human malignancies, and pursuit of new therapeutic targets for treatment has been a major research focus. Cyclin-dependent kinase 9 (CDK9), which plays a crucial role in transcription, has emerged as a target for cancer treatment. CDKI-73, one of the most potent and pharmacologically superior CDK9 inhibitors, has demonstrated excellent anti-tumour efficacy against several types of cancers. In this study, we evaluated its therapeutic potential against CRC. CDKI-73 elicited high cytotoxicity against all colon cancer cell lines tested. Cell cycle and apoptosis analysis in HCT 116 and HT29 cells revealed that CDKI-73 induced cell death without accumulation of DNA at any phase of the cell cycle. Moreover, it caused depolarisation of mitochondrial membrane, leading to caspase-independent apoptosis. Knockdown by shRNA demonstrated the CDK9-targeted mechanism of CDKI-73, which also affected the Mnk/eIF4E signalling axis. In addition, RT-qPCR analysis showed that CDKI-73 down-regulated multiple pro-survival factors at the mRNA level. Its in vivo anti-tumour efficacy was further evaluated in Balb/c nude mice bearing HCT 116 xenograft tumours. CDKI-73 significantly inhibited tumour growth (***P < 0.001) without overt toxicity. Analysis of the tumour tissues collected from the xenografted animals confirmed that the in vivo anti-tumour efficacy was associated with CDK9 targeting of CDKI-73. Overall, this study provides compelling evidence that CDKI-73 is a promising drug candidate for treating colorectal cancer.

Structural studies of the Alzheimer's amyloid precursor protein copper-binding domain reveal how it binds copper ions.

Alzheimer's disease (AD) is the major cause of dementia. Amyloid beta peptide (Abeta), generated by proteolytic cleavage of the amyloid precursor protein (APP), is central to AD pathogenesis. APP can function as a metalloprotein and modulate copper (Cu) transport, presumably via its extracellular Cu-binding domain (CuBD). Cu binding to the CuBD reduces Abeta levels, suggesting that a Cu mimetic may have therapeutic potential. We describe here the atomic structures of apo CuBD from three crystal forms and found they have identical Cu-binding sites despite the different crystal lattices. The structure of Cu(2+)-bound CuBD reveals that the metal ligands are His147, His151, Tyr168 and two water molecules, which are arranged in a square pyramidal geometry. The site resembles a Type 2 non-blue Cu center and is supported by electron paramagnetic resonance and extended X-ray absorption fine structure studies. A previous study suggested that Met170 might be a ligand but we suggest that this residue plays a critical role as an electron donor in CuBDs ability to reduce Cu ions. The structure of Cu(+)-bound CuBD is almost identical to the Cu(2+)-bound structure except for the loss of one of the water ligands. The geometry of the site is unfavorable for Cu(+), thus providing a mechanism by which CuBD could readily transfer Cu ions to other proteins.

A phase 2 study of bortezomib in relapsed, refractory myeloma.

BACKGROUND: Bortezomib, a boronic acid dipeptide, is a novel proteasome inhibitor that has been shown in preclinical and phase 1 studies to have antimyeloma activity. METHODS: In this multicenter, open-label, nonrandomized, phase 2 trial, we enrolled 202 patients with relapsed myeloma that was refractory to the therapy they had received most recently. Patients received 1.3 mg of bortezomib per square meter of body-surface area twice weekly for 2 weeks, followed by 1 week without treatment, for up to eight cycles (24 weeks). In patients with a suboptimal response, oral dexamethasone (20 mg daily, on the day of and the day after bortezomib administration) was added to the regimen. The response was evaluated according to the criteria of the European Group for Blood and Marrow Transplantation and confirmed by an independent review committee. RESULTS: Of 193 patients who could be evaluated, 92 percent had been treated with three or more of the major classes of agents for myeloma, and in 91 percent, the myeloma was refractory to the therapy received most recently. The rate of response to bortezomib was 35 percent, and those with a response included 7 patients in whom myeloma protein became undetectable and 12 in whom myeloma protein was detectable only by immunofixation. The median overall survival was 16 months, with a median duration of response of 12 months. Grade 3 adverse events included thrombocytopenia (in 28 percent of patients), fatigue (in 12 percent), peripheral neuropathy (in 12 percent), and neutropenia (in 11 percent). Grade 4 events occurred in 14 percent of patients. CONCLUSIONS: Bortezomib, a member of a new class of anticancer drugs, is active in patients with relapsed multiple myeloma that is refractory to conventional chemotherapy.

Structure of Alzheimer's disease amyloid precursor protein copper-binding domain at atomic resolution.

Amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease, as its cleavage generates the Abeta peptide that is toxic to cells. APP is able to bind Cu2+ and reduce it to Cu+ through its copper-binding domain (CuBD). The interaction between Cu2+ and APP leads to a decrease in Abeta production and to alleviation of the symptoms of the disease in mouse models. Structural studies of CuBD have been undertaken in order to better understand the mechanism behind the process. Here, the crystal structure of CuBD in the metal-free form determined to ultrahigh resolution (0.85 A) is reported. The structure shows that the copper-binding residues of CuBD are rather rigid but that Met170, which is thought to be the electron source for Cu2+ reduction, adopts two different side-chain conformations. These observations shed light on the copper-binding and redox mechanisms of CuBD. The structure of CuBD at atomic resolution provides an accurate framework for structure-based design of molecules that will deplete Abeta production.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of chloride intracellular channel 2 (CLIC2).

The chloride intracellular channel (CLIC) family of proteins are unusual in that they can exist in either an integral membrane-channel form or a soluble form. Here, the expression, purification, crystallization and preliminary diffraction analysis of CLIC2, one of the least-studied members of this family, are reported. Human CLIC2 was crystallized in two different forms, both in the presence of reduced glutathione and both of which diffracted to better than 1.9 A resolution. Crystal form A displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 44.0, b = 74.7, c = 79.8 A. Crystal form B displayed P2(1) symmetry, with unit-cell parameters a = 36.0, b = 66.9, c = 44.1 A. Structure determination will shed more light on the structure and function of this enigmatic family of proteins.

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1.

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.