Diverse alterations associated with resistance to KRAS(G12C) inhibition.

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.

A nanoparticle vaccine that targets neoantigen peptides to lymphoid tissues elicits robust antitumor T cell responses.

Cancer vaccines using synthetic long peptides (SLP) targeting tumor antigens have been tested in the clinic but the outcomes have been unimpressive, perhaps because these peptides elicit predominantly CD4+ T cell responses. We hypothesized that enhanced delivery of peptide antigens to, and uptake in, secondary lymphoid tissues should elicit more robust CD8+ and CD4+ T cell responses and improved anti-tumor responses. Here, we have designed SLP-containing cationic lipoplexes (SLP-Lpx) that improve delivery of peptides to myeloid cells in the spleen and lymphatics. Using the G12D KRAS mutations as neoantigens, we found that vaccination of mice with naked synthetic peptides harboring the G12D mutation with CpG adjuvant stimulated mainly CD4+ T cell responses with limited tumor growth inhibition. On the other hand, immunization with SLP-Lpx stimulated both CD4+ and CD8+ T cells and suppressed tumor growth in a CD8+ T cell-dependent manner. Combination of the SLP-Lpx vaccines with a checkpoint inhibitor led to profound growth suppression of established tumors. These studies suggest that preferential targeting of peptides derived from neoantigens to the spleen via lipoplexes elicits potent CD4+ and CD8+ T cell responses that inhibit tumor growth.

Drug Repurposing: The Anthelmintics Niclosamide and Nitazoxanide Are Potent TMEM16A Antagonists That Fully Bronchodilate Airways.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor ß-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated Cl- channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ∼580,000 compounds. The anthelmintics niclosamide, nitazoxanide, and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting ß-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the ß-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

Discovery and in Vivo Evaluation of the Potent and Selective PI3Kδ Inhibitors 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-6-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-0687) and 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-5-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-1430).

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors.

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

IL-17RA Signaling in Airway Inflammation and Bronchial Hyperreactivity in Allergic Asthma.

Asthma is a heterogeneous disease characterized by airway inflammation and hyperreactivity. IL-17 receptor A (IL-17RA) is a shared receptor subunit required for activity of IL-17 family cytokines, including IL-17A and IL-25. IL-17A and IL-25 induce different proinflammatory responses, and concentrations are elevated in subjects with asthma. However, the individual contributions of IL-17A and IL-25 to disease pathogenesis are unclear. We explored proinflammatory activities of the IL-17 pathway in models of pulmonary inflammation and assessed its effects on contractility of human bronchial airway smooth muscle. In two mouse models, IL-17RA, IL-17RB, or IL-25 blockade reduced airway inflammation and airway hyperreactivity. Individually, IL-17A and IL-25 enhanced contractility of human bronchial smooth muscle induced by methacholine or carbachol. IL-17A had more pronounced effects on methacholine-induced contractility in bronchial rings from donors with asthma compared with donors without asthma. Blocking the IL-17 pathway via IL-17RA may be a useful therapy for some patients with asthma by reducing pulmonary inflammation and airway hyperreactivity.

Synthesis and SAR study of potent and selective PI3Kδ inhibitors.

2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.

Discovery and in vivo evaluation of (S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (AMG319) and related PI3Kδ inhibitors for inflammation and autoimmune disease.

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Discovery and in vivo evaluation of dual PI3Kß/δ inhibitors.

Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kß/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kß and δ isoforms in the treatment of a number of inflammatory diseases.

Defining dose-response relationships in the therapeutic blockade of B7RP-1-dependent immune responses.

The ICOS (Inducible T cell Co-Stimulator)/B7RP-1 (B7-related protein 1) interaction is critical for the proper activation of a T lymphocyte. In this manuscript we describe a systematic in vivo approach to determine the level of blockade required to impair the generation of a T cell-dependent antibody response. We have developed an overall strategy for correlating drug exposure, target saturation, and efficacy in a biological response that can be generalized for most protein therapeutics. Using this strategy, we determined that low levels of B7RP-1 blockade are still sufficient to inhibit the immune response. These data suggest that contact between the T cell and the antigen-presenting cell during antigen presentation is much more sensitive to inhibition than previously believed and that ICOS/B7RP-1 blockade may be efficacious in the treatment of autoimmune diseases.

Structure-guided design of aminopyrimidine amides as potent, selective inhibitors of lymphocyte specific kinase: synthesis, structure-activity relationships, and inhibition of in vivo T cell activation.

The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).

Discovery of aminoquinazolines as potent, orally bioavailable inhibitors of Lck: synthesis, SAR, and in vivo anti-inflammatory activity.

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.

Novel 2-aminopyrimidine carbamates as potent and orally active inhibitors of Lck: synthesis, SAR, and in vivo antiinflammatory activity.

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.

Mouse embryonic explant culture system for analysis of hematopoietic and vascular development.

In vertebrates, the earliest differentiated cell types (hematopoietic and endothelial) arise from mesoderm induced during the process of gastrulation. These cells become organized into the blood islands of the extraembryonic yolk sac and are morphologically apparent by around d 7.5 in the mouse. Additional waves of hematopoietic and vasculogenic/angiogenic activity later result in the development of definitive hematopoietic lineages and in the formation of the allantois and cardiovascular system of the embryo proper. In part because of the limited accessibility of the mammalian embryo to experimental manipulation in vivo, regulation of these events is still not well understood. Both in the yolk sac and within the embryo proper, prospective hematovascular mesoderm differentiates in the vicinity of endodermal tissues. Here we describe an embryonic explant culture system that permits the dissection of mesodermal and endodermal contributions to hematopoietic and endothelial cell formation during gastrulation. This system can be used to assay for soluble or endodermal cell-associated molecules involved in mediating critical interactions between mesoderm and endoderm in the formation of the hematopoietic and endothelial lineages during embryonic development.

Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon.

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.

Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos.

In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm. However, in mouse, only a single Mix gene (mMix) has been identified to date and its function is unknown. We have analyzed the expression of mouse Mix RNA and protein in embryos, embryoid bodies formed from embryonic stem cells and F9 teratocarcinoma cells, as well as several differentiated cell types. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. In F9 cells induced by retinoic acid to differentiate to VE, mMix is coordinately expressed with three other endodermal transcription factors, well before AFP, and its protein product is localized to the nucleus. In a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. Intriguingly, mMix mRNA is detected in a population (T+Flk1+) of cells which may contain hemangioblasts, before the onset of hematopoiesis and activation of hematopoietic markers. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. Injection of mouse mMix RNA into early frog embryos results in axial truncation of developing tadpoles and, in animal cap assays, mMix alone is sufficient to activate expression of several endodermal (but not mesodermal) markers. Although these observations do not exclude a possible cell-autonomous function for mMix in mesendodermal progenitor cells, they do suggest an additional, non-cell autonomous role in nascent mesoderm in the formation and/or patterning of adjacent definitive endoderm.