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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. Multiple treatment-emergent alterations appear in patients with advanced-stage cancer who were treated with a KRAS inhibitor.

Sotorasib is an approved KRASG12C-selective inhibitor for the treatment of KRAS p.G12C-mutant advanced and previously treated non-small cell lung cancers (NSCLC). Acquired resistance due to genomic alterations following sotorasib treatment has been observed in 28% of lung cancer patients (CodeBreaK100, ASCO 2022) and can include bypass signaling, inactivation of negative feedback loops, or compensatory mutations in KRASG12C including amplification of KRAS. Preclinical studies on the mechanisms of acquired resistance are critical for understanding these resistance patterns and can reveal new therapeutic or combination strategies in the clinic. For that reason, we developed an NCI-H358 lung cancer model of acquired resistance to KRASG12C inhibition in vitro. NCI-H358 cells were continuously cultured in the presence of elevated concentrations of sotorasib (IC90) until resistance to KRASG12C inhibitor was achieved. Expression analyses of the resistant cells by ddPCR and immunoblotting showed increases in both KRAS gene and protein levels, resulting in enhanced MAPK signaling. siRNA knockdown of KRAS G12C expression re-sensitized the resistant cells to an analog of sotorasib. Further characterization of this cell line by whole exome sequencing (WES) and fluorescence in situ hybridization (FISH) showed that resistance was not due to genetic co-alterations but amplification of the mutant allele specifically. In comparison to DMSO-treated and parental controls, the sotorasib-resistant NCI-H358 cells contained an approximately 50-fold increase in KRAS G12C mutant allele copy numbers. Preclinical data generated using a KRASG12C inhibitor-resistant lung cancer cell line is consistent with clinical observations of acquired KRAS amplification following KRASG12C inhibitor treatment as a mechanism of resistance. This model further provides an opportunity to study acquired KRAS amplification and investigate combination treatment options in vitro. Citation Format: Deanna Mohn, Anne Y. Saiki, Pragathi Achanta, Andres Plata Stapper, J. Russell Lipford, Rati Verma. KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3871.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor β-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 β-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 β-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.

Sotorasib (formerly known as AMG 510), the first-in-class KRASG12C inhibitor, has demonstrated promising clinical efficacy in KRAS p.G12C mutant cancers. Sotorasib binds to KRASG12C through a unique interaction with a surface groove created by side-chain rotation of histidine 95 (His95). Characterization of sotorasib and other His95-groove binders revealed enhanced potency and selectivity as compared to other KRASG12C inhibitor scaffolds, which bind in the P2 pocket via hydrogen bonding with His95. The novel binding mode of sotorasib also translated to similar biochemical and cellular potencies against both NRASG12C and HRASG12C, which encode leucine and glutamine at position 95, respectively. In contrast, other KRASG12C inhibitor scaffolds demonstrated a dramatic loss of potency against NRASG12C and HRASG12C, suggesting that the alternate residues impacted the binding of these molecules in the P2 pocket. To extend characterization of the cellular effects of RAS ‘His95-groove' binders, we analyzed the expression of major histocompatibility complex (MHC) class I proteins and other inflammatory markers in multiple human and murine KRAS p.G12C cell lines. These studies revealed a partial dependency on the cytosolic DNA-sensing (cGAS/STING) pathway for the effects observed with some markers. Finally, His95-groove binders were evaluated for potential mechanisms of resistance to this class of KRASG12C inhibitors. In the mouse syngeneic Lewis Lung Carcinoma (LL/2) cell line, which carries both KRAS p.G12C and NRAS p.Q61H mutations, intrinsic resistance to KRASG12C inhibition was observed, but combination treatment with the MEK inhibitor trametinib demonstrated synergistic improvement in the effects on viability. MIA PaCa-2 and NCI-H358 models of acquired resistance to KRASG12C inhibition were also developed through long-term exposure to high concentrations of sotorasib. Characterization of these resistant cell lines indicated a requirement for constant exposure to sotorasib and also showed that the resistance was not due to genetic alterations but involved either overexpression of KRAS or bypass signaling through alternative pathways. Taken together, these data demonstrate that His95-binders like sotorasib display superior potency and off-target selectivity, as well as unique activity against all versions of RASG12C. In addition, characterization of potential resistance mechanisms to sotorasib will inform combination strategies in the clinic. Citation Format: Anne Y. Saiki, Deanna Mohn, Yu Li, Tao Osgood, Karen Rex, Hui-Ling Wang, Ivonne Archibeque, Christopher Mohr, Pragathi Achanta, Andres Plata Stapper, Aaron S. Rapaport, Jude Canon, Victor J. Cee, Brian A. Lanman, J. Russell Lipford. In vitro characterization of sotorasib and other RAS ‘His95-groove' binders and investigation of resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1285.

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.

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.

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.

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.

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.

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 PI3Kβ/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 PI3Kβ and δ isoforms in the treatment of a number of inflammatory diseases.

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.

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).

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 inbibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

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.

Angel is the first miniature inverted-repeat transposable element (MITE) isolated from fish. Angel elements are imperfect palindromes with the potential to form stem-loop structures in vitro. Despite sequence divergence of elements of up to 55% within and between species, their inverted repeat structures have been maintained, implying functional importance. We estimate that there are about 10(3)-10(4) Angels scattered throughout the zebrafish genome, evidence that this family of transposable elements has been significantly amplified over the course of evolution. Angel elements and Xenopus MITEs carry common sequence motifs at their termini, indicating common origin and/or related mechanisms of transposition. We present a model in which MITEs take advantage of the basic cellular mechanism of DNA replication for their amplification, which is dependent on the characteristic inverted repeat structures of these elements. We propose that MITEs are genomic parasites that transpose via a DNA intermediate, which forms by a folding-back of a single strand of DNA, that borrow all of the necessary factors for their amplification from products encoded in the genomes in which they reside. DNA polymorphisms in different lines of zebrafish were detected by PCR using Angel-specific primers, indicating that such elements, combined with other transposons in vertebrate genomes, will be useful molecular tools for genome mapping and genetic analyses of mutations.

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.

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.

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.

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. Developmental Dynamics 226:000–000, 2003. © 2003 Wiley-Liss, Inc.

During gastrulation in the mouse, mesoderm is induced and patterned by secreted signaling molecules, giving rise first to primitive erythroblasts and vascular endothelial cells. We have demonstrated previously that development of these lineages requires a signal(s) secreted from the adjacent primitive endoderm. We now show that Indian hedgehog (Ihh) is a primitive endoderm-secreted signal that alone is sufficient to induce formation of hematopoietic and endothelial cells. Strikingly, as seen with primitive endoderm, Ihh can respecify prospective neural ectoderm (anterior epiblast) along hematopoietic and endothelial (posterior) lineages. Downstream targets of the hedgehog signaling pathway (the genes encoding patched, smoothened and Gli1) are upregulated in anterior epiblasts cultured in the presence of Ihh protein, as is Bmp4, which may mediate the effects of Ihh. Blocking Ihh function in primitive endoderm inhibits activation of hematopoiesis and vasculogenesis in the adjacent epiblast, suggesting that Ihh is an endogenous signal that plays a key role in the development of the earliest hemato-vascular system. To our knowledge, these are the earliest functions for a hedgehog protein in post-implantation development in the mouse embryo.