Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, ...an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.
Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-α catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the ...helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-α mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-α H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.
We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models.
The activity of ganetespib was ...compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model.
Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA.
Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.
Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The ...assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.
Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. ...Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.
The
EGFR T790M mutation has been identified in tumors from lung cancer patients that eventually develop resistance to erlotinib. In this study, we generated a mouse model with doxycycline-inducible ...expression of a mutant
EGFR containing both L858R, an erlotinib-sensitizing mutation, and the T790M resistance mutation (
EGFR TL). Expression of
EGFR TL led to development of peripheral adenocarcinomas with bronchioloalveolar features in alveoli as well as papillary adenocarcinomas in bronchioles. Treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI), HKI-272, shrunk only peripheral tumors but not bronchial tumors. However, the combination of HKI-272 and rapamycin resulted in significant regression of both types of lung tumors. This combination therapy may potentially benefit lung cancer patients with the
EGFR T790M mutation.
The epidermal growth factor receptor (EGFR) secondary kinase domain T790M non-small cell lung cancer (NSCLC) mutation enhances receptor catalytic activity and confers resistance to the reversible ...tyrosine kinase inhibitors gefitinib and erlotinib. Currently, irreversible inhibitors represent the primary approach in clinical use to circumvent resistance. We show that higher concentrations of the irreversible EGFR inhibitor CL-387,785 are required to inhibit EGFR phosphorylation in T790M-expressing cells compared with EGFR mutant NSCLC cells without T790M. Additionally, CL-387,785 does not fully suppress phosphorylation of other activated receptor tyrosine kinases (RTK) in T790M-expressing cells. These deficiencies result in residual Akt and mammalian target of rapamycin (mTOR) activities. Full suppression of EGFR-mediated signaling in T790M-expressing cells requires the combination of CL-387,785 and rapamycin. In contrast, Hsp90 inhibition overcomes these limitations in vitro and depletes cells of EGFR, other RTKs, and phospho-Akt and inhibits mTOR signaling whether or not T790M is present. EGFR-T790M-expressing cells rendered resistant to CL-387,785 by a kinase switch mechanism retain sensitivity to Hsp90 inhibition. Finally, Hsp90 inhibition causes regression in murine lung adenocarcinomas driven by mutant EGFR (L858R) with or without T790M. However, efficacy in the L858R-T790M model requires a more intense treatment schedule and responses were transient. Nonetheless, these findings suggest that Hsp90 inhibitors may be effective in T790M-expressing cells and offer an alternative therapeutic strategy for this subset of lung cancers.
The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes ...systemic antigen-specific CD8
T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher
potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.
Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) ...pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene ...expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.
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