Discovery and Characterization of a Novel Aryl Hydrocarbon Receptor Inhibitor, IK-175, and Its Inhibitory Activity on Tumor Immune Suppression.

Aryl hydrocarbon receptor (AHR) is a transcription factor that regulates the activity of multiple innate and adaptive immune cells subsequent to binding to numerous endogenous and exogenous ligands. For example, AHR is activated by the metabolite kynurenine, which is secreted into the tumor microenvironment by cancer cells leading to broad immunosuppression. Therefore, AHR inhibition provides a novel and ideal approach to stimulate immune-mediated recognition and subsequent eradication of tumor cells. We report here the discovery and characterization of IK-175, a novel, potent and selective AHR antagonist with favorable ADME and pharmacokinetic profiles in preclinical species. IK-175 inhibits AHR activity in experimental systems derived from multiple species including mouse, rat, monkey, and humans. In human primary immune cells, IK-175 decreased AHR target gene expression and anti-inflammatory cytokine release and increased proinflammatory cytokine release. Moreover, IK-175 led to a decrease in suppressive IL17A-, IL-22+ expressing T cells in a Th17 differentiation assay. IK-175 dose dependently blocks ligand-stimulated AHR activation of Cyp1a1 transcription in mouse liver and spleen, demonstrating on-target in vivo activity. IK-175 increases proinflammatory phenotype of the tumor microenvironment in mouse syngeneic tumors and in adjacent tumor-draining lymph nodes. As a monotherapy and combined with an anti-PD-1 antibody, IK-175 demonstrates antitumor activity in syngeneic mouse models of colorectal cancer and melanoma. IK-175 also demonstrates antitumor activity combined with liposomal doxorubicin in syngeneic mouse tumors. These studies provide rationale for targeting AHR in patients with cancer. IK-175 is being evaluated in a phase I clinical trial in patients with advanced solid tumors.

Blockade of the AHR restricts a Treg-macrophage suppressive axis induced by L-Kynurenine.

Tryptophan catabolism by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2 (IDO/TDO) promotes immunosuppression across different cancer types. The tryptophan metabolite L-Kynurenine (Kyn) interacts with the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) to drive the generation of Tregs and tolerogenic myeloid cells and PD-1 up-regulation in CD8+ T cells. Here, we show that the AHR pathway is selectively active in IDO/TDO-overexpressing tumors and is associated with resistance to immune checkpoint inhibitors. We demonstrate that IDO-Kyn-AHR-mediated immunosuppression depends on an interplay between Tregs and tumor-associated macrophages, which can be reversed by AHR inhibition. Selective AHR blockade delays progression in IDO/TDO-overexpressing tumors, and its efficacy is improved in combination with PD-1 blockade. Our findings suggest that blocking the AHR pathway in IDO/TDO expressing tumors would overcome the limitation of single IDO or TDO targeting agents and constitutes a personalized approach to immunotherapy, particularly in combination with immune checkpoint inhibitors.

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.

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.

The impact of the MYB-NFIB fusion proto-oncogene in vivo.

Recurrent fusion of the v-myb avian myelobastosis viral oncogene homolog (MYB) and nuclear factor I/B (NFIB) generates the MYB-NFIB transcription factor, which has been detected in a high percentage of individuals with adenoid cystic carcinoma (ACC). To understand the functional role of this fusion protein in carcinogenesis, we generated a conditional mutant transgenic mouse that expresses MYB-NFIB along with p53 mutation in tissues that give rise to ACC: mammary tissue, salivary glands, or systemically in the whole body. Expression of the oncogene in mammary tissue resulted in hyperplastic glands that developed into adenocarcinoma in 27.3% of animals. Systemic expression of the MYB-NFIB fusion caused more rapid development of this breast phenotype, but mice died due to abnormal proliferation in the glomerular compartment of the kidney, which led to development of glomerulonephritis. These findings suggest the MYB-NFIB fusion is oncogenic and treatments targeting this transcription factor may lead to therapeutic responses in ACC patients.

Targeting the oncogenic MUC1-C protein inhibits mutant EGFR-mediated signaling and survival in non-small cell lung cancer cells.

PURPOSE: Non-small cell lung cancers (NSCLC) that express EGF receptor with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known. EXPERIMENTAL DESIGN: Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival, and tumorigenicity. RESULTS: Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation, and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC → AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK → ERK activation, colony formation, and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT, and MEK → ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants. CONCLUSIONS: These findings indicate that targeting MUC1-C inhibits mutant EGFR signaling and survival, and thus represents a potential approach alone and in combination for the treatment of NSCLCs resistant to EGFR kinase inhibitors.

Kinase domain activation of FGFR2 yields high-grade lung adenocarcinoma sensitive to a Pan-FGFR inhibitor in a mouse model of NSCLC.

Somatic mutations in FGFR2 are present in 4% to 5% of patients diagnosed with non-small cell lung cancer (NSCLC). Amplification and mutations in FGFR genes have been identified in patients with NSCLCs, and clinical trials are testing the efficacy of anti-FGFR therapies. FGFR2 and other FGFR kinase family gene alterations have been found in both lung squamous cell carcinoma and lung adenocarcinoma, although mouse models of FGFR-driven lung cancers have not been reported. Here, we generated a genetically engineered mouse model (GEMM) of NSCLC driven by a kinase domain mutation in FGFR2. Combined with p53 ablation, primary grade 3/4 adenocarcinoma was induced in the lung epithelial compartment exhibiting locally invasive and pleiotropic tendencies largely made up of multinucleated cells. Tumors were acutely sensitive to pan-FGFR inhibition. This is the first FGFR2-driven lung cancer GEMM, which can be applied across different cancer indications in a preclinical setting.

Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression.

Lung squamous cell carcinoma (SCC) is a deadly disease for which current treatments are inadequate. We demonstrate that biallelic inactivation of Lkb1 and Pten in the mouse lung leads to SCC that recapitulates the histology, gene expression, and microenvironment found in human disease. Lkb1;Pten null (LP) tumors expressed the squamous markers KRT5, p63 and SOX2, and transcriptionally resembled the basal subtype of human SCC. In contrast to mouse adenocarcinomas, the LP tumors contained immune populations enriched for tumor-associated neutrophils. SCA1(+)NGFR(+) fractions were enriched for tumor-propagating cells (TPCs) that could serially transplant the disease in orthotopic assays. TPCs in the LP model and NGFR(+) cells in human SCCs highly expressed Pd-ligand-1 (PD-L1), suggesting a mechanism of immune escape for TPCs.

D-2-hydroxyglutarate produced by mutant IDH2 causes cardiomyopathy and neurodegeneration in mice.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2(R140Q) and IDH2(R172K) alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span, recapitulating the abnormalities observed in D2HGA patients. The diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant up-regulation of genes involved in glycogen biosynthesis. Notably, mild cardiac hypertrophy was also observed in nude mice implanted with IDH2(R140Q)-expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion. Finally, we show that silencing of IDH2(R140Q) in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors.

UNLABELLED: The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. SIGNIFICANCE: We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.

Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer.

PURPOSE: Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non-small cell lung cancer (NSCLC) with BET inhibition. EXPERIMENTAL DESIGN: We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cell lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. RESULTS: Although JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knockdown of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. CONCLUSION: Bromodomain inhibition comprises a promising therapeutic strategy for KRAS-mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. Clin Cancer Res; 19(22); 6183-92. ©2013 AACR.

Inhibitor-sensitive FGFR2 and FGFR3 mutations in lung squamous cell carcinoma.

A comprehensive description of genomic alterations in lung squamous cell carcinoma (lung SCC) has recently been reported, enabling the identification of genomic events that contribute to the oncogenesis of this disease. In lung SCC, one of the most frequently altered receptor tyrosine kinase families is the fibroblast growth factor receptor (FGFR) family, with amplification or mutation observed in all four family members. Here, we describe the oncogenic nature of mutations observed in FGFR2 and FGFR3, each of which are observed in 3% of samples, for a mutation rate of 6% across both genes. Using cell culture and xenograft models, we show that several of these mutations drive cellular transformation. Transformation can be reversed by small-molecule FGFR inhibitors currently being developed for clinical use. We also show that mutations in the extracellular domains of FGFR2 lead to constitutive FGFR dimerization. In addition, we report a patient with an FGFR2-mutated oral SCC who responded to the multitargeted tyrosine kinase inhibitor pazopanib. These findings provide new insights into driving oncogenic events in a subset of lung squamous cancers, and recommend future clinical studies with FGFR inhibitors in patients with lung and head and neck SCC.

Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer.

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1-wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.

αvß8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion.

The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvß8 is a major driver of GBM cell invasion. ß8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing ß8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. ß8 integrin coimmunoprecipitates with Rho-GDP dissociation inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvß8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvß8 integrin, via interactions with RhoGDI1, regulates activation of Rho proteins to promote GBM cell invasiveness. Hence targeting the αvß8 integrin-RhoGDI1 signaling axis might be an effective strategy for blocking GBM cell invasion.

Glioblastoma angiogenesis and tumor cell invasiveness are differentially regulated by ß8 integrin.

Glioblastoma multiforme (GBM) is a highly invasive brain tumor that develops florid microvascular proliferation and hemorrhage. However, mechanisms that favor invasion versus angiogenesis in this setting remain largely uncharacterized. Here, we show that integrin ß8 is an essential regulator of both GBM-induced angiogenesis and tumor cell invasiveness. Highly angiogenic and poorly invasive tumors expressed low levels of ß8 integrin, whereas highly invasive tumors with limited neovascularization expressed high levels of ß8 integrin. Manipulating ß8 integrin protein levels altered the angiogenic and invasive growth properties of GBMs, in part, reflected by a diminished activation of latent TGFßs, which are extracellular matrix protein ligands for ß8 integrin. Taken together, these results establish a role for ß8 integrin in differential control of angiogenesis versus tumor cell invasion in GBM. Our findings suggest that inhibiting ß8 integrin or TGFß signaling may diminish tumor cell invasiveness during malignant progression and following antivascular therapies.

Beta8 integrin regulates neurogenesis and neurovascular homeostasis in the adult brain.

Central nervous system (CNS) neurovascular units are multicellular complexes consisting of neural cells, blood vessels and a milieu of extracellular matrix (ECM) proteins. ECM-mediated adhesion and signaling events within neurovascular units probably contribute to proper CNS development and physiology; however, the molecular mechanisms that control these events remain largely undetermined. Previous studies from our group and others showed that ablation of the ECM receptor, alphavbeta8 integrin, in neural progenitor cells (NPCs) of the embryonic mouse brain results in severe developmental neurovascular pathologies and premature death. Here, we have investigated the functions for this integrin in the adult brain by studying mice harboring a homozygous-null beta8 gene mutation generated on an outbred background that permits survival for several months. We show that adult beta8-/- mice display widespread defects in neurovascular unit homeostasis, including increased numbers of intracerebral blood vessels with pronounced perivascular astrogliosis. Furthermore, in neurogenic regions of the adult brain, where NPCs cluster around blood vessels in neurovascular niches, beta8 integrin is essential for normal control of NPC proliferation and survival. Analysis of NPCs cultured ex vivo reveals that the growth and survival defects correlate, in part, with diminished integrin-mediated activation of latent transforming growth factor beta1 (TGFbeta1), which is an ECM protein ligand for alphavbeta8 integrin. Collectively, these data identify essential functions for beta8 integrin in regulating neurovascular unit physiology in the post-natal mouse brain.