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PDF - 62K, Legends for Supplementary Figures 1 and 2.

PDF - 71K, A list of the solid tumor xenografts tested for regulation of human or murine Gli1 following IPI-926 treatment, as measured by q-RT-PCR on whole tumor RNA extracts.

PDF - 516K, Hh signaling in normal mouse chondrocytes is inhibited by IPI-926 treatment and leads to growth plate closure. A. Active control growth plate with a normal appearance. B. Corresponding growth plate from an IPI-926 treated mouse shows premature closure (indicated by black arrow).

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

PDF - 427K, Representative image of a hematoxylin and esosin stained primary chondrosarcoma xenograft. The image shows a typical appearance of low cellularity (blue nuclei) with considerable extracellular matrix between the cells and no obvious tumor stroma.

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

Hedgehog (Hh) pathway inhibition in cancer has been evaluated in both the ligand-independent and ligand-dependent settings, where Hh signaling occurs either directly within the cancer cells or within the nonmalignant cells of the tumor microenvironment. Chondrosarcoma is a malignant tumor of cartilage in which there is ligand-dependent activation of Hh signaling. IPI-926 is a potent, orally delivered small molecule that inhibits Hh pathway signaling by binding to Smoothened (SMO). Here, the impact of Hh pathway inhibition on primary chondrosarcoma xenografts was assessed. Mice bearing primary human chondrosarcoma xenografts were treated with IPI-926. The expression levels of known Hh pathway genes, in both the tumor and stroma, and endpoint tumor volumes were measured. Gene expression profiling of tumors from IPI-926–treated mice was conducted to identify potential novel Hh target genes. Hh target genes were studied to determine their contribution to the chondrosarcoma neoplastic phenotype. IPI-926 administration results in downmodulation of the Hh pathway in primary chondrosarcoma xenografts, as demonstrated by evaluation of the Hh target genes GLI1 and PTCH1, as well as inhibition of tumor growth. Chondrosarcomas exhibited autocrine and paracrine Hh signaling, and both were affected by IPI-926. Decreased tumor growth is accompanied by histopathologic changes, including calcification and loss of tumor cells. Gene profiling studies identified genes differentially expressed in chondrosarcomas following IPI-926 treatment, one of which, ADAMTSL1, regulates chondrosarcoma cell proliferation. These studies provide further insight into the role of the Hh pathway in chondrosarcoma and provide a scientific rationale for targeting the Hh pathway in chondrosarcoma. Mol Cancer Ther; 13(5); 1259–69. ©2014 AACR.

Contains fulltext : 291567.pdf (Publisher’s version ) (Open Access) The aryl hydrocarbon receptor (AHR) is a sensor of low-molecular-weight molecule signals that originate from environmental exposures, the microbiome, and host metabolism. Building upon initial studies examining anthropogenic chemical exposures, the list of AHR ligands of microbial, diet, and host metabolism origin continues to grow and has provided important clues as to the function of this enigmatic receptor. The AHR has now been shown to be directly involved in numerous biochemical pathways that influence host homeostasis, chronic disease development, and responses to toxic insults. As this field of study has continued to grow, it has become apparent that the AHR is an important novel target for cancer, metabolic diseases, skin conditions, and autoimmune disease. This meeting attempted to cover the scope of basic and applied research being performed to address possible applications of our basic knowledge of this receptor on therapeutic outcomes.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >10(9) variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

The Hippo signaling cascade regulates cell proliferation and survival, as well as overall tissue homeostasis. These functions are mediated by the TEAD family of transcription factors which, when bound to the co-activators YAP or TAZ, induce expression of pro-growth and anti-apoptotic genes. This pathway is frequently dysregulated across tumor types, with genetic alterations along with other mechanisms driving hyper-active YAP/TAZ-TEAD. In addition, Hippo signaling mediates resistance to therapies targeting key oncogenic pathways such as EGFR and RAS. As a result, TEAD transcription factors are promising therapeutic targets. The TEAD family comprises four paralogs (TEAD1-4) that have both over-lapping and non-redundant functions. Given the essential roles of the Hippo pathway in normal physiology, it may be beneficial to target a subset of TEADs to minimize potential on-target toxicity while maintaining anti-tumor efficacy. Although highly homologous, the lipid-binding pocket of TEADs harbor some sequence divergence, highlighting an opportunity to design paralog-specific compounds. We developed IK-930 as a novel, selective inhibitor that potently blocks TEAD transcriptional activity by disrupting the auto-palmitoylation required for its interaction with YAP/TAZ. To demonstrate the selectivity profile of IK-930, a suite of biochemical assays was employed, all of which showed preferential compound binding to an individual paralog. A novel NanoBRETTM system that enabled quantitative, high-throughput measurements of cellular TEAD engagement also documented selective inhibitor interaction with the same family member. To assess how differences in selectivity affect therapeutic index, the in vivo efficacy and toxicity of IK-930 was compared to a pan-TEAD inhibitor. In several Hippo-mutated xenograft models, both compounds displayed potent anti-tumor activity. Moreover, IK-930 exhibited synergy with targeted agents, including EGFR inhibitors, indicating that its selectivity profile could drive robust efficacy in diverse tumor types. Previous studies in genetically engineered mouse models reported that perturbation of YAP/TAZ results in kidney toxicity. Treatment with a pan-TEAD inhibitor in rats and non-human primates led to substantial proteinuria and kidney pathology at exposures similar to those needed for antitumor activity in mouse models. In contrast, IK-930 demonstrated limited kidney toxicity in rats above efficacious doses and no signs of renal problems in non-human primates. Collectively, these results suggest that paralog selectivity with IK-930 broadens the therapeutic window of this novel compound class. With its distinct TEAD inhibitory profile, IK-930 offers unique therapeutic index advantages, further supporting its development as a first-in-class paralog-selective TEAD inhibitor. Citation Format: Nathan Young, George Punkosdy, Jill Cavanaugh, Collin Bantle, Alex Constan, Bin Li, James Conley, Marta Sanchez-Martin, Lan Xu, Karen McGovern, Alfredo Castro, Michelle Zhang, Jeffrey Ecsedy. IK-930, a paralog-selective TEAD inhibitor for treating YAP/TAZ-TEAD dependent cancers [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 1646.

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.

BackgroundAryl Hydrocarbon Receptor (AHR) is a ligand-activated transcription factor that regulates the activities of multiple innate and adaptive immune cell types. Multiple ligands such as kynurenine bind to AHR driving its nuclear translocation and transcriptional activation, leading to an immunosuppressive tumor microenvironment.1 2 AHR activation is implicated in tumor development in multiple cancer types. In addition, high levels of serum kynurenine are associated with resistance to checkpoint inhibitors.3 To overcome AHR-mediated immunosuppression in cancers, we developed a selective oral AHR inhibitor IK-175 and took a combined computational and tissue-based approach to select cancer indications for its clinical development.MethodsThe aim of this work is to identify tumor indications dependent on AHR signaling and design patient selection strategies based on a proprietary transcriptional signature, mRNA and protein detection assays to evaluate AHR pathway activation in tumors.ResultsGenomic profiling of solid and hematological cancers from TCGA and Project GENIE databases identified bladder and esophageal tumors among others, as frequently harboring AHR gene amplifications.A proprietary gene signature of AHR activation was developed integrating literature, pathway analysis, RNAseq and nanostring data from PBMC, T-cells and cell lines upon AHR inhibition. Transcriptional analysis of the TCGA data using this signature demonstrated bladder cancer has the highest expressions of AHR and AHR signature genes, suggesting increased pathway activity in bladder cancer relative to other cancer types. Increased AHR signature gene expression was associated with worse overall survival in the TCGA bladder cancer cohort. Furthermore, RNAscope analysis of a tissue microarray containing 10 different tumor types revealed bladder cancer had one of the highest AHR transcript expression in the tumor compartment.Finally, nuclear localization of AHR protein was assessed as an indicator of pathway activation through the development of a novel IHC method. Extensive TMA screening of AHR protein in 15 different indications demonstrated bladder cancer as the tumor type with the highest prevalence of AHR nuclear expression.ConclusionsIn summary, we demonstrated high prevalence of nuclear AHR protein expression, AHR gene amplification and target gene expression in bladder cancer, suggesting aberrant AHR activation may play an important role in the progression of this tumor type. This study provides rationale for therapeutic targeting of AHR in bladder cancer patients. Ikena is currently evaluating the anti-tumor activity of IK-175 as a single agent and in combination with nivolumab in bladder cancer in a Phase 1a/1b clinical study (NCT04200963).ReferencesQuintana FJ, Sherr DH. Aryl hydrocarbon receptor control of adaptive immunity. Pharmacol Rev 2013 Aug 1;65(4):1148–61.Murray IA, Patterson AD, Perdew GH. Aryl hydrocarbon receptor ligands in cancer: friend and foe. Nat Rev Cancer 2014 Dec;14(12):801–14.Li, Haoxin et al. ‘Metabolomic adaptations and correlates of survival to immune checkpoint blockade.’ Nature Communications 2019 Sep 25;10:1–4346.

The Hippo pathway is critical to cancer progression, biogenesis, metastasis, and therapeutic resistance. Many cancer indications have a high frequency of mutations in the Hippo pathway. These pro-tumor mutations lead to constitutive TEAD transcription factor activation, which drives gene expression involved in cell growth and pro-survival signaling. To target cancers harboring mutations in the Hippo pathway, we have discovered and are developing IK-930, a novel small molecule that selectively inhibits TEAD-dependent transcription by directly blocking autopalmitoylation. IK-930 prevents this critical post-translational modification that is required for the functional interaction of TEAD with two transcriptional activators, YAP1 and TAZ (WWTR1). IK-930 inhibits in vitro proliferation of Hippo pathway-deficient cancer cell lines, but not Hippo pathway wild type cells. In human mesothelioma xenografts, IK-930 downregulates TEAD-dependent genes. Daily oral administration of IK-930 resulted in antitumor activity in Hippo-dysregulated mesothelioma xenograft models. In EGFR or KRAS mutated tumors, IK-930 enhanced apoptosis and in vivo antitumor activity in combination with EGFR and MEK inhibitors, respectively. IK-930 is inactive in a broad panel of kinases, receptors, and transporters, furthering evidence of selectivity. Multispecies pharmacokinetic analysis and additional in vitro ADME properties imply favorable pharmacokinetic properties, with a low potential for clinically significant drug-drug interactions. To identify indications that may be dependent on TEAD we evaluated tumor types for the incidence of gene alterations in the Hippo pathway, as well as for YAP1 and TAZ activation. YAP1 and TAZ activation was assessed by evaluating nuclear protein expression in tumor tissue microarrays. These analyses showed high YAP1 nuclear expression in tumors with frequent genetic alterations, that may help guide the development of IK-930. Mesothelioma ranks top among tumor types evaluated, due to high prevalence of YAP1 nuclear expression and Hippo pathway genetic alterations, which are predominantly mutations and copy number alterations in the tumor suppressor NF2. In summary, Hippo pathway dysregulation has been implicated in the etiology of multiple tumor types, including mesothelioma. IK-930 is a selective and potent TEAD inhibitor expected to enter the clinic in early 2022. Our analysis pointed to tumors with genetic alterations driving aberrant Hippo signaling. IK-930 demonstrates efficacy in tumor models representing these indications and beneficial combination activity with other targeted therapies. Taken together, these data have informed the clinical development plan for IK-930. Citation Format: Benjamin S. Amidon, Marta Sanchez-Martin, Wilmin Bartolini, Sakeena Syed, Karen McGovern, Lan Xu, Jeffrey Ecsedy, X. Michelle Zhang, Alex Constan, Alfredo C. Castro. IK-930 is a novel TEAD inhibitor for the treatment of cancers harboring mutations in the Hippo signal transduction pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2156.

Background Aryl Hydrocarbon Receptor (AHR) is a transcription factor that regulates the activity of multiple innate and adaptive immune cells subsequent to binding to a diverse set of endogenous and exogenous ligands. One such endogenous AHR ligand is kynurenine, generated from the precursor tryptophan by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2). Binding of kynurenine to AHR leads to a net immunosuppressive tumor microenvironment. In addition, increased levels of serum kynurenine are associated with resistance to checkpoint inhibitors. Given that kynurenine can be generated by both IDO1 and TDO2 and that AHR is activated by multiple other endogenous ligands, AHR inhibition provides a novel and ideal approach to overcome immunosuppression in a broad range of cancers. Methods We sought to identify an orally active AHR antagonist as an immunomodulatory agent for the treatment of solid tumors. Lead optimization efforts identified IK-175 as an AHR antagonist with a favorable ADME and pharmacokinetic profile in preclinical species. Results IK-175 inhibits AHR activity in rodent and human cancer cell lines as well as human and nonhuman primate primary immune cells, with concentration dependent effects on AHR target gene expression and cytokine release. IK-175 is inactive in a broad panel of kinases, receptors, and transporters. Orally administered IK-175 dose-dependently blocks ligand stimulated-AHR activation of Cyp1a1 transcription in liver and spleen, demonstrating on-target in vivo activity in mice. IK-175 alone and in combination with an anti-PD-1 antibody demonstrates significant antitumor activity in syngeneic mouse models of colorectal cancer (CT26.WT) and melanoma (B16-IDO1). In addition, IK-175 in combination with liposomal doxorubicin demonstrates antitumor activity in syngeneic mouse models of colorectal cancer (CT26.WT and MC38). Conclusions These studies provide rationale for targeting AHR in cancer patients. Ikena will evaluate the anti-tumor activity of IK-175 as a single agent in cancers with activated AHR and in combination with other therapies. Overall, our data demonstrates that IK-175 is a selective orally active AHR antagonist that inhibits tumor growth and reverses immune suppression in mouse tumors models. IK-175 is currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors and urothelial carcinoma (Clinicaltrials.gov NCT04200963).

Significance Standard of care for metastatic castration-resistant prostate cancer (mCRPC) mainly relies on suppression of androgen receptor (AR) signaling. This approach has no lasting benefit due to the emergence of resistance mechanisms, such as ligand-independent splicing variant AR-V7. A metabolic feature of mCRPC is the upregulation of de novo lipogenesis to provide substrates and fuel for metastatic spread. Whether increased levels of fats affect AR signaling to promote an aggressive disease remains to be determined. Using a selective and potent inhibitor of fatty acid synthase we demonstrate that suppression of this key enzyme inhibits AR, most importantly AR-V7, and reduces mCRPC growth. Our findings offer a therapeutic opportunity for mCRPC and a potential mechanism to overcome resistance to AR inhibitors., 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.

The Hippo signaling cascade is an important pathway that plays a role in controlling cell proliferation and limiting apoptosis, but when dysregulated can contribute to cancer initiation, progression, and therapeutic resistance. The TEAD family of transcription factors, in conjunction with YAP1 or WWTR1/TAZ, are regulated by Hippo pathway signaling and modulate cell growth and proliferation. IK-930 inhibits the growth of TEAD-dependent human cancer xenografts. By exploiting synthetic lethality, the IK-930 single agent activity in TEAD-dependent cancers could expand to other indications upon inhibition of certain oncogenic pathways. Literature indicates that activation of TEAD-dependent transcription by other oncogenes such as mutant EGFR or KRAS mediates resistance to multiple targeted therapies. Additionally, genetic loss of YAP1 leads to increased apoptosis in osimertinib and trametinib treated mutant EGFR NSCLC cell lines (Kurppa, 2020). Here, we present preclinical data that support the use of IK-930 combination therapies to enhance anti-tumor impact of EGFR and MEK blockade in mutant EGFR NSCLC, or with MEK inhibition in several BRAF and KRAS mutant cancers. In these studies, EGFR and MEK1/2 were inhibited and IK-930 used to enhance anti-tumor activity in EGFR-mutant NSCLC cells. These NSCLC cell lines that are largely insensitive to osimertinib and/or trametinib, were observed to have a marked increase in apoptosis in vitro when treated with the combination of osimertinib, trametinib, and IK-930. We also observed increased nuclear accumulation of YAP1 after treatment of NSCLC cells with osimertinib alone, or osimertinib and trametinib and upregulation of TEAD-dependent transcription. In NSCLC xenografts, the combination of osimertinib and IK-930 prevented tumor growth and the triple combination of osimertinib, IK-930, and trametinib drove complete tumor regression and demonstrated greater efficacy than any single agent or double combination. A second set of studies tested the synthetic lethality effect of IK-930 and MEK inhibition in RAF- and RAS-mutant tumors. The combination of TEAD inhibition with MEK inhibition enhanced apoptosis in several KRAS-mutant NSCLC, PDAC, CRC KRAS-mutant cell lines and BRAF-mutant melanoma. The combination of trametinib and IK-930 prevented tumor growth in these xenografts, where either single agent had either modest or little activity. In summary, the Hippo pathway has been implicated in multiple tumor types, including those with resistance mechanisms to EGFR and MEK blockade. IK-930-mediated TEAD inhibition has been investigated to suppress the bypass pathway activation mechanism to the targeted therapies. These data demonstrated the potential to further expand single agent patient benefit by combining IK-930 with MEK and/or EGFR inhibition in a wide variety of cancer indications, including mutant EGFR-driven NSCLC and mutant KRAS colon, lung, and pancreatic cancers. Taken together, these data are informing the clinical development plan and combination strategy for IK-930, a novel TEAD inhibitor. Citation Format: Benjamin Amidon, Hyejin Frosch, Sakeena Syed, Jill Cavanaugh, Chelsea Turcotte, Katie O'Callaghan, Prabitha Natarajan, Jeffrey Ecsedy, Karen McGovern, Alfredo Castro. IK-930 mediated TEAD inhibition decreases and delays tumor growth and enhances targeted apoptosis in lung and colon cancer xenografts when combined with MEK or EGFR inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P216.

TEAD transcription factors are the final effectors of the Hippo pathway, a signaling cascade comprising multiple tumor suppressors (NF2, MST1/2, LATS1/2) critical in regulating proliferation, survival, and tissue homeostasis. Activated Hippo signaling suppresses TEAD-dependent transcription through the phosphorylation and degradation of TEAD co-activators YAP1 and TAZ in the cytosol. Consistently, genetic alterations in NF2, LATS1/2, YAP1 or TAZ lead to aberrant TEAD activation and are implicated in tumor initiation, progression, and therapeutic resistance in cancer. Here we present a unique systems biology approach integrating genomic, transcriptional and tissue-based analysis, to inform clinical development, indication and patient selection biomarkers for a novel inhibitor of TEAD being developed by Ikena. To identify cancers dependent on TEAD activity, tumor types were evaluated based on the cumulative incidence of genetic alterations in Hippo pathway genes including NF2, LATS1/2, YAP1, TAZ and others. This novel analysis pointed to a subset of tumors with high frequency of genetic alterations driving aberrant Hippo signaling including mesothelioma and NSCLC. This same subset was found to highly express a transcriptional signature indicative of YAP1/TEAD-dependency, confirming increased activation of TEAD transcription and pointing to the dependency of these tumors on TEAD activity. A proprietary IHC method was used to further assess YAP1/TAZ activation by assessing expression of either protein in the nucleus in multiple tumor tissue microarrays. Tissue-based analysis showed high YAP1 nuclear expression in tumors with frequent genetic alterations and high YAP1/TEAD-signature. Mesothelioma in particular ranks top among these indications, due to high frequency of NF2 deficiency and other Hippo pathway alterations. Consistently, TEAD inhibition showed single agent activity in two xenograft models of mesothelioma with NF2 deficiency and LAST1/2 alterations respectively. Hippo pathway alterations frequently co-occur with mutations in other oncogenic signaling pathways, e.g. EGFR. Importantly, YAP1/TAZ have been implicated in acquired resistance to targeted therapies in cancer including EGFR inhibitors in EGFR mutant tumors. Indeed, PDX models derived from patients who relapsed on osimertinib treatment showed high YAP1 protein expression in the nucleus. In addition, combination of TEAD and EGFR inhibitors induced apoptosis in vitro and greater antitumor activity than either drug as a single agent in vivo in EGFR mutant cancer models. In summary, the integration of multi-disciplinary bioinformatics, pharmacologic and tissue-based approaches enabled the identification of cancer types with high dependency on Hippo signaling. Moreover, these studies support the for monotherapy and combination of TEAD inhibitors with other targeted therapies including EGFR inhibitors. Altogether this unique approach has identified cancer patients who may benefit from TEAD inhibition and has informed the clinical development plan of a novel TEAD inhibitor. Citation Format: Marta Sanchez-Martin, Sakeena Syed, Hyejin Frosch, Chelsea Turcotte, Benjamin Amidon, Karen McGovern, Jeffrey Ecsedy, Michelle X. Zhang. Systems biology-guided indication selection to inform the clinical development of a novel TEAD inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P212.

Background: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor critical for cellular metabolism, stem cell differentiation and immune control. HLA-E is a non-classical HLA molecule that is critical for NK cell activation. HLA-E is expressed on some AML blasts but genomic characterization is limited. IK-364, a tool compound generated by Ikena Oncology, is an AHR inhibitor in preclinical investigation. We set out to identify the genomic signatures associated with activation of the AHR pathway in AML samples utilizing the Beat AML dataset. Methods: Whole bone marrow or peripheral blood samples were procured from AML patients under IRB-approved protocol (eIRB# 4422). Utilizing the Beat AML database, all samples were examined based on RNA AHR expression levels (RPKM) and correlated with genomic expression. High vs. low AHR expression was based on 90 th and 10 th percentiles. Pathway enrichment from upregulated genes was identified and compared between high vs. low AHR expression. Single cell RNA sequencing by 10x genomics Chromium system by the Massively Parallel Sequencing Shared Resource at OHSU was performed with previously frozen FLT3 positive AML samples (4 high AHR and 4 low AHR). Immunophenotyping was conducted by flow cytometry. The AHR antagonist IK-364 was utilized to study the in vitro effects of AHR modulation. Cytotoxicity was measured by flow cytometric detection of caspase-3/7 and SytoxAAD activation. Results: The highest and lowest 10% expression levels of AHR in all samples in Beat AML were evaluated for mutation profiles. Ranking the top 18 mutations based on frequency we found an enrichment of FLT3 mutations in the high AHR expression group compared to the lower expression group (Figure 1A). ELN risk did not correlate with AHR expression however FAB classification M0/M1 and M4/M5 monocytic subtypes had higher AHR expression than M3 FAB subtype (Figure 2A). AHR gene expression was highly correlated with FLT3-ITD mutations (p=0.0007) (Figure 2B) and HLA-E expression (p= Discussion: Overall, we found that high AHR gene expression in AML correlates with FLT3-ITD expression and HLA-E as well as monocytic subtype. Pathway enrichment highlight the important role of AHR in immune regulation. Furthermore, AHR antagonism by IK-364, downregulates HLA-E on AML blasts and augments NK cell mediated killing of a mutant FLT3-ITD positive AML cell line. Our results highlight a continued interest in targeting the AHR pathway to augment immune-based therapies in AML.uture studies are underway to determine the mechanism behind this critical immune regulation. Figure 1 Figure 1. Disclosures Saultz: IKENA: Research Funding. Lind: IKENA: Research Funding. Tyner: Seattle Genetics: Research Funding; Constellation: Research Funding; Agios: Research Funding; Incyte: Research Funding; Petra: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Array: Research Funding; Schrodinger: Research Funding; Astrazeneca: Research Funding. Kosaka: IKENA: Research Funding. McGovern: IKENA: Current Employment. Wang: IKENA: Current Employment. Sanchez-Martin: IKENA: Current Employment.

The Hippo signaling cascade is an important pathway for cancer biogenesis and tumor maintenance. The Hippo pathway is heavily mutated across many cancer indications through loss of function mutations in genes such as NF2. These pro-tumor mutations lead to the constitutive activation of the downstream transcriptional coactivators YAP and TAZ that drive the expression of many pro-survival and proliferation genes through the essential interaction with a TEAD protein family member. In addition, this unrestrained transcriptional program drives enhanced immune suppression in the tumor microenvironment. To target this oncogenic pathway we identified novel small molecule inhibitors that selectively bind to TEAD and disrupt their interaction with YAP and TAZ thereby downregulating YAP- and TAZ-dependent transcription. These inhibitors prevent TEAD palmitoylation which is essential for the interaction between YAP and TEAD. Furthermore, they inhibit in vitro proliferation of YAP-dependent (i.e. Hippo pathway-deficient cancer cell lines), but not Hippo pathway wild type cancer cell lines. An attraction of targeting this pathway is that many differentiated cells and tissues do not rely on the Hippo pathway, but notable exceptions have been demonstrated with tissue specific mouse knockouts in the bile duct and kidney podocytes. Importantly, our compounds did not affect survival of a differentiated mouse podocyte cell line or compromise mouse kidney histology. Subsequent experiments in vivo demonstrate these inhibitors downregulate YAP-dependent genes in human tumor xenografts after oral dosing. In addition, these inhibitors exhibit single agent tumor growth inhibition of human tumor xenografts in mice at well tolerated oral doses. These data demonstrate the potential for targeting this critical pathway in cancers with small molecules. Additional studies to identify tumor types that are Hippo pathway-driven and dependent on TEAD function are in progress. Citation Format: Ben Amidon, Sakeena Syed, Jill Cavanaugh, Hyejin Frosch, Prabitha Natarajan, Jeffrey Ecsedy, Karen McGovern, Alfredo C. Castro. Potent small molecule TEAD inhibitors targeting the Hippo pathway exhibit antiproliferation in vitro and anti-tumor effect in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2474.

Immune checkpoint blockade (ICB) has been shown to convey significant clinical activity across a spectrum of malignancies, yet there is now recognition that multiple mechanisms of resistance can impair response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by enzymes such as indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) plays a major suppressive role. Recently it was shown that Kyn acts as an endogenous agonist of the Aryl hydrocarbon receptor (AhR). In order to gain a better understanding of this pathway, we sought to characterize the mechanisms of immunosuppression associated with AhR and evaluate its potential as therapeutic target. Gene-expression analysis of IDO-overexpressing melanomas (B16-IDO vs. B16-WT) demonstrated reduced expression levels of Type 1 inflammatory genes, including IFNy, TNF, GzmB, and CD40. In addition, B16-IDO presents higher infiltration of tumor-associated macrophages TAMs, which upregulate the AHR as well as classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2 phenotype. Tumor-antigen specific CD8+T cells show reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated antigen-presenting cells. In addition, TAMs from B16-IDO tumors suppressed activation of CD8+T cells in vitro and their depletion delayed tumor growth. When B16-IDO cells are implanted in mice depleted of Foxp3 expressing cells, TAMs do not accumulate. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) upregulates MHC II in APCs, activation markers in CD8s, and reduced frequency of T-regs in B16-IDO tumors. AhR inhibition slows tumor growth and prolongs survival of tumors with active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and this is enhanced when PD-1 blockade is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AhR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB. This abstract is also being presented as Poster A57. Citation Format: Luis F. Campesato, Sadna Budhu, Mathieu Gigoux, Jeremy Tchaicha, Stephane Pourpe, Cailian Liu, Dmitriy Zamarin, Mark G. Manfredi, Karen McGovern, Jedd D. Wolchok, Taha Merghoub. Blockade of AHR activation by IDO/TDO-derived kynurenine restricts cancer immune suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR05.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

Hedgehog (Hh) pathway inhibition in cancer has been evaluated in both the ligand-independent and ligand-dependent settings, where Hh signaling occurs either directly within the cancer cells or within the nonmalignant cells of the tumor microenvironment. Chondrosarcoma is a malignant tumor of cartilage in which there is ligand-dependent activation of Hh signaling. IPI-926 is a potent, orally delivered small molecule that inhibits Hh pathway signaling by binding to Smoothened (SMO). Here, the impact of Hh pathway inhibition on primary chondrosarcoma xenografts was assessed. Mice bearing primary human chondrosarcoma xenografts were treated with IPI-926. The expression levels of known Hh pathway genes, in both the tumor and stroma, and endpoint tumor volumes were measured. Gene expression profiling of tumors from IPI-926–treated mice was conducted to identify potential novel Hh target genes. Hh target genes were studied to determine their contribution to the chondrosarcoma neoplastic phenotype. IPI-926 administration results in downmodulation of the Hh pathway in primary chondrosarcoma xenografts, as demonstrated by evaluation of the Hh target genes GLI1 and PTCH1, as well as inhibition of tumor growth. Chondrosarcomas exhibited autocrine and paracrine Hh signaling, and both were affected by IPI-926. Decreased tumor growth is accompanied by histopathologic changes, including calcification and loss of tumor cells. Gene profiling studies identified genes differentially expressed in chondrosarcomas following IPI-926 treatment, one of which, ADAMTSL1, regulates chondrosarcoma cell proliferation. These studies provide further insight into the role of the Hh pathway in chondrosarcoma and provide a scientific rationale for targeting the Hh pathway in chondrosarcoma. Mol Cancer Ther; 13(5); 1259–69. ©2014 AACR.

Checkpoint inhibitors (CPIs) have become the cornerstone of immune-based oncology therapy; still many cancer patients do not benefit from these agents. Resistance to checkpoint inhibitors is due in part to the presence of immunosuppressive molecules which prevent immune activation despite T cell checkpoint inhibition. Aryl Hydrocarbon Receptor (AHR) is a transcription factor that mediates the immune response in multiple innate and adaptive immune cells subsequent to binding of a diverse set of endogenous and exogenous ligands. One such AHR agonist is kynurenine. Kynurenine, metabolized from tryptophan by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), binds to AHR and leads to a net immunosuppressive tumor microenvironment. Given that kynurenine can be synthesized by both IDO1 and TDO2 and that AHR is activated by endogenous ligands other than kynurenine, AHR inhibition provides a novel approach to reverse immunosuppression in a broad range of tumor types. We demonstrated that AHR antagonism affects multiple immune cell types and can lead to pro-inflammatory phenotypes in human T cells and myeloid cells in vitro and in murine tumor models. AHR antagonism inhibits growth in the B16-IDO, CT26 and MC38 models and reverses the immunosuppressive microenvironment as indicated by changes in T cell and myeloid cell populations. Immune signature changes characterized via Nanostring in multiple tumor models treated with AHR antagonists were also assessed. Oral dosing of AHR antagonists led to tumor growth inhibition as a single agent and increased anti-tumor activity when combined with checkpoint inhibitors. In addition, beneficial anti-tumor activity occurred with AHR antagonists combined with chemotherapy or with radiation therapy in syngeneic mouse tumor models. Our data indicate that reversing AHR-mediated immune suppression in the tumor microenvironment drives anti-tumor activity alone and in combination with other therapeutic modalities. Overall, our data demonstrates that AHR is an attractive target for reversing immune suppression in tumors. Therefore, we are developing AHR antagonists and translational insights to treat patients most likely to benefit from this approach. Citation Format: Jeremy Tchaicha, Silvia Coma, Meghan Walsh, Jill Cavanaugh, Marta Sanchez-Martin, X. Michelle Zhang, Alfredo Castro, Jeff Ecsedy, Mark Manfredi, Karen McGovern. Overcoming aryl hydrocarbon receptor mediated tumor immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4131.

Immune checkpoint blockade (ICB) results in clinical benefit for a subset of cancer patients, yet multiple mechanisms of resistance can impair optimal response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by the expression of enzymes such as IDO or TDO is a frequent phenomenon that plays a suppressive role in tumor immunity. Recently it was shown that Kyn acts as agonist of the aryl hydrocarbon receptor (AHR). Here we sought to characterize the mechanisms of immune suppression associated with the AHR pathway and to evaluate its potential as therapeutic target. RNAseq analysis of human cancers revealed a correlation between the expressions of AHR-related genes with markers associated with immunotherapy resistance (PD-1, FOXP3, CD206). By using IDO or TDO-overexpressing variants of a melanoma cell model (B16-F10), we found that myeloid cells, such as tumor-associated macrophages (TAMs) and dendritic cells (DCs), present up-regulation of the AHR. IDO-expressing tumors (B16-IDO) show higher myeloid cell infiltration, which present a tolerogenic phenotype. Tumor-antigen specific CD8T cells present reduced expression of activation markers and proliferation rate when primed by Kyn-treated BMDCs. Treatment of B16-IDO-bearing mice with an AHR-specific antagonist (CH-223191) leads to an increase of MHC II in TAMs, of activation markers in CD8 T cells and reduced frequency of T-regs. AHR inhibition delays progression of tumors with an active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and efficacy is further improved when ICB is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AHR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB.

BACKGROUND During development, the Hedgehog pathway plays important roles regulating the proliferation and differentiation of chondrocytes, providing a template for growing bone. In this study, the authors investigated the components of dysregulated Hedgehog signaling as potential therapeutic targets for osteosarcoma. METHODS Small-molecule agonists and antagonists that modulate the Hedgehog pathway at different levels were used to investigate the mechanisms of dysregulation and the efficacy of Hedgehog blockade in osteosarcoma cell lines. The inhibitory effect of a small-molecule Smoothened (SMO) antagonist, IPI-926 (saridegib), also was examined in patient-derived xenograft models. RESULTS An inverse correlation was identified in osteosarcoma cell lines between endogenous glioma-associated oncogene 2 (GLI2) levels and Hedgehog pathway induction levels. Cells with high levels of GLI2 were sensitive to GLI inhibition, but not SMO inhibition, suggesting that GLI2 overexpression may be a mechanism of ligand-independent activation. In contrast, cells that expressed high levels of the Hedgehog ligand gene Indian hedgehog (IHH) and the target genes patched 1 (PTCH1) and GLI1 were sensitive to modulation of both SMO and GLI, suggesting ligand-dependent activation. In 2 xenograft models, active autocrine and paracrine, ligand-dependent Hedgehog signaling was identified. IPI-926 inhibited the Hedgehog signaling interactions between the tumor and the stroma and demonstrated antitumor efficacy in 1 of 2 ligand-dependent models. CONCLUSIONS The current results indicate that both ligand-dependent and ligand-independent Hedgehog dysregulation may be involved in osteosarcoma. It is the first report to demonstrate Hedgehog signaling crosstalk between the tumor and the stroma in osteosarcoma. The inhibitory effect of IPI-926 warrants additional research and raises the possibility of using Hedgehog pathway inhibitors as targeted therapeutics to improve treatment for osteosarcoma. Cancer 2014;120:537–547. © 2013 American Cancer Society.

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.

Targeting tumour-infiltrating suppressive myeloid cells with a selective PI3Kγ inhibitor overcomes resistance to checkpoint blockade therapy in various mouse myeloid-rich tumour models. Therapeutic blockade of immune checkpoints with antibodies against CTLA-4 and PD-1 has proved effective against some cancer types, but clinical benefit has been limited to a subset of patients. Here Olivier De Henau et al. show that resistance to checkpoint blockade is associated with a high level of infiltration by suppressive myeloid cells in various mouse tumour models. In addition, targeting the myeloid-derived suppressor cells with a selective inhibitor of the γ isoform of phosphoinositide 3-kinase (PI3Kγ) increases sensitivity to checkpoint blockade therapy in a melanoma mouse model. 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 cancers1. 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 immunity2,3,4. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance5,6. 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.

Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer1,2,3,4,5. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors1,2,3,4,5,6,7. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPβ activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPβ activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

The Sonic Hedgehog (Shh) pathway drives a subset of medulloblastomas, a malignant neuroectodermal brain cancer, and other cancers. Small-molecule Shh pathway inhibitors have induced tumor regression in mice and patients with medulloblastoma; however, drug resistance rapidly emerges, in some cases via de novo mutation of the drug target. Here we assess the response and resistance mechanisms to the natural product derivative saridegib in an aggressive Shh-driven mouse medulloblastoma model. In this model, saridegib treatment induced tumor reduction and significantly prolonged survival. Furthermore, the effect of saridegib on tumor-initiating capacity was demonstrated by reduced tumor incidence, slower growth, and spontaneous tumor regression that occurred in allografts generated from previously treated autochthonous medulloblastomas compared with those from untreated donors. Saridegib, a known P-glycoprotein (Pgp) substrate, induced Pgp activity in treated tumors, which likely contributed to emergence of drug resistance. Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutation-dependent nor Gli2 amplification-dependent, and saridegib was found to be active in cells with the D473H point mutation that rendered them resistant to another Smo inhibitor, GDC-0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of genetic mutations that confer resistance distinguishes saridegib from other Smo inhibitors.

Despite the sustained clinical benefit demonstrated by immune checkpoint inhibitors, a majority of patients derive minimal or no appreciable benefit, indicating the urgent need to incorporate novel immunomodulatory targets and therapeutic strategies. Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) catalyze the first and rate-limiting step in the immunosuppressive tryptophan/kynurenine pathway and are both upregulated in a number of tumor types. Although small-molecule IDO1 inhibitors are being clinically evaluated in several tumor types, so far they have not demonstrated significant clinical benefits either as a single agent or in combination with immune checkpoint inhibition. We are developing pegylated kynureninase (Kynase), a kynurenine degrading enzyme, to treat a broader population with IDO1 and/or TDO2 expressing tumors. We believe that a more robust antitumor immune response can be achieved by depleting kynurenine, produced by both IDO1 and TDO2, with Kynase, than by inhibiting only IDO1. The human Kynase (HsKYN) has been successfully engineered to exhibit vastly improved catalytic activity and stability toward kynurenine over the wild-type human enzyme. HsKYN achieved durable and near complete plasma kynurenine depletion in mice, rats and non-human primates. HsKYN demonstrated single agent efficacy in CT26, MC38 and B16-IDO syngeneic mouse models. Tumor gene expression analysis using NanoString revealed that HsKYN treatment upregulated T-cell and NK cell activation signature. More importantly, HsKYN significantly increased the tumor-infiltrating CD8 T-cells and their activation/polyfunctionality, and reduced the Treg population. As a direct comparison, the lead IDO1 inhibitor epacadostat did not impose any meaningful effects on the same immune cell populations. Furthermore, HsKYN showed beneficial combination efficacy with anti-PD-1 that was superior to combined Epacadostat / anti-PD-1. Evidence to date suggest that HsKYN is well tolerated in multiple species. Therefore, immunoprofiling, efficacy and safety results strongly support that Kynase is a more effective therapeutic approach than IDO1 inhibition. HsKYN is moving toward clinical development for treatment of cancers where IDO1 and/or TDO2 pathways play a significant immunosuppressive role. Citation Format: Silvia Coma, Jillian Cavanaugh, James Nolan, Jeremy Tchaicha, Karen McGovern, Everett Stone, John Blazeck, Candice Lamb, George Georgiou, Mark G Manfredi, Michelle Zhang. Treatment of IDO1 and TDO2 positive tumors with a kynurenine-degrading enzyme: A highly differentiated approach from IDO1 inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B008.

Though checkpoint inhibitors have become the cornerstone of immune-based oncology therapy, the majority of cancer patients do not benefit from these agents. Resistance to checkpoint inhibitors is due in part to the presence of immunosuppressive molecules which prevent immune activation despite T cell checkpoint inhibition. One such immunosuppressive molecule is kynurenine. Kynurenine, metabolized from tryptophan by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), binds to the Aryl hydrocarbon receptor (AHR) in multiple immune cell types which leads to a net immunosuppressive environment. IDO1 inhibitors combined with checkpoint inhibitors have led to substantial clinical benefit in early clinical trials, validating disruption of the kynurenine pathway as a therapeutic strategy and substantiating that metabolites within human cancers induce immune cell tolerance. Given that kynurenine can be synthesized by both IDO1 and TDO2 and that AHR is activated by endogenous ligands other than kynurenine, AHR inhibition provides a novel approach to reverse immunosuppression in a broad range of tumor types. We will present data demonstrating that AHR antagonism can affect multiple immune cell types within the tumor microenvironment, reverse immune suppression and lead to tumor growth inhibition in tumor models. In addition, AHR inhibition combined with checkpoint inhibitors leads to enhanced anti-tumor activity and improved survival in these models relative to either agent alone. Kynurenine can lead to increased levels of many immunosuppressive cytokines including IL22 in human T-cells which is reversed with AHR inhibition. In addition, we have previously shown that kynurenine addition during T-cell maturation leads to a more quiescent T-effector cells while AHR antagonism during maturation maintains T-cells in a more naïve-like, T-central memory state. In vivo studies in the B16 and the B16-IDO overexpressing models demonstrate that kynurenine-mediated activation of the AHR pathway leads to faster tumor growth and a more immunosuppressive tumor microenvironment, with FOXP3 T-cells playing a major role. There are also more suppressive myeloid cells in the IDO overexpressing model. AHR antagonism inhibits growth in the B16-IDO model and reverses the immunosuppressive microenvironment as indicated by changes in T cell and myeloid cell populations as well as AHR-dependent gene expression. Similar activity is also seen in the CT26 model. In addition, enhanced tumor growth inhibition and survival benefit is seen in combination with checkpoint inhibitors. Our data indicate that AHR is a viable target for reversing immune suppression in the tumor microenvironment and could have activity in many tumor types due to activation via kynurenine and multiple other potential ligands. Citation Format: Jeremy Tchaicha, Karen McGovern, Luis Felipe Campesato, Silvia Coma, Xiaoyan Michelle Zhang, Meghan Walsh, Jill Cavanaugh, Taha Merghoub, Jedd Wolchok, Mark Manfredi. Targeting the IDO and TDO pathway through inhibition of the aryl hydrocarbon receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4723.

The tryptophan/kynurenine pathway has been clinically validated in several tumor types with small-molecule IDO1 inhibitors in combination with checkpoint inhibition. Indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) are upregulated in a number of tumor types, metabolizing tryptophan to form immunosuppressive kynurenine. We are developing Kynureninase (Kynase), a kynurenine depleting enzyme, to treat IDO1 and TDO2 positive tumors. The human Kynase has been successfully engineered to vastly increase catalytic activity and stability toward kynurenine over the wild type enzyme. In mice, Kynase achieved prolonged Kynurenine degradation (≥5 days) in plasma and tumor draining lymph node (TDLN), leading to anti-tumor activity as a single agent and in combination with check point inhibitors in mouse syngeneic tumor models. Kynase demonstrated superior tumor growth inhibition and survival benefit relative to a leading IDO1 inhibitor epacadostat in these models. The effects of Kynase on a number of immune cell types, both in vitro and in vivo, are being investigated. Human Kynase has also shown a favorable PK profile and kynurenine degradation in non-human primates, and Kynase variants are now moving toward development candidate selection for treatment of cancers where both IDO/TDO pathways play a significant immunosuppressive role. Citation Format: Silvia Coma, Jill Cavanaugh, James Nolan, Jeremy Tchaicha, Karen McGovern, Everett Stone, Candice Lamb, Christos Karamitros, John Blazek, Kendra Garrison, George Georgiou, Mark Manfredi, Xiaoyan Michelle Zhang. Targeting the IDO/TDO pathway through degradation of the immunosuppressive metabolite kynurenine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3757.

Although with notable impact on cancer treatment, immune-checkpoint blockade (ICB) have multiple mechanisms of resistance. The catabolism of Tryptophan into metabolites known as Kynurenines (Kyn) by enzymes such as IDO or TDO plays a major suppressive role in different tumor types. Recently it was shown that Kyn acts as an endogenous agonist of the Aryl-hydrocarbon Receptor (AhR). In order to gain a better understanding of this pathway, we sought to characterize the mechanisms of immunosuppression associated with AhR and evaluate its potential as therapeutic target. Gene-expression analysis of IDO-overexpressing melanomas (B16-IDO vs B16-WT) present reduced expression levels of Type-1 inflammatory genes, including IFNy, TNFa, Granzyme B and CD40. In addition, B16-IDO presents higher infiltration of TAMs, which up-regulate classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2-phenotype. Tumor-antigen specific CD8+T cells present reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated BMDMs. Also, B16-IDO TAMs suppressed activation of CD8+T cells in vitro and their depletion abrogated tumor growth. Implantation of B16-IDOs in FoxP3-depleted mice prevents TAMs accumulation. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) up-regulates MHC II in APCs, activation markers in CD8s and reduced frequency of T-regs. AhR inhibition slows down tumor growth and prolongs survival, which is improved in combination with PD-1 blockade. In summary, our findings demonstrate that targeting the Kyn pathway through AhR-inhibition represents a promising approach in cancer patients who are resistant to ICB.

Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamine. The acid sensitive D-ring of cyclopamine was homologated utilizing a sequence of chemoselective cyclopropanation and stereoselective acid-catalyzed rearrangement. Further modification of the A/B-ring homoallylic alcohol to the conjugated ketone led to the discovery of new cyclopamine analogues with improved pharmaceutical properties and in vitro potency (EC 50) ranging from 10 to 1000 nM.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML.

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

A requisite step for canonical Hedgehog (Hh) pathway activation by Sonic Hedgehog (Shh) ligand is accumulation of Smoothened (Smo) to the primary cilium (PC). Activation of the Hh pathway has been implicated in a broad range of cancers, and several Smo antagonists are being assessed clinically, one of which is approved for the treatment of advanced basal cell carcinoma. Recent reports demonstrate that various Smo antagonists differentially impact Smo localization to the PC while still exerting inhibitory activity. In contrast to other synthetic small molecule Smo antagonists, the natural product cyclopamine binds to and promotes ciliary accumulation of Smo and "primes" cells for Hh pathway hyper-responsiveness after compound withdrawal. We compared the properties of IPI-926, a semi-synthetic cyclopamine analog, to cyclopamine with regard to potency, ciliary Smo accumulation, and Hh pathway activity after compound withdrawal. Like cyclopamine, IPI-926 promoted accumulation of Smo to the PC. However, in contrast to cyclopamine, IPI-926 treatment did not prime cells for hyper-responsiveness to Shh stimulation after compound withdrawal, but instead demonstrated continuous inhibition of signaling. By comparing the levels of drug-induced ciliary Smo accumulation with the degree of Hh pathway activity after compound withdrawal, we propose that a critical threshold of ciliary Smo is necessary for "priming" activity to occur. This "priming" appears achievable with cyclopamine, but not IPI-926, and is cell-line dependent. Additionally, IPI-926 activity was evaluated in a murine tumor xenograft model and a pharmacokinetic/pharmacodynamic relationship was examined to assess for in vivo evidence of Hh pathway hyper-responsiveness. Plasma concentrations of IPI-926 correlated with the degree and duration of Hh pathway suppression, and pathway activity did not exceed baseline levels out to 96 hours post dose. The overall findings suggest that IPI-926 possesses unique biophysical and pharmacological properties that result in Hh pathway inhibition in a manner that differentiates it from cyclopamine.

During development, the Hedgehog pathway plays important roles regulating the proliferation and differentiation of chondrocytes, providing a template for growing bone. In this study, the authors investigated the components of dysregulated Hedgehog signaling as potential therapeutic targets for osteosarcoma.Small-molecule agonists and antagonists that modulate the Hedgehog pathway at different levels were used to investigate the mechanisms of dysregulation and the efficacy of Hedgehog blockade in osteosarcoma cell lines. The inhibitory effect of a small-molecule Smoothened (SMO) antagonist, IPI-926 (saridegib), also was examined in patient-derived xenograft models.An inverse correlation was identified in osteosarcoma cell lines between endogenous glioma-associated oncogene 2 (GLI2) levels and Hedgehog pathway induction levels. Cells with high levels of GLI2 were sensitive to GLI inhibition, but not SMO inhibition, suggesting that GLI2 overexpression may be a mechanism of ligand-independent activation. In contrast, cells that expressed high levels of the Hedgehog ligand gene Indian hedgehog (IHH) and the target genes patched 1 (PTCH1) and GLI1 were sensitive to modulation of both SMO and GLI, suggesting ligand-dependent activation. In 2 xenograft models, active autocrine and paracrine, ligand-dependent Hedgehog signaling was identified. IPI-926 inhibited the Hedgehog signaling interactions between the tumor and the stroma and demonstrated antitumor efficacy in 1 of 2 ligand-dependent models.The current results indicate that both ligand-dependent and ligand-independent Hedgehog dysregulation may be involved in osteosarcoma. It is the first report to demonstrate Hedgehog signaling crosstalk between the tumor and the stroma in osteosarcoma. The inhibitory effect of IPI-926 warrants additional research and raises the possibility of using Hedgehog pathway inhibitors as targeted therapeutics to improve treatment for osteosarcoma.

Nature 539, 437–442 (2016); doi:10.1038/nature19834 In this Letter, the Gene Expression Omnibus (GEO) accession number for the RNA sequencing data (for in vivo tumour and tumour-associated macrophage samples) was incorrectly listed in the ‘Data availability’ section as GSE84318; the correct GEO accession number is GSE84535.

The PI3 kinases (PI3K) belong to a family of signal-transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-gamma (γ) isoform plays an important role in macrophage/myeloid cell function and migration, and a role for PI3K-γ in tumor growth and immune tolerance has been established in studies utilizing PI3K-γ knockout (KO) mice (Schmid et al., Cancer Cell, 2011; Gunderson et al., Cancer Discovery, 2015). We propose that pharmacological inhibition of PI3K-γ in myeloid cells can alter the tumor-immune microenvironment leading to enhanced antitumor T-cell responses. IPI-549 is an oral, potent, and selective inhibitor of PI3K-γ. Prior studies showed single agent antitumor activity in multiple murine tumor models, and enhanced antitumor activity and improved survival when combined with immune-checkpoint blockade. This antitumor activity is dependent on the presence of both immune-suppressive tumor-associated CD11b+ myeloid cells and CD8+ cytotoxic T cells. IPI-549 can reduce the T-cell-suppressive activity of both murine and human myeloid-derived suppressor cells in vitro (Kutok et al, 2015 CRI-CIMT-EATI-AACR Cancer Immunotherapy Meeting; De Henau et al, 2016 AACR Annual Meeting). We now show that IPI‑549 treatment of tumor‑bearing mice leads to a shift in tumor-associated myeloid cells from an immunosuppressive M2 phenotype to a proinflammatory M1 phenotype, characterized by reduced CD206 expression and enhanced expression of MHC class II and NOS2. Compared to vehicle-treated controls, short-term (9 days) treatment of CT26 tumor‑bearing animals with IPI‑549 revealed an increased frequency of circulating tumor-specific T cells, an increased percentage of tumor-infiltrating CD8+IFNγ+ T cells, and a reduced percentage of CD4+Foxp3+ regulatory T cells, leading to a trend towards increasing the CD8+/T-reg cell ratio. Treatment of 4T1 and B16GM tumor-bearing mice with IPI-549 for 14 days led to a significant increase in the CD8+/T-reg cell ratio. Together these data show that IPI-549 treatment leads to a proinflammatory tumor microenvironment. Importantly, gene and protein expression analysis of whole tumor tissue collected from IPI-549-treated mice revealed a cytotoxic T-cell signature characterized by increased production of proinflammatory cytokines, and enhanced expression costimulatory and coinhibitory genes relative to vehicle-treated animals. These findings indicate that IPI-549 increases antitumor immunity by remodeling the tumor-immune microenvironment via blockade of tumor-associated myeloid cells. In addition, the up-regulation of costimulatory and coinhibitory genes with IPI-549 treatment provides a mechanistic rationale for the observed combination activity with immune checkpoint inhibition. IPI-549 is currently in Phase I development, both as a single agent and in combination with an anti-PD-1 antibody, in solid tumors (ClinicalTrials.gov NCT02637531). Citation Format: Matthew Rausch, Jeremy Tchaicha, Thomas Tibbitts, Olivier De Henau, Sujata Sharma, Melissa Pink, Joseph Gladstone, Jennifer Proctor, Mark Douglas, Howard Stern, Taha Merghoub, Jedd Wolchok, Karen McGovern, Jeff Kutok, David Winkler. The PI3K-γ inhibitor, IPI-549, increases antitumor immunity by targeting tumor-associated myeloid cells and remodeling the immune-suppressive tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B032.

The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is predominantly expressed in leukocytes and not expressed in most epithelial tumors or sarcomas. Genetic studies highlight an important role for PI3K-γ in myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Schmid et al. Canc Cell 2011). Targeting PI3K-γ could therefore alter the immune tumor microenvironment, enabling the immune system to attack tumor cells more effectively. We are developing IPI-549, an investigational small molecule inhibitor of PI3K-γ, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. IPI-549 is a potent and selective inhibitor of PI3K-γ with favorable pharmacological properties. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization, but did not affect M1 polarization. Oral administration of IPI-549 to tumor-bearing mice resulted in significant tumor growth inhibition in multiple syngeneic solid tumor models at PI3K-γ selective doses. Analysis of the tumor-associated immune cells demonstrated that IPI-549 treatment results in decreased immune suppressive myeloid cells and increased CD8+ T cells, suggesting enhanced anti-tumor immunity. To address the requirement for targeting myeloid cells by IPI-549, CD11b+ cells were depleted from a transplanted whole tumor Lewis Lung Carcinoma model and the effect of IPI-549 on limiting tumor growth was abrogated. In addition, a myeloid-infiltrated B16-GMCSF model, but not the isogenic B16 model without GMCSF, was responsive to IPI-549. Studies in immune-deficient mice or CD8 T-cell depleted tumor bearing mice demonstrated the T-cell dependence of IPI-549-mediated tumor growth inhibition. IPI-549 treatment also led to a significant reduction in lung metastases in the 4T1 and B16-GMCSF models. Importantly, in vivo studies with IPI-549 in combination with the immune checkpoint inhibitors anti-PD-1, anti-PDL-1 and anti-CTLA-4 showed increased tumor growth inhibition in multiple models compared to monotherapies alone. These data can inform combinations for future clinical trials. Our studies support a role for PI3K-γ in immune suppressive myeloid cells in the tumor microenvironment and provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit tumor growth in preclinical models. A Phase 1 study evaluating IPI-549 as an orally administered therapeutic, as a single agent and in combination with an anti-PD-1 antibody therapy, in patients with selected solid tumors is expected to begin in early 2016. Citation Format: Olivier De Henau, Taha Merghoub, David Winkler, Sujata Sharma, Melissa Pink, Jeremy Tchaicha, Matthew Rausch, Jennifer Proctor, Nicole Kosmider, John Soglia, Vito Palombella, Jeffery L. Kutok, Jedd D. Wolchok, Karen McGovern. Checkpoint blockade therapy is improved by altering the immune suppressive microenvironment with IPI-549, a potent and selective inhibitor of PI3K-gamma, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 554.