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Supplementary Video Legends and Supplementary Figures 1-10

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Cell lines used in this study.

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mp3 file (11 MB). In the May edition of the Cancer Discovery podcast, Science Writer Elizabeth McKenna talks with Ingo K. Mellinghoff about his paper, which suggests that the disappointing clinical activity of first-generation EGFR inhibitors in glioblastoma versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types.

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Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance anti-tumor immunity and augment cancer immunotherapies. By screening a small molecule library of epigenetics-based therapeutics, BET bromodomain inhibitors (BETi) were identified as agents that promote the anti-tumor activity of CD8+ T-cells. BETi sensitized diverse tumor types to the cytotoxic effects of the pro-inflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of pro-survival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific (TCB) antibodies increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune oncology agents.STATEMENT OF SIGNIFICANCEManipulating the epigenome is an evolving strategy to enhance anti-tumor immunity. We demonstrate that BET bromodomain inhibitors potently sensitize solid tumors to CD8+ T-cell killing in a TNF-dependent manner. This immunomodulatory mechanism can be therapeutically leveraged to augment immuno-oncology therapies, including TCB antibodies and immune checkpoint blockade.

MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF. Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.

Bruton tyrosine kinase (BTK) links the B-cell antigen receptor (BCR) and Toll-like receptors with NF-κB. The role of BTK in primary central nervous system (CNS) lymphoma (PCNSL) is unknown. We performed a phase I clinical trial with ibrutinib, the first-in-class BTK inhibitor, for patients with relapsed or refractory CNS lymphoma. Clinical responses to ibrutinib occurred in 10 of 13 (77%) patients with PCNSL, including five complete responses. The only PCNSL with complete ibrutinib resistance harbored a mutation within the coiled-coil domain of CARD11, a known ibrutinib resistance mechanism. Incomplete tumor responses were associated with mutations in the B-cell antigen receptor–associated protein CD79B. CD79B-mutant PCNSLs showed enrichment of mammalian target of rapamycin (mTOR)-related gene sets and increased staining with PI3K/mTOR activation markers. Inhibition of the PI3K isoforms p110α/p110δ or mTOR synergized with ibrutinib to induce cell death in CD79B-mutant PCNSL cells. Significance: Ibrutinib has substantial activity in patients with relapsed or refractory B-cell lymphoma of the CNS. Response rates in PCNSL were considerably higher than reported for diffuse large B-cell lymphoma outside the CNS, suggesting a divergent molecular pathogenesis. Combined inhibition of BTK and PI3K/mTOR may augment the ibrutinib response in CD79B-mutant human PCNSLs. Cancer Discov; 7(9); 1018–29. ©2017 AACR. See related commentary by Lakshmanan and Byrd, p. 940. This article is highlighted in the In This Issue feature, p. 920

Bromodomain and extra-terminal family (BET) proteins bind to acetylated lysine residues on histone tails to modulate transcription. While the focus of BET inhibitors (BETi) has been to attenuate the transcription of oncogenes, recent work has shown that BETi suppress PD-L1 expression thereby possibly increasing anti-tumor immunity. Upon activation through immune-oncology (IO) agents, cytotoxic T-cells release pro-inflammatory cytokines such as IFNγ, TNF and Granzyme B leading to direct cancer cell cytolysis at the immunological synapse and bystander cancer cell death in the surrounding tumor microenvironment. However, rapid genetic and epigenetic tumor evolution can lead to immune escape and clinical resistance against IO agents. Therefore, our work examined how BETi reprogram cancer cells to become more sensitive to T-cell derived tumor necrosis factor (TNF) leading to increased bystander killing in combination with IO agents. Using TNF as a surrogate for activated T-cells, we tested a large panel of cell lines for enhanced sensitivity to TNF in the presence of BETi, RG6146. In a subset of cancer cells, BETi treatment sensitized the cells to TNF induced cell death irrespective of their histology or genetic background. The combination of TNF and RG6146 led to complete proliferation arrest and induction of cell death. We identified that RG6146 in this context suppressed the expression of important signaling partners in the pro-survival NF-κB pathway leading to potent Caspase-8 activation and induction of the extrinsic apoptotic pathway. In order to further confirm the phenotype, we activated T-cells using a tumor antigen targeted approach. The CEATCB is a (2:1) T-cell bispecific (TCB) antibody connecting cancer cells expressing carcinoembryonic antigen (CEA) on their cell surface with CD3 on the surface of T cells. This interaction induces T-cell activation, release of cytokines and subsequent killing of cancer cells. Addition of RG6146 to the supernatant of the CEATCB assay containing TNF, could significantly decrease viability of cancer cells compared to control treatment indicating a synergistic effect of the CEATCB and RG6146. We could verify these results in a co-culture experiment with a mixture of cancer cells expressing high and low levels of CEA and PBMCs. Even though treatment of this co-culture with the CEATCB alone increased bystander killing of cancer cells expressing low CEA levels, addition of RG6146 significantly enhanced this effect. We used syngeneic recipient mice to validate our findings in vivo. While single agent treatment of CEATCB or BETi decreased tumor growth, the combination of both molecules caused tumor regression. Taken together this data establishes a paradigm where BETi can rewire NF-κB signaling, leading to enhanced sensitivity to cytotoxic lymphocyte-derived TNF and therapeutically augmenting the anti-tumor activity of IO agents. Citation Format: Lisa C. Wellinger, Simon J. Hogg, Dane Newman, Thomas Friess, Daniela Geiss, Marina Bacac, Tanja Fauti, Astrid Ruefli-Brasse, Ricky W. Johnstone, Daniel Rohle. Sensitizing cancer cells to TNF induced cell death by the BET-inhibitor RG6146 [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 1703.

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway., The epidermal growth factor receptor (EGFR) signalling is regulated at multiple levels. Here the authors show that the importin RanBP6 acts as a tumor suppressor in Glioblastoma and regulates EGFR signalling through promoting translocation of STAT3 to the nuclei and repressing EGFR transcription.

The BET family of proteins bind to acetylated lysine residues on histone proteins and transcription factors to co-activate gene expression. BET proteins regulate the expression of oncogenes and can control the activity of various oncogenic transcription programs and have thereby emerged as therapeutic targets for the treatment of cancer. RG6146 is a novel non-covalent inhibitor of BET proteins that is in early phase clinical trials for the treatment of haematological and solid malignancies. The anti-tumor activity of BET inhibitors has primarily been attributed to tumor cell intrinsic effects, however increasing evidence suggests BET inhibitors modulate anti-tumor immune responses. Here, we examined the anti-solid tumor activity of RG6146 and evaluated the ability of RG6146 to enhance anti-tumor CD8+ T-cell responses. To model anti-tumor CD8+ T-cell responses in vitro, syngeneic colon and breast tumor cells expressing ovalbumin (Ova) antigen were co-cultured with activated CD8+ T-cells derived from OT-1 transgenic mice. RG6146 functionally increased the activity of both wild-type and perforin-deficient OT-1 T-cells, leading to significantly enhanced T cell-mediated tumor cell death in a time- and dose-dependent manner. Mechanistic studies revealed that enhanced tumor cell death induced by RG6146 was dependent upon CD8+ T-cell derived tumor necrosis factor-α (TNF-α), independent of perforin/granzyme-dependent granule exocytosis. As RG6146 did not increase TNF-α production in CD8+ T-cells, we hypothesized RG6146 may sensitize tumors cells to TNF-α. Indeed, screening of cell lines revealed that BET inhibition significantly enhanced TNF-α-induced cell death in solid tumors of diverse origin. Underlying this response, we demonstrate using RNA- and ChIP-sequencing that BET inhibition suppresses transcription of pro-survival NF-kB target genes to elicit a potent pro-apoptotic phenotype. Finally, using syngeneic solid tumor models, we demonstrated that the adaptive immune system promotes the efficacy of RG6146 and evaluated the ability of RG6146 to therapeutically augment cancer immunotherapies in vivo. Taken together, these data demonstrate that RG6146 is a potent BET bromodomain inhibitor with multi-faceted anti-cancer activity against solid tumors. We have identified a novel immunological TNF-α-dependent mechanism of bystander tumor killing by which BET inhibitors promote anti-tumor responses in vivo. Finally, we provide evidence that BET inhibition will augment the activity of cancer immunotherapies, establishing a strong rationale to evaluate these combinations in the clinic. Citation Format: Simon J. Hogg, Lisa Wellinger, Daniel Rohle, Ricky W. Johnstone. Enhancing antitumor immune responses with clinical BET bromodomain inhibitor RG6146 [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 4485.

Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. Significance: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain. Cancer Discov; 2(5); 458–71. ©2012 AACR. Read the Commentary on this article by Park and Lemmon, p. 398. This article is highlighted in the In This Issue feature, p. 377.

Glucose and glutamine are the two principal nutrients that cancer cells use to proliferate and survive. Many cancers show altered glucose metabolism, which constitutes the basis for in vivo positron emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)F-FDG). However, (18)F-FDG is ineffective in evaluating gliomas because of high background uptake in the brain. Glutamine metabolism is also altered in many cancers, and we demonstrate that PET imaging in vivo with the glutamine analog 4-(18)F-(2S,4R)-fluoroglutamine ((18)F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced (18)F-FGln tumor avidity, corresponding with decreased tumor burden. (18)F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where (18)F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that (18)F-FGln is avidly taken up by gliomas, can be used to assess metabolic nutrient uptake in gliomas in vivo, and may serve as a valuable tool in the clinical management of gliomas.

Both genome-wide genetic and epigenetic alterations are fundamentally important for the development of cancers, but the interdependence of these aberrations is poorly understood. Glioblastomas and other cancers with the CpG island methylator phenotype (CIMP) constitute a subset of tumours with extensive epigenomic aberrations and a distinct biology1–3. Glioma CIMP (G-CIMP) is a powerful determinant of tumour pathogenicity, but the molecular basis of G-CIMP remains unresolved. Here we show that mutation of a single gene, isocitrate dehydrogenase 1 (IDH1), establishes G-CIMP by remodelling the methylome. This remodelling results in reorganization of the methylome and transcriptome. Examination of the epigenome of a large set of intermediate-grade gliomas demonstrates a distinct G-CIMP phenotype that is highly dependent on the presence of IDH mutation. Introduction of mutant IDH1 into primary human astrocytes alters specific histone marks, induces extensive DNA hypermethylation, and reshapes the methylome in a fashion that mirrors the changes observed in G-CIMP-positive lower-grade gliomas. Furthermore, the epigenomic alterations resulting from mutant IDH1 activate key gene expression programs, characterize G-CIMP-positive proneural glioblastomas but not other glioblastomas, and are predictive of improved survival. Our findings demonstrate that IDH mutation is the molecular basis of CIMP in gliomas, provide a framework for understanding oncogenesis in these gliomas, and highlight the interplay between genomic and epigenomic changes in human cancers.

Glutamine is the most abundant plasma amino acid and many cancers show altered glutamine metabolism. We evaluated glutamine uptake and metabolism in gliomas using PET imaging and biochemical approaches. We demonstrate that glutamine is a key TCA cycle anaplerotic substrate and is metabolized to generate 2-HG in IDH1-mutant gliomas. PET imaging with18F-labeled glutamine (18F-FGln) showed high uptake in gliomas in vivo but low background uptake in the surrounding brain in RCAS-PDGF/PTEN null and IDH1-mutant glioma animal models, facilitating clear tumor delineation in contrast to that seen with 18F-FDG. We did not observe 18F-FGln uptake in animals with neuroinflammation or animals with a disrupted BBB. Further, 18F-FGln uptake was specifically reduced on chemo/radiation therapy. Finally, 18F-FGln showed high avidity in human glioma with low uptake in the surrounding brain. These data suggest that 18F-FGln is specifically taken up by gliomas, can be used to assess the metabolic state of gliomas in vivo and may serve as a valuable tool in the clinical management of gliomas.