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BackgroundDiffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy.MethodsImmunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function.ResultsGD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo.ConclusionOur study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

The adoptive transfer of the chimeric antigen receptor (CAR) expressing T-cells has produced unprecedented successful results in the treatment of B-cell malignancies. However, the use of this technology in other malignancies remains less effective. In the setting of solid neoplasms, CAR T-cell metabolic fitness needs to be optimal to reach the tumor and execute their cytolytic function in an environment often hostile. It is now well established that both tumor and T cell metabolisms play critical roles in controlling the immune response by conditioning the tumor microenvironment and the fate and activity of the T cells. In this review, after a brief description of the tumoral and T cell metabolic reprogramming, we summarize the latest advances and new strategies that have been developed to improve the metabolic fitness and efficacy of CAR T-cell products.

Background Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC. Methods The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. Results TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation. Conclusions Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

3D models are increasingly used to study mechanisms driving tumor progression and mimicking in vitro processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems that allow for downstream molecular biology investigations. Materials & methods: Here we present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established by comparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification. Results and conclusion: The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.

Diffuse hemispheric glioma H3G34-mutant (DHG-G34) is a pediatric-type high-grade glioma affecting children and young adults. Despite surgery and radio/chemotherapy, patients have a dismal prognosis. The intratumoural heterogeneity and the high infiltrative nature of DHG-G34 cells limit the development of effective therapies. Analysing single-cell RNA sequencing data from a publicly available dataset, we identified a large and distinct sub-population of cells displaying high stem and low differentiation marker expression levels. Gene ontology analyses revealed a gene signature related to cell migration/invasion. This observation is supported by our data on in vitro 3D invasion assay and in vivo orthotopic xenograft models, showing that DHG-G34 disseminating cells are characterised by high expression level of the stem-cell marker NESTIN and low expression level of the differentiation marker GFAP. Following these findings, we developed high-throughput cell-based assays with the aim to screen a library of 1300 FDA-approved compounds and identify drugs able to induce DHG-G34 cell differentiation and inhibit their invasive phenotype. The screen, a co-immunofluorescence assay for NESTIN and GFAP, followed by dose response assays on 3D growth and 3D invasion, led to the identification of 3 FDA-approved drugs, the MEK inhibitor Cobimetinib and 2 HMG-CoA reductase inhibitors, Rosuvastatin and Pitavastatin. These 3 drugs potently induced cell differentiation (decreased Nestin and increased GFAP expression) and inhibited invasion with minimal effect on the proliferation of our DHG-G34 cell line. We are currently extending these findings to additional patient-derived DHG cell lines and we are using these drugs and different omics and imaging technologies to characterize the regulatory networks associated to DHG-G34 stemness, (de)-differentiation and invasiveness. Our work may lead to the identification of new therapeutic approaches for targeting the stem/invasive properties of these aggressive diseases.

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.

Kabuki syndrome (KS) is an extremely rare genetic disorder, mainly caused by germline mutations at specific epigenetic modifier genes, including KMT2D. Because the tumor suppressor gene KMT2D is also frequently altered in many cancer types, it has been suggested that KS may predispose to the development of cancer. However, KS being a rare disorder, few data are available on the incidence of cancer in KS patients. Here, we report the case of a 5-year-old boy affected by KS who developed Burkitt lymphoma (BL). Genetic analysis revealed the presence of a novel heterozygous mutation in the splice site of the intron 4 of KMT2D gene in both peripheral blood-extracted DNA and tumour cells. In addition, the tumour sample of the patient was positive for the classical somatic chromosomal translocation t(8;14) involving the c-MYC gene frequently identified in BL. We propose that the mutated KMT2D gene contributes to the development of both KS and BL observed in our patient and we suggest that strict surveillance must be performed in KS patients.

Low-grade gliomas (LGG) are the most common central nervous system tumors in children. Prognosis depends on complete surgical resection. For patients not amenable of gross total resection (GTR) new approaches are needed. The BRAF mutation V600E is critical for the pathogenesis of pediatric gliomas and specific inhibitors of the mutated protein, such as Vemurafenib, are available. We investigated the safety and efficacy of Vemurafenib as single agent in pediatric patients with V600E+ LGG. From November 2013 to May 2018, 7 patients have been treated in our Institution; treatment was well-tolerated, the main concern being dermatological toxicity. The best responses to treatment were: 1 complete response, 3 partial responses, 1 stable disease, only one patient progressed; in one patient, the follow-up is too short to establish the clinical response. Two patients discontinued treatment, and, in both cases, immediate progression of the disease was observed. In one case the treatment was discontinued due to toxicity, in the other one the previously assessed BRAF V600E mutation was not confirmed by further investigation. Two patients, after obtaining a response, progressed during treatment, suggesting the occurrence of resistance mechanisms. Clinical response, with improvement of the neurologic function, was observed in all patients a few weeks after the therapy was started. Despite the limitations inherent to a small and heterogeneous cohort, this experience, suggests that Vemurafenib represents a treatment option in pediatric patients affected by LGG and carrying BRAF mutation V600E.

Diffuse intrinsic pontine glioma (DIPG) and pediatric glioblastoma (pGBM) are heterogeneous brain tumors characterized by different anatomical and molecular subgroups and the presence of genetically and phenotypically distinct subclonal cell populations. It is recognized that exosomes mediate cross-talk among tumor cells. We hypothesize that there are different exosome-mediated paracrine signaling promoting tumour progression in DIPG and pGBM. Our aim was to determine the specific DIPG and pGBM-derived exosome oncogenic signatures. We used a panel of fifteen patient primary-derived cell lines, which included nine DIPG (seven H3.3 K27M, one H3.3 K27M/ACVR1 and one H3.1 K27M/ACVR1), one diffuse midline glioma H3.3 K27M and three GBM (one H3.3 G34R and two histone WT). Conditioned medium was collected from cells maintained under stem-cell culture condition, adherent on laminin and/or as neurospheres (NS), and exosomes harvested through serial centrifugations. Electron microscopy demonstrated that the isolated microvescicles are exosomes sized between 50–80 nm. DIPG derived-exosomes appeared to have a variable cargo of total protein (µg)/10(6) cells, which was higher than for pGBM-exosomes. Proteomic analysis revealed that proteins associated with vesicle docking, exocytosis and synaptic transmission were exclusively enriched in pontine-derived exosomes, while cell-cell and cell-matrix interaction proteinswere exclusive tohemispheric ones. Proteins in common to the two locations were involved in metabolism and energy pathways. Interestingly, principle component analysis on the different molecular subgroups suggests that ACVR1 may be not implicated in the exosomal proteomic signature. Exosomal miRNA profile appeared to be driven by the two main histone mutated subgroups H3.3 K27M and H3.1 K27M with the latter overexpressing hypoxia and angiogenic-associated miRNAs, leading to distinct oncogenic programs with different specific potential therapeutic targets. This study aimed to development new diagnostic/prognostic tools for DIPG and pGBM patients. Further investigations are aimed to identify new therapeutic strategies to inhibit the cross-talk among glioma subpopulations.

Diffuse midline gliomas (DMG) are aggressive paediatric brain tumors for which there is no effective treatment. Recent pre-clinical studies suggest that adoptive transfer of chimeric antigen receptor (CAR) T-cells targeting the disialoganglioside antigen GD2 (GD2-CAR) has a significant therapeutic potential for H3K27M-mutant DMG. Still, some tumor cells resist to treatment suggesting that a multimodal approach may be necessary to treat more efficiently the disease. Our aim was to identify chemical compounds that, in combination with CAR T-cells, would enhance anti-tumor efficacy. After having confirmed the GD2 expression in tissue samples and patient-derived H3K27M-mutant DMG cells, we developed a high throughput cell-based assay to screen 40 kinase inhibitors in combination with T-cells expressing the GD2-CAR.CD28.4-1BB.z construct. The screening led to the identification of the dual IGF1R/IR antagonists, BMS-754807 and linsitinib, which, in combination with GD2-CAR T-cells, improved antitumor activity by 25% (p

Pediatric Glioblastoma (pGBM) and Diffuse Intrinsic Pontine Glioma (DIPG) are highly heterogeneous brain tumors which we demonstrated are comprised by distinct sub-clones interacting in a functional network. Exosomes are known to mediate the crosstalk between tumor and its microenvironment. Based on this, we aimed to investigate the role of exosomes in mediating pGBM and DIPG inter-clonal communication. By using optical barcoding for single cell-tracking, we generated two bulk multicolor patient derived-cell lines (one DIPG H3.3K27M and one pGBM histone WT) from which we obtained two and five single cell-derived clones respectively. The sub-clones demonstrated significantly phenotypic differences in terms of morphology, growth, adhesion, migration and invasion properties. In particular, co-culture experiments, with the two most different clones for both cell-lines, confirmed the cell-cell interaction key role in driving their more aggressive phenotype. Furthermore, we found that pGBM and DIPG sub-clones release exosomes which are actively and differentially up-taken by individual clones. Analysis of the exosomal microRNAs showed a different profile between the two selected clones in each cell-line. In particular, we found a pool of five upregulated microRNAs in 1C5 clone (DIPG cell-line) strongly associated to Wnt-signaling and PI3K-AKT pathway. Similarly, a pool of five upregulated microRNAs for 5E2 clone (pGBM cell-line) were found associated with focal adhesion and PI3K-AKT pathway. Our study may provide novel therapeutic strategies by interfering with the exosome-mediated inter-clonal communication in pGBM and DIPG.

Extraventricular neurocytoma (EVN) is an extremely rare tumor of neuroglial origin with a tendency toward ganglionic or glial differentiation. In the 2016 World Health Organization Classification, EVN was classified as a grade II tumor and described as a neoplasm with good outcome. However, the presence of cellular atypia is an important unfavorable prognostic factor. Here, we describe the first case of a patient with a congenital EVN localized in the brainstem. After a sub-total resection, his disease rapidly progressed despite several chemotherapies, including molecular targeting approaches. He died 13 months after diagnosis. In conclusion, we report an atypical case of EVN presenting an extremely aggressive behavior, despite the absence of cellular atypia. The brainstem origin and the age of the patient may have represented two important prognostic factors for our patient.

A series of 4,6-disubstituted pyrimidines from a phenotypic screen targeting the HSF1 pathway are also potent inhibitors of CDK9., Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

A unifying characteristic of aggressive cancers is a profound anabolic shift in metabolism to enable sustained proliferation and biomass expansion. The ribosome is centrally situated to sense metabolic states but whether it impacts systems that promote cellular survival is unknown. Here, through integrated chemical-genetic analyses, we find that a dominant transcriptional effect of blocking protein translation in cancer cells is complete inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for tumorigenesis. Translational flux through the ribosome reshapes the transcriptional landscape and links the fundamental anabolic processes of protein production and energy metabolism with HSF1 activity. Targeting this link deprives cancer cells of their energy and chaperone armamentarium thereby rendering the malignant phenotype unsustainable.

Heat shock factor 1 (HSF1) was originally identified as a master regulator of the classical ‘cytoprotective’ heat shock response. However, a large body of evidence has now verified the importance of HSF1 to tumorigenesis and cancer progression. HSF1 is activated by various elements of the cancer state, reprogramming the transcriptome in a way that is overlapping with, but distinct from, the canonical heat-shock response. Also, there is a strong correlation between the expression of activated HSF1 in tumors and adverse clinical outcomes. This evidence indicates that the inhibition of HSF1-mediated transcription could be a viable strategy in cancer treatment. Inhibiting the HSF1 stress pathway represents an attempt at targeting non-oncogene addiction and proteotoxic stress, which has been proposed to be advantageous. However, HSF1 is a ligandless transcription factor and is unlikely to be amenable to standard drug discovery strategies and direct inhibition with small molecules. Therefore, we proposed that inhibitors of HSF1-mediated transcription, which antagonize the HSF1 pathway but without necessarily binding directly to HSF1, could be discovered and developed via a cell-based phenotypic screen. We carried out a high throughput Arrayscan assay of 200,000 compounds to measure the inhibition of HSF1-mediated HSP72 expression stimulated by pre-treatment with an HSP90 inhibitor. We identified a singleton hit with a bisamide core, CCT245232. This compound showed potent growth inhibition in a range of human cancer cell lines but had poor physicochemical properties leading to an unacceptable pharmacokinetic profile. Improvement of the physicochemical properties of CCT245232 whilst maintaining potency versus our cell-based assays led to the orally bioavailable tool compound CCT251236. This compound shows potent growth inhibition (GI50 values in low nanomolar range) of human ovarian cancer cell lines in vitro and good efficacy against human ovarian cancer xenografts in nude mice in vivo. We applied chemo-proteomic strategies to identify the molecular target using a probe based on CCT251236 and discovered pirin as a high affinity molecular target. Binding of CCT251236 to recombinant pirin was confirmed in biophysical assays. CCT251236 recapitulates the reported anti-migratory phenotype for a pirin ligand although binding to pirin alone does not explain the cellular phenotype observed with our chemical tool. We are currently using CCT251236 as a chemical probe while further optimizing its properties to identify a clinical candidate. Citation Format: Matthew D. Cheeseman, Nicola E. Chessum, Carl S. Rye, Elisa A. Pasqua, Michael J. Tucker, Birgit Wilding, Lindsay E. Evans, Susan Lepri, Meirion Richards, Swee Y. Sharp, Salyha Ali, Martin Rowlands, Lisa O'Fee, Asadh Miah, Angela Hayes, Alan T. Henley, Marissa Powers, Robert te Poele, Emmanuel De Billy, Loredana Pellegrino, Florence Raynaud, Rosemary Burke, Robert L. van Montfort, Suzanne A. Eccles, Keith Jones, Paul Workman. Discovery of chemical probe CCT251236: An orally bioavailable efficacious pirin ligand from an HSF1 phenotypic screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-304. doi:10.1158/1538-7445.AM2017-LB-304

Two studies highlight the role of the heat shock response in initiating and maintaining cancer.

Correction for ‘Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9’ by Carl S. Rye et al., Med. Chem. Commun., 2016, 7, 1580–1586.

The transcription factor heat shock factor 1 (HSF1) is the master regulator of the heat shock response. It is crucial for cell homeostasis and implicated in aging, neurodegenerative disease and cancer [1]. Although induction by HSF1 of the expression of molecular chaperones and other regulators of protein quality control, both folding and degradation, is well established, the precise and detailed transcriptional network that HSF1 regulates in cancer is poorly understood. An important new study identifies an HSF1-regulated transcriptional program in highly malignant cells that is surprisingly distinct from the traditional heat shock response [2]. The results have significant implications for our molecular understanding of cancer and the development of new therapies. The heat shock response, mediated by activation of HSF1, is an ancient, highly conserved mechanism that protects organisms against various adverse environmental and pathological conditions that damage cellular proteins [3]. In cancer, such proteotoxic stress is caused by accumulation of mutated proteins, aneuploidy, reactive oxygen species and the challenging tumor microenvironment (eg hypoxia, low pH), which malignant cells must counteract by activating HSF1 to survive [4, 5]. In healthy, non-stressed cells, HSF1 is located in the cytoplasm in an inhibitory complex with the molecular chaperone heat shock protein 90 (HSP90) – another key player in proteostasis and malignant progression. Upon proteotoxic stress as classically induced by heat shock, HSF1 dissociates from HSP90, trimerizes, translocates to the nucleus and associates with its cognate DNA sequence localized at the promoters of its target genes to modulate their transcription (Figure ​(Figure1).1). HSF1 activation by proteotoxic stress is tightly regulated by many post-translational modifications, including phosphorylation, sumoylation and acetylation, the pattern and extent of which varies depending on the nature and the intensity of the stress [3]. Figure 1 Distinct and overlapping genes are regulated by activation of the heat shock response when induced by cancer versus other cell stresses Deregulated proteostasis in cancer is a vulnerability, exploitable by targeting protein folding and degradation. The proteasome inhibitor bortezomib (approved in multiple myeloma) and HSP90 inhibitors (showing promise in breast and non small cell lung cancer) enhance proteotoxic stress in cancer cells, causing cell cycle arrest and apoptosis. However, their efficacy is limited by activation of HSF1 and hence induction of numerous cytoprotective proteins, including chaperones like HSP90 and HSP70 family members that ameliorate the proteostatic damage, causing drug resistance [6-8]. Accumulating evidence shows that HSF1 is critically involved in oncogenesis. Key studies using HSF1-knockout mice demonstrate the requirement for HSF1 in tumorigenesis by oncogenic RAS or mutant P53 [9]. Furthermore, HSF1 silencing blocks proliferation and survival of cancer cells driven by diverse oncogenic factors. HSF1 supports the malignant phenotype by promoting oncogenic signal transduction pathways, proliferation, survival, protein synthesis and glucose metabolism [9]. Thus HSF1 is not only a critical factor in drug resistance and regulation of proteotoxic stress, but also a crucial driver in cancer and a potential therapeutic target [10, 11]. The common view is that the key effects of HSF1 in oncogenesis are mediated via increased HSP expression with oncogenic-support properties [10]. However, Medillo et al. now demonstrate that HSF1 rewires the transcriptome in cancer in a surprising new way – one that is quite distinct from the classical heat shock response. First, Medillo et al demonstrate that HSF1 protein expression is up-regulated constitutively and also activated – as shown by its nuclear localization and its phosphorylation at serine 326 – in oncogene-transformed breast cancer cells that are highly tumorigenic and metastatic, as compared to their less malignant isogenic counterparts. Also, the most aggressive cancer cell lines are more dependent on HSF1 for their growth and survival, indicating a stronger ‘addiction’ and therapeutic vulnerability. Next, Medillo et al. use chromatin immunoprecipitation linked to next-generation sequencing (ChIP-Seq) to identify the genes bound by HSF1. Under basal conditions HSF1 binds many more genes in the highly malignant compared to less aggressive cancer cells or immortalized but non-transformed cells. Binding occurs at both promoter regions and distal sites. Following heat shock, HSF1 binding to genes is enhanced in all the cell lines. Surprisingly, however, there are marked differences in the genes bound by HSF1 in cancer under basal conditions versus normal cells following heat shock. Many genes bound specifically in the aggressive breast cancer cells are not classical heat shock proteins and there is enrichment for genes involved in protein translation, RNA binding, metabolism and adhesion. Examples of cancer-specific genes highlighted by Medillo et al. include CKS2 encoding cyclin-dependent kinase interacting protein; LY6K coding for glycophosphatidyl-inositol-anchored membrane protein with cancer connections; and RBM23 encoding an RNA-binding protein implicated in estrogen-mediated transcription. On the other hand, another set of genes bound by HSF1 in the cancer cell lines under basal conditions are common to those binding in non-transformed cell lines after heat shock. Thus the results indicate both interesting differences and a level of overlap between the HSF1 gene signature regulated by thermal stress and that associated with malignancy – raising the possibility that different molecular mechanisms (eg epigenetic regulation and various post-translational modifications) may be preferentially involved under these two stress-activating conditions (Figure ​(Figure1).1). Importantly, Medillo et al. use RNA interference to confirm the role of HSF1 in regulating transcription of its identified bound genes. Following on, Medillo et al. confirm the identified cancer-specific HSF1 gene occupancy profile in cancer cells from diverse tumor origins including breast, colon and lung. Of high translational significance, they then use ChIP-Seq to confirm that the cancer-specific HSF1-mediated gene transcription program is active in breast, colon and lung tumors obtained directly from human patients. Finally and of direct clinical relevance, Medillo et al. demonstrate that an ‘HSF1-cancer gene signature’ comprising 456 cancer HSF1-regulated genes – which include the cancer-specific set and the cancer-regulated genes overlapping with the heat shock set – is associated with poor clinical outcome in diverse human cancers including breast, colon and lung cancer and is more predictive than other commonly used prognostic markers such as MYC, Ki67 and the MammaPRINT gene signature. This important new study provides a much clearer understanding of the role of HSF1 in progression to the highly malignant and metastatic state – as well as much food for thought for future research. Most significant and surprising is the specific rewiring of the transcriptome by HSF1 in cancer cells. The new work also highlights the complexity of the mechanisms implicated in HSF1 regulation and function. Differences in the genes bound by HSF1 between the highly aggressive versus less malignant cell lines could reflect disparities in the amount of active HSF1 present in the nucleus or may result from the deregulation of several key oncogenic pathways (eg EGFR/HER2, RAS/MAPK and insulin/IGF1) and/or epigenetic states. In addition to the implications for basic research there are important translational consequences. Understanding the functional relevance of the epigenetic factors and post-translational modifications that modulate HSF1 activity, as well as identifying the proteins involved and the cofactors associated with HSF1 transcriptional activity, is likely to provide both new cancer biomarkers and novel ways to target more specifically and efficiently (eg Figure ​Figure1)1) the megalomaniac role played by HSF1 in malignancy. Thus, although HSF1 has been around for a billion years of evolution, it continues to surprise us. In the near future we will very likely see more shocks about heat shock in cancer.

Heat Shock Factor 1 (HSF1) is a key transcription factor involved in proteostasis and response to stress, as well as being implicated in many diseases including cancer. Up-regulation of its activity by environmental stress or oncogenesis leads to transcriptional induction of genes involved in diverse cellular processes supporting the cancer state, including proteostasis, proliferation, survival and metastasis. Inhibition of HSF1 is therefore potentially beneficial for cancer treatment, but HSF1 is not technically druggable. We developed a high-throughput cell-based reporter gene assay to screen a library of small-molecule kinase inhibitors and identified 3 compound series as validated inhibitors of HSF1 activation by the HSP90 inhibitor 17-AAG. Further characterization of imidazo[1,2-b]pyridazine compounds showed inhibition of HSF1 target gene expression and the ability to mimic other features of the HSF1 knockdown phenotype. Using these tool compounds and by siRNA knockdown of HSF1 expression we demonstrate that HSF1 inhibition leads to cell cycle arrest and enhances the antiproliferative effect of 17-AAG. In addition, we show that HSP90 inhibition induces HSF1 phosphorylation at two serine residues, Ser326 and Ser320, highlighting the importance of Ser320 phosphorylation in HSP72 up-regulation by 17-AAG-induced HSF1 activation, and show that this activation is inhibited by our tool compounds. Our findings support the optimization and development of small-molecule inhibitors of the HSF1 pathway for cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Emmanuel de Billy, Nicola Chessum, Robert Te Poele, Jennifer Smith, Lorenzo Zani, Swee Sharp, Mark Stubbs, Wynne Aherne, Keith Jones, Paul Workman. Identification of small molecule inhibitors of HSF1 stress pathway activation in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2014-1775