Glioblastoma (GBM) is the most lethal and aggressive adult brain tumor, requiring the development of efficacious therapeutics. Towards this goal, we screened five genetically distinct patient-derived brain-tumor initiating cell lines (BTIC) with a unique collection of small molecule epigenetic modulators from the Structural Genomics Consortium (SGC). We identified multiple hits that inhibited the growth of BTICs in vitro, and further evaluated the therapeutic potential of EZH2 and HDAC inhibitors due to the high relevance of these targets for GBM. We found that the novel SAM-competitive EZH2 inhibitor UNC1999 exhibited low micromolar cytotoxicity in vitro on a diverse collection of BTIC lines, synergized with dexamethasone (DEX) and suppressed tumor growth in vivo in combination with DEX. In addition, a unique brain-penetrant class I HDAC inhibitor exhibited cytotoxicity in vitro on a panel of BTIC lines and extended survival in combination with TMZ in an orthotopic BTIC model in vivo. Finally, a combination of EZH2 and HDAC inhibitors demonstrated synergy in vitro by augmenting apoptosis and increasing DNA damage. Our findings identify key epigenetic modulators in GBM that regulate BTIC growth and survival and highlight promising combination therapies.

MYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.

Medulloblastoma (MB), the most prevalent malignant childhood brain tumour, consists of at least 4 distinct subgroups each of which possesses a unique survival rate and response to treatment. To rapidly determine MB subgroup affiliation in a manner that would be actionable during surgery, we subjected murine xenograft tumours of two MB subgroups (SHH and Group 3) to Mass Spectrometry (MS) profiling using a handheld Picosecond InfraRed Laser (PIRL) desorption probe and interface developed by our group. This platform provides real time MS profiles of tissue based on laser desorbed lipids and small molecules with only 5-10 seconds of sampling. PIRL-MS analysis of ex vivo MB tumours offered a 98% success rate in subgroup determination, observed over 194 PIRL-MS datasets collected from 19 independent tumours (∼10 repetitions each) utilizing 6 different established MB cell lines. Robustness was verified by a 5%-leave-out-and-remodel test. PIRL ablated tissue material was collected on a filter paper and subjected to high resolution LC-MS to provide ion identity assignments for the m/z values that contribute most to the statistical discrimination between SHH and Group 3 MB. Based on this analysis, rapid classification of MB with PIRL-MS utilizes a variety of fatty acid chains, glycerophosphates, glycerophosphoglycerols and glycerophosphocholines rapidly extracted from the tumours. In this work, we provide evidence that 5-10 seconds of sampling from ex vivo MB tissue with PIRL-MS can allow robust tumour subgroup classification, and have identified several biomarker ions responsible for the statistical discrimination of MB Group 3 and the SHH subgroup. The existing PIRL-MS platform used herein offers capabilities for future in vivo use.

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

The prognosis for patients diagnosed with glioblastoma multiforme (GBM) remains dismal, with current treatment prolonging survival only modestly. As such, there remains a strong need for novel therapeutic strategies. The janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 pathway regulates many cellular processes in GBM, including survival, proliferation, invasion, anti-apoptosis, and immune evasion. Here, we evaluated the preclinical efficacy of pacritinib, a novel compound targeting JAK2, using a collection of diverse patient-derived brain tumor initiating cells (BTICs).

Picosecond infrared laser mass spectrometry (PIRL-MS) is shown, through a retrospective patient tissue study, to differentiate medulloblastoma cancers from pilocytic astrocytoma and two molecular subtypes of ependymoma (PF-EPN-A, ST-EPN-RELA) using laser-extracted lipids profiled with PIRL-MS in 10 s of sampling and analysis time. The average sensitivity and specificity values for this classification, taking genomic profiling data as standard, were 96.41 and 99.54%, and this classification used many molecular features resolvable in 10 s PIRL-MS spectra. Data analysis and liquid chromatography coupled with tandem high-resolution mass spectrometry (LC-MS/MS) further allowed us to reduce the molecular feature list to only 18 metabolic lipid markers most strongly involved in this classification. The identified 'metabolite array' was comprised of a variety of phosphatidic and fatty acids, ceramides, and phosphatidylcholine/ethanolamine and could mediate the above-mentioned classification with average sensitivity and specificity values of 94.39 and 98.78%, respectively, at a 95% confidence in prediction probability threshold. Therefore, a rapid and accurate pathology classification of select pediatric brain cancer types from 10 s PIRL-MS analysis using known metabolic biomarkers can now be available to the neurosurgeon. Based on retrospective mining of 'survival' versus 'extent-of-resection' data, we further identified pediatric cancer types that may benefit from actionable 10 s PIRL-MS pathology feedback. In such cases, aggressiveness of the surgical resection can be optimized in a manner that is expected to benefit the patient's overall or progression-free survival. PIRL-MS is a promising tool to drive such personalized decision-making in the operating theater.

Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens.

Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009, an analogue of the previously reported ALK2 inhibitor LDN-214117. Although highly selective for ALK2 over the TGF-βR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives (26a-c), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.