Discovery of TNG908: A Selective, Brain Penetrant, MTA-Cooperative PRMT5 Inhibitor That Is Synthetically Lethal with MTAP-Deleted Cancers.

It has been shown that PRMT5 inhibition by small molecules can selectively kill cancer cells with homozygous deletion of the MTAP gene if the inhibitors can leverage the consequence of MTAP deletion, namely, accumulation of the MTAP substrate MTA. Herein, we describe the discovery of TNG908, a potent inhibitor that binds the PRMT5·MTA complex, leading to 15-fold-selective killing of MTAP-deleted (MTAP-null) cells compared to MTAPintact (MTAP WT) cells. TNG908 shows selective antitumor activity when dosed orally in mouse xenograft models, and its physicochemical properties are amenable for crossing the blood-brain barrier (BBB), supporting clinical study for the treatment of both CNS and non-CNS tumors with MTAP loss.

Targeting oncoproteins with a positive selection assay for protein degraders.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

Peptidic degron for IMiD-induced degradation of heterologous proteins.

Current systems for modulating the abundance of proteins of interest in living cells are powerful tools for studying protein function but differ in terms of their complexity and ease of use. Moreover, no one system is ideal for all applications, and the best system for a given protein of interest must often be determined empirically. The thalidomide-like molecules (collectively called the IMiDs) bind to the ubiquitously expressed cereblon ubiquitin ligase complex and alter its substrate specificity such that it targets the IKZF1 and IKZF3 lymphocyte transcription factors for destruction. Here, we mapped the minimal IMiD-responsive IKZF3 degron and show that this peptidic degron can be used to target heterologous proteins for destruction with IMiDs in a time- and dose-dependent manner in cultured cells grown ex vivo or in vivo.

BRCA1-IRIS promotes human tumor progression through PTEN blockade and HIF-1α activation.

BRCA1 is an established breast and ovarian tumor suppressor gene that encodes multiple protein products whose individual contributions to human cancer suppression are poorly understood. BRCA1-IRIS (also known as "IRIS"), an alternatively spliced BRCA1 product and a chromatin-bound replication and transcription regulator, is overexpressed in various primary human cancers, including breast cancer, lung cancer, acute myeloid leukemia, and certain other carcinomas. Its naturally occurring overexpression can promote the metastasis of patient-derived xenograft (PDX) cells and other human cancer cells in mouse models. The IRIS-driven metastatic mechanism results from IRIS-dependent suppression of phosphatase and tensin homolog (PTEN) transcription, which in turn perturbs the PI3K/AKT/GSK-3ß pathway leading to prolyl hydroxylase-independent HIF-1α stabilization and activation in a normoxic environment. Thus, despite the tumor-suppressing genetic origin of IRIS, its properties more closely resemble those of an oncoprotein that, when spontaneously overexpressed, can, paradoxically, drive human tumor progression.

Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma.

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.

Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine.

The HIF transcription factor promotes adaptation to hypoxia and stimulates the growth of certain cancers, including triple-negative breast cancer (TNBC). The HIFα subunit is usually prolyl-hydroxylated by EglN family members under normoxic conditions, causing its rapid degradation. We confirmed that TNBC cells secrete glutamate, which we found is both necessary and sufficient for the paracrine induction of HIF1α in such cells under normoxic conditions. Glutamate inhibits the xCT glutamate-cystine antiporter, leading to intracellular cysteine depletion. EglN1, the main HIFα prolyl-hydroxylase, undergoes oxidative self-inactivation in the absence of cysteine both in biochemical assays and in cells, resulting in HIF1α accumulation. Therefore, EglN1 senses both oxygen and cysteine.

Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma.

The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.

Cancer-related epigenome changes associated with reprogramming to induced pluripotent stem cells.

The ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome-wide assays, we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These include hundreds of abnormal gene silencing events, patterns of aberrant responses to epigenetic-modifying drugs resembling those for cancer cells, and presence in iPS and partially reprogrammed cells of cancer-specific gene promoter DNA methylation alterations. Our findings suggest that by studying the process of induced reprogramming, we may gain significant insight into the origins of epigenetic gene silencing associated with human tumorigenesis, and add to means of assessing iPS for safety.

Just say no to ATOH: how HIC1 methylation might predispose medulloblastoma to lineage addiction.

Hypermethylated in cancer-1 (HIC1) is a tumor suppressor frequently targeted for promoter hypermethylation in medulloblastoma, an embryonal tumor of the cerebellum. Recently, we showed that HIC1 is a direct transcriptional repressor of ATOH1, a proneural transcription factor required for normal cerebellar development, as well as for medulloblastoma cell viability. Because demethylating agents can induce reexpression of silenced tumor suppressors, restoring HIC1 function may present an attractive therapeutic avenue in medulloblastoma by exploiting an apparent addiction to ATOH1.

Cooperation between the Hic1 and Ptch1 tumor suppressors in medulloblastoma.

Medulloblastoma is an embryonal tumor thought to arise from the granule cell precursors (GCPs) of the cerebellum. PATCHED (PTCH), an inhibitor of Hedgehog signaling, is the best-characterized tumor suppressor in medulloblastoma. However, <20% of medulloblastomas have mutations in PTCH. In the search for other tumor suppressors, interest has focused on the deletion events at the 17p13.3 locus, the most common genetic defect in medulloblastoma. This chromosomal region contains HYPERMETHYLATED IN CANCER 1 (HIC1), a transcriptional repressor that is a frequent target of epigenetic gene silencing in medulloblastoma. Here we use a mouse model of Ptch1 heterozygosity to reveal a critical tumor suppressor function for Hic1 in medulloblastoma. When compared with Ptch1 heterozygous mutants, compound Ptch1/Hic1 heterozygotes display a fourfold increased incidence of medulloblastoma. We show that Hic1 is a direct transcriptional repressor of Atonal Homolog 1 (Atoh1), a proneural transcription factor essential for cerebellar development, and show that ATOH1 expression is required for human medulloblastoma cell growth in vitro. Given that Atoh1 is also a putative target of Hh signaling, we conclude that the Hic1 and Ptch1 tumor suppressors cooperate to silence Atoh1 expression during a critical phase in GCP differentiation in which malignant transformation may lead to medulloblastoma.