BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX’s ‘on switch’. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequencespecific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D V12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.R AS signaling is a critical control point for a host of cellular functions ranging from cellular survival and proliferation to cellular endocytosis and motility (1). The on or off state of RAS is dictated by nucleotide exchange. GTP-bound RAS is the activated form that engages its downstream effectors with high avidity. The endogenous GTPase activity of RAS hydrolyzes GTP to GDP and inactivates signaling. This biochemical process is further regulated by GTPase-activating proteins (GAPs) that impair RAS signaling through increasing endogenous GTPase activity and guanine-nucleotide exchange factors (GEFs) that enhance RAS signaling by facilitating GDP release and, thus, GTP association. Given the central roles of RAS in cellular growth and metabolism, it is not surprising that cancer cells usurp its prosurvival activities to achieve immortality.Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N-, and H-RAS, and are associated with poor prognosis and chemoresistance (2). KRAS mutations are present in ∼30% of human tumors and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. For example, in pancreatic ductal adenocarcinomas (PDAC) that carry a 5-y survival rate of less than 5%, activating KRAS mutations are present in more than 90% of tumors (3). Thus, therapeutic inhibition of RAS is among the highest priority goals of the cancer field. Because oncogenic forms of KRAS typically harbor single-point mutants that stabilize its active GTP-bound form, a host of recent small molecule and peptide development efforts have been aimed at disarming this pathologic biochemical state. The extremely high affinity of KRAS for its GTP substrate has hampered the development of competitive GTP inhibitors. However, a GDP mimetic that covalently modifies the mutant cysteine of KRAS G12C represents a promising approach to plugging the nucleotid...

Head and neck cancer (HNC) includes a diverse range of malignancies arising commonly from mucosal epithelia of the upper aerodigestive tract. Head and neck squamous cell carcinoma (HNSCC), the most common form of HNC, develops in the oral cavity, pharynx, and larynx and is associated with tobacco exposure, alcohol abuse, and infection with oncogenic viruses. Despite global advances in cancer care, HNSCC often presents with advanced disease and is associated with poor 5-year survival of ~50%. Genotyping tumor tissue to guide clinical decision-making is becoming commonplace in modern oncology, but in the management of HNSCC, tissue biopsies with cytopathology or histopathology remain the mainstay for diagnosis. Furthermore, conventional biopsies are temporally and spatially limited, often providing a brief snapshot of a single region of a heterogeneous tumor. In the absence of a useful biomarker, both primary and recurrent HNSCCs are diagnosed with conventional imaging and clinical examination. As a result, many patients are diagnosed with advanced disease. Tumor DNA is an emerging biomarker in HNSCC. DNA fragments are constantly being shed from tumors and metastatic lesions, and can therefore be detected in blood and other bodily fluids. Utilizing next-generation sequencing techniques, these tumor DNA can be characterized and quantified. This can serve as a minimally invasive liquid biopsy allowing for specific tumor profiling, dynamic tumor burden monitoring, and active surveillance for disease recurrences. In HNSCC, analysis of tumor DNA has the potential to enhance tumor profiling, aid in determining patient prognosis, and guide treatment decisions.

Exposure to metallic dental hardware has increased in the past few decades given the rise of orthodontic braces and older adults retaining more teeth. Although this study does not prove a causal relationship between oral cavity SCC and dental hardware, this is a step toward identifying and investigating their role.

Objective: To highlight a case from 1960 connecting endolymphatic sac tumour and von Hippel-Lindau disease.Case report: In 1960, a 24-year-old woman presented with unilateral hearing loss, pulsatile tinnitus and a mass visible on otoscopy. The patient underwent surgical biopsy, which was complicated by haemorrhage, and ultimately resulted in death. At autopsy, a destructive temporal bone neoplasm with cystic and papillary architecture was observed that had eroded into the otic capsule. Intra-abdominal lesions consistent with von Hippel-Lindau disease were also observed, and the surgeon postulated a connection between endolymphatic sac tumour and von Hippel-Lindau disease.Method: A review of the literature was carried out using PubMed. Conclusion: Endolymphatic sac tumours are rare neoplasms of the temporal bone that can occur sporadically or as part of von Hippel-Lindau disease. The connection between endolymphatic sac tumour and von Hippel-Lindau disease was first proposed in 1988 and formalised in 1997. We believe that this case represents the first documented connection between endolymphatic sac tumour and von Hippel-Lindau disease.

Therapeutic manipulation of the BCL-2 family using BH3 mimetics is an emerging paradigm in cancer treatment and immune modulation. For example, peptides mimicking the BIM BH3 helix can directly target the full complement of anti- and pro-apoptotic BCL-2 proteins to trigger apoptosis. This study has incorporated the potent BH3 α-helical death domain of BIM into peptide amphiphile (PA) nanostructures designed to facilitate cellular uptake and induce cell death. This study shows that these PA nanostructures are quickly incorporated into cells, are able to specifically bind BCL-2 proteins, are stable at physiologic temperatures and pH, and induce dose-dependent apoptosis in cells. The incorporation of a cathepsin B cleavable linker between the BIM BH3 peptide and the hydrophobic tail resulted in increased intracellular accumulation and mitochondrial co-localization of the BIM BH3 peptide while also improving BCL-2 family member binding and apoptotic reactivation. This PA platform represents a promising new strategy for intracellular therapeutic peptide delivery for the disruption of intracellular protein:protein interactions.

Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N- and H-RAS, and are associated with poor prognosis and chemoresistance. KRAS mutations are present in approximately 30% of tumors, and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. In pancreatic ductal adenocarcinomas (PDAC), which carries a 5-year survival rate of less than 5%, activating KRAS mutations are present in over 90% of tumors. Moreover, these mutations have been causally linked to the initiation and progression of PDAC. Despite the mechanistic insights into KRAS-mediated oncogenesis, development of high affinity targeted inhibitors remains a formidable challenge. Here, we report the synthesis and application of hydrocarbon-stapled peptides as prototype therapeutics for blocking wild type and mutant KRAS in vitro and in cancer cells. Stabilized Alpha-Helices of SOS1 (SAH-SOS1) were generated by inserting hydrocarbon staples into SOS1 peptide sequence to recapitulate the alpha-helical structure of the native KRAS-interaction domain. SAH-SOS1 peptides, but not negative control analogs, bound to a variety of KRAS constructs with nanomolar affinity, inhibiting nucleotide exchange. This sequence-specific biochemical activity correlated with impairment of KRAS-driven cancer cell viability and signal transduction. These studies provide proof-of-concept for the utility of SAH-SOS1 peptides in dissecting and targeting the oncogenic KRAS pathway in human cancer. Citation Format: Elizaveta S. Leshchiner, Joseph Bellairs, Gregory H. Bird, Kwadwo Opoku-Nsiah, Marina Godes, Loren D. Walensky. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B41. doi: 10.1158/1557-3125.RASONC14-B41

There are no approved therapeutics for the prevention of hearing loss and vestibular dysfunction from drugs like aminoglycoside antibiotics. While the mechanisms underlying aminoglycoside ototoxicity remain unresolved, there is considerable evidence that aminoglycosides enter inner ear mechanosensory hair cells through the mechanoelectrical transduction (MET) channel. Inhibition of MET-dependent uptake with small molecules or modified aminoglycosides is a promising otoprotective strategy. To better characterize mammalian ototoxicity and aid in the translation of emerging therapeutics, a biomarker is needed. In the present study we propose that neonatal mice systemically injected with the aminoglycosides G418 conjugated to Texas Red (G418-TR) can be used as a histologic biomarker to characterize in vivo aminoglycoside toxicity. We demonstrate that postnatal day 5 mice, like older mice with functional hearing, show uptake and retention of G418-TR in cochlear hair cells following systemic injection. When we compare G418-TR uptake in other tissues, we find that kidney proximal tubule cells show similar retention. Using ORC-13661, an investigational hearing protection drug, we demonstrate in vivo inhibition of aminoglycoside uptake in mammalian hair cells. This work establishes how systemically administered fluorescently labeled ototoxins in the neonatal mouse can reveal important details about ototoxic drugs and protective therapeutics.