Simultaneous screening of overexpressed genes in breast cancer for oncogenic drivers and tumor dependencies.

There are hundreds of genes typically overexpressed in breast cancer cells and it's often assumed that their overexpression contributes to cancer progression. However, the precise proportion of these overexpressed genes contributing to tumorigenicity remains unclear. To address this gap, we undertook a comprehensive screening of a diverse set of seventy-two genes overexpressed in breast cancer. This systematic screening evaluated their potential for inducing malignant transformation and, concurrently, assessed their impact on breast cancer cell proliferation and viability. Select genes including ALDH3B1, CEACAM5, IL8, PYGO2, and WWTR1, exhibited pronounced activity in promoting tumor formation and establishing gene dependencies critical for tumorigenicity. Subsequent investigations revealed that CEACAM5 overexpression triggered the activation of signaling pathways involving ß-catenin, Cdk4, and mTOR. Additionally, it conferred a growth advantage independent of exogenous insulin in defined medium and facilitated spheroid expansion by inducing multiple layers of epithelial cells while preserving a hollow lumen. Furthermore, the silencing of CEACAM5 expression synergized with tamoxifen-induced growth inhibition in breast cancer cells. These findings underscore the potential of screening overexpressed genes for both oncogenic drivers and tumor dependencies to expand the repertoire of therapeutic targets for breast cancer treatment.

Induction of Multiple Alternative Mitogenic Signaling Pathways Accompanies the Emergence of Drug-Tolerant Cancer Cells.

Drug resistance can evolve from a subpopulation of cancer cells that initially survive drug treatment and then gradually form a pool of drug-tolerant cells. Several studies have pinpointed the activation of a specific bypass pathway that appears to provide the critical therapeutic target for preventing drug tolerance. Here, we take a systems-biology approach, using proteomics and genomics to examine the development of drug tolerance to EGFR inhibitors in EGFR-mutant lung adenocarcinoma cells and BRAF inhibitors in BRAF-mutant melanoma cells. We found that there are numerous alternative mitogenic pathways that become activated in both cases, including YAP, STAT3, IGFR1, and phospholipase C (PLC)/protein kinase C (PKC) pathways. Our results suggest that an effective therapeutic strategy to prevent drug tolerance will need to take multiple alternative mitogenic pathways into account rather than focusing on one specific pathway.

Safety, pharmacokinetics and sialic acid production after oral administration of N-acetylmannosamine (ManNAc) to subjects with GNE myopathy.

GNE myopathy is a rare, autosomal recessive, inborn error of sialic acid metabolism, caused by mutations in GNE, the gene encoding UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase. The disease manifests as an adult-onset myopathy characterized by progressive skeletal muscle weakness and atrophy. There is no medical therapy available for this debilitating disease. Hyposialylation of muscle glycoproteins likely contributes to the pathophysiology of this disease. N-acetyl-D-mannosamine (ManNAc), an uncharged monosaccharide and the first committed precursor in the sialic acid biosynthetic pathway, is a therapeutic candidate that prevents muscle weakness in the mouse model of GNE myopathy. We conducted a first-in-human, randomized, placebo-controlled, double-blind, single-ascending dose study to evaluate safety and pharmacokinetics of ManNAc in GNE myopathy subjects. Single doses of 3 and 6g of oral ManNAc were safe and well tolerated; 10g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma unconjugated free sialic acid (Neu5Ac) (Tmax of 8-11h). Neu5Ac levels remained above baseline 48h post-dose in subjects who received a dose of 6 or 10g. Given that Neu5Ac is known to have a short half-life, the prolonged elevation of Neu5Ac after a single dose of ManNAc suggests that intracellular biosynthesis of sialic acid was restored in subjects with GNE myopathy, including those homozygous for mutations in the kinase domain. Simulated plasma concentration-time profiles support a dosing regimen of 6g twice daily for future clinical trials.

Sialylation of Thomsen-Friedenreich antigen is a noninvasive blood-based biomarker for GNE myopathy.

AIM: The exact pathomechanism of GNE myopathy remains elusive, but likely involves aberrant sialylation. We explored sialylation status of blood-based glycans as potential disease markers. METHODS: We employed immunoblotting, lectin histochemistry and mass spectrometry. RESULTS: GNE myopathy muscle showed hyposialylation of predominantly O-linked glycans. The O-linked glycome of patients' plasma compared with controls showed increased amounts of desialylated Thomsen-Friedenreich (T)-antigen, and/or decreased amounts of its sialylated form, ST-antigen. Importantly, all patients had increased T/ST ratios compared with controls. These ratios were normalized in a patient treated with intravenous immunoglobulins as a source of sialic acid. DISCUSSION:  GNE myopathy clinical trial data will reveal whether T/ST ratios correlate to muscle function.  CONCLUSION: Plasma T/ST ratios are a robust blood-based biomarker for GNE myopathy, and may also help explain the pathology and course of the disease.

Mutation update for GNE gene variants associated with GNE myopathy.

The GNE gene encodes the rate-limiting, bifunctional enzyme of sialic acid biosynthesis, uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Biallelic GNE mutations underlie GNE myopathy, an adult-onset progressive myopathy. GNE myopathy-associated GNE mutations are predominantly missense, resulting in reduced, but not absent, GNE enzyme activities. The exact pathomechanism of GNE myopathy remains unknown, but likely involves aberrant (muscle) sialylation. Here, we summarize 154 reported and novel GNE variants associated with GNE myopathy, including 122 missense, 11 nonsense, 14 insertion/deletions, and seven intronic variants. All variants were deposited in the online GNE variation database (http://www.dmd.nl/nmdb2/home.php?select_db=GNE). We report the predicted effects on protein function of all variants well as the predicted effects on epimerase and/or kinase enzymatic activities of selected variants. By analyzing exome sequence databases, we identified three frequently occurring, unreported GNE missense variants/polymorphisms, important for future sequence interpretations. Based on allele frequencies, we estimate the world-wide prevalence of GNE myopathy to be ∼4-21/1,000,000. This previously unrecognized high prevalence confirms suspicions that many patients may escape diagnosis. Awareness among physicians for GNE myopathy is essential for the identification of new patients, which is required for better understanding of the disorder's pathomechanism and for the success of ongoing treatment trials.