DNA replication stress and mitotic catastrophe mediate sotorasib addiction in KRASG12C-mutant cancer.

BACKGROUND: Sotorasib is the first KRASG12C inhibitor approved by the US Food and Drug Administration for treating KRASG12C-mutant non-small-cell lung cancer (NSCLC). Clinical trials on the therapeutic use of sotorasib for cancer have reported promising results. However, KRASG12C-mutant cancers can acquire resistance to sotorasib after treatment. We incidentally discovered that sotorasib-resistant (SR) cancer cells are addicted to this inhibitor. In this study, we investigated the mechanisms underlying sotorasib addiction. METHODS: Sotorasib-resistant cells were established using KRASG12C-mutant pancreatic cancer and NSCLC cell lines. Cell viability in the presence or absence of sotorasib and in combination with multiple inhibitors was assessed through proliferation assay and annexin V/propidium iodide (PI) flow cytometry assays. The mechanisms underlying drug addiction were elucidated through 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, immunofluorescence staining, time-lapse microscopy, and comet assay. Furthermore, a subcutaneous xenograft model was used to demonstrate sotorasib addiction in vivo. RESULTS: In the absence of sotorasib, the sotorasib-resistant cells underwent p21Waf1/Cip1-mediated cell cycle arrest and caspase-dependent apoptosis. Sotorasib withdrawal resulted in robust activation of mitogen-activated protein kinase (MAPK) pathway, inducing severe DNA damage and replication stress, which activated the DNA damage response (DDR) pathway. Persistent MAPK pathway hyperactivation with DDR exhaustion led to premature mitotic entry and aberrant mitosis, followed by micronucleus and nucleoplasmic bridge formation. Pharmacologic activation of the MAPK pathway with a type I BRAF inhibitor could further enhance the effects of sotorasib withdrawal on sotorasib-resistant cancer cells both in vitro and in vivo. CONCLUSIONS: We elucidated the mechanisms underlying the sotorasib addiction of cancer cells. Sotorasib addiction appears to be mediated through MAPK pathway hyperactivity, DNA damage, replication stress, and mitotic catastrophe. Moreover, we devised a therapeutic strategy involving a type I BRAF inhibitor to strengthen the effects of sotorasib addiction; this strategy may provide clinical benefit for patients with cancer.

SIN3-HDAC complex-associated factor, a chromatin remodelling gene located in the 12p amplicon, is a potential germ cell tumour-specific oncogene.

A genetic hallmark of malignant germ cell tumours (GCTs) is isochromosome 12p, but oncogenes located in 12p that are specifically expressed in GCT have not yet been identified. SIN3-HDAC complex-associated factor (SINHCAF) is a subunit of the Sin3/histone deacetylase (HDAC) complex, and it defines a Sin3a-Hdac complex variant that is required for the self-renewal of mouse embryonic stem cells. This study demonstrated that SINHCAF is expressed in a vast majority of malignant GCTs and is rarely expressed in somatic malignancy. Fluorescence in situ hybridisation revealed SINHCAF amplification in malignant GCTs. SINHCAF silencing using shRNA reduced anchorage-dependent cell proliferation and tumoursphere formation and inhibited tumour cell migration and invasion in GCT cell lines. Moreover, in the GCT cell line NTERA2/D1, SINHCAF silencing inhibited the expression of genes associated with embryonic stem cells and induced the expression of genes associated with neuronal and white fat cell differentiation. Compared with somatic cell lines, GCT cell lines were more susceptible to HDAC inhibitor treatment. Thus, we identified SINHCAF to be a potential oncogene located in the amplicon of chromosome 12p and showed that SINHCAF was specifically expressed in malignant GCTs. HDAC inhibitor treatment may counteract the oncogenic activity of SINHCAF and is a promising therapeutic approach for GCTs. © 2022 The Pathological Society of Great Britain and Ireland.

Microinsertions in PRKACA cause activation of the protein kinase A pathway in cardiac myxoma.

Cardiac myxoma is the most common cardiac tumour. Most lesions occur sporadically, but occasional lesions develop in patients with Carney complex, a syndrome characterized by cardiac myxoma, spotty pigmentation, and endocrine overactivity. Two-thirds of patients with Carney complex harbour germline mutations in PRKAR1A, which encodes the type I regulatory subunit of protein kinase A (PKA). Most studies have not found a mutation in PRKAR1A in sporadic cardiac myxoma cases. Recent studies identified frequent mutations in PRKACA, which encodes the catalytic subunit of PKA, in cortisol-secreting adrenocortical adenoma cases. To determine whether the PRKACA mutation is involved in the tumourigenesis of cardiac myxoma, we performed Sanger sequencing of 41 specimens of sporadic cardiac myxoma to test for the presence of mutations in the coding regions and intron-exon boundaries of PRKACA. Mutations were identified in four cases (9.7%). In contrast to the point mutations identified in adrenocortical adenoma, all mutations were in-frame microinsertions of 18-33 bp clustered in exons 7 and 8. The mutated PRKACA proteins lost their ability to bind to PRKAR1A, and thereby lead to constitutive activation of the PKA pathway. Together with previous reports of PRKAR1A mutations in syndromic cardiac myxoma, our study demonstrates the importance of the PKA pathway in the tumourigenesis of cardiac myxoma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased Trimethylation of histone H3K36 associates with biliary differentiation and predicts poor prognosis in resectable hepatocellular carcinoma.

INTRODUCTION: Trimethylation of histone H3K36 (H3K36me3), an epigenetic marker of transcription-associated histone modification and stem cell regulation, is expressed in a variety of human cancers. This study elucidated the prognostic significance of H3K36me3 in patients with resectable hepatocellular carcinoma (HCC). METHODS: Expression of H3K36me3 was retrospectively evaluated through immunohistochemistry in 152 surgically resected primary HCCs. RESULTS: In nontumorous liver parenchyma, H3K36Me3 was detected in bile ducts but not in hepatocytes. H3K36me3 was positive in 104 (68.4%) of the HCCs. Positivity for H3K36me3 was associated with high level of serum α-fetoprotein (>200 ng/mL, P = 0.0148), high tumor grade (P = 0.0017), and high tumor stage (P = 0.0008). Patients with H3K36me3-positive tumors were more likely to have lower 5-year disease-free survival and 5-year overall survival than those with H3K36me3-negative tumors (P = 0.0484 and P = 0.0213, respectively). Multivariate analysis showed that H3K36me3 positivity was an independent predictor of high tumor grade (P = 0.0475) and high tumor stage (P = 0.0114) and thus contributed to poor prognosis. Furthermore, H3K36me3 positivity was significantly correlated with the expression of biliary markers cytokeratin 19 (CK19) and hepatocyte nuclear factor 1ß (HNF1ß) (P < 0.0001 and P = 0.0005, respectively). Combinatorial analysis revealed that CK19 and HNF1ß expression individually exerted additive prognostic adverse effects on HCCs with H3K36me3 positivity. CONCLUSIONS: Our study indicates that H3K36me3 positivity is associated with the expression of biliary markers and is a crucial predictor of poor prognosis in resectable HCC.