Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.

The ubiquitin receptor ADRM1/Rpn13 governs the specificity of eukaryotic protein degradation. By SMRT sequencing, we first discovered a novel spliced variant of ADRM1 with a skipped exon 9, termed ADRM1-ΔEx9, in human hepatocellular carcinoma (HCC). This study aimed to elucidate this novel ubiquitin receptor's underlying biology and clinical implications in HCC. The role of ADRM1-ΔEx9 in early liver carcinogenesis was studied using human liver-derived non-tumoral organoids and a murine model with hydrodynamic in vivo transfection. ADRM1-ΔEx9 biology in HCC and its potential as a biomarker for predicting Olaparib response were investigated using patient-derived tumor organoids and xenograft models. The underlying mechanism was delineated using the Proteome Profiler Human Ubiquitin Array. ADRM1-ΔEx9, not its full-length counterpart, conferred human liver organoids with pro-survival advantages and a more profound tumor formation in a hydrodynamic transfected murine model. Functional knockdown resulted in spontaneous apoptosis in cell lines and patient-derived organoids, highlighting a pivotal role for ADRM1-ΔEx9 in HCC oncogenicity. Mechanistically, the shortened C-terminus of ADRM1-ΔEx9 signified a specific deubiquitinase partner BAP1 and navigated proteasome specificity. The new exon 8-10 fusion in ADRM1-ΔEx9 created a de novo binding to tumor suppressor protein FBXW7, resulting in its selective proteasome-mediated degradation. The loss of FBXW7 protein in ADRM1-ΔEx9 expressing tumors underscores their sensitivity to PARP inhibitor Olaparib. Notably, findings on ADRM1-ΔEx9 in primary HCC tumors denote its overexpression in a subgroup of patients with inferior survival and a window of therapeutic opportunity through its synthetic lethality association with Olaparib. ADRM1-ΔEx9 redirects ubiquitin proteasome specificity to degrade the tumor suppressor protein FBXW7 selectively. This promotes HCC tumor formation and provides a synthetic lethal link for PARPi therapy. Reduced tumor suppressor protein FBXW7 expression is pivotal in HCC pathogenesis and other liver diseases. However, the regulatory mechanism governing FBXW7 protein expression remains elusive. Herein, we unveil a non-canonical spliced isoform of the ubiquitin receptor ADRM1 that selectively degrades FBXW7 protein, thereby promoting the premalignant transformation of hepatic cells and conferring growth advantages to HCC tumors. Furthermore, our results demonstrate that ADRM1-ΔEx9-expressing HCC tumors exhibited sensitivity to Olaparib in a dose-dependent manner, implicating the potential use of Olaparib in targeting ADRM1-ΔEx9-driven HCC growth.

Cerebral ischemia is a leading cause of ischemic stroke, which may lead to severe disability and mortality worldwide. There are some key factors concerned in cardioprotection, such as peroxisome proliferator‑activated receptor γ (PPARγ), a ligand binding transcription factor involved in various biological functions including atherosclerosis, vascular dysfunction and hypertension, and baculoviral IAP repeat‑containing 5 (BIRC5), which may protect human brain endothelial cells from ischemia‑induced apoptosis. To determine the potential roles of PPARγ in brain microvascular endothelial (bEnd.3) cells during cerebral ischemia and the relationship between PPARγ and BIRC5, a cerebral ischemia model was established with bEnd.3 cells cells by oxygen‑glucose deprivation (OGD) treatment. OGD treatment reduced proliferation and enhanced apoptosis of bEnd.3 cells in a time‑dependent manner. PPARγ expression levels were decreased in bEnd.3 cells following OGD treatment. Upregulation of PPARγ expression protected bEnd.3 cells from ischemia injury and also upregulated BIRC5 expression. PPARγ‑specific binding sites in the BIRC5 promoter were predicted bioinformatically and verified by luciferase reporter experiments. Results from electrophoretic mobility shift/supershift and chromatin immunoprecipitation assays suggested that BIRC5 may be a novel target of PPARγ transcriptional regulation during ischemic injury. The present results indicated that PPARγ may serve a protective role on bEnd.3 cells and that BIRC5 may be a downstream target of PPARγ regulation during cerebral ischemia.

Metabolic reprogramming, notably alterations in the tricarboxylic acid (TCA) cycle, has emerged as a hallmark of cancer that supports tumor growth and metastasis. Despite the TCA cycle being a classical central metabolic pathway, further exploration is needed to fully elucidate the intricate manifestations and contributory mechanisms of TCA cycle rewiring in colorectal carcinogenesis. In this study, we identified a splicing isoform of citrate synthase (CS), CS-ΔEx4, and unveiled its role in TCA cycle dysregulation in colorectal cancer. CS-ΔEx4 was distinctly upregulated in colorectal cancer tumors compared with the canonical CS full-length (CS-FL) isoform. Clinical analyses established a strong correlation between elevated CS-ΔEx4 expression and cancer recurrence as well as inferior survival outcomes in patients with colorectal cancer. Functional experiments revealed the active contribution of CS-ΔEx4 to the aggressive phenotype of colorectal cancer cells both in vitro and in vivo. Mechanistically, CS-ΔEx4 formed a heterocomplex with CS-FL within the mitochondria that influenced the enzymatic function of canonical CS and accelerated TCA cycle flux, thereby promoting the accumulation of the oncometabolite 2-hydroxyglutarate. The CS-ΔEx4–mediated metabolic alterations engendered epigenomic modulations that drove the upregulation of oncogenic gene signatures. In silico screening identified a small molecule with potent antiproliferative effects in colorectal cancer cell line and organoid models that selectively antagonized the CS-ΔEx4 and CS-FL heterocomplex activity while sparing the CS-FL homodimers. Together, this study discovered the presence of a spliced CS isoform that promotes colorectal cancer progression and identified a molecule that holds potential for targeting the CS-ΔEx4 and CS-FL heterocomplex. Significance: The citrate synthase variant CS-ΔEx4 augments TCA metabolic flux to facilitate epigenetic reprogramming and colorectal carcinogenesis and can be targeted with a small molecule, providing a promising treatment strategy for colorectal cancer. See related commentary by Schatton and Frezza, p. 4293

The glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) has emerged as a vital oncogene in many cancer types as a result of its metabolic function and signaling pathways. However, the effects of alternative splicing variants of PFKFB4 remain largely unexplored. Clinical hepatocellular carcinoma (HCC) specimens were used to established the clinical implications of PFKFB4 variants. HCC cell lines and xenograft models were applied to characterize the function of PFKFB4 variants. The underlying mechanisms involved were investigated through the use of human proteome phosphate arrays, in vitro kinase assays, and immunoprecipitation. We identified a previously uncharacterized splicing variant of PFKFB4 in human HCC. This variant displayed a skipping exon 6 (ΔEx6), resulting in an in-frame 19-amino acid deletion. Functionally, PFKFB4-ΔEx6 demonstrated more pronounced effects compared with its canonical PFKFB4-full length (FL) counterpart on HCC cell proliferation, migration, and in vivo tumorigenicity (n = 8, p <0.01). Mechanistically, Human Phospho-Kinase Array profiling revealed the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway as the major downstream effector of PFKFB4-ΔEx6. Protein immunoprecipitation and in vitro kinase assays revealed that PFKFB4-ΔEx6 could trigger AKT activation through direct binding to the kinase domain, whereas such effects were not detected for PFKFB4-FL. Moreover, the presence of PFKFB4-ΔEx6 notably sensitized HCC cells to everolimus both in vitro and in subcutaneous models (n = 8, p <0.01). Clinically, common upregulation of PFKFB4-ΔEx6 in HCC tumors correlated with poorer overall survival of patients (n = 116, p = 0.001). Our study highlights the oncogenic characteristics of the novel PFKFB4-ΔEx6 variant in HCC. The direct effect of PFKFB4-ΔEx6 on AKT activation underscores its role in HCC progression and as a therapeutic biomarker for mTOR/AKT inhibitors. Our study identified a non-canonical variant of PFKFB4 as a crucial activator of the AKT/mTOR signaling pathway in HCC. This discovery reveals a novel oncogenic mechanism that extends beyond the conventional role of PFKFB4 in metabolism. The PFKFB4-ΔEx6 variant enhances tumor progression and renders HCC cells sensitive to everolimus, indicating its potential as both a prognostic biomarker and a therapeutic target. Given the clinical importance of mTOR/AKT inhibitors, PFKFB4-ΔEx6 could act as a predictive marker for treatment response, paving the way for personalized therapeutic strategies in HCC. These findings provide new insights into the functional diversity of glycolytic enzymes in cancer biology and targeted therapy.

Tafazzin is a mitochondrial enzyme necessary for the remodeling of the phospholipid cardiolipin. Seneviratne and Xu et al. demonstrated that Tafazzin-mediated phospholipid production regulates stemness in Acute Myeloid Leukemia (AML). Tafazzin influenced intracellular levels of phospholipids to control AML stemness. Thus, inhibiting mitochondrial phospholipid production could be a new therapeutic strategy for AML.