Drugging mutant KRAS by disrupting binding to effectors and GTP
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Oncogenic RAS induces a distinctive form of non-canonical autophagy mediated by the P38-ULK1-PI4KB axis
Cancer cells with RAS mutations exhibit enhanced autophagy, essential for their proliferation and survival, making it a potential target for therapeutic intervention. However, the regulatory differences between RAS-induced autophagy and physiological autophagy remain poorly understood, complicating the development of cancer-specific anti-autophagy treatments. In this study, we identified a form of non-canonical autophagy induced by oncogenic KRAS expression, termed RAS-induced non-canonical autophagy via ATG8ylation (RINCAA). RINCAA involves distinct autophagic factors compared to those in starvation-induced autophagy and incorporates non-autophagic components, resulting in the formation of non-canonical autophagosomes with multivesicular/multilaminar structures labeled by ATG8 family proteins (e.g., LC3 and GABARAP). We have designated these structures as RAS-induced multivesicular/multilaminar bodies of ATG8ylation (RIMMBA). A notable feature of RINCAA is the substitution of the class III PI3K in canonical autophagy with PI4KB in RINCAA. We identified a regulatory P38-ULK1-PI4KB-WIPI2 signaling cascade governing this process, where ULK1 triggers PI4KB phosphorylation at S256 and T263, initiating PI4P production, ATG8ylation, and non-canonical autophagy. Importantly, elevated PI4KB phosphorylation at S256 and T263 was observed in RAS-mutated cancer cells and colorectal cancer specimens. Inhibition of PI4KB S256 and T263 phosphorylation led to a reduction in RINCAA activity and tumor growth in both xenograft and KPC models of pancreatic cancer, suggesting that targeting ULK1-mediated PI4KB phosphorylation could represent a promising therapeutic strategy for RAS-mutated cancers.
Multiomic quantification of the KRAS mutation dosage improves the preoperative prediction of survival and recurrence in patients with pancreatic ductal adenocarcinoma
Most cancer mutation profiling studies are laboratory-based and lack direct clinical application. For clinical use, it is necessary to focus on key genes and integrate them with relevant clinical variables. We aimed to evaluate the prognostic value of the dosage of the KRAS G12 mutation, a key pancreatic ductal adenocarcinoma (PDAC) variant and to investigate the biological mechanism of the prognosis associated with the dosage of the KRAS G12 mutation. In this retrospective cohort study, we analyzed 193 surgically treated patients with PDAC between 2009 and 2016. RNA, whole-exome, and KRAS-targeted sequencing data were used to estimate the dosage of the KRAS G12 mutant. Our prognostic scoring system included the mutation dosage from targeted sequencing ( > 0.195, 1 point), maximal tumor diameter at preoperative imaging ( > 20 mm, 1 point), and carbohydrate antigen 19-9 levels ( > 150 U/mL, 1 point). The KRAS mutation dosage exhibited comparable performance with clinical variables for survival prediction. High KRAS mutation dosages activated the cell cycle, leading to high mutation rates and poor prognosis. According to prognostic scoring systems that integrate mutation dosage with clinical factors, patients with 0 points had superior median overall survival of 97.0 months and 1-year, 3-year, and 5-year overall survival rates of 95.8%, 70.8%, and 66.4%, respectively. In contrast, patients with 3 points had worse median overall survival of only 16.0 months and 1-year, 3-year, and 5-year overall survival rates of 65.2%, 8.7%, and 8.7%, respectively. The incorporation of the KRAS G12 mutation dosage variable into prognostic scoring systems can improve clinical variable-based survival prediction, highlighting the feasibility of an integrated scoring system with clinical significance.
Nelarabine in T-cell acute lymphoblastic leukemia: intracellular metabolism and molecular mode-of-action
T-cell acute lymphoblastic leukemia (T-ALL) patients often have a poor 5-year event-free survival. The only T-ALL specific drug in clinical practice is nelarabine. A prodrug of the deoxyguanosine analog ara-G, nelarabine is a rationally designed agent selective for the treatment of T-cell malignancies. Originally approved for relapsed/refractory T-ALL, it is increasingly used in T-ALL therapy and is currently being evaluated in upfront treatment. Whilst the clinical use of nelarabine has been the topic of multiple review articles, a thorough overview of the preclinical data detailing the molecular underpinnings of its anti-leukemic activity is lacking, which is critical to inform mechanism-based use. Thus, in the present article we conducted a semi-systematic review of the literature and critically evaluated the preclinical knowledge on the molecular pharmacology of nelarabine. Whilst early studies identified ara-G triphosphate to be the principal active metabolite and nuclear DNA synthesis to be a key target, many fundamental questions remain that could inform upon future use of this therapy. These include the nature of nelarabine-induced DNA lesions and their repair, together with additional cellular targets of ara-G metabolites and their role in efficacy and toxicity. A critical avenue of research in need of development is investigation of nelarabine combination therapies, both in the context of current T-ALL chemotherapy regimens and with emerging anti-leukemic agents, and we highlight some areas to pursue. Altogether, we discuss what we can learn from the preclinical literature as a whole and present our view for future research regarding nelarabine treatment in T-ALL.
Menin maintains lysosomal and mitochondrial homeostasis through epigenetic mechanisms in lung cancer
Lysosome-mediated autophagy (including mitophagy) is crucial for cell survival and homeostasis. Although the mechanisms of lysosome activation during stress are well recognized, the epigenetic regulation of lysosomal gene expression remains largely unexplored. Menin, encoded by the MEN1 gene, is a chromatin-related protein that is widely involved in gene transcription via histone modifications. Here, we report that menin regulates the transcription of specific lysosomal genes, such as CTSB, CTSE, and TFE3, through MLL-mediated H3K4me3 reprogramming, which is necessary for maintaining lysosomal homeostasis. Menin also directly controls the expression of SQSTM1 and MAP1LC3B to maintain autophagic flux in a manner independent of AMPK/mTORC1 pathways. Furthermore, loss of menin led to mitochondrial dysfunction, elevated levels of reactive oxygen species (ROS), and genome instability. In genetically engineered mouse models, Men1 deficiency resulted in severe lysosomal and mitochondrial dysfunction and an impaired self-clearance ability, which further led to metabolite accumulation. SP2509, a histone demethylase inhibitor, effectively reversed the downregulation of lysosomal and mitochondrial genes caused by loss of Men1. Our study confirms the previously unrecognized biological and mechanistic importance of menin-mediated H3K4me3 in maintaining organelle homeostasis.
Disrupting Amh and androgen signaling reveals their distinct roles in zebrafish gonadal differentiation and gametogenesis
Sex determination and differentiation in zebrafish involve a complex interaction of male and female-promoting factors. While Dmrt1 has been established as a critical male-promoting factor, the roles of Anti-Müllerian hormone (Amh) and androgen signaling remain less clear. This study employed an estrogen-deficient zebrafish model (cyp19a1a-/-) to dissect individual and combined roles of Amh and androgen receptor (Ar) signaling in gonadal differentiation and gametogenesis. Loss of amh, but not ar, could rescue all-male phenotype of cyp19a1a-/-, leading to female or intersex, confirming the role of Amh in promoting male differentiation. This rescue was recapitulated in bmpr2a-/- but not bmpr2b-/-, supporting Bmpr2a as the type II receptor for Amh in zebrafish. Interestingly, while disruption of amh or ar had delayed spermatogenesis, the double mutant (amh-/-;ar-/-) exhibited severely impaired spermatogenesis, highlighting their compensatory roles. While Amh deficiency led to testis hypertrophy, likely involving a compensatory increase in Ar signaling, Ar deficiency resulted in reduced hypertrophy in double mutant males. Furthermore, phenotype analysis of triple mutant (amh-/-;ar-/-;cyp19a1a-/-) provided evidence that Ar participated in early follicle development. This study provides novel insights into complex interplay between Amh and androgen signaling in zebrafish sex differentiation and gametogenesis, highlighting their distinct but cooperative roles in male development.
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