GRAIL1 Stabilizes Misfolded Mutant p53 through a Ubiquitin Ligase-Independent, Chaperone Regulatory Function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's esophagus progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL-gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But, how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here, we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for heat shock protein 40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain or a cell-permeable peptide (Pep-J) encoding the above 10 amino acids can bind and inhibit DNAJ-Hsp70 co-chaperone activity, thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic Barrett's esophagus and EAC cells and inhibit the growth of patient-derived organoids of dysplastic Barrett's esophagus in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J are comparable with simvastatin, a cholesterol-lowering drug that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase-independent, chaperone-regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

Isoform alterations in the ubiquitination machinery impacting gastrointestinal malignancies.

The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote tumorigenesis. An active area in cancer research is uncovering the roles of ubiquitination on spliceosome assembly contributing to transcript diversity and expression of alternative isoforms. However, the effects of isoform changes on functionality of ubiquitination machineries (E1, E2, E3, E4, and deubiquitinating (DUB) enzymes) influencing onco- and tumor suppressor protein stabilities is currently understudied. Characterizing these changes could be instrumental in improving cancer outcomes via the identification of novel biomarkers and targetable signaling pathways. In this review, we focus on highlighting reported examples of direct, protein-coded isoform variation of ubiquitination enzymes influencing cancer development and progression in gastrointestinal (GI) malignancies. We have used a semi-automated system for identifying relevant literature and applied established systems for isoform categorization and functional classification to help structure literature findings. The results are a comprehensive snapshot of known isoform changes that are significant to GI cancers, and a framework for readers to use to address isoform variation in their own research. One of the key findings is the potential influence that isoforms of the ubiquitination machinery have on oncoprotein stability.

A model-based clustering algorithm with covariates adjustment and its application to lung cancer stratification.

Usually, the clustering process is the first step in several data analyses. Clustering allows identify patterns we did not note before and helps raise new hypotheses. However, one challenge when analyzing empirical data is the presence of covariates, which may mask the obtained clustering structure. For example, suppose we are interested in clustering a set of individuals into controls and cancer patients. A clustering algorithm could group subjects into young and elderly in this case. It may happen because the age at diagnosis is associated with cancer. Thus, we developed CEM-Co, a model-based clustering algorithm that removes/minimizes undesirable covariates' effects during the clustering process. We applied CEM-Co on a gene expression dataset composed of 129 stage I non-small cell lung cancer patients. As a result, we identified a subgroup with a poorer prognosis, while standard clustering algorithms failed.

CircSMARCA5 silencing impairs cell proliferation and invasion via the miR-17-3p-EGFR signaling in lung adenocarcinoma.

AIMS: Circular RNAs are widely expressed in various cancers and play important roles in tumorigenesis and tumor progression. The function and mechanism of circSMARCA5 in lung adenocarcinoma however remains unclear. MAIN METHODS: QRT-PCR analysis was applied for determining circSMARCA5 expression in lung adenocarcinoma patient tumor tissues and cells. Molecular biological assays were used for investigating the role of circSMARCA5 in lung adenocarcinoma progression. Luciferase reporter and bioinformatics assays were used for identifying the underlying mechanism. KEY FINDINGS: In this study, we observed that circSMARCA5 expression was decreased in lung adenocarcinoma tissues but silencing of circSMARCA5 in lung adenocarcinoma cells inhibited cell proliferation, colony formation, migration and invasion. Mechanistically, we found EGFR, c-MYC and p21 were down-regulated upon circSMARCA5 knockdown. MiR-17-3p efficiently down- regulated EGFR expression via directly binding to EGFR mRNA. SIGNIFICANCE: These studies suggest that circSMARCA5 functions as an oncogene via targeting miR-17-3p-EGFR axis and may represent a promising therapeutic target for lung adenocarcinoma.

Characterizing isoform switching events in esophageal adenocarcinoma.

Isoform switching events with predicted functional consequences are common in many cancers, but characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was used to detect levels of RNA transcripts and identify specific isoforms in treatment-naïve esophageal tissues ranging from premalignant Barrett's esophagus (BE), BE with low- or high-grade dysplasia (BE.LGD, BE.HGD), and EAC. Samples were stratified by histopathology and TP53 mutation status, identifying significant isoform switching events with predicted functional consequences. Comparing BE.LGD with BE.HGD, a histopathology linked to cancer progression, isoform switching events were identified in 75 genes including KRAS, RNF128, and WRAP53. Stratification based on TP53 status increased the number of significant isoform switches to 135, suggesting switching events affect cellular functions based on TP53 mutation and tissue histopathology. Analysis of isoforms agnostic, exclusive, and shared with mutant TP53 revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. Nearly half of isoform switching events were identified without significant gene-level expression changes. Importantly, two TP53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC.

FAM83A antisense RNA 1 (FAM83A-AS1) silencing impairs cell proliferation and induces autophagy via MET-AMPKɑ signaling in lung adenocarcinoma.

Studies demonstrate that long non-coding RNAs (lncRNAs) play vital roles in cancer progression. However, the expression pattern and molecular mechanisms of lncRNA FAM83A-AS1 in lung cancer remain largely unclear. Here, we analyzed FAM83A-AS1 expression in lung cancer tissues from three RNA-sequencing (RNA-Seq) datasets and validated these results using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in an independent set of lung adenocarcinoma. Cell proliferation, migration, invasion, and autophagy were analyzed after knockdown FAM83A-AS1 with siRNAs. The underlying molecular mechanisms of FAM83A-AS1 were performed by Western blot, qRT-PCR, and RNA-seq analysis. We found that FAM83A-AS1 was up-regulated in lung cancer and elevated expression was associated with poor patient survival. These results were confirmed using RT-PCR in an independent set of lung cancer. Functional study indicated that FAM83A-AS1 knockdown reduced cell proliferation, migration, invasion, and colony formation in cancer cells. FAM83A-AS1 silencing induced autophagy and cell cycle arrest at G2. Mechanistically, serval oncogenic proteins such as EGFR, MET, PI3K, and K-RAS were decreased upon FAM83A-AS1 silencing, while phosphor AMPKα and ULK1 were increased. Based on the above results, we believe that FAM83A-AS1 may have potential as a diagnosis/prognosis marker and its oncogenic role and autophagy regulation may be through MET-AMPKα signaling, which could lead to potential targeting for lung cancer therapy.

Detection of Barrett's neoplasia with a near-infrared fluorescent heterodimeric peptide.

BACKGROUND: Esophageal adenocarcinoma (EAC) is a molecularly heterogeneous disease with poor prognosis that is rising rapidly in incidence. We aimed to demonstrate specific binding by a peptide heterodimer to Barrett's neoplasia in human subjects. METHODS: Peptide monomers specific for EGFR and ErbB2 were arranged in a heterodimer configuration and labeled with IRDye800. This near-infrared (NIR) contrast agent was topically administered to patients with Barrett's esophagus (BE) undergoing either endoscopic therapy or surveillance. Fluorescence images were collected using a flexible fiber accessory passed through the instrument channel of an upper gastrointestinal endoscope. Fluorescence images were collected from 31 BE patients. A deep learning model was used to segment the target (T) and background (B) regions. RESULTS: The mean target-to-background (T/B) ratio was significantly greater for high grade dysplasia (HGD) and EAC versus BE, low grade dysplasia (LGD), and squamous epithelium. At a T/B ratio of 1.5, sensitivity and specificity of 94.1 % and 92.6 %, respectively, were achieved for the detection of Barrett's neoplasia with an area under the curve of 0.95. No adverse events attributed to the heterodimer were found. EGFR and ErbB2 expression were validated in the resected specimens. CONCLUSIONS: This "first-in-human" clinical study demonstrates the feasibility of detection of early Barrett's neoplasia using a NIR-labeled peptide heterodimer.

UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma.

BACKGROUND & AIMS: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. METHODS: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. RESULTS: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate ß-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. CONCLUSIONS: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

CSE1L silencing impairs tumor progression via MET/STAT3/PD-L1 signaling in lung cancer.

CSE1L is involved in the cancer progression of several types of cancer. Its expression status, potential oncogenic role and underlying mechanism in lung cancer, however, are unclear. Here, we investigated CSE1L expression in primary lung adenocarcinoma based on multiple datasets and then investigated its oncologic role in lung cancer. We also examined the potential molecular mechanisms of CSE1L in cancer progression. CSE1L levels were increased in cancer as compared to normal lung tissues. CSE1L expression was higher in poorly-differentiated late stage and lymph node positive metastatic tumors. Higher CSE1L level was correlated with worse patient outcome. Knockdown of CSE1L using siRNAs impaired cell proliferation, invasion, migration and induced cell apoptosis. Mechanistically, MET, STAT3 and PD-L1 proteins were decreased upon CSE1L silencing. These results suggest that CSE1L may affect tumor progression through MET/STAT3/PD-L1 signaling. CSE1L may have potential as a biomarker and therapeutic target for lung cancer.

Collagen modifying enzyme P4HA1 is overexpressed and plays a role in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related deaths globally and is histologically defined as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), with the latter accounting for 80% of all lung cancers. The 5-year overall survival rate for lung cancer patients is low as it is often discovered at advanced stages when potential cure by surgical resection is no longer an option. To identify a biomarker and target for lung cancer, we performed analysis of multiple datasets of lung cancer gene expression data. Our analyses indicated that the collagen-modifying enzyme Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1) is overexpressed in NSCLC. Furthermore, our investigation found that overexpression of enzymes involved in this pathway predicts poor outcome for patients with lung adenocarcinoma. Our functional studies using knockdown strategies in lung cancer cell lines in vitro indicated that P4HA1 is critical for lung cancer growth, migration, and invasion. Additionally, diethyl pythiDC (PythiDC), a small molecule inhibitor, decreased the malignant phenotypes of lung cancer cells. Moreover, we found that miR-124 regulates and targets P4HA1 in lung cancer cells. Thus, our study suggests that collagen-modifying enzymes play an important role in lung cancer aggressiveness. Furthermore, our studies showed that P4HA1 is required for lung cancer cell growth and invasion, suggesting its potential as a valid therapeutic target in lung adenocarcinoma.

MiR-22-3p suppresses cell growth via MET/STAT3 signaling in lung cancer.

MiR-22-3p has been reported to be down-regulated in several cancers, but its expression pattern and roles in lung cancer is unclear. Given the crucial role of microRNAs in cancer progression, we examined the expression and function of miR-22-3p in lung adenocarcinoma. MiR-22-3p expression in lung cancer tissues and cell lines was measured by qRT-PCR. Cell proliferation was measured by WST-1 and colony formation assays were used to reveal the role of miR-22-3p in lung cancer in vitro. MiR-22-3p was notably down-regulated in lung cancer tissues as compared to normal lung tissues, but it was not associated with the clinical characteristics of tumor stage, differentiation and patient's smoking status. Colony formation ability and cell proliferation were suppressed by miR-22-3p mimics in lung cancer cell lines. Mechanistically, miR-22-3p mimics could reduce MET and STAT3 protein expression and induce apoptosis as measured by PARP protein. We conclude that miR-22-3p may play a tumor suppressor role via inhibiting MET-STAT3 signaling and have potential to be a therapeutic target and biomarker in lung adenocarcinoma.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett's progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro-B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett's progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

Chaperones and Ubiquitin Ligases Balance Mutant p53 Protein Stability in Esophageal and Other Digestive Cancers.

The incidence of esophageal adenocarcinoma (EAC) and other gastrointestinal (GI) cancers have risen dramatically, thus defining the oncogenic drivers to develop effective therapies are necessary. Patients with Barrett's Esophagus (BE), have an elevated risk of developing EAC. Around 70%-80% of BE cases that progress to dysplasia and cancer have detectable TP53 mutations. Similarly, in other GI cancers higher rates of TP53 mutation are reported, which provide a significant survival advantage to dysplastic/cancer cells. Targeting molecular chaperones that mediate mutant p53 stability may effectively induce mutant p53 degradation and improve cancer outcomes. Statins can achieve this via disrupting the interaction between mutant p53 and the chaperone DNAJA1, promoting CHIP-mediated degradation of mutant p53, and statins are reported to significantly reduce the risk of BE progression to EAC. However, statins demonstrated sub-optimal efficacy depending on cancer types and TP53 mutation specificity. Besides the well-established role of MDM2 in p53 stability, we reported that individual isoforms of the E3 ubiquitin ligase GRAIL (RNF128) are critical, tissue-specific regulators of mutant p53 stability in BE progression to EAC, and targeting the interaction of mutant p53 with these isoforms may help mitigate EAC development. In this review, we discuss the critical ubiquitin-proteasome and chaperone regulation of mutant p53 stability in EAC and other GI cancers with future insights as to how to affect mutant p53 stability, further noting how the precise p53 mutation may influence the efficacy of treatment strategies and identifying necessary directions for further research in this field.

LINC00857 Interacting with YBX1 to Regulate Apoptosis and Autophagy via MET and Phosphor-AMPKa Signaling.

Long noncoding RNA (lncRNA) LINC00857 has been reported to be upregulated in lung cancer and related to poor patient survival. It can regulate cell proliferation and tumor growth in lung cancer as well as several other cancers. However, the underlying molecular mechanisms that are regulated by LINC00857 are unclear. In this study, we found that LINC00857 silencing can impair cell proliferation in 14 different genomic alterations of lung cancer cell lines. These alterations are EGFR, KRAS, TP53, MET, and LKB1 mutations. The cell apoptosis and autophagy were induced upon LINC00857 silencing in lung cancer cells. Mechanistically, LINC00857 can bind to the Y-box binding protein 1 (YBX1) protein, prevent it from proteasomal degradation, and increase its nuclear translocation. LINC00857 regulated MET expression via YBX1 at a transcriptional level. Induced cell autophagy by LINC00857 knockdown was mainly through increased phosphor-AMP-activated protein kinase (p-AMPK)a. Collectively, LINC00857-YBX1-MET/p-AMPKa signaling is critical to regulate cell proliferation, apoptosis, and autophagy, which may provide a potential clinically therapeutic target in lung cancer.

Silencing of LOC389641 impairs cell proliferation and induces autophagy via EGFR/MET signaling in lung adenocarcinoma.

High-throughput RNA-sequencing studies of tumor samples have identified a large number of long non-coding RNAs (lncRNAs) which are associated with various types of cancer. LncRNAs play key roles in regulating chromatin dynamics, gene expression, growth, differentiation and development. However, the role of LOC389641 in non-small cell lung cancer (NSCLC) tumorigenesis is not clear. Here, we investigated the expression pattern, roles and mechanism of LOC389641 in lung cancer. LOC389641 expressions in tumor tissues and cell lines were measured by qRT-PCR. Functional studies including colony formation, cell proliferation and invasion were performed in lung cancer cell lines and Western blot was used to exam the protein changes upon siRNA treatment. We found that LOC389641 was highly expressed in lung adenocarcinomas and was associated with poor patient survival. Silencing of LOC389641 reduced colony formation, cell proliferation and invasion, as well as induced autophagy and apoptosis of lung adenocarcinoma cell lines in vitro. Mechanistically, downregulation of LOC389641 was found to decrease EGFR, MET and STAT3 proteins expression in lung cancer cells. LOC389641 is highly expressed and plays an oncogenic role in this type of NSCLC. Because of its specificity, LOC389641 may be a potential biomarker for prognosis and a possible target for lung adenocarcinoma.

Multi-Institutional Prospective Validation of Prognostic mRNA Signatures in Early Stage Squamous Lung Cancer (Alliance).

INTRODUCTION: Surgical resection is curative for some patients with early lung squamous cell carcinoma. Staging and clinical factors do not adequately predict recurrence risk. We sought to validate the discriminative performance of proposed prognostic gene expression signatures at a level of rigor sufficient to support clinical use. METHODS: The two-stage validation used independent core laboratories, objective quality control standards, locked test parameters, and large multi-institutional specimen and data sets. The first stage validation confirmed a signature's ability to stratify patient survival. The second-stage validation determined which signature(s) optimally improved risk discrimination when added to baseline clinical predictors. Participants were prospectively enrolled in institutional (cohort I) or cooperative group (cohort II) biospecimen and data collection protocols. All cases underwent a central review of clinical, pathologic, and biospecimen parameters using objective criteria to determine final inclusion (cohort I: n = 249; cohort II: n = 234). Primary selection required that a signature significantly predict a 3-year survival after surgical resection in cohort I. Signatures meeting this criterion were further tested in cohort II, comparing risk prediction using baseline risk factors alone versus in combination with the signature. RESULTS: Male sex, advanced age, and higher stage were associated with shorter survival in cohort I and established a baseline clinical model. Of the three signatures validated in cohort I, one signature was validated in cohort II and statistically significantly enhanced the prognosis relative to the baseline model (C-index difference 0.122; p < 0.05). CONCLUSIONS: These results represent the first rigorous validation of a test appropriate to direct adjuvant treatment or clinical trials for patients with lung squamous cell carcinoma.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.