Alterations In Human Cancers Research Articles

Mitochondrial Plasticity and Glucose Metabolic Alterations in Human Cancer under Oxidative Stress-From Viewpoints of Chronic Inflammation and Neutrophil Extracellular Traps (NETs).

Oxidative stress elicited by reactive oxygen species (ROS) and chronic inflammation are involved both in deterring and the generation/progression of human cancers. Exogenous ROS can injure mitochondria and induce them to generate more endogenous mitochondrial ROS to further perpetuate the deteriorating condition in the affected cells. Dysfunction of these cancer mitochondria may possibly be offset by the Warburg effect, which is characterized by amplified glycolysis and metabolic reprogramming. ROS from neutrophil extracellular traps (NETs) are an essential element for neutrophils to defend against invading pathogens or to kill cancer cells. A chronic inflammation typically includes consecutive NET activation and tissue damage, as well as tissue repair, and together with NETs, ROS would participate in both the destruction and progression of cancers. This review discusses human mitochondrial plasticity and the glucose metabolic reprogramming of cancer cells confronting oxidative stress by the means of chronic inflammation and neutrophil extracellular traps (NETs).

Abstract 2093: Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well

Abstract Background: Both colorectal and pancreatic cancers are major global health issues and among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic malignancies. The KRAS G12D mutation subtype is present in more than 40% of pancreatic ductal adenocarcinoma (PDAC). To this date there is no available targeted therapy options for patients with KRAS subtype mutations. MRTX1133 has been identified as a non-covalent, potent, and selective inhibitor of KRASG12D. This small molecule inhibitor has been shown to suppress KRASG12D signaling in cells and in vivo. Tumor Treating Fields (TTFields) therapy is a novel approach to treating cancer, the electric fields alter the behavior of cancer cells and prevent them from growing and dividing. We hypothesize that the co-application of MRTX1133 with TTFields will enhance the effects of MRTX1133 in PDAC and colorectal cancer (CRC). Martials and Methods: PDAC cell line CFPAC1 with KRAS G12 V mutation and LS513 CRC cell line with KRAS G12D Mutation were treated with same dose of MRTX1133 (500 nM) and were co-treated with 150kHz TTFields. After 48 hours western blot was used to probe for cleaved PARP, cleaved C3, phosphor-ERK and DUSP6. Results: In both KRAS G12D & KRAS G12V cell lines, synergistic upregulation of cPARP was observed following 48-hour co-treatment. Synergistic inhibition of pERK happened in KRAS G12D cell line but no change in pERK level following TTFields or MRTX1133 treatment was seen. Upregulation of cC3 in KRAS G12D happened in only co-treatment conditions. DUSP6 levels increased following MRTX1133 treatment in KRAS G12V cell line. Conclusions: The surprising synergistic upregulation of cPARP in both KRAS G12V and G12D cells following co-treatment of KRAS G12D inhibitor MRTX1133 and TTFields can bring hope for other KRAS mutation subtypes as well as G12D. the increase in cPARP in KRAS G12V with no effect on pERK is particularly interesting and demonstrates the need of more studies to investigate the mechanisms of this synergy. Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian PInho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2093.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1634.

Genetic and epigenetic alterations in human cancers

Genetic and epigenetic alterations in human cancers

Isoprenylcysteine carboxyl methyltransferase (ICMT) promotes invadopodia formation and metastasis in cancer cells

Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN, which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors.

Abstract B014: KDM4B mutations in human cancers

Abstract Homology-dependent-repair (HDR) is an essential process for the error-free correction of double-strand-breaks (DSBs). The histone demethylase KDM4B plays a role in the HDR repair process, wherein its H3K9 demethylase activity aids recruitment of repair factors such as the ATM checkpoint activator and KAT5 histone acetyltransferase and remodeler to the break site. Inhibition of KDM4B has been shown to disrupt the recruitment of ATM, and KAT5, affecting other downstream HDR factors and leading to widespread unrepaired DNA damage. Both over- and under-expression of KDM4B have implications for cancer severity, and changes in KDM4B expression have been shown to affect the expression of other oncogenes outside of the HDR pathway. We performed a pan-cancer analysis of the effects of KDM4B alterations in human cancers using data from the Catalogue of Somatic Mutations in Cancers (COSMIC). We find that KDM4B mutations occur in nearly every cancer. Further, there is a tendency of co-occurring mutations between KDM4B and several HDR genes the most significant being BRCA2. Machine learning tools did not predict any of the observed KDM4B mutations to be drivers. Our data suggests that although KDM4B does not display a driver phenotype it contributes to the destabilization of DNA damage repair machinery that causes cellular transformation and immortalization. Citation Format: Wesley A. Bush, Ruben C. Petreaca. KDM4B mutations in human cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B014.

Combination of 5-FU plus KRAS G12D inhibitor MRTX1133 against human colorectal and pancreatic cancer cells and the affects on inhibition of pERK and immune-stimulatory cytokine patterns in in KRAS G12D and KRAS G12V tumor cells.

e16301 Background: Colorectal cancer (CRC) & pancreatic cancer are among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic, colorectal malignancies. KRAS is considered an especially difficult, if not "undruggable" target. The KRAS G12D is present in more than 40% of PDAC & 34.2% of CRC cases. KRAS mutation subtypes are associated with different downstream pathways and varied clinical outcomes in cancer. MRTX1133 has been identified as a noncovalent, potent, and selective inhibitor of KRASG12D which has been shown to suppress KRASG12D signaling in cells and in vivo. We hypothesized that combination of MRTX1133 with 5-FU will be more effective at slowing or stopping tumor growth, and prolonging overall survival time for patients while allowing lower dose 5-FU with less side effects. Methods: We investigated synergistic effects on pERK inhibition and changes in immune tumor killing with 5-FU plus KRAS G12D inhibitor MRTX1133 in pancreatic and colon cancer cell lines. Human CRC (N=6) and pancreatic (N=4) cancer cell lines with different KRAS mutations were used in experiments evaluating drug effects on pERK at 6 and 24 hours. We evaluated changes at 48 hours in cytokine profiles in treated cells using a custom panel of 62 cytokines, chemokines, and growth factors associated with tumor growth, immune stimulation or immune suppression. Results: Colorectal and pancreatic cancer cell lines had IC50 sensitivities ranging from 7 to 12 microM 5-FU, and >100 nM to >5 microM for MRTX1133 (G12D N=4: LS513, 120 nM; HPAF-II, 1.8 microM; SNUC2B, 5.7 microM; PANC-1, 2.8 microM). For non-G12D, the range of IC50 for MRTX1133 was in the range of 2 to 9 microM for CRC lines with G12V, G13D, or wild- type KRAS (N=7). Synergies between 5-FU and MRTX1133 were observed regardless of presence of KRAS G12D mutation with combination indices of <0.5 indicating strong synergy. pERK was suppressed by MRTX1133 treatment of tumor cells regardless of KRAS G12D mutation. IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha were reduced while IL-18/IL-1F4 increased. Conclusions: Our studies reveal strong synergy between MRTX1133 & 5-FU in human pancreatic & colon cancer models at much lower than IC50 dosage which is important for avoiding side effects. This is the first description that effect of KRAS G12D inhibitor MRTX1133 is active on KRAS G12V. The surprising synergy in KRAS G12V samples with combination therapy and the important synergistic change in cytokine patterns suggests potential strong immune stimulatory anti-cancer effects of MRTX1133 & 5FU against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation which should be considered when including patients with respective mutations in clinical trials.

BET Inhibition Sensitizes Immunologically Cold Rb-Deficient Prostate Cancer to Immune Checkpoint Blockade.

Non-T-cell-inflamed immunologically "cold" tumor microenvironments (TME) are associated with poor responsiveness to immune checkpoint blockade (ICB) and can be sculpted by tumor cell genomics. Here, we evaluated how retinoblastoma (Rb) tumor-suppressor loss-of-function (LOF), one of the most frequent alterations in human cancer and associated with lineage plasticity, poor prognosis, and therapeutic outcomes, alters the TME, and whether therapeutic strategies targeting the molecular consequences of Rb loss enhance ICB efficacy. We performed bioinformatics analysis to elucidate the impact of endogenous Rb LOF on the immune TME in human primary and metastatic tumors. Next, we used isogenic murine models of Rb-deficient prostate cancer for in vitro and in vivo mechanistic studies to examine how Rb loss and bromodomain and extraterminal (BET) domain inhibition (BETi) reprograms the immune landscape, and evaluated in vivo therapeutic efficacy of BETi, singly and in combination with ICB and androgen deprivation therapy. Rb loss was enriched in non-T-cell-inflamed tumors, and Rb-deficient murine tumors demonstrated decreased immune infiltration in vivo. The BETi JQ1 increased immune infiltration into the TME through enhanced tumor cell STING/NF-κB activation and type I IFN signaling within tumor cells, resulting in differential macrophage and T-cell-mediated tumor growth inhibition and sensitization of Rb-deficient prostate cancer to ICB. BETi can reprogram the immunologically cold Rb-deficient TME via STING/NF-κB/IFN signaling to sensitize Rb-deficient prostate cancer to ICB. These data provide the mechanistic rationale to test combinations of BETi and ICB in clinical trials of Rb-deficient prostate cancer.

Abstract 2607: Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by comprehensive mRNA expression analysis

Abstract TP53 mutations are the most common single gene alteration in human cancer with diagnostic and prognostic implications in some cancer types. While no TP53-targeting therapeutics have been approved in the USA or Europe yet, drugs tailored to specific TP53 mutations, restoring the functionality of mutated TP53 (TP53mut), and protecting TP53 from negative regulation are being explored in preclinical studies and clinical trials. We performed a comprehensive mRNA expression analysis in 24 cancer types of the TCGA to extract (i) a consensus expression signature shared across TP53 mutation types and cancer types, (ii) differential gene expression patterns between different TP53 mutation types such as LOF, GOF, as well as dominant-negative mutations, and (iii) cancer types specific gene expression patterns. Mutational hotspots showed a similar pattern across cancer types, but at the same time prevalence was significantly different between cancer types for about half of the most prevalent hotspots. Both can be explained in part by the mutational processes operational for example the clock-like process behind SBS1 that is operational across cancer types and aflatoxin exposure assocated with SBS24 that ist operational in liver hepatocellular carcinoma. Virtually no genes were differential between tumors harboring different types of TP53 mutations in none of the cancer types, while hundreds of genes were over- and underexpressed in TP53mut compared to TP53wt tumors. A consensus gene list of 178 common over- and 32 common underexpressed genes was shared between at least two-thirds of the 24 cancer types. Analysis of the immune tumor microenvironment revealed exclusively decreased immune cell populations in the TP53mut tumors in six, a mixed pattern in four, exclusively increased immune cell populations in two, and no significant alterations in 20 cancer subtypes. The analysis of a large cohort of human tumors complements results from experimental studies and support the development of novel strategies for therapeutic targeting of TP53mut. Citation Format: Jan Budczies, Eva Romanovsky, Klaus Kluck, Iordanis Ourailidis, Michael Menzel, Susanne Beck, Markus Ball, Daniel Kazdal, Petros Christopoulos, Peter Schirmacher, Thorsten Stiewe, Albrecht Stenzinger. Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by comprehensive mRNA expression analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2607.

ARID1A in cancer: Friend or foe?

ARID1A belongs to a class of chromatin regulatory proteins that function by maintaining accessibility at most promoters and enhancers, thereby regulating gene expression. The high frequency of ARID1A alterations in human cancers has highlighted its significance in tumorigenesis. The precise role of ARID1A in cancer is highly variable since ARID1A alterations can have a tumor suppressive or oncogenic role, depending on the tumor type and context. ARID1A is mutated in about 10% of all tumor types including endometrial, bladder, gastric, liver, biliopancreatic cancer, some ovarian cancer subtypes, and the extremely aggressive cancers of unknown primary. Its loss is generally associated with disease progression more often than onset. In some cancers, ARID1A loss is associated with worse prognostic features, thus supporting a major tumor suppressive role. However, some exceptions have been reported. Thus, the association of ARID1A genetic alterations with patient prognosis is controversial. However, ARID1A loss of function is considered conducive for the use of inhibitory drugs which are based on synthetic lethality mechanisms. In this review we summarize the current knowledge on the role of ARID1A as tumor suppressor or oncogene in different tumor types and discuss the strategies for treating ARID1A mutated cancers.

The repertoire of copy number alteration signatures in human cancer.

Copy number alterations (CNAs) are a predominant source of genetic alterations in human cancer and play an important role in cancer progression. However comprehensive understanding of the mutational processes and signatures of CNA is still lacking. Here we developed a mechanism-agnostic method to categorize CNA based on various fragment properties, which reflect the consequences of mutagenic processes and can be extracted from different types of data, including whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) array. The 14 signatures of CNA have been extracted from 2778 pan-cancer analysis of whole genomes WGS samples, and further validated with 10851 the cancer genome atlas SNP array dataset. Novel patterns of CNA have been revealed through this study. The activities of some CNA signatures consistently predict cancer patients' prognosis. This study provides a repertoire for understanding the signatures of CNA in cancer, with potential implications for cancer prognosis, evolution and etiology.

COX-2/PGE2 upregulation contributes to the chromosome 17p-deleted lymphoma

Deletions of chromosome 17p, where TP53 gene locates, are the most frequent chromosome alterations in human cancers and associated with poor outcomes in patients. Our previous work suggested that there were p53–independent mechanisms involved in chromosome 17p deletions-driven cancers. Here, we report that altered arachidonate metabolism, due to the deficiency of mouse Alox8 on chromosome 11B3 (homologous to human ALOX15B on chromosome 17p), contributes to the B cell malignancy. While the metabolites produced from lipoxygenase pathway reduced, chromosome 11B3 deletions or Alox8 loss, lead to upregulating its paralleling cyclooxygenase pathway, indicated by the increased levels of oncometabolite prostaglandin E2. Ectopic PGE2 prevented the apoptosis and differentiation of pre-B cells. Further studies revealed that Alox8 deficiency dramatically and specifically induced Cox-2(Ptgs2) gene expression. Repressing Cox-2 by its shRNAs impaired the tumorigenesis driven by Alox8 loss. And, in turn, tumor cells with Alox8 or 11B3 loss were sensitive to the COX-2 inhibitor celecoxib. This correlation between COX-2 upregulation and chromosome 17p deletions was consistent in human B-cell lymphomas. Hence, our studies reveal that the arachidonate metabolism abnormality with unbalanced ALOX and COX pathways underlies human cancers with 17p deletions and suggest new susceptibility for this disease.

The High Expression of p53 Is Predictive of Poor Survival Rather TP53 Mutation in Esophageal Squamous Cell Carcinoma.

TP53 is a well-known tumor suppressor gene and one of the most common genetic alterations in human cancers. However, the role of p53 as a prognostic marker of esophageal squamous cell carcinoma (ESCC) is controversial in the association between TP53 alterations and clinical outcomes. To address this issue, we evaluated TP53 mutations, p53 protein expression, clinicopathological parameters, and survivals rates in a large scale of patients with ESCC. Two cohorts were included in this study: TP53 mutations were detected by next-generation sequencing in 316 ESCC patients, and p53 protein expression was tested by immunohistochemistry in 6,028 ESCC patients. Survival analysis was performed using the Kaplan-Meier curve and the Cox proportional hazards model. TP53 mutations were found in ESCC patients from 241 of 316 (76.3%), and the rate of positive expression of p53 protein was 59.1% in 6,028 ESCC patients (including 1819 with high expression of p53 protein), respectively. Most mutations were missense, which has a high expression of p53 protein. Compared with wild-typeTP53, TP53 gene mutations were not significantly associated with survival time (p=0.083). In multivariate analysis, the p53 protein expression was an independent prognostic factor for ESCC. The high-expression group of p53 protein has poor survival (p < 0.001) compared to low-expression group in patients with ESCC. The high expression of the p53 protein, not the TP53 mutation, is predictive of poor survival in patients with ESCC, and p53 protein expression might have the potential to be a prognosis biomarker and therapy target in ESCC.

DrOGA: An Artificial Intelligence Solution for Driver-Status Prediction of Genomics Mutations in Precision Cancer Medicine

Precision cancer medicine suggests that better cancer treatments would be possible guiding therapies by tumor’s genomics alterations. This hypothesis boosted exome sequencing studies, collection of cancer variants databases and developing of statistical and Machine Learning-driven methods for alterations’ analysis. In order to extract relevant information from huge exome sequencing data, accurate methods to distinguish driver and neutral or passengers mutations are vital. Nevertheless, traditional variant classification methods have often low precision in favour of higher recall. Here, we propose several traditional Machine Learning and new Deep Learning techniques to finely classify driver somatic non-synonymous mutations based on a 70-features annotation, derived from medical and statistical tools. We collected and annotated a complete database containing driver and neutral alterations from various public data sources. Our framework, called Driver-Oriented Genomics Analysis (DrOGA), presents the best performances compared to individual and other ensemble methods on our data. Explainable Artificial Intelligence is used to provide visual and clinical explanation of the results, with a particular focus on the most relevant annotations. This analysis and the proposed tool, along with the collected database and the feature engineering pipeline suggested, can help the study of genomics alterations in human cancers allowing precision oncology targeted therapies based on personal data from next-generation sequencing.

A Rare Event of Liver Dysfunction on Sotorasib and Management Strategy.

KRAS mutations are the most common alteration in human cancers, accounting for approximately 30% of mutations in multiple cancer types, including colorectal, pancreatic, non-small lung cancer, and ovarian. Of these, the KRAS p.G12C mutation occurs in 13% of non-small lung cancers and 1% to 3% of colorectal and other cancers (Hong et al., 2020). With the approval of the direct KRAS p.G12C inhibitor sotorasib in early 2021, this first-in-class small-molecule agent has increased progression-free survival by 6.3 months in patients with p.G12C non-small cell lung cancer. Side effects associated with sotorasib have been mild, with the most frequent being diarrhea and nausea, but grade 3 to 4 toxicity has also been observed, which is clinically significant. Grade 3 toxicity related to aspartate aminotransferase and alanine aminotransferase is defined as an increase of more than 5 to 20 times the upper limit of normal (ULN), while grade 4 is more than 20 times the ULN. This is significant and requires withholding treatment as it can be life-threatening in some cases. The following case study outlines a patient who developed abnormal liver enzyme elevation while on the phase I clinical trial of sotorasib, and the management of this event.

164 (PB044) - Enhancement of anti-tumor immunity in immunogenic and immune-refractory RAS mutant tumors with tri-complex RAS(ON) inhibitors

RAS mutations (RASMUT), the most frequent oncogenic alterations in human cancers, promote carcinogenesis through sustained cellular proliferation and also shape the tumor microenvironment to allow cancer cells to evade an anti-tumor immune response. Some RASMUT tumors are responsive to immune checkpoint inhibition but many, such as most colorectal and pancreatic cancers, are not. A rational approach to treating RASMUT tumors likely would be to combine optimized targeted therapy, including direct inhibition of RASMUT itself, with effective immunologic therapy.

Ufl1 deficiency causes skin pigmentation by up-regulation of Endothelin-1.

Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.

Extracellular 5′-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5′-deoxy-5′-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

Signatures of copy number alterations in human cancer

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage–fusion–bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3–5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Abstract 3938: Discovery of a novel small molecule inhibitor of the YAP1/TAZ-TEAD transcriptional complex

Abstract Hippo pathway alterations in human cancers often result in dephosphorylation of yes-associated protein (YAP1) and its paralog TAZ (WWTR1), allowing the formation of an active complex with transcriptional enhanced associate domain transcription factors (TEADs). This complex formation results in the activation of pro-survival and pro-proliferative transcriptional programs in cancer cells. Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of tumors are driven by YAP1/TEAD activity. Although traditionally difficult to drug with small molecules, identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs necessary for YAP1 interaction has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TAZ-TEAD complex formation and transcriptional activity. We report the discovery and characterization of the novel YAP1/TAZ-TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TAZ-TEAD activity. In biochemical thermal shift assays, MRK-A caused a concentration-dependent melting temperature shift of 8-12.5 and 0.6-1.5 degrees for TEAD1 and TEAD2, respectively, indicating direct binding to TEAD protein. In cellular assays, MRK-A demonstrated inhibition of a TEAD-based reporter assay, with little to no activity in multiple orthogonal off-target reporter assays such as WNT, NF-KB, TGFB and PPARG (8.4 nM vs. &amp;gt;10000 nM), which is consistent with the exquisite selectivity profile of this molecule (&amp;gt;1000x selectivity against 350+ measured kinases and other common off-targets). In the NF2-deficient mesothelioma cell line H226, MRK-A suppressed the transcription of endogenous YAP/TAZ-TEAD target genes CYR61, ERBB3, ANKRD1 and CTGF (50-75% inhibition at 100 nM), but not LATS1, a non-TEAD regulated Hippo pathway gene. In co-immunoprecipitation assays, MRK-A disrupted the interaction of YAP1 and TEAD in H226 cells at concentrations consistent with inhibition of target genes. In addition, MRK-A potently blocked the clonogenic growth and viability of H226 cells in a dose-dependent manner (maximal response at 1 µM compound &amp;gt;90% growth inhibition), while sparing the Hippo wild-type mesothelioma cell line H28. Furthermore, structurally similar control compounds, MRK-B and MRK-C, without the ability to block TEAD-mediated transcription (TEAD reporter MCF7 assay IC50 &amp;gt; 10000 nM), did not impact the clonogenic growth of H226 cells. In vivo, MRK-A did not show acute tolerability signals in mice and demonstrated pharmacokinetics suitable for daily oral dosing in efficacy studies. In summary, we report the structure and characterization of MRK-A demonstrating potent and specific inhibition of YAP1/TAZ-TEAD mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling. Citation Format: Casey J. Moure, Christopher Sondey, Mangeng Cheng, My Mansueto, Rafael Fernandez, Sebastian E. Schneider, Julia V. Ramirez, Brian Long, Erin DiMauro, Brandon Vara, Charles Yeung, Abe Achab, Jongwon Lim, Ronald Kim, Cayetana Zarate, Jonathan Bennett, Rachel Palte, Robert Foti, Vladimir Simov, Evan Barry. Discovery of a novel small molecule inhibitor of the YAP1/TAZ-TEAD transcriptional complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3938.