Ras MAPK Research Articles

ADT-1004: A First-in-Class, Orally Bioavailable Selective pan-RAS Inhibitor for Pancreatic Ductal Adenocarcinoma.

ADT-1004 displayed robust antitumor activity in aggressive and clinically relevant PDAC models with unique tumor specificity to block RAS activation and MAPK signaling in RAS mutant cells. As a pan-RAS inhibitor, ADT-1004 has broad activity and potential efficacy advantages over allele-specific KRAS inhibitors by averting resistance. These findings support clinical trials of ADT-1004 for KRAS mutant PDAC.

Abstract C036: Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma

Abstract PDAC growth is characterized by dependencies on both mutant KRAS signaling and autophagy. Our group and others previously demonstrated that inhibition of the RAF/MEK/ERK pathway in PDAC induces an increased dependence on autophagy, a metabolic nutrient scavenging process by which cellular components are recycled in times of nutrient stress. Combined inhibition of the ERK MAPK pathway and autophagy inhibited the growth of multiple preclinical models of PDAC. Based on these findings, combined ERK/MEK inhibition and hydroxychloroquine (HCQ) is currently under clinical evaluation (NCT03825289, NCT04386057). However, resistance to this combination has already been described. To identify sensitizers to CQ treatment, our lab performed a CRISPR-Cas9 mediated loss-of- function screen using an sgRNA library targeting genes associated with cancer signaling pathways. Surprisingly, we identified multiple autophagy related genes, indicating that concurrent inhibition of two nodes of the autophagy pathway may be a more effective method of inhibiting autophagy in PDAC. For example, we identified, PIK3C3, the gene that encodes for VPS34, a protein essential for the nucleation of autophagosomes, as a potential sensitizer to CQ treatment. Inhibition of VPS34 with the compound SAR405, resulted in both decreased autophagic flux and impaired proliferation. Additionally, VPS34 inhibition sensitized cells to CQ treatment, leading to further reduced proliferation. These results prompted us to hypothesize that inhibition of ULK1, a serine/threonine kinase critical for the initiation of autophagy, could also sensitize PDAC cells to CQ treatment. We found that ULK1 inhibition further reduced CQ-mediated anti-proliferative effects. Additionally, both ULK1 and VPS34 inhibitor treatment sensitized cells to PIKfyve inhibition with apilimod, a chemically and mechanistically distinct inhibitor of the termination phase of the autophagic pathway. Previous studies have indicated that autophagy inhibition alone is not a viable therapeutic avenue for the treatment of PDAC. Thus, we next determined whether vertical inhibition of the autophagy pathway improves the efficacy of combined inhibition of the RAS and autophagy pathway. We found that both VPS34 inhibition and ULK1 inhibition can decrease RAS inhibitor induced autophagic flux. Finally, vertical inhibition of the autophagic pathway via VPS34 inhibition and CQ sensitized PDAC cells to RAS inhibition to further reduce PDAC proliferation. Ongoing and future studies are aimed at elucidating the mechanism leading to decreased proliferation of combined anti-autophagy and RAS inhibitor therapies, as well as mechanistically understanding the effect of autophagy inhibition at multiple nodes on autophagic flux. Citation Format: Mallory K Roach, Jonathan M DeLiberty, Elyse G Schechter, Runying Yang, Noah L Pieper, Clint A Stalnecker, Kirsten L Bryant. Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C036.

Phenotypic Expansion of Autosomal Dominant LZTR1-Related Disorders with Special Emphasis on Adult-Onset Features.

Leucine zipper-like transcription regulator 1 (LZTR1) acts as a negative factor that suppresses RAS function and MAPK signaling; mutations in this protein may dysregulate RAS ubiquitination and lead to impaired degradation of RAS superfamily proteins. Germline LZTR1 variants are reported in Noonan syndrome, either autosomal dominant or autosomal recessive, and in susceptibility to schwannomatosis. This article explores the genetic and phenotypic diversity of the autosomal dominant LZTR1-related disorders, compiling a cohort of previously published patients (51 with the Noonan phenotype and 123 with schwannomatosis) and presenting two additional adult-onset cases: a male with schwannomatosis and Parkinson's disease and a female with Noonan syndrome, generalized joint hypermobility, and breast cancer. This review confirms that autosomal dominant LZTR1-related disorders exhibit an extreme phenotypic variability, ranging from relatively mild manifestations to severe and multi-systemic involvement, and offers updated frequences of each clinical feature. The aim is to precisely define the clinical spectrum of LZTR1-related diseases, using also two new emblematic clinical cases. Gaining insight into the mechanisms underneath this variability is crucial to achieve precision diagnostics and the development of therapeutic interventions.

NFS-13. UNUSUAL NEUROFIBROMATOSIS TYPE 1-ASSOCIATED PILOCYTIC ASTROCYTOMAWITH MALIGNANT TRANSFORMATION IN A 7-YEAR-OLD GIRL

Abstract INTRODUCTION Neurofibromatosis type 1(NF1) is a tumor predisposition syndrome with germline NF 1 gene aberrations with overactivation of the RAS–MAPK signaling pathway. NF1-associated gliomas in children are usually low-grade Glioma with indolent course. Malignant transformation in NF1 associated LGG is predominated in adults is scarce in children METHOD A 7-year-old girl diagnosed as NF1-based on diagnostic criteria and germline NF1 gene mutation, presented with progressive RT side hemiplegia, Brain MRI showed a large left thalamic cystic and solid mass and obstructive hydrocephalus she had VPS insertion and stereotactic biopsy.pathological examination revealed aggressive High-grade Glioma HGG features diffuse brain infiltration, endothelial hyperplasia, increased mitotic activity necrosis, and High Ki67. Immunohistochemically and molecular analysis showed NF1 mutation in addition to TP53 mutation, retained ATRX, negative CDKN2A/B, and high tumor mutational burden (6 Muts/Mb). Tumor methylation profiling (DKFZ CNS v12.5) classified as Supratentorial midline pilocytic astrocytoma (score 0.6878). debulking of the tumor and ommaya reservoir insertion was done with recurrent aspiration of the cystic component. The second biopsy demonstrated low-grade pilocytic astrocytoma, confirmed by methylation profiling (score 0.9377). Immunohistochemically and molecular analysis showed NF1 mutation, negative TP53, and Retained ATRX RESULT She was treated with low-grade glioma chemotherapy carboplatin/ vincristine, with improved clinical and neurological condition and stable MRI evaluation. One year after diagnosis while on maintenance LGG chemotherapy MRI imaging showed tumor progression Subtotal resection of the mass was performed, morphology and molecular studies confirmed HGG, and she received focal radiation therapy. Up till now, the patient is currently stable clinically and by imaging surveillance. CONCLUSION Our case characterized a transformed glioma in a child with NF1 illustrating the intratumoral heterogeneity and presence of transformed/anaplastic clone within NF1-associated pilocytic astrocytoma and demonstrate the complexity of molecular genetics of gliomas in children with NF1.

Environmental enrichment and the combined interventions of EE and metformin enhance hippocampal neuron survival and hippocampal-dependent memory in type 2 diabetic rats under stress through the BDNF-TrkB signaling pathways

BackgroundType 2 diabetes (T2D) with depression causes severe cognitive impairments. The devastating conditions will further compromise the overall quality of life. The overconsumption of high-fat and high-sucrose (HFS) diet is one of the modifiable risk factors for T2D, depression, and cognitive impairments. Thus, it is essential to identify effective therapeutic strategies to overcome the cognitive impairments in T2D with depression. We proposed environmental enrichment (EE) which encompasses social, cognitive, and physical components as the alternative treatment for such impairments. We also investigated the potential neuroprotective properties of the antidiabetic drug metformin. This study aimed to investigate the effects of EE and metformin interventions on hippocampal neuronal death, and hippocampal-dependent memory impairment in T2D rats under stress. MethodsThirty-two male rats (200–250 g) were divided into four groups: C group (standard diet + conventional cage), DS group [HFS-induced T2D + restraint stress (RS)], DSE group [HFS-induced T2D + RS + EE] and DSEM group [HFS + RS + EE + metformin]. Serum corticosterone (CORT) was measured to evaluate stress levels. The serum Free Oxygen Radicals Testing (FORT) and Free Oxygen Radicals Defence Test (FORD) were measured to evaluate the systemic oxidative status (OS). Serum brain-derived neurotrophic factor (BDNF) and T-maze tasks were performed to evaluate cognitive functions. Rats were humanely sacrificed to collect brains for histological, morphometric, and hippocampal gene expression studies. ResultsThe CORT and the serum FORT levels in the DSE and DSEM groups were lower than in the DS group. Meanwhile, the serum BDNF, T-maze scores, histological, and morphometric analysis were improved in the DSE and DSEM groups than in the DS group. These findings supported that EE and the combined interventions of EE and metformin had neuroprotective properties. The hippocampal gene expression analysis revealed that the DSE and DSEM groups showed improved regulation of BDNF-TrkB signalling pathways, including the BDNF/TrkB binding, PI3K – Akt pathway, Ras–MAPK pathway, PLCγ–Ca2+ pathway, and CREB transcription. ConclusionEE and the combined interventions of EE and metformin improved hippocampal neuron survival and hippocampal-dependent memory in T2D rats under stress by enhancing gene expression regulation of neurogenesis and synaptic plasticity.

The CNK–HYP scaffolding complex promotes RAF activation by enhancing KSR–MEK interaction

The RAS–MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1–BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR–MEK interaction, which in turn enhances RAF–KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR–MEK–CNK–HYP complex reveals a ring-like arrangement of the CNK–HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR–MEK interaction.

Divarasib plus cetuximab in KRAS G12C-positive colorectal cancer: a phase 1b trial

KRAS G12C mutation is prevalent in ~4% of colorectal cancer (CRC) and is associated with poor prognosis. Divarasib, a KRAS G12C inhibitor, has shown modest activity as a single agent in KRAS G12C-positive CRC at 400 mg. Epidermal growth factor receptor has been recognized as a major upstream activator of RAS–MAPK signaling, a proposed key mechanism of resistance to KRAS G12C inhibition in CRC. Here, we report on divarasib plus cetuximab (epidermal growth factor receptor inhibitor) in patients with KRAS G12C-positive CRC (n = 29) from arm C of an ongoing phase 1b trial. The primary objective was to evaluate safety. Secondary objectives included preliminary antitumor activity. The safety profile of this combination was consistent with those of single-agent divarasib and cetuximab. Treatment-related adverse events led to divarasib dose reductions in four patients (13.8%); there were no treatment withdrawals. The objective response rate was 62.5% (95% confidence interval: 40.6%, 81.2%) in KRAS G12C inhibitor-naive patients (n = 24). The median duration of response was 6.9 months. The median progression-free survival was 8.1 months (95% confidence interval: 5.5, 12.3). As an exploratory objective, we observed a decline in KRAS G12C variant allele frequency associated with response and identified acquired genomic alterations at disease progression that may be associated with resistance. The manageable safety profile and encouraging antitumor activity of divarasib plus cetuximab support the further investigation of this combination in KRAS G12C-positive CRC.ClinicalTrials.gov identifier: NCT04449874

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS–MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

P23 Stabilization of aggressive disease in NRAS kaposiform lymphangiomatosis with sirolimus

Abstract Kaposiform lymphangiomatosis (KLA) is a rare disorder of lymphatic vasculature characterized by infiltrative and multifocal lymphatic abnormalities. The disease course is often complicated by a severe coagulopathy and frequently involves the central conducting lymphatic vessels leading to large-volume chylous pleural/pericardial effusions and ascites. There is a significant morbidity and mortality with 5-year survival from time of diagnosis estimated to be 50%. The genetic basis of KLA has recently been described as somatic activating mutations of the RAS–MAPK pathway. A 14-month-old girl, previously well, presented with a massive pericardial effusion leading to cardiac tamponade and cardiac arrest, requiring emergency pericardial drainage and extracorporeal membrane oxygenation. The patient had a profound thrombocytopenia not responding to repeat transfusion. MRI of the thorax revealed extensive cystic lesions consistent with diffuse lymphatic disease. Clinical suspicion of KLA was confirmed by mediastinal biopsy revealing characteristic kaposiform spindle-shaped lymphatic endothelial cells. Targeted next-generation sequencing revealed a mutation in NRAS (p.Q61R) in 2% of sequenced reads. Treatment with mTOR inhibitor sirolimus plus vincristine rapidly stabilized her refractory thrombocytopenia and led to a rapid clinical improvement enabling extubation within 72 h. Repeat MRI 2 months later confirmed reduced lymphatic disease. Lymphatic disease remains stable after 18 months of sirolimus monotherapy with no significant adverse effects. KLA is an aggressive mosaic disease of the lymphatic vasculature with high morbidity and mortality. Early diagnosis is key to enabling targeted medical therapies in this cohort. Clinicians should be aware of this differential when presented with high-volume effusions and refractory thrombocytopenia.

Analysis of core mutation and TET2/ASXL1 mutations DNA methylation profile in myelodysplastic syndrome

ABSTRACTObjectivesThe study aims to analyze genetic mutation and clinical characteristics and study their correlation with survival prognosis of patients with myelodysplastic syndromes (MDS). Moreover, the differential DNA methylation profiles between TET2 mutated (Mut)/ASXL1 wild-type (WT) and TET2-Mut/ASXL1-Mut MDS samples were investigated to explore the mechanism of MDS patients with TET2/ASXL1 mutations.MethodsThe clinical data of 195 patients diagnosed with MDS were selected and statistically analyzed. The DNA methylation sequencing data set was obtained from the GEO and bioinformatics analyzed.ResultsOf the 195 MDS patients, 42 (21.5%) carried TET2 mutations. 81% of TET2-Mut patients could detect comutated genes. The most commonly comutated gene in MDS patients with TET2-Mut was ASXL1, which had a tendency towards poorer prognosis (P = 0.08). GO analysis showed that highly methylated differentially methylated genes (DMGs) was mainly enriched in biological processes such as cell surface receptor signal pathway and cell secretion. Hypomethylated DMGs was mainly enriched in cell differentiation and cell development. KEGG analysis showed that hypermethylated DMGs was mainly enriched in Ras signal pathway and MAPK signal pathway. Hypomethylated DMGs was mainly enriched in extracellular matrix receptor interaction and focal adhesion. PPI network analysis identified 10 hub genes of hypermethylated and hypomethylated DMGs that may be associated with patients with TET2-Mut/ASXL1-Mut respectively.ConclusionsOur results illustrate the interrelationships between genetic mutations and clinical phenotypes and disease outcomes, with substantial potential for clinical application. Differentially methylated hub genes might represent potential biomarkers and provide novel insights and possible targets for MDS with double TET2/ASXL1 mutations.

A phase 2 basket trial of an ERK1/2 inhibitor (LY3214996) in combination with abemaciclib for patients whose tumors harbor pathogenic alterations in BRAF, RAF1, MAP2K1/2 ERK1/2, and NF1.

e15088 Background: LY3214996 (L) is a potent inhibitor of ERK1/2 kinases that can potentially obviate resistance mechanisms in cancers that harbor BRAF, RAS, NF1, MAP2K1/2, and other MAPK alterations. Abemaciclib (A) is a CDK 4/6 inhibitor. Studies have shown that concomitant inhibition of the MAPK pathway exemplified by ERK inhibitors may augment the antitumor activity of CDK4/6 inhibitors. In this biomarker-informed phase 2 study, we assess the clinical efficacy of L in combination with A. Methods: The study enrolled patients with advanced unresectable or metastatic cancer with no other standard of care options available. The malignancy must harbor pathogenic alterations in BRAF, RAF1, MAP2K1/2, ERK1/2, or NF1, in whom standard treatments failed to provide clinical benefit. L was administered at a dose of 200 mg orally QD in combination with A at 150 mg BID hours on Days 1 through 28 of a 28-day cycle until progression, toxicity, or withdrawal. The primary endpoint is the objective response rate. Secondary outcomes include evaluation of safety and tolerability, progression-free survival (PFS), and duration of the overall response. Additionally, tumor samples were collected for organoid development. Results: Between September 1, 2020, and February 1, 2023, 12 patients were enrolled. Primary malignancies included colorectal (6), lung (1), breast (2), pancreas (1), uterus (1), and thyroid (1). The pathogenic alterations included NF1 (4), BRAF (6), and MAP2K1/2 (2). There were no CR or PR observed. Stable disease was achieved in 4 patients (33%) with a median disease control rate of 6.5 months. Pathogenic alterations in patients with stable disease included BRAF, MAP2K1/2, and NF1. Most common treatment-related adverse events of any grade included fatigue (41%), rash (41%), nausea (33%), vomiting (33%), anemia (25%), anorexia (25%), and increased creatinine (25%). There were no grade 4 or 5 adverse events. Conclusions: The combination of L+A is safe and promising in patients whose tumors harbor pathogenic alterations in BRAF, MAP2K1/2, and NF1. Enrollment is ongoing, and efficacy and toxicity outcomes continue to be assessed. (NCT04534283). Clinical trial information: NCT04534283 .

IPSC‐derived neurons with amyloid‐β treatment reflect features of Alzheimer’s disease patients

AbstractBackgroundThere is increasing evidence showing that induced pluripotent stem cells (iPSC)‐derived brain cells recapitulate disease‐relevant processes in patients with Alzheimer’s disease (AD). However, it remains unclear to what extent iPSC‐derived neurons treated with amyloid‐β (Aβ) reflect patient Aβ burden and other endophenotypes as determined by changes in protein expression.MethodTo investigate this, we firstly generated iPSC lines from 14 individuals with AD from the Deep and Frequent Phenotyping (DFP) pilot cohort. All of these individuals had undergone clinical measures (i.e. Mini Mental Score Examination) and neuropathological measures (i.e. Aβ, p‐tau and tau). We then differentiated these lines into cortical neurons and treated them with Aβ oligomers. We collected both cell media and cell lysates and measured over 5000 proteins in these samples using SOMAscan assay. In addition, proteins in the cerebrospinal fluid (CSF) and plasma of the same individuals were also measured using the same assay.ResultUsing Wilcox test, we identified 627 proteins that were differentially expressed in neuronal cultures under Aβ oligomers treatment after multiple correction. These proteins were significantly enriched in 14 pathways such as TGF‐β signaling pathway, Ras signaling pathway and MAPK signaling pathway etc. From protein co‐expression network analysis, we identified 12 modules (ranging from 45 to 2029 proteins) in neuronal cultures. Of them, eight were significantly associated with at least one endophenotype of the amyloid/Tau/Neurodegeneration/Cognition AT(N)C framework. We further checked the replication of these modules in human. We found that eight and nine modules were significantly preserved in CSF and plasma, respectively. Furthermore, some preserved modules were also significantly associated with some AD endophenotypes including Aβ.ConclusionThese results suggests that iPSC‐derived neurons with Aβ treatment reflect the patients’ endophenotypes, indicating that their use in capturing disease‐relevant processes in AD.

Environmental Enrichment and Metformin Improve Metabolic Functions, Hippocampal Neuron Survival, and Hippocampal-Dependent Memory in High-Fat/High-Sucrose Diet-Induced Type 2 Diabetic Rats

Background: The Western-style diet-induced type 2 diabetes mellitus (T2D) may eventually trigger neurodegeneration and memory impairment. Thus, it is essential to identify effective therapeutic strategies to overcome T2D complications. This study aimed to investigate the effects of environmental enrichment (EE) and metformin interventions on metabolic dysfunctions, hippocampal neuronal death, and hippocampal-dependent memory impairments in high-fat/high-sucrose (HFS) diet-induced T2D rats. Methods: Thirty-two male rats (200-250 g) were divided into four groups: C group (standard diet + conventional cage); D group (HFS diet + conventional cage); DE group (HFS diet + EE cage/6hr daily); and DM group (HFS diet + metformin + conventional cage). Body weight was measured every week. T-maze tasks, anthropometric, biochemical, histological, and morphometric parameters were measured. The expression changes of hippocampal genes were also analyzed. Results: The anthropometric and biochemical parameters were improved in DE and DM groups compared with the D group. DE and DM groups had significantly higher T-maze percentages than the D group. These groups also had better histological and morphometric parameters than the D group. The interventions of EE and metformin enhanced the expression of hippocampal genes related to neurogenesis and synaptic plasticity (BDNF/TrkB binding, PI3K-Akt, Ras-MAPK, PLCγ-Ca2+, and LTP). Conclusion: Environmental enrichment (EE) and metformin improved metabolic functions, hippocampal neuron survival, and hippocampal-dependent memory in HFS diet-induced T2D rats. The underlying mechanisms of these interventions involved the expression of genes that regulate neurogenesis and synaptic plasticity.

Comprehensive characterization of mesothelin expression in colorectal cancer.

245 Background: Mesothelin (MSLN) is highly expressed in several malignancies including colorectal cancer (CRC) compared to normal tissue and is associated with tumor cell proliferation and worse patient survival. MSLN could be a potential target for antigen-specific therapy. Here, we characterized the MSLN expression in CRC and its association with molecular and immune-related markers. Methods: 15,673 CRC samples were tested by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Microsatellite-instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Tumors with MSLN-high(H) and MSLN-low(L) expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q<0.05). Results: There was no difference in median age or gender distribution between MSLN-H and MSLN-L CRC tumors. Right-sided tumors had increased median MSLN expression compared to rectal, transverse, and left-sided tumors (6.12 vs 5.29 vs 5.35 vs 4.81 TPM, q<0.05). Median MSLN expression was higher in metastatic compared to primary tumors (1.21-fold, q<0.05). Median MSLN expression was highest in CMS4 (10.17 TPM), but, in MSS CRC, MSLN expression was highest in CMS1 (10.46 TPM, all q<0.05). MSLN-H tumors had higher frequency of KRAS (59.8% vs 36.4% ) and BRAF (13.1% vs 9.2% ) mutations but lower frequency of APC mutations (67.5% vs 77.2%), TMB High (9.8% vs 12%) and dMMR/MSI-H (6.6% vs 8.9%) compared to MSLN-L tumors (q<0.05). Further, MSLN-H tumors had higher MAPK activation score (MPAS) compared to MSLN-L tumors (1.72-fold, q<0.05). Similarly, in the MSS only cohort, MSLN-H tumors had increased frequency of KRAS (62.2% vs 37.9%) BRAF (10.5% vs 5.0%) mutations but decreased APC mutations (70% vs 80.9%) (q<0.05). MSLN-H tumors had higher expression of immune checkpoint genes ( CD274, PDCD1, PDCD1LG2, CTLA4, LAG3, HAVC2 and FOXP3, FC: 1.1-1.8) and T cell inflamed score (3.3-fold) (all q<0.05). MSLN-H was associated with worse OS compared to MSLN-L in all CRC (934 vs 1253 days; HR: 1.21, 95% CI (1.10-1.32), p<0.0001) and MSS CRC (972 vs 1168 days; HR: 1.15, 95% CI (1.06-1.23), p<0.001) but not in MSI-H CRC (p=0.67). Conclusions: Our data suggest a strong association between MSLN expression and increased mutations in the RAS pathway and MAPK activation score, decreased mutations in WNT pathway genes, a differentially modulated immune tumor microenvironment, and poor overall survival. These findings support MSLN as a potential prognostic biomarker candidate in colorectal cancers pending further prospective validation.

Comparative genetic profiling of neuroblastoma at the onset vs recurrence or progression of the tumor and determination of the prognostic role of genetic alterations

Neuroblastoma (NB) is the most prevalent extracranial solid embryonal tumor in children, capable of both spontaneous regression and extremely aggressive course. The clinical heterogeneity of NB feeds the continual interest in its molecular genetic profiles as a potential key to the variable tumor behaviors and treatment personalization. To date, a number of studies indicate unfavorable prognostic roles of p53 and RAS–MAPK pathway activation, as well as that of telomere maintenance mechanisms. The increase in mutational load observed in recurrent NB has been shown to reduce the degree of subclonal heterogeneity, leading to pronounced distinction between genetic profiles of the tumor as recorded at primary and recurrent manifestations. This study aimed to analyze the alterations in genetic profiles of NB over time from the initial verification of the diagnosis till a disease-related adverse event (relapse or progression), as well as the potential prognostic significance of such alterations. The retrospective-prospective study enrolled 46 patients with morphologically verified peripheral neurogenic tumors, receiving treatment entirely or partially at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology from July 2013 till December 2021 in accordance with the modified NB-2004 protocol. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The cohort included an observation group of 25 patients, 1 intermediate-risk case and 20 high-risk cases. The main inclusion criterion concerned the availability of tumor tissue suitable for molecular examination, obtained both at the onset of the disease and at relapse/progression. We used the multiplex ligation-dependent probe amplification assay (MLPA) for the assessment of segmental and copy number chromosome aberrations, next-generation sequencing (NGS) to identify nucleotide variants, and reverse transcription polymerase chain reaction to assess the TERT gene expression levels. The statistical analysis used data available by April 01, 2022. In the studied cohort, the analysis confirmed prognostic contribution of MYCN amplifications (p < 0.001) detected at the onset of the disease or at its progression, as well as of 1p deletions (p = 0.01) detected in primary tumors. The expression of TERT in primary NBs was shown to correlate with the clinical course of the disease in the intermediate-risk and observation group, including local recurrence/progression (n = 3; –5 (–10.5…–3.5)), systemic relapses (n = 3; –2 (–2.5…–1)) and the lack of second adverse event (n = 20; –7 (–9…–5.5)), р = 0.037. Pairwise comparison of mutational profiles at the onset of the disease and at relapse revealed extreme degree of instability and significant variations: 38.5% (10/26) of the patients had no clinically significant genetic variants in the analyzed genomic region of interest in paired samples, 19.2% (5/26) of the patients had identical oncogenic variants in primary and relapsed tumor tissues, albeit with altered variant allele fraction (2/5), loss of one of the variants identified in the primary tumor (1/5), or acquisition of additional mutations upon recurrence (2/5), in 19.2% (5/26) of the patients, oncogenic genetic variants detected in primary NB were subsequently lost, in 7.7% (2/26) of patients, a loss of an oncogenic genetic variant identified at the onset on the disease was accompanied by the emergence of new clinically significant variants, and 15.4% (4/26) of the patients presented with new mutations in relapsed tumor tissue. Genetic variants leading to RAS–MAPK pathway activation and р53 pathway inhibition in primary tumors were identified in 31% (9/29) and 20.7% (6/29) of primary tumors and 29% (9/31) and 6.5% (2/31) of relapsed tumors, respectively. Risk group stratification based solely on cytogenetic aberrations fails to account for all cases of aggressive course of NB. Advanced molecular genetic approaches including MLPA and NGS, as well as the TERT mRNA quantitation, provide expanded characterization of the tumor biology and aggressiveness. Although prognostic markers are typically found in primary tumors, for NB, the use of such markers can be hampered by extremely high intratumoral heterogeneity. The data on acquisition or loss of prognostically significant molecular genetic markers in the course of the disease is useful for long-term prognosis and selection of targets for specific therapies.

Inhibition of angiotensin II type 1 receptor partially prevents acute elevation of pulmonary arterial pressure induced by endovascular ethanol injection.

This study aimed to examine whether the administration of losartan can prevent acute elevation of pulmonary arterial pressure (AEPAP) induced by endovascular ethanol injection and to assess its related mechanisms. Male swine were selected and performed with absolute ethanol endovascular injection. Saline was used as the negative control. Losartan was administered preoperatively. Pulmonary arterial pressure (PAP), femoral arterial pressure (FAP) and heart rate (HR) were monitored during operations. Venous plasma and pulmonary artery (PA) tissue were harvested for analyses. Protein level was detected by Western blotting and ELISA, whereas qRT-PCR was used in mRNA detection. H & E staining and immunohistochemistry were conducted to evaluate histopathology. Ethanol injection elevated PAP in swine. The concentration of RAS ligands was elevated in plasma (all P < 0.0001) but not in PA. The level of oxidative stress increased in both plasma and PA. MRNA level of AT1R (P < 0.01, 95% CI: 0.251-1.006), not AT2R increased in PA. Losartan failed to inhibit AEPAP after all sessions of ethanol injection, and partially reversed the ethanol-induced PA remodeling. The P38 MAPK was activated after ethanol injection and could be inhibited by losartan (P < 0.01, 95% CI: -0.391 to -0.164). Ethanol also promoted the translocation of the P40-PHOX/P47-PHOX/P67-PHOX complex and the activation of NOX, which was independent from RAS. Endovascular ethanol injection can induce AEPAP mainly by activating RAS and P38 MAPK signaling. Losartan can partially prevent AEPAP and vascular remodeling owing to the promotion of NOX activity by ethanol. Mechanism diagram of endovascular ethanol injection-induced acute elevation of pulmonary arterial pressure (AEPAP) partially prevented by losartan. RAS: Renin-angiotensin system; AGT: angiotensinogen; Ang I: angiotensin I; ACE: angiotensin I converting enzyme; Ang II: angiotensin II. AT1R: angiotensin II type 1 receptor. NOX2: NADPH oxidase 2. PA:pulmonary artery.

In This Issue

In This Issue

Ras drives malignancy through stem cell crosstalk with the microenvironment

Squamous cell carcinomas are triggered by marked elevation of RAS–MAPK signalling and progression from benign papilloma to invasive malignancy1–4. At tumour–stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFβ signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)–AKT–mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

The Role of Relapse Specific over-Expression of S100A8/A9 B Leukemic Cells in Promoting Chemoresistance

Background: We previously discovered a novel superenhancer (SE) adjacent to S100A8/9 at relapse in a majority of patient samples indicating a role in clonal evolution. In agreement with these findings, a metanalysis of gene expression studies demonstrated that S100A8 is the top overexpressed transcript at relapse in childhood ALL. S100A8 is a member of the S100 calcium-binding protein family and some studies suggest a role in mediating intrinsic drug resistance. However, secreted S100A8/9 proteins play a major role in inflammation and it is possible that their role in clonal evolution occurs by altering the tumor microenvironment (TME). Design/Method Isogenic B-ALL cell lines (697 and RS4;11) were created to overexpress an empty vector control (EV), S100A8 or S100A9 alone, or S100A8+S100A9 together. Protein expression and secretion were measured using an ELISA for human S100A8/9 (Abcam). Proliferation over a 7-day period was measured using the Beckman Coulter Vi-CELL counter. Chemoresistance was measured using Promega's CellTiter-Glo® assay following exposure to chemotherapeutic agents used in the treatment of ALL including mercaptopurine, prednisolone, methotrexate, vincristine, cytarabine, and etoposide for 72-120 hours. Colony forming potential was assessed after 14 days in MethoCult (STEMCELL Technologies). To determine that expression originated from leukemia cells one sample pair (SGZ) known to overexpress S100A8 at relapse using bulk RNA expression, was compared to 3 paired patient samples without overexpression using single cell RNAseq (scRNAseq). CD45+CD19+ (blasts) and CD45+CD19- (microenvironment) were sorted and then pooled to increase the fraction of cells from the microenvironment before scRNAseq using 10X Genomics. CellPhoneDB analysis was used to predict ligand-receptor interactions between different cell types. Results Western blot demonstrated stable expression of S100A8 and S100A9 proteins when co-expressed but protein was not detected when either was alone. Overexpression of S100A8+S100A9 was confirmed via ELISA using both cell lysate and condition media suggesting secretion. No differences in proliferation, colony formation, or chemoresistance were observed between cells overexpressing S100A8/9 compared to the empty vector control line, suggesting no cell intrinsic effect of S100A8/9 overexpression ssRNAseq analysis validated upregulation of S100A8/9 specifically in leukemic blasts (cluster 12, L2FC of 5.6 and 5.3 (p<.0001) for S100A8 and S100A9, respectively). In contrast, two myeloid monocytic-like populations showed minimal to no change in expression level from diagnosis to relapse (L2FC 1.13 and 0.18 for S100A8 and S100A9, respectively (p<.01). Pathway analysis of differentially regulated genes in SGZ (L2FC cutoff of 1 and p<.001, Enrichr, Panther 2016) revealed known relapse pathways, including Ras, p53, and p38 MAPK, but also those known to impact the TME including Inflammation mediated by chemokine and cytokine signaling, Toll receptor signaling, and TFG-beta signaling. Interestingly, we also observed an increase in, and higher percentage of non-classical monocytes within the non-leukemic cell population at relapse in SGZ (>20%) compared to minimal (<5%) in the other three patients. In SGZ, we found 455 and 573 significantly upregulated (p<.05, abs L2FC>.32) and 686 and 362 significantly downregulated genes (p <.05, absolute L2FC <.32) in classical monocytes and non-classical monocytes, respectively. In contrast, we found less than 20 differential genes within these clusters in any of the other patient pairs, suggesting a profound impact of S100A8/A9 expression on the transcriptome of these myeloid populations. Using CellPhone DB we studied the cell-cell communication mediated by ligand-receptor unique to SGZ by comparing the blast and the two monocytic populations. A number of unique interactions were seen at relapse, including CCL7-CCL3/CCR1, CCL3/IDE and CCL5/CCR5 known to induce the migration of leukocytes and myeloid cells. Other interactions involved in tumorigenesis and inflammation upregulated at relapse were seen: NRP2/VEGFA, LGALS9/SLC1A5. Conclusion Our results indicate that S100A8/A9 overexpression in B-ALL cells at relapse has no cell intrinsic impact but triggers remodeling of the TME by inducing an increase in myeloid subpopulations and unique cell to cell interactions.

In This Issue

In This Issue