Mutant Allele Research Articles

Abstract 6252: Driver indel discovery and allelic imbalance in >9,000 tumor RNA-Seq samples from the Cancer Genome Atlas (TCGA)

Abstract High-throughput DNA sequencing technologies have enabled unbiased screening of genomic alterations such as single nucleotide variants (SNVs) and small insertions/deletions (indels). However, variant analysis using transcriptomic sequencing (RNA-seq) data has not become standard due to challenges in distinguishing true variants, in particular indels, from artifacts that can arise from RNA-seq mapping and library preparation. We have previously developed a tool, RNAIndel, which classifies indels as somatic, germline, or artifacts using a machine learning-based model. Here, we present enhanced somatic indel discovery by incorporating tumor RNA-Seq data with conventional paired tumor-normal DNA-Seq by re-analyzing the Cancer Genome Atlas (TCGA) data using RNAIndel. Our analytic process involves running RNAIndel on 9,101 TCGA tumor RNA-Seq samples across 33 cancer types hosted on Cancer Genomics Cloud by Seven Bridges Genomics (https://www.cancergenomicscloud.org) and comparing the result with the reference somatic indel set generated from paired tumor/normal whole-exome sequencing (WES) data using an ensemble caller developed by NCI Genomic Data Commons (GDC). The comparison validates the RNAIndel variants by matching to the corresponding WES dataset. We also use the GDC indel set to test their expression in RNA-Seq. Indel alignments across sequencing platforms were compared by indelPost, an algorithm that we developed, to perform indel realignment to overcome the differences in mapping algorithms (BWA vs. STAR) and read lengths (100-bp vs. 50-bp) in WES versus RNA-Seq data.The joint DNA/RNA indel analysis provides important insights into the expression profile of indels in known cancer driver genes versus non-drivers. First, indels in driver genes are more likely expressed than those in non-drivers (79% vs. 50%, p < 2.2 × 10-16). Second, mutant allele expression is frequently upregulated for truncating indels in tumor-suppressor genes (TSG) as allelic imbalance was detected in 6% of such variants in TSGs vs. 2% non-drivers. This may indicate a potential second hit leading to loss of the wild-type allele of in the TSGs. Third, allelic imbalance for in-frame indels was also frequent for oncogenes (20%) compared with the non-drivers (7%). The high expression likelihood of driver genes and the enriched indel allele expression for TSGs and oncogenes both support the use of RNA-Seq to enhance driver indel discovery. Indeed, our analysis reveals ~10% additional driver indels by RNA-Seq analysis which are absent from the GDC reference indel set. These indels affect prominent driver genes, most frequently CDKN2A, TP53 and ARID1A. Our experience argues for incorporating de novo indel analysis in RNA-Seq as a standard approach for future cancer genomic analysis. Citation Format: Kohei Hagiwara, Andrew Thrasher, Jinghui Zhang. Driver indel discovery and allelic imbalance in >9,000 tumor RNA-Seq samples from the Cancer Genome Atlas (TCGA) [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 6252.

Abstract 6557: Longitudinal cell-free tumor load dynamics represent an early endpoint for immunotherapy response in non-small cell lung cancer

Abstract Background: Liquid biopsies can inform treatment strategies by rapidly and accurately assessing tumor burden; with emerging data supporting the clinical utility of circulating cell-free tumor DNA (ctDNA) as an early endpoint of immunotherapy response. Methods: We performed targeted error-correction sequencing of ctDNA (n=330) and matched white blood cell (WBC) DNA (n=109) from 109 patients with advanced non-small cell lung cancer (NSCLC) who received immune checkpoint inhibitor monotherapy (n=79) or immunotherapy-containing combination regimens (n=30). Variant cellular origin was determined using a tumor-independent WBC DNA-informed approach. The maximum mutant allele fraction (maxMAF) of tumor-derived mutations was tracked longitudinally to assess changes in total cell-free tumor load (cfTL). Results: Among 3,418 variants with resolved cellular origin, 2,452 (72%) were tumor-derived, 125 (4%) were germline, while 841 (25%) were related to clonal haematopoiesis (CH). Cell-free DNA fragments harbouring tumor-derived mutations were significantly shorter than fragments containing wild-type alleles; however, fragment length alone was insufficient to distinguish CH variants from wild-type. Overall, 97 (89%) patients with >1 detectable tumor mutation were considered for cfTL tracking and molecular response assessment. Clearance of cfTL indicated molecular response (mR) whereas persistence indicated molecular progression (mPD). Time-course analyses, aiming to establish the optimal timing for molecular response assessment, revealed that early cfTL clearance within 3-9 weeks was significantly linked to improved survival outcomes. Notably, blood tumor mutation burden above or below 16 mutations/megabase showed no association with overall survival (OS; log rank p=0.98) or progression-free survival (PFS; log rank p=0.95). Of 81 patients evaluated for early on-therapy molecular responses, 28 (35%) were classified as having an mR, while 53 (65%) patients were classified in the mPD category. The median time to mR was 5 weeks in patients receiving immune checkpoint monotherapy and 6 weeks in patients receiving chemo-immunotherapy combination regimens. Patients in the mR group achieved longer OS (log rank p=0.001) and PFS (log rank p=0.0003) compared to those with mPD. Assessment of landmark PFS at 6 months demonstrated a significant association between ctDNA molecular response and durable clinical benefit (DCB) in patients receiving immune checkpoint monotherapy (p < 0.001, Fisher’s exact test), and a numerical association with DCB in patients on chemo-immunotherapy (p=0.07, Fisher’s exact test). Conclusions: Longitudinal tracking of ctDNA provides a real-time and accurate approach for monitoring immunotherapy response. These findings underscore the clinical utility of employing ctDNA molecular response to inform clinical decision-making. Citation Format: Lavanya Sivapalan, Bahar Alipanahi, Archana Balan, Jamie E. Medina, Noushin Niknafs, Erica Peters, Jaime Wehr, Alissa Konicki, Mimi Najjar, Bryan Chesnick, Gavin Pereira, Sarah Rongione, Nisha Rao, Michael Rongione, Brittany Shavatt, Lorenzo Rinaldi, Benjamin Levy, Victor E. Velculescu, Patrick M. Forde, Valsamo Anagnostou. Longitudinal cell-free tumor load dynamics represent an early endpoint for immunotherapy response in non-small cell lung 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 6557.

Abstract 447: Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC

Abstract Background: Pre-clinical studies identified creatine metabolism to be activated in KRAS mutant metastatic colorectal cancer (CRC) as a mechanism to fuel the increased energy demands of RAS mutant tumors. Metastatic colon cancer cells overexpress and subsequently release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high energy metabolite phosphocreatine. Ompenaclid is a small molecule inhibitor of the creatine transporter SLC6A8. Inhibition of SLC6A8 by ompenaclid prevents import of phosphocreatine/creatine, resulting in depletion of intracellular creatine, phosphocreatine and ATP levels. This results in tumor apoptosis and robust anti-tumor efficacy in CRC models with a wide range of KRAS mutant alleles. Ompenaclid is currently in a Phase 2 trial, in combination with FOLFIRI and bevacizumab, in patients with RAS-mutated second-line advanced/metastatic colorectal cancer. Here, we assessed efficacy of ompenaclid in tumors beyond CRC, including KRAS WT and KRAS mutant NSCLC. Methods: Athymic mice were inoculated with CRC, pancreatic, gastric or NSCLC cancer cell lines and treated with ompenaclid once tumors reached ~150 mm3, until termination. Plasma metabolites were quantified by LC-MS/MS and tumoral expression of CKB and SLC6A8 in NSCLC and CRC tumors was measured by qPCR after treatment with a control or ompenaclid-supplemented diet for ~10 days. Combination treatment with MRTX849, a KRAS G12C inhibitor, was conducted in a pancreatic MiaPaca xenograft model. Results: Our studies demonstrate ompenaclid anti-tumor efficacy in NSCLC and pancreatic xenografts. Notably, and consistent with findings in CRC, efficacy in NSCLC was more robust in RAS mutant cancer models, relative to those bearing KRAS WT tumors. Metabolite analysis of the plasma of tumor-bearing mice at baseline revealed that mice harboring RAS mutant tumors had higher levels of p-creatine and lower levels of ATP, indicating that RAS mutant tumors utilize higher amounts of ATP, which may be converted to phosphocreatine by tumoral CKB, as confirmed by qPCR analysis. This finding is consistent with metastatic RAS mutant CRC tumors utilizing extracellular ATP to generate phosphocreatine and thus exhibiting greater dependency on creatine metabolism relative to RAS WT tumors. Treatment with ompenaclid also demonstrated additive efficacy in combination with KRAS G12C inhibitors. In summary, these data support development of ompenaclid in indications beyond CRC, including NSCLC, as well as in combination with KRAS G12C inhibitors. Citation Format: PuiChi Lo, Victor Sanz Chavez, Shugaku Takeda, Masoud Tavazoie, Isabel Kurth. Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC [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 447.

Abstract 2406: Circulating tumor DNA as a potential indicator of treatment response in patients with surgically unresectable PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly heterogeneous and extremely aggressive neoplasm. Most patients with PDAC present with symptomatic, surgically unresectable disease at initial diagnosis. Systemic chemotherapy is the standard care for patients diagnosed with unresectable PDAC, despite the dismal survival rate due to disease recurrence. In this context, liquid biopsy based ctDNA monitoring may aid to improve detection accuracy and allow real time disease prognosis. Here we aimed to investigate the utility of ctDNA in longitudinal tumor monitoring to assess treatment response and to predict disease recurrence. We performed targeted NGS sequencing to investigate over 900 mutations (SNVs) and 12 copy number variations (CNVs) in 53 genes, using the Ion S5 Oncomine PanCancer gene panel in 40 PDAC patients with unresectable disease. Serial plasma samples were obtained from patients in the course of 24 months, including samples collected before the first intervention (baseline) as well as within 2 months after initiation of chemotherapy. Patients were followed for a period of up to 2-years from diagnosis through treatment, to death or final follow-up. Genomic DNA from paired plasma-buffy coat samples were sequenced to exclude clonal hematopoiesis. Overall detection of ctDNA alterations in baseline samples was 55% (22/40) with increasingly higher detection rates and mutation loads in advanced disease (88% in stage IV patients). Alterations including SNVs and CNVs were preferentially distributed among 9 genes; mutations in TP53 (45%), KRAS (38%) and SMAD4 (5%) were most commonly identified. To asses response to chemotherapy, the presence of residual ctDNA after clinical intervention was evaluated by comparing total ctDNA MAF (mutant allele frequency) detected in plasma at diagnosis and within 2 months of chemotherapy. 71% (17/24) of patients who reached progressive disease (PD) had detectable baseline mutations and 79% (19/24) had persistent residual mutations at 2 months of treatment. Conversely, 94% (15/16) of non-PD patients had no detectable ctDNA or had a significant reduction in total MAF after 2 months of chemotherapy. Absence of residual ctDNA after 2 months of chemotherapy was associated with a better OS (p=0.031; HR = 3.48). On the other hand, residual ctDNA after 2 months of chemotherapy was associated with a shorter PFS (p=0.042; HR = 3.28). Our study demonstrates that monitoring ctDNA levels derived from plasma provides valuable insights into the therapeutic response in patients with unresectable PDAC, even in the absence of matched tumor genotyping. We found a significant correlation between increased ctDNA levels following the initiation of treatment and poorer survival outcomes. Citation Format: Fabio Pittella Silva, Yasutoshi Kimura, Masayo Motoya, Toru Nakamura, Takeshi Murakami, Kazunori Watanabe, Siew-Kee Low, Yusuke Nakamura. Circulating tumor DNA as a potential indicator of treatment response in patients with surgically unresectable PDAC [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 2406.

Abstract 3317: KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications

Abstract Alterations in KRAS are a main cancer driver. Amplifications of the KRAS wild-type (wt) allele account for ~7% of all KRAS-driven cancers and are frequent in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). KRASG12V mutations account for ~28% of pancreatic cancers, 9% of colorectal cancers and 6% of lung adenocarcinoma. There is an urgent medical need to identify targeted therapies for these frequent KRAS alterations. Herein, we describe BI 3706674, a novel, potent and orally bioavailable small molecule inhibitor of the KRAS oncogene for clinical testing in patients with tumors harboring KRASG12V mutations and KRAS wild-type amplifications. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Importantly, BI 3706674 is highly selective for KRAS vs HRAS or NRAS, and it is therefore expected to be well-tolerated in normal tissues. In two large cancer cell line panels (PRISM and Horizon), BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wt amplifications and KRAS mutations e.g., G12A/C/D/V, G13D and Q61H). The strongest sensitivity was observed for cell lines with KRAS wt amplifications (relative copy number (CN) of > 10) followed by cell lines with KRASG12V mutations along with significant pharmacodynamic (PD) biomarker modulation (e.g., down-regulation of DUSP6 mRNA). In vivo, BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models of human gastric cancer with KRAS wt CN > 10 and diverse tumor types with KRASG12V mutations. A twice daily oral dose of 30 mg/kg induced significant tumor regression and PD biomarker modulation. Combination of BI 3706674 with standard of care therapies and novel agents are being tested in preclinical models to guide clinical development. Results of the ongoing pre-clinical analysis will be shared. A Phase I clinical trial is in preparation in patients with advanced solid cancers harboring KRASG12V mutations and KRAS wt amplifications to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and efficacy of BI 3706674. Citation Format: Antonio Tedeschi, David H. Peng, Fiorella Schischlik, Lorenz Herdeis, Otmar Schaaf, Valeria Santoro, Daniel Gerlach, Fabio Savarese, Jesse Lipp, Christian Haslinger, Francesca Rocchetti, Johannes Popow, Heinrich J. Huber, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broeker, Tobias Wunber, Michael Gmachl, Klaus Rumpel, Matthew Rees, Melissa Ron, Jennifer Roth, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Scott Kopetz, Michael Kim, Don L. Gibbons, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications [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 3317.

Abstract 3321: KRASmulti inhibitor BI 3706674, an orally bioavailable, direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRASG12V-driven preclinical models

Abstract Alterations of KRAS are observed in approximately one in seven of all human tumors, making KRAS one of the most prevalent oncogenic drivers. Gain-of-function missense mutations in KRAS, leading to its aberrant activation, are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma, with KRASG12V mutations accounting for ~28%, 9% and 6% of the cases, respectively. The poor outcome associated with these tumor types as well as the lack of targeted inhibitors for the KRASG12V-mutant allele calls for the urgent need to identify therapies able to effectively address this allele. BI 3706674 is a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including KRASG12V, in the GDP-bound state, and blocks downstream oncogenic signalling. Here, we show that BI 3706674 has a strong anti-proliferative activity in a panel of KRASG12V-mutant cancer cell lines in vitro, along with significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and down-regulation of DUSP6 mRNA). In vivo, a twice daily oral dose of 30 mg/kg was well tolerated, while inducing tumour regression in several mutant KRASG12V patient-derived xenograft (PDX) models across multiple different tumor types. Feedback activation of upstream signalling pathways including receptor tyrosine kinases, such as the EGFR, has been suggested to limit the efficacy of GDP-KRAS-targeting compounds in preclinical studies. Clinical combination trials involving KRASG12C inhibitors such as adagrasib and sotorasib and anti-EGFR modalities are further supporting these findings. Here we show that a combination with anti-EGFR antibodies (e.g., Cetuximab) potently enhances the response observed for BI 3706674 in models of KRASG12V-mutant colorectal cancer. The deeper response observed upon combination of BI 3706674 with Cetuximab across multiple xenograft models provides a strong rationale for the clinical investigation of this combination therapy. Moreover, based on our in vitro mechanistic studies and ex vivo organoid platform we have identified prospective mechanisms of resistance and opportunity for novel drug combinations with BI 3706674 that can potentially translate into clinical trials. Citation Format: David Hwa Peng, Antonio Tedeschi, Lorenz Herdeis, Otmar Schaaf, Fabio Savarese, Francesca Rocchetti, Johannes Popow, Heinrich J. Huber, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Bröeker, Tobias Wunberg, Firoella Schischlik, Jesse Lipp, Mariah Williams, Vaness Chandler, Charles E. Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Scott Kopetz, Michael Kim, Don L. Gibbons, Christopher P. Vellano, Joseph R. Marszalek, TImothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. KRASmulti inhibitor BI 3706674, an orally bioavailable, direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRASG12V-driven preclinical models [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 3321.

Abstract 7295: A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR

Abstract The Janus kinase 2 (JAK2) gene is located on chromosome 9p24.1 and encodes a non-receptor tyrosine kinase that plays a central role in cytokine and growth factor signaling. The JAK2 protein is especially important for controlling the production of blood cells from hematopoietic stem cells. In fact, the JAK2 mutation at amino acid 617 (Valine 617 to Phenylalanine, V617F) is the most frequently detected mutation in myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). This dominant gain-of-function V617F mutation results in constitutive tyrosine phosphorylation activity, leading to uncontrolled blood cell production, including increased numbers of erythrocytes, neutrophils, and platelets. To detect the JAK2 V617F mutation in DNA from patient blood samples, we developed a unique real-time PCR assay using our proprietary XNA technology that enables the assay to selectively amplify the mutant sequence by using a synthetic DNA analog XNA (Xenonucleic Acid). The JAK2 V617F mutation detection assay is designed to detect a valine-to-phenylalanine mutation at amino acid 617 (V617F). PCR primers, TaqMan probes, and XNA were designed for the JAK2 V617F mutation, along with primers and probes for an internal control gene in the duplex setting. The analytical performance of the assay was verified and validated using a variety of control samples. The results revealed that the XNA completely suppressed the amplification of wildtype sequence while only the V617F mutant sequence was amplified. The JAK2 V617F mutation detection assay is highly reproducible with intra- and inter-assay coefficients of variation of less than 10%. Results for the assay’s analytical sensitivity indicated that V617F could be detected with about 0.25% mutant allele frequency in 10 ng DNA input. In addition, no significant cross-amplification between V617F and V617I was observed. Thus, the JAK2 V617F mutation detection assay is specific for the V617F mutation. The JAK2 V617F mutation detection assay, using XNA-based technology, provides high sensitivity and specificity to detect the JAK2 V617F mutation. Thus, this assay would be a useful tool for clinical decision-making in determining the presence of the JAK2 V617F mutation in DNA from patient blood samples. Citation Format: Shuo Shen, Robert Brown, Daniel Kim, Larry Pastor, Andrew Y. Fu, Pushpinder Kaur, Alisha Babu, Wei Liu, Aiguo Zhang, Hiromi Tanaka, Michael Y. Sha. A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR [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 7295.

Abstract 4564: Therapeutic potential of SOS1 and KRAS inhibitors in malignant peripheral nerve sheath tumors

Abstract Neurofibromatosis type 1 (NF1) is a genetic disorder that affects the nervous system. It is caused by mutations in the NF1 gene. NF1 loss of function leads to elevated RAS signaling, which plays a crucial role in cell growth and proliferation. Due to the dysregulated RAS signaling, individuals with NF1 are prone to the development of neurofibromas. These neurofibromas are typically benign and called Plexiform Neurofibromas (PNFs), in some cases, they can transform into malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are highly aggressive and invasive cancers that contribute to a significant proportion of soft tissue sarcomas. The exact mechanisms behind this transformation are not fully understood, however it involves additional genetic mutations and alterations in cellular signaling pathways. With limited effective targeted therapies available, surgical removal remains the primary treatment option. However, due to tumor size and location, surgery may not always be feasible. The MEK inihibitor, selumetinib, has received approval for the treatment of PNFs, however some patients who were treated with selumetinib continued to develop MPNSTs suggesting that MEK inhibitors may have limitations in addressing MPNSTs. To target the RAS pathway and mitigate the effects of NF1 gene alterations, we aim to explore the therapeutic potential of two novel, potent and orally bioavailable small molecule inhibitors targeting SOS1 (BI 1701963) or KRAS (KRASmulti inhibitor BI 3706674), as alternative treatment options for MPNSTs. The KRASmulti inhibitor BI 3706674 binds non-covalently to the KRAS wild type allele as well as multiple KRAS mutant alleles in the GDP-bound state and thereby disrupts KRAS signaling. Therefore, we hypothesize that both inhibitors will work in NF1 models by reducing activated KRAS levels. In this study, a series of in vitro and in vivo experiments were conducted to evaluate the efficacy of BI 1701963 and BI 3706674 inhibitors, both individually and in combination. The studies were done in comparison to the MEK inhibitor selumetinib. In vitro analysis revealed a synergistic anti-proliferative effect when combining BI 1701963 and BI 3706674 inhibitors both in 2D and 3D growth conditions, which led to the downregulation of the RAS pathway, specifically the MAPK and PI3K signaling cascade, and an increase in apoptosis. The in vivo efficacy of either BI 1701963 or BI 3706674 inhibitor alone or in combination was tested in four different in vivo MPNST models. We observed significant tumor growth stabilization as well as tumor regression in all tested models in vivo. In-depth pathway modulation and pharmaco-dynamic studies are ongoing. In conclusion, this study highlights the combined effects of SOS1 (BI 1701963) and KRAS (BI 3706674) inhibition in MPNSTs. The combination therapy of these novel therapeutics may hold promise as a potential treatment strategy for patients with MPNSTs. Citation Format: Özlem Yüce Petronczki, Laura Pisarsky, Petra Lujza Szabó, Julia Brunmair, Antonia Geyer, Norbert Kraut, Darryl McConnell, Irene Waizenegger. Therapeutic potential of SOS1 and KRAS inhibitors in malignant peripheral nerve sheath tumors [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 4564.

Abstract 988: Genome-wide repeat landscapes in cancer and cell-free DNA

Abstract Introduction: Repeat sequences comprise >50% of the human genome and structural and epigenetic changes in these regions are implicated in cancer. However, no systematic analysis of the compendium of repeat sequences has ever been performed in human cancer or cell-free DNA (cfDNA), largely due to the inability to identify and quantify repeat sequences genome-wide. We describe here the first comprehensive analysis of genome-wide repeat landscapes in cancer and demonstrate their utility in cfDNA liquid biopsies. Methods: We developed ARTEMIS (Analysis of RepeaT EleMents in dISease) an alignment-free, genome-wide approach for analyzing repeat landscapes in short read sequencing. This approach uses a de novo search of short sequences (kmers) in the telomere to telomere (chm13) reference genome to identify 1.2 billion 24-mers uniquely defining 1280 individual repeat types occurring genome-wide across 57 subfamilies and 6 families. We analyzed ARTEMIS kmers in whole genome sequences of 525 matched tumor/normal pairs from breast, colorectal, liver, lung, ovarian, cervical, prostate, thyroid, head and neck, gastric, and bladder cancers in the Pan Cancer Analysis of Whole Genomes (PCAWG), and in low coverage (1-2x) whole genome sequences of 1450 cfDNA samples from individuals with and without 8 types of cancer. Results: Analysis of ARTEMIS kmer repeat landscapes in 525 PCAWG tumors identified changes in all 1280 repeat element types, including 820 novel elements not previously known to be altered in cancer. A median of 807 repeat elements (range 246-1280) were altered in each tumor compared to its matched normal. The majority of changes were in repeat elements not previously described as altered in tumorigenesis and were most frequently found within Satellites, LINEs and SINEs, though changes were also observed in LTRs, Transposable Elements, and RNA Elements. A cross-validated cfDNA model using repeat landscapes (ARTEMIS) and fragmentation features (DELFI) detected individuals in a diagnostic cohort (n=287) across all stages of lung cancer with high performance (AUC 0.91, 95% CI 0.88-0.95) and was externally validated in a separate population (n=513). The locked model generated scores that correlated with circulating tumor mutant allele fractions for patients (n=19) undergoing targeted lung cancer therapy (r=0.80, p<2.2e-16), and stratified progression-free survival (p<0.001). ARTEMIS repeat landscape analyses of cfDNA also detected liver cancer in a high-risk cohort (n=208) of individuals with cirrhosis or viral hepatitis (AUC 0.91, 95% CI 0.87-0.95), and identified tissue of origin among seven tumor types (n=423). Conclusions: ARTEMIS reveals genome-wide repeat landscapes in human cancer, including in 820 novel elements not previously known to be altered in tumorigenesis. These repeat landscapes that can now be described are evaluable in the circulation and provide an avenue for noninvasive detection and characterization of cancer. Citation Format: Akshaya Annapragada, Noushin Niknafs, James R. White, Daniel C. Bruhm, Christopher Cherry, Jamie E. Medina, Vilmos Adleff, Carolyn Hruban, Dimitrios Mathios, Zachariah H. Foda, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Genome-wide repeat landscapes in cancer and cell-free DNA [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 988.

Abstract 2422: Liquid biopsy approaches for monitoring metastatic pancreatic cancer in immunotherapy treated patients

Abstract Introduction: Pancreatic cancer has a poor prognosis especially when identified at advanced stages. Globally in 2020, >450,000 people died from the disease. For those with locally advanced or metastatic cancer, the standard of care treatment is chemotherapy. Preliminary studies show that some patients with advanced-stage disease respond to immune checkpoint blockade treatment. Determining response to therapy using imaging techniques can be challenging. There is a clinical unmet need for noninvasive approaches that can provide a real-time assessment of response to therapy in these patients. We evaluated two methods for assessing response to therapy using circulating cell-free DNA (cfDNA) in patients with metastatic pancreatic cancer treated with immune checkpoint inhibition and radiation as part of the CheckPAC study (NCT02866383). Methods: For all patients with available samples (n=40), we evaluated one pre-treatment blood draw and one on-treatment blood draw (26-65 days after treatment start). We extracted cfDNA from each sample and performed low-coverage 1-2x whole genome sequencing (WGS). We evaluated tumor and matched buffy coat for the 34 patients who had > 10% tumor purity using WGS with at least 60x and 30x coverage, respectively. We compiled the mutated bases in the tumor genome and examined these positions in the cfDNA. We counted the number of mutant observations from cfDNA WGS and divided them by the total number of observations at these bases to calculate whole-genome mutant allele frequency (MAF). We categorized responders based on >30% drop in MAF from baseline. In a tissue independent approach, we detected changes in genome-wide cfDNA fragmentation patterns using the DNA evaluation of fragments for early interception (DELFI) approach. We evaluated survival using DELFI scores of on-treatment samples. Samples were designated DELFI high (above median) or DELFI low (below median.) Log-Rank tests were used to compare survival curves. Results: For the mutation based approach, molecular responders (n=7) had a median progression free survival (PFS) of 485 days compared to 57 days for non-responders (n=9) (p=0.02). Molecular responders had a median overall survival (OS) of 694 days compared to 200 days for non-responders (p=0.062). Patients with DELFI low scores after therapy (n=14) had a longer median PFS than those with DELFI high scores (n=14) (307 days versus 70 days, respectively) (p=0.013). DELFI low patients also had a longer median OS than DELFI high patients (627 vs 158 days, respectively) (p<0.0001). Conclusions: These analyses suggest that WGS mutation-based and fragmentation cfDNA approaches can identify individuals with metastatic pancreatic cancer who respond to immune checkpoint inhibition. Incorporation of these molecular methods for evaluation of tumor burden may provide information for patient-physician decision making to improve patient quality of life. Citation Format: Carolyn Anna Hruban, Inna Chen, Daniel C. Bruhm, Shashikant Koul, Jennie Yao, Susann Theile, Kavya Boyapati, Nicholas A. Vulpescu, Akshaya Annapragada, Leonardo Ferreira, Stephen Cristiano, Zachariah H. Foda, Vilmos Adleff, Julia Johansen, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Liquid biopsy approaches for monitoring metastatic pancreatic cancer in immunotherapy treated patients [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 2422.

Abstract 3695: Monitoring response to immunotherapy using cell free DNA fragmentomes

Abstract BACKGROUND: The rapid detection of disease progression in patients receiving immune checkpoint inhibitors (ICIs) is challenging since there are no reliable biomarkers of clinical response. Liquid biopsy assays may address this unmet need by using cell-free DNA (cfDNA) to monitor treatment responses. Current targeted next-generation sequencing cfDNA assays are costly and have limited assay sensitivity for detection of cancer-specific mutations at low mutant allele frequency (MAF). Here, we demonstrate the utility of DELFI Tumor Fraction (DELFI-TF), a tumor- and mutation-independent cfDNA fragmentome approach to monitor treatment response in patients with metastatic non-small cell lung cancer (mNSCLC). METHODS: Overall, 422 longitudinal blood samples were collected from 141 mNSCLC patients of which 109 were treated with immunotherapy and 32 with targeted therapy (TT). Plasma-derived cfDNA was processed with whole genome sequencing at low coverage (~4x). Circulating tumor burden was quantified as the maximum MAF (maxMAF) of tumor-derived variants detected using a 500+gene panel. Matched white blood cells were used to filter out germline and clonal hematopoietic variants. A random forest regression model was trained with maxMAF as the response using fragmentation-derived features. The accuracy of DELFI-TF was assessed using leave one patient out cross-validation in the ICI cohort and holdout validation in the cohort of patients receiving TT. For survival analyses, we used the median baseline scores to define low/high DELFI-TF and maxMAF. RESULTS: In the ICI cohort, DELFI-TF scores were strongly correlated with the maxMAF (r=0.94, p<0.001, Pearson). At baseline, DELFI-TF and maxMAF both differentiate responding from non-responding tumors (p = 0.01 and p = 0.001 for DELFI-TF and maxMAF respectively; Wilcoxon). Patients with high DELFI-TF or maxMAF had significantly shorter progression-free survival (PFS) compared to low DELFI-TF or maxMAF (17.0 vs 4.7 months, p < 0.001; 17.0 vs 4.6 months, p < 0.001; Log-Rank respectively) and similar overall survival (OS) (DELFI-TF high 11.4 vs low 34.1 months, p<0.001; maxMAF high 10.9 vs low 34.1 months, p < 0.001, Log-Rank). Changes in DELFI-TF and maxMAF at the earliest timepoint were highly correlated (n=109; r=0.90; Pearson). In the validation cohort of TT patients, DELFI-TF at baseline outperformed maxMAF in distinguishing patients with PFS and OS benefits (DELFI-TF PFS 36.4 vs 11.5 months, p = 0.03; maxMAF PFS 21.9 vs 15.9 months, p=0.6; Log-Rank) (DELFI-TF OS high 37 months vs low not reached (NR), p < 0.05; Max MAF OS high 37 vs NR, p = 0.1, Log-Rank). The majority of TT patients (81%) had consistent changes with DELFI-TF and maxMAF from baseline to the earliest timepoint. CONCLUSIONS: DELFI-TF is a tumor and mutation independent approach that is cost-efficient with high performance comparable to current ctDNA assays for estimating tumor burden and monitoring response in cancer patients. Citation Format: Bahar Alipanahi, Lavanya Sivapalan, Jamie Medina, Zachary L. Skidmore, Paola Ghanem, Erica Peters, Gavin Pereira, Nisha Rao, Kavya Velliangiri, Alissa Konicki, Stephen Cristiano, Laurel Millberg, Jacob Carey, Keith Lumbard, Noushin Niknafs, Bryan Chesnick, Jennifer Tom, Alessandro Leal, Benjamin Levy, Patrick Forde, Peter Bach, Nicholas C. Dracopoli, Robert B. Scharpf, Victor Velculescu, Lorenzo Rinaldi, Valsamo Anagnostou. Monitoring response to immunotherapy using cell free DNA fragmentomes [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 3695.

Abstract 3659: Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer

Abstract Biliary tract cancer (BTC) is a rare type of abdominal cancer and is more frequent in Asian countries than in Western. The 5-year survival rate for BTC is very low 7 to 20% and only 20% of BTC patients could undergo curative-intent surgery. BTC diagnosis based on biopsy and cytology are low in sensitivity that prompted the necessity to identify alternative biopsy. Liquid biopsy, specifically circulating tumor DNA (ctDNA) is known for the accessibility and low invasiveness received great interest for their potential uses in various clinical applications for patients with insufficient tumor tissue, including mutation profiling, treatment monitoring, and cancer detection. However, the panel available in the market up-to-date focused mainly targeted, actionable alterations and thus not suitable for minimal residual disease (MRD) monitoring. In this study, we aimed to develop a customized ctDNA panel for MRD monitoring in BTC. In this study, 124 BTC patients undergone curative resections were enrolled at Hokkaido University and Sapporo Medical University from May 2019 to April 22. All enrolled patients have BTC diagnosis confirmed by pathological diagnosis after surgery, and without other malignant tumors. Genomic profiles of FFPE tissue were conducted using Ampliseq CCP. 13 patients were excluded for high fragmentation. Referring to the genomic profiles of 111 patients in our study and 1924 patients in TCGA established a customized panel using Ion Ampliseq HD technology. The Ampliseq HD panel claims to detect variants at 0.1% LOD for ctDNA detection. It also integrates molecular barcodes to reduce NGS-related errors. The designed panel was 258 Amplicons and 20 genes including eleven therapeutically targetable genes. Clinical characteristics of ctDNA detection were also assessed. Results:The panel validation: concordance of the mutation allele frequency (MAF) between the NGS standard control and Ampliseq HD panel showed a coefficient of determination (R2) of 0.96. Also, concordance of MAF Ampliseq CCP and Ampliseq HD panel on FFPE tumor profiling showed R2 of 0.78. When assessing 70% of BTC patients were covered to have at least one tumor-derived Ampliseq HD panel. When assessing using plasma cell-free DNA, 55% of BTC patients were detected to have at least one tumor-derived ctDNA. Distribution of detected altered top4 genes using Ampliseq HD panels shows TP53 35%, KRAS 10%, SMAD4 5%, ARID1A 2.5%. Clinical characteristics may affected the concordance of plasma and FFPE tumor tissue; stage (P=0.035), size of tumors (P=0.0088), subtype (P=0.013), LN metastasis (P=0.11). Conclusions: A highly sensitive ctDNA customized panel for BTC was established. We also found that clinical characteristics of the BTC affects the detection of ctDNA from blood liquid biopsy. Future study: MRD evaluation using this customized panel. Citation Format: Yoshihito Shinohara, Siew-Kee Low, Toru Nakamura, Yasutoshi Kimura, Takeshi Murakami, Kazuma Kiyotani, Satoshi Hirano. Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract 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 3659.

Abstract 2410: Comparative genomic profiling of unresectable NSCLC patients in the U.S. and China using a globally harmonized liquid biopsy assay platform

Abstract Background: Non-Small Cell Lung Cancer (NSCLC), accounting for over 80% of all lung cancers, benefits from targeted therapies based on genetic tests like EGFR, KRAS, ALK, and ERBB2. While prior studies focused on molecular characteristics through tissue biopsies, limited research has explored NSCLC molecular profiles via liquid biopsy, especially across different human races. This study presents a comprehensive genomic profiling analysis of unresectable NSCLC patients in the U.S. and China, using a globally harmonized liquid biopsy assay. Methods: As part of Predicine's Phoenix Program, a global molecular biomarkers screening initiative, 61 patients in the U.S. and 352 patients in China with unresectable advanced NSCLC were enrolled. All were treatment-naïve or recurred after 1st-line targeted therapies. Blood samples (10ml) were tested using PredicineCARE, an NGS-based liquid biopsy assay, to profile somatic mutations, copy number variations, and gene fusions. Results: The assay, validated in both regions using the same reference materials, achieved a Limit of Detection (LOD) of 0.25% mutation allele frequency, with a positive predictive value exceeding 99%. Profiling NSCLC patients in the U.S. and China revealed common genes like TP53, CDKN2A, EGFR, KRAS, RB1, and PIK3CA. TP53 and PIK3CA variations showed equivalent prevalence. However, EGFR variations were significantly higher in China (p<0.05), while CDKN2A (p<0.001), KRAS (p<0.01), and RB1 (p<0.01) variations were notably higher in the U.S. Conclusions: This study unveils the mutational landscape of advanced NSCLC through liquid biopsy. Unique prevalence patterns between U.S. and China cohorts suggest novel biomarkers for clinical diagnosis and provide insights for targeted therapy mechanism studies. Citation Format: Haoran Tang, Cancan Jia, Feng Xie, Yue Zhang, Xiaoxi Dong, Yong Huang, Shading Jia. Comparative genomic profiling of unresectable NSCLC patients in the U.S. and China using a globally harmonized liquid biopsy assay platform [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 2410.

Abstract 5015: Genomic profiling of colorectal cancer - insights from liquid biopsy comparisons between U.S. and China cohorts

Abstract Background: Molecular characteristics play a pivotal role in cancer diagnosis, treatment selection, and disease monitoring across various tumor types. While previous research has elucidated the molecular classification of colorectal cancer using tissue NGS, limited investigation has explored the molecular profile of colorectal cancer through liquid biopsy, especially across diverse human races. This study presents a comprehensive genomic profiling analysis of colorectal cancer patients, using a globally harmonized liquid biopsy assay to compare patient cohorts from both the U.S. and China. Methods: The prospective study is part of Predicine’s Phoenix Program, a global molecular biomarker screening initiative across multiple solid tumors. At present, the study has enrolled 57 patients in the U.S. and 62 patients in China, all presenting with advanced colorectal cancer. A 10ml blood sample was collected from each patient and tested using PredicineCARE, an NGS-based liquid biopsy assay, to profile somatic mutations, copy number variations, and gene fusions among these patients. Results: The assay, validated in both the U.S. and China using the same reference materials, achieved a LOD of 0.25% mutation allele frequency, with a positive predictive value exceeding 99%. Subsequently, the assay was applied to profile molecular aberrations in colorectal patients in both the U.S. and China. Among U.S. patients, the most commonly altered genes were APC (60%), KRAS (56%), TP53 (54%), MYC (25%), PIK3CA (16%), and EGFR (14%). In Chinese patients, the predominant altered genes were TP53 (48%), APC (42%), KRAS (32%), GNAS (21%), EGFR (16%), and PIK3CA (11%). The prevalence of GNAS variations was significantly higher in China than in the U.S. (p < 0.05), while the prevalence of APC (p < 0.05), KRAS (p < 0.01), and MYC (p < 0.001) variations was significantly higher in the U.S. than in China. Conclusions: This study reveals the extensive mutational landscape in advanced colorectal cancer patients through liquid biopsy. Distinct prevalence patterns in certain genes between the U.S. and China cohorts offer novel biomarkers for clinical diagnosis and provide insights for targeted therapy mechanism studies. Citation Format: Haoran Tang, Cancan Jia, Feng Xie, Yue Zhang, Xiaoxi Dong, Yong Huang, Shidong Jia. Genomic profiling of colorectal cancer - insights from liquid biopsy comparisons between U.S. and China cohorts [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 5015.

Abstract 1612: Timing ecDNA acquisition across cancer

Abstract Extrachromosomal DNA (ecDNA) are acentric, circular DNA fragments which form after DNA double strand breaks. They segregate randomly during mitosis, promoting high-level amplifications of positively selected oncogenes such as EGFR and MYC. The prevalence of ecDNA across several cancer types, such as glioblastoma and sarcoma, has made promising therapeutic targets. However, while DNA damage is a hallmark of nearly all cancers, the formation of ecDNA is not, and the determinants for ecDNA acquisition are unclear. Furthermore, whether the timing of tumors’ acquisition of ecDNA (i.e., early or late clonal or sub-clonal) affects patient survival and drug resistance is unclear. Here, we leverage orthoganol information from ecDNA amplicons, such as mutation allele frequency and sequence synteny, to develop computational methods for elucidating the evolutionary history of ecDNA. Applying these to several cancer subtypes in TCGA, we find that most ecDNAs arise early in tumor evolution. We expect these results will help identify determinants of ecDNA acquisition and its temporal influence on patient survival. Citation Format: Andrew R. Lynch, Peter Van Loo. Timing ecDNA acquisition across 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 1612.

Impact of IRGM gene promoter polymorphisms on susceptibility to chronic HBV infection.

The autophagy gene immunity-related GTPase M (IRGM) can affect the immune response against intracellular pathogens. The study was performed to determine any possible association between three IRGM single-nucleotide polymorphisms (SNPs) (rs4958842, rs4958843 and rs4958846) and chronic hepatitis B virus (HBV) infection. A total of 171 chronic HBV-infected individuals and 171 healthy controls were collected. Peripheral blood cells and Sanger sequencing were used to extract genomic DNA and determine the SNP genotypes, respectively. The C allele of rs4958843 is a risk factor for chronic HBV infection in various genetic models, including allelic, codominant and dominant models, with the following respective statistical data: allelic (T vs. C: OR=1.371, 95% CI=1.009-1.863, p=.043), codominant (TT vs. CC: OR=2.137, 95% CI=1.104-4.138, p=.024) and dominant (TT+TC vs. CC: OR=1.976, 95% CI=1.106-3.533, p=.021) models. The genotype and allele distributions of rs4958842 and rs4958846 showed no significant differences between chronic HBV infection patients and healthy controls. IRGM rs4958843 CC genotype carriers had significantly elevated values of alanine transaminase, aspartate transaminase alpha-fetoprotein and total bilirubin (OR=3.467, 95%CI=1.167-10.298), which was positively associated with the disease progression of HBV infection. Mutant allele C of IRGM rs4958843 polymorphism is associated with the risk of chronic HBV infection in the Han people in central China and contributes to the disease progression.

Role of IL-27 in Epstein-Barr virus infection revealed by IL-27RA deficiency.

Epstein-Barr virus (EBV) infection can engender severe B cell lymphoproliferative diseases1,2. The primary infection is often asymptomatic or causes infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder3. Selective vulnerability to EBV has been reported in association with inherited mutations impairing T cell immunity to EBV4. Here we report biallelic loss-of-function variants in IL27RA that underlie an acute and severe primary EBV infection with a nevertheless favourable outcome requiring a minimal treatment. One mutant allele (rs201107107) was enriched in the Finnish population (minor allele frequency = 0.0068) and carried a high risk of severe infectious mononucleosis when homozygous. IL27RA encodes the IL-27 receptor alpha subunit5,6. In the absence of IL-27RA, phosphorylation of STAT1 and STAT3 by IL-27 is abolished in T cells. In in vitro studies, IL-27 exerts a synergistic effect on T-cell-receptor-dependent T cell proliferation7 that is deficient in cells from the patients, leading to impaired expansion of potent anti-EBV effector cytotoxic CD8+ T cells. IL-27 is produced by EBV-infected B lymphocytes and an IL-27RA-IL-27 autocrine loop is required for the maintenance of EBV-transformed B cells. This potentially explains the eventual favourable outcome of the EBV-induced viral disease in patients with IL-27RA deficiency. Furthermore, we identified neutralizing anti-IL-27 autoantibodies in most individuals who developed sporadic infectious mononucleosis and chronic EBV infection. These results demonstrate the critical role of IL-27RA-IL-27 in immunity to EBV, but also the hijacking of this defence by EBV to promote the expansion of infectedtransformed B cells.

Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F–positive myeloproliferative neoplasms

AbstractPegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F–positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α–treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F–positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F–positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms.

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants.

The CPLANE protein Fuzzy regulates ciliogenesis by suppressing actin polymerization at the base of the primary cilium via p190A RhoGAP.

The primary cilium decorates most eukaryotic cells and regulates tissue morphogenesis and maintenance. Structural or functional defects of primary cilium result in ciliopathies, congenital human disorders affecting multiple organs. Pathogenic variants in the ciliogenesis and planar cell polarity effectors (CPLANE) genes FUZZY, INTU and WDPCP disturb ciliogenesis, causing severe ciliopathies in humans and mice. Here, we show that the loss of Fuzzy in mice results in defects of primary cilia, accompanied by increased RhoA activity and excessive actin polymerization at the basal body. We discovered that, mechanistically, Fuzzy interacts with and recruits the negative actin regulator ARHGAP35 (also known as p190A RhoGAP) to the basal body. We identified genetic interactions between the two genes and found that a mutant ArhGAP35 allele increases the severity of phenotypic defects observed in Fuzzy-/- mice. Based on our findings, we propose that Fuzzy regulates ciliogenesis by recruiting ARHGAP35 to the basal body, where the latter likely restricts actin polymerization and modifies the actin network. Our study identifies a mechanism whereby CPLANE proteins control both actin polymerization and primary cilium formation.