Somatic Genetic Events Research Articles

Somatic mutations in arteriovenous malformations in hereditary hemorrhagic telangiectasia support a bi-allelic two-hit mutation mechanism of pathogenesis

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder of vascular malformations characterized by mucocutaneous telangiectases and arteriovenous malformations (AVMs) in internal organs. HHT is caused by inheritance of a loss of function mutation in one of three genes. Although individuals with HHT are haploinsufficient for one of these genes throughout their entire body, rather than exhibiting a systemic vascular phenotype, vascular malformations occur as focal lesions in discrete anatomic locations. The inconsistency between genotype and phenotype has provoked debate over whether haploinsufficiency or a different mechanism gives rise to the vascular malformations. We previously showed that HHT-associated skin telangiectases develop by a two-hit mutation mechanism in an HHT gene. However, somatic mutations were identified in only half of the telangiectases, raising the question whether a second-hit somatic mutation is a necessary (required) event in HHT pathogenesis. Here, we show that another mechanism for the second hit is loss of heterozygosity across the chromosome bearing the germline mutation. Secondly, we investigate the two-hit mutation mechanism for internal organ AVMs, the source of much of the morbidity of HHT. Here, we identified somatic molecular genetic events in eight liver telangiectases, including point mutations and a loss of heterozygosity event. We also identified somatic mutations in one pulmonary AVM and two brain AVMs, confirming that mucocutaneous and internal organ vascular malformations undergo the same molecular mechanisms. Together, these data argue that bi-allelic loss of function in an HHT gene is a required event in the pathogenesis of HHT-associated vascular malformations.

Targeted panel sequencing of pharmacogenes and oncodrivers in colorectal cancer patients reveals genes with prognostic significance

BackgroundColorectal cancer is still the second leading cause of cancer-related deaths and thus biomarkers allowing prediction of the resistance of patients to therapy and estimating their prognosis are needed. We designed a panel of 558 genes with pharmacogenomics records related to 5-fluorouracil resistance, genes important for sensitivity to other frequently used drugs, major oncodrivers, and actionable genes. We performed a target enrichment sequencing of DNA from tumors and matched blood samples of patients, and compared the results with patient prognosis stratified by systemic adjuvant chemotherapy.ResultsThe median number of detected variants per tumor sample was 18.5 with 4 classified as having a high predicted functional effect and 14.5 moderate effect. APC, TP53, and KRAS were the most frequent mutated genes (64%, 59%, and 42% of mutated samples, respectively) followed by FAT4 (23%), FBXW7, and PIK3CA (16% for both). Patients with advanced stage III had more frequently APC, TP53, or KRAS mutations than those in stages I or II. KRAS mutation counts followed an increasing trend with grade (G1 < G2 < G3). The response to adjuvant therapy was worse in carriers of frameshift mutations in APC or 12D variant in KRAS, but none of these oncodrivers had prognostic value. Carriage of somatic mutations in any of the genes ABCA13, ANK2, COL7A1, NAV3, or UNC80 had prognostic relevance for worse overall survival (OS) of all patients. In contrast, mutations in FLG, GLI3, or UNC80 were prognostic in the same direction for patients untreated, and mutations in COL6A3, LRP1B, NAV3, RYR1, RYR3, TCHH, or TENM4 for patients treated with adjuvant therapy. The first association was externally validated. From all germline variants with high or moderate predicted functional effects (median 326 per patient), > 5% frequency and positive Manhattan plot based on 3-year RFS, rs72753407 in NFACS, rs34621071 in ERBB4, and rs2444274 in RIF1 were significantly associated with RFS, OS or both.ConclusionsThe present study identified several putative somatic and germline genetic events with prognostic potential for colorectal cancer that should undergo functional characterization.

Ubtf- Tandem Duplications Are Recurrent Somatic Genetic Events in Adult AML and Associate with Additional Structural Variants and a Distinct Gene Expression Profile (Alliance)

Background The gene upstream binding transcription factor ( UBTF ) encodesa nucleolar protein that is a key regulator of the epigenetic status of ribosomal DNA and RNA transcription. Tandem duplications (TDs) of the UBTF gene have been initially identified in pediatric patients (pts) with acute myeloid leukemia (AML) and were recently also reported as relatively rare, but recurrent genetic events in adult AML based on targeted re-sequencing efforts (1.2%-3%). Their presence was associated with dysplastic bone marrow features, and co-occurrence of select genetic features, including FLT3-internal tandem duplications (ITDs) and WT1 mutations, and inferior outcome. However, associated biologic features and affected pathways have not yet been thoroughly investigated, and may provide further insights into leukemogenesis and identify potentially targetable vulnerabilities. Thus, we set out to assess the frequency of UBTF-TDs in a large cohort of adult AML pts (n=807) treated on frontline protocols of the Alliance for Clinical Trials in Oncology with complete RNA sequencing data, comprehensive targeted mutation profiling, centrally reviewed cytogenetic data and clinical information available, and to gain further insights into UBTF-TDs via comprehensive integrated genomic profiling. Methods UBTF-TD detection was performed via the RNAseq-based RNAmut and EnFusion algorithm. To gain insights into associated molecular features, all UBTF-TD + pts underwent paired tumor-normal whole-exome sequencing. To understand associated pathways and downstream effects, differential gene expression (DGE) and gene set enrichment analyses (GSEA) were performed. Results UBTF-TDs were detected in 6/807 pts (0.7%). In line with previous studies, these 6 UBTF-TD + pts were young, with a median age of 35y (range: 19-59y). Three pts had extramedullary disease, 2 with skin and 1 with gingival involvement. All but 1 of the 6 pts were cytogenetically normal. Clinically, pts did poorly after treatment with intensive chemotherapy induction, with a CR rate of only 33%. No pt was alive 2.5 y after diagnosis. In paired tumor/normal exome sequencing, UBTF-TD + pts had a median mutation burden of 21 non-synonymous variants in coding genes (range: 17-27), with a notable presence of larger insertions/deletions (indels), loss of heterozygosity (LOH) or TDs as co-occurring somatic genetic lesions (median: 4, range: 2-5). There was a 100% co-occurrence of UBTF-TDs with FLT3-ITDs. In addition, pts harbored larger indels in WT1 (n=2), BPTF, CEBPA, Corf53, KRTAP5-7, NFE2, PTPRN2, RNF144B, SOWAHC and ZCCHC14. Also, analysis of copy-number variations and LOH revealed LOH in 5 pts, in chromosomes 13 (n=3), 15 (n=1) and 18 (n=1), suggestive of a propensity to the acquisition of structural variants. In unsupervised clustering analysis of the RNAseq data, UBTF-TD + pts clustered with FLT3-ITD + pts but formed a relatively distinct subcluster in principal component and t-distributed stochastic neighbor embedding analyses. In supervised clustering comparing 6 UBTF-TD +/ FLT3-ITD + and 196 UBTF-TD -/ FLT3-ITD + pts, 2 UBTF-TD - pts clustered together with the UBTF-TD +/ FLT3-ITD + group, suggesting either presence of an undetected TD or genetic phenocopy. UBTF expression was higher in UBTF-TD pts. DGE analysis of UBTF-TD +/ FLT3-ITD + and UBTF-TD -/ FLT3-ITD + pts (Fig 1) showed distinct upregulation of the locus of the stem cell and chemoresistance marker ALDH1A3, including the associated long non-coding RNAs AC015712.7/AC015712.2 in UBTF-TD + pts, which was confirmed by a comparison of those pts to AML pts who had neither TD (n=605). GSEA indicated a downregulation of inflammation pathways and several metabolic pathways of UBTF-TD +/ FLT3-ITD + compared to FLT3-ITD + pts (Fig 2). Conclusions UBTF-TDs are rare, but recurrent genetic events in adult AML, associated with high chemotherapy resistance. Our results highlight a susceptibility of those pts to the acquisition of structural variants and suggest a distinct associated biology, with upregulation of ALDH1A3 as possible contributing resistance mechanism. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202); https://acknowledgments.alliancefound.org

Comprehensive genomic characterization of sporadic synchronous colorectal cancer: Implications for treatment optimization and clinical outcome

Sporadic synchronous colorectal cancer (SCRC) refers to multiple primary CRC tumors detected simultaneously in an individual without predisposing hereditary conditions, which accounts for the majority of multiple CRCs while lacking a profound understanding of the genomic landscape and evolutionary dynamics to optimize its treatment. In this study, 103 primary tumor samples from 51 patients with SCRC undergo whole-exome sequencing. The germline and somatic mutations and evolutionary and clinical features are comprehensively investigated. Somatic genetic events are largely inconsistent between paired tumors. Compared with solitary CRC, SCRCs have higher prevalence of tumor mutation burden high (TMB-H; 33.3%) and microsatellite-instability high (MSI-H; 29.4%) and different mutation frequencies in oncogenic signaling pathways. Moreover, neutrally evolving SCRC tumors are associated with higher intratumoral heterogeneity and better prognosis. These findings unveil special molecular features, carcinogenesis, and prognosis of sporadic SCRC. Strategies for targeted therapy and immunotherapy should be optimized accordingly.

TSC2 inactivation, low mutation burden and high macrophage infiltration characterise hepatic angiomyolipomas.

Although TSC1 or TSC2 inactivating mutations that lead to mTORC1 hyperactivation have been reported in hepatic angiomyolipomas (hAML), the role of other somatic genetic events that may contribute to hAML development is unknown. There are also limited data regarding the tumour microenvironment (TME) of hAML. The aim of the present study was to identify other somatic events in genomic level and changes in TME that contribute to tumorigenesis in hAML. In this study, we performed exome sequencing in nine sporadic hAML tumours and deep-coverage targeted sequencing for TSC2 in three additional hAML. Immunohistochemistry and multiplex immunofluorescence were carried out for15 proteins to characterise the tumour microenvironment and assess immune cell infiltration. Inactivating somatic variants in TSC2 were identified in 10 of 12 (83%) cases, with a median allele frequency of 13.6%. Five to 18 somatic variants (median number: nine, median allele frequency 21%) not in TSC1 or TSC2 were also identified, mostly of uncertain clinical significance. Copy number changes were rare, but detection wasimpaired by low tumour purity. Immunohistochemistry demonstrated numerous CD68+ macrophages of distinct appearance from Küpffer cells. Multiplex immunofluorescence revealed low numbers of exhausted PD-1+/PD-L1+, FOXP3+ and CD8+ T cells. hAML tumours have consistent inactivating mutations in TSC2 and have a low somatic mutation rate, similar to other TSC-associated tumours. Careful histological review, standard IHC and multiplex immunofluorescence demonstrated marked infiltration by non-neoplastic inflammatory cells, mostly macrophages.

Somatic genetic reversion of large duplication event in IL2RG in siblings

BackgroundDeleterious mutations in the common gamma chain (γC, CD132) are associated with X-linked severe combined immunodeficiency (X-SCID) characterized by a T-B+NK- phenotype. Hypomorphic variants in the IL2RG gene have also been associated with a milder or atypical SCID phenotype. Milder phenotypes can also occur when somatic genetic rescue occurs in the setting of severe IL2RG mutations. Spontaneous somatic variants particularly in hematologic disorders can occur particularly in genes with known selection pressure including IL2RG. ResultsHere, we report the clinical and molecular findings from two brothers with atypical X-SCID phenotype including hypogammaglobulinemia, elevated IgM, T cell lymphopenia and extensive verruca plana. One brother required splenectomy secondary to a benign vascular tumor and had recurrent pancreatitis consistent with primary sclerosing cholangitis. Both brothers developed recurrent suppurative pneumonia resulting in early bronchiectasis in their first decade. Next generation sequencing initially was negative, yet reanalysis found a large duplication event in exon 3 of IL2RG gene with somatic genetic rescue events that occurred twice in two brothers. Functional studies revealed a unique mosaic pattern in each affected sibling when stimulating peripheral blood mononuclear cells with IL-2 or IL-21 resulting in bimodal phospho-STAT5 or phospho-STAT3 respectively. The genetic analysis revealed the maternal allele was heterozygous as expected, although the ratio of these events and clonal analysis revealed cell type skewing. ConclusionOur data demonstrate an atypical SCID phenotype in two brothers secondary to the combination of a large duplication event in the IL-2RG gene partially rescued by somatic genetic rescue. Although previous somatic reversion cases have been reported involving the IL2RG genetic locus, our cases demonstrate a large duplication reversion event with recurrence. Our case highlights the unique selection pressure to amplify rare genetic events at the IL2RG locus.

Abstract 2712: Establishing the early genetic progression of cancers with unobtainable premalignant disease

Abstract Premalignant tissue samples collected at different timepoints along the path to invasive tumors have been used to establish the typical order of somatic genetic events in several cancer types. For many other cancer types, however, precancer tissue is unobtainable, leaving the genetic progression unknown. Here, we demonstrate an approach that can reconstruct progression histories for such cancers from exome sequencing of primary tumors. We validated our recent computational method, PhylogicNDT, on classic, HPV-negative head and neck squamous cell carcinoma (HNSCC), recapitulating the previous experimentally determined sequence of genetic events that occur during progression. We uncovered progression of HPV-positive HNSCC tumors, which lack premalignant tissue, identifying subtype specific early drivers. We associated the timing of major changes in ploidy with the development of intra-tumor genetic heterogeneity and shorter overall survival. In cancers with unobtainable premalignant tissue, our approach can fill the knowledge gap about early progression, providing the basis for development of experimental models and methods for early detection, interception, and prognostication. Citation Format: Ignaty Leshchiner, Edmund A. Mroz, Justin Cha, Daniel Rosebrock, Oliver Spiro, Juliana Bonilla-Velez, William Faquin, Armida Lefranc-Torres, Derrick Lin, William Michaud, Gad Getz, James W. Rocco. Establishing the early genetic progression of cancers with unobtainable premalignant disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2712.

Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue

AbstractHuman telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

P24.04 Concordance of Tissue and Cell-Free DNA-Based Next-Generation Sequencing in Patients With Lung Adenocarcinoma

Panel-based next-generation sequencing (NGS) to detect targetable somatic genetic events is guideline-recommended initial diagnostic testing in lung adenocarcinoma. Tissue biopsy has a number of limitations, including expense and biopsy-related adverse events. “Liquid biopsy” uses blood-based NGS on cell-free DNA (cfDNA). Prior investigations have demonstrated high rates of concordance at initial diagnosis. However, concordance at time of progression remains unknown.

Somatic genetic rescue of a germline ribosome assembly defect

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.

Molecular Pathogenesis of Progressive Refractoriness in MLL -Rearranged AML

BackgroundDespite advanced therapeutics, many leukemia patients become refractory to additional therapy, accounting for a major cause of leukemic deaths. Among major human acute myeloid leukemias (AMLs), MLL -rearranged AML is characteristic of progressive refractoriness to chemotherapy and shorter period to relapse. To clarify the molecular pathogenesis of such characteristics, we analyzed genetic abnormalities accumulated in MLL -rearranged AML in additional to the genetic events like MLL -fusion and MLL -partial tandem duplication (MLL-PTD).MethodWe exploited a mouse model of recurrent leukemia generated by mouse progenitors transduced with human MLL / AF9. Mouse leukemic cells with MLL / AF9 were serially transplanted in a total of 58 mice in 4 generations. We investigated somatic genetic events in leukemic cells from mouse models of MLL -rearranged AML and patients with MLL -rearranged AML. Whole exome and targeted-capture sequencing results were combined and analyzed to detect somatic genetic events and whole transcriptome sequencing and microarray dataset to assess gene expression.ResultsIn two independent experiments using mouse with transduced MLL/AF9, leukemic cells obtained in the 1st (N=2) and 4th (N=9) transplants were analyzed by whole exome sequencing. The onset of leukemia became progressively earlier in later generation transplant. Median periods to relapse were 57 and 16 days in the 1st and the 4th transplants, respectively (P < 0.001). With regard to molecular pathogenesis, leukemic cells in the 4th transplants had 12−31 more mutations, in addition to the mutations found in those in the 1st transplants (6-44 mutations). Significantly, two RNA polymerases (Polr2a and Polr3d), which are functionally related to Mll, were mutated in two experiments during the 4th transplants, suggesting that genetic lesions in MLL-associated complex could play a synergetic role in survival advantage of MLL/AF9 positive cells.In addition, mutations in Ptpn11 (G60R) and Gnb2 (G77R), which correspond to the same residues in human proteins, were also detected. In the cohort of human hematological neoplasms analyzed by targeted-capture sequencing, PTPN11 (G60R) is located at the very well documented hotspot in MLL -rearranged human AML and GNB2 G77 residue is present close to that affected by other recurrent mutations in human AML. Of note is that a germline mutation of the identical position of orthologous protein GNB1 was most recently reported in a case with acute lymphoblastic leukemia. To confirm pathological significance of such a novel GNB2 mutation, we analyzed gene expression in human AML. When compared between human MLL -rearranged (N=176) and MLL -rearrangement (−) (N=692) AML cases, GNB2 transcripts were significantly upregulated in human MLL -rearranged AML cases. Functionally, we induced wild-type and G77R mutant GNB2 in human hematopoietic cell lines. Overexpression of wild-type GNB2 conferred cytokine independency to granulocyte macrophage colony-stimulating factor dependent leukemic cell line TF1. Induced GNB2 (G77R)and knocked down wild-type GNB2 in MLL -rearranged AML cell lines were used to assess and confirm the molecular pathogenesis associated with a novel candidate protein GNB2.ConclusionsOur results in serial transplant experiments provide insights into the molecular mechanism of progression and progressive refractoriness of human MLL -rearranged AML. This study confirms that GNB2 is a novel candidate protein whose overexpression and activated mutation might play a significant role in the pathogenesis of this leukemia. DisclosuresTakaori-Kondo:celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria. Makishima:Yasuda Medical Foundation: Research Funding.

Abstract PO-010: Kidney angiomyolipomas are defined by a unique transcriptomic profile and H3K27ac chromatin state

Abstract Background: Kidney angiomyolipomas (AML) are benign mesenchymal tumors that are commonly seen in Tuberous Sclerosis Complex (TSC), a rare genetic neurocutaneous disorder, but also occur sporadically. Kidney AML are due to either TSC1 or TSC2 biallelic loss, whereas other somatic genetic events are rare and do not contribute to tumor development. We hypothesized that the chromatin state and master transcription factors are also drivers of kidney angiomyolipoma growth, alongside mTORC1 hyperactivation. Material and Methods: We performed whole transcriptome RNA-sequencing on 28 kidney AML, and ChIP-seq for H3K27ac (a histone modification that marks open chromatin and regulates high transcription of nearby genes) on 25 kidney AML, a human kidney AML derived TSC2 null cell line (621-101), and a pigmented melanoma cell line (SK-MEL30). ChIP-Seq for MITF (Microphthalmia-associated transcription factor) was also carried out on three kidney AML tumors and SK-MEL30 cells. Functional studies were performed to assess the oncogenic role of MITF in vitro and in vivo. Results: Differential expression analyses of kidney AML compared to both The Cancer Genome Atlas (TCGA) tumors and GTEx normal tissues revealed 347 differentially expressed genes (DEGs), including 18 transcription factors (TFs; FDR/adjusted p-value&amp;lt;0.05). MITF (the isoform A) and PPARγ, known oncogenes, were highly expressed in kidney AML (4th and 1st out of 27 TCGA tumor types, respectively). In addition, 6 of 10 top DEGs in kidney AML are known MITF targets including CTSK, PMEL, and GPNMB. ROSE (Ranking of Super Enhancers) and regulatory potential (RP) analysis of H3K27ac ChIP-seq data compared to human normal tissues (Epigenome Roadmap project), identified MITF (near Transcription Start Site of isoform A), PPARγ, CTSK and GPNMB as genes with extended open regulatory chromatin regions, known as ‘super-enhancers’, suggesting they are critical for kidney AML development. Gene set enrichment analysis (GSEA) of all 347 DEGs showed enrichment in pathways for epithelial-mesenchymal transition, myogenesis, adipogenesis, and estrogen response (all q-values&amp;lt; 6.54 × 10−9). Immunohistochemistry demonstrated positive staining for nuclear MITF and cytoplasmic GPNMB in kidney AML, lymphangioleiomyomatosis (LAM), a lung tumor entity similar to kidney AML, and hepatic AML (n&amp;gt;=3 sections per tumor) compared to adjacent normal tissue. Knock out of MITF-A by CRISPR/Cas9 showed reduction in cell growth (82%, p&amp;lt;0.01), invasion (48%, p&amp;lt;0.01) and migration (70%, p&amp;lt;0.001) in vitro, whereas stable overexpression of MITF-A in 601-101 cells enhanced xenograft tumor formation in vivo. Conclusions: Our studies have identified unique chromatin signatures, and several highly-expressed TFs, including MITF-A and PPARγ, which likely are essential for kidney AML development, enabling potential novel treatment strategies. Citation Format: Krinio Giannikou, Clemens K. Probst, Mahsa Zarei, Xintao Qiu, Melissa Duarte, Nikolas Kesten, Zachary Hebert, Raga Vadhi, Alba Font-Tello, Paloma Cejas, Charles H. Yoon, Chin-Lee Wu, Myles Brown, Elizabeth P. Henske, Henry Long, David J. Kwiatkowski. Kidney angiomyolipomas are defined by a unique transcriptomic profile and H3K27ac chromatin state [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-010.

Defining eligible patients for allele-selective chemotherapies targeting NAT2 in colorectal cancer

Therapies targeting somatic bystander genetic events represent a new avenue for cancer treatment. We recently identified a subset of colorectal cancer (CRC) patients who are heterozygous for a wild-type and a low activity allele (NAT2*6) but lack the wild-type allele in their tumors due to loss of heterozygosity (LOH) at 8p22. These tumors were sensitive to treatment with a cytotoxic substrate of NAT2 (6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine, APA), and pointed to NAT2 loss being a therapeutically exploitable vulnerability of CRC tumors. To better estimate the total number of treatable CRC patients, we here determined whether tumor cells retaining also other NAT2 low activity variants after LOH respond to APA treatment. The prevalent low activity alleles NAT2*5 and NAT2*14, but not NAT2*7, were found to be low metabolizers with high sensitivity to APA. By analysis of two different CRC patient cohorts, we detected heterozygosity for NAT2 alleles targetable by APA, along with allelic imbalances pointing to LOH, in ~ 24% of tumors. Finally, to haplotype the NAT2 locus in tumor and patient-matched normal samples in a clinical setting, we develop and demonstrate a long-read sequencing based assay. In total, > 79.000 CRC patients per year fulfil genetic criteria for high sensitivity to a NAT2 LOH therapy and their eligibility can be assessed by clinical sequencing.

Abstract A25: In vivo evidence validating the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic cancers

Abstract A dynamic cycle of palmitoylation and depalmitoylation stably localizes N-Ras, but not K-Ras-4b, at the plasma membrane. This selective dependency suggests that targeting palmitoylation could inhibit the growth of NRAS-mutant cancers while leaving normal cells unaffected, due to their intact K-Ras-4b function. We sought to clarify the dependency of oncogenic N-Ras signaling on palmitoylation in a model relevant to human cancer. We used the lox-STOP-lox (LSL) knock-in system (Haigis et al., Nat Genet 2008) to express an NrasG12D oncogene at physiologic levels, and generated a second site mutation abrogating the palmitoylation site (C181S). We then compared Mx1-Cre; NrasLSL-G12D,C181S/LSL-G12D,C181S to Mx1-Cre; NrasWT/WT and Mx1-Cre; NrasLSL-G12D/LSL-G12D (hereafter G12D,C181S, WT, and G12D, respectively). We injected mice with poly(I:C) at weaning to excise the LSL cassette in hematopoietic cells. As reported (Wang et al., Blood 2011; Xu et al., Cancer Disc 2013), G12D mice died early (119-267 days, n=14) from an aggressive myeloproliferative disorder (MPD). In contrast, no mortality was observed in the G12D,C181S and WT cohorts until 18-19 months of age (p=3x10-6 by Kaplan-Meier analysis). Pathologic analysis of 6-month-old mice showed that key features of MPD were fully rescued in the G12D,C181S cohort. Specifically, whereas white blood cell (WBC) counts and spleen sizes were in the normal range in G12D,C181S and WT mice, G12D mice had profound leukocytosis (median WBC count: 52.19 k/μL), splenomegaly (median spleen weight: 0.884 g), and myeloid expansion (n=6). Biochemical analysis revealed that N-RasG12D,C181S accumulated in the GTP-bound conformation, but mostly localized in the cytosol, and failed to hyperactivate the MAPK pathway. We next generated NrasG12D,C181S/G12D compound heterozygous mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S mutation in vivo. Over half of these mice developed hematologic disease and died prematurely, although later than homozygous G12D mutant mice (median survivals: 429 and 225 days; n = 22 and 14, respectively). Common findings in moribund mice were splenomegaly, expansion of myeloid cells, and different degrees of leukocytosis and anemia. Importantly, genetic analysis from diseased mice revealed reduced frequency or absence of the NrasG12D,C181S allele (allelic burden 0-30%) due to secondary somatic genetic events. This observation supports the existence of high selective pressure to overcome growth-suppressive properties of unpalmitoylated N-Ras during leukemogenesis. In conclusion, these studies provide strong genetic evidence validating palmitoylation as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model for addressing this question in other cancers. Citation Format: Noemi A. Zambetti, Ari J. Firestone, Jasmine C. Wong, Amanda M. Long, Anagha Inguva, Jarrett R. Remsberg, Radu M. Suciu, Benjamin F. Cravatt, Kevin M. Haigis, Kevin Shannon. In vivo evidence validating the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A25.

Abstract IA31: Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo

Abstract Despite remarkable progress over the past few years, developing chemical inhibitors that directly and selectively target mutant Ras proteins remains a fundamental challenge. Given this, inhibitors of proteins that function upstream or downstream of oncogenic Ras are a promising alternative therapeutic strategy for RAS mutant tumors. However, the complexity of Ras signaling networks and adaptive mechanisms that result in pathway “rewiring” complicates identifying genes and proteins that are essential for the growth and survival of these cancers. Genetically engineered mice that express mutant Ras alleles from the endogenous locus are a robust system for uncovering pathway dependencies and potential therapeutic vulnerabilities. We have generated novel strains of mice to address two specific questions. First, we collaborated with Kevin Haigis to generate a “second site” C181S substitution within a conditional NrasLSL-G12D mutant allele (Nat Genet 2008;40:600) to ask if palmitoylation is essential for myeloid transformation in vivo. We intercrossed mice carrying this mutation with the Mx1-Cre strain and induced Cre recombinase expression in the hematopoietic compartment by injecting compound mutant pups with polyI:polyC. As previously reported, homozygous NrasLSL-G12D mice uniformly developed an aggressive myeloproliferative disorder (MPD) characterized by markedly elevated blood leukocyte counts, splenomegaly, and death by age 9 months. By contrast, homozygous NrasLSL-G12D,C181S mice remained healthy with no evidence of hematologic disease despite constitutively elevated levels of RasGTP in bone marrow cells that were similar to those observed in NrasLSL-G12D mice. Whereas normal NRas and N-RasG12D proteins are predominantly in the plasma membrane, N-RasG12D, C181S was largely mis-localized in the cytosol. We also generated Mx1-Cre, NrasG12D,C181S/G12D compound heterozygous mutant mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S substitution. Over half of these mice developed aggressive hematologic malignancies, albeit with prolonged latency compared to homozygous NrasG12D mice. Importantly, genetic analysis of diseased tissues revealed reduced frequency or complete absence of the NrasG12D,C181S allele due to secondary somatic genetic events, which indicates strong selective pressure to overcome the growth-suppressive properties of unpalmitoylated N-Ras. Together, these studies validate the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model system for addressing this question in other cancers. In another ongoing project, we collaborated with Pedro Perez-Macera and David Tuveson to modify a LSL-KrasG12D knock-in allele to introduce a second-site amino acid Y64G substitution that severely impairs binding to PI3 kinase. Surprisingly, mice expressing KrasG12D,Y64G in the germline are viable and phenotypically normal, although they are born at a lower than expected Mendelian frequency. Whereas Mx1-Cre, KrasG12D/+ mice uniformly develop aggressive MPD and die by 4 months of age, KrasG12D,Y64G animals have normal blood counts at 12-18 months of age. However, 100% of KrasG12D,Y64G mice develop lung lesions by 1 year of age, and ultimately succumb from lung tumors with a median survival of 496 days. The majority of these lesions are classified pathologically as atypical lymphoid proliferation or papillary adenomas, with a few mice developing adenocarcinomas. These studies demonstrating that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis are generally consistent with and also extend elegant studies of Pi3kca mutant mice from the Downward lab (Cell 2007;129:957; Cancer Cell 2013;24:617). Beyond the bone marrow and lung, KrasG12D,Y64G mice are a potent genetic tool for dissecting the role of aberrant PI3 kinase signaling in lung, pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and these data have implications for treating human cancers with KRAS mutations. Citation Format: Kevin M. Shannon. Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA31.

Tumor sequencing is useful to refine the analysis of germline variants in unexplained high-risk breast cancer families

BackgroundMultigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene.MethodsWhole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate.ResultsSixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation.ConclusionTumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.

Somatic genetic rescue in Mendelian haematopoietic diseases.

Somatic mutations occur spontaneously in normal individuals and accumulate throughout life. These genetic modifications contribute to progressive ageing phenotypes and are directly involved in cancer development. However, a growing number of studies of Mendelian haematopoietic disorders indicate that somatic genetic events can offset the pathogenic effect of germline mutations at the cellular level, leading to genetic mosaicism and, in some cases, resulting in a milder disease phenotype. Notably, spontaneous genetic events that confer a positive effect on cells do not always benefit the individual, for whom the effects can be neutral or even clinically detrimental. These somatic genetic rescue events have important diagnostic, therapeutic and clinical consequences and constitute valuable models for studying the differentiation and/or homeostasis of haematopoietic lineages.

Analysis of Genomic Predispositions to Sporadic Myeloid Neoplasms Mediated By DDX41 in Japan

Analysis of Genomic Predispositions to Sporadic Myeloid Neoplasms Mediated By DDX41 in Japan

Abstract P3-04-06: Genomic analysis of cancers arising in breasts with different mammographic density

Abstract Background: High mammographic density (MD) is a known risk factor for breast cancer, but it is currently unknown whether this altered microenvironment leads to the development of genomically different tumours. Method: We used lifepool, an Australian prospective population-based cohort of over 53,000 women currently in a mammographic screening program, to identify &amp;gt;1000 cases of breast carcinoma (in situ and invasive breast cancer) for analysis. Pathology reports, detection modality (screen-detected/interval), MD data (AutoDensity, Nickson et al., 2013 DOI:10.1186/bcr3474), germline DNA and tumor tissue were available for a subset of cases. Tumours were classified into quintiles, depending on the relative MD ranking of the most recent normal mammogram. Tumour DNA was analysed using Affymetrix OncoScan MIP arrays or using a targeted Agilent SureSelect and Illumina sequencing panel of 105 genes relevant to breast cancer. Copy number and mutation data were compared between tumours from the highest (high-MD) and lowest (low-MD) quintiles of MD. Results: Preliminary analysis found no significant differences in clinico-pathological features between tumours from breasts with high-MD (n=38) and low-MD (n=35). Genomic analysis of 8 high-MD tumours and 8 low-MD tumours identified chromosome 15 loss as being significantly enriched in low-MD tumours. Only Low-MD tumours to date contained TP53 mutation (3/8). Data will be presented from at least an additional 9 low-MD and 15 high-MD tumours currently undergoing genomic assays. Conclusion: When complete, our data will be the first to evaluate the somatic genetic events in tumours arising in low-MD and high-MD breasts. Although our cohort to date is small, it may be possible that tumours from extremes of MD have different genomic characteristics. Citation Format: Gorringe KL, Hughes S, Nickson C, Devereux L, Pridmore V, Rowley SM, Byrne DJ, Cheasley D, Kavanagh AM, Mann GB, Fox SB, Campbell IG. Genomic analysis of cancers arising in breasts with different mammographic density [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-06.

Pheochromocytoma and paraganglioma: genotype versus anatomic location as determinants of tumor phenotype.

To date, germline or somatic genetic events can be detected for at least 60% of paragangliomas. Strong genotype-phenotype associations have been recognized and become increasingly refined. Characteristics closely linked with genotype include syndromic presentation, age of onset, risk of metastatic disease and predominant anatomic site. In contrast, profiles of catecholamine secretion appear to be largely determined by anatomic location or cell type of origin. This review summarizes current knowledge of genotype-phenotype correlations for paragangliomas in different locations and scrutinizes previous publications on the respective tissues of origin to find potential explanations for site-related differences. We hypothesize that differential sensitivities of distinct chromaffin cell populations to hypoxia are major determinants of these differences, with increased sensitivity to hypoxia likely exacerbating vulnerability to mutation-derived disruption of hypoxic signaling pathways. Potential involvement of endothelin-1, tumor necrosis factor type 1 receptor-associated protein and the hypoxia-inducible miR-210 in the development of abdomino-thoracic or head and neck paragangliomas are discussed. Recognition of factors that predispose to chromosomal losses, or amplify sub-threshold molecular alterations towards tumorigenic events in different (chromaffin) cell types, may facilitate the leap from developing targeted therapies towards establishment of tumor preventative measures.