Gene Copy Number Loss Research Articles

The integrate profiling of single-cell and spatial transcriptome RNA-seq reveals tumor heterogeneity, therapeutic targets, and prognostic subtypes in ccRCC.

Clear-cell renal cell carcinoma (ccRCC) is the most common type of RCC; however, the intratumoral heterogeneity in ccRCC remains unclear. We first identified markers and biological features of each cell cluster using bioinformatics analysis based on single-cell and spatial transcriptome RNA-sequencing data. We found that gene copy number loss on chromosome 3p and amplification on chromosome 5q were common features in ccRCC cells. Meanwhile, NNMT and HILPDA, which are associated with the response to hypoxia and metabolism, are potential therapeutic targets for ccRCC. In addition, CD8+ exhausted T cells (LAG3+ HAVCR2+), CD8+ proliferated T cells (STMN+), and M2-like macrophages (CD68+ CD163+ APOC1+), which are closely associated with immunosuppression, played vital roles in ccRCC occurrence and development. These results were further verified by whole exome sequencing, cell line and xenograft experiments, and immunofluorescence staining. Finally, we divide patients with ccRCC into three subtypes using unsupervised cluster analysis. and generated a classifier to reproduce these subtypes using the eXtreme Gradient Boosting algorithm. Our classifier can help clinicians evaluate prognosis and design personalized treatment strategies for ccRCC. In summary, our work provides a new perspective for understanding tumor heterogeneity and will aid in the design of antitumor therapeutic strategies for ccRCC.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling

BackgroundCholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy.MethodsThe expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA.ResultsWe identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth.ConclusionsThis study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Clinicopathological characterisation of MTAP alterations in gastrointestinal cancers

BackgroundMethylthioadenosine phosphorylase (MTAP) is an essential metabolic enzyme in the purine and methionine salvage pathway. In cancer, MTAP gene copy number loss (MTAP loss) confers a selective dependency on the...

Abstract 1049: Liquid biopsy-based comprehensive genomic profiling reveal mutational landscape in advanced breast cancer

Abstract Introduction: Breast cancer is the most common malignancy worldwide in females, with more than 2.26 million new cases and 680 thousand of deaths in 2020. Advanced breast cancer is characterized by complicated genomic landscape and requires comprehensive genomic profiling. However, tissue biopsy is hard to get in patients with recurrent metastasis. Meanwhile, limited studies have been reported to use liquid biopsy and were not capable of reporting gene copy number loss. Here we reported a comprehensive genomic profiling study in advanced breast cancer patients using liquid biopsy. Methods: The prospective study is part of the Predicine’s Phoenix Program, a global molecular biomarkers screening program in multiple solid tumors. Currently, the study enrolled 220 advanced breast cancer patients from 8 centers in China, who were naïve to the 1st line treatment. 10ml of blood was collected from each patient and delivered to a central lab for ctDNA analysis. The study applied PredicineCARE, an NGS-based liquid biopsy assay, to profile somatic mutations, copy number variations, and gene fusions among these patients. Results: The study identified 793 somatic mutations and 283 copy number variants among blood samples from 220 patients. The most common altered genes were TP53(101/220, 46%), PIK3CA(77/220, 35%), BRCA2(37/352, 17%), PTEN(26/220, 12%), and ATM(24/220, 11%). Gene copy number gain incidents were identified on MYC(n=28), AKT3(n=23), FGFR1(n=18), and PIK3CA(n=16), etc. The study also reported gene copy number loss incidents through liquid biopsy, such as CDKN2A(n=17), BRCA1(n=12), TP53(n=11), RB1(n=11), BRCA2(n=10), PTEN(n=9), and ATM(n=8). Conclusions: This study revealed the comprehensive mutational landscape of advanced breast cancer patients through liquid biopsy, providing novel biomarkers for clinical diagnosis and targeted therapy mechanism studies. Citation Format: Haoran Tang, Feng Xie, Yue Zhang, Shidong Jia. Liquid biopsy-based comprehensive genomic profiling reveal mutational landscape in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1049.

1390P Circulating tumor DNA (ctDNA) and prognosis with PSMA-targeted radionuclide therapy (TRT)

PSMA-TRT is approved for mCRPC based upon demonstrated overall survival (OS) advantage. However, even after pre-selection by PSMA imaging, not all patients respond (54% without PSA50 response in the VISION study). In addition to dose (which is likely related to both target expression and amount of radioactivity administered), prognostic genomic factors that may relate to radiosensitivity exist. Collection of real-time tissue biopsies in the setting of mCRPC presents challenges that might be overcome with the use of ctDNA. Germline control + plasma DNA were obtained prior to treatment in 2 prospective trials of PSMA-TRT and assayed utilizing the PCF SELECT (Specific Evaluation in Liquid biopsies of Established prostate Cancer Targets) platform. Variables of interest included AR copy number (wild-type vs gain), homologous recombination repair (HRR) gene copy number (wild-type vs loss), and allele-specific ploidy (asP; diploid vs high). asP is a novel metric for genomic instability and loss of heterozygosity, computed as the weighted mean of the allele-specific copy number of homologous chromosomes (Ciani et al. Cell Systems 2022). Cox regression modelling was used to assess the association of these variables with clinical outcome measures of >50% PSA response (PSA50) and OS. 80 patients received PSMA-TRT (177Lu-PSMA-617 n=50; 225Ac-J591 n=30). High asP was associated with poorer OS (HR 2.14, 95% CI 1.15-4, p=0.017) and a trend to lower rates of achieving PSA50 (OR 0.48, 95% CI 0.16-1.47, p=0.20). AR copy number gain was associated with poorer OS (HR 2.69, 95% CI 1.40-5.18, p=0.003) and lower rates of PSA50 (OR 0.47, 95% CI 0.14-1.49, p=0.20). In this study, HRR gene copy number loss was not associated with differential responses or OS. Similar results as this combined study were obtained when individual studies of 177Lu-PSMA-617 or 225Ac-J591 were analyzed for AR copy number and asP. ctDNA analysis, a non-invasive technique, in patients undergoing PSMA-TRT demonstrate that AR copy number gain and high allele-specific ploidy, reflecting genomic instability and complex karyotype, are associated with poorer prognosis.

Features of chromosome instability in Chinese early-onset colorectal cancers.

e15519 Background: In recent years, the etiology and molecular mechanism of early-onset colorectal cancers (EOCRC) has evoking more and more attention. Previous studies suggested that EOCRC had unique somatic mutational characteristics, such as frequent mutations in genes of WNT signaling pathway. However, the features of chromosome instability in this particular subgroup of colorectal cancer remain unclear. Somatic copy number alteration (SCNA) is the main manifestation of chromosome instability. We performed a systematic analysis in regard to SCNAs in a large cohort of Chinese colorectal. Since the microsatellite instable (MSI-H) cancers rarely occurred SCNAs, only microsatellite stable (MSS) patients were included. Methods: A total of 1696 Chinese MSS CRC patients diagnosed in 2018-2020 were included. Early-onset were defined as patients diagnosed under 50 year-olds and the others were late-onset (LO). Genomic alterations and SCNAs were elucidated in all patients’ tumor specimen using next-generation sequencing (NGS) testing. Results: All patients were stratified as EOCRC (n = 439) and LOCRC (n = 1214). 3.87% EOCRC harbored germline cancer susceptibility genes (mainly APC and ATM) mutations while in the LOCRC counterpart the proportion was only 2.06% (mainly MUTYH and BRCA2). Of note, MYC amplification was detected in 71.43% EOCRC with copy-number gain and presented a trend to correlated with APC germline mutations (Fisher’s exact test, p-value = 0.07). In the other CRC (n = 1654) with no germline cancer susceptibility genes, genomic amplifications were detected in 831 (50.2%) cases. The frequency of 8q and 13q amplification were equal in EOCRC and LOCRC. However, EOCRC had higher proportion of 8p and 17q amplification, lower proportion of 20q amplification than LOCRC. Moreover, a higher ratio of EOCRC had high-level amplifications, mainly concentrated in 17q ( ERBB2, CDK12 and RARA) and 13q ( FLT3, CDK8 and FLT1), than LOCRC (23.7% vs 19.1%). Other high-level amplified genes include MYC, CCND2 and EGFR. EGFR high-level amplifications occurred more in EOCRC than LOCRC (13.5 % 2.6%), while CCND2 high-level amplifications were only detected in LOCRC. Gene copy number losses were detected in 195 (11.8%) cases. Loss of SMAD4, CDKN2A, CDKN2B, MAP2K4, PTEN and AMER1 genes were most recurrent. The incidence of SMAD4 (43.1% vs 22.9%), CDKN2B (11.8% vs 6.2%), MAP2K4 (7.8% vs 4.2%) gene losses were higher in EOCRC while the incidence of PTEN (13.9% vs 7.8%) and SMAD3 (6.9% vs 1.9%) gene losses were higher in LOCRC. Conclusions: EOCRC present different SCNA characteristics from LOCRC, including unique focal amplifications, distinct recurrent genes with copy number losses, and more high-level amplifications events mainly distributed in 17q and 13q. It can be reasonably speculated that MYC gene gain was a critical event correlated with familial mutations in EOCRC.

Loss of Integrin α9β1 on Tumor Keratinocytes Enhances the Stromal Vasculature and Growth of Cutaneous Tumors

Angiogenesis is critical to tumor progression, and the function of integrins in tumor angiogenesis is complex. In this study, we report that loss of integrin α9β1 expression from epidermal tumor cells is critical to maintaining persistent stromal vessel density. Forced expression of α9 in transformed mouse keratinocytes dramatically reduces vessel density in allograft tumors invivo compared with that in the same cells lacking α9β1. Moreover, α9 mRNA expression is dramatically reduced in mouse and human epidermal tumors as is α9β1-dependent gene regulation. Loss of tumor cell α9β1 occurs through at least two mechanisms: (i) ITGA9 gene copy number loss in human tumors and (ii) epigenetic silencing in mouse and human tumors. Importantly, we show that reversal of epigenetic silencing of Itga9 restores α9 expression in mouse keratinocytes and that human tumors without ITGA9 copy number loss have increased promoter methylation. Our data suggest that for epidermal tumorigenesis to occur, tumor cells must avoid the tumor and angiogenic suppressive effects of α9β1 by repressing its expression through deletion and/or epigenetic silencing, thereby promoting stromal development and tumor growth.

Integrated Multiomics Analyses Revealing Different Molecular Profiles Between Early- and Late-Stage Lung Adenocarcinoma.

The molecular differences in genetic and epigenetic profiling between early-stage (ES) and late-stage (LS) lung adenocarcinoma (LUAD), which might help to understand cancer progression and biomarker guided precision treatment, need further be investigated. In this study, we performed comprehensive analysis using multi-omics next-generation sequencing (NGS) on tissue samples from 7 ES (stage I) and 10 LS (stage III/IV) LUAD patients to study molecular characteristics between the two groups. Characterization of the genomic and transcriptomic profiles showed stage-specific somatic mutations, copy number variations (CNVs) and differentially expressed genes (DEGs). LS samples tend to have more TP53, ERBB2 and CHD4 mutations. Gene copy number loss occurs in immune-related gene pathways in the late stage of LUAD. ATAC-seq analysis showed that LS samples harbored more open chromatin peaks around promoter regions and transcription start sites (TSS) than ES samples. We then identified the known transcription factor (TF) binding motifs for the differentially abundant ATAC-seq peaks between the ES and LS samples and found distinct regulatory mechanisms related to each stage. Furthermore, integrative analysis of ATAC-seq with WGS and RNA-seq data showed that the degree of chromatin accessibility is related to copy number changes, and the open chromatin regions could directly regulate the expression of some DEGs. In conclusion, we performed a comprehensive multi-omics analysis of the early and late stages of LUAD and highlighted some important molecular differences in regulatory mechanisms during cancer progression. Those findings help to further understand mechanism and biomarker related targeted therapy.

Circulating Cell-Free DNA-Based Detection of Tumor Suppressor Gene Copy Number Loss and Its Clinical Implication in Metastatic Prostate Cancer.

Current liquid biopsy assays lack sufficient sensitivity to detect copy number loss, which limits the interrogation of critical tumor suppressor gene deletions during cancer progression and treatment. Here we describe a liquid biopsy assay with improved sensitivity for detection of copy number loss in blood samples with low levels of circulating tumor DNA, and demonstrate its utility by profiling PTEN, RB1, and TP53 genetic loss in metastatic prostate cancer patients.

Abstract 551: Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer

Abstract Loss of expression of the PTEN gene is associated with poorer prognosis in many solid tumors. While cfDNA-based liquid biopsy testing are gradually adopted as the routine testing method in the clinical setting, most of them are unable to reliably detect copy number loss and/or are limited reporting copy number amplification events only. Method: We have developed a capture-based CLIA-certified NGS assay, PredicineCARE, to robustly identify tumor-derived aberrations including SNVs, Indels, fusions, copy number amplifications and deletions from blood samples. Analytical validation of the assay was performed with commercial standard reference materials and contrived cell lines. Copy number loss of PTEN was also confirmed by Immunohistochemistry (IHC) assay in 15 clinical samples. Clinical utility of PTEN copy number loss was further established by its significant association with poor outcome in clinical patients with metastatic castration-resistant prostate cancer (mCRPC). Results: Analytical validation of the PredicineCARE assay demonstrated its super sensitivity and robustness of detecting homozygous copy number loss at approximately 10% tumor fraction. Both the sensitivity and Positive Prediction Value reached 100% when overall gene copy number is below 1.75 copies. When comparing to tissue IHC results reviewed by two independent pathologists, PredicineCARE liquid biopsy assay revealed 86.7% concordance with IHC in detecting PTEN loss (80% sensitivity, 100% specificity, and 100% PPV). The copy number loss was further confirmed by low-pass whole-genome sequencing of cfDNA samples. When the assay was applied to profile mCRPC patients, copy number loss of PTEN was detected at the prevalence rate of 19.2% (10/52) and 35.9% (23/64) respectively. Notably, patients harboring PTEN copy number loss showed significantly worse overall survival in the two cohorts independently (log-rank P = 0.0001 and 0.0004 respectively). Conclusion: We have developed PredicineCARE for clinical use, a highly sensitive cfDNA-based assay to detect gene copy number variants in addition to gene mutations and fusions. Its ability to detect gene copy number loss in low fraction of tumor contents as a valuable molecular biomarker holds great potential for clinical applications and patient selections in personalized precision oncology, particularly for investigational drugs targeting the AKT-PI3K-PTEN pathway. Citation Format: Xiaoxi Dong, Minghua Zhang, Tiantian Zheng, Lu Tan, Feng Xie, Amy Xiaohong Wang, Pan Du, Shidong Jia, Chris Lu, Jianjun Yu. Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 551.

Genomic Landscape of Chinese Clear Cell Renal Cell Carcinoma Patients With Venous Tumor Thrombus Identifies Chromosome 9 and 14 Deletions and Related Immunosuppressive Microenvironment.

BackgroundClear cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) is associated with a poor clinical outcome. Although several studies have examined the genomic features of ccRCC, the genetic profile of VTT along with its matched primary tumor has not been fully elucidated.Materials and methodsSamples of VTT tissues and matched primary tumor tissues from ccRCC patients (n = 25), as well as primary tumor tissues from patients without VTT (n = 25) were collected and analyzed using whole-exome sequencing. Four additional ccRCC patients who were unfit for surgery were treated with an anti-programmed death receptor-1 (PD-1) monoclonal antibody (Toripalimab, 240 mg, Q3W, IV).ResultsBy comparing the primary kidney tumors from ccRCC patients with or without VTT, a relatively higher prevalence of BAP1 and KDM5C alterations were found in ccRCC patients with VTT, and these alterations were associated with worse overall survival in the kidney renal clear cell carcinoma (KIRC) database. Based on subclone analysis, VTT was predicted to primarily originate directly from the primary renal mass. A significantly higher prevalence of CELSR2 and TET2 alterations were identified in the VTTs compared with the matched primary tumors. An increased prevalence of DNA damage repair genes, especially those involved in homologous recombination repair and non-homologous end joining, was found in ccRCC patients with VTT. Notably, VTT was characterized by the increase incidence of copy number loss in the whole exome (p < 0.05), particularly in the chromosome 9 and 14 regions. Deletion of chromosome 9 and 14 was associated with worse survival, unfavorable clinical features, and the presence of an immunosuppressive microenvironment, which was characterized by higher infiltration of regulatory T cells, follicular helper T cells, and resting mast cells, but lower counts of resting CD4 memory T cells and CD8 positive T cells. A significantly lower count of CD4+ and CD8+ tumor-infiltrated lymphocytes was identified in the VTT samples comparing with matched primary tumor. Of note, three out of the four ccRCC patients with VTT in our cohort who were treated with the anti-PD-1 therapy exhibited remarkable remission in the renal mass but no notable shrinkage in the VTT mass.ConclusionOur study revealed the genetic profile of Chinese ccRCC patients with VTT, and identified multiple features associated with known poor outcomes, including gene alterations and copy number loss. The deletions in chromosomes 9 and 14, and the associated immunosuppressive microenvironment may indicate limited sensitivity to anti-PD-1/PD-L1 monotherapy in VTT.

Genetic instabilities as potential biomarkers for pregnancy-associated breast cancer.

e12559 Background: Pregnancy associated breast cancer (PABC), defined as breast cancer diagnosed during or within one year after pregnancy, occurs approximately once in every 3,000 pregnancies and accounts for up to 6.9% of all breast cancers in women ≤ 45 years. Compared to age-matched non-PABC patients, PABC is generally characterized by a particularly aggressive histopathologic profile (more ER-, PR- and HER2 negative tumors and tumors of higher grade) with a higher mortality rate. Whether these cancers arise before or during pregnancy, and whether their genetic profile differs from non-PABC tumors, is currently unknown. This study assesses the genetic background of PABC by detection of specific DNA copy number alterations (CNA). Methods: We assembled 29 triple-negative PABC patients from the Dutch nationwide pathology database (PALGA). From their formalin-fixed and paraffin embedded (FFPE) tumor tissue blocks, DNA was extracted. Multiplex Ligation-Dependent probe Amplification (MLPA) analysis was performed for detection of CNAs in 20 oncogenes and 2 tumor suppressor genes using the P078-D2 kit (MRC Holland). Individual gene copy number loss (MLPA ratio &lt; 0.7), gain (ratio 1.3-2.0) and amplification (ratio &gt; 2.0) was determined and unsupervised hierarchical cluster analysis was performed to identify differences in copy number (CN) patterns between PABC patients. Chi square statistics were used to interrogate associations with clinicopathologic characteristics. Log-Rank test was used to compare Kaplan Meier survival curves between CN subgroups. Results: Triple negative PABC tumors showed frequent copy number loss on chromosome 17q ( CPD, MED1, TOP2A, MAPT, PPM1D) and 8p ( ZNF703 and ADAM9), as well as frequent copy number gain/amplification of MYC (8q) and CCND1 (11q). Cluster analysis identified 2 clusters of PABC patients based on their tumor copy number patterns: a CN-neutral cluster and a CN-high cluster, the latter demonstrating significantly lower chromosome 17 copy numbers than the former (for PPM1D, ERBB2, CPD, MED1, TOP2A, CDC6 and MAPT). Patients with CN-neutral tumors more often showed lymph node metastases (p = 0.028), were of significantly higher stage (p = 0.031), and demonstrated a worse overall survival (p = 0.036) compared with PABC patients harboring CN-high tumors. Conclusions: We demonstrate different tumor CN patterns within a small group of triple negative PABC-patients, associated with distinct aggressiveness. This will form the basis for further in-depth genetic profiling within a larger Dutch PABC cohort, rendering insight in the disease progression and contributing to the development of more targeted and personalized treatments.

Abstract PO-10: Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture

Abstract Linker histone H1 binds to nucleosomes and causes chromatin compaction, yet little is known about their biologic function. Somatic missense mutations in histone 1 genes (HIST1H1B-E) occur in ~30% of follicular lymphomas and DLBCL, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. Examining whole exomes in 547 DLBCL patients, we found significant enrichment for H1 gene SNVs and copy number loss in the MCD class of ABC-DLBCLs as compared to other subtypes. Next we performed a genetic driver analysis in 101 DLBCLs with matched germline control to identify mutations significantly enriched over background somatic variation. We find that lymphoma-associated H1 alleles H1C and H1E are true genetic driver mutations in lymphomas. Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to bind to chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRAP assays in live cells. Hence both types of mutation confer loss of function. Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T cell-dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments, indicating that they have superior fitness (p=0.0086). We performed RNA-seq in H1C/EDKO GC B-cells, which revealed an aberrant gene derepression signature. Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming and are normally silenced during early development by the PRC2 complex (p &amp;lt;0.05 to 1e-05). Indeed, histone mass spectrometry showed reduced H3K27me3 (p=0.0003) and increased H3K36me2 (p =0.001) in H1C/EDKO GC B cells. This prompted us to characterize the epigenome of purified H1C/EDKO vs WT GC B cells using Hi-C, ATAC-seq, and ChIP-seq for multiple histone marks. H1 deficiency causes profound architectural remodeling of the genome characterized by large-scale yet focal compartment shifts from compartment B to compartment A. The degree of decompaction results in distinct epigenetic states, primarily due to gain of H3K36me2 and/or loss of repressive H3K27me3. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR&amp;lt;0.001) in the RNA-seq profiles of independent cohorts of DLBCL patients with H1C and H1E mutations. We crossed H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Consistent with acquisition of stem cell characteristics, H1C+/-H1E+/-;VavP-Bcl2 but not VavP-Bcl2 primary lymphoma cells manifested lymphoma-initiating functionality after secondary transplantation into recipient animals. Citation Format: Nevin Z. Yusufova, Matt Teater, Andreas Kloetgen, Alexey Soshnev, Adewola Osunade, Christopher Chin, Ashley Doane, Louis Staudt, David Scott, Neil Kelleher, Aristotelis Tsirigos, Marcin Imielinski, Yael David, David Allis, Ethel Cesarman, Ari Melnick. Histone 1 deficiency drives lymphoma through disruption of 3D chromatin architecture [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-10.

Pathway-Specific Genome Editing of PI3K/mTOR Tumor Suppressor Genes Reveals that PTEN Loss Contributes to Cetuximab Resistance in Head and Neck Cancer.

Cetuximab, an mAb targeting EGFR, is a standard of care for the treatment for locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC). However, despite overexpression of EGFR in more than 90% of HNSCC lesions, most patients with HNSCC fail to respond to cetuximab treatment. In addition, there are no available biomarkers to predict sensitivity or resistance to cetuximab in the clinic. Here, we sought to advance precision medicine approaches for HNSCC by identifying PI3K/mTOR signaling network-specific cetuximab resistance mechanisms. We first analyzed the frequency of genomic alterations in genes involved in the PI3K/mTOR signaling circuitry in the HNSCC TCGA dataset. Experimentally, we took advantage of CRISPR/Cas9 genome editing approaches to systematically explore the contribution of genomic alterations in each tumor suppressor gene (TSG) controlling the PI3K-mTOR pathway to cetuximab resistance in HNSCC cases that do not exhibit PIK3CA mutations. Remarkably, we found that many HNSCC cases exhibit pathway-specific gene copy number loss of multiple TSGs that normally restrain PI3K/mTOR signaling. Among them, we found that both engineered and endogenous PTEN gene deletions can mediate resistance to cetuximab. Our findings suggest that PTEN gene copy number loss, which is highly prevalent in HNSCC, may result in sustained PI3K/mTOR signaling independent of EGFR, thereby representing a promising mechanistic biomarker predictive of cetuximab resistance in this cancer type. Further prospective studies are needed to investigate the impact of PTEN loss on cetuximab efficacy in the clinic.

Tumour kinome re-wiring governs resistance to palbociclib in oestrogen receptor positive breast cancers, highlighting new therapeutic modalities

Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of ‘classical’ oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M–checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.

GABPA-dependent down-regulation of DICER1 in follicular thyroid tumours.

Mutations in the miRNA enzyme gene DICER1 have been reported in several endocrine malignancies and is associated with the rare tumour-predisposing DICER1 syndrome. DICER1 mutations have been reported in subsets of follicular thyroid carcinoma (FTC), but the role of DICER1 in follicular thyroid tumorigenesis has not been extensively studied. In this study, we investigate the role of DICER1 in 168 follicular thyroid tumours and in an FTC cell line. We found rare DICER1 mutations in paediatric FTC cases and a general DICER1 down-regulation in FTCs visualized both on mRNA and protein level, especially pronounced in Hürthle cell carcinoma (HuCC). The down-regulation was also evident in follicular thyroid adenomas (FTAs), suggesting a potential early step in tumorigenesis. The expression of DICER1 was lower in FTCs of older patients in which TERT promoter mutations are more frequent. In FTCs, DICER1 down-regulation was not caused by gene copy number loss but significantly correlated to expression of the transcription factor GABPA in clinical cases. GABPA was found to bind to the DICER1 promoter and regulate DICER1 expression in vitro, as GABPA depletion in FTC cell lines reduced DICER1 expression. This in turn stimulated cell proliferation and affected the miRNA machinery, evident by altered miRNA expression. To conclude, we show that GABPA directly regulates DICER1 in FTC, acting as a tumour suppressor and displaying down-regulation in clinical samples. We also show reduced expression of DICER1 in benign and malignant follicular thyroid tumours, suggesting a potentially early tumorigenic role of this gene aberrancy.

Reduced SKP1 Expression Induces Chromosome Instability through Aberrant Cyclin E1 Protein Turnover.

Chromosome instability (CIN), or progressive changes in chromosome numbers, is an enabling feature of many cancers; however, the mechanisms giving rise to CIN remain poorly understood. To expand our mechanistic understanding of the molecular determinants of CIN in humans, we employed a cross-species approach to identify 164 human candidates to screen. Using quantitative imaging microscopy (QuantIM), we show that silencing 148 genes resulted in significant changes in CIN-associated phenotypes in two distinct cellular contexts. Ten genes were prioritized for validation based on cancer patient datasets revealing frequent gene copy number losses and associations with worse patient outcomes. QuantIM determined silencing of each gene-induced CIN, identifying novel roles for each as chromosome stability genes. SKP1 was selected for in-depth analyses as it forms part of SCF (SKP1, CUL1, FBox) complex, an E3 ubiquitin ligase that targets proteins for proteolytic degradation. Remarkably, SKP1 silencing induced increases in replication stress, DNA double strand breaks and chromothriptic events that were ascribed to aberrant increases in Cyclin E1 levels arising from reduced SKP1 expression. Collectively, these data reveal a high degree of evolutionary conservation between human and budding yeast CIN genes and further identify aberrant mechanisms associated with increases in chromothriptic events.

Reduced Expression of Genes Regulating Cohesion Induces Chromosome Instability that May Promote Cancer and Impact Patient Outcomes

Chromosome instability (CIN), or continual changes in chromosome complements, is an enabling feature of cancer; however, the molecular determinants of CIN remain largely unknown. Emerging data now suggest that aberrant sister chromatid cohesion may induce CIN and contribute to cancer. To explore this possibility, we employed clinical and fundamental approaches to systematically assess the impact reduced cohesion gene expression has on CIN and cancer. Ten genes encoding critical functions in cohesion were evaluated and remarkably, each exhibits copy number losses in 12 common cancer types, and reduced expression is associated with worse patient survival. To gain mechanistic insight, we combined siRNA-based silencing with single cell quantitative imaging microscopy to comprehensively assess the impact reduced expression has on CIN in two karyotypically stable cell lines. We show that reduced expression induces CIN phenotypes, namely increases in micronucleus formation and nuclear areas. Subsequent direct tests involving a subset of prioritized genes also revealed significant changes in chromosome numbers with corresponding increases in moderate and severe cohesion defects within mitotic chromosome spreads. Collectively, our clinical and fundamental findings implicate reduced sister chromatid cohesion, resulting from gene copy number losses, as a key pathogenic event in the development and progression of many cancer types.

Identification of De Novo Enhancers Activated by TGFβ to Drive Expression of CDKN2A and B in HeLa Cells.

Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFβ selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFβ signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFβ induced ARF mRNA and protein, and ARF knockdown limited TGFβ-mediated growth suppression. TGFβ-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFβ by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFβ, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

4E-BP1 Is a Tumor Suppressor Protein Reactivated by mTOR Inhibition in Head and Neck Cancer.

Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. SIGNIFICANCE: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.