Allelic Loss Research Articles

P-802 Distinct genetic impact of CRISPR/Cas9 gene correction in human embryos compared to induced pluripotent stem cells

Abstract Study question Does CRISPR/Cas9 gene editing have a distinct genetic impact in human embryos compared to induced pluripotent stem cells (iPSCs)? Summary answer IPSCs display a difference in repair events, such as template usage, compared to the germline which indicates a distinct genetic impact by CRISPR/Cas9 gene editing. What is known already CRISPR/Cas9 is utilized to induce targeted DNA editing. To introduce specific changes, such as mutational correction, a DNA template is generally administered, stimulating homology-directed repair. Recent human germline editing studies aiming for mutational repair created a moderate amount of embryos which solely carried wild-type alleles. Remarkably, no template use was observed, but instead loss-of-heterozygosity (LOH) was demonstrated. This is the presence of only one (the wild-type) allele, either caused by gene conversion events using the wild type allele or chromosome loss. Up to date, no iPSCs studies have evaluated LOH events when attempting to correct a heterozygous mutation. Study design, size, duration A guide RNA targeting the mutant allele, and a repair template containing the wild-type allele and a synonymous variant to track template usage were designed. For iPSCs targeting, the components were nucleofected after which DNA was extracted from the whole well (n = 3) or from individual colonies (n = 33). For human embryo targeting, the components were injected simultaneously with sperm into donated spare oocytes (n = 32). DNA was extracted from embryos after 3-6 days of in vitro culture. Participants/materials, setting, methods Sperm and renal cells, from which the iPSCs were derived, were donated by a patient with a heterozygous base pair substitution in PLCZ1 (c.136-1G>C) causing fertilization failure. To overcome fertilization failure, assisted oocyte activation was employed during ICSI. For the embryos, next-generation sequencing (NGS), short tandem repeat (STR) analysis and a whole genome single nucleotide polymorphism (SNP) assay were performed. For the iPSCs, NGS and a targeted SNP assay were carried out. Main results and the role of chance The genetic events in embryos originating from mutant sperm (n = 32) displayed following distribution: 19% showed the untargeted mutant allele, 56% showed additional mutagenesis and 25% showed only the wild-type allele. In the latter group, the template was never utilized, pointing to LOH. STR analysis indeed revealed LOH events of different lengths in these embryos. SNP analysis of one embryo, originating from mutant sperm but displaying the wild-type allele, did not demonstrate LOH (with a detection limit of 500bp). These findings suggest the occurrence of gene conversion, which rejects the formerly stated hypothesis that the observed LOH in human embryos can mostly be attributed to chromosome loss. In the iPSCs, whole-well (n = 3) and single colony (n = 33) data demonstrated a similar trend in genetic event distribution compared to the embryos, with the main difference that in 30% of the corrected reads/colonies, template use was observed. LOH was further analysed in nineteen single colonies. In all the colonies (3/3) corrected with the template, no LOH was present. When the colonies displayed additional mutagenesis, 7% (1/13) contained LOH events. In 33% (1/3) of the colonies showing only the wild-type allele (without signs of template use), LOH was observed. Limitations, reasons for caution SNP assays have a higher resolution compared to STR analysis, and enable distinction of LOH events between ‘gene conversion’ and ‘chromosome loss’. Therefore, more embryos and iPSC colonies will be analysed with SNP assays. However, informative SNPs define the resolution of our assay (currently 500 bp from Cas9 cut site). Wider implications of the findings Our results show that template usage seems to differ between human embryos and iPSCs, but that iPSCs do not solely rely on template use as well. This demonstrates that embryo experiments to study the genetic impact of CRISPR/Cas9 cannot fully be replaced by iPSCs experiments. Trial registration number Not Applicable

PB1954: COMPARING STR PROFILES OF DNA ISOLATED FROM DIVERSE TUMOR SITES IN MULTIPLE MYELOMA

Background: Multiple myeloma (MM) course is often complicated by extramedullary lesions - plasmacytomas, which pathogenesis is still not completely understood. Genome instability underlies dissemination in various neoplasias, including MM. An important manifestation of genetic instability is the loss of heterozygosity, i.e., the loss of the second allele in the regions of the genome featuring inherited polymorphisms. STR profiling is a method that allows simultaneously assessing the degree of DNA degradation and giving an integral assessment of the stability of the tumor genome. To assess the heterogeneity of MM, not only the tumor substrate in the bone marrow and plasmacytoma but also in circulating cell-free DNA (cfDNA) present in the peripheral blood plasma could be effectively analyzed. Aims: To compare STR profiles of DNA isolated from various anatomical locations (plasma, bone marrow, plasmacytoma) in MM patients. Methods: The prospective study included 21 patients with newly diagnosed MM (11 women, 10 men) aged 39 to 82 years (median 53 years). MM was diagnosed according to the IMWG-2014 criteria. In 15 patients, MM was complicated by plasmacytoma, in 6 of them a tumor biopsy was performed and tumor DNA was isolated. In all patients, plasma circulating cfDNA and control DNA were isolated from buccal epithelium using a commercial kit (Qiagen, Germany) according to the manufacturer’s protocol. In 11 patients, positive immunomagnetic selection of CD138+ bone marrow cells was performed using a monoclonal antibody to CD138 (Miltenyi Biotec, Germany). STR-profiling using COrDIS Plus panel (Gordiz LLC, Russia) of isolated DNA samples (21 plasma circulating cfDNA samples, 11 CD138+ DNA samples of bone marrow cells, 6 plasmacytoma DNA samples) was performed. STR profiles were analyzed using GeneMapper v.4 software (Applied Biosystems, USA). Results: In 10 out of 11 patients, analysis of STR profiles in CD138+ bone marrow cells revealed loss of heterozygosity (LOH) variants with different allelic load, suggesting either a deletion/copy number neutral LOH, or a duplication of one of the two alleles affecting from one to six STR loci for every patient. In all patients for whom plasmacytoma DNA was isolated (n=6), various variants of LOH were also identified (from 1 to 6 loci) (Table 1). Of the 21 plasma circulating cfDNA samples, LOH variants were detected in ten cases (48%) (from 1 to 4 loci), in the other 10 cases, no tumor DNA was detected, the samples matched the control DNA from the buccal epithelium; only in one case, the result turned out to be non-informative due to a significant degree of degradation of the cfDNA. STR profiles of DNA isolated from different tumor sites of the same patient in most cases were identical. In one case aberrant STR loci in plasma circulating cfDNA and bone marrow CD138+ cells were different (patient No. 20). In this patient, MM was complicated by plasmacytomas of various anatomical locations (Th7, 8th rib). One can speculate that the discrepancy between plasma circulating cfDNA and bone marrow CD138+ STR profiles indicates the presence of various MM clones in different tumor loci. Image:Summary/Conclusion: Evaluation of plasma circulating cfDNA might be an effective approach for molecular diagnosis in MM. In 48% of MM patients, it is possible to detect tumor cfDNA in the plasma at the onset of the disease. Comparison of DNA STR profiles from different lesions might reveal the diversity of tumor clones in MM.

Abstract 3818: Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma

Abstract Introduction: Loss-of-heterozygosity (LOH) events in chromosome 6p, comprising the human leukocyte antigen (HLA) genes, have been reported in about 10% of cutaneous melanoma (compared to 20-40% of squamous cell carcinomas), while copy number gains in this region have been observed in over 50% of melanoma. Recent studies focused in HLA allelic loss have been restricted to DNA-based approaches, and have not been validated orthogonally at the RNA or protein levels. Here, using clinical melanoma biopsies and patient-derived melanoma cell lines, we show that genetic alterations in HLA genes results in imbalanced allele expression, subsequently skewing antigen presentation by melanoma cells. Methods: Whole exome and RNA sequencing (WES, RNAseq) analyses were performed on 760 melanoma biopsies and 60 patient-derived melanoma cell lines. Patient-matched normal WES was used to perform HLA haplotyping across Class I and II HLA genes. Tumor WES was analyzed for copy number alterations in chromosome 6, identifying which alleles were lost or gained. Differential expression of HLA alleles was quantified in tumor RNAseq, correlating the allelic imbalance at the DNA and RNA levels. Melanoma cell lines heterozygous for HLA-A*02, A*03, and A*24 were analyzed by flow cytometry for surface-level HLA protein expression using allele-specific antibodies to quantify allelic densities and compare the imbalance of HLA-A alleles at the DNA, RNA, and protein levels. Results: Across 760 melanoma biopsies, copy number alterations in chromosome 6p were identified in 76% of tumors; 12% had LOH, and 54% had copy number gains that resulted in imbalanced copies of alleles. In paired tumor WES and RNAseq (N=682), genetic imbalance was correlated with imbalanced expression of HLA alleles in the classical Class I HLA genes (Spearman rho=0.64-0.7; p=2.2e-16); this association was strengthened in tumors with high tumor cellularity, and was not associated with the total expression of the HLA genes.These patterns were explored in a 60 patient-derived melanoma cell lines with matched tumor WES and RNAseq, confirming that alleles gained at the genetic level were also expressed at higher levels than alleles that were not gained or lost. In 10 cell lines heterozygous for either HLA-A*02, A*03, or A*24, allelic imbalance at the DNA and RNA level resulted in correlative imbalanced surface presentation of alleles at the protein level. Conclusions: Evaluation of paired tumor WES and RNAseq revealed orthogonal validation of HLA allelic imbalance, and analysis in cell lines suggested that these patterns were likely tumor intrinsic. Experimental validation of these findings at the protein level suggests that antigen presentation density can be modulated by chromosomal gains, and not just allelic loss, in HLA genes. This knowledge is important for the design of cancer vaccines or T cell therapies targeting neoantigens presented by HLA class I complexes. Citation Format: Katie M. Campbell, Justin Saco, Egmidio Medina, Meelad Amouzgar, Shannon M. Pfeiffer, Cynthia R. Gonzalez, Gabriela Steiner, Ameya Champhekar, Cristina Puig Saus, Jesse Zaretsky, Gabriel Abril Rodriguez, Agustin Vega-Crespo, Ignacio Baselga Carretero, Mito Tariveranmoshabad, Anusha Kalbasi, Christine Spencer, Zachary L. Skidmore, Malachi Griffith, Obi L. Griffith, Daniel K. Wells, Antoni Ribas. Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma [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 3818.

Abstract 2196: Oncogenic context drives the landscape of tumor suppression in lung cancer

Abstract Cooperative interactions between genomic alterations in oncogenes and tumor suppressors are a hallmark of cancer. While molecular characterization of tens of thousands of primary tumors has solidified estimates of how frequently specific genomic alterations occur, identifying genetic interactions from co-mutation rates alone remains limited by the vast number of possible combinations as well as inherent biases and confounding effects. Conversely, genetically engineered mouse models can reliably uncover causal effects of specific genetic alterations but are not sufficiently high throughput to generate broad insights into the interplay between genetic alterations in tumors. Therefore, how the effects of one alteration on tumor growth change in the context of other alterations remains largely unknown. We previously addressed these technical limitations by combining CRISPR/Cas9-mediated gene editing, tumor barcoding, and ultra-deep barcode sequencing methods with genetically engineered mouse models of human lung cancer, which together enable multiplexing of tumor genotypes in individual mice and precise, high-throughput quantification of in vivo tumor fitness. Here, we leverage this platform to systematically quantify the growth effects of over 45 known and putative tumor suppressor gene alterations across different common oncogenic point mutations in Kras and Braf. While the set of genes we identified as tumor suppressive in KrasG12D- and KrasG12C-driven lung cancers was largely consistent, inactivation of those genes typically resulted in larger tumor growth advantages in the KrasG12D model. Inactivation of most genes in BrafV600E-driven lung tumors had a strikingly different growth effect than in tumors with either Kras variant, and effects were generally far less pronounced in the BrafV600E context. Interestingly, loss of genes upstream of Braf within the Ras pathway often had strong effects in the KrasG12D or KrasG12C models—loss of Nf1, a negative regulator of Kras, or loss of the wildtype Kras allele enhanced oncogenic Kras-driven tumor growth, while loss of Shp2, a positive regulator of Kras, was mildly detrimental—but had little or no effects in the BrafV600E model. We assessed translatability of our experimental cause-and-effect data through comparisons to correlative data from publicly available human lung cancer genomics databases, and found that genetic interactions predicted by our models and gene co-mutation rates in patient tumors largely align. Together, these findings underscore the need for a contextual understanding of the ways in which tumors are influenced by their genetics and highlight the utility of high-throughput, quantitative autochthonous mouse models in pursuing this endeavor. Citation Format: Ian Winters, Lily Blair, Lafia Sebastian, Gabriel Grenot, Edwin Apilado, Wensheng Nie, Vy Tran, Ian Lai, Gregory Wall, Dmitri Petrov, Monte Winslow, Joseph Juan, Michael Rosen. Oncogenic context drives the landscape of tumor suppression in lung cancer [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 2196.

Abstract 823: ABL1 kinase regulates leukemia phenotype and response to therapy

Abstract The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase. Cytoplasmic ABL1 stimulates, whereas nuclear ABL1 inhibits cell growth. Analysis of the CGAP Mitelman database detected loss of ABL1 allele in hematologic malignancies. Among the top genetic aberrations detected in acute myeloid leukemias were t(8;21) generating fusion protein AML1-ETO and NUP98 translocations, e.g., t(1;11) encoding NUP98-PMX1.Expression of AML1-ETO and NUP98-PMX1 in Lin-cKit+ stem cell enriched bone marrow cells from Abl1-/-;Vav-Cre and Abl1+/+;Vav-Cre mice revealed that oncogene-expressing Abl1-/- cells gained tremendous proliferation advantage. Conversely, overexpression of ABL1 and DPH (ABL1 kinase agonist)-mediated activation of ABL1 kinase resulted in reduced proliferation of AML1-ETO and NUP98-PMX1 cells. Since the role of ABL1 in DNA damage response (DDR) is well established, we applied the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi) to identify potential therapeutic vulnerabilities. AML1-ETO;Abl1-/- cells were more sensitive to all three inhibitors when compared to AML1-ETO;Abl1+/+ cells. Remarkably, NUP98-PMX1;Abl1-/- cells were exceptionally sensitive to ATRi, but not to ATMi and DNA-PKi when compared to NUP98-PMX1;Abl+/+ cells. ABL1 did not affect the sensitivity of transformed cells to DNA damaging agent doxorubicin. To pinpoint mechanisms collaborating with Abl1 deletion in transformation of hematopoietic cells, Abl1 was knockout in 32Dcl3 cells by CRISPR/Cas9 to obtain 32Dcl3-Abl1ko cells. 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells were challenged to induce transformation by starving them from growth factors (IL3). Only 32Dcl3-Abl1ko cell populations generated growth factor-independent cells. This indicates that the absence of Abl1 may facilitate malignant transformation of hematopoietic cells. RNA-seq was performed to compare gene expression in 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells in the presence or absence of IL-3. Pathway analysis of FPKM reads (top 30 significant canonical pathways) and Venn diagram analysis followed by KEGG pathway analysis (291 genes uniquely expressed in growth factor-independent 32Dcl3-Abl1ko cells) implicated PI3K-AKT pathway signaling including mTOR and sirtuins (SITR) in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib, mTOR inhibitor rapamycin and SIRT2 inhibitor thiomyristoyl exerted selective activity against Lin-cKit+ AML1-ETO;Abl1-/- and/or NUP98-PMX1;Abl1-/- cells when compared to Abl1+/+ counterparts. Moreover, inhibition of ABL1 kinase by imatinib in Lin-cKit+ AML1-ETO;Abl1+/+ and/or NUP98-PMX1;Abl+/+ cells induced the sensitivity to PI3K-ATK pathway inhibitors. In conclusion, we showed that normal ABL1 kinase plays a tumor suppressor role and is a potential therapeutic target in hematological malignancies induced by AML1-ETO and NUP98-PMX1. The latter statement is being currently tested in animal models. Citation Format: Konstantin Golovine, Zhaorui Lian, Kumaraswamy Naidu Chitrala, Gleb Ablakov, Margaret Nieborowska-Skorska, Jian Huang, Tomasz Skorski. ABL1 kinase regulates leukemia phenotype and response to therapy [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 823.

Abstract 6080: The heterogeneity and evolution of lung neuroendocrine tumors within TRACERx

Abstract Introduction: Primary pulmonary neuroendocrine tumors (NETs) contribute to 20-25% of all lung cancer diagnoses and lie on a spectrum of malignant behavior, ranging from low grade classical carcinoid tumors to high grade, aggressive small cell carcinomas. The evolutionary relationships between NETs of different grades remain poorly understood, here we seek to use the TRACERx study to explore the genomic evolution of these lung cancers. Methods: Tissue and blood samples were taken from a cohort of 20 patients with pulmonary NETs in a sub-study within TRACERx. Multi-region sampling (84 total regions) and whole exome sequencing was performed on fresh frozen tissue from each tumor. Histological classification of each tumor region was confirmed and scoring of tumor infiltrating lymphocytes (TILs) was performed. The data were then analyzed using the established TRACERx bioinformatics pipeline. Results: Phylogenetic evolutionary histories were generated, illustrating the genomic divergence in each tumor and identifying points at which specific known drivers were acquired during tumor evolution. Lower grade classical and atypical carcinoids were characterized by a low mutational and copy number burden with a high fraction of subclonal mutations, with 5/12 cases having no apparent somatic mutational driver mutation. In contrast higher grade large cell neuroendocrine carcinomas and small cell carcinomas had higher mutational and copy number burden, as well as higher rates of genome instability and whole genome doubling. We identified no evolutionary link between subtypes such as commonly shared clonal drivers, suggesting that atypical or classical carcinoids later evolve into higher grade carcinomas. Dn/ds was used to investigate for evidence of selection in the carcinoid tumors; while there was no evidence for selection in known cancer driver genes associated with NETs or lung cancer, there was a high global dn/ds value suggesting the existence of unknown drivers for this cancer type. Furthermore, there was evidence of parallel evolution in one atypical carcinoid case with two distinct subclonal MEN1 frameshift mutations co-occurring with loss of the other allele. One small cell carcinoma patient was identified as having two genetically unrelated tumors. A germline predisposition for this tumor was suspected since we identified a variant in BRCA2 (rs11571833) that was maintained under loss of heterozygosity in one tumor with a corresponding somatic BRCA2 mutation in the other, suggesting two independent double hit events. Conclusions: Genomic differences are identifiable between different members of the pulmonary NET family, with high grade tumors showing increased whole genome instability indices and phylogenetic divergence than low grade tumors. This work has shed light on the genomic landscape of pulmonary NETs and enabled the identification of distinct evolutionary trajectories in the different subtypes. Citation Format: Robert Bentham, David A. Moore, Lorelle Brownlee, Raju Veeriah, Lizi Manzano, Fiona Blackhall, Colin R. Lindsay, TRACERx Consortium, Nicholas McGranahan, Charles Swanton, Mariam Jamal-Hanjani. The heterogeneity and evolution of lung neuroendocrine tumors within TRACERx [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 6080.

Abstract 1549: The tumorigenesis model in DGCR8 associated schwannomatosis

Abstract Purpose: Schwannomatosis is an inherited disorder that affects Schwann cells from peripheral nerves. It is diagnosed when multiple schwannomas occur in the absence of bilateral vestibular schwannomas. The two main genes associated with this disorder are SMARCB1 and LZTR1 both on chromosome 22q (Chrm22q). Somatic inactivation of NF2, downstream of SMARCB1, is observed in most schwannomas. The accepted model of schwannomatosis involves multiple hits over three steps to inactivate LZTR1 or SMARCB1 together with NF2. Following this pattern, most of the LZTR1/SMARCB1-schwannomas acquire a somatic loss of Chrm22 (thus deleting the three genes) and a somatic mutation affecting the remaining wild-type NF2 copy on the GPV allele. Several studies have postulated the plausible existence of other susceptibility genes predisposing to schwannomatosis and the likelihood of those being localized in Chrm22q. Last year we identified a GPV in the microprocessor DGCR8 (c.1552G>A; p.E518K) located in the Chrm22q11 region, as responsible for a familial form of schwannomatosis and multinodular goiter (Rivera et al JCI, 2020). Our goal is to clarify the role of DGCR8 as a novel tumor susceptibility gene and the tumorigenic mechanisms that lead to DGCR8-schwannomatosis. Methods: We searched for patients affected of schwannoma and thyroid tumors. By whole exome sequencing we identified the same DGCR8 (c.1552G>A; p.E518K) variant in one patient. We then collected a total of 13 DGCR8-schwannomas from carriers. Eleven tumors were subjected to WES and two tumors were subjected to a NGS targeted panel covering all known schwannoma genes in Chrm22q. Results: We report the second case of a patient with peripheral schwannomatosis and thyroid alterations caused by the germline pathogenic variant E518K in DGCR8. Loss of Chrm22q was seen in all 13 tumors analyzed. While all tumors had at least one alteration of NF2, 4 tumors had no somatic mutations on the retained (not deleted) allele (30.8%). Given that DGCR8 localizes 5’ of LZTR1, the second step (LOH) leads to the deletion of DGCR8 and the three bona fide schwannoma genes (LZTR1, SMARCB1 and NF2) adding up to a total of 6 hits in a 3-step model. Suggesting that the path to tumorigenesis driven by DGCR8 requires the loss of the wild type allele of Chrm22q and in more than two thirds of the tumors a complete inactivation of NF2 occurs. Conclusion: Our findings highlight DGCR8 as a schwannomatosis gene mapping to the Chrm22 cluster of tumor suppressors that cooperate to promote tumorigenesis in Schwann cell and pinpoints an important role of miRNA regulation in this process. Citation Format: Clara Nogué, Anne-Sophie Chong, Elia Grau, HyeRim Han, Eduard Dorca, Carla Roca, Jose Luis Mosquera, Conxi lazaro, William D. Foulkes, Joan Brunet, Bárbara Rivera Polo. The tumorigenesis model in DGCR8 associated schwannomatosis [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 1549.

Abstract LB039: VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition

Abstract Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability, but lacks mutations in canonical DNA damage response (DDR) genes. Bi-allelic loss of VHL is a near ubiquitous, truncal event in ccRCC development. Herein, we performed preclinical and clinical analysis to understand how VHL loss impacts DDR pathway activity and how specific DDR deficiencies (DDR-D) may be targeted in ccRCC. Methods: We performed DNA whole exome sequencing (WES) and reverse phase protein array (RPPA) early stage ccRCC tumors. All patients were consented under an IRB-approved protocol for the use of tissue. Transcriptomic and protein analyses of select DDR pathways was performed on in silico data (TCGA). DDR functional assays and ATR inhibitor treatment were performed on preclinical cell line models, and in vivo ATR inhibitor treatment in ccRCC xenografts with varying VHL status. Results: Bi-allelic loss of VHL via mutation in one allele and copy number loss of chromosome 3p was observed in 13/13 patient tumors. Mutations in other genes associated with ccRCC and/or canonical DDR genes were not observed. Despite VHL loss being the only driver mutation, these early stage tumors displayed tumor mutational burden (TMB) similar to ccRCC across all tumor stages (1.1 ± 0.5 mut/Mb). Proteomic analysis of patient tumor versus normal tissue (N=12) revealed significantly different (P<0.05) mean expression level of 218 proteins, including significantly lower phospho-ATM at the autophosphorylated S1981 site and lower phospho-CHK2 at the downstream T68 site. VHL-/- cells display defective binding to and repressed activation of Tip60 (KAT5), a key acetyltransferase that activates ATM kinase and aids in the selection of DNA repair pathways following damage. Lastly, treatment with ATRi (VE-822) of 786-O and 786-VHL overexpressing tumor xenografts showed ATRi was safe and significantly inhibited tumor growth preferentially in the 786-O VHL null model. Conclusions: Here we show in patient tissue and preclinical models that VHL biallelic loss results in genomic instability and functional impairment of ATM kinase activity; furthermore, we provide preclinical evidence for selectively targeting the ATR signaling cascade in patients with VHL-deficient kidney cancer. Clinical trials of ATR inhibitor based therapy in patients with advanced cancer, including ccRCC, are underway (e.g. NCT04266912). Citation Format: Patrick G. Pilie, Lijun Zhou, Yan-Ting Zhang, Christine Peterson, Daniel McGrail, Yang Peng, Guang Peng, Xiande Liu, Xuesong Zhang, Eric Jonasch. VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition [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 LB039.

Abstract 4028: Menin inhibitor-based combinations to improve efficacy and overcome resistance in AML

Abstract In MLL1 rearranged (MLL1r) AML (~10%), N-terminus of MLL1 gene is fused to the C-terminus of a fusion partner, e.g. AF9, AF4, ENL and ELL, creating MLL1 fusion protein (MLL-FP), which increases expression of leukemogenic HOXA9 and its co-factor MEIS1. In AML with mutant (mt) NPM1 (NPM1c), MLL1 is the main driver of HOXA9, MEIS1 and FLT3, promoting self-renewal and growth of AML stem-progenitor cells (LSCs). Treatment with Menin inhibitor (MI), SNDX-50469 or SNDX-5613, disrupts binding of Menin to its binding pocket in MLL1/2 and MLL1-FP, reducing expressions of their targets and inducing differentiation and apoptosis. In the phase I/II AUGMENT-101 clinical trial, SNDX-5613 monotherapy was well tolerated and achieved objective remissions in patients with previously treated relapsed/refractory AML harboring MLL1r or NPM1c. However, majority of patients either fail to respond or eventually relapse. A minority of MLL1r AML (~9%) also exhibit a co-mutation in TP53, which is known to confer therapy resistance and poor outcome in AML. This creates the need to develop MI-based synergistic combinations with superior efficacy against patient-derived (PD) AML cells harboring MLL-FP or NPM1c. Present studies found that, following MI treatment, CyTOF analysis of PD MLL1-r and NPM1c AML cells showed decline in protein levels of Menin, MEIS1, MEF2C, PBX3, FLT3, CDK6 and BCL2 in phenotypically characterized AML LSCs expressing CLEC12A, CD123, CD244 and CD99. Notably, in vitro co-treatment with SNDX-50469 in combination with venetoclax, OTX015 (pan-BET inhibitor) or abemaciclib (CDK6 inhibitor) induced synergistic (determined by SynergyFinder V2) loss of viability in AML cell lines (MV4-11, MOLM13 and OCI-AML3), as well as in MOLM13 cells with CRISPR-Cas9-mediated knock-in of mutant or allelic loss of TP53 and in PD AML cells with MLL-r or NPM1c, but not in normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c. A CRISPR screen with a validated, domain-specific gRNA library against chromatin regulators revealed BRD4, p300, MOZ and KDM1A as druggable co-dependencies with treatment with MI. Consistent with this, co-treatment with SNDX-50469 and OTX015, or the p300/CBP inhibitor GNE049 was synergistically lethal in vitro in AML cells with MLL1r or NPM1c, either sensitive to MI or tolerant-resistant (induced in vitro) to MI (MITR cells). Co-treatment with SNDX-5613 and venetoclax or OTX015 compared to each drug or vehicle control, administered orally for 3 to 4 weeks to NSG mice engrafted with either MOLM13-GFP/Luciferase xenograft or with PD NPM1c and mtFLT3 AML xenograft, caused significantly greater reduction in AML burden and increased overall survival without weight loss or other toxicities (p < 0.005). These preclinical findings highlight novel MI-based combinations exhibiting superior in vitro and in vivo anti-AML efficacy against AML cells harboring MLL1-FP or NPM1c that are sensitive to MI or the MITR cells. Citation Format: Warren C. Fiskus, Christopher P. Mill, Christine Birdwell, John A. Davis, Qi Jin, Tapan M. Kadia, Courtney D. DiNardo, Koichi Takahashi, Gerard M. McGeehan, Naval Daver, Kapil N. Bhalla. Menin inhibitor-based combinations to improve efficacy and overcome resistance in AML [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 4028.

Abstract 5852: Doxorubicin increases the level of a BRCA2 exon 3-skipping mRNA isoform in MCF7

Abstract Introduction: BRCA2 is a tumor suppressor that participates in DNA repair. Inherited pathogenic mutations in BRCA2 are associated with a 38%-84% risk for breast cancer and a 16.5%-27% risk for ovarian cancer. It is therefore critical to distinguish pathogenic mutations from sequence variants of unknown clinical significance (VUSs). Among the VUSs that require clinical classification are those that could potentially disrupt normal splicing patterns. The challenge in determining the spliceogenic nature of a VUS is the ubiquity of naturally occurring alternative splicing variants. BRCA2 exon 3, which encodes a binding site for the interacting proteins EMSY and PALB2, is frequently skipped during alternative splicing events to different levels in different cell types under different conditions. It is not yet known whether BRCA2 exon 3-skipping is a regulated event contributing to the spectrum of BRCA2 functions or is a stochastic occurrence. A genomic deletion that results in complete allelic loss of exon 3 has been shown to be pathogenic, yet VUSs that result in increased frequency of exon 3-skipping have been shown to be non-pathogenic. Thus, the allele-specific ability of BRCA2 to contribute to DNA repair is affected by the level of exon 3-skipping. Alternative splicing patterns have also been observed in response to DNA damaging agents, potentially linking some therapies to risks from genome-wide aberrant splicing. In this study we explore the effects of the topoisomerase inhibitor doxorubicin on the frequency of BRCA2 exon 3-skipping. Isoform-specific end-point RT-PCR shows that doxorubicin increases the frequency of BRCA2 exon 3-skipping in the breast cancer cell line MCF7. Methods: MCF7 cells were treated with doxorubicin according to standard protocols. RNA was prepared using Qiagen reagents and amplified with Invitrogen One-Step RT-PCR reagents. Isoform-specific RT-PCR primers were designed to amplify the full-length region surrounding BRCA2 exon 3 independently from the region left by an exon-skipping event (Δ3). RT-PCR products were visualized using agarose gel electrophoresis. Results and Conclusion: Preliminary results suggest that doxorubicin increases the level of exon 3-skipping in MCF7. These results suggest doxorubicin, used to treat breast and other cancers, may compromise the repair functions of BRCA2 in addition to other transcriptome-wide effects from aberrant splicing patterns. However, it is not known whether this difference in isoform accumulation between treated and untreated cells is the result of increased frequency of alternative splicing events or changes in relative stabilities of the full-length and Δ3 isoforms, so it cannot yet be determined whether any potential physiological defects associated with alternative splicing of BRCA2 exon 3 result from insufficiency of the full-length isoform. Citation Format: Zoha Hameedi, Talia Ishfaq, Zaain Ahmad, James D. Fackenthal. Doxorubicin increases the level of a BRCA2 exon 3-skipping mRNA isoform in MCF7 [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 5852.

Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus

The active form of vitamin B6, pyridoxal 5′-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. In this work, we investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. Here, we found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, we identified the mitotic recombination as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

Complex functional redundancy of Tbx2 and Tbx3 in mouse limb development

The limb phenotypes of Tbx2 and Tbx3 mutants are distinct: loss of Tbx2 results in isolated duplication of digit 4 in the hindlimb while loss of Tbx3 results in anterior polydactyly and posterior oligodactly in the forelimb. In the face of such disparate phenotypes, we sought to determine whether Tbx2 and Tbx3 have functional redundancy during development of the mouse limb. We found that sequential loss of alleles generates defects that are not simply additive of those observed in single mutants and that multiple structures in both the forelimb and hindlimb display compound sensitivity to decreased gene dosage.

Bifurcations in the symmetric viability model with weak selection

In this paper we discuss bifurcations of equilibria in the symmetric viability model with weak selection. Overcoming the difficulties from irrational fractions of the 4 parameters in determining the signs of determinant and trace, we find that the system has at most 5 interior equilibria, 4 edge equilibria and 4 corner equilibria and give a complete qualitative analysis for all equilibria in both hyperbolic cases and non-hyperbolic cases, including the case of fully null degeneracy. On this basis we display all bifurcations of equilibria such as pitchfork bifurcation, transcritical bifurcation and bifurcations at corner equilibria of fully null degeneracy, which is of codimension 2 but none of known results of codimension 2 can be applied to. We further discuss the unfolding system of fully null degeneracy, displaying all its bifurcations and giving bifurcation curves analytically. Our results provide an intuitive basis for us to predict the genetic composition of a population and provide the basic mathematical theory for the protection and loss of alleles.

Identification of Germline Mutations in Genes Involved in Classic FAP in Patients from Northern Brazil.

Colorectal cancer is a common cancer worldwide, with 5-10% of cases being hereditary. Familial adenomatous polyposis syndrome (FAP) is caused by germline mutations in the APC gene or rarely in the MUTYH gene. This work did not identify germline mutations in the MUTYH, NTHL1, POLD1 and POLE genes in 15 individuals belonging to five families with classic FAP, who had the mutation in the APC gene confirmed in a previous study. Our results support mutations in the APC gene as the main genetic contribution of classical FAP with severe phenotype. In the family that had the most aggressive form of the disease, we performed an array-based Comparative Genomic Hybridization analysis and identified the germinal loss of an allele of the NOTCH2 and BMPR2 genes in the mother (proband) and daughter. In order to validate the involvement of these genes in the other four families of this study, we analyzed the DNA copy number variation in the peripheral blood of the 15 participants. FAP is a syndrome with considerable genetic and phenotypic heterogeneity and this phenomenon may explain the presence of secondary genetic alterations, such as the allelic loss of NOTCH2 and BMPR2 genes, found only in one family in this study. The CNV analysis confirmed that only the two members of the FAP2 family (patient 02H and 02F) had a deletion of these two genes, as the aCGH methodology had found. The other study participants did not show allelic loss for these two genes. Validation in a larger number of families could confirm the presence of these new genetic alterations in classic FAP and improve understanding of the different types of aggressiveness of the disease.

Population differentiation of polygenic score predictions under stabilizing selection.

Given the many small-effect loci uncovered by genome-wide association studies (GWAS), polygenic scores have become central to genomic medicine, and have found application in diverse settings including evolutionary studies of adaptation. Despite their promise, polygenic scores have been found to suffer from limited portability across human populations. This at first seems in conflict with the observation that most common genetic variation is shared among populations. We investigate one potential cause of this discrepancy: stabilizing selection on complex traits. Counterintuitively, while stabilizing selection constrains phenotypic evolution, it accelerates the loss and fixation of alleles underlying trait variation within populations (GWAS loci). Thus even when populations share an optimum phenotype, stabilizing selection erodes the variance contributed by their shared GWAS loci, such that predictions from GWAS in one population explain less of the phenotypic variation in another. We develop theory to quantify how stabilizing selection is expected to reduce the prediction accuracy of polygenic scores in populations not represented in GWAS samples. In addition, we find that polygenic scores can substantially overstate average genetic differences of phenotypes among populations. We emphasize stabilizing selection around a common optimum as a useful null model to connect patterns of allele frequency and polygenic score differentiation.This article is part of the theme issue ‘Celebrating 50 years since Lewontin's apportionment of human diversity’.

Schinus terebinthifolia Raddi: a comparative framework on population genetic structure in a restored area after 12 years

The success of restoration projects depends on the genetic diversity of the implanted species. It is a limiting factor, often because the seed sources are immersed in highly fragmented landscapes. In this work, we compare the genetic diversity of the juveniles, and the adult trees of Schinus terebinthifolia Raddi in a native mixed-species planting, both in the restoration process and in the remaining natural vegetation Atlantic Forest Biome. Polymorphic DNA fragments using five SSR primers were used to estimate the restored population that showed a higher genetic diversity index (He) (0.553 adults and 0.505 juveniles) compared to the wild population (0.487 adults and 0.483 juveniles). The forested area was established with individuals of high genetic diversity. There is a reduced genetic diversity for juveniles, with the loss of exclusive alleles and maintenance of endogamy and coancestry in reforested populations, and we can infer that there was a low gene flow inter fragments. The effective population size in both (adults and juveniles) was lower than the value recommended for the sustainability of populations in the short and long term. The results indicated that continuous monitoring of this particular area is of absolute necessity and should use techniques that promote the connectivity of the fragments. It would allow for a more significant reduction of genetic drift and the persistence of the planted populations.

Case Review: Whole-Exome Sequencing Analyses Identify Carriers of a Known Likely Pathogenic Intronic BRCA1 Variant in Ovarian Cancer Cases Clinically Negative for Pathogenic BRCA1 and BRCA2 Variants.

Background: Detecting pathogenic intronic variants resulting in aberrant splicing remains a challenge in routine genetic testing. We describe germline whole-exome sequencing (WES) analyses and apply in silico predictive tools of familial ovarian cancer (OC) cases reported clinically negative for pathogenic BRCA1 and BRCA2 variants. Methods: WES data from 27 familial OC cases reported clinically negative for pathogenic BRCA1 and BRCA2 variants and 53 sporadic early-onset OC cases were analyzed for pathogenic variants in BRCA1 or BRCA2. WES data from carriers of pathogenic BRCA1 or BRCA2 variants were analyzed for pathogenic variants in 10 other OC predisposing genes. Loss of heterozygosity analysis was performed on tumor DNA from variant carriers. Results: BRCA1 c.5407-25T>A intronic variant, identified in two affected sisters and one sporadic OC case, is predicted to create a new splice effecting transcription of BRCA1. WES data from BRCA1 c.5407-25T>A carriers showed no evidence of pathogenic variants in other OC predisposing genes. Sequencing the tumor DNA from the variant carrier showed complete loss of the wild-type allele. Conclusions: The findings support BRCA1 c.5407-25T>A as a likely pathogenic variant and highlight the importance of investigating intronic sequences as causal variants in OC families where the involvement of BRCA1 is highly suggestive.

Polygenic sex determination produces modular sex polymorphism in an African cichlid fish

For many vertebrates, a single genetic locus initiates a cascade of developmental sex differences in the gonad and throughout the organism, resulting in adults with two phenotypically distinct sexes. Species with polygenic sex determination (PSD) have multiple interacting sex determination alleles segregating within a single species, allowing for more than two genotypic sexes and scenarios where sex genotype at a given locus can be decoupled from gonadal sex. Here we investigate the effects of PSD on secondary sexual characteristics in the cichlid fish Metriaclima mbenjii, where one female (W) and one male (Y) sex determination allele interact to produce siblings with four possible sex classes: ZZXX females, ZWXX females, ZWXY females, and ZZXY males. We find that PSD in M. mbenjii produces an interplay of sex linkage and sex limitation resulting in modular variation in morphological and behavioral traits. Further, the evolution or introgression of a newly acquired sex determiner creates additional axes of phenotypic variation for varied traits, including genital morphology, craniofacial morphology, gastrointestinal morphology, and home tank behaviors. In contrast to single-locus sex determination, which broadly results in sexual dimorphism, polygenic sex determination can induce higher-order sexual polymorphism. The modularity of secondary sexual characteristics produced by PSD provides context for understanding the evolutionary causes and consequences of maintenance, gain, or loss of sex determination alleles in populations.

Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression.

ObjectivesMammalian DNA methyltransferases are essential to re‐establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl‐CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)—a zinc finger type of MBP—is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear.Materials and MethodsThis study used the Cre‐loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real‐time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms.ResultsGermline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity.ConclusionsThese findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Autophagy Blockage Reduces the Incidence of Pancreatic Ductal Adenocarcinoma in the Context of Mutant Trp53.

Macroautophagy (hereafter referred to as autophagy) is a homeostatic process that preserves cellular integrity. In mice, autophagy regulates pancreatic ductal adenocarcinoma (PDAC) development in a manner dependent on the status of the tumor suppressor gene Trp53. Studies published so far have investigated the impact of autophagy blockage in tumors arising from Trp53-hemizygous or -homozygous tissue. In contrast, in human PDACs the tumor suppressor gene TP53 is mutated rather than allelically lost, and TP53 mutants retain pathobiological functions that differ from complete allelic loss. In order to better represent the patient situation, we have investigated PDAC development in a well-characterized genetically engineered mouse model (GEMM) of PDAC with mutant Trp53 (Trp53 R172H ) and deletion of the essential autophagy gene Atg7. Autophagy blockage reduced PDAC incidence but had no impact on survival time in the subset of animals that formed a tumor. In the absence of Atg7, non-tumor-bearing mice reached a similar age as animals with malignant disease. However, the architecture of autophagy-deficient, tumor-free pancreata was effaced, normal acinar tissue was largely replaced with low-grade pancreatic intraepithelial neoplasias (PanINs) and insulin expressing islet β-cells were reduced. Our data add further complexity to the interplay between Atg7 inhibition and Trp53 status in tumorigenesis.