Biology subjects

Andrejka, L.

Publications and source records attributed to Andrejka, L..

12 recordsLinked to original sources

bioRxiv2024

A STAG2-PAXIP1/PAGR1 axis suppresses lung tumorigenesis

The cohesin complex is a critical regulator of gene expression. STAG2 is the most frequently mutated cohesin subunit across several cancer types and is a key tumor suppressor in lung cancer. Here, we coupled somatic CRISPR-Cas9 genome editing and tumor barcoding with an autochthonous oncogenic KRAS-driven lung cancer model and show that STAG2 is uniquely tumor suppressive among all core and auxiliary cohesin components. The heterodimeric complex components PAXIP1 and PAGR1 have highly correlated effects with STAG2 in human lung cancer cell lines, are tumor suppressors in vivo, and are epistatic to STAG2 in oncogenic KRAS-driven lung tumorigenesis in vivo. STAG2 inactivation elicits changes in gene expression, chromatin accessibility and 3D genome conformation that impact cancer cell state. Gene expression and chromatin accessibility similarities between STAG2- and PAXIP1-deficient neoplastic cells further relates STAG2-cohesin to PAXIP1/PAGR1. These findings reveal a STAG2-PAXIP1/PAGR1 tumor-suppressive axis and uncover novel PAXIP1-dependent and PAXIP1-independent STAG2-cohesin mediated mechanisms of lung tumor suppression. SUMMARYSTAG2 is a frequently mutated cohesin subunit across several cancers and one of the most important functional suppressors of lung adenocarcinoma. Our findings underscore important roles of STAG2 in suppressing lung tumorigenesis and highlight a STAG2-PAXIP1/PAGR1 tumor-suppressive program that may transcend cancer type.

cancer biology↗

bioRxiv2024

EML4-ALK variant-specific genetic interactions shape lung tumorigenesis

Oncogenic fusions of EML4 and ALK occur in [~]5% of lung adenocarcinomas. More than 15 EML4-ALK variants with distinct breakpoints within EML4 have been identified, but the functional differences between these variants remain poorly understood. Here we use CRISPR/Cas9 somatic genome editing to generate autochthonous mouse models of the two most common EML4-ALK variants, V1 and V3, and show that V3 is more oncogenic than V1. By integrating these models with multiplexed genome editing, we quantify the effects of 29 putative tumor suppressor genes on V1- and V3-driven lung cancer growth in vivo and show that many tumor suppressor genes have dramatically variant-specific effects on tumorigenesis. Analysis of a novel dataset representing the largest human EML4-ALK lung cancer cohort to date identified alterations in the genomic landscape depending on the EML4-ALK variant. These findings demonstrate functional heterogeneity among EML4-ALK variants, suggesting that EML4-ALK variants behave more like distinct oncogenes than a uniform entity. More broadly, these findings highlight the dramatic impact of oncogenic fusions partner proteins on tumor biology.

cancer biology↗

bioRxiv2024

Functional mapping of epigenetic regulators uncovers coordinated tumor suppression by the HBO1 and MLL1 complexes

Epigenetic dysregulation is widespread in cancer. However, the specific epigenetic regulators and the processes they control to drive cancer phenotypes are poorly understood. Here, we employed a novel, scalable and high-throughput in vivo method to perform iterative functional screens of over 250 epigenetic regulatory genes within autochthonous oncogenic KRAS-driven lung tumors. We identified multiple novel epigenetic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and the MLL1 complex are among the most impactful tumor suppressive epigenetic regulators in lung. The histone modifications generated by the HBO1 complex are frequently absent or reduced in human lung adenocarcinomas. The HBO1 and MLL1 complexes regulate chromatin accessibility of shared genomic regions, lineage fidelity and the expression of canonical tumor suppressor genes. The HBO1 and MLL1 complexes are epistatic during lung tumorigenesis, and their functional correlation is conserved in human cancer cell lines. Together, these results demonstrate the value of quantitative methods to generate a phenotypic roadmap of epigenetic regulatory genes in tumorigenesis in vivo.

cancer biology↗

bioRxiv2024

Aging represses lung tumorigenesis and alters tumor suppression

Most cancers are diagnosed in persons over the age of sixty, but little is known about how age impacts tumorigenesis. While aging is accompanied by mutation accumulation -- widely understood to contribute to cancer risk -- it is also associated with numerous other cellular and molecular changes likely to impact tumorigenesis. Moreover, cancer incidence decreases in the oldest part of the population, suggesting that very old age may reduce carcinogenesis. Here we show that aging represses tumor initiation and growth in genetically engineered mouse models of human lung cancer. Moreover, aging dampens the impact of inactivating many, but not all, tumor suppressor genes with the impact of inactivating PTEN, a negative regulator of the PI3K/AKT pathway, weakened to a disproportionate extent. Single-cell transcriptomic analysis revealed that neoplastic cells from tumors in old mice retain many age-related transcriptomic changes, showing that age has an enduring impact that persists through oncogenic transformation. Furthermore, the consequences of PTEN inactivation were strikingly age-dependent, with PTEN deficiency reducing signatures of aging in cancer cells and the tumor microenvironment. Our findings suggest that the relationship between age and lung cancer incidence may reflect an integration of the competing effects of driver mutation accumulation and tumor suppressive effects of aging.

cancer biology↗

bioRxiv2024

Modeling the genomic complexity of human cancer using Cas12a mice

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo.

cancer biology↗

bioRxiv2024

Combinatorial in vivo genome editing identifies widespread epistasis during lung tumorigenesis

Lung adenocarcinoma, the most common subtype of lung cancer, is genomically complex, with tumors containing tens to hundreds of non-synonymous mutations. However, little is understood about how genes interact with each other to enable tumorigenesis in vivo, largely due to a lack of methods for investigating genetic interactions in a high-throughput and multiplexed manner. Here, we employed a novel platform to generate tumors with all pairwise inactivation of ten tumor suppressor genes within an autochthonous mouse model of oncogenic KRAS-driven lung cancer. By quantifying the fitness of tumors with every single and double mutant genotype, we show that most tumor suppressor genetic interactions exhibited negative epistasis, with diminishing returns on tumor fitness. In contrast, Apc inactivation showed positive epistasis with the inactivation of several other genes, including dramatically synergistic effects on tumor fitness in combination with Lkb1 or Nf1 inactivation. This approach has the potential to expand the scope of genetic interactions that may be functionally characterized in vivo, which could lead to a better understanding of how complex tumor genotypes impact each step of carcinogenesis.

cancer biology↗

bioRxiv2023

Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with additional tumor suppressor mutations. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations required for the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

cancer biology↗

bioRxiv2022

A multiplexed in vivo approach to identify driver genes in small cell lung cancer

Small cell lung cancer (SCLC) is a highly lethal form of lung cancer. The high mutation burden in SCLC cells makes it challenging to predict key drivers of SCLC from genome sequencing data, thereby hindering the identification of possible therapeutic targets. Here we develop a quantitative multiplexed approach based on lentiviral barcoding with somatic CRISPR/Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. Lentiviral vector-mediated SCLC initiation was greatly enhanced by naphthalene pre-treatment, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Based on a meta-analysis across multiple human SCLC genomic datasets, we quantified the impact of inactivating 39 genes across many candidate pathways and captured both positive and detrimental effects on SCLC initiation and progression upon gene inactivation. This analysis and subsequent validation in human SCLC cells identified TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This new approach should illuminate novel drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify new therapeutic targets.

cancer biology↗

bioRxiv2021

Combinatorial tumor suppressor inactivation efficiently initiates lung adenocarcinoma with therapeutic vulnerabilities

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Through the generation of hundreds of diverse combinations of tumor suppressor alterations, we demonstrate that the inactivation of suppressors of the RAS and PI3K pathways drive the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common event in oncogene- negative human lung adenocarcinomas. We demonstrate that these Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacological inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma.

cancer biology↗

bioRxiv2021

Multiplexed identification of RAS paralog imbalance as a driver of lung cancer growth

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we used multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung tumors, we identified HRAS and NRAS as key suppressors of KRASG12D-driven tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogs interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signaling. HRAS mutations identified in KRAS-driven human tumors partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRAS- and BRAF-driven lung cancer models confirmed that RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven lung cancer.

cancer biology↗

bioRxiv2020

Genetic determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo

Cancer genome sequencing has uncovered substantial complexity in the mutational landscape of tumors. Given this complexity, experimental approaches are necessary to establish the impact of combinations of genetic alterations on tumor biology and to uncover genotype-dependent effects on drug sensitivity. In lung adenocarcinoma, EGFR mutations co-occur with many putative tumor suppressor gene alterations, however the extent to which these alterations contribute to tumor growth and their response to therapy in vivo has not been explored experimentally. By integrating a novel mouse model of oncogenic EGFR-driven Trp53-deficient lung adenocarcinoma with multiplexed CRISPR-Cas9-mediated genome editing and tumor barcode sequencing, we quantified the effects of inactivation of ten putative tumor suppressor genes. Inactivation of Apc, Rb1, or Rbm10 most strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2 - which are the strongest drivers of tumor growth in an oncogenic Kras-driven model - reduced EGFR-driven tumor growth. These results are consistent with the relative frequency of these tumor suppressor gene alterations in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, Keap1 inactivation reduces the sensitivity of EGFR-driven Trp53-deficient tumors to the EGFR inhibitor osimertinib. Importantly, in human EGFR/TP53 mutant lung adenocarcinomas, mutations in the KEAP1 pathway correlated with decreased time on tyrosine kinase inhibitor treatment. Our study highlights how genetic alterations can have dramatically different biological consequences depending on the oncogenic context and that the fitness landscape can shift upon drug treatment.

cancer biology↗

bioRxiv2019

A Long Lost Key Opens an Ancient Lock: Drosophila Myb Causes a Synthetic Multivulval Phenotype in Nematodes

The five-protein MuvB core complex (LIN9/Mip130, LIN37/Mip40, LIN52, LIN54/Mip120, and LIN53/p55CAF1/RBBP4) has been highly conserved during the evolution of animals. This nuclear complex interacts with proteins encoded by the RB tumor suppressor gene family and its associated E2F-DP transcription factors to form DREAM complexes that repress the expression of genes that regulate cell cycle progression and cell fate. The MuvB core complex also interacts with proteins encoded by the Myb oncogene family to form the Myb-MuvB complexes that activate many of the same target genes. We show that animal-type Myb genes and proteins are present in Bilateria, Cnidaria, and Placozoa, the latter including some of the simplest known animal species. However, bilaterian nematode worms appear to have lost their animal-type Myb genes hundreds of millions of years ago. Nevertheless, the amino acids in the LIN9 and LIN52 proteins that directly interact with the MuvB-binding domains of human B-Myb and Drosophila Myb are conserved in C. elegans. Here we show that, despite greater than 500 million years since their last common ancestor, the Drosophila melanogaster Myb protein can bind to the nematode LIN9 and LIN52 family proteins in vitro and can cause a synthetic multivulval (synMuv) phenotype in vivo. This phenotype is similar to that caused by loss-of-function mutations in C. elegans synMuvB class genes including those that encode homologs of the MuvB core, RB, E2F, and DP. Furthermore, amino acid substitutions in the MuvB-binding domain of Drosophila Myb that disrupt its functions in vitro and in vivo also disrupt its activity in C. elegans. We speculate that nematodes and other animals may contain another protein that can bind to LIN9 and LIN52 in order to activate transcription of genes repressed by DREAM complexes.

molecular biology↗