Search

To gain insight into how ERG translocations cause prostate cancer, we performed single cell transcriptional profiling of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in prostate epithelial cells, we observed enhanced proliferation primarily in an intermediate subpopulation with a basal identity based on transcriptomics but with luminal morphology and cytokeratin expression. Through a series of lineage tracing and primary prostate epithelial transplantation experiments, we find that tumor initiating activity resides in a subpopulation of basal cells that also express luminal genes such asTmprss2andNkx3.1, but not in the larger population of classical luminal cells. Upon ERG activation, these basal-luminal hybrid cells expand into a highly proliferative population of intermediate basal-luminal identity then transition to fully differentiated luminal cells that reflect the lineage of ERG-positive cancers. These findings help resolve a preexisting debate about the cell of origin of ERG-driven prostate cancer and narrow the focus for future mechanistic studies to a basal-luminal subpopulation of tumor initiating cells.

The phosphoinositide 3–kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer.Significance:Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer.See related commentary by Claridge and Hopkins, p. 2216

Supplemental Tables 1-4. Table S1. Sequences of sgRNAs and oligonucleotides; Table S2. GSEA pathway enrichment analysis of enriched and dropout genes in MCF7 and T47D cells; Table S3. The top 2.5% (3745) enriched sgRNAs of whole-genome CRISPR/Cas9 sgRNA knockout screen in MCF7 cells; Table S4. The top 2.5% (3745) enriched sgRNAs of whole-genome CRISPR/Cas9 sgRNA knockout screen in T47D cells.

The phosphoinositide 3–kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer. Significance: Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer. See related commentary by Claridge and Hopkins, p. 2216

Genomic analysis of targeted patient tumor sequencing identified frequent mutations, 41% in prostate cancer (Li, et al., 2020) in the gene FOXA1, a developmentally important pioneer transcription factor (TF) in mammary and prostate tissues. Previous work by our group and others has shown that these FOXA1 mutations alter global chromatin accessibility and promote growth in prostate cells (Adams, 2019), but the underlying molecular details, including the identity of partner TFs, remain unclear. To address this topic, we generated mouse prostate organoids expressing Foxa1 alleles harboring three distinct classes of mutations: (i) overexpression of WT Foxa1 (reflecting focal gene amplification seen in tumors), (ii) a series of mutants within the Wing2region of the forkhead binding domain (FHBD) and (iii) a mutant bearing a stop codon after the FHBD to represent a series of C-terminal truncation mutants. We performed single nucleus multiome sequencing to obtain gene expression (snRNA-seq) and chromatin accessibility (snATAC-seq) readouts from the same individual nuclei. Whereas each Foxa1 mutant has distinct, often mutant-specific features, several themes emerge. These include alterations in the relative proportion of stem-like (L2) luminal cells vs secretory (L1) luminal cells as well as changes in luminal or basal gene signatures, increased androgen receptor signaling output, and enrichment for motifs of distinct classes of partner TFs. For example, cells expressing the truncation mutant show gain in the accessibility of Gata3 and Pou2f1 TF binding motifs, as well as enhanced numbers of L1-like luminal cells. Functional studies demonstrate that Pou2f1 is specifically required for the pro-luminal phenotype in cells expressing the truncation mutant whereas Gata3 plays a more general pro-luminal role. Correlations in motif accessibility and transcription factor expression across single cells further revealed a composite androgen receptor (AR)-FOXA1 motif enriched in the pro-luminal truncation mutant, while the canonical AR motif was enriched in pro-basal cell mutants. Finally, Foxa1 mutants cooperative with Trp53 and Pten loss in orthotopic prostate tumorigenicity assays, most strikingly manifest by reversion of the basal-like features characteristic of Trp53/Pten loss tumors to Ck8+ luminal adenocarcinoma histology, mirroring that seen in Foxa1-mutant human prostate cancer tumors in mice. Thus, mutant Foxa1 alleles cooperate with canonical prostate cancer tumor suppressors and alter the histologic phenotype of prostate cancers in mice through the activation of basal or luminal lineage differentiation programs. Citation Format: Erik Ladewig, Abbas Nazir, Christina Leslie, Charles Sawyers. Mutations in FOXA1 alter chromatin remodeling and cell fate in prostate organoids [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 2048.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER(+)) breast cancer. We identify that ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER(+) breast cancer. An epigenome CRISPR-CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the eR degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARIDIA-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER-FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER(+) breast cancer.

Seeing double can be a good thingMany human breast cancers harbor activating mutations inPIK3CA, the gene coding for the catalytic subunit of phosphoinositide 3-kinase (PI3K). Clinical trials are underway to evaluate the efficacy of PI3K inhibitors in cancer patients. Vasanet al.found unexpectedly that a subset of breast cancers harbor not one—but two—PIK3CAmutations, and the mutations occur on the same allele (see the Perspective by Toker). In model systems, the double mutations hyperactivate PI3K signaling and enhance tumor growth. Preliminary analysis of clinical trial data suggests that breast cancers with double mutations are more responsive to PI3K inhibitors than those with a single mutation.PIK3CAmutational status could help identify the breast cancer patients most likely to benefit from these drugs.Science, this issue p.714; see also p.685

Significance Fifty percent of diffuse large B cell lymphoma (DLBCL) evade immune-surveillance via somatic genetic lesions abrogating the expression of the class I major histocompatibility complex (MHC-I) complex on the cell surface, thus preventing the presentation of tumor neoantigens to the immune system. The results herein significantly extend these findings by showing that an additional 40% of DLBCL cases, despite expressing MHC-I, carry monoallelic HLA-I genetic alterations that limit the repertoire of neoantigens for presentation to immune cells. Both MHC-INEG and MHC-IPOS/monoallelically disrupted cases have significantly higher mutational load. Notably, homozygosis of HLA-I loci is significantly and preferentially enriched in the germline of DLBCL patients, suggesting a stepwise process by which limited neoantigen presentation is selected during DLBCL development., Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) breast cancer patients. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2 and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacological inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance.

Background Despite extensive efforts on the genomic characterization of gliomas, very few studies have reported the genetic alterations of cerebellar glioblastoma (C-GBM), a rare and lethal disease. Here, we provide a systematic study of C-GBM to better understand its specific genomic features. Methods We collected a cohort of C-GBM patients and compared patient demographics and tumor pathologies with supratentorial glioblastoma (S-GBM). To uncover the molecular characteristics, we performed DNA and mRNA sequencing and DNA methylation arrays on 19, 6, and 4 C-GBM cases, respectively. Moreover, chemical drug screening was conducted to identify potential therapeutic options for C-GBMs. Results Despite differing anatomical origins of C-GBM and S-GBM, neither histological, cytological, nor patient demographics appeared significantly different between the 2 types. However, we observed striking differences in mutational patterns, including frequent alterations of ATRX, PDGFRA, NF1, and RAS and absence of EGFR alterations in C-GBM. These results show a distinct evolutionary path in C-GBM, suggesting specific therapeutic targeted options. Targeted-drug screening revealed that C-GBMs were more responsive to mitogen-activated protein kinase kinase (MEK) inhibitor and resistant to epidermal growth factor receptor inhibitors than S-GBMs. Also, differential expression analysis indicated that C-GBMs may have originated from oligodendrocyte progenitor cells, suggesting that different types of cells can undergo malignant transformation according to their location in brain. Master regulator analysis with differentially expressed genes between C-GBM and proneural S-GBM revealed NR4A1 as a potential therapeutic target. Conclusions Our results imply that unique gliomagenesis mechanisms occur in adult cerebellum and new treatment strategies are needed to provide greater therapeutic benefits for C-GBM patients. Key points 1. Distinct genomic profiles of 19 adult cerebellar GBMs were characterized. 2. MEK inhibitor was highly sensitive to cerebellar GBM compared with supratentorial GBM. 3. Master regulator analysis revealed NR4A1 as a potential therapeutic target in cerebellar GBM.

Precision medicine in cancer proposes that genomic characterization of tumors can inform personalized targeted therapies. However, this proposition is complicated by spatial and temporal heterogeneity. Here we study genomic and expression profiles across 127 multisector or longitudinal specimens from 52 individuals with glioblastoma (GBM). Using bulk and single-cell data, we find that samples from the same tumor mass share genomic and expression signatures, whereas geographically separated, multifocal tumors and/or long-term recurrent tumors are seeded from different clones. Chemical screening of patient-derived glioma cells (PDCs) shows that therapeutic response is associated with genetic similarity, and multifocal tumors that are enriched with PIK3CA mutations have a heterogeneous drug-response pattern. We show that targeting truncal events is more efficacious than targeting private events in reducing the tumor burden. In summary, this work demonstrates that evolutionary inference from integrated genomic analysis in multisector biopsies can inform targeted therapeutic interventions for patients with GBM.

Activating mutations in

PIK3CA mutations represent the most frequent oncogenic driver lesion found across all human cancers. Given that single PIK3CA mutations are a predictive biomarker for response to PI3K inhibition, and that these clinical benefits are small, we hypothesized that additional genomic factors may cooperate with single PIK3CA mutations in oncogenesis and response to PI3K inhibitors. We analyzed several large pan-cancer datasets and have discovered a phenomenon of dual PIK3CA mutations in 15% of all PIK3CA-mutant cancers, including breast cancer. Dual PIK3CA mutations are clonal, are found in both primary untreated and metastatic tumors, and are enriched at conserved amino acid positions. We have proved that the majority of dual PIK3CA mutations—including the most frequent dual PIK3CA mutant combinations in breast cancer—are in cis compound mutations (i.e., on the same allele, resulting in a protein molecule with two mutations) in patient samples and cell lines, as shown through multiple sequencing techniques including long-range single-molecule real-time sequencing (SMRT-seq). Compound PIK3CA mutations increase PI3K pathway signaling and growth proliferation in nontransformed cells and cancer cells in vitro and in vivo. Biochemical experiments using recombinant PI3K complexes reveal a mechanism where compound mutants increase PI3K complex thermal instability, resulting in increased kinase activity and liposome binding as compared to single mutants. Compound PIK3CA mutations render cells more sensitive to the PI3Kα inhibitors BYL719 and GDC-0077 than single PIK3CA mutations. These data translate to the clinic, where patients with dual PIK3CA-mutant metastatic breast cancer exhibit more durable responses to PI3K inhibition versus patients with single PIK3CA-mutant breast tumors. Our data support a novel mutational dose-response model for oncogene activation, where compound mutations amplify PI3K signaling and growth to a greater degree than single mutants, resulting in increased sensitivity to PI3Kα inhibitor targeted therapy. These findings also have therapeutic relevance, where compound PIK3CA mutations may be a superior predictive biomarker for response to PI3Kα inhibitors than single PIK3CA mutations, across PIK3CA-mutant cancers. Citation Format: Neil Vasan, Jared Johnson, Hong Shao, Pedram Razavi, Alexander Gorelick, Erik Ladewig, Alesia Antoine, Hardik Shah, Eneda Toska, Guotai Xu, Abiha Kazmi, Barry Taylor, Komal Jhaveri, Maura Dickler, Elisa de Stanchina, Eduard Reznik, Raul Rabadan, Melissa Smith, Robert Sebra, Lewis Cantley, Maurizio Scaltriti, Jose Baselga. Compound PIK3CA mutations support a mutational dose-response model for oncogene activation and response to PI3K inhibitor targeted therapy in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A21.

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER(+)) breast cancers. Examining FOXA1 in ~5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third β strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response.

PIK3CA is the most frequently mutated oncogene across all human cancers, and codes for p110α, the catalytic subunit of the PI3K complex. PI3K catalyzes the phosphorylation of the lipid PIP2 to PIP3, which initiates a downstream signaling cascade involving the activation of AKT and mTOR. Hotspot E545K and H1047R mutations constitutively activate PI3K and are oncogenic in multiple cancer histologies including breast cancer, where PIK3CA mutations are present in 40% of tumors and are a target for cancer therapy. Recently, the PI3Kα inhibitor alpelisib has demonstrated improved PFS in patients with ER+ PIK3CA mutant metastatic breast cancer on a phase 3 randomized clinical trial, resulting in its FDA approval. In early clinical trials, we observed a population of patients with displayed prolonged clinical benefit to alpelisib, with double PIK3CA mutant tumors. This prompted us to undertake a comprehensive analysis of the prevalence of multiple PIK3CA mutations and to investigate their potential biological relevance and correlation with sensitivity to PI3Kα inhibitors.We analyzed multiple independent primary and metastatic tumor cohorts and found that 10-15% of all PIK3CA mutant tumors across all cancer histologies contain multiple PIK3CA mutations, the vast majority of which carry exactly two mutations. Double mutations occur at specific amino acid positions by codon enrichment analysis, where the most frequent combinations in breast cancer are comprised of a canonical “major mutant” hotspot (involving either E542, E545, or H1047) combined with a second “minor mutant” site (involving either E453, E726, or M1043). Double PIK3CA mutations are enriched in ER+/HER2- breast cancers and occur at similar frequencies in therapy-naïve primary tumors and metastatic tumors. To study the allelic configuration of double mutations, we leveraged single molecule real time sequencing (SMRT-seq) on fresh breast tumor samples from patients known to carry two PIK3CA mutations in their tumors by NGS. Six tumors representative of the most frequent double mutants in breast cancer were obtained from patients, and were analyzed by SMRT-seq. All contained double mutations in cis. We overexpressed double cis mutants and constituent single mutants in nontransformed and ER+ breast cancer cells. Double PIK3CA mutations in cis increased downstream PI3K pathway signaling and cell/tumor proliferation in vitro and in vivo, when compared to single mutations. In contrast, mutations in trans do not increase cell signaling or growth proliferation more than single mutations.The prevailing model of PI3K activation occurs through the engagement of the p85α regulatory subunit with phosphotyrosines on RTK signaling complexes, which relieves catalytic inhibition. Single oncogenic mutations recapitulate these events by weakening the interactions between p110α and p85α (“disrupters”), or by promoting binding to membrane (“binders”). We purified recombinant full length PI3Kα complexes containing single and double cis p110α mutations to dissect the biochemical mechanisms by which these double PIK3CA mutations in cis modulate kinase activity. We demonstrate that cis mutants have increased kinase activity compared to single mutants and that this activation is due to both increased p85α disruption and increased lipid binding, compared to single mutants.We tested whether cis mutant cells exhibit differential sensitivity to PI3Kα inhibitors. While in the absence of treatment, cis mutant signaling is increased compared to single mutants, treatment with the PI3Kα inhibitors alpelisib or GDC-0077 results in a similar PI3K pathway inhibition in nontransformed and ER+ breast cancer cells. With respect to cell viability, E545K and H1047R major hotspot mutants are more sensitive to alpelisib and GDC-0077 compared to minor mutants and WT. In turn, all cis mutants are synergistically more sensitive to alpelisib and GDC-0077 compared to single major and minor hotspots.We analyzed response data from SANDPIPER, a randomized phase 3 clinical trial that tested the efficacy of the ER degrader fulvestrant with or without the PI3Kα/γ/δ inhibitor taselisib in metastatic ER+ PIK3CA mutant breast cancer. We used ctDNA to detect the presence of PIK3CA mutations. PIK3CA mutant patients on the taselisib arm had an objective response rate (ORR) of 20.3% vs 9.7% compared to the placebo arm, which was statistically significant, confirming that PIK3CA mutation predicts response to PI3Kα inhibition. We then compared responses of patients with single vs multiple mutations. Single mutant patients on the taselisib arm had an ORR of 18.1% vs 10.0% compared to the placebo arm, which was not statistically significant. On the contrary, multiple mutant patients on the taselisib arm achieved an ORR of 30.2% vs 8.7% compared to the placebo arm, which was statistically significant. These findings confirm that breast cancer patients with multiple mutant tumors achieve higher clinical benefit to PI3Kα inhibition compared to single mutant tumors.In this work, we have identified double mutations in cis as a novel, and relatively frequent, genomic alteration in PIK3CA, which translates into a clinically meaningful number of patients who may derive additional benefit from targeted therapy. Our results implicate a model of oncogene addiction to double mutant PIK3CA in breast cancer, with graded levels of activation and inhibition for single vs double mutants. PI3Kα inhibitors are now a standard of care in PIK3CA mutant ER+ metastatic breast cancer and are being explored in other PIK3CA mutant tumor histologies. Our findings provide a rationale for testing whether patients with multiple PIK3CA mutant tumors are exquisitely sensitive to PI3Kα inhibitors. Citation Format: Neil Vasan, Pedram Razavi, Jared L. Johnson, Hong Shao, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Ting-Yu Lin, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T. Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Sarat Chandarlapaty, Raul Rabadan, Ed Reznik, Melissa L. Smith, Robert Sebra, Frauke Schimmoller, Timothy R. Wilson, Lori S. Friedman, Lewis C. Cantley, Maurizio Scaltriti, Jose Baselga. Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG16.

Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide-3-kinase (PI3K), are the most frequent oncogenic alterations in estrogen receptor-positive (ER+) breast cancer and are also prevalent in other tumor types. PI3Kα inhibitors including alpelisib have recently been shown to be clinically active in ER+ PIK3CA mutant breast cancer. To characterize determinants of sensitivity to these agents, we undertook a comprehensive analysis of PIK3CA mutant cancer genomes and observed the presence of double PIK3CA mutations in 12-15% of breast cancer and other tumor types. These double PIK3CA mutations are clonal, located in cis on the same allele, and are composed of a single hotspot mutation combined with a recurrent second-site mutation. Double PIK3CA mutations in cis result in increased PI3K activity and downstream signaling together with enhanced cell proliferation and tumor growth compared to single hotspot mutations. The biochemical mechanisms underlying this increased oncogenicity include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased membrane lipid binding. Finally, these double PIK3CA mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations (e.g. E545K or H1047R) in experimental models and in patients with ER+ PIK3CA mutant metastatic breast cancer from the SANDPIPER randomized phase III clinical trial. These findings implicate double PIK3CA mutations in cis as a novel mechanism of oncogene addiction relative to single hotspot mutations, providing a rationale to develop PI3Kα inhibitors for the therapy of double PIK3CA mutant cancers. Citation Format: Neil Vasan, Pedram Razavi, Jared L Johnson, Hong Shao, Timothy Wilson, Frauke Schimmoller, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Ting-Yu Lin, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Sarat Chandarlapaty, Raul Rabadan, Ed Reznik, Melissa L Smith, Robert Sebra, Lori Friedman, Lewis C Cantley, Maurizio Scaltriti, José Baselga. Double PIK3CA mutations in cis drive oncogene addiction and enhance sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-01.

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features1, 2, 3, 4, 5, 6. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a ‘universal model’ based on a single set of organism-independent parameters. In particular, this work was funded by a contract from the National Human Genome Research Institute modENCODE Project, contract U01 HG004271 and U54 HG006944, to S.E.C. (principal investigator) and P.C., T.R.G., R.A.H. and B.R.G. (co-principal investigators) with additional support from R01 GM076655 (S.E.C.) both under Department of Energy contract no. DE-AC02-05CH11231, and U54 HG007005 to B.R.G. J.B.B.’s work was supported by NHGRI K99 HG006698 and DOE DE-AC02-05CH11231. Work in P.J.B.’s group was supported by the modENCODE DAC sub award 5710003102, 1U01HG007031-01 and the ENCODE DAC 5U01HG004695-04. Work in M.B.G.’s group was supported by NIH grants HG007000 and HG007355. Work in Bloomington was supported in part by the Indiana METACyt Initiative of Indiana University, funded by an award from the Lilly Endowment, Inc. Work in E.C.L.’s group was supported by U01-HG004261 and RC2-HG005639. P.J.P. acknowledges support from the National Institutes of Health (grant no. U01HG004258). We thank the HAVANA team for providing annotation of the human reference genome, whose work is supported by National Institutes of Health (grant no. 5U54HG004555), the Wellcome Trust (grant no. WT098051). R.G. acknowledges support from the Spanish Ministry of Education (grant BIO2011-26205). We also acknowledge use of the Yale University Biomedical High Performance Computing Center. R.W.'s lab was supported by grant no. U01 HG 004263.

Activating mutations in PIK3CA, the gene encoding for the catalytic subunit (p100a), are the most common oncogenic alterations in estrogen receptor-positive (ER+). The majority of PIK3CA mutations occur within two hot spots; exons 9 and exon 20 which encode the helical (E545K) and kinase domains (H1047R), respectively. These mutations result in hyperactivation of the PI3K/AKT/mTOR pathway and provide the rationale for the development of inhibitors targeting the PI3K pathway. To this end, PI3K α-specific inhibitors are showing antitumor activity in patients with PIK3CA-mutant, ER-positive breast cancer. Evidence of alternative mRNA regulation and splicing in various cancers has been described in the literature. Although, the majority of events appear to have unknown clinical significance, there is evidence alternative splicing can lead to drug resistance. The role of the PI3K pathway on transcriptional regulation and mRNA processing is not well studied. Through transcriptome analysis and cell assays in mouse MEF and human MCF10A cells we demonstrate splicing defects accrue as a result of the PI3K pathway activation by the PIK3CA H1047R mutation. PI3Ka pathway inhibitors were able to restore wildtype exon inclusion levels in these mutants, suggesting a potential clinical benefit. We propose that PIK3CA H1047R imposes mRNA differential isoform regulation by acting through the most commonly mutated PI3K pathway in ER+ breast cancers and that such mutations are targetable with PI3K pathway inhibitors. Citation Format: Erik Ladewig, Lauren Fairchild, Maurizio Scaltriti, Christina Leslie, Eneda Toska, Jose Baselga. PI3K pathway mediated splicing defects in ER+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4346.

Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide 3-kinase (PI3K), are the most frequent oncogenic alterations in all cancers, including estrogen receptor-positive (ER+) breast cancer. There are many distinct oncogenic PIK3CA mutations including major hotspot mutations (e.g. E542K, E545K, H1047R) which are predictive of response to PI3Kα inhibitors. While responses to PI3Kα inhibitors can be variable, some patients with single hotspot mutant tumors derive a deep and prolonged clinical benefit. In a comprehensive analysis of the genomic data available in breast cancer, we observed double PIK3CA mutations and investigated their potential biological relevance and potential correlation with sensitivity to PI3Kα inhibitors. We detected double PIK3CA mutations in 10-15% of all PIK3CA-mutant cancers including breast cancer. Double PIK3CA mutations in breast cancer are defined by a pattern of co-expression of a major hotspot mutation associated with a recurrent second-site PIK3CA mutation (‘minor mutation’). We have found that these double PIK3CA mutations are compound, that is in cis on the same allele, using single molecule real time sequencing (SMRT-seq) of fresh breast tumor samples. Double compound PIK3CA mutations result in increased PI3K activity and downstream signaling compared to single hotspot mutants in nontransformed cells and in ER+ breast cancer cells. Double compound mutations also increase cell proliferation and xenograft growth compared to single hotspot mutants. Biochemical experiments using recombinant PI3K compound mutant protein complexes reveal a combination mechanism of action, with simultaneous PI3K destabilization and increased membrane binding compared to single hotspot mutants, leading to increased kinase activity. Importantly, these compound mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutants in both preclinical models and also in patients with breast cancer. Together, our data support that double compound PIK3CA mutations enhance the oncogenicity of single hotspot PIK3CA mutations and this increased dependency results in increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations. We propose that double compound activating mutations are an additional mechanism of oncogenic transformation in addition to other known mechanisms such as gene amplification, point mutation, or gene translocation. We speculate that double compound mutant PIK3CA may function as a clinical biomarker of increased sensitivity to PI3K-directed targeted therapies and may improve the therapeutic window of PI3K inhibitors in ER+ breast cancer and other PIK3CA-mutant tumor histologies. Citation Format: Neil Vasan, Pedram Razavi, Jared Johnson, Hong Shao, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T. Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Raul Rabadan, Ed Reznik, Melissa L. Smith, Robert Sebra, Lewis C. Cantley, Maurizio Scaltriti, Jose Baselga. Double PIK3CA mutations in cis enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3917.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. To better understand how GBM evolves, we analyzed longitudinal genomic and transcriptomic data from 114 patients. The analysis shows a highly branched evolutionary pattern in which 63% of patients experience expression-based subtype changes. The branching pattern, together with estimates of evolutionary rate, suggests that relapse-associated clones typically existed years before diagnosis. Fifteen percent of tumors present hypermutation at relapse in highly expressed genes, with a clear mutational signature. We find that 11% of recurrence tumors harbor mutations in LTBP4, which encodes a protein binding to TGF-β. Silencing LTBP4 in GBM cells leads to suppression of TGF-β activity and decreased cell proliferation. In recurrent GBM with wild-type IDH1, high LTBP4 expression is associated with worse prognosis, highlighting the TGF-β pathway as a potential therapeutic target in GBM.

Atypical miRNA substrates do not fit criteria often used to annotate canonical miRNAs, and can escape the notice of miRNA genefinders. Recent analyses expanded the catalogs of invertebrate splicing-derived miRNAs (“mirtrons”), but only a few tens of mammalian mirtrons have been recognized to date. We performed meta-analysis of 737 mouse and human small RNA data sets comprising 2.83 billion raw reads. Using strict and conservative criteria, we provide confident annotation for 237 mouse and 240 human splicing-derived miRNAs, the vast majority of which are novel genes. These comprise three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 5′-tailed mirtrons, and 3′-tailed mirtrons. In addition, we segregated several hundred additional human and mouse loci with candidate (and often compelling) evidence. Most of these loci arose relatively recently in their respective lineages. Nevertheless, some members in each of the three mirtron classes are conserved, indicating their incorporation into beneficial regulatory networks. We also provide the first Northern validation for mammalian mirtrons, and demonstrate Dicer-dependent association of mature miRNAs from all three classes of mirtrons with Ago2. The recognition of hundreds of mammalian mirtrons provides a new foundation for understanding the scope and evolutionary dynamics of Dicer substrates in mammals.

Nucleotide modifications to microRNAs or their precursors can influence their processing and/or activity. A challenge to their analysis is the lack of independent references for the termini generated by primary processing; typically, these are empirically assigned as the most abundant mapped reads. Mirtrons offer such an independent measure since these microRNA hairpins are defined by splicing. Consequently, mirtron-derived reads that deviate from splice sites reflect modification following primary processing. Analysis in Drosophila revealed multiple modification patterns, including select alterations of 5′ termini, many 3′ resection events, and unexpectedly abundant 3′ untemplated monouridylation. Resections occur on mature AGO1-loaded species, whereas uridylation occurs on pre-miRNAs but is compatible with dicing and AGO1 loading. Strikingly, we found many mirtrons whose modified reads are more abundant than those produced by primary processing. In some cases, these abundant modified reads matched the genome owing to fortuitous uridines in downstream flanking exons, thus highlighting the value of an independent reference for the primary-processed sequence. We could further extend the principle of abundant and preferred uridylation of mirtrons, relative to canonical pre-miRNAs, to Caenorhabditis elegans, mouse, and human. Finally, we found that 3′ resection occurs broadly across AGO1-loaded canonical miRNA and star species. Altogether, these findings substantially broaden the complexity of terminal modification pathways acting upon small regulatory RNAs.

The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and Senataxin) with the noncoding RNA processing function of RNA exosome determine the strand specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.

Dissecting the genetics of classical Hodgkin lymphoma (cHL), a common lymphoma derived from germinal center B (GCB) cells, has remained a difficult task compared to other lymphomas, due to the rarity (usually Thus, we microdissected 1200-1800 single HRS cells per case from frozen biopsies of 36 patients, along with a similar number of adjacent non-neoplastic cells, and performed in duplicate whole-genome DNA amplification (WGA) followed by independent whole-exome sequencing, thus allowing to control for possible biases introduced by the WGA step. The putative somatic protein-changing mutations identified in a major tumor clone (i.e., ≥20% variant allele frequency) were then validated by targeted deeper sequencing of the same WGA DNA, as well as by ultra-deep sequencing and digital PCR of unamplified whole-biopsy DNA. We found highly recurrent (11/36 patients; 31%), heterozygous, STAT6 missense mutations in exons encoding for the DNA binding domain, identical to the activating STAT6 variants known in primary mediastinal B-cell lymphoma (PMBCL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. ShRNA-induced STAT6 knockdown caused marked apoptosis in the STAT6-mutated cHL cell line L1236, which expresses phosphorylated STAT6 (pSTAT6), while it showed little or no effect in the STAT6-unmutated/pSTAT6+ cHL lines L428 and HDLM2, and in the STAT6-unmutated/STAT6-unphosphorylated cHL line L540. Thus, STAT6 mutations in cHL appear to confer a survival advantage distinct from (and beyond that of) STAT6 phosphorylation, possibly due to their aberrant DNA binding activity following pSTAT6-dependent entry into the nucleus. Interestingly, STAT6 mutations were often associated with disruptive somatic mutations of the JAK-STAT signaling inhibitor SOCS1 (frequency in the evaluable STAT6-mutated versus unmutated patients: 6/7, i.e. 87%, versus 4/14, i.e. 29%; p=0.02), suggesting a genetic interaction. Indeed, transduction of wild-type SOCS1 induced apoptosis of STAT6-mutated L1236 cells to a much greater extent than STAT6-unmutated L428 cells, despite both carry a SOCS1disruptive mutation and express pSTAT6. Furthermore, the presence in additional cases of genetic lesions of other JAK-STAT pathway members (mutations activating STAT3, STA5B, JAK1 or inactivating the inhibitor PTPN1; JAK2 copy number gains) attests to the pervasive role of JAK-STAT signaling in the genetics of cHL (up to ~70% of patients affected). GNA13, encoding Gα13, carried mutations, mostly heterozygous, in 8/36 (22%) patients. By transmitting signals from the S1PR2 and P2RY8 receptors, Gα13 ensures the confinement of proliferating GCB cells within secondary lymphoid follicles, while constraining at the same time their expansion by facilitating apoptosis in this potentially dangerous niche (Nature 2014;516:254). GNA13 mutations in cHL included disruptive (frame-shifting and non-sense) variants and were similar to those identified in GC B cell-derived Burkitt lymphoma and DLBCL of the GCB-type. The apparent histogenetic selectivity of inactivating GNA13mutations, almost never found in post-germinal center ABC-DLBCL, suggests they may facilitate cHL lymphomagenesis by rescuing from apoptosis crippled GC B cells (the proposed HRS cell precursors) and by promoting their dissemination outside the follicles, where HRS cells are indeed observed in lymph nodes with early, partial involvement by cHL (Am J Surg Pathol 1983;7:145). Heterozygous missense mutations of XPO1 occurred in 6/36 patients (17%) at the hot-spot amino acid residue E571, which is recurrently targeted also in chronic lymphocytic leukemia and PMBCL. XPO1encodes exportin-1, which shuttles outside the nucleus more than 200 proteins, including tumor suppressors involved in cHL pathogenesis and functioning in the nucleus (e.g., NFKBIA, FOXOs, TP53) (Blood 2012;120:4621). XPO1 inhibition by the clinical compound selinexor caused growth inhibition and apoptosis of cHL cell lines in a mutation-dependent manner. By providing a global characterization of the cHL coding genome, this works uncovered several new genes implicated in the genetics of this lymphoma, and most prominently in the JAK-STAT pathway, that likely play a significant role in its biology. #Shared first authorship °Shared last authorship Disclosures No relevant conflicts of interest to declare.