Search

Purpose:Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk FLT3-ITD–positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of FLT3-ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of FLT3-ITD mutations, we investigated the effects of FLT3-ITD structural features on the response of AML patients to treatment.Experimental Design: We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in FLT3-ITD–positive AML patients.Results:HeatITup accurately identifies and classifies ITDs into newly defined categories of “typical” or “atypical” based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type FLT3, whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type FLT3. Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical FLT3-ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypical FLT3-ITDs.Conclusions:These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software.See related commentary by Gallipoli and Huntly, p. 460

Figure S1: Characterization of FLT3-ITD clones in the FLT3i cohort. Table S1: Confusion matrix of HeatITup and genetic reviewers manual annotation for sequences in Figure 3. Table S2: Univariate Cox regression analyses for the FLT3-ITD-positive UPENN cohort. Table S3: Cox regression analysis of FLT3-ITD duplication length for the non-FLT3i-treated cohort. Table S4: Cox regression analysis of FLT3-ITD classification for the non-FLT3i-treated cohort. Table S5: Cox regression analysis of FLT3-ITD duplication length with classification for the non-FLT3i-treated cohort. Table S6: Cox regression analysis of FLT3-ITD classification for the top third longest ITD duplications in the non-FLT3i-treated cohort (Benjamini-Hochberg adjusted p-values from long non-FLT3i-treated cohort analyses). Table S7: Cox regression analysis of FLT3-ITD spacer sequence exogenous fraction for the non-FLT3i-treated cohort. Table S8: Cox regression analysis of FLT3-ITD spacer sequence exogenous fraction for the top third longest ITD duplications in the non-FLT3i-treated cohort. Table S9: Cox regression analysis of FLT3i treatment for the UPENN cohort. Table S10: Cox regression analysis of FLT3-ITD duplication length for the FLT3i-treated cohort (Benjamini-Hochberg adjusted p-values from FLT3i cohort analyses). Table S11: Cox regression analysis of FLT3-ITD classification for the FLT3i-treated cohort (Benjamini-Hochberg adjusted p-values from FLT3i cohort analyses). Table S12: Cox regression analysis of FLT3-ITD spacer sequence exogenous fraction for the FLT3i-treated cohort.

T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While exhausted T cells are hypofunctional, some exhausted T cells retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR+T cells may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using scRNA-seq, flow cytometry, RNA velocity, and scTCR-seq, we demonstrate that deleting the pseudokinase Trib1 shifts TEXtowards CX3CR1+intermediates (TINT) with robust enrichment of KLR+CD8+T cells (TKLR) via clonal T cell expansion. These changes are associated with globally increased KLR gene expression throughout the exhaustion program. Further, Trib1 loss augments anti-PD-L1 blockade to improve viral clearance by expanding the TKLRpopulation. Together, these data identify Trib1 as an important regulator of T cell exhaustion whose targeting enhances the KLR+effector state and improves the response to checkpoint inhibitor therapy.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs, we developed AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX show the superior performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulated known islet pathobiology and showed differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+T cells infiltration in islets during type 1 diabetes progression.

The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.

Purpose: Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk FLT3-ITD–positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of FLT3-ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of FLT3-ITD mutations, we investigated the effects of FLT3-ITD structural features on the response of AML patients to treatment. Experimental Design: We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in FLT3-ITD–positive AML patients. Results: HeatITup accurately identifies and classifies ITDs into newly defined categories of “typical” or “atypical” based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type FLT3, whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type FLT3. Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical FLT3-ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypical FLT3-ITDs. Conclusions: These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software. See related commentary by Gallipoli and Huntly, p. 460

Chromatin misfolding has been implicated in cancer pathogenesis; yet, its role in therapy resistance remains unclear. Here, we systematically integrated sequencing and imaging data to examine the spatial and linear chromatin structures in targeted therapy-sensitive and -resistant human T cell acute lymphoblastic leukemia (T-ALL). We found widespread alterations in successive layers of chromatin organization including spatial compartments, contact domain boundaries, and enhancer positioning upon the emergence of targeted therapy resistance. The reorganization of genome folding structures closely coincides with the restructuring of chromatin activity and redistribution of architectural proteins. Mechanistically, the derepression and repositioning of the B-lineage-determining transcription factor EBF1 from the heterochromatic nuclear envelope to the euchromatic interior instructs widespread genome refolding and promotes therapy resistance in leukemic T cells. Together, our findings suggest that lineage-determining transcription factors can instruct changes in genome topology as a driving force for epigenetic adaptations in targeted therapy resistance.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells destroy insulin-producing beta cells. The aetiology of this complex disease is dependent on the interplay of multiple heterogeneous cell types in the pancreatic environment. Here, we provide a single-cell atlas of pancreatic islets of 24 T1D, autoantibody-positive and nondiabetic organ donors across multiple quantitative modalities including ~80,000 cells using single-cell transcriptomics, ~7,000,000 cells using cytometry by time of flight and ~1,000,000 cells using in situ imaging mass cytometry. We develop an advanced integrative analytical strategy to assess pancreatic islets and identify canonical cell types. We show that a subset of exocrine ductal cells acquires a signature of tolerogenic dendritic cells in an apparent attempt at immune suppression in T1D donors. Our multimodal analyses delineate cell types and processes that may contribute to T1D immunopathogenesis and provide an integrative procedure for exploration and discovery of human pancreatic function.

Type 1 diabetes (T1D) is an autoimmune disease of only partially defined etiology in which immune cells destroy insulin-producing beta cells. Using single-cell transcriptomics and an advanced analytical strategy to assess pancreatic islets of T1D, autoantibody-positive, and non-diabetic organ donors, we identified both canonical cell types and rare insulin-expressing cells with a hybrid mixture of endocrine and exocrine gene signatures within all donors. We further found elevated expression of MHC Class II pathway genes in exocrine ductal cells of T1D donors, which we confirmed through CyTOF, in situ imaging mass cytometry, and immunofluorescence analysis. Taken together, our multimodal analyses identify novel cell types and processes that may contribute to T1D immunopathogenesis and provide new cellular and molecular insights into human pancreas function.

Tumors depend on a blood supply to deliver oxygen and nutrients, making tumor vasculature an attractive anticancer target. However, only a fraction of patients with cancer benefit from angiogenesis inhibitors. Whether antiangiogenic therapy would be more effective if targeted to individuals with specific tumor characteristics is unknown. To better characterize the tumor vascular environment both within and between cancer types, we developed a standardized metric - the endothelial index (EI) - to estimate vascular density in over 10,000 human tumors, corresponding to 31 solid tumor types, from transcriptome data. We then used this index to compare hyper- and hypovascular tumors, enabling the classification of human tumors into 6 vascular microenvironment signatures (VMSs) based on the expression of a panel of 24 vascular "hub" genes. The EI and VMS correlated with known tumor vascular features and were independently associated with prognosis in certain cancer types. Retrospective testing of clinical trial data identified VMS2 classification as a powerful biomarker for response to bevacizumab. Thus, we believe our studies provide an unbiased picture of human tumor vasculature that may enable more precise deployment of antiangiogenesis therapy.

The response to Poly (ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair mechanisms and the abundance of lesions that trap PARP enzymes on chromatin. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR based screen, we identify the PAR-binding Snf2-like ATPase, ALC1/CHD1L, as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of BRCA mutant cells and enhanced their sensitivity to PARPi by up to 250-fold, while overcoming several known resistance mechanisms. ALC1 loss was not epistatic to other repair pathways that execute the PARPi response. Instead, ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of repair factors. This resulted in an accumulation of replication associated DNA damage and a reliance on HR. These findings establish PAR-dependent chromatin remodeling as a mechanistically distinct aspect of PARPi responses, implicating ALC1 inhibition as a new approach to overcome therapeutic resistance in HR-deficient cancers.

Summary The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.

Rome et al. show that Trib1 controls effector versus exhausted T cell differentiation and function by restraining effector programming during persistent infection, revealing a new regulatory axis that could be targeted to recover waning T cell responses during chronic disease., In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity., Graphical Abstract

A fundamental requirement for growth of rapidly proliferating cells is metabolic adaptation to promote synthesis of biomass1. ATP citrate lyase (ACLY) is a critical enzyme responsible for synthesis of cytosolic acetyl-CoA, the key building component for de novo fatty acid synthesis and links vital pathways such as carbohydrate and lipid metabolism2. The mechanisms of ACLY regulation are not completely understood and the regulation of ACLY function by tyrosine phosphorylation is unknown. Here we show using mass-spectrometry-driven phosphoproteomics and metabolomics that ACLY is phosphorylated and functionally regulated at an evolutionary conserved residue, Y682. Physiologic signals promoting rapid cell growth such as epidermal growth factor stimulation in epithelial cells and T-cell receptor activation in primary human T-cells result in rapid phosphorylation of ACLY at Y682. In vitro kinase assays demonstrate that Y682 is directly phosphorylated by multiple tyrosine kinases, including ALK, ROS1, SRC, JAK2 and LTK. Oncogenically activating structural alterations such as gene-fusions, amplification or point mutations of ALK tyrosine kinase result in constitutive phosphorylation of ACLY in diverse forms of primary human cancer such as lung cancer, anaplastic large cell lymphoma (ALCL) and neuroblastoma. Expression of a phosphorylation-defective ACLY-Y682F mutant in NPM-ALK+ ALCL decreases ACLY activity and attenuates lipid synthesis. Metabolomic analyses reveal that ACLY-Y682F expression results in increased β-oxidation of 13C-oleic acid-labeled fatty acid with increased labeling of +2-citrate (pin vivo. Our results reveal a novel mechanism for direct ACLY regulation that is subverted by multiple oncogenically-activated tyrosine kinases in diverse human cancers. These findings have significant implications for novel therapies targeting ACLY in cancer and metabolism.

The transformed state in acute leukemia requires gene regulatory programs involving transcription factors (TFs) and chromatin modulators. Here, we uncover an IRF8-MEF2D TF regulatory circuit as an acute myeloid leukemia (AML)-specific dependency. We discover and characterize the mechanism by which the chromatin ‘reader' ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their AML-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcriptional coactivator essential for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential selective therapeutic target for modulating essential transcriptional programs in AML. Citation Format: Zhendong Cao, Krista A. Budinich, Hua Huang, Bin Lu, Zhen Zhang, Diqiu Ren, Yeqiao Zhou, Yuhan Huang, Bianca Pingul, Molly C. Kingsley, Alexandra K. Lenard, Jun Qi, Martin P. Carroll, Gerd A. Blobel, Robert B. Faryabi, Kathrin M. Bernt, Shelley L. Berger, Junwei Shi. The IRF8-MEF2D transcription factor circuit regulated by a druggable multiple post-translational modification (PTM) reader ZMYND8 in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB205.

Summary Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC , which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.

Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is currently incurable with conventional chemotherapy. Some TNBC patients harbor either recurrent somatic gain-of-function mutations or focal amplification in genes coding for Notch receptors. Recent findings suggest that Notch signals, in the absence of Notch mutations are also important to maintain chemotherapy-resistant circulating tumor cells. Importantly, elevated Notch signaling is specifically enriched in the TNBC and is associated with worse survival in this subtype. These findings underscore the importance of Notch signaling in pathogenesis of TNBC and suggest potential new avenue for therapeutic approaches. Despite this, the target genes and oncogenic mechanisms downstream of aberrant Notch signaling are poorly understood. To create a high confidence map of the oncogenic Notch-driven regulome in TNBC, we performed an integrative, comprehensive and functional genome-wide analysis of Notch-driven targets in Notch-mutated TNBC cell lines by combining high-resolution genome-wide chromatin topology data (HiChIP), dynamic mapping of Notch transcriptional complex members and active and repressive chromatin marks (ChIP-seq), as well as evaluation of dynamic transcription (RNAseq) in presence and absence of Notch signals. One important oncogene and critical Notch target in TNBC is MYC. Our integrative analysis identified detailed map of Notch-dependent MYC regulatory network. The TNBC-specific MYC regulatory region, located 5' of MYC, is much more complex compared to other known Notch-driven malignancies and constitutes of a broad domain of active enhancers marked by H3K27ac that are not present in Notch-driven lymphoma and leukemia. Integrative analysis of ChIP-seq and HiChIP revealed multiple enhancers within this broad domain of regulatory elements that are directly bound by the members of Notch transcriptional complex, sensitive to acute changes in Notch activation and that contact MYC promoter via tumor specific chromatin loops. We showed that genetic perturbation of MYC by CRISPR/Cas9 approach was sufficient to kill TNBC cells. Since, MYC is difficult to target pharmacologically an alternative strategy is to use drugs that inhibit factors regulating MYC expression. To this end, elucidating the Notch regulatory landscape, and in particular, the mechanism by which Notch regulates MYC expression in TNBC may reveal new therapeutic strategies to precisely target MYC in Notch-dependent breast cancer. Citation Format: Petrovic J, Zhou Y, Georgakilas G, Vahedi G, Pear WS, Faryabi RB. Triple negative breast cancer-specific chromatin conformation links Notch signal to tumor-specific transcriptional program [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-07.

Summary Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice. Multi-enhancer interactions formed at genomic regions harboring genes with prominent roles in T cell development in both strains. However, diabetes risk-conferring loci coalesced enhancers and promoters in NOD, but not C57BL/6 thymocytes. 3D genome mapping of NODxC57BL/6 F1 thymocytes revealed that genomic misfolding in NOD mice is mediated in cis. Moreover, immune cells infiltrating the pancreas of humans with T1D exhibited increased expression of genes located on misfolded loci in mice. Thus, genetic variation leads to altered 3D chromatin architecture and associated changes in gene expression that may underlie autoimmune pathology.

Transcriptional programs contribute to phenotypic and functional cell states. While elucidation of cell state heterogeneity and its role in biology and pathobiology has been advanced by studying single cell level measurements, the underlying assumptions of current analytical methods limit the identification and exploration of cell clades. Unlike other methods, which produce a single uni-layer partition of cells ignoring echelons of cell states, we presentTooManyCells, a software consisting of a suite of graph-based tools for efficient, global, and unbiased identification and visualization of cell clades while maintaining and presenting the relationship between cell states.TooManyCellsprovides a set of tools based on a matrix-free efficient divisive hierarchical spectral clustering algorithm wholly different from the prevalent Louvain-based methods.BirchBeer, the visualization component ofTooManyCells, introduces a new approach for single cell analysis that is built on a concept intentionally orthogonal to the widely used dimensionality reduction methods. Together, this suite of tools provide a paradigm shift in the analysis and interpretation of single cell data by enabling simultaneous comparisons of cell states at context-and application-dependent scales. A byproduct of this shift is the immediate detection and visualization of rare populations that outperforms previous algorithms as demonstrated by applying these tools to existing single cell RNA-seq data sets from various mouse organs.

SUMMARY Emerging single-cell epigenomic assays are used to investigate the heterogeneity of chromatin activity and its function. However, identifying cells with distinct regulatory elements and clearly visualizing their relationships remains challenging. To this end, we introduce TooManyPeaks to address the need for the simultaneous study of chromatin state heterogeneity in both rare and abundant subpopulations. Our analyses of existing data from three widely used single-cell assays for transposase-accessible chromatin using sequencing (scATAC-seq) show the superior performance of TooManyPeaks in delineating and visualizing pure clusters of rare and abundant subpopulations. Furthermore, the application of TooManyPeaks to new scATAC-seq data from drug-naive and drug-resistant leukemic T cells clearly visualizes relationships among these cells and stratifies a rare “resistant-like” drug-naive sub-clone with distinct cis-regulatory elements., Graphical Abstract, In brief Schwartz et al. present TooManyPeaks, a suite of algorithms to explore and visualize heterogeneity of regulatory elements by using single-cell ATAC-seq data. Using TooManyPeaks’s functionalities, they find evidence that heterogeneity of the chromatin accessibility state contributes to the propensity of Notch-mutated T leukemic cells to develop resistance to Notch inhibitors.

Chromatin loops enable transcription factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors, such as active forms of Notch, can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3-dimensional (3D) organization of the cancer genome is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B-cell lymphoma, we show that far beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes through establishing new long-range regulatory interactions. Moreover, a large fraction of Notch-promoted regulatory loops forms highly interacting enhancer and promoter spatial clusters, termed “3D cliques”. Loss-and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML). ITD mutations of Fms-like tyrosine kinase 3 (FLT3) receptor increase kinase activity, and are associated with poor prognostic outcomes. Currently, several small-moleculeFLT3inhibitors (FLT3i) are in clinical trials for targeted therapy of high-risk FLT3-ITD-positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence ofFLT3-ITD mutations in a patient might not guarantee effective clinical response to targeted inhibition ofFLT3kinase. Motivated by the heterogeneity ofFLT3-ITD mutations, we sought to investigate the effects ofFLT3-ITD structural features on response to treatment in AML patients. To this end, we developed HeatITup (HEAT diffusion for Internal Tandem dUPlication), an algorithm to efficiently and accurately identify ITDs and classify them based on their nucleotide composition into newly defined categories of “typical” or “atypical”. Typical ITDs insert sequences are entirely endogenous to theFLT3locus whereas atypical ITDs contain nucleotides exogenous to the wildtypeFLT3. We applied HeatITup to our cohort ofde novoand relapsed AML patients. Individuals with AML carrying typical ITDs benefited significantly more from FLT3i than patients with atypical ITDs, regardless of whether FLT3i was used after initial induction or at relapse. Furthermore, analysis of the TCGA AML cohort demonstrated improved survival for patients with typical ITDs treated with induction chemotherapy. These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing towards personalized treatment for AML patients.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including MYC and HES1 , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

SummaryThe DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.

Summary The biological and functional heterogeneity between tumors—both across and within cancer types—poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

In contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development., Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3′ ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP−/− mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.

Gain-of-function mutations in Notch receptor genes occur in 10-15% of cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and are associated with inferior clinical outcomes. Nearly all Notch mutations reported in B cell tumors lead to loss of the C-terminal negative regulatory PEST domain and result in stabilization of the activated form of Notch (intracellular Notch [ICN]), whereas mutations that lead to ligand-independent Notch activation (which are common in T cell acute lymphoblastic leukemia [T-ALL]) are rare. ICN can be detected in tumor cells within lymph nodes of >80% of patients with CLL, suggesting that Notch may have a broader oncogenic role than the incidence of Notch mutations would suggest. However, the downstream targets of Notch in B-cell tumors have not been identified. We used a gamma-secretase inhibitor (GSI) washout strategy to determine the immediate, direct effects of Notch activation in three MCL cell lines with Notch gain-of-function mutations, including two cell lines with unusual Notch gene rearrangements that lead to ligand-independent Notch activation, as well as a third line with a Notch PEST domain mutation in which signaling was activated with recombinant Notch ligand. Using these models, we identified likely direct target genes and their associated genomic Notch response elements using RNA-seq and ChIP-Seq in the Notch-on and Notch-off states. Most of these response elements corresponded to long-range enhancers that showed Notch-dependent changes in H3K27 acetylation, and were bound by components of the Notch transcription complex (NTC) in both cell lines. We confirmed these associations by performing ChIP-Seq on primary CLL and MCL biopsies, and by identifying specific looping interactions with Notch target gene promoters in public genome-wide proximity ligation datasets (RNA Pol2 ChIA-PET) from a lymphoblastoid cell line expressing the EBV-encoded Notch surrogate protein EBNA2. MYC was among the most strongly Notch-activated genes in Notch-dependent MCL cell lines and was associated with NTC binding at two B cell-specific 5' enhancers distinct from the Notch-dependent MYC enhancer previously identified in T-ALL. MCL cell line proliferation was blocked by Cas9 nuclease or epigenetic repressors targeting the 5' MYC enhancers, whereas cells were rescued from Notch inhibition by GSI via transduction with MYC. Gene set enrichment analysis of other direct Notch target genes identified in MCL models showed enrichment for regulators of B cell receptor (BCR) signaling, including the Src family kinase genes FYN, LYN, and BLK, and the signaling complex adaptor BLNK, as well as regulators of CD40 and cytokine signaling. RNA-seq analysis of primary CLL lymph node biopsies revealed significantly higher expression of many Notch target genes in biopsies with high levels of ICN. To functionally validate Notch target genes in primary tumors, we co-cultured CLL and MCL cells obtained from peripheral blood with Notch ligand-expressing stromal cells in the presence ("notch off") or absence ("notch on") of GSI, and demonstrated increased expression of Notch target genes, including MYC, in the "notch-on" cells. Furthermore, "notch-on" CLL cells showed increased phosphorylation of the BCR signaling intermediates SYK and PLCg2 upon BCR crosslinking compared to GSI-treated cells. Finally, we validated Notch-dependent regulation of target genes in vivo in a patient-derived xenograft model of NOTCH1-mutant MCL. Notch target gene expression was significantly higher in MCL cells within the spleen versus bone marrow or blood, but was markedly reduced in animals treated for five days with GSI. Additional xenograft studies are ongoing, and will be described at the meeting. Our data link active Notch signaling to two well-characterized oncogenic drivers in B cell lymphoma, MYC and BCR signaling, and may have important implications for the development of treatment strategies involving Notch antagonists and other targeted therapeutics, such as BCR targeting agents. Disclosures Weinstock: Novartis: Consultancy, Research Funding.

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.

Summary Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4 + CD8 + DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3′ Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene ( Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αβ T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer T cell development.