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BackgroundSTK11 loss-of-function mutations, found in approximately 20% of non-small cell lung cancers (NSCLC), are associated with primary resistance to immune checkpoint therapy. TNG260 is a small molecule inhibitor of CoREST, a chromatin complex that regulates gene expression through histone deacetylase (HDAC) activity. Preclinical work demonstrated that CoREST inhibition by TNG260 sensitizes STK11-mutant tumors to anti-PD1 treatment. TNG260, in combination with pembrolizumab, is being evaluated in a phase 1/2 clinical trial (NCT05887492).MethodsPatients with STK11-mutated, locally advanced or metastatic solid tumors received TNG260 at 40 mg, 80 mg or 120 mg administered once daily (QD) in combination with pembrolizumab at the standard dose of 200 mg IV every 3 weeks. Patients were evaluated to determine the TNG260 pharmacokinetics, safety, tolerability, and preliminary efficacy of the combination therapy, as well as the recommended phase 2 dose (RP2D).ResultsForty-one patients with STK11-mutant, locally advanced or metastatic solid tumors were enrolled into 4 dose escalation cohorts. TNG260 achieved exposures in the range predicted to be efficacious by preclinical studies. Paired biopsies from patients that received 80 mg and 120 mg QD doses of TNG260 showed CoREST inhibition in tumor tissue, increased PD-L1 expression, and increased intratumoral infiltration of cytotoxic T cells, providing proof-of-mechanism that TNG260 can remodel the immune microenvironment of STK11-mutant tumors. Dose-limiting toxicities were on-target and consistent with HDAC inhibition, including cytopenias and fatigue. Adverse events with TNG260 were dose-dependent. The maximal tolerated dose (MTD) was determined to be 80 mg. Patients with STK11 mutant/KRAS wild-type NSCLC (representing approximately 50% of STK11 mutant NSCLC patients) receiving clinically active doses of TNG260 plus pembrolizumab exhibited a median PFS of 27 weeks, a marked improvement over the SOC PFS of approximately 10 weeks. Limited clinical activity was observed in patients with KRAS mutant NSCLC and in other solid tumors.ConclusionsTNG260 in combination with pembrolizumab is a promising new treatment strategy for reversing immune evasion in STK11 mutant NSCLC tumors, demonstrating a marked improvement in PFS over the current standard of care (SOC). Dose expansion is ongoing at the RP2D of 80 mg TNG260 plus standard dose pembrolizumab for patients with advanced KRAS wild-type/STK11 mutant NSCLC.Trial RegistrationNCT05887492Ethics ApprovalThe study protocol was approved by the appropriate institutional review board at each participating site. All study subjects provided written informed consent prior to their participation in the trial.

Changes in gene expression drive differentiation along distinct cell lineages, and these shifts in gene expression are associated with alterations in chromatin accessibility and modifications reflecting activation or repression. We used deep sequencing of polyA+ RNA to map the transcriptomes of the megakaryocyte-erythroid progenitor (MEP) and cells of its two daughter lineages, erythroblasts (ERY) and megakaryocytes (MEG) in mice to reveal insights into differentiation. Transcriptome comparisons revealed that MEPs already expressed much of the MEG program while continuing to express genes associated with parallel myeloid lineages. By contrast, ERY underwent an extensive program of gene induction along with repression of pan-hematopoietic and MEG genes. Maps of transcription factor (TF) occupancy also indicated distinct modes of regulation for the MEG and ERY programs, with MEG genes preferentially occupied by hematopoietic TFs in multipotent progenitors and continued occupancy post-commitment, in contrast to erythroid genes that were primarily occupied in committed ERY. Previous work had indicated a surprising discordance in the clustering of MEP with other hematopoietic cell types by RNA-seq chromatin states. We combined the differential expression data with chromatin accessibility across blood cell types to identify trends that contribute to this discordance. Specifically, candidate cis-regulatory elements (cCREs) in some ERY-specific genes were precociously actuated in the bipotential cell populations, and some other genes were expressed in both the MEP population and MEG but their cCREs have less chromatin accessibility in MEP. This discordance in cell type clustering by different modalities of functional genomics may reflect the different contributions of subpopulations in the MEP to the different modalities measured.

Attenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.Attenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.

Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators' direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.

Motivation: Ribosomal RNAs (rRNAs) form a majority of cellular RNA, and the loci from which they are transcribed (rDNA gene repeats) are among the most intensively transcribed regions of the genome. Due to the repetitive nature of rDNA, there has been a historical lack of genome assemblies and bioinformatic pipelines capable of yielding rDNA signal from high-throughput sequencing datasets. The study of rDNA regulation has been stunted by the lack of such tools, leading most investigators to ignore rDNA in genome wide studies, creating a significant knowledge gap in our understanding of how the most abundant RNA in the cell is regulated. Our recent work1 revealed previously-unnoticed binding of numerous mammalian transcription factors and chromatin proteins to rDNA. Several of these factors were known to play critical roles in development, tissue function, immunity, and malignancy, and had been dissected in detail for decades, but their potential rDNA roles had remained unexplored. Our work demonstrates the extent of the blind spot into which rDNA has fallen in modern genetic and epigenetic studies, and highlights an unfulfilled need for rDNA-optimized public genome assemblies and accessible mapping workflows. Results: We customized the human hg38 and mouse mm39 genomes to render them suitable for rDNA mapping. The standard builds for these genomes contain numerous partial or fragmented rDNA loci. We identified and masked all rDNA-like regions in the hg38 and mm39 assemblies, added a single rDNA reference sequence of the appropriate species as a ~45kb chromosome R. We also created annotation files to aid visualization of rDNA features in genome browser tracks. We validated the utility of these customized genomes for mapping of known rDNA binding proteins, and we present in this paper a simple workflow for mapping ChIP-seq datasets to rDNA and generating browser tracks. These resources make rDNA mapping and visualization readily accessible to a broad audience. Availability and Implementation: Customized genome assemblies (hg38-rDNA v1.0 and mm39-rDNA v1.0), annotation files, and positive and negative control mapping tracks are freely available through GitHub at: https://github.com/vikramparalkar/rDNA-Mapping-GenomesCompeting Interest StatementThe authors have declared no competing interest.Footnotes* https://github.com/vikramparalkar/rDNA-Mapping-Genomes

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA Polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2200 ChIP-Seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA, and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of CEBPA (C/EBP alpha), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced Pol I occupancy. Our work identifies numerous potential rRNA regulators, and provides a template for dissection of TF roles in rRNA transcription. Competing Interest Statement Jeremy E Wilusz serves as a consultant for Laronde. Footnotes * Title changed; Figure 3C updated to show that ChIP-Seq peak is lost after CEBPA degradation; Figure 5 western blot images and bargraph updated; Figure 6 and associated discussion text revised to clarify overall model; Figure S5C added to show that CEBPA does not bind rDNA; Fig S6D updated to include 28S rRNA as loading control; Fig S6E-G added to show that Pol I degradation leads to reduced 47S-FISH-Flow signal; Fig S7 added to show effect of CEBPA degradation on nucleolar size, p53 protein and p21 mRNA; Formatting changed for Methods section

Relapse of acute myeloid leukemia (AML) after allogeneic bone marrow transplantation (alloSCT) has been linked to immune evasion due to reduced expression of major histocompatibility complex class II (MHC-II) proteins through unknown mechanisms. We developed CORENODE, a computational algorithm for genome-wide transcription network decomposition, that identified the transcription factors (TFs) IRF8 and MEF2C as positive regulators and MYB and MEIS1 as negative regulators of MHC-II expression in AML cells. We show that reduced MHC-II expression at relapse is transcriptionally driven by combinatorial changes in the levels of these TFs, acting both independently and through the MHC-II coactivator CIITA. Beyond the MHC-II genes, MYB and IRF8 antagonistically regulate a broad genetic program responsible for cytokine signaling and T-cell stimulation that displays reduced expression at relapse. A small number of cells with altered TF levels and silenced MHC-II expression are present at the time of initial leukemia diagnosis, likely contributing to eventual relapse. Our findings reveal an adaptive transcriptional mechanism of AML evolution after allogenic transplantation whereby combinatorial fluctuations of TF levels under immune pressure result in selection of cells with a silenced T-cell stimulation program. Competing Interest Statement KS has consulted for KronosBio and Auron Therapeutics, has stock options with Auron Therapeutics, and received grant funding from Novartis on topics unrelated to this manuscript. NVD is a current employee of Genentech, Inc., a member of the Roche Group. KE has consulted for Third Rock Ventures on topics unrelated to this manuscript. The other authors have no competing interests to report.

A small set of lineage-restricted transcription factors (TFs), termed core regulatory circuitry (CRC), control cell identity and malignant transformation. Here, we integrated gene dependency, chromatin architecture and TF perturbation datasets to characterize 31 core TFs in acute myeloid leukemia (AML). Contrary to a widely accepted model, we detected a modular CRC structure with hierarchically organized, partially redundant and only sparsely interconnected modules of core TFs controlling distinct genetic programs. Rapid TF degradation followed by measurement of genome-wide transcription rates revealed that core TFs directly regulate dramatically fewer genes than previously assumed. Leukemias carrying KMT2A (MLL) rearrangements depend on the IRF8/MEF2 axis to directly enforce expression of the key oncogenes MYC, HOXA9 and BCL2. Our datasets provide an evolving model of CRC organization in human cells, and a resource for further inquiries into and therapeutic targeting of aberrant transcriptional circuits in cancer. Competing Interest Statement K. Stegmaier has funding from Novartis Institute of Biomedical Research, consults for and has stock options in Auron Therapeutics, and has consulted for Kronos Bio and AstraZeneca on topics unrelated to this work. N.V. Dharia is a current employee of Genentech, Inc., a member of the Roche Group. J. Xavier Ferrucio is a current employee of Vor Biopharma. C.Y. Lin is a current employee of Kronos Bio. B. Nabet is an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148440, WO/2018/148443 and WO/2020/146250). K. Eagle has consulted for Third Rock Ventures and Flare Therapeutics on topics unrelated to this manuscript. All other authors declare no potential conflict of interest.

In the last years, neural networks have proven to be a powerful framework for various image analysis problems. However, some application domains have specific limitations. Notably, digital pathology is an example of such fields due to tremendous image sizes and quite limited number of training examples available. In this paper, we adopt state-of-the-art convolutional neural networks (CNN) architectures for digital pathology images analysis. We propose to classify image patches to increase effective sample size and then to apply an ensembling technique to build prediction for the original images. To validate the developed approaches, we conducted experiments with Breast Cancer Histology Challenge dataset and obtained 90\\% accuracy for the 4-class tissue classification task.