Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens.

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.

Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition.

Here we show that acute myeloid leukemia (AML) cells require the BRD9 subunit of the SWI-SNF chromatin-remodeling complex to sustain MYC transcription, rapid cell proliferation and a block in differentiation. Based on these observations, we derived small-molecule inhibitors of the BRD9 bromodomain that selectively suppress the proliferation of mouse and human AML cell lines. To establish these effects as on-target, we engineered a bromodomain-swap allele of BRD9 that retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the antiproliferative effects of our compound series, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. To our knowledge, our study provides the first evidence for a role of BRD9 in cancer and reveals a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells.

BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements.

The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance.

Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor.

Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer.

Most mammalian transcription factors (TFs) and cofactors occupy thousands of genomic sites and modulate the expression of large gene networks to implement their biological functions. In this study, we describe an exception to this paradigm. TRIM33 is identified here as a lineage dependency in B cell neoplasms and is shown to perform this essential function by associating with a single cis element. ChIP-seq analysis of TRIM33 in murine B cell leukemia revealed a preferential association with two lineage-specific enhancers that harbor an exceptional density of motifs recognized by the PU.1 TF. TRIM33 is recruited to these elements by PU.1, yet acts to antagonize PU.1 function. One of the PU.1/TRIM33 co-occupied enhancers is upstream of the pro-apoptotic gene Bim, and deleting this enhancer renders TRIM33 dispensable for leukemia cell survival. These findings reveal an essential role for TRIM33 in preventing apoptosis in B lymphoblastic leukemia by interfering with enhancer-mediated Bim activation.

A rationale to target the SWI/SNF complex for cancer therapy.

SWI/SNF is a multisubunit chromatin-remodeling complex that performs fundamental roles in gene regulation, cell lineage specification, and organismal development. Mutations that inactivate SWI/SNF subunits are found in nearly 20% of human cancers, which indicates that the proper functioning of this complex is necessary to prevent tumor formation in diverse tissues. Recent studies show that SWI/SNF-mutant cancers depend on residual SWI/SNF complexes for their aberrant growth, thus revealing synthetic lethal interactions that could be exploited for therapeutic purposes. Other studies reveal that certain acute leukemias and small cell lung cancers, which lack SWI/SNF mutations, can be vulnerable to inhibition of the SWI/SNF ATPase subunit BRG1, whereas several normal and malignant cell types do not show this sensitivity. Here, we review the emerging evidence that implicates SWI/SNF as a tumor-dependency and candidate drug target in human cancer.

Atrial arrhythmogenicity in aged Scn5a+/DeltaKPQ mice modeling long QT type 3 syndrome and its relationship to Na+ channel expression and cardiac conduction.

Recent studies have reported that human mutations in Nav1.5 predispose to early age onset atrial arrhythmia. The present experiments accordingly assess atrial arrhythmogenicity in aging Scn5a+/KPQ mice modeling long QT3 syndrome in relationship to cardiac Na(+) channel, Nav1.5, expression. Atrial electrophysiological properties in isolated Langendorff-perfused hearts from 3- and 12-month-old wild type (WT), and Scn5a+/KPQ mice were assessed using programmed electrical stimulation and their Nav1.5 expression assessed by Western blot. Cardiac conduction properties were assessed electrocardiographically in intact anesthetized animals. Monophasic action potential recordings demonstrated increased atrial arrhythmogenicity specifically in aged Scn5a+/DeltaKPQ hearts. These showed greater action potential duration/refractory period ratios but lower atrial Nav1.5 expression levels than aged WT mice. Atrial Nav1.5 levels were higher in young Scn5a+/DeltaKPQ than young WT. These levels increased with age in WT but not Scn5a+/DeltaKPQ. Both young and aged Scn5a+/DeltaKPQ mice showed lower heart rates and longer PR intervals than their WT counterparts. Young Scn5a+/DeltaKPQ mice showed longer QT and QTc intervals than young WT. Aged Scn5a+/DeltaKPQ showed longer QRS durations than aged WT. PR intervals were prolonged and QT intervals were shortened in young relative to aged WT. In contrast, ECG parameters were similar between young and aged Scn5a+/DeltaKPQ. Aged murine Scn5a+/DeltaKPQ hearts thus exhibit an increased atrial arrhythmogenicity. The differing Nav1.5 expression and electrocardiographic indicators of slowed cardiac conduction between Scn5a+/DeltaKPQ and WT, which show further variations associated with aging, may contribute toward atrial arrhythmia in aged Scn5a+/DeltaKPQ hearts.