Group 3 medulloblastoma transcriptional networks collapse under domain specific EP300/CBP inhibition.

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.

A CRISPR-drug perturbational map for identifying compounds to combine with commonly used chemotherapeutics.

Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers.

Collateral lethality between HDAC1 and HDAC2 exploits cancer-specific NuRD complex vulnerabilities.

Transcriptional co-regulators have been widely pursued as targets for disrupting oncogenic gene regulatory programs. However, many proteins in this target class are universally essential for cell survival, which limits their therapeutic window. Here we unveil a genetic interaction between histone deacetylase 1 (HDAC1) and HDAC2, wherein each paralog is synthetically lethal with hemizygous deletion of the other. This collateral synthetic lethality is caused by recurrent chromosomal deletions that occur in diverse solid and hematological malignancies, including neuroblastoma and multiple myeloma. Using genetic disruption or dTAG-mediated degradation, we show that targeting HDAC2 suppresses the growth of HDAC1-deficient neuroblastoma in vitro and in vivo. Mechanistically, we find that targeted degradation of HDAC2 in these cells prompts the degradation of several members of the nucleosome remodeling and deacetylase (NuRD) complex, leading to diminished chromatin accessibility at HDAC2-NuRD-bound sites of the genome and impaired control of enhancer-associated transcription. Furthermore, we reveal that several of the degraded NuRD complex subunits are dependencies in neuroblastoma and multiple myeloma, providing motivation to develop paralog-selective HDAC1 or HDAC2 degraders that could leverage HDAC1/2 synthetic lethality to target NuRD vulnerabilities. Altogether, we identify HDAC1/2 collateral synthetic lethality as a potential therapeutic target and reveal an unexplored mechanism for targeting NuRD-associated cancer dependencies.

Proteasome inhibition targets the KMT2A transcriptional complex in acute lymphoblastic leukemia.

Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.

Analysis of rare driving events in pediatric acute myeloid leukemia.

Elucidating genetic aberrations in pediatric acute myeloid leukemia (AML) provides insight in biology and may impact on risk-group stratification and clinical outcome. This study aimed to detect such aberrations in a selected series of samples without known (cyto)genetic aberration using molecular profiling. A cohort of 161 patients was selected from various study groups: DCOG, BFM, SJCRH, NOPHO and AEIOP. Samples were analyzed using RNA sequencing (n=152), whole exome (n=135) and/or whole genome sequencing (n=100). In 70 of 156 patients (45%), of whom RNA sequencing or whole genome sequencing was available, rearrangements were detected, 22 of which were novel; five involving ERG rearrangements and four NPM1 rearrangements. ERG rearrangements showed self-renewal capacity in vitro, and a distinct gene expression pattern. Gene set enrichment analysis of this cluster showed upregulation of gene sets derived from Ewing sarcoma, which was confirmed comparing gene expression profiles of AML and Ewing sarcoma. Furthermore, NPM1-rearranged cases showed cytoplasmic NPM1 localization and revealed HOXA/B gene overexpression, as described for NPM1 mutated cases. Single-gene mutations as identified in adult AML were rare. Patients had a median of 24 coding mutations (range, 7-159). Novel recurrent mutations were detected in UBTF (n=10), a regulator of RNA transcription. In 75% of patients an aberration with a prognostic impact could be detected. Therefore, we suggest these techniques need to become standard of care in diagnostics.

SIX1 reprograms myogenic transcription factors to maintain the rhabdomyosarcoma undifferentiated state.

Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription.

EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2ß, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. SIGNIFICANCE: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2ß. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.

Integrative Genomic Analysis of Pediatric Myeloid-Related Acute Leukemias Identifies Novel Subtypes and Prognostic Indicators.

Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell-like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. SIGNIFICANCE: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549.

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia.

Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3 ITD , FLT3 N676K , and NRAS G12D accelerate KMT2A-MLLT3 leukemia onset. Further, also subclonal FLT3 N676K mutations accelerate disease, possibly by providing stimulatory factors. Herein, we show that one such factor, MIF, promotes survival of mouse KMT2A-MLLT3 leukemia initiating cells. We identify acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 in KMT2A-MLLT3 leukemia cells that favored clonal expansion. During clonal evolution, we observe serial genetic changes at the Kras G12D locus, consistent with a strong selective advantage of additional Kras G12D . KMT2A-MLLT3 leukemias with signaling mutations enforce Myc and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlight the importance of activated signaling as a contributing driver.

T-bet controls severity of hypersensitivity pneumonitis.

Hypersensitivity Pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease is characterized by alveolitis, granuloma formation and in some patients' fibrosis. IFNγ plays a critical role in HP; in the absence of IFNγ granuloma formation does not occur. However, recent studies using animal models of HP have suggested that HP is a Th17 disease calling into question the role of IFNγ. In this study, we report that initially IFNγ production is dependent on IL-18 and the transcription factor T-bet, however as the disease continues IFNγ production is IL-18-independent and partially T-bet dependent. Although IFNγ production is required for granuloma formation its role is distinct from that of T-bet. Mice that are deficient in T-bet and exposed to S. rectivirgula develop more severe disease characterized by an exacerbated Th17 cell response, decreased Th1 cell response, and increased collagen production in the lung. T-bet-mediated protection does not appear to be due to the development of a protective Th1 response; shifting the balance from a Th17 predominant response to a Th1 response by inhibition of IL-6 also results in lung pathology. The results from this study suggest that both Th1 and Th17 cells can be pathogenic in this model and that IFNγ and T-bet play divergent roles in the disease process.

MyD88 is necessary for neutrophil recruitment in hypersensitivity pneumonitis.

Hypersensitivity pneumonitis is an interstitial lung disease that is characterized by alveolitis, granuloma formation, and in some patients, fibrosis. Using the Saccharopolyspora rectivirgula animal model of Farmer's lung disease, our laboratory has demonstrated that neutrophils play a critical role in IFN-gamma production during the acute phase of the disease. As IFN-gamma is necessary for granuloma formation, it is important to identify the factors that lead to neutrophil recruitment during disease. To begin to identify the pattern recognition receptors (PRRs) that initiate chemokine production, leading to neutrophil recruitment following S. rectivirgula exposure, we examined the role of MyD88 and TLR2. Our results demonstrate that neutrophil recruitment, as measured by flow cytometry and the myeloperoxidase assay, was abolished in the absence of MyD88 following S. rectivirgula exposure. The decrease in neutrophil recruitment was likely a result of a significant decrease in production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine. These results suggest that S. rectivirgula interacts with PRRs that are upstream of the MyD88 pathway to initiate cytokine and chemokine production. In vitro studies suggest that S. rectivirgula can interact with TLR2, and stimulation of adherent cells from TLR2 knockout (KO) mice with S. rectivirgula resulted in a significant decrease in MIP-2 production. However, TLR2 KO mice did not have a reduction in neutrophil recruitment compared with wild-type mice following S. rectivirgula exposure. The results from our studies suggest that one or more PRR(s) upstream of MyD88 are necessary for neutrophil recruitment following S. rectivirgula exposure.

IFN-gamma production by innate immune cells is sufficient for development of hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled particulate antigens. The disease is characterized by lymphocytic alveolitis, granuloma formation and fibrosis. IFN-gamma is required for the formation of granulomas in HP, and we therefore focused on identifying the cellular sources of IFN-gamma during the disease. Using the Saccharopolyspora rectivirgula (SR) animal model of HP, we demonstrated that the majority of IFN-gamma(+) cells in the lung following SR exposure are neutrophils. Ab-mediated depletion of neutrophils in mice prior to exposure to SR resulted in a decrease in the level of IFN-gamma mRNA and protein compared to isotype Ab-treated mice, suggesting that neutrophils are an important source of IFN-gamma during HP. To determine the contribution of T and non-T cell sources of IFN-gamma to granuloma formation, we performed adoptive transfer studies. RAG-1(-/-) mice reconstituted with spleen cells from IFN-gamma(-/-) mice developed granulomas similarly to RAG-1(-/-) mice reconstituted with normal spleen cells. Therefore innate immune cell IFN-gamma production in the absence of T cell IFN-gamma production is sufficient for granuloma formation. These results provide new insight into the pathogenesis of HP and demonstrate the important contribution of innate immune cells to the disease process.

Chemokine production during hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled particulate antigens. Individuals with HP develop lymphocytic alveolitis,granuloma formation, and fibrosis. HP is categorized as a Th1 disease, and granuloma formation is dependent on T cells and the Th1 cytokine IFN-gamma. We therefore hypothesized that the IFN-gamma-inducible chemokines IP-10, Mig, and I-TAC, which are frequently associated with Th1 diseases, would play an important role in the pathogenesis of disease. We analyzed the expression of multiple chemokines in the lungs of wild-type (WT) and IFN-gamma-knockout (GKO) mice exposed to the particulate antigen Saccharopolyspora rectivirgula (SR). Our results demonstrate the production of IP-10, Mig, and I-TAC in WT mice during the development of HP, whereas GKO mice have reduced levels of IP-10 and no Mig or I-TAC mRNA in the lungs in response to SR exposure. The production of these chemokines is associated with an influx of CXCR3+/CD4+ T cells into lungs of WT mice, which is reduced in GKO mice. These results suggest that IFN-gamma mediates the recruitment of CXCR3+/CD4+ T cells into the lung via production of the chemokines IP-10, Mig, and I-TAC, resulting in granuloma formation.