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Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

SummaryDirect reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression ofGATA4,HAND2,MEF2C,TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-β) inhibition efficiently promotes reprogramming. However, the mechanisms by which TGF-β blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) – demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-β signaling regulates the interaction between GATA4 and JMJD3. TGF-β activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation inGATA4(V267M) exhibits reduced binding to JMJD3 and impaired cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-β pathway activation impairs cardiac gene programming by interfering with GATA4-JMJD3 interactions.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, demonstrating clinical success in B-cell malignancies that are refractory to standard therapies. Relapses after CAR therapy has demonstrated the limitations of current CAR T cell therapies to target antigen-low tumors and induce consistent persistence. Furthermore, the success of CAR T cell therapy in B-lineage malignancies has not yet been translated to acute myeloid leukemia (AML). The targeting of myeloid-restricted antigens, such as CD33, with standard 2nd generation CAR T cells has not yielded similar clinical responses as ALL. Therefore, we hypothesize that novel CAR T cell strategies will be required to improve responses in non-B cell malignancies. We identified that CAR T cells inefficiently activate the scaffolding protein LAT in response to low levels of antigen, leading to diminished efficacy. We designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART) to overcome this deficiency of signaling in CAR T cells. In xenograft models, 22ALA-CART cells completely eradicated CD22-low leukemia, which is not responsive to standard CD22-BBz CAR T cells. We have extended these findings to CD19-directed ALA-CART cells, which eradicate CD19-low leukemia that is otherwise resistant to standard CD19-BBz CAR T cells. To understand how ALA-CART improves responses to antigen-low leukemia, we evaluated the in vivo potency of ALA-CART relative to 2nd generation CARs and found ALA-CART cells could eradicate ALL in xenograft models at lower doses than standard CAR T cells. RNA-Seq analysis demonstrated global differences in ALA-CART cells in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Stimulation through the ALA-CART receptor led to widespread changes in transcription factor activity in T cells which were distinct from standard CAR activation, particularly inducing significantly lower interferon response factor activity which has been associated with CAR T cell failure in patients. As strategies targeting myeloid lineage-restricted antigens with 2nd generation CAR T cells have not yet produced the clinical success seen with ALL-directed CAR therapy, we tested whether the ALA-CART platform could improve the activity of CD33-directed CAR T cells. CD33 ALA-CART cells demonstrated improved in vivo potency and exhibited enhanced memory potential like our results with ALL-directed ALA-CART. Thus, through the reversal of signaling deficits intrinsic to 2nd generation CARs, the ALA-CART platform improves the potency and persistence of CAR T cell therapy against both lymphoid and myeloid acute leukemias. Citation Format: Catherine Danis, Lillie Leach, Christopher Ebmeier, Amanda Novak, Etienne Danis, M. Eric Kohler. ALA-CART induces global transcriptional changes leading to enhanced potency and persistence of CAR T cells against both lymphoid and myeloid acute leukemias [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 3190.

Invasive lobular carcinoma of the breast (ILC) affects ∼40,000 US women annually and is a top ten most diagnosed cancer in women. ∼95% of ILC express estrogen receptor alpha (ER), but ILC is associated with poor long-term outcomes and anti-estrogen resistance, suggesting ER function is distinct in ILC cells. We reported that MDC1 (mediator of DNA damage checkpoint 1) is an ILC-specific ER co-regulator required for ER-driven proliferation, and that MDC1 knockdown dysregulates the ER transcriptome in ILC. Mechanisms of MDC1 co-regulator activity are unknown, but must be defined to understand endocrine response and resistance in ILC. To understand MDC1 co-regulator functions, we profiled ER/MDC1 genomic binding (ie. "cistrome") using CUT&RUN in ER+ ILC cell line MDA MB 134VI (MM134). We identified ∼2500 binding sites each for ER and MDC1, but only ∼8% of sites were bound by both factors. However, using BETA to integrate cistrome and transcriptome data predicted that both ER and MDC1 activate estrogen-regulated genes with over half of direct ER target genes also direct MDC1 targets (p=9.8×10-375). Among shared ER: MDC1 direct target genes (n=760), 58% had MDC1 binding in the promoter region with ER bound at nearby enhancers, while only 15% were linked to shared ER: MDC1 binding sites, suggesting MDC1 primarily facilitates promoter access for ER-bound enhancers. Further, MDC1 knockdown reduced ER binding at enhancer sites at ER: MDC1 target genes, implicating MDC1 in regulating chromatin access. Importantly, MDC1 does not have known enzymatic activity to directly regulate chromatin structure. We profiled MDC1-interacting proteins in ILC cells by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins), and determined whether knockdown of putative ER: MDC1 partners disrupted ER function. This identified the SWI/SNF complex protein BRG1 (SMARCA4) as a critical ER: MDC1 partner. BRG1 was required for ILC cell growth, and BRG1 knockdown specifically suppressed ER regulation of MDC1-dependent ER target genes. Taken together, our data suggest that in ILC cells, MDC1 facilitates ER access to and activity at target gene promoters. The BRG1 SWI/SNF complex is a putative ER: MDC1 partner and mediator of MDC1 co-regulator functions. Ongoing research is focused on understanding how MDC1 regulates ILC-specific ER activity by licensing access to ILC-specific target genes, and the role of the SWI/SNF complex in mediating genomic ER: MDC1 activity. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT sub‑groups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase‑3‑positive cells in the idasanutlin treatment group, confirming the induction of apoptosis

Background While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood. Method To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs). Results We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors. Conclusions We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.

High-grade gliomas (HGG) afflict both children and adults and respond poorly to current therapies. Epigenetic regulators have a role in gliomagenesis, but a broad, functional investigation of the impact and role of specific epigenetic targets has not been undertaken. Using a two-step, in vitro/in vivo epigenomic shRNA inhibition screen, we determine the chromatin remodeler BPTF to be a key regulator of adult HGG growth. We then demonstrate that BPTF knockdown decreases HGG growth in multiple pediatric HGG models as well. BPTF appears to regulate tumor growth through cell self-renewal maintenance, and BPTF knockdown leads these glial tumors toward more neuronal characteristics. BPTF’s impact on growth is mediated through positive effects on expression of MYC and MYC pathway targets. HDAC inhibitors synergize with BPTF knockdown against HGG growth. BPTF inhibition is a promising strategy to combat HGG through epigenetic regulation of the MYC oncogenic pathway.

Background Treatment for pediatric posterior fossa group A (PFA) ependymoma with gain of chromosome 1q (1q+) has not improved over the past decade owing partially to lack of clinically relevant models. We described the first 2 1q+ PFA cell lines, which have significantly enhanced our understanding of PFA tumor biology and provided a tool to identify specific 1q+ PFA therapies. However, cell lines do not accurately replicate the tumor microenvironment. Our present goal is to establish patient-derived xenograft (PDX) mouse models. Methods Disaggregated tumors from 2 1q+ PFA patients were injected into the flanks of NSG mice. Flank tumors were then transplanted into the fourth ventricle or lateral ventricle of NSG mice. Characterization of intracranial tumors was performed using imaging, histology, and bioinformatics. Results MAF-811_XC and MAF-928_XC established intracranially within the fourth ventricle and retained histological, methylomic, and transcriptomic features of primary patient tumors. We tested the feasibility of treating PDX mice with fractionated radiation or chemotherapy. Mice tolerated radiation despite significant tumor burden, and follow-up imaging confirmed radiation can reduce tumor size. Treatment with fluorouracil reduced tumor size but did not appear to prolong survival. Conclusions MAF-811_XC and MAF-928_XC are novel, authentic, and reliable models for studying 1q+ PFA in vivo. Given the successful response to radiation, these models will be advantageous for testing clinically relevant combination therapies to develop future clinical trials for this high-risk subgroup of pediatric ependymoma.

SummaryRhabdomyosarcoma (RMS) is a pediatric skeletal muscle sarcoma characterized by the expression of the myogenic-lineage transcription factors (TF) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their function. Here, we demonstrate that the developmental TF, SIX1, is highly expressed in RMS and is critical to maintain a muscle progenitor-like state. SIX1 loss induces terminal differentiation of RMS cells into myotube-like cells and dramatically 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 terminal muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status in RMS and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role for SIX1 in the repression of RMS differentiation via genome-wide alterations in MYOD1-mediated transcription.HighlightsSIX1 prevents differentiation in RMS while it promotes differentiation during normal developmentFN-RMS are highly dependent on SIX1 for growth in both zebrafish and mouse xenograft modelsLoss of SIX1 alters the transcriptional landscape of RMS cells, inducing a growth to differentiation switchSIX1 knockdown in FN-RMS causes reduced super enhancer-based activity at stem-related genes and enhanced MYOD1 binding to differentiation loci, resulting in the activation of a myogenic differentiation programA gene signature derived from SIX1 loss strongly correlates with myogenic differentiation status and is predictive of advanced RMS.

Prostate cancer genomic subtypes that stratify aggressive disease and inform treatment decisions at the primary stage are currently limited. Previously, we functionally validated an aggressive subtype present in 15% of prostate cancer characterized by dual deletion of MAP3K7 and CHD1. Recent studies in the field have focused on deletion of CHD1 and its role in androgen receptor (AR) chromatin distribution and resistance to AR-targeted therapy; however, CHD1 is rarely lost without codeletion of MAP3K7. Here, we show that in the clinically relevant context of co-loss of MAP3K7 and CHD1 there are significant, collective changes to aspects of AR signaling. Although CHD1 loss mainly impacts the expansion of the AR cistrome, loss of MAP3K7 drives increased AR target gene expression. Prostate cancer cell line models engineered to cosuppress MAP3K7 and CHD1 also demonstrated increased AR-v7 expression and resistance to the AR-targeting drug enzalutamide. Furthermore, we determined that low protein expression of both genes is significantly associated with biochemical recurrence (BCR) in a clinical cohort of radical prostatectomy specimens. Low MAP3K7 expression, however, was the strongest independent predictor for risk of BCR over all other tested clinicopathologic factors including CHD1 expression. Collectively, these findings illustrate the importance of MAP3K7 loss in a molecular subtype of prostate cancer that poses challenges to conventional therapeutic approaches. Implications: These findings strongly implicate MAP3K7 loss as a biomarker for aggressive prostate cancer with significant risk for recurrence that poses challenges for conventional androgen receptor–targeted therapies.

Higa et al. show that chronic interleukin-1β exposure primes multipotent hematopoietic progenitor cells for myelopoiesis by upregulating myeloid differentiation programs and repressing stem gene programs in a Cebpa-dependent manner. Consequently, interleukin-1 potently selects for Cebpa loss in hematopoietic stem and progenitor cells., The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1β (IL-1β), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell–intrinsic competitive advantage; rather chronic IL-1β exposure engendered potent selection for Cebpa loss. Chronic IL-1β augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1β, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention., Graphical Abstract

BACKGROUND: Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated promoter proximal pause-release of Pol II is a conserved regulatory mechanism for synchronous transcriptional induction best described in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. DESIGN/METHOD: In order to examine the dynamics of chromatin reorganization following radiotherapy, we performed a combination of ChIP-, ATAC-, and RNA-seq in model systems of diffuse intrinsic pontine glioma (DIPG) and other pediatric high-grade gliomas (pHGG) following IR exposure. We interrogated IR-induced gene expression in the presence or absence of PTEFb blockade, including both mechanistic and functional consequences of concurrent inhibition or genetic depletion. We utilized culture models with live cell imaging to assess the therapeutic synergy of PTEFb inhibition with IR, as well as the therapeutic index of this intervention relative to normal controls. Finally, we employed orthotopic models of pHGG treated with conformal radiotherapy and CNS-penetrant PTEFb inhibitors in order to assess tolerability and anti-tumor effect in vivo. RESULTS: Rapid genome-wide redistribution of active chromatin features and PTEFb facilitates Pol II pause-release to drive nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb imparts a transcription elongation defect, abrogating canonical adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. CONCLUSION: These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

Atypical Teratoid Rhabdoid Tumor is a highly aggressive pediatric brain tumor with poor prognosis driven by loss of the chromatin remodeling factor SMARCB1 that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. We investigated the effect of SIRT2 inhibition in ATRT which was identified as a primary dependency in ATRT. SIRT2 inhibition with shRNA or Thiomyristoyl (TM) decreased ATRT cell growth, inhibited clonogenic potential and leaded to the cell cycle arrest. SIRT2 inhibition effectively suppresses pluripotency-associated genomic programs, significantly changed stem cell frequency, decreased tumor-sphere formation of ATRT cells and attenuated tumor cell self-renewal. In vivo SIRT2 inhibition decreased oncogenic markers and increased accumulation neuronal differentiation markers. Furthermore, SIRT2 induced apoptosis, decreased tumor growth and prolonged survival in orthotopic xenograft models. Single-cell RNA transcriptome analysis of xenoftaft tumors reveals elimination of tumor cells expressing stem cell genes and expansion of tumor cells expressing differentiated genes following TM treatment in ATRT. We demonstrated that SIRT2 inhibition is a molecular vulnerability in SMARCB1-deleted ATRT.

MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. CDK7 was identified as a mediator of medulloblastoma tumorigenesis. Using covalent inhibitors and genetic depletion of CDK7 we observe the cessation of tumor growth in xenograft mouse models and increase in apoptotic mechanisms. The results are attributed to repression of a core set of Myc-driven transcriptional programs mediating DNA repair. We further establish that blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage and extended survival and tumor latency in medulloblastoma xenograft mouse models. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

Loss of SMARCB1 is the hallmark genetic event that characterizes rhabdoid tumors in children. Rhabdoid tumors of the brain (ATRT) occur in young children and are particularly challenging with poor long-term survival. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. Recent studies demonstrate that ATRT consists of 3 genomic subgroups with a subset of poor outcome tumors expressing high BMP and MYC pathway activation. Here we show that MYC occupies distinct promoter loci in ATRT compared to embryonic stem (ES) cells. Furthermore, using human ATRT cell lines, patient-derived cell culture, ex vivo patient-derived tumor, and orthotopic xenograft models, we show that MYC inhibition is a molecular vulnerability in SMARCB1-deleted tumors and that such inhibition effectively suppresses BMP and pluripotency-associated genomic programs, attenuates tumor cell self-renewal, promotes senescence, and inhibits ATRT tumor growth in vivo. Transgenic expression of Omomyc (a bona-fide MYC dominant negative) or chemical inhibition of MYC transcriptomic programs with the BET inhibitor JQ1 phenocopy genetic depletion of MYC, effectively restricting ATRT tumor growth and opening a promising therapeutic avenue for rhabdoid tumors in children.

Atypical Teratoid Rhabdoid Tumor is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, the children usually have poor survival. A functional genomic screen identified the TP53-MDM2 axis as a therapeutic vulnerability in ATRT. Gene expression demonstrates that all ATRT sub-groups have high level of MDM2 a negative regulator of p53. We demonstrate that MDM2 inhibition by shRNA or with small-molecule drugs, Nutlin3 and Idasanutlin resulted in decreased ATRT cell growth, inhibition of clonogenic potential and induction of apoptosis in vitro and in vivo. MRI imaging of intracranial tumors shows that Apparent Diffusion Coefficient (ADC), a good marker for successful treatment, significantly increased with Idasanutlin treatment showing tumor necrosis. Moreover, Idasanutlin significantly decreased growth of intracranial orthotopic ATRT brain tumors as evaluated by T2 MRI imaging and prolonged survival compared to control animals. Further idasanutlin potentiated radiation induced DNA damage and increased sensitivity to radiaton of ATRT cells. These findings highlight the TP53-MDM2 axis as a rational therapeutic target in ATRT.

Loss of SMARCB1 is the hallmark genetic event that characterizes ATRT. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. To identify co-operating epigenetic factors, we performed an unbiased shRNA screen targeting 408 epigenetic/chromatin molecules in patient-derived ATRT cell lines and identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as essential for ATRT cell viability. Genetic and Chemical inhibition of BMI1 inhibited clonogenic potential and induced apoptosis in vitro. In vivo PTC 596 significantly decreased growth of intracranial orthotopic ATRT tumors as evaluated by T2 MRI imaging and significantly prolonged survival compared to control animals. Using RNA-seq and ChIP-Seq our studies show that BMI1 co-operates with SMARCB1 loss to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. We then used a doxycycline-inducible SMARCB1 expression system and performed Immunoprecipitation for BMI1, followed by and mass spectrometry analysis. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. Re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. CBX4 is implicated DNA damage response, tumor angiogenesis and self-renewal. CBX8 activates lineage-specific genes during differentiation of ESC. Our data suggest that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex. These studies identify the mechanistic basis of BMI1 co-operation with SMARCB1 loss in ATRT and establish BMI1 inhibition as a novel therapeutic approach in ATRT.

Diffuse Intrinsic pontine glioma is an incurable brain tumor of childhood characterized by histone mutations at lysine 27 which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. We performed a combined RNAi and chemical screen targeting epigenetic regulatory genes and identified the polycomb (PcG) repressive complex 1, component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy was enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 results in decreased cell self-renewal and attenuation of tumor growth due to induction of senescence. Prolong BMI1 inhibition induces a senescence associated secretory phenotype which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to synergistically attenuate tumor growth in vivo.

Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse intrinsic pontine gliomas (DIPGs), aggressive pediatric brain tumors for which no curative therapy currently exists. In order to identify specific epigenetic dependencies which arise as a consequence of the H3K27M mutation, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DIPG cell lines. This identified AFF4, a component of the super elongation complex (SEC), as both highly expressed and necessary for DIPG cells to maintain growth and self-renewal. We hypothesized that SEC overexpression occurs as a direct consequence of the H3K27M mutation and that this relative abundance overcomes repressive transcriptional regulation in order to suppresses differentiation and promote self-renewal of DIPG tumor stem cells. We found that AFF4 expression is consistently elevated in both DIPG patient samples and established cell lines relative to normal pediatric pons. We interrogated the role of AFF4 in H3K27M-mutant DIPG using an shRNA lentiviral approach. Using live cell imaging, we demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate differentiation programs under AFF4 regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DIPG. Using ChIP-seq following CDK9 pharmacologic inhibition, we demonstrate that the loss of regulatory transcriptional input in DIPG may be reversed via CDK9 inhibition and that this acts as a potent inhibitor of DIPG cell growth both in vitro and in vivo orthotopic xenograft models. These studies represent the first pre-clinical validation of SEC inhibition as a novel therapeutic approach in DIPG.

Simple Summary Group 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a poor prognosis. FBXW7, a critical tumor suppressor in many types of cancer, regulates the proteasome-mediated degradation of oncoproteins including MYC. However, the role of FBXW7 in the tumorigenesis of group 3 MB has not been well studied. In this study, we show that FBXW7 is downregulated in group 3 MB patient samples, and FBXW7 stabilization is crucial for inhibiting c-MYC. We identified a FBXW7-MYC-PLK1 regulatory loop in MYC-driven MB, which provides a mechanism of using protein kinase inhibitors for translation in the future. Abstract Polo-like kinase 1 (PLK1) is highly expressed in group 3 medulloblastoma (MB), and it has been preclinically validated as a cancer therapeutic target in medulloblastoma. Here, we demonstrate that PLK1 inhibition with PCM-075 or BI6727 significantly reduces the growth of MB cells and causes a decrease of c-MYC mRNA and protein levels. We show that MYC activates PLK1 transcription, while the inhibition of PLK1 suppresses MB tumor development and causes a decrease in c-MYC protein level by suppressing FBXW7 auto poly-ubiquitination. FBXW7 physically interacts with PLK1 and c-MYC, facilitating their protein degradation by promoting ubiquitination. These results demonstrate a PLK1-FBXW7-MYC regulatory loop in MYC-driven medulloblastoma. Moreover, FBXW7 is significantly downregulated in group 3 patient samples. The overexpression of FBXW7 induced apoptosis and suppressed proliferation in vitro and in vivo, while constitutive phosphorylation mutation attenuated its tumor suppressor function. Altogether, these findings demonstrated that PLK1 inhibition stabilizes FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing medulloblastoma progression.

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc overexpression results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. The cyclin-dependent kinase, CDK7, was identified as a top candidate. CDK7 phosphorylates the c-terminal domain of RNA Pol II facilitating transcriptional initiation and elongation. We subjected Myc-amplified cells treated with CDK7 inhibitors to whole transcriptomic analysis. The resultant data revealed gene networks mediating DNA repair were functionally repressed. Consistent with this data, ChIP-sequencing showed the most significant reduction in RNA Pol II and Myc promoter occupancy within a subset of DNA repair genes including BRCA2 and RAD51 but not across the whole genome. These data suggest that inhibition of CDK7 mechanistically limits Myc driven transcriptional processivity of DNA repair networks. Further, evaluation of genes mediating DNA repair show a muted response to DNA damage and increased cell death with CDK7 inhibition. We next evaluated Myc-amplified MB cell response to ionizing radiation in vitro and in vivo with CDK7 inhibition. Inhibition of CDK7 enhanced radiation sensitivity of Myc MB cells by potentiating DNA damage. Further, cotreatment produced decreased MRI T2 tumor volumes and enhanced survival benefit in orthotopic PDX xenografted mice compared to radiation alone. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) driven by mutations in the histone 3 (H3) gene (H3K27M) are aggressive pediatric brain tumors for which there is no curative therapy. METHODS To identify novel therapeutic targets we performed a high throughput drug screen combined with an epigenetically targeted RNAi screen using H3K27M and H3.3 WT DIPG cells. RESULTS Chemical and genetic depletion of BMI1 in vitro resulted in inhibition of clonogenicity and cell self-renewal consistent with previous studies. We show for the first time that clinically relevant BMI1 inhibitors attenuates growth of orthotopic DIPG xenografts as measured by MRI and prolong survival in vivo. We found that BMI1 inhibition drives phenotypic cellular senescence and that the senescent cells were able reactivate to form new neurospheres in vitro and tumor growth in vivo. RNA-seq, ChIP-Seq and immuno-proteomic analysis revealed that the senescent cells induced the expression of the Senescence Associated Secretory Phenotype (SASP) cytokines by increasing occupancy of activated histone marks at SASP factor promoters. The SASP results in increased expression of anti-apoptotic BH3 proteins including BCLxl, and BCL2. Treatment of the PTC028 treated senescent DIPG cells with BH3 mimetics induces apoptosis and clears the senescent cells. Combining BH3 mimetics with BMI1 inhibition attenuates tumor growth in vivo synergistically and significantly prolongs survival of DIPG bearing mice compared to BMI1 inhibition alone. CONCLUSION These data inform the current trial of BMI1 inhibition as a monotherapy and predict the need for adding BH3 mimetics to achieve efficacy.

Mutations in the histone 3 gene (H3K27M) are the eponymous driver in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain tumors for which no curative therapy currently exists. To identify specific epigenetic dependencies within the context of the H3K27M mutation, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This identified AFF4, a component of the super elongation complex (SEC), as necessary for DMG cells to maintain growth and self-renewal. We hypothesized that aberrant SEC expression occurs as a consequence of the H3K27M mutation and that this dysregulated SEC signaling overcomes repressive transcriptional regulation in order to suppresses differentiation and promote self-renewal of DMG tumor stem cells. We interrogated the role of AFF4 in DMG using an shRNA lentiviral approach. We demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate differentiation programs under SEC regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DMG. Using RNA polymerase II ChIP-seq, we demonstrate that CDK9 pharmacologic inhibition restores regulatory Pol II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for the translational exploration of CDK9 inhibition as a promising therapeutic approach.

Myc is highly expressed in group 3 medulloblastoma (Myc-MB) and influences cell growth, proliferation and oncogenesis by directly promoting the activity of RNA polymerases (RNA Pol). Myc driven RNA Pol II activity is mediated by Positive Transcription Elongation Factor b (pTEFb). pTEFb’s catalytic core consists of cyclin dependent kinase-9 (CDK9) and Cyclin T, that phosphorylate and release RNA Pol II into active elongation. CDK9 is over expressed in group 3 MB suggesting that MB may be vulnerable to inhibition of CDK9 (CDK9i). The exact mechanism is not completely known in MB. Genetic depletion of CDK9 suppressed Myc-MB cell clonogenicity in vitro and tumor growth in vivo. CDK9i by two clinically relevant inhibitors, Atuveciclib and AZD4573, suppressed clonogenicity and cell self-renewal of Myc-MB cell lines. CDK9i in Myc-MB cell lines downregulated Myc and RNA Pol II phosphorylation at Ser2 and Ser5, and, upregulated P21. Further, mice with orthotopic xenografts treated with CDK9 inhibitors survived significantly longer than control mice. RNA-Seq-based gene set enrichment analysis showed that CDK9i decreased c-Myc driven transcriptomic programs and enhanced differentiation networks. ChIP-Seq for Pol2 and Myc, demonstrated that the Myc-driven aberrant transcriptional input can be reversed via CDK9i. These findings highlight the role of CDK9 in Myc-driven pathogenesis and that its inhibition is critical to the treatment of Myc-MB.

Group 3 medulloblastoma tumors (Myc-MB), and particularly the 3γ subtype, have the worst prognosis and show a 5-year overall survival of less than 40%. Group 3 tumors are often accompanied by Myc amplification and have a higher rate of metastatic disease and relapse. Unfortunately, therapeutic strategies to target Mychave remained elusive. Further, the relapse of the MB has been linked to DNA replication stress. Ataxia telangiectasia and Rad3-related protein (ATR) senses persistent DNA damage, which arises due to replication stress, and activates damage checkpoints, thereby leading to increased cell survival. ATR is highly expressed in MB and is thought to contribute to undisturbed DNA replication to protect genomic integrity. Yet, the exact underlying mechanisms involving ATR are still unclear in MB. Inhibition of ATR (ATRi) using the ATR inhibitor, AZD6738, suppressed clonogenicity and cell self-renewal in Myc-MB. ATRi in Myc-MB cell lines downregulated Chk1 and upregulated P21. ATRi also induced cell cycle arrest and increased apoptosis in Myc-MB cell lines. Further, mice with orthotopic xenografts treated with ATR inhibitor survived significantly longer than control mice. High-throughput drug screening showed ATRi to be synergistic with chemotherapeutic agents including gemcitabine, cisplatin and topotecan. The treatment of Myc-MB cells with ATR inhibitor in combination with gemcitabine and with radiation increased in expression of DNA damage markers. These findings emphasize the role of ATR in alleviating DNA replication stress and that its inhibition is critical to the treatment of Myc-MB.

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children, accounting for 20% of all childhood brain tumors. The molecular profiling of MB into 4 major subgroups (WNT, SHH, Grp3, and Grp4) emphasizes the heterogeneity of MB and opens paths in which treatments may be targeted to molecularly aggressive and distinct tumors. Current therapeutic strategies for Group 3 MB are challenging and can be accompanied by long-term side effects from treatment. The involvement of altered epigenetic machinery in neoplastic transformation in MB has become more evident. Thus, we performed an epigenomic RNAi and chemical screen and identified SETD8/PRE-SET7/KMT5a as a critical player in maintaining proliferation and cell survival of MB cells. We have found that inhibition of SETD8 effects the migration/invasive ability of MB cells. SETD8 alters H4K20me chromatin occupancy at key genes involved in tumor invasiveness and pluripotency. Interestingly, these results link the aggressive and metastatic behavior of MYC-driven MB with SETD8 activity. Based on our results, we suggest that SETD8 has a critical role mediating Group 3 MB tumorigenesis. Establishing a role for SETD8 as a factor in MYC-driven MB has potential to lead to more effective therapies needed to improve outcomes in high-risk patients.

Mutations in oncogenes and tumor suppressor genes engender unique metabolic phenotypes crucial to the survival of tumor cells. EGFR signaling has been linked to the rewiring of tumor metabolism in non–small cell lung cancer (NSCLC). We have integrated the use of a functional genomics screen and metabolomics to identify metabolic vulnerabilities induced by EGFR inhibition. These studies reveal that following EGFR inhibition, EGFR-driven NSCLC cells become dependent on the urea cycle and, in particular, the urea cycle enzyme CPS1. Combining knockdown of CPS1 with EGFR inhibition further reduces cell proliferation and impedes cell-cycle progression. Profiling of the metabolome demonstrates that suppression of CPS1 potentiates the effects of EGFR inhibition on central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism, coinciding with reduced glycolysis and mitochondrial respiration. We show that EGFR inhibition and CPS1 knockdown lead to a decrease in arginine levels and pyrimidine derivatives, and the addition of exogenous pyrimidines partially rescues the impairment in cell growth. Finally, we show that high expression of CPS1 in lung adenocarcinomas correlated with worse patient prognosis in publicly available databases. These data collectively reveal that NSCLC cells have a greater dependency on the urea cycle to sustain central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism to meet cellular energetics upon inhibition of EGFR. Implications: Our results reveal that the urea cycle may be a novel metabolic vulnerability in the context of EGFR inhibition, providing an opportunity to develop rational combination therapies with EGFR inhibitors for the treatment of EGFR-driven NSCLC.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Despite treatment with surgery, radiation, and chemotherapy, outcomes are unsatisfactory. MB is a heterogenous disease with four major and distinct molecular subgroups (WNT, SHH, Group 3 and Group 4) characterized by unique genomic and clinical features. Group 3 tumors have the worst outcomes and are frequently characterized by MYC amplification. Among the features of many of the group 3 tumors are the overexpression of EZH2, gains of the chromosome 7 and increased global H3K27me3 chromatin-repressive marks. EZH2, the histone methyltransferase responsible for H3K27me3 and located on 7q36.1 is critical during embryonic development to establish cell identities. In many cancers, both oncogenic and tumor suppressor functions have been observed for EZH2. More studies are required to better understand the role of EZH2 overexpression in medulloblastomas. Using RNAi, chromatin immunoprecipitation and pharmacological approaches, we were able to show that EZH2 inhibition impairs the pluripotency and self-renewal properties of human medulloblastoma cells and reactivates a differentiation program associated with differentiated cerebellar cells. Combinatorial drug screenings have been developed to identify drugs synergizing with EZH2 inhibition in human MB cell lines. Using human cerebellar stem cells and medulloblastoma cell lines, we also evaluated the effects of both EZH2 overexpression and pharmacological inhibition in the context of MYC overexpression in immunocompromised mice. The results of our in vitro and in vivo experiments will be presented.

The original version of this Article omitted the following from the Acknowledgements: This work was supported by the Luke's Army Pediatric Cancer Research Fund St. Baldrick's Scholar Award. This has now been corrected in both the PDF and HTML versions of the Article.

SummaryThe Twist1 transcription factor is known to promote tumor metastasis and induce Epithelial-Mesenchymal Transition (EMT). Here, we report that Twist1 is capable of promoting the formation of invadopodia, specialized membrane protrusions for extracellular matrix degradation. Twist1 induces PDGFRα expression, which in turn activates Src, to promote invadopodia formation. We show that Twist1 and PDGFRα are central mediators of invadopodia formation in response to various EMT-inducing signals. Induction of PDGFRα and invadopodia is essential for Twist1 to promote tumor metastasis. Consistent with PDGFRα being a direct transcriptional target of Twist1, coexpression of Twist1 and PDGFRα predicts poor survival in breast tumor patients. Therefore, invadopodia-mediated matrix degradation is a key function of Twist1 in promoting tumor metastasis.

SummaryAnimal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.

The importance of stem cell and self-renewal programs in Acute Myeloid Leukemia (AML) is generally accepted, but the molecular details are incompletely understood. The master transcriptional regulator GATA2 is highly expressed in hematopoietic stem cells (HSCs) and has critically important roles in the hematopoietic system. Gata2 is required for murine HSC development and maintenance, and heterozygous loss of Gata2 compromises murine HSC- and progenitor cell-function. High levels of GATA2-expression have been correlated with adverse prognosis in human AML. GATA2 is also overexpressed in human chronic myeloid leukemia. These data suggest an important role for GATA2 in normal stem cells and in leukemia. However, genetic lesions resulting in compromised GATA2 function can lead to MDS and in some cases AML. In a murine AML model driven by Flt3-ITD and inactivation of Tet2, Gata2 is strongly downregulated. Furthermore, mouse models of leukemia suggest that high-level forced expression of Gata2 can have a tumor suppressor role. To clarify the role of Gata2in AML we used homozygous genetic inactivation in established murine models of leukemia, using a a conditional allele. We initially tested the role of Gata2 in a murine leukemia mediated by forced expression of Meningioma1 (MN1). This model has a HoxA9/Meis1 transcriptional program. We recently found that MN1-driven leukemia depends on the histone methyltransferase Dot1l (J Clin Invest. 2016 Feb 29. pii: 80825). Lineage marker negative (Lin-), Sca1+, Kit-positive (LSK) bone marrow cells from mice with a floxed exon 5 in the Gata2 gene, and a ROSA26-YFP Cre-reporter allele were transduced with an MSCV-based ecotropic retroviral vector expressing MN1 and linked via an internal ribosomal entry site (IRES) the selectable marker GFP. Floxed Gata2-sequences were excised using transduction with a self-excising Cre-expressing vector (HR-Cre). Cells were sorted and plated in methylcellulose. The GFP/YFP double positive Gata2ko cells showed a replating defect compared to GFP single positive Gata2-floxed cells, both with regard to colony number and colony size. Next, we tested the role of Gata2 in disease maintenance in vivo. We established MN1 Gata2ff leukemias in primary recipients. Primary leukemias were transduced with Cre-expressing vector and Gata2ko and Gata2ff MN1 cells were transplanted. While all mice in the Gata2ffgroup developed leukemia with a median survival of 35 days, the mice in the Gata2ko cohort developed leukemia with incomplete penetrance with a latency of 249 days (p=0.0005). These data suggest an important role for Gata2in MN1 leukemia in vitro and in vivo. Genetic inactivation of Gata2 resulted in increased protein levels of p53 in vitro as detected by Western blot. Furthermore, MN1-transduced cells showed accentuated p53 stabilization and apoptosis in response to the Mdm2-antagonist and p53 stabilizer Nutlin3. We next tested the role of Gata2 in leukemia driven by the oncogenic fusion MLL-AF9. In contrast to the MN1 model, recipients of Gata2koleukemias only showed a trend towards prolonged latency in secondary recipients (median survival Gata2ff=48 days vs. Gata2ko=62 days, p=0.09). In this model, we also did not observe a substantial effect of Gata2-inactivation on p53 activation. We are currently characterizing the underlying molecular mechanisms. Our data document an important role for Gata2 in AML mediated by MN1 and to a lesser degree, MLL-AF9. The role of Gata2 in leukemia is complex and depends on expression levels and cellular context. A more detailed understanding of leukemic self-renewal, including the role of Gata2, will inform the development of more efficacious and less toxic therapies for this difficult-to-treat malignancy. Disclosures Bernt: Epizyme: Patents & Royalties: patent filed. Neff:Epizyme: Patents & Royalties: patent filed; Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel.

Previous observations led to the conclusion that in Xenopus eggs and during early development, DNA replication initiates at regular intervals but with no apparent sequence specificity. Conversely, here, we present evidence for site-specific DNA replication origins in Xenopus egg extracts. Using lambda DNA, we show that DNA replication origins are activated in clusters in regions that contain closely spaced adenine or thymine asymmetric tracks used as preferential initiation sites. In agreement with these data, AT-rich asymmetric sequences added as competitors preferentially recruit origin recognition complexes and inhibit sperm chromatin replication by increasing interorigin spacing. We also show that the assembly of a transcription complex favors origin activity at the corresponding site without necessarily eliminating the other origins. Thus, although Xenopus eggs have the ability to replicate any kind of DNA, AT-rich domains or transcription factors favor the selection of DNA replication origins without increasing the overall efficiency of DNA synthesis. These results suggest that asymmetric AT-rich regions might be default elements that favor the selection of a DNA replication origin in a transcriptionally silent complex, whereas other epigenetic elements linked to the organization of domains for transcription may have further evolved over this basal layer of regulation.

Polycomb repressive complex 2 (PRC2) is a chromatin regulator with central roles in development and cancer. The canonical function of PRC2 is the trimethylation of histone 3 on lysine residue 27. This epigenetic modification is associated with gene silencing. Both tumor suppressor and oncogenic functions have been reported for PRC2, depending on cellular context. In leukemia mediated by the leukemogenic fusion MLL-AF9, complete ablation of canonical PRC2 function by genetic inactivation of the core component embryonic ectoderm development (Eed) or by combined pharmacologic inhibition of the PRC2 methyltransferases EZH2 and EZH1 has a strong anti-leukemic effect, and this effect has been linked to de-repression of the PRC2 target locus Cdkn2a. We asked whether inactivation of Cdkn2a is sufficient to restore leukemic activity of Eed-inactivated MLL-AF9 leukemia cells, using combined genetic inactivation of Cdkn2a and Eed. We found that Cdkn2a inactivation partially rescues in vitro and in vivo growth of Eed-inactivated MLL-AF9 cells. However, the growth of Eed-null Cdkn2a-null MLL-AF9 cells in the absence of Cdkn2a remained severely compromised in vitro and in vivo, compared with that of their Eed-floxed Cdkn2a-null counterparts. RNA sequencing analysis revealed that several genes previously implicated in inefficient growth of MLL-AF9-transformed cells, including Gata2, Egr1, and Cdkn2b were de-repressed as a consequence of Eed inactivation. Furthermore, we found that direct binding targets of MLL fusion proteins are negatively enriched in Eed-inactivated Cdkn2a-null MLL-AF9-transformed cells. Our data indicate that interference with PRC2 function affects MLL-AF9-mediated leukemogenesis by both Cdkn2a-dependent and Cdkn2a-independent mechanisms.

Summary MCM2-7 proteins are replication factors required to initiate DNA synthesis and are currently the best candidates for replicative helicases. We show that the MCM2-7-related protein MCM8 is required to efficiently replicate chromosomal DNA in Xenopus egg extracts. MCM8 does not associate with the soluble MCM2-7 complex and binds chromatin upon initiation of DNA synthesis. MCM8 depletion does not affect replication licensing or MCM3 loading but slows down DNA synthesis and reduces chromatin recruitment of RPA34 and DNA polymerase-α. Recombinant MCM8 displays both DNA helicase and ATPase activities in vitro. Reconstitution experiments show that ATP binding in MCM8 is required to rescue DNA synthesis in MCM8-depleted extracts. MCM8 colocalizes with replication foci and RPA34 on chromatin. We suggest that MCM8 functions in the elongation step of DNA replication as a helicase that facilitates the recruitment of RPA34 and stimulates the processivity of DNA polymerases at replication foci.

Polycomb Repressive Complex 2 (PRC2) is a multi-protein complex with important roles in development and cancer. Both hyper- and hypoactivity of PRC2 are associated with blood-related malignancies. Activating mutations of the PRC2 methyltransferase EZH2 have been found in human B-lineage lymphomas. Inactivating mutations of PRC2 components EZH2, EED and SUZ12 have been described in early T-cell Precursor ALL (ETP-ALL) and inactivating PRC2-alterations are found in Myelodysplastic and Myeloproliferative Syndromes. The mechanisms underlying this paradox are incompletely understood. We here investigate the context dependent role of PRC2 in murine models. We initially studied PRC2 in normal hematopoiesis: Chip-seq analysis of the PRC2-mediated H3K27me3 chromatin mark demonstrates that many genes highly expressed in immature hematopoiesis gain H3K27me3 in the developmental transition from more immature Lin-Sca1+Kit+ (LSK) cells to lineage committed Granulocyte Macrophage Progenitors (GMPs). Transcription of these genes is enriched in EZH2ko GMP compared to EZH2ffGMP by Gene Set Enrichment Analysis (GSEA). These data suggest that PRC2 is important for the silencing of immature gene expression programs in the developmental transition from LSK to GMP. We next analyzed the role of PRC2 in two murine models of acute leukemia: MLL-AF9 driven leukemia, and a model of early T-cell precursor T-ALL (ETP-ALL). In MLL-AF9 leukemia, we previously found that inactivation of Eed completely abrogate leukemogenesis in vitro and in vivo. We now report that genetic inactivation of the tumor suppressor Cdkn2a (a canonical PRC2 target) partially rescued MLL-AF9 mediated leukemia in vitro and in vivo. However, Cdkn2akoEEDko MLL-AF9 leukemia remained compromised. In vitro growth was reduced to approximately 10% of Eedff controls. While control MLL-AF9 leukemia developed in vivo in 100% of the recipients, Cdkn2akoEEDkoMLL-AF9 leukemia developed with significantly prolonged latency and incomplete penetrance (25%). RNAseq analysis revealed that high level expression of genes with established roles in MLL-AF9 leukemia such as HoxA9, Cdk6 and Jmjd1c unexpectedly depends on Eed. These data are in keeping with the absence of alterations in PRC2-components in human MLL-rearranged leukemia. In contrast, PRC2 core components (EZH2/EED/SUZ12) are deleted or mutated in > 40% of ETP-ALL. ETP-ALL also often has direct or indirect activation of the RAS-pathway, and carries frequent deletions of the CDKN2A locus. To model the effects of EED and EZH2-inactivation in ETP-ALL, we established Cdkn2akoEedff vs Cdkn2akoEedko, and Cdkn2akoEzh2ff vs Cdkn2akoEZH2koleukemias by transduction with NRASQ61K followed by expansion on OP9DL1 stroma cells to activate T-lineage differentiation via Notch-signaling. Cdkn2ako NRASQ61K leukemia showed an immunophenotype similar to human ETP-ALL (positive for c-Kit, CD5 and myeloid markers and mostly negative for CD4/8). Inactivation of Eed or Ezh2 in this model led to a shortening of latency (p=0.03 for Eed, p=0.0001 for Ezh2). RNAseq revealed enrichment of genes associated with murine DN1 thymocytes and with human ETP-ALL in Eedko vs Eedff Cdkn2ako NRASQ61K leukemia. These genesets showed even more pronounced enrichment in Ezh2kocompared to Ezh2ff Cdkn2ako NRASQ61K leukemia. Genes highly expressed in early hematopoiesis were enriched in Eedko and Ezh2ko cells in both, the MLL-AF9 and NRASQ61K leukemia models. However, there was an opposing effect on HoxA9 gene expression, with PRC2 inactivation leading to decreased HoxA9 expression in MLL-AF9, and increased HoxA9 expression in Cdkn2ako NRASQ61K leukemia. Decreased HoxA9 has been shown to impair MLL-AF9 leukemia growth. To test the functional significance of elevated HoxA9-levels in the Eedko and EZH2koNRASQ61K leukemias, we co-expressed HoxA9 and NRASQ61K in the presence of intact Eed and Ezh2 loci. Preliminary data suggest that HoxA9 accelerates leukemia development in this setting. Alterations in chromatin modifiers, including PRC2, are frequent in leukemia and lymphoma. Our data demonstrate that manipulation of PRC2 can have opposite effects on leukemia phenotype and expression of key PRC2-repressed genes such as HoxA9 in the context of different tumors. We are currently characterizing the mechanisms leading to divergent outcomes of PRC2 manipulation in MLL-AF9 leukemia compared to NRASQ61K ETP-like leukemia. Disclosures Armstrong: Epizyme : Consultancy.

Tumor cells acquire the ability to degrade basement membrane and extracellular matrix (ECM), thus allowing them to invade and metastasize to distant organs. The molecular triggers for ECM degradation and invasion are largely unknown. The Twist1 transcription factor plays key roles in tumor metastasis and is known to induce Epithelial-Mesenchymal Transition (EMT). In addition to Twist1, a number of studies show that other EMT-inducing transcription factors, including Snail1/Snail2 and ZEB1/ZEB2, are also important players in tumor metastasis. One question unanswered is what are the specific cellular functions and transcriptional targets of individual EMT-inducing transcription factors required for EMT and tumor metastasis. Here we report a novel function of Twist1 in promoting extracellular matrix degradation by inducing the formation of invadopodia. Invadopodia are specialized actin-based membrane protrusions that degrade ECM via clustering of proteases. Invadopodia contain a F-actin core and actin regulatory proteins, such as cortactin, the unique adaptor proteins Tks4 and Tks5 in clustering components of invadopodia, and a large number of proteases for matrix degradation. Twist1 induces PDGFRα expression, which in turn activates Src kinase in various breast tumor cells. Phosphorylation of multiple invadopodia components by Src kinase promotes invadopodia assembly and matrix degradation. We show that Twist1 and PDGFRα are central mediators of invadopodia formation and ECM degradation in response to various EMT-inducing signals, including TGFβ and Snail1. Induction of PDGFRα and invadopodia is essential for Twist1 to promote invadopodia formation, local tumor invasion, and distant metastasis in vivo. Consistent with PDGFRα being a direct transcriptional target of Twist1, expression of Twist1 and PDGFRα is highly correlated and associated with poor survival in breast cancer patients. Together, our study demonstrates that invadopodia-mediated matrix degradation is a key function of Twist1 in promoting tumor metastasis. Furthermore, it reveals that matrix degradation and loss of cell adhesion are regulated by different EMT-inducing transcription factors. This explains why multiple factors need to be activated coordinately to promote carcinoma cells to undergo EMT and invade. We also identify PDGFRα as a direct transcriptional target of Twist1 in promoting invadopodia formation and tumor metastasis, therefore suggesting that PDGFRs might be potential targets for anti-metastasis therapies. Grant acknowledgements: NIH Director's New Innovator Award (DP2 OD002420-01), Kimmel Scholar Award, and California Breast Cancer Research Program(12IB-0065) to J.Y. a DOD Breast Cancer Pre-doctoral fellowship to M.A.E, a DOD Breast Cancer Era of Hope Postdoctoral Fellowship to E.D., Susan G. Komen Foundation grant FAS0703850 to J.K. and L.O. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2011-4747