Transcriptional Regulatory Complex Research Articles

Transcriptional Plasticity Compensates for Ikaros and Aiolos Proteasomal Degradation and Mediates Resistance to IMiDs in Multiple Myeloma (MM)

Background: Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, TXNDC5, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, MAFB, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in MM. However, the identity of the transcription factors (TFs) or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. As such the discovery of the BET family member BRD4 as the master histone acetyl mark reader at enhancers loci regulating MYC lead to promising therapeutic developments in MM and numerous other cancers. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKAROS (IKZF1) and AIOLOs (IKZF3) leading to the transcriptional repression of MYC and the suppression of MM cells survival and proliferation. However, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes.Methods and Results: In order to define how IMiDs repress MYC transcription, we first defined IKZF1, BRD4, the lysine acetyl transferase P300 and the mediator complex subunit MED1 mapping within the MM genome using ChIPseq. In MM cell lines (MM1S, RPMI8226, ARP1 and AMO1), IKZF1 predominantly mapped to intronic and intergenic loci which are typically enriched with enhancer and superenhancer elements. Indeed, IKZF1 mapping to the genome nearly completely (96.5%) overlapped that of P300, MED1 and BRD4 co-occupied enhancer and superenhancer loci. We also confirmed that in the MM1S sensitive cell lines IMiDs (lenalidomide 10 μM, 24h) exposure efficiently depleted IKZF1, BRD4, P300 and MED1 at enhancer loci with ensuing MYC (and MAF) downregulation. In contrast, in resistant cell lines (RPMI8226) and in spite efficient IKZF1 displacement, BRD4, P300 and MED1 were retained at the oncogenic enhancer (IGLL5) driving MYC (and MAF). These findings lead us to postulate that in IMiDs resistant cells retention of BRD4 and MED1 at oncogenic enhancers in the absence of IKZF1 likely results from rewiring of the TFs regulating MYC. To identify TFs that may co-localize with BRD4 and IKZF1, we analyzed the enrichment of DNA motifs at IKZF1and BRD4 co-occupied enhancers using the MEME suite motif-finding algorithms. This computational analysis revealed a strong enrichment at these MM enhancers of the GGAA motif recognized by the ETS family of TFs (P= 3.2 e-743) and other motifs boxes for the RUNX (P= 9.6 e-725), MYC/MYB (P= 8.8 e-52) and interferon regulatory (IRF) (P= 3.1 e-293) TFs. We next confirmed that the ETS family TF ETV4 was indeed expressed in IMiDs resistant, but not sensitive, MM cell lines. ChiPseq occupancy profiles in IMiDs resistant RPMI8226 cell line revealed co-localization of ETV4 with IKZF1, P300 and BRD4. As predicted, lenalidomide treatment induced global depletion of IKZF1 but not ETV4 at BRD4 occupied enhancers in resistant cell lines (RPMI8226 and ARP1). Importantly, Cas9-mediated knock out of ETV4 in RPMI8226 cells sensitized them to lenalidomide with ensuing MYC downregulation and cell death.Confirming its role in MM, ETV4 transcript was indeed detectable in primary patients' samples in the CoMMpass data repository (ETV4 FPKM >1.0 in 112/724) and its expression was associated with significantly reduced survival outcomes (HR 0.64; P=0.0008). Similarly, high expression (top quartiles) of RUNX2 or MYB, TFs with enriched motifs at IKZF1 co-occupied enhancer loci, was also associated with decreased survival. Of note RNAseq analysis of paired patient samples pre- and post-IMiDs treatment (n=14 pairs) revealed significant upregulation of ETV4 at the time of acquired IMiDs resistance (7/14). Lastly transcriptome analysis of 101 patients enrolled in the RD arm (lenalidomide and dexamethasone) of the POLLUX trial (NCT02076009) confirmed the reduced survival of patients with top quartiles expression of ETV4 as well as MYB and RUNX2 (Fig.1)Conclusion: Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. [Display omitted] DisclosuresNeri:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Soong:Jannsen: Employment. Chiu:Janssen: Employment. Bahlis:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana, disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 (ref4-3) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex.

Enhancer remodeling regulates epigenetic adaptation and resistance to MEK1/2 inhibition in triple-negative breast cancer

ABSTRACTKinase inhibitors targeting the mitogen/extracellular signal-regulated kinase kinase (MEK)- extracellular signal related kinase (ERK) signaling pathway have limited durability in inhibiting growth of triple-negative breast cancer. We defined genome wide enhancer remodeling following MEK inhibition capable of driving adaptive gene transcription. Targeting positive elongation factor (P-TEFb) transcriptional regulatory complex members can block enhancer remodeling making the response to MEK-ERK inhibition durable.

Global profiling of lysine reactivity and ligandability in the human proteome.

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

Abstract 5083: A novel and highly efficacious small-molecule degrader of BET-BRD proteins for the treatment of acute leukemia

Abstract The bromodomain and extra-terminal domain (BET-BRD) family of lysine acetylation readers comprises BRD2, BRD3, BRD4, and BRDT that contain two conserved N-terminal bromodomains (BD1 and BD2) and a long C-terminal region containing the extraterminal (ET) protein-protein interaction domain. These proteins have key roles in the assembly of transcriptional regulatory complexes containing RNA polymerase II. BET-BRDs are new therapeutic targets for cancer treatment and several small-molecule BET-BRD inhibitors are currently in clinical development for a diverse set of cancers including leukemia. Based on the proteolysis targeting chimera (PROTAC) concept, we have developed novel and highly potent BET degraders, ZBC246 and ZBC260, with exceptional selectivity based upon a new class of BET inhibitor, ZBC11. ZBC260 effectively degrades all BET-BRD proteins at concentrations as low as 30 pM within a few hours of treatment in the RS4;11 leukemia cell line and achieves IC50 values of 50 pM in inhibition of RS4;11 cell growth. ZBC260 was tested in a panel of 10 acute leukemia cell lines generating IC50s ranging from 20 pM to 1 nM in 4-days cell growth inhibition assays. In contrast to the cytostatic effect observed with BET-BRD inhibition, degradation of BET-BRDs by ZBC246 and ZBC260 induced robust apoptosis demonstrating a differential biology between BET-BRD inhibitors and BET-BRD degraders. Significantly, ZBC260 induces rapid regression of RS4;11 xenograft tumors without overt signs of toxicity in mice. More importantly, treatment of leukemia cells obtained from 10 patients with ZBC260 demonstrated efficient degradation of BET-BRDs within 5 h at 0.3 nM, while treatment with the inhibitor (300 nM) induced up-regulation of BET-BRDs. Treatment of patients’ peripheral blasts with ZBC260 at 1 to 10 nM induced remarkable levels of apoptosis within 24 h, even in relapsed and refractory patient samples. In conclusion, ZBC260 represents a highly potent and efficacious BET-BRD degrader undergoing extensive preclinical evaluation for the treatment of acute leukemias. Citation Format: Ester Fernandez-Salas, Zhuo Chen, Mei Lin, Bing Zhou, Donna McEachern, Sally Przybranowski, Karson Kump, Luke F. Peterson, Malathi Kandarpa, Jiantao Hu, Fuming Xu, Liu Liu, Longchuan Bai, Bo Wen, Duxin Sun, Moshe Talpaz, Shaomeng Wang. A novel and highly efficacious small-molecule degrader of BET-BRD proteins for the treatment of acute leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5083. doi:10.1158/1538-7445.AM2017-5083

Abstract 5080: BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models

Abstract Prostate cancer is a leading cause of cancer death worldwide. Deregulated transcriptional factors and epigenetic effectors contribute to prostate cancer progression and castration resistance providing ideal targets for developing new therapeutic strategies. Bromodomain and extra-terminal (BET) proteins act transcriptional co-activators interacting with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors (BETi) disrupt transcriptional regulatory complexes and have broad anticancer activity. INCB054329 and INCB057643 are BETi with proven pre-clinical activity in hematological and solid tumors. Both compounds are currently in clinical trials (NCT02431260 and NCT02711137) in advanced cancer patients. In this study we explored for the first time the activity of these two BETi in prostate cancer, comparing their effects in androgen-dependent and independent models in vitro and in vivo. We assessed the effect of INCB054329 and INCB057643 on cell proliferation, colony formation and tumor-sphere assays in androgen-dependent (LNCaP and VCaP) and androgen-independent (DU145, PC3, 22Rv1) cells. The in vivo antitumor activity was evaluated in mice with 22Rv1 derived subcutaneous xenografts with drugs administered orally (BID, daily for 3 weeks). INCB054329 and INCB057643 inhibited proliferation of prostate cancer cell lines. In short-term cell proliferation assays the BETi appeared more effective against androgen-dependent (VCaP and LNCaP) than androgen-independent (DU145 and PC3) cells (GI50 of ≤100 nM and ≥500 nM, respectively). 22Rv1 cells, which express androgen-independent AR splice variants, exhibited an intermediate level of sensitivity to both compounds (GI50: 150-250 nM). Interestingly, in colony and tumor-sphere forming assays all the cell lines showed substantially greater sensitivity to the BETi than in short-term assays. Notably, androgen-independent 22Rv1 cells were highly responsive with GI50 values ≤100 nM similar to those seen in androgen-sensitive LNCaP cells. Treatment of mice bearing 22Rv1 tumor xenografts with INCB054329 (50 mg/kg) and INCB057643 (3 mg/kg) led to significant inhibition of tumor growth (T/C%: 42 and 45%, respectively) and consistent reduction of tumor weight relative to vehicle-treated mice. These results provide evidence of activity of INCB054329 and INCB057643 in prostate cancer cell lines. Although short term assays suggest a preferential anti-proliferative effect in androgen sensitive prostate cancer cells, both compounds also exhibit significant activity in an androgen-independent model both in vitro and in vivo suggesting that they might represent a valid therapeutic option for treatment of castration-resistant prostate cancer. Citation Format: Ramiro Vázquez, Gianluca Civenni, Martina Marchetti, Sabrina Zadic, Phillip Liu, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2017-5080

SET domain-containing protein 5 is required for expression of primordial germ cell specification-associated genes in murine embryonic stem cells.

Primordial germ cell (PGC) specification is one of the most fundamental processes in developmental biology. Because PGCs are a common source of both gametes, generation of PGCs from embryonic stem cells (ESCs) is a useful model for analysing the germ line lineage. Although several studies focused on the role of epigenetic regulation on PGC differentiation from ESCs in vitro have been published, germ line commitment remains poorly understood. Here, we show that SET domain-containing protein (Setd5), which has a previously unknown function, is essential for regulating germ cell-associated genes in murine ESCs (mESCs). Even though Setd5 knockdown with 3 distinct shRNAs did not affect expression of pluripotency genes or levels of global histone methylation, all 3 shRNAs significantly diminished the expression of early and late-stage PGC-associated genes. Furthermore, our immunoprecipitation assay showed that Setd5 can interact with Tbl1xr1 and Ctr9, which are components of 2 different transcriptional regulatory complexes, namely, NcoR1 corepressor complex and Paf1 complex, respectively, in mESCs. Taken together, our data suggest that Setd5 is required for maintaining PGC-associated genes and Setd5-associated protein complexes containing Tbl1xr1 and Ctr9, which in turn are likely involved in regulating germ cell-related genes in mESCs.

IFIT1 exerts opposing regulatory effects on the inflammatory and interferon gene programs in LPS-activated human macrophages

Abstract Activation of the TLR4 signaling pathway by LPS leads to activation of both inflammatory and interferon stimulated genes, however the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remains poorly understood. In a genome-wide siRNA screen of the LPS-induced TNF-a response in macrophages, we identified the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identified an unexpected positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced inflammatory and interferon gene expression. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3a-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate an unappreciated function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. IFIT1 depletion led to a significant increase in the replication of B. cenocepacia. Prior treatment of IFIT1 depleted cells with recombinant IFN-β reversed the enhanced replication of B. cenocepacia, indicating a role for autocrine IFN-β in controlling B. cenocepacia infection and a requirement for IFIT1 in interferon induction in response to bacterial infection. We also observed reduced expression of IFNβ1 in response to viral and bacterial infection in IFIT1 depleted cells, suggesting a broad role for IFIT1 in supporting IFNβ1 expression.

RIP140 in monocytes/macrophages regulates osteoclast differentiation and bone homeostasis.

Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage-specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases.

Functional specialization of two paralogous TAF12 variants by their selective association with SAGA and TFIID transcriptional regulatory complexes

TATA box-binding protein (TBP)-associated factors (TAFs), evolutionarily conserved from yeast to humans, play a central role during transcription initiation. A subset of TAF proteins is shared in transcription factor II D (TFIID) and SAGA transcription regulatory complexes. Although higher eukaryotes contain multiple TAF variants that specify tissue- and developmental stage-specific organization of TFIID or SAGA complexes, in unicellular genomes, however, each TAF is encoded by a single gene. Surprisingly, we found that the genome of Candida albicans, the predominant human fungal pathogen, contains two paralogous TAF12 genes, CaTAF12L and CaTAF12, encoding H2B-like histone-fold domain-containing variants. Of the available fungal genome sequences, only seven other closely related diploid pathogenic Candida genomes encode the two TAF12 paralogs. Using affinity purifications from C. albicans cell extracts, we demonstrate that CaTAF12L uniquely associates with the SAGA complex and CaTAF12 associates with the TFIID complex. We further show that CaTAF12, but not CaTAF12L, is essential for C. albicans growth. Conditional depletion of the two TAF12 variant proteins caused distinct cellular and colony phenotypes. Together our results define a specialized organization of the TAF12 variants and non-redundant roles for the two TAF12 variants in the unicellular C. albicans genome.

Epigenetic Regulation of Chromatin Dynamics

The physical organization of all eukaryotic genomes is evolutionarily conserved where octamers of histone proteins repeatedly wrap DNA into nucleosomes to form long chromatin fibers. Post translational modifications (PTMs) of histone protein octamers regulate essential DNA processing, including RNA transcription and DNA repair. I will discuss our recent work to understand how histone PTMs, and the proteins that bind these PTMs, combine to regulate DNA accessibility to transcription regulatory complexes through controlling chromatin structural dynamics.

Glucocorticoid receptor control of transcription: precision and plasticity via allostery.

The glucocorticoid receptor (GR) is a constitutively expressed transcriptional regulatory factor (TRF) that controls many distinct gene networks, each uniquely determined by particular cellular and physiological contexts. The precision of GR-mediated responses seems to depend on combinatorial, context-specific assembly of GR-nucleated transcription regulatory complexes at genomic response elements. In turn, evidence suggests that context-driven plasticity is conferred by the integration of multiple signals, each serving as an allosteric effector of GR conformation, a key determinant of regulatory complex composition and activity. This structural and mechanistic perspective on GR regulatory specificity is likely to extend to other eukaryotic TRFs.

Generation of a Cellular Reporter for Functional BRD4 Inhibition.

The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many tumours. Even though BRD4 is more and more studied, its mechanism of action has not been fully described yet. Therefore, we aimed at generating a cellular reporter system to monitor BRD4 inhibition. Such reporter can be potentially used in high throughput chemical and genetic screenings, in order to uncover new possible BRD4 functional pathways. The deeper understanding of the mechanism of action of BRD4 activity will certainly help in developing new therapy strategies for those cancers so called BRD4-dependent.

The TAL1 Short Isoform Generated By PRMT1-Mediated Alternative RNA Splicing Promotes Erythroid Differentiation

The isoforms of key transcription factors in hematopoiesis such as TAL1, GATA1 and RUNX1 are generated through alternative RNA splicing regulated by the PRMT1-RBM15 axis (Zhang et al. 2015). The functions of short isoforms of GATA1 (GATA1s) and RUNX1 (RUNX1a) are well characterized, yet it is unknown how the short isoform of TAL1 (TAL1s) regulates hematopoiesis. In this presentation, we report that the short isoform of TAL1, i.e. TAL1s, is generated via alternative RNA splicing as detected by isoform specific real-time PCR reactions using RNA isolated from leukemia cell lines and primary human cord blood cells. RBM15, an RNA binding protein, which is involved in chromosome translocation to produce RBM15-MKL1 fusion protein in acute megakaryocytic leukemia, regulates the alternative RNA splicing of TAL1. RBM15 promotes the production of full-length TAL1 mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway favors the production of TAL1s. RBM15 directly binds to intronic regions on TAL1 pre-mRNA. Binding of RBM15 is responsible for recruiting SF3B1-associated RNA splicing complex. Given that PRMT1 senses the hypoxia status of hematopoietic cells, the changing of TAL1s/TAL1fl ratio by PRMT1 activity may be an adaptive response of hematopoietic cells to hypoxia status. The short form TAL1s still contains the helix-loop-helix DNA binding domain but not the N terminal regions upstream of the DNA binding domain. Thus, the TAL1s may act as a dominant negative mutant of the full-length TAL1fl to block TAL1fl-regulated transcription. We demonstrated that overexpression of TAL1s not the full-length TAL1promotes the erythroid differentiation of K562 cells. Although TAL1 gene is required for both erythroid and megakaryocyte differentiation at early stage of hematopoiesis, TAL1s does not promote megakaryocyte differentiation. Therefore, fine-tuning the TAL1 isoforms by the PRMT1-RBM15 axis determine the cell fate of a MEP progenitor cell. Using immunoprecipitation assays and mass spectrometry analysis, we identified proteins specifically associated with the N terminal region of TAL1. How unique TAL1s-associated transcriptional regulatory complex plays in erythroid differentiation will be discussed in the presentation in comparison with the Tal1fl-asociated protein complex. In summary, our findings stratify another new layer of regulation by PRMT1, which relays extracellular signals (such as hypoxia signal) to transcriptional regulatory program. Given that PRMT1 is often constitutively highly expressed in leukemia cells, how overproduction of short form TAL1 interferes with normal hematopoiesis may help to explain the molecular mechanisms of many hematological malignancies associated with dysregulation of TAL1 expression. DisclosuresNo relevant conflicts of interest to declare.

The DNA-Binding Protein High Mobility Group Box 1 (HMGB1) Interacts with RelB to Maintain Non-Canonical NF-κb Signaling, Cell Survival and Immune Evasion in Infant Acute Lymphoblastic Leukemia

Disordered NF-κB signaling is a hallmark of many hematologic malignancies but has not been well-studied in MLL-rearranged infant lymphoblastic leukemia (iMLL-ALL), a subtype of pediatric ALL with an extremely poor prognosis in need of new therapy. Given the stable genome and short latency of iMLL-ALL, targeting of oncoproteins that play a coordinated role in regulation of the leukemic transcriptome represent ideal targets. Here, we present one such potential target: a complex formed between the nucleosome-stabilizing protein high mobility group box 1 (HMGB1) and RelB, a transcriptionally active subunit of the noncanonical NF-κB pathway.Disruptions in the non-canonical NF-κB pathway, which normally functions to support lymphogenesis, are known to drive lymphoid malignancies. Specifically, changes in proteasomal processing of the p100 subunit modulate nuclear p52/RelB regulation of the NF-κB2 gene. In stable short-term culture, we observed that primary infant MLL-ALL cells (derived from the diagnostic pheresis specimen of a 9 month old with t(4;11)) express an endogenous NF-κB subunit profile consistent with constitutive non-canonical activation, including significantly increased levels of IKKa, RelB, and NF-κB2 by Western Blot and qPCR compared to healthy human B cells and cytogenetically normal ALL. First, we asked whether exogenous "drivers" of the canonical (p65/RelA) pathway could alter this expression profile by treating iMLL-ALL with TNFalpha and bacterial LPS. Exogenous cytokine treatment did not alter expression levels of IKKa, RelB or NF-κB2, nor did it increase p65RelA, likely due to the lack of pattern recognition receptors on precursor lymphoblastic cells.We have previously demonstrated that infant MLL-ALL express high intracellular levels of the nucleosome-stabilizing protein high mobility group box-1 (HMGB1). During infection, HMGB1 forms a transcription regulatory complex with RelB that, by epigenetic means, silences inflammatory cytokine promoters as a negative feedback. In this study, we hypothesized that HMGB1-RelB interactions stabilize and maintain a pro-leukemic NF-κB program. Because the function of HMGB1 is determined by post-translational modifications, we first verified the DNA-binding disulfide form of the protein in the nuclear fraction of iMLL-ALL cells by immunoprecipitation. Next, we proved a physiologically relevant interaction between HMGB1 and RelB by co-immunoprecipitation; both HMGB1 and RelB could be identified by immunoblot from iMLL-ALL extracts probed for either for RelB or HMGB1. Detection of the complex required minimal stabilization, suggesting a durable interaction. Preliminary studies suggest that the complex associates with the acetylated form of histone 3 (H3), rather than histone 1 as in sepsis models.We then determined the effect of HMGB1 siRNA knockdown on the NF-κB subunit expression profile, proliferation and survival of iMLL-ALL, nonleukemic control PBMC and 293T cells. HMGB1 siRNA produced 30-50% spontaneous apoptosis in MLL-ALL, but not nonleukemic control cells, measured by Annexin/PI. iMLL-ALL transfected with a nonsense siRNA sequence did not apoptose. HMGB1 siRNA also silenced expression of RelB and NF-κB 2, measured by Western Blot and qPCR. Unexpectedly, HMGB1 siRNA restored expression of critical canonical NF-κB subunits: p65/RelA, the TNF-associated factor TRAF3, and the proteasome regulator NEMO. Further, HMGB1 siRNA knockdown resulted in a 51.3% (+/- 3.47, p<0.0001) increase in engulfment by activated bone marrow macrophages in a dual-label phagocytosis assay, compared to nonsense siRNA and control iMLL-ALL- suggesting that HMGB1 expression plays a role in evasion of iMLL-ALL by the innate immune system.Taken together, these data indicate that HMGB1-RelB interactions represent an important regulatory component of the NF-κB signaling program in human infant MLL-ALL cells that support survival and leukemogenesis. Current studies are comparing p100 processing and proteasome activity in iMLL-ALL with competent and disrupted HMGB1-RelB interactions, as well as comparing the effectiveness of proteasome inhibition with bortezomib. Near-future studies will screen other high-risk ALL’s for HMGB1-Rel activity and elaborate the downstream effects of complex binding on the MLL-ALL transcriptome to establish it as a rational target for drug development. DisclosuresNo relevant conflicts of interest to declare.

AZD5153: A Novel Bivalent BET Bromodomain Inhibitor Highly Active against Hematologic Malignancies.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.

Histone acetyl transferase GCN5 promotes human hepatocellular carcinoma progression by enhancing AIB1 expression.

BackgroundGeneral control non-depressible 5 (GCN5) is a crucial catalytic component of a transcriptional regulatory complex that plays important roles in cellular functions from cell cycle regulation to DNA damage repair. Although GCN5 has recently been implicated in certain oncogenic roles, its role in liver cancer progression remains vague.ResultsIn this study, we report that GCN5 was overexpressed in 17 (54.8 %) of 31 human hepatocellular carcinoma (HCC) specimens. Down-regulation of GCN5 inhibited HCC cell proliferation and xenograft tumor formation. GCN5 knockdown decreased the protein levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and amplified in breast cancer 1 (AIB1), but increased the protein levels of cell cycle inhibitor p21Cip1/Waf1 in HepG2 cells. GCN5 regulated AIB1 expression, at least in part, by cooperating with E2F1 to enhance AIB1 transcription. Consistently, GCN5 expression was positively correlated with AIB1 expression in human HCC specimens in two GEO profile datasets.ConclusionSince AIB1 plays a promoting role in HCC progression, our results propose that GCN5 promotes HCC progression at least partially by regulating AIB1 expression. This study implicates that GCN5 might be a potential molecular target for HCC diagnosis and treatment.

Abstract 4705: Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers

Abstract The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Across a number of tumor models pharmacological targeting of BRD4 bromodomains by small-molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor. AZD5153 is a candidate drug that possesses an unprecedented bivalent binding mode among reported BET inhibitors, which allows AZD5153 to ligate the tandem bromodomains in BRD4. The avidity resulted from the bivalent binding interaction translates into markedly enhanced cellular potency. Although AZD5153 demonstrates broad activity across a cancer cell line panel comprising solid and hematologic subtypes, there is enriched antitumor activity against hematologic cell lines, including acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL). The activity of AZD5153 in hematologic tumors was further confirmed in five selected xenograft models of AML, MM, and DLBCL where AZD5153 treatment led to tumor stasis or regression, accompanied by concomitant modulation of BRD4 pharmacodynamic markers, such as MYC and HEXIM1. In order to characterize the transcriptional consequences elicited by AZD5153, we carried out transcriptional profiling of 11 hematologic tumor lines and identified the robust modulation of MYC, and E2F transcriptional programs. Moreover, our transcriptional data was used to identify candidate clinical PD biomarkers. The suitability and dynamic range of the top two candidate biomarkers for AZD5153 was confirmed using human whole blood from normal healthy volunteers. To identify protein biomarkers associated with sensitivity to AZD5153 treatment, we deployed reverse-phase protein array (RPPA) technology to quantitatively examine the level of 182 proteins following AZD5153 treatment. Our findings indicate that cell lines sensitive to AZD5153 uniquely exhibit a marked decrease in the level of mTOR-pathway associated proteins following AZD5153 treatment. Conversely, MYC modulation was observed in both sensitive and resistant groups. Thus, these data suggest that in hematologic malignancies, mTOR pathway downregulation may serve as an appropriate biomarker of sensitivity to BRD4 inhibitors such as AZD5153. Our study establishes AZD5153 as a novel and potent BRD4/BET inhibitor possessing a unique bivalent binding property. We have characterized the pharmacological consequences of BRD4/BET inhibition by AZD5153 via unbiased transcriptional and proteome profiling. These efforts are the first to identify mTOR modulation as a putative biomarker of sensitivity to BET bromodomain inhibition in hematologic tumors and may help to inform future clinical evaluation of AZD5153 and other BET bromodomain inhibitors. Citation Format: Huawei (Ray) Chen, Maureen Hattersley, Garrett Rhyasen, Austin Dulak, Wendy Wang, Phil Petteruti, Ian Dale, Tony Cheung, Shenghua Wen, Lilian Castriotta, Deborah Lawson, Mike Collins, Miika Ahdesmaki, Graeme Walker, Al Rabow, Jonathan Dry, Corinne Reimer, Paul Lyne, Steve Fawell, MIke Waring, Mike Zinda, Ed Clark, Ed Clark. Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4705.

Mutant p53 and ETS2, a Tale of Reciprocity.

TP53 is one of the most frequently inactivated tumor suppressor genes in human cancer. However, unlike other tumor suppressor genes whose expression is lost, TP53 is usually inactivated as a result of a single nucleotide change within the coding region. Typically, these single nucleotide mutations result in a codon change that creates an amino acid substitution. Thus, unlike other tumor suppressor genes whose expression is lost due to genetic or epigenetic changes, the p53 gene primarily suffers missense mutations, and therefore, the cells retain and express a mutant form of the p53 protein (mtp53). It is now well established that mtp53 contributes to tumor development through its gain-of-function (GOF) activities. These GOF activities can arise from novel protein–protein interactions that can either disable other tumor suppressors (e.g., p63 and p73) or enable oncogenes such as ETS2, an ETS family member. In this review, I will focus on the identification of the mtp53/ETS2 complex and outline the diverse activities that this transcriptional regulatory complex controls to promote cancer.

Abstract B21: Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC

Abstract The RAF-MEK-ERK signaling pathway regulates the proliferation of Triple Negative Breast Cancer (TNBC). Although MEK inhibition by trametinib in TNBC cell lines and mouse models inhibits growth, the response is not durable and the tumor develops resistance. The lack of durability of targeted kinase inhibitors is a common problem in many tumor types due to induction of alternative bypass signaling pathways that lead to resistance. We have profiled the response of TNBC to MEK inhibition using trametinib in order to identify adaptive signaling pathways which drive resistance. We used a chemical proteomics method [multiplexed inhibitor beads coupled to mass spectrometry (MIB/MS)], to quantify global changes in kinase activity coupled with transcriptional changes in response to trametinib using RNAseq. Our studies revealed that adaptive resistance to trametinib occurs through increased expression and activation of multiple receptor tyrosine kinases, leading to reactivation of ERK. These adaptive kinases are heterogeneous across TNBC including PDGFRβ, VEGFR2, DDR1, AXL, KIT and FGFR2. There is no single targeted kinase inhibitor able to block all the adaptive kinases induced and activated, making the design of combination targeted kinase inhibitor therapies impractical. We therefore hypothesized an alternative therapeutic approach was required to inhibit adaptive reprogramming. Our goal was to block the initial transcriptional response to trametinib which upregulates adaptive kinase expression. Several studies in the lab determined that we could block adaptive kinase expression by inhibiting BET family bromodomain proteins that interact with acetylated histones and facilitate the formation of transcriptional regulatory complexes. Our work demonstrated that BET family bromodomain inhibitors (JQ1 and iBET151), synergize with trametinib to prevent TNBC growth in short-term (4 days) and long-term (4 weeks) growth assays. Using MIB/MS we found that combination trametinib/BET bromodomain inhibitor treatment blocks upregulation of adaptive kinase expression and activation. RNAseq demonstrated that BET bromodomain inhibitors synergize with trametinib to block the induction of kinases expressed as part of the adaptive bypass response. ChIP-seq of the BET bromodomain protein BRD4 and H3K27ac, a marker of active enhancers, showed BRD4 is recruited to super-enhancer regions proximal to adaptive response kinases as early as 4hr following trametinib treatment. Furthermore, the recruitment of BRD4 to enhancer regions is maintained in cells made resistant to trametinib by constant culture in low-dose drug. In sum, these results suggest that BRD4 recruitment to enhancer regions, following trametinib treatment, leads to the formation of transcriptional complexes which maintain adaptive gene expression. Ongoing studies seek to understand the mechanism by which BRD4 is recruited to super-enhancers following trametinib treatment in TNBC. Mouse xenograft studies also show promising evidence of enhanced tumor growth suppression in vivo using combination trametinib/iBET151 treatment. The results presented provide a novel treatment strategy to block adaptive resistance and have the potential to lead to durable clinical responses of TNBC. Citation Format: Samantha M. Miller, Jon S. Zawistowski, Timothy J. Stuhlmiller, Noah Sciaky, Charlene M. Santos, David B. Darr, Gary L. Johnson. Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B21.