RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Abstract Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML. Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS. Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio. Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. Disclosures Logan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

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Genome-Wide Association Study Identifies an Immune-Related Etiology for Severe Aplastic Anemia

Blood ◽

10.1182/blood-2019-128235 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1224-1224

Author(s):

Sharon A. Savage ◽

Mathias Viard ◽

Weiyin Zhou ◽

Meredith Yeager ◽

Shengchao Li ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Association Study ◽

Research Funding ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Class I ◽

Severe Aplastic Anemia ◽

Genome Wide ◽

Validation Set

Introduction. Acquired severe aplastic anemia (SAA) is a life-threatening disorder characterized by severe progressive pancytopenia and hypocellular bone marrow. The etiology of acquired SAA is not understood but believed to be related to abnormal immune responses to environmental exposures. We conducted a genome-wide association study (GWAS) to identify common germline variants associated with SAA. Methods. We identified 895 patients with SAA who underwent related or unrelated hematopoietic cell transplant (HCT) with clinical data and pre-HCT blood samples available in the Center for International Blood and Marrow Transplant Research (CIBMTR) database and biorepository. Pre-HCT DNA was extracted from blood of patients with SAA and genome-wide genotyping was conducted using the Illumina OmniExpress array. We excluded 93 inherited bone marrow failure cases. The SAA cases were grouped cases into discovery and validation sets based on time of batch sample receipt. Analyses were limited to patients of European ancestry based on principal component analyses to minimize the potential effect of population stratification. Controls were genomically matched and selected from previously scanned cancer-free subjects at the Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, NCI. The final analysis included 534 acquired SAA cases (359 in the discovery set and 175 in the validation set), and 2,455 controls (1,396 in the discovery set, and 1,059 in the validation set). Results. Patients with SAA in this study received HCT between 1989-2015 at a median age of 21 years, 56% were male, and the median time between SAA diagnosis and HCT was 11 months. Strong genome-wide association signals were identified across the human leukocyte antigen (HLA) genes encoded at the major histocompatibility complex (MHC) on chromosome 6p21 (Figure 1). The top SNP was located in the P4 binding pocket of the HLA class II gene HLA-DPB1(rs1042151A>G, p.Met105Val, pooled-odds ratio [OR]=1.75, 95% confidence interval [CI]=1.50-2.03, p=1.94x10-13). The expression of HLA-DP in CD19+ cells from 175 healthy donors was significantly different by rs1042151 A>G genotype (p=2.04x10-6) (Figure 2). A second SNP near HLA-B, rs28367832G>A, also reached genome-wide significance (pooled-OR=1.49, 95% CI=1.22-1.78, p=7.27x10-9). Copy-number variant analysis and next generation sequencing also identified somatic, clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes in 8.6% of the SAA cases and none of the controls. Conclusion. This SAA GWAS identified strong association signals between common germline genetic variants in HLA class I and II genes and SAA. The main SNP is associated with changes in HLA-DP expression suggesting a key role for this locus in SAA etiology. This study adds further evidence to the connection between SAA and immune dysregulation. Disclosures Cerhan: Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; Celgene: Research Funding. Lee:Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; AstraZeneca: Research Funding.

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Yeats4 drives ILC lineage commitment via activation of Lmo4 transcription

Journal of Experimental Medicine ◽

10.1084/jem.20182363 ◽

2019 ◽

Vol 216 (11) ◽

pp. 2653-2668

Author(s):

Benyu Liu ◽

Liuliu Yang ◽

Xiaoxiao Zhu ◽

Huimu Li ◽

Pingping Zhu ◽

...

Keyword(s):

Bone Marrow ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Lineage Commitment ◽

Rna Pol Ii ◽

Hematopoietic System ◽

Pol Ii ◽

Effector Functions ◽

Lineage Differentiation ◽

Mucosal Homeostasis

Innate lymphoid cells (ILCs) play critical roles in defending infections and maintaining mucosal homeostasis. All ILCs arise from common lymphoid progenitors (CLPs) in bone marrow. However, how CLPs stratify and differentiate into ILC lineages remains elusive. Here, we showed that Yeats4 is highly expressed in ILCs and their progenitors. Yeats4 conditional KO in the hematopoietic system causes decreased numbers of ILCs and impairs their effector functions. Moreover, Yeats4 regulates α4β7+ CLP differentiation toward common helper ILC progenitors (CHILPs). Mechanistically, Yeats4 recruits the Dot1l–RNA Pol II complex onto Lmo4 promoter through recognizing H3K27ac modification to initiate Lmo4 transcription in α4β7+ CLPs. Additionally, Lmo4 deficiency also impairs ILC lineage differentiation and their effector functions. Collectively, the Yeats4–Lmo4 axis is required for ILC lineage commitment.

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Promoter-Wide Transcriptional Deregulation In Waldenstrom Macroglobulinemia

Blood ◽

10.1182/blood.v116.21.3620.3620 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3620-3620

Author(s):

Yang Liu ◽

Min Ni ◽

Aldo M. Roccaro ◽

Xavier Leleu ◽

Yong Zhang ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Board Of Directors ◽

Binding Sites ◽

Research Funding ◽

Enrichment Analysis ◽

Promoter Regions ◽

Advisory Committees ◽

Pol Ii ◽

Normal Controls

Abstract Abstract 3620 Introduction: Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results: A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion: The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Genome Wide DNA Methylation Profiling In Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v116.21.3622.3622 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3622-3622

Author(s):

Yang Liu ◽

Shenghua Duan ◽

Xavier Leleu ◽

Yong Zhang ◽

Abdel Kareem A. Azab ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Cell Lines ◽

Promoter Region ◽

Genomic Dna ◽

Research Funding ◽

Plasma Cells ◽

Genome Wide

Abstract Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Copy Number Variants Underlying Inherited Bone Marrow Failure Syndromes

Blood ◽

10.1182/blood.v126.23.2416.2416 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2416-2416

Author(s):

Nicolas Waespe ◽

Santhosh Dhanraj ◽

Manju Wahala ◽

Tom Enbar ◽

Bozana Zlateska ◽

...

Keyword(s):

Bone Marrow ◽

Short Stature ◽

Copy Number ◽

Research Funding ◽

Bone Marrow Failure ◽

Organ System ◽

Severe Phenotype ◽

Nucleotide Level ◽

Bone Marrow Failure Syndromes ◽

Genome Wide

Abstract Background. Inherited bone marrow failure syndromes (IBMFSs) comprise a genetically heterogeneous group of diseases with hematopoietic failure and varying degrees of physical malformations. The diagnosis of an IBMFS and categorizing the specific syndrome critically impact on clinical care; however, these are commonly challenging and rely on genetic testing. Since over 80 genes have been associated with IBMFSs and might be affected by different types of DNA aberrations, the best strategy to establish a diagnosis in a timely and cost effective manner is unknown. The aims of this study were to evaluate the role of genome-wide copy number variant (CNV) analysis in unraveling causal genetic alterations in IBMFS patients with unknown genotype and determine whether correlation exists between large CNVs and more severe phenotype. Methods. Patients from the Canadian Inherited Marrow Failure Registry (CIMFR) who were genetically investigated were included in this analysis. Genetic and clinical data were extracted and analyzed. Mann-Whitney test and Fisher's exact test were used to assess statistical significance. Results. Among 328 patients from the CIMFR who underwent molecular investigation, a causal genotype was identified in 185 cases (56.4%). 69 patients had genome-wide CNV analysis by SNP/CGH arrays, among which ten (14.5%) had positive results. In four out of ten cases who were genotyped by SNP/CGH array, genome-wide CNV analysis was critical for establishing the diagnosis. Among 308 patients who were tested for nucleotide-level mutations by either targeted gene analysis or next generation sequencing panels, casual mutations were found in 169 (54.9%). Three patients had compound heterozygosity for a CNV and nucleotide-level mutation. To determine whether large deletions are correlated with more severe phenotype we included nine additional patients with causal CNVs whose genotype was identified by MLPA (n=1), targeted FISH (n=1), DNA-qPCR analysis (n=1), Southern blotting (n=1) or metaphase cytogenetics (n=5). The causal CNVs among patients in our cohort ranged from 0.02 to 145.5 Mb in size. The most common disease associated with causal CNVs was Diamond-Blackfan anemia (four patients). Patients with CNVs tended to have significantly more non-hematological organ system involvement (p=0.03), developmental delay (mean=56% vs. 28%, p=0.03) and short stature (mean=67% vs. 40%, p=0.04) than patients with nucleotide-level mutations. The difference remained significant when we compared all patients with mutations that are predicted to result in truncation or lack of protein from the respective allele (large CNV, nonsense, and indel/ frameshift) to patients with mutations that are predicted to be hypomorphic or affect function (splicing, indel/ inframe and missense). There was no correlation between CNVs and the severity of the hematological disease. Conclusions. Most patients with IBMFSs have nucleotide-level mutations. However, a significant proportion of patients without such mutations have large CNVs that are not efficiently detected by current nucleotide-level testing methods. Therefore, genome-wide CNV analysis should be considered in IBMFS cases, where nucleotide-level sequencing does not reveal the causal mutation. Patients with IBMFSs and large CNVs had more non-hematological organ system involvement, a higher prevalence of developmental delay and short stature. This might be related to an additional impact of the CNVs on other genes close to the affected IBMFS gene or the severe damaging effect of the CNVs. Disclosures Lipton: Teva: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

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Genome-wide mapping of RNA Pol-II promoter usage in mouse tissues by ChIP-seq

Nucleic Acids Research ◽

10.1093/nar/gkq775 ◽

2010 ◽

Vol 39 (1) ◽

pp. 190-201 ◽

Cited By ~ 37

Author(s):

Hao Sun ◽

Jiejun Wu ◽

Priyankara Wickramasinghe ◽

Sharmistha Pal ◽

Ravi Gupta ◽

...

Keyword(s):

Rna Pol Ii ◽

Pol Ii ◽

Genome Wide ◽

Mouse Tissues ◽

Promoter Usage ◽

Rna Pol Ii Promoter

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NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

10.1101/246827 ◽

2018 ◽

Author(s):

Constantine Mylonas ◽

Peter Tessarz

Keyword(s):

Splicing Factors ◽

Elongation Factors ◽

Single Nucleotide ◽

Rna Pol Ii ◽

Pol Ii ◽

Genome Wide ◽

Regulatory Complexity ◽

Pol Ii Pausing ◽

Nucleotide Resolution ◽

The Impact

ABSTRACTThe advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. However, little is known, at such high resolution, how transcription factors directly influence RNA Pol II pausing and directionality. To map the impact of transcription/elongation factors on transcription dynamics genome-wide at base pair resolution, we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). Application of NET-prism on elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals their inherent command on transcription dynamics, with regards to directionality and pausing over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity during gene expression.

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Nutrient-Driven O-GlcNAcylation at Promoters Impacts Genome-Wide RNA Pol II Distribution

Frontiers in Endocrinology ◽

10.3389/fendo.2018.00521 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Michael W. Krause ◽

Dona C. Love ◽

Salil K. Ghosh ◽

Peng Wang ◽

Sijung Yun ◽

...

Keyword(s):

Rna Pol Ii ◽

Pol Ii ◽

Genome Wide

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Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803275115 ◽

2018 ◽

Vol 115 (30) ◽

pp. 7777-7782 ◽

Cited By ~ 9

Author(s):

Hemanth Tummala ◽

Arran D. Dokal ◽

Amanda Walne ◽

Alicia Ellison ◽

Shirleny Cardoso ◽

...

Keyword(s):

Bone Marrow ◽

Dna Repair ◽

Rna Polymerase Ii ◽

Nuclear Dna ◽

Genome Instability ◽

Excision Repair ◽

Bone Marrow Failure ◽

Rna Pol Ii ◽

Pol Ii ◽

Repair Defect

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients’ lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients’ LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients’ LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA–RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

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Transcriptional and Post-Translational Regulation Of The Bcl-2 Family By IL-6 Mediates Resistance To ABT-737 In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.1924.1924 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1924-1924 ◽

Cited By ~ 1

Author(s):

Vikas A. Gupta ◽

Shannon M. Matulis ◽

Jason E. Conage-Pough ◽

Ajay K. Nooka ◽

Jonathan L. Kaufman ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Induced Resistance ◽

Research Funding ◽

Alternative Pathway ◽

Mek Inhibitor ◽

Conditioned Media ◽

Bone Marrow Niche

Abstract While direct inhibition of Bcl-2 and/or Bcl-xL is an exciting new approach in the treatment of hematologic malignancies, current agents in clinical testing including navitoclax (ABT-263) and ABT-199 are not predicted to be efficacious in most cases of multiple myeloma. Navitoclax and the related molecule ABT-737 promote apoptosis by releasing the pro-apoptotic BH3 only protein Bim from Bcl-2 and Bcl-xL, but are incapable of disrupting the interaction between Bim and Mcl-1, the predominant anti-apoptotic protein in both normal and malignant plasma cells. However, despite their dependence on Mcl-1, some human myeloma cell lines (HMCL) as well as freshly isolated patient-derived myeloma cells are sensitive to ABT-737 in vitro. Knowing that myeloma normally requires the bone marrow niche for survival, we hypothesized that a stromal derived factor might mediate the resistance to ABT-737 in vivo and identified IL-6 as a key resistance factor. In our initial experiments, the HS-5 stromal cell line induced resistance to ABT-737 in the HMCL MM.1s, as did conditioned media from both HS-5 cells and patient derived bone marrow stromal cells. Blocking IL-6 with an IL-6 neutralizing antibody reversed the protective effect of conditioned media, while addition of 10 ng/ml IL-6 protected cells to the same degree as conditioned media. In order to understand the mechanisms of IL-6 mediated resistance to ABT-737, we have focused on the effect of IL-6 on the Bcl-2 family of proteins. In previous work from our lab, the ABT-737 sensitive HMCLs KMS18, MM.1s, and 8226 all showed increased binding of Bim to Bcl-2 and Bcl-xL compared to ABT-737 resistant lines. We therefore examined the effect of IL-6 on the distribution of Bim among Bcl-2, Bcl-xL, and Mcl-1. In KMS18, stimulation with 10 ng/ml IL-6 for 24 hours increases binding of Bim to Mcl-1. The increased binding correlates with a 2 fold increased Mcl-1 expression at both the RNA and protein level. The increased Mcl-1 expression in response to IL-6 may be limited to KMS18 as it was not observed in MM.1s, 8226, or KMS11. IL-6 also does not prevent ABT-737 from disrupting the interaction between Bim and Bcl-xL. We also examined Bim for IL-6 induced post-translational modifications that could alter its binding to Bcl-2 proteins. Bim is known to be phosphorylated on serine 69 by Erk in response to growth factor stimulation. We observed Bim serine 69 phosphorylation within 5 minutes of IL-6 stimulation in both KMS18 and MM.1s cells. Phosphorylation was reversible with 10 μM of the MEK inhibitor U0126. Although serine 69 phosphorylation has been reported to result in Bim degradation, we do not observe any change in Bim levels over the course of 24 hours. Interestingly, the MEK inhibitor sensitized both KMS18 and MM.1s to ABT-737 and was able to partially overcome IL-6 induced resistance. Inhibition of Akt with the PI3K inhibitor LY294002 had no effect on Bim serine 69 phosphorylation or IL-6 induced resistance. Of note, the MEK inhibitor failed to prevent upregulation of Mcl-1 in KMS18, suggesting an alternative pathway mediates this effect. We are currently studying the role of JAK signaling in Mcl-1 expression with the inhibitors AZD1480 and ruxolitinib, and are also extending our results to additional cell lines and patient samples. These results suggest that targeting IL-6 or its downstream pathways may sensitize myeloma to Bcl-2 antagonists such as ABT-199 and navitoclax. Disclosures: Kaufman: Onyx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy; Jansenn: Consultancy; Merck: Research Funding. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.

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