scholarly journals Integrated Epigenetic and Transcriptional Single Cell Analysis of t(11;14) Myeloma and Its BCL2 Dependency

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 725-725
Author(s):  
Noemie Leblay ◽  
Sungwoo Ahn ◽  
Ranjan Maity ◽  
Holly Lee ◽  
Elie Barakat ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Single Cell ◽  
Plasma Cell ◽  
Copy Number ◽  
Acquired Resistance ◽  
Copy Number Gain ◽  
Chromatin Accessibility ◽  
Immunoglobulin Gene ◽  
Cell Markers

Abstract Multiple myeloma is characterized by recurrent chromosomal translocations that involve the immunoglobulin gene enhancers and partners such as the cyclin D genes (CCND1, CCND2 or CCND3) or other genes like WHSC1 and MAF. t(11;14) results in upregulation of CCND1 with unique morphological, phenotypic markers, and drug sensitivity profiles with exquisite sensitivity to BCL2 inhibitors. It is evident now that this unique sensitivity profile is driven by the BH3-proapoptotic protein priming of BCL2 with high BCL2/MCL1 or BCL2/BCL2L1 ratios. However, the epigenetic mechanisms associated with t(11;14) and their impact on genes regulation and clinical response to venetoclax remains elusive. In the present study we compared the transcriptomics (scRNA-seq) and chromatin accessibility (scATAC-seq) of single plasma cells of MM patients with and without t(11;14) as well as pre- and post-venetoclax exposure in order to establish the epigenomic signature of t(11;14) and/or BCL2-sensitivity in myeloma. Serial BM aspirates (n=24) were collected from 15 relapsed or refractory myeloma patients (RRMM); harboring t(11;14) (n=6 pairs) and 9 without this translocation prior to initiation of salvage therapy and at time of relapse. All t(11;14) MM patients were treated with venetoclax. Unbiased chromatin accessibility and mRNA profiling of CD138 pos cells were performed using the chromium single cell ATAC and RNA-Seq 3' solution (10x Genomics), respectively. Cell Ranger, Seurat and ArchR were used for sample de-multiplexing, barcode processing, single-cell 3' gene, peaks counting, and data analysis. We first compared the scATAC-seq and scRNA-seq profiles found in CD138 pos MM cell isolated from patients harboring t(11;14) with the one obtained in patients without this translocation. As expected, t(11:14) patients had high chromatin accessibility at the CCND1 locus and high mRNA expression. Differentially accessible chromatin analysis identified 147518 peaks that were specific to t(11;14) patients. Of interest, motifs enrichment analysis of accessible peaks identified a "B cell-like" motifs signature with enriched TFs motifs such as TCF4 and PAX5 in t(11;14) patients compared to non t(11:14) enriched for IRF and STAT family of motifs. The integration of the scATAC-seq and scRNA-seq data confirmed the B cell signature of t(11;14) patients with upregulation of B cell markers such as MS4A1, VPREB3, CD79A, CD19, and down-regulation of plasma cell markers such as TDO2, EFEMP1, CD28, SLAMF7, and IL6R. Additionally, we found PAX1, PAX5, TCF3, TCF5, and SPI1 transcription factors to be highly expressed in t(11;14) while the non t(11:14) were enriched for IRF1-9 transcription factors. Of interest, the clustering analysis performed on scATAC-seq data identified 3 non t(11;14) patients with a chromatin accessibility profile similar to that of t(11;14) patients. They expressed B cell markers (PAX5, VPREB3 or FCRLA), overexpressed BCL2 and we are currently examining whether this B cell-like epigenetic signature determines sensitivity to venetoclax. In order to define the epigenetic contribution to the acquired resistance to venetoclax in t(11;14) myeloma, we compared the chromatin accessibility profiles of t(11;14) patients pre- vs. post-venetoclax treatment. Enriched motifs within accessible peaks differed significantly between pre- and post-venetoclax with RELA, REL, RELB and EGR1 motifs predominantly enrichmed in the pre-samples in contrast to JUN, JUNB, JUND and FOSL1/L2 motifs enrichment in the post-samples. Of note, integration analysis of scRNAseq (differentially expressed genes) and ATACseq data (differentially accessible peaks) identified MCL1 and ENSA (a gene 60 Kb centromeric to MCL1 on chr1q) as the top enriched genes and peaks in resistant samples suggesting that copy number gain at the MCL1 locus (which we confirmed by single cell CNV analysis) rather than epigenetic modifications is likely the main determinant of acquired resistant to venetoclax in t(11;14) MM. In the current study we have defined the epigenetic regulome and transcriptome associated with t(11;14) myeloma and its relatedness to B cell rather than plasma cell biology. Our studies also suggest that acquired resistance to venetoclax is largely driven by copy number gain at the MCL1 locus. Disclosures Bahlis: Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria. Neri: BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

scholarly journals IGLL5-BCL2L1 Rearrangement with Loss of BCL2 Dependency As Mechanism of Venetoclax Resistance in Multiple Myeloma (MM)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 686-686
Author(s):  
Jean-Baptiste Alberge ◽  
Sarthak Sinha ◽  
Ranjan Maity ◽  
Arzina Jaffer ◽  
Justin Donovan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Single Cell ◽  
Copy Number ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Chromatin Accessibility ◽  
Cell Clusters ◽  
Chromosome 20 ◽  
Apoptotic Genes

Background: Targeting the anti-apoptotic BCL2 protein in haematological malignancies has demonstrated significant anti-tumoral activity in a subset of multiple myeloma patients harbouring rearrangements involving the CCND1 and the immunoglobulin heavy chain enhancers (Eμ and α1/2). The mechanisms underlying the dependency of this subgroup of MM patients on BCL2 remains to be elucidated as well as the mechanisms of resistance to BCL2 inhibition with BH3 mimetic venetoclax. Methods and Results: Sorted bone marrow plasma cells from a cohort of t(11;14) myeloma patients treated with venetoclax were profiled through multi-omics single cell mRNA expression (scRNAseq), copy number profiling (scCNVseq) as well as chromatin accessibility with single cell ATAC-seq. Sequenced reads were aligned to hg38 reference genome. Samples were processed with CellRanger suite v3.0 and downstream analyses were realized with Seurat, Monocle, Signac, and Cicero R packages. Single plasma cells exhibited differential chromatin accessibility landscapes within and across individual patients as well as pre- and post-venetoclax with enrichment of MYC:MAX, RELA, IRF family, RUNX1/3 and ETS motifs. Integration of mRNA and ATAC data revealed a dynamic change of regulatory motifs across individual cell clusters with evidence of selective pressures driven by venetoclax treatment. Similarly mRNA profiling of the apoptotic genes pre- and post-venetoclax exposure showed loss of BCL2 and upregulation of MCL1 and/or BCL2L1 as well as loss of the BH3-only pro-apoptotic genes PMAIP1 and BCC3 in single cell clusters. mRNA levels mirrored open chromatin at the gene bodies and their respective promoter loci consistent with a direct transcriptional regulation. In a patient with several fold upregulation of the BCL2L1 transcript in the post-venetoclax sample (Figure A-B), scATACseq identified a gain in the chromatin accessibility mapping to a genomic region centromeric to BCL2L1 locus on chromosome 20 (chr20:31,617,200-31,619,900). Single cell CNV analysis identified a 5q loss (chr5:142,400,001-156,240,000) mapping to NR3C1 locus explaining with the clinical resistance to dexamethasone. Importantly scCNV also revealed a copy number gain mapping to the same locus with the newly acquired chromatin accessibility on chromosome 20. Mate-pair analysis of the sequencing reads identified the potent IGLL5 B-cell enhancer on chromosome 22 (chr22:22,960,001-22,980,000) as the mate partner juxtaposed the BCL2L1 locus (Figure C). This finding explains the robust upregulation of BCL2L1 mRNA observed in this patient and the shift in BCL2 dependency detected by ex vivo BH3 sensitivity profiling. Of note, while scCNV analysis also depicted a gain in 1q21 (chr1:149,940,001-169,980,001) MCL1 locus at the time of venetoclax resistance the acquisition of t(20,22) shifted the plasma cells dependency to BCL-xL rather than MCL1. This finding was corroborated by the plasma cells ex vivo resistance to dual BCL2 and MCL1 inhibition. Conclusion: Dynamic single cell epigenome and transcriptome profiling of pre- and post-venetoclax of primary plasma cells identified a de novo translocation driving BCL-xL transcription with the IGLL5 B-cell enhancer. This demonstrates that in addition to canonical TF-promoter regulation, restructuring of immunoglobulin regulatory sequences (i.e., enhancers) can also drive aberrant malignant circuitry endowing resistance to anti-BCL2 agents. Figure. Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Bahlis:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

DNA Copy Number Gains of TCF4 (E2-2) Are Associated with Poor Outcome in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2686-2686 ◽  
Author(s):  
Keenan Hartert ◽  
Saber Tadros ◽  
Alyssa Bouska ◽  
Dalia Moore ◽  
Christine Pak ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
B Cell ◽  
Copy Number ◽  
Research Funding ◽  
Copy Number Gain ◽  
Cell Receptor ◽  
Dna Copy Number ◽  
B Cell Malignancy ◽  
Bcr Signaling

Abstract The development of B-cells is a complex process that proceeds through multiple stages and is regulated by a hierarchy of transcription factors and other physiologic signals. Each unique B-cell malignancy can be aligned with a 'normal counterpart' at one or more of these discrete developmental stages. However, with the exception of translocations of transcription factor genes, the genetic basis for this is not well defined. We performed an analysis of high-resolution single nucleotide polymorphism (SNP) microarrays from 694 diffuse large B-cell (DLBCL) tumors to identify significant somatic copy number alterations (SCNA). Through integrative analysis of 249 tumors with matched gene expression profiling (GEP) data, we identified the likely targets of these alterations and found that genes that were targeted by DNA copy number gain were significantly enriched for DNA binding activity and transcription factor function. We extended upon this observation by analyzing SNP microarray data of a further 2,716 tumors from 7 additional subtypes of B-cell malignancy. Through this analysis, we identified patterns of transcription factor alterations that aligned with the differentiation state of the 'normal B-cell counterpart' of each malignancy. This provides evidence that SCNA of B-cell transcription factors may underlie the differentiation state of B-cell malignancies. DLBCL can be divided into two subtypes based upon gene expression profiles that align with either the germinal center B-cell differentiation state (GCB-like) or a post-GCB activated B-cell state (ABC-like). Having observed an enrichment for transcription factor SCNAs in DLBCL, and an alignment between transcription factor alterations and differentiation states in other B-cell malignancies, we hypothesized that SCNAs of transcription factors may also underlie the etiology of these molecular subtypes. By testing for associations between SCNAs and cell of origin subtype, we identified three co-segregating DNA copy number gains that were significantly enriched in the ABC-like subtype. These included gains of the BCL6 and SPIB genes that have been previously observed to be associated with the ABC-like subtype. In addition, we found gains of the TCF4 (E2-2) gene to be significantly enriched in ABC-like tumors. In line with this, TCF4 alterations were significantly associated with reduced overall survival in cohorts of patients treated with either CHOP (n=232, P=0.009) or R-CHOP (n=197, P=0.041). B-cell receptor (BCR) signaling is a key survival pathway in ABC-like DLBCL, and the TCF4 gene has a defined role in promoting the expression of immunoglobulin (Ig) genes that encode the B-cell receptor (BCR). The analysis of paired SCNA and GEP data revealed a significantly higher expression of Ig genes in tumors with TCF4 DNA copy number gain compared to those without, suggesting that normal BCR expression may be deregulated by this genetic alteration. In addition, chromatin-immunoprecipitation sequencing (ChIP-seq) for TCF4 in ABC-like DLBCL cell lines also revealed binding of TCF4 to an Ig gene enhancer region. As BCR signaling can be altered by somatic mutations in the CARD11, CD79B and MYD88 genes, we evaluated the relative representation of these mutations and TCF4 DNA copy number gains using targeted deep sequencing of 124 DLBCL tumors. This revealed that TCF4 DNA copy number gains largely mutually excluded CARD11 mutations, but significantly co-segregated with both MYD88 (FDR=0.005) and CD79B (FDR=0.053) mutations. In addition, we observed significant co-segregation between CD79B and MYD88 mutations (FDR<0.001). This is particularly notable due to the preliminary associations between combined CD79B and MYD88 mutation status and response to an inhibitor of BCR signaling, Ibrutinib. Together these data highlight an association between SCNA of B-cell transcription factors and the differentiation state of the 'normal counterpart' of the respective malignant B-cell. In line with this, we show that DNA copy number gains of the TCF4 transcription factor are associated with the ABC-like subtype of DLBCL, significantly worse overall survival, and increased Ig expression. These characteristics, in addition to the co-association between TCF4 DNA copy number gains and somatic mutations of CD79B and MYD88, suggest that TCF4 may be an important modifier of BCR signaling and contribute to the etiology of ABC-like DLBCL. Disclosures Rosenquist: Gilead Sciences: Speakers Bureau. Lunning:TG Therapeutics: Consultancy; AbbVie: Consultancy; Gilead: Consultancy; Bristol-Myer-Squibb: Consultancy; Juno: Consultancy; Genentech: Consultancy; Spectrum: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

Activation of the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) in Aggressive B-Cell Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2038-2038
Author(s):  
Olga Balague ◽  
Luis Colomo ◽  
Armando Lopez-Guillermo ◽  
Elias Campo ◽  
Antonio Martinez
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factors ◽  
Western Blot ◽  
Er Stress ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Lymphoid Tissues ◽  
B Cell Lymphomas

Abstract BACKGROUND The UPR is a prosurvival pathway activated in cells under ER stress induced by the accumulation of unfolded proteins. UPR activation in B cells normally occurs during the differentiation to antibody secreting plasma cells and requires XBP1activation. XBP-1 is a member of the TREB family of transcription factors that exists in the endoplasmic reticulum (ER) as a 33kDa protein, and in the nucleus as an active 50kDa transcription factor. The UPR stimulates two different ER proteins, ATF-6 and Ire-1, to increase XBP-1 transcription and XBP-1 mRNA splicing resulting in the accumulation of the active 50kDa nuclear protein. Moreover XBP1 is a target of proteosome inhibitors and is related to the aggressive behaviour of some carcinomas. The role of the activation of XBP-1 in lymphomas is still unknown. DESIGN: Reactive lymphoid tissues and 25 neoplastic human B-cell lines representing different stages of B-cell development were studied for XBP-1 expression by western blot and XBP-1, PAX-5, Blimp-1/prdm1, MUM-1/IRF-4 and ICSBP1/IRF-8 by immunohistochemistry. XBP-1 activation was assessed in 225 B-cell lymphomas from the archives of the laboratory of pathology by western blot, RT-PCR and immunohistochemistry . To further evaluate whether XBP-1 activation was related to the plasmacytic program or to ER stress signals we analyzed the cell lines by Western blot for XBP-1 and ATF-6 expression. RESULTS We characterize XBP-1 expression in reactive lymphoid tissues, 25 human cell lines and 225 B-cell tumors. In nearly all tonsillar lymphoid cells XBP-1 was detected as a cytoplasmic protein with a paranuclear dot pattern. Nuclear positivity was observed only in scattered centrocytes in the light zone of the germinal centers and in plasma cells, always coexpressed with plasma cell related transcription factors as MUM-1/IRF-4 and Blimp1/prdm1. Active p50XBP-1 was found in 24/25 cell lines by western blot regardless ATF-6 expression and confirmed by immunohistochemistry . Moreover p50XBP1 was found in 27/31(87%) plasmacytomas, 36/64(56%) DLBCL-ABC and in 3/10(30%) DLBCL-GCB and 22/43(51%) plasmablastic lymphomas. Intriguingly, p50XBP1 was detected also in 2/11(18%)BL and 4/25(16%)MCL with blastic features. CONCLUSIONS.XBP-1 is activated in a subset of follicular centre cells committed to plasma cell differentiation and in plasma cells.UPR prosurvival pathways in the neoplastic cell lines are activated independently of the extent of the ATF-6 activation.p50XBP1 is mostly activated in aggressive B-cell lymphomas regardless to the plasmacytic differentiation of the tumours. Thus, p50XBP-1 may be a new molecular target in the treatment of aggressive B-cell malignancies.

Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 293-293 ◽  
Author(s):  
ChristoPher B Miller ◽  
Charles G Mullighan ◽  
Xiaoping Su ◽  
Jing Ma ◽  
Michael Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Point Mutation ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Lymphoid Development ◽  
B Lymphoid

Abstract Genes regulating B lymphoid development are somatically mutated in over 40% of B-progenitor acute lymphoblastic leukemia (ALL) cases, with the most common targets being the transcription factors PAX5, IKZF1 (encoding Ikaros), and EBF1. Notably, BCR-ABL1 ALL is characterized by a high frequency of mutations of IKZF1 (85%), PAX5 (55%), and CDKN2A/B (encoding INK4/ARF, 55%), suggesting that these lesions cooperate with BCR-ABL1 in lymphoid leukemogenesis. To examine cooperativity between Pax5 haploinsufficiency and BCR-ABL1, we transplanted Pax5+/+ and Pax5+/− bone marrow cells transduced with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 transduced Pax5+/− marrow developed B progenitor cell ALL with significantly higher penetrance and decreased latency when compared to animals transplanted with BCR-ABL1 transduced Pax5+/+ marrow (median survival 36 vs. 60 days, P=0.0003). The latency of tumor onset was further decreased in the presence of Arf haploinsufficiency (Pax5+/+Arf+/+ 60 days, Pax5+/−Arf+/+ 36 days, Pax5+/−Arf+/− 21 days, P&lt;0.0001). All leukemias were of B cell lineage and were transplantable to secondary recipients. In addition, Southern blot analysis revealed the Pax5+/−Arf+/+ leukemias to be monoclonal, where as the Pax5+/−Arf+/− leukemias were oligoclonal. Importantly, the Pax5+/− leukemias exhibited a more immature B cell immunophenotype than Pax5 wild type leukemias. Moreover, a proportion of the Pax5+/− leukemias (19%) exhibited a very immature early pro B cell immunophenotype (Cd19−, Bp1−), suggesting the possibility of acquired lesions in other key regulators of normal B cell differentiation. To explore this possibility and to identify the total complement of genetic lesions required to generate overt leukemia, we performed genome-wide copy number analysis on 30 murine leukemias (15 Pax5+/+, 15 Pax5+/−) using a custom CGH microarray (Agilent) that interrogated 477,000 autosomal loci, including 18,000 probes covering 20 genes encoding B lymphoid transcription factors and genes targeted by recurring copy number abnormalities (CNAs) in human BCR-ABL1 ALL (Bcl11a, Cdkn2a, Ebf1, Ikzf1, Ikzf2, Ikzf3, Il7r, Lef1, Mdm2, Mef2c, Myb, Pax5, Pten, Rb1, Sfpi1, Sox4, Stat5a, Tcf3, Tcf4, and Trp53). This analysis identified focal recurring CNAs in multiple genes including Cdkn2a/b, Ebf1, Ikzf1, Ikzf2, Ikzf3, and Pax5, each of which is a target of mutation in human B-ALL. Overall, there were on average 3.5 CNAs in Pax5+/+ leukemias versus 0.7 CNAs in Pax5+/− leukemias. Genomic resequencing was also performed on Pax5 and revealed three missense mutations in the DNA binding paired domain (R38H, P80R and G85R), one of which (P80R) is the most common PAX5 point mutation in human B-ALL. All three point mutations are predicted to impair DNA binding of Pax5. Interestingly, the majority of the pro-B cell leukemias that arose in the Pax5+/−Arf+/+ animals were found to harbor mutations (CNAs or point mutation) of the retained Pax5 allele, consistent with the immature immunophenotype. To further explore the relationship between our murine model and human BCR-ABL1 ALL, we performed gene expression profiling of Pax5+/+ and Pax5+/− leukemias and compared their signatures to those of human BCR-ABL1 ALL and stage-specific murine B lymphoid developmental signatures using gene set enrichment analysis (GSEA). This analysis identified significant similarity between murine and human BCR-ABL1 leukemias, thus providing further evidence that this model closely recapitulates human BCR-ABL1 ALL. Notably, Pax5+/− leukemias, or Pax5+/+ leukemias that acquired additional mutations of B-lymphoid regulators exhibited a less mature gene expression profile than leukemias lacking B-lymphoid regulatory mutations. These data indicate that loss of Pax5 contributes to leukemogenesis, that additional genomic alterations in genes regulating B lymphoid development and cell cycle regulators/tumor suppressors (Arf) are frequent events in BCR-ABL1 acute lymphoblastic leukemia, and that these lesions result in impaired B-lymphoid maturation in B-ALL. The genetic complexity of BCR-ABL1 ALL is likely to have important therapeutic implications for this poor prognosis subtype of leukemia.

A Targeted Functional Clone Tracking Assay for the Identification of Tumour Suppressor Genes in BCP- ALL Implicates the Transcription Factors FOXO3 and PRDM1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2449-2449
Author(s):  
Paul Sinclair ◽  
Joanna Cheng ◽  
Prahlad Raninga ◽  
Rebecca Hanna ◽  
Shaun Hollern ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Candidate Gene ◽  
Copy Number ◽  
B Cell Development ◽  
Tumour Suppressor ◽  
Tumour Suppressor Genes ◽  
Suppressor Genes

Abstract B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is accompanied by genomic mutations and rearrangements that commonly affect cytokines, transcription factors or signalling molecules that drive B-cell development or contribute to the pre-B cell receptor (pre-BCR) checkpoint. Deletions of the long arm of chromosome 6 [del(6q)] occur in ~10% of BCP-ALL and are also frequent in mature B and T-cell malignancies. Loss of function of the 6q genes EPHA7 and PRDM1, have been implicated in the genesis of lymphoma and BACH2, as a mediator of pre-BCR negative selection, is functionally a candidate tumour suppressor gene. However loss of these or other 6q genes have not been demonstrated, for example through biallelic inactivation, to contribute to BCP-ALL. Analysis of our own and published SNP6.0 data from ALL patients defined 5 focal recurrent regions of deletion on 6q, 4 mapping to 6q15-6q21, coincident with previously published common regions of deletion in ALL. These 4 regions contain 22 candidate genes, including EPHA7 but not BACH2 or PRDM1, which nevertheless mapped close to focal deletions and were also classed as candidate tumour suppressors. To develop the clone tracking assay, we adapted the SIN-SIEW lentiviral construct that expresses EGFP under the control of a spleen focus forming virus (SFFV) promoter. Candidate gene consensus coding sequence (CCDS) or a control luciferase cDNA were cloned between the promoter and an internal ribosomal entry site immediately upstream of EGFP. Transduction of the control (pSLIEW) or candidate gene SIN-SIEW-CCDS constructs consistently expressed EGFP in 697, a BCP-ALL cell line with del(6)(q14.1-22.3). For clone tracking, SIN-SIEW-CCDS constructs were assigned to 4 pools that also included pSLIEW. Pools were transduced into 697 cells that were both cultured in vitro and transplanted by intra-femoral injection into NOD/LtSz-scid IL2Rƴ null (NSG) mice. DNA was isolated from transduced cells immediately before transplant and then at 3 to 5 day intervals from cultured cells or from cells recovered from mouse bone marrow, spleen or liver at end stage disease. The pSLIEW construct facilitated monitoring of disease progression by in vivo imaging and also served as a control to measure CCDS construct copy number changes against. To quantify changes in integrated SIN-SIEW-CCDS, we developed a multiplex targeted Illumina sequencing approach. In vitro, highly significant (p<0.01) reductions in copy number relative to pSLIEW over time, occurred for constructs expressing FOXO3, POU3F2, SIM1, PRDM13, C6orf168 and both α and β isoforms of PRDM1 (Fig 1a). With the exception of C6orf168, these genes also strongly suppressed leukemia development in vivo in all tissues analysed (Fig 1b). The known tumour suppressor genes, BACH2 and EPHA7, had no effect on cell growth in vitro. In vivo a moderate reduction for one of two EPHA7 CCDS was observed though curiously cells expressing BACH2 increased in relative copy number by approximately 3 fold. RNA sequencing data from 697 and published array data for normal pre-B cells and cases of BCP-ALL showed no, or extremely low, levels of expression for POU3F2, SIM1, PRDM13 and C6orf168 making it unlikely that they function as tumour suppressor genes in BCP-ALL. However significant expression of the transcription factors FOXO3 and PRDM1 were seen across data sets. Western blot confirmed expression of FOXO3 and PRDM1 in 697 and other BCP-ALL cell lines and demonstrated substantial increases in the corresponding proteins after transduction of 697 with FOXO3 and PRDM1 SIN-SIEW CCDS constructs. Over-expression of FOXO3 and both isoforms of PRDM1 decreased the proportion of cells in S and G2 phases of the cell cycle, but failed to induce apoptosis as measured by Annexin-5 staining. Comparison of total mRNA sequencing profiles of 697 cells, FACS sorted for ectopic expression of FOXO3, PRDM1 or control construct, showed distinctive patterns of up or down regulated mRNA. The roles of FOXO3 and PRDM1 in early B-cell development are currently undefined but notably our data suggests they influence expression of components of the pre-BCR and related signalling pathways and therefore may contribute to the pre-BCR checkpoint. Disclosures No relevant conflicts of interest to declare.

Quantitative analysis of CKS1B mRNA expression and copy number gain in patients with plasma cell disorders

2014 ◽  
Vol 53 (3) ◽  
pp. 110-117 ◽  
Author(s):  
Flavia Stella ◽  
Estela Pedrazzini ◽  
Edgardo Baialardo ◽  
Dorotea Beatriz Fantl ◽  
Natalia Schutz ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Mrna Expression ◽  
Plasma Cell ◽  
Copy Number ◽  
Copy Number Gain ◽  
Plasma Cell Disorders

scholarly journals Chromatin Accessibility Profiling Reveals Cis-Regulatory Heterogeneity and Novel Transcription Factor Dependencies in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1313-1313
Author(s):  
Christopher J. Ott ◽  
Raphael Szalat ◽  
Matthew Lawlor ◽  
Mehmet Kemal Samur ◽  
Yan Xu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Board Of Directors ◽  
Plasma Cell ◽  
Therapeutic Target ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by clinical and genomic heterogeneity. Recurrent IgH translocations, copy number abnormalities and somatic mutations have been reported to participate in myelomagenesis; however no universal driver of the disease has been identified. Here, we hypothesize that transcriptional deregulation is critical for MM pathogenesis and the maintenance of the MM cell state. In order to capture signatures of transcription factor engagement with the myeloma epigenome, we performed the assay for transposase-accessible chromatin sequencing (ATAC sequencing), deep RNA sequencing in 23 primary myeloma samples and 5 normal plasma cell samples (NPC) from healthy donors along with whole genome sequencing and H3K27ac ChIP-seq in a cohort of these primary MM samples. We identified 22,603 variable accessible loci between MM and NPC and correlated impact of these on expression of associated genes using RNA-seq data. Together with robust differential analysis of open chromatin regions and nuclease-accessibility footprints to identify discrete transcription factor binding events, we have discerned the myeloma-specific open chromatin landscape, identified transcription factor dependencies and potential new myeloma drivers. In our dataset we observe a vast number of loci with heterogeneous chromatin states across the sample cohort, and the majority of the open chromatin sites identified are unique to a single sample. However, distinct variable chromatin accessibility signatures indicative of the MM chromatin state when compared to normal plasma cells were observed. Remarkably, we observed more frequent recurrent loss of variable accessible loci compared to gains. In addition, specific open chromatin profiles evident in hyperdiploid and non-hyperdiploid MM were also identified. Accessibility footprinting revealed MM-specific enrichment for transcription factors known to be essential for MM cell survival including Interferon Regulatory Factors (IRFs), Nuclear Factor Kappa B (NFkB), Ikaros, and Sp1. Interestingly, we also identify the myocyte enhancer factor 2 (MEF2) family of transcription factors as being specifically enriched in open chromatin regions in MM cells. Using a CRISPR-Cas9 knockout system, we identify the MEF2 family member MEF2C as essential for MM cell proliferation and survival. MEF2C is significantly overexpressed at the RNA level in our study as well as in several independent cohorts and is a central enhancer-localized transcription factor in MM core regulatory circuitry as determined by H3K27ac ChIP-sequencing profiles of primary MM samples. In order to evaluate MEF2C as a therapeutic target, we used small molecule inhibitors targeting MEF2C activity via inhibition of MEF2C phosphorylation using inhibitors of salt-induced kinases (SIK) and microtubule-associated protein/microtubule affinity regulating kinases (MARK). SIK/MARK have been described to specifically activate MEF2C. SIK and MARK inhibition resulted in both dose- and time-dependent inhibition of MM cell growth and survival in a panel of 12 MM cell lines with various genotypic and phenotypic characteristics, revealing a potential approach to targeting the dysregulated gene regulatory state of myeloma. To conclude, here we identify here an altered chromatin accessibility landscape in multiple myeloma that likely contributes to oncogenic transcription states through the activity of transcription factors such as MEF2C, representing a new MM dependency and potential therapeutic target. Disclosures Anderson: Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder. Young:Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director.

scholarly journals Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 861-865
Author(s):  
J Epstein ◽  
B Barlogie ◽  
J Katzmann ◽  
R Alexanian
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Tumor Cells ◽  
Plasma Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Markers ◽  
Aneuploid Tumor ◽  
Beta 2 Microglobulin ◽  
Normal Differentiation ◽  
Mature Plasma Cell

The expression of early and mature B cell markers, surface beta 2- microglobulin (B2M) and cytoplasmic immunoglobulin (clg) by aneuploid tumor cells in bone marrow aspirates from 44 patients with multiple myeloma was evaluated by correlated DNA immunofluorescence flow cytometry. Myeloma tumor cells of almost 90% of the patients contained monoclonal clg and expressed the mature plasma cell antigen R1–3 as well as surface B2M; common acute lymphoblastic leukemia antigen (CALLA) was present in 55%, B2 in 17%, and B4 in 23% of samples studied. Coexpression of CALLA and clg in 46% of all patients identified a novel myeloma phenotype without known counterpart in the normal differentiation of B cells. CALLA and clg were independently expressed and gave rise to CALLA+/clg-, CALLA+/clg+, and CALLA-/clg+ cells. The association of CALLA and mature plasma cell markers may define discrete stages of neoplastic plasma cell differentiation.

scholarly journals Chromatin Content Capture Reveals Acute Leukaemia Oncogenic Vulnerability Point in Human B Cell Development

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 673-673
Author(s):  
Reema Baskar ◽  
Patricia Favaro ◽  
Warren D. Reynolds ◽  
Pablo Domizi ◽  
Albert G Tsai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
High Activity ◽  
Chromatin Structure ◽  
Chromatin Accessibility ◽  
Cell Development ◽  
B Cell Development ◽  
Human B Cell

Abstract Human B cell development in adult human bone marrow (BM) is tightly regulated through well-defined stages to produce adaptive immune cells with assembled and functional B cell antigen receptor (BCR)(Martin et al., 2016). To produce mature B cells with functional immunoglobulin receptors, B cell progenitors must undergo multiple stages of highly regulated chromatin remodelling and transcriptional reprogramming which correspond to unique patterns of surface protein expression (Nutt and Kee, 2007). This complex process is frequently dysregulated in B cell neoplasia such as B cell Acute Lymphoblastic Leukemia (B-ALL). B-ALL is highly heterogenous in its phenotypic and clinical presentation, as well as in its underlying molecular features such as DNA methylation patterns and genetic aberrations (Cobaleda and Sánchez-García, 2009). The lack of general mechanism of leukemogenesis has made it difficult to identify when and where adult and pediatric B-ALL blasts diverge from normal B cell development. Here we show that across 5 B-ALL patients and 3 cell lines with diverse phenotypic and clinical presentations, blasts are epigenetically arrested at a conserved point within healthy human B cell development. First, we sought to establish a trajectory of normal B cell development to delineate the phenotypic and concomitant epigenetic changes occurring in BM progenitors as they differentiate into naïve B cells. To capture phenotype, function, and epigenetic state via single cell chromatin content (chromotype) of developing B cells in BM, we developed a multiplexed, high throughput, single cell proteomic method (chromotyping) to simultaneously measure cell surface markers, intracellular regulators such as transcription factors and chromatin structure regulators such as histone post-translational modifications (i.e. H3K4me3, H3K27me3, H2AK119ubi) and chromatin re-modelers (i.e. CTCF, DNMT1, MLL1). Using these surrogates for single cell, global chromatin content, we notably identified 3 coordinated epigenetic inflection or switch (S) points in healthy B cell development corresponding to previously characterized phenotypic landmarks of STAT5 signalling and active re-arrangement of IgH loci (S1), CD24 expression-linked high translation and proliferation (S2), and IgM and CD20 expression-linked BCR assembly completion (S3) (Bendall et al., 2014). To determine how these coordinated chromotypes translated to chromatin accessibility and primed gene regulation networks, we isolated BM B cell population from these chromatin content transition points and analysed them with our modified ATAC-seq protocol, InTAC-seq (Baskar et al., 2021). Strikingly, the chromatin accessibility landscape revealed putative oncogenic priming with high activity of leukemic TFs such as PAX5, TCF3, ZEB1 and ID4 predominantly at S2 and some at S3 switch points. By integrating our InTAC-seq data with publicly available single cell ATAC and RNA seq data on BM, we located this oncogenic primed state as existing from S2 to before S3 (IgH rearranged, late pro- / Pre-B cell stage) in healthy B cell development. This integration further associated this state with high activity of ASCL1 (role in chromatin remodelling) and high expression of STMN1 (Leukaemia-associated phosphoprotein 18). Finally we showed that across B-ALL patients (n=5) and cell lines (REH, NALM6, SUBP15), chromatin accessibility of neoplastic B cells indeed continue to occupy this point of oncogenic vulnerability in the B cell developmental space from S2 to right before S3 in our integrated scATAC map, despite variable immunophenotypes. This corresponds to a coordinated minima in our chromotyping map (lowest, coordinated abundance of chromatin structure regulators across trajectory). Further analysis of B-ALL patients reinforced the divergence between immunophenotypic and epigenetic heterogeneity within and between samples. Taken together, our findings identify key epigenetic switch points in B cell development and their underlying chromatin accessibility and gene expression patterns. Consequently, we reveal a point of epigenetic vulnerability in healthy B cell development that could be predisposed to leukemic transformation. This work opens up the possibility for new diagnostic strategies for B-ALL utilizing chromatin content and could pave the way for epigenetic modulation-based treatments beyond DNA methylation inhibition. Disclosures Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

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