B-cell Identity Research Articles

Pax5 Heterozygosity Affects B-Cell Differentiation and Generates a Deregulated Precursor Population in the Bone Marrow

Introduction: PAX5 is a well-known master regulator for B-cell development, commitment and identity. Hence, germline or somatic deregulation of PAX5 facilitates the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Previously, our group has described B-cell development abnormalities in three families carrying PAX5 germline variants ( Escudero et al., Leukemia, 2022) as well as established a link between Pax5 heterozygosity and infection exposure, thereby recapitulating the incomplete penetrance of the human BCP-ALL ( Martin-Lorenzo et al., Cancer Discovery, 2015). Nevertheless, the molecular single-cell characteristics of the susceptible precursor population mediated by reduced Pax5 levels is still poorly understood. Methods: Here, we deeply characterized the susceptible precursor population in Pax5+/- mice utilizing single-cell RNA Sequencing (scRNA-Seq), multicolor flow cytometry analyses and in-vivo transplantation models. Results: Our data show, that Pax5 heterozygosity leads to an aberrant pre-BII population (B220 +CD19 +IgM -CD25 +) in the bone marrow (BM) of Pax5+/- mice (p=0.0026, Student's t-test) (Fig. 1A). This population is stable over time and characterized by higher CD25 and IL-7-Receptor expression (p<0.0001, Student's t-test). Furthermore, bulk RNA-Sequencing of the pre-BII population revealed B-cell receptor (BCR) signaling as one of the top downregulated pathways (including CD79a/b, Lyn and CD72), while DNA replication and cell cycle signaling pathways were upregulated in Pax5+/- vs. Wildtype (WT) pre-BII cells. ScRNA-Seq analyses confirmed these results (Fig. 1B) and further showed that pre-BII cells of Pax5+/- mice are skewed toward the less commonly used lambda light chain BCR-rearrangements. The observed delay in the transition to IgM-positivity was additionally validated in a murine in-vivo transplantation model. Here, after 72 hours pre-BII cells from Pax5+/- mice preferably homed to the BM, while matured transplanted WT cells were predominantly found in the spleen (p=0.0023 and p=0.0140, respectively, Student's t-test). To depict the full spectrum of malignant transformation based on the described deregulated precursor B-cell population, we additionally performed scRNA-Seq on different stages of Pax5-mediated BCP-ALL evolution. Our data show that in a first step, arrested pre-leukemic cells of Pax5+/- mice lose their B-cell identity and display high Myc levels. Sequentially, full blown BCP-ALL arises in a second step after acquisition of additional oncogenic driver mutations (including Jak1 and Jak3). Interestingly, although the analyzed BCP-ALLs of Pax5+/- mice were all based on the same predisposition, they greatly varied in their transcriptional profile and cell-cycle state, depending on their oncogenic 2 nd hit. Conclusion: In summary, we deeply characterized how reduced Pax5 transcriptional activity in the BM generates a predisposed precursor B-cell environment, which is susceptibility for malignant transformation. These findings are important for understanding the molecular mechanisms to prevent or treat a significant proportion of childhood BCP-ALLs.

Functional inhibition of MEF2 by C/EBP is a possible mechanism of leukemia development by CEBP-IGH fusion gene.

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n=8) compared with other B-ALL (n=315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.

Determinants of Resistance to Engineered T-Cell Therapies Targeting CD19 in Large B-Cell Lymphomas

Background: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have activity in patients with relapsed/refractory large B-cell lymphoma (rrLBCL), but over half of patients ultimately relapse. We originally applied cell-free DNA (cfDNA) analysis to a cohort (n=65) of patients receiving axicabtagene ciloleucel (axi-cel) to identify determinants of resistance and characterize molecular thresholds predictive of treatment failure. Here, we extend these analyses to an additional independent cohort (n=73) of patients to validate these thresholds and refine genomic alterations associated with resistance. Methods: We developed a novel hybrid capture strategy targeting 187 protein coding genes, all T-cell receptor (TCR) loci, and axi-cel retroviral vector. This approach allows for simultaneous evaluation of circulating tumor-derived DNA (ctDNA), CAR19-derived cfDNA (cfCAR) and T-cell receptor rearrangements (cfTCR) from blood plasma. We applied this platform to profile serial samples from discovery (n=65) and validation (n=73) cohorts of rrLBCL patients receiving CAR19 therapy (total n=128). Pretreatment and relapse tumor tissue was also profiled when available. A previously determined optimized pretreatment (Day 0) ctDNA threshold to stratify event free survival (EFS) was revalidated in the discovery cohort using bootstrap resampling (2.5 log10 haploid genome equivalents per milliliter). Results: The median follow-up was 36.4 months in the discovery cohort, and 12.5 months in the validation cohort. 55% (36/65) and 58% (42/73) of patients in the discovery and validation cohorts had progressed following CAR19, respectively. In both cohorts, patients with high pretreatment ctDNA demonstrated inferior EFS (Discovery: p<0.001, Validation, p=0.002). Similarly, a ~300-fold (2.5 log10) decrease in ctDNA levels at Day 28 (Major Molecular Response - MMR) post CAR19 infusion was identified as the optimal post-treatment change to stratify EFS, and patients in both cohorts who achieved MMR had significantly superior EFS (Discovery: p<0.001, Validation, p=0.028). When considering ctDNA, cfTCR and cfCAR as continuous variables in the discovery cohort, higher pre- and post-treatment ctDNA levels were prognostic of EFS at all assayed time-points, as were pre-treatment LDH, week 1 cfCAR19 and week 4 cfTCR levels (Fig. A). Forward stepwise multivariable regressions identified week 4 ctDNA level (HR=2.2, p<0.001) and week 1 cfCAR19 level (HR=0.6, p=0.017) as the optimal independent covariates for predicting outcomes (Fig. A). A Cox proportional hazards model incorporating these two variables was trained on the discovery cohort (CAR Outcome Score) and was validated to be prognostic for outcomes in the validation cohort (HR=2.7, p<0.001). Genomic alterations in several genes were significantly associated with inferior outcomes in both cohorts, including PAX5, IRF8, TMEM30A, PPM1D and TP53 (Fig. B). Additional, non-stereotyped, emergent alterations were noted in relapsing patients in other genes associated with immunological escape, including mutations in CD19 and amplifications in the PD-L1 locus. CIBERSORTx was used to characterize intratumoral cell type composition, which was found to be influenced by tumor genotypes. Further, CAR19 persistence in tumors at relapse was associated with specific changes in the tumor microenvironment, including regulatory T-cell expansion and B-cell depletion. Conclusions: Pre-treatment and dynamic ctDNA levels predict treatment failure of LBCL after CAR19 therapy, and a novel CAR outcome score incorporating week 4 ctDNA and week 1 cfCAR19 levels is strongly predictive of outcome. Genomic determinants of resistance to CAR19 therapy are diverse, including recurrent mutations in genes controlling B-cell identity (IRF8, PAX5), and those influencing the composition of the tumor microenvironment and heterotypic cell-cell interactions (TMEM30A). Additional immunological escape mechanisms arise under selective pressure from CAR19 cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of PHF6 in Hematopoiesis and Hematologic Malignancies.

Epigenetic regulation of gene expression represents an important mechanism in the maintenance of stem cell function. Alterations in epigenetic regulation contribute to the pathogenesis of hematological malignancies. Plant homeodomain finger protein 6 (PHF6) is a member of the plant homeodomain (PHD)-like zinc finger family of proteins that is involved in transcriptional regulation through the modification of the chromatin state. Germline mutation of PHF6 is the causative genetic alteration of the X-linked mental retardation Borjeson-Forssman-Lehmann syndrome (BFLS). Somatic mutations in PHF6 are identified in human leukemia, such as adult T-cell acute lymphoblastic leukemia (T-ALL, ~ 38%), pediatric T-ALL (~ 16%), acute myeloid leukemia (AML, ~ 3%), chronic myeloid leukemia (CML, ~ 2.5%), mixed phenotype acute leukemia (MPAL, ~ 20%), and high-grade B-cell lymphoma (HGBCL, ~ 3%). More recent studies imply an oncogenic effect of PHF6 in B-cell acute lymphoblastic leukemia (B-ALL) and solid tumors. These data demonstrate that PHF6 could act as a double-edged sword, either a tumor suppressor or an oncogene, in a lineage-dependent manner. However, the underlying mechanisms of PHF6 in normal hematopoiesis and leukemogenesis remain largely unknown. In this review, we summarize current knowledge of PHF6, emphasizing the role of PHF6 in hematological malignancies. Epigenetic regulation of PHF6 in B-ALL. PHF6 maintains a chromatin structure that is permissive to B-cell identity genes, but not T-cell-specific genes (left). Loss of PHF6 leads to aberrant expression of B-cell- and T-cell-specific genes resulting from lineage promiscuity and binding of T-cell transcription factors (right).

Pharmacological targeting EZH2 to modulate chronic graft-versus-host disease

Pharmacological targeting EZH2 to modulate chronic graft-versus-host disease

Ikaros Mediates Antigen Escape Following CD19 CAR T Cell Therapy in r/r B-ALL

Despite impressive response to front-line treatment, relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) remains one of the most common causes of death in children with cancer. In pediatric and adult r/r B-ALL, CD19 CAR T cell (CART19) therapy has shown impressive results with over 80% of patients achieving complete remission. However, as an unintended consequence of long-term persistence of CAR T cells and the resulting sustained pressure against CD19 expressing cells, 30 - 70% of initial responders will eventually relapse with CD19 loss. Early identification of patients that will respond successfully to CART19 therapy vs. those at risk for CD19 Neg relapse would support clinical decision making and improve patient care.To address this problem, we profiled by mass cytometry (CyTOF) 25 pre-CART19 samples including patients that achieved a durable complete remission (CR, n = 6), patients that underwent CD19 Neg (n = 11) or CD19 Pos relapse (n = 4) and patients that were refractory to CART19 (n = 4). For 14 of those patients, we also analyzed paired post-CART19 samples: 8 from CD19 Neg relapse, 2 from CD19 Pos relapse, and 4 from refractory tumors. We observed a significant increase in the proportion of leukemic early progenitor non-B cells (called early-non-BI) after CD19 Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. In addition, in the pre-CART19 samples we identified a subpopulation of CD19 Pos pro-B cells that resemble early-non-BI cells. We hypothesized that these pro-B “discordant” cells are able to lose CD19 expression and escape the immune pressure exerted by the CART19, leading to CD19 Neg relapse. Closer analysis revealed lower levels of IKAROS in pro-B discordant cells from patients that underwent CD19 Neg relapse, than in those that achieved a durable CR.To further explore the difference between pro-B discordant cells from patients that achieved a durable CR or underwent CD19 Neg relapse, we performed single cell REAP-seq (RNA expression and protein sequencing) from 3 pre-CART19 CR and 3 pre-CART19 CD19 Neg relapse r/r B-ALL samples. From this analysis, we confirmed a subpopulation of leukemic pro-B like cells with decreased expression of IKZF1 in those patients that underwent CD19 Neg relapse.IKAROS is a transcription factor that plays a crucial role in B cell lineage specification and commitment and is a known B-ALL tumor suppressor. Genetic alterations affecting the IKAROS gene, IKZF1, have been associated with poor response to front-line treatment and they are a factor in relapse risk stratification. However, a role for IKAROS in CART19 failure has not been described. To assess if there is a correlation between the levels of IKAROS and CD19 surface expression, we manipulated IKAROS levels in B-ALL cell lines through RNA silencing and targeted protein degradation approaches. Consistently, we observed that CD19 surface expression diminishes upon IKAROS downregulation. To determine how IKAROS regulates CD19 expression, we performed CyTOF, RNA-seq and ATAC-seq analysis of isogenic B-ALL cell lines with wild-type or reduced (IKAROS KD) IKAROS levels. While the chromatin accessibility to CD19 regulatory regions did not change, CD19 mRNA levels were lower in IKAROS KD cells. Interestingly, we found that IKAROS regulates several genes associated with mRNA splicing. In particular, CD19 mRNA preferentially retains intron 10 in IKAROS KD cells. The inclusion of intron 10 introduces several premature stop codons. Moreover, IKAROS KD B-ALL cells more closely resemble myeloid cells based on their gene expression profiles, suggesting a loss in their B cell identity.In summary, this is the first description of a leukemia-specific subpopulation of CD19 Pos pro-B discordant cells in patients prior CART19 administration. Furthermore, lower levels of IKAROS in this subpopulation correlates with poor response to CART19 therapy. Preliminary molecular analysis suggests that low IKAROS result in retention of CD19 intron 10 and loss of B-cell identity (Figure 1). Future research will focus on in-depth characterization of the myeloid-like phenotype induced by low levels of IKAROS expression, and the impact that intron 10 retention has on CD19 mRNA stability and surface expression. [Display omitted] DisclosuresMullighan: Pfizer: Research Funding; Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Barrett: Tmunity Therapeutics: Current Employment. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers.

BackgroundThe most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping “cell-of-origin”. Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry.MethodsWe compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naïve and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays).FindingsFrom the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation.InterpretationOur study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma.FundingNational Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation.

The diversity of the plasmablast signature across species and experimental conditions: A meta-analysis.

Antibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states.

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

Loss of Synergistic Transcriptional Feedback Loops Drives Diverse B-Cell Cancers

Background: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping “cell-of-origin”. Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. Methods: We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naive and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). Findings: From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. Interpretation: Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. Funding: National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation. Declaration of Interest: None to declare. Ethical Approval: De-identified peripheral blood, lymph node biopsies, and excised tonsils were obtained from FL, DLBCL, or CLL or healthy tonsillectomy patients (Table 1 and Fig. S1A) seen at Washington University School of Medicine who provided informed consent under IRB-approved protocols in concordance with the Declaration of Helsinki. Due to yield constraints, some samples were not subjected to all assays.

Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

AbstractHematopoietic cell kinase (HCK) is an SRC family member that is aberrantly upregulated in B-cell neoplasms dependent on MYD88-activating mutations and supports their growth and survival. We showed herein that activation of Toll-like receptor (TLR) signaling in MYD88 wild-type B cells also triggered HCK expression, denoting on path regulatory function for HCK by MYD88. To clarify the signaling cascades responsible for aberrant HCK expression in MYD88-mutated B-cell lymphomas, we performed promoter-binding transcription factor (TF) profiling, PROMO weighted TF consensus binding motif analysis, and chromatin immunoprecipitation studies. We identified PAX5, and the mutated MYD88 downstream signaling mediators STAT3, NF-κB, and AP-1, as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88-mutated lymphoma cells. Among AP-1 complex components, JunB showed greatest relevance to TLR/MYD88 signaling and HCK transcription regulation. In MYD88-mutated Waldenström macroglobulinemia and activated B-cell-diffuse large B-cell lymphoma cells, knockdown of MYD88 reduced phosphorylation of JunB but not c-Jun, and knockdown of JunB reduced HCK protein levels. Deletion of STAT3, NF-κB, and AP-1 binding sites reduced corresponding TFs binding and HCK promoter activity. Moreover, inhibitors to STAT3, NF-κB, and AP-1 reduced HCK promoter activity and messenger RNA levels, particularly in combination, in MYD88-mutated lymphoma cells. The findings provide new insights into the transcriptional regulation of HCK prosurvival signaling by mutated MYD88, and the importance of JunB as a downstream mediator of the MYD88-directed signaling apparatus.

Mutated MYD88 Regulates HCK Pro-Survival Signaling through JunB in MYD88 Mutated B-Lymphoma Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies. Disclosures Hunter: Janssen: Consultancy. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

1028 - B-Cell Identity as A Metabolic Barrier Against Malignant Transformation

1028 - B-Cell Identity as A Metabolic Barrier Against Malignant Transformation

‘Big bang’ of B-cell development revealed

Earlier studies have identified transcription factors that specify B-cell fate, but the underlying mechanisms remain to be revealed. Two new studies by Miyai and colleagues (pp. 112-126) and Li and colleagues (pp. 96-111) in this issue of Genes & Development provide new and unprecedented insights into the genetic and epigenetic mechanisms that establish B-cell identity.

Pax5 as a potential candidate marker for canine B-cell lymphoma

Immunophenotyping is a valuable method for prognosis in canine malignant lymphoma. The general B-cell marker is CD79a; however, Pax5 or B-cell specific activator protein, a transcription factor that controls B-cell identity and cell maturation, could also be used as a B-cell indicator in canine lymphomas. This study aimed to use Pax5, CD79a and CD3 expression in immunohistochemistry of spontaneous canine lymphomas, in order to carry out diagnosis and histopathological classification according to the World Health Organization guidelines. Forty-six retrospective cases including 33 multicentric, eight extranodal, and five alimentary lymphomas in dogs were immunostained by anti-Pax5 and anti-CD79a antibodies for B-cell identification, and anti-CD3 antibody for T-cell identification. T-cell lymphomas (CD3+/Pax5–/CD79a–) accounted for 30.43% of cases (14/46), and four of the lymphomas (28.57%) presented with CD3+/Pax5–/CD79a+. Conversely, B-cell lymphomas (CD3–/Pax5+/CD79a+) accounted for 69.57% of cases (32/46) and 12.5% of these (4/32) showed only Pax5-positive cells (CD3–/Pax5+/CD79a–). Therefore, in dogs, Pax5 appears to be a more useful marker for staining all B-cell subtypes compared to CD79a. Immunophenotyping with both Pax5 and CD3 are necessary for lymphoid lineage identification in canine lymphomas.

Transcriptional Control of Glucose and Energy Supply Prevents Oncogenic Signaling and B Cell Transformation

Background and Hypothesis: B-cell identity is determined by a set of B-cell transcription factors including PAX5, IKZF1 and EBF1. However, B-lineage leukemia clones often carry secondary genetic lesions that result in reduced activity or inactivation of these transcription factors. Studying patient samples from clinical trials for B-lineage childhood (P9906; n=187) and adult (MDACC; n=92) leukemia, we found that genetic defects in one or more B-cell transcription factors represent near-obligate lesions in human acute lymphoblastic leukemia (209 of 279 B-lineage ALL cases). While previously of unknown significance, we found that adult ALL cases with known lesions in one or more of these transcription factors had higher activity of lactate dehydrogenase, and phospho-states indicated higher activity of IRS1, PDK1, and AKT, which contribute to glucose uptake. For this reason, we investigated whether B-cell transcription factors set metabolic constraints of oncogenic signaling in leukemia.Results: Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells carrying PAX5 and IKZF1deletions decreased phospho-AKT levels. Furthermore, protein levels of multiple positive regulators involved in glucose uptake and metabolism (including insulin receptor, glucose transporters and hexokinases) were downregulated upon reconstitution of PAX5 and IKZF1. Conversely, protein levels of negative regulators of glucose uptake and metabolism including NR3C1 and TXNIP were upregulated upon reinstatement of PAX5 and IKZF1 function. Analysis of ChIPseq data of human B cells revealed binding of multiple B-cell transcription factors including PAX5, IKZF1 and EBF1 to promoter regions of genes encoding positive regulators of glucose uptake and metabolism. Binding peaks for B-cell transcription factors were also observed at genes that encode negative regulators of glucose uptake and metabolism (NR3C1 and TXNIP). In addition, direct recruitment of PAX5 was confirmed by single-locus quantitative chromatin immunoprecipitation (qChIP).Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells caused depletion from the cell culture in competitive growth assays, in parallel with reduced glucose consumption and depletion of cellular ATP levels. On the contrary, overexpression of dominant-negative PAX5-ETV6 and IK6 in patient-derived B-lineage ALL cells expressing wildtype PAX5 and IKZF1 resulted in a net survival advantage, concomitant with increases in both glycolytic activity and cellular ATP levels. PAX5-mediated impaired survival fitness was significantly rescued by CRISPR-based activation of gene expression of insulin receptor, glucose transporter and hexokinases in patient-derived B-lineage ALL cells. Conversely, CRISPR/Cas9-mediated deletion of NR3C1 and TXNIP largely reversed the effects of PAX5. Finally, reduced survival fitness upon reconstitution of wild-type PAX5 and IKZF1 in patient-derived pre-B ALL cells was mostly rescued by metabolites that can enter the TCA cycle and thus provide ATP.Conclusions: In summary,B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation. Our findings suggest a causative link between impaired glucose uptake and metabolism caused by B-cell transcription factors as well as cell death, and the known tumor suppressive function of PAX5 and IKZF1 in B-lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Abstract A44: The role of PHF6 in maintaining pre-B cell commitment in B-cell acute lymphoblastic leukemia

Abstract Loss of function mutations in the plant homeodomain factor 6 (PHF6) are responsible for the Börjeson–Forssman–Lehmann syndrome, a familial X-linked intellectual disability disorder, and are observed in approximately 20% of adult T-cell acute lymphoblastic leukemias (T-ALLs) and 3% of adult acute myeloid leukemias (AMLs). However, mutations in B-cell lineage malignancies are notably absent. Interestingly, our recent work has uncovered PHF6 as a positive growth regulator in B-cell acute lymphoblastic leukemia (B-ALL) through a genome-scale in vivo loss-of-function screen. To identify the molecular mechanism by which PHF6 acts to promote B-ALL growth in vivo, we utilized CRISPR-Cas9 to delete Phf6 in murine B-ALL cells. Transplantation of Phf6 knockout cells (Phf6KO) into immunocompetent syngeneic recipients significantly extends disease latency and survival, therefore validating PHF6 as a bona fide positive growth regulator of B-ALL in vivo. Strikingly, these mice develop lymphoma-like disease with complete penetrance, characterized by significantly enlarged lymph nodes, decreased disease burden in the spleen and increased expression of the canonical T-cell marker CD4, suggesting that Phf6KO B-ALL cells transdifferentiated to cells resembling those of the T-cell lineage. To dissect the mechanism by which PHF6 regulates this lineage decision, we carried out a combination of RNA sequencing and chromatin immunoprecipitation (ChIP) analyses in Phf6WT and Phf6KO cells. RNA sequencing analysis revealed many differentially expressed genes in Phf6KO B-ALL cells , including gene sets involved in pathways important for B-cell development. ChIP-sequencing analysis of PHF6 and several histone marks (H3K27Ac, H3K27me3, H3K4me3) in Phf6WT B-ALL cells revealed that PHF6 and H3K27Ac signals co-localize close to the transcription start site of a significant proportion of differentially expressed genes. Transcription factor binding motif analysis revealed significant enrichment for several well-described master regulators of B-cell development, including PU.1, EGR-1, EBF-1, NF-κB and TCF3/TCF12. Notably, a number of these predicted transcription factors co-immunoprecipitated with PHF6 in Phf6WT B-ALL cells. These findings reveal an essential role for PHF6 in the maintenance of B-cell identity in B-ALL by activating, directly or indirectly, genes that are crucial for B-cell lineage commitment. Collectively, these results indicate that loss-of-function of PHF6 in B-ALL leads to transdifferentiation to the T-cell lineage, potentially explaining the apparent absence of PHF6 mutations in human B cell-lineage malignancies. Citation Format: Yadira M. Soto-Feliciano, Jordan ME Bartlebaugh, Yunpeng Liu, Francisco J. Sánchez-Rivera, Abraham S. Weintraub, Arjun Bhutkar, Tyler E. Jacks, Richard A. Young, Michael T. Hemann. The role of PHF6 in maintaining pre-B cell commitment in B-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A44.

ZNF423 and ZNF521: EBF1 Antagonists of Potential Relevance in B-Lymphoid Malignancies.

The development of the B-lymphoid cell lineage is tightly controlled by the concerted action of a network of transcriptional and epigenetic regulators. EBF1, a central component of this network, is essential for B-lymphoid specification and commitment as well as for the maintenance of the B-cell identity. Genetic alterations causing loss of function of these B-lymphopoiesis regulators have been implicated in the pathogenesis of B-lymphoid malignancies, with particular regard to B-cell acute lymphoblastic leukaemias (B-ALLs), where their presence is frequently detected. The activity of the B-cell regulatory network may also be disrupted by the aberrant expression of inhibitory molecules. In particular, two multi-zinc finger transcription cofactors named ZNF423 and ZNF521 have been characterised as potent inhibitors of EBF1 and are emerging as potentially relevant contributors to the development of B-cell leukaemias. Here we will briefly review the current knowledge of these factors and discuss the importance of their functional cross talk with EBF1 in the development of B-cell malignancies.

The regulatory network of B-cell differentiation: a focused view of early B-cell factor 1 function.

During the last decades, many studies have investigated the transcriptional and epigenetic regulation of lineage decision in the hematopoietic system. These efforts led to a model in which extrinsic signals and intrinsic cues establish a permissive chromatin context upon which a regulatory network of transcription factors and epigenetic modifiers act to guide the differentiation of hematopoietic lineages. These networks include lineage-specific factors that further modify the epigenetic landscape and promote the generation of specific cell types. The process of B lymphopoiesis requires a set of transcription factors, including Ikaros, PU.1, E2A, and FoxO1 to ‘prime’ cis-regulatory regions for subsequent activation by the B-lineage-specific transcription factors EBF1 and Pax-5. The expression of EBF1 is initiated by the combined action of E2A and FoxO1, and it is further enhanced and maintained by several positive feedback loops that include Pax-5 and IL-7 signaling. EBF1 acts in concert with Ikaros, PU.1, Runx1, E2A, FoxO1, and Pax-5 to establish the B cell-specific transcription profile. EBF1 and Pax-5 also collaborate to repress alternative cell fates and lock cells into the B-lineage fate. In addition to the functions of EBF1 in establishing and maintaining B-cell identity, EBF1 is required to coordinate differentiation with cell proliferation and survival.

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.