Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma.

Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.

The RNA helicases DDX19A/B modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export in leukemia cells.

Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.

Oncogenic dependency on SWI/SNF chromatin remodeling factors in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy arising from immature thymocytes. Unlike well-known oncogenic transcription factors, such as NOTCH1 and MYC, the involvement of chromatin remodeling factors in T-ALL pathogenesis is poorly understood. Here, we provide compelling evidence on how SWI/SNF chromatin remodeling complex contributes to human T-ALL pathogenesis. Integrative analysis of transcriptomic and ATAC-Seq datasets revealed high expression of SMARCA4, one of the subunits of the SWI/SNF complex, in T-ALL patient samples and cell lines compared to normal T cells. Loss of SMARCA protein function resulted in apoptosis induction and growth inhibition in multiple T-ALL cell lines. ATAC-Seq analysis revealed a massive reduction in chromatin accessibility across the genome after the loss of SMARCA protein function. RUNX1 interacts with SMARCA4 protein and co-occupies the same genomic regions. Importantly, the NOTCH1-MYC pathway was primarily affected when SMARCA protein function was impaired, implicating SWI/SNF as a novel therapeutic target.

Acquired copy number amplification at the MYC enhancer in human B-precursor acute lymphoblastic leukemia cell lines.

Our study highlights the discovery of recurrent copy number alterations in noncoding regions, specifically blood enhancer cluster (BENC-CNA), in B-precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. We demonstrate that BENC-CNA acts as a super-enhancer, driving MYC expression and possibly contributing to the immortalization and proliferative advantage of BCP-ALL cells in vitro.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Deciphering the regulatory landscape of murine splenic response to anemic stress at single-cell resolution.

ABSTRACT: Stress erythropoiesis can be influenced by multiple mediators through both intrinsic and extrinsic mechanisms in early erythroid precursors. Single-cell RNA sequencing was conducted on spleen tissue isolated from mice subjected to phenylhydrazine and serial bleeding to explore novel molecular mechanisms of stress erythropoiesis. Our results showed prominent emergence of early erythroblast populations under both modes of anemic stress. Analysis of gene expression revealed distinct phases during the development of emerging erythroid cells. Interestingly, we observed the presence of a "hiatus" subpopulation characterized by relatively low level of transcriptional activities that transitions between early stages of emerging erythroid cells, with moderate protein synthesis activities. Moreover, single-cell analysis conducted on macrophage populations revealed distinct transcriptional programs in Vcam1+ macrophages under stress. Notably, a novel marker, CD81, was identified for labeling central macrophages in erythroblastic islands (EBIs), which is functionally required for EBIs to combat anemic stress. These findings offer fresh insights into the intrinsic and extrinsic pathways of early erythroblasts' response to stress, potentially informing the development of innovative therapeutic approaches for addressing anemic-related conditions.

ASCL1 characterizes adrenergic neuroblastoma via its pioneer function and cooperation with core regulatory circuit factors.

Neuroblastoma originates from developing neural crest and can interconvert between the mesenchymal (MES) and adrenergic (ADRN) states, each of which are controlled by different sets of transcription factors forming the core regulatory circuit (CRC). However, the roles of CRC factors in induction and maintenance of specific state are poorly understood. Here, we demonstrate that overexpression of ASCL1, an ADRN CRC factor, in MES neuroblastoma cells opens closed chromatin at the promoters of key ADRN genes, accompanied by epigenetic activation and establishment of enhancer-promoter interactions, initiating the ADRN gene expression program. ASCL1 inhibits the transforming growth factor ß-SMAD2/3 pathway but activates the bone morphogenetic protein SMAD1-ID3/4 pathway. ASCL1 and other CRC members potentiate each other's activity, increasing the expression of the original targets and inducing a new set of genes, thereby fully inducing the ADRN program. Our results demonstrate that ASCL1 serves as a pioneer factor and cooperates with CRC factors to characterize the ADRN gene expression program.

Regulatory mechanisms and context-dependent roles of TAL1 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy derived from thymic T-cell precursors. Approximately 40-60% of T-ALL cases exhibit aberrant overexpression of the TAL1 oncogenic transcription factor. Here, we provide a comprehensive view of the TAL1-induced transcriptional program in human T-ALL cells using a rapid protein degradation system coupled with integrative approaches. Our study demonstrates that TAL1 targets can be classified into several groups, each of which exhibits unique gene expression kinetics, chromatin features, and regulatory mechanisms. Group A genes are highly dependent on TAL1, many of which are not expressed in normal T-cells or TAL1-negative T-ALL cells, representing an oncogenic TAL1 signature. The TAL1 complex predominantly activates Group A genes. TAL1's effect is not replaceable with its regulatory partners GATA3 or RUNX1. In contrast, Group B genes, many of which are generally expressed across different T-ALL subgroups, exhibit densely-connected chromatinchromatin interactions and demonstrate the collaborative roles played by TAL1 with other transcription factors. Interestingly, TAL1 cooperates with NOTCH1 to regulate gene expression in TAL1-positive T-ALL cells, whereas it potentially antagonizes the NOTCH1-MYC pathway and leads to lethality in TAL1-negative/TLX3-positive cells, demonstrating the context-dependent roles of TAL1.

TAL1 hijacks MYCN enhancer that induces MYCN expression and dependence on mevalonate pathway in T-cell acute lymphoblastic leukemia.

A hallmark of T-cell acute lymphoblastic leukemia (T-ALL) is the dysregulated expression of oncogenic transcription factors (TFs), including TAL1, NOTCH1 and MYC. Rewiring of the transcriptional program disrupts the tightly controlled spatiotemporal expression of downstream target genes, thereby contributing to leukemogenesis. In this study, we first identify an evolutionarily conserved enhancer element controlling the MYCN oncogene (named enhMYCN) that is aberrantly activated by the TAL1 complex in T-ALL cells. TAL1-positive T-ALL cells are highly dependent on MYCN expression for their maintenance in vitro and in xenograft models. Interestingly, MYCN drives the expression of multiple genes involved in the mevalonate pathway, and T-ALL cells are sensitive to inhibition of HMG-CoA reductase (HMGCR), a rate-limiting enzyme of this pathway. Importantly, MYC and MYCN regulate the same targets and compensate for each other. Thus, MYCN-positive T-ALL cells display a dual dependence on the TAL1-MYCN and NOTCH1-MYC pathways. Together, our results demonstrate that enhMYCN-mediated MYCN expression is required for human T-ALL cells and implicate the TAL1-MYCN-HMGCR axis as a potential therapeutic target in T-ALL.

Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma.

BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

Case report: Genomic analysis of a therapy-related chronic myelomonocytic leukemia with KMT2A rearrangement that progressed to acute myeloid leukemia with acute promyelocytic leukemia-like features.

We report a 69-year-old female who was a human T-cell leukemia virus type 1 carrier and exhibited a unique clinical course of developing three hematological malignancies within a short period: diffuse large B-cell lymphoma (DLBCL), chronic myelomonocytic leukemia (CMMoL), and acute myeloid leukemia (AML). Although the blast cells in AML showed typical morphological and immunophenotypical features of acute promyelocytic leukemia (APL), it did not harbor RARα gene fusion and thus initially diagnosed as APL-like leukemia (APLL). The patient developed heart failure with a fulminant clinical course and died soon after the diagnosis of APLL. Retrospective analysis with whole-genome sequencing detected a chromosomal rearrangement between KMT2A and ACTN4 gene loci both in CMMoL and APLL samples, but not in the DLBCL sample. Therefore, CMMoL and APLL were considered to be derived from the same clone with KMT2A translocation associated with prior immunochemotherapy. However, KMT2A rearrangement is rarely found in CMMoL in general and ACTN4 is also a rare partner of KMT2A translocation. Thus, this case did not follow typical transformational process of CMMoL or KMT2A-rearranged leukemia. Importantly, additional genetic alterations, including NRAS G12 mutation, were found in APLL, but not in CMMoL samples, suggesting that they might contribute to leukemic transformation. This report highlights the diverse effects of KMT2A translocation and NRAS mutation on the transformation of hematological cells as well as the importance of upfront sequencing analysis to detect genetic backgrounds for a better understanding of therapy-related leukemia.

Targeting dual oncogenic machineries driven by TAL1 and PI3K-AKT pathways in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of thymic T-cell precursors. Overexpression of oncogenic transcription factor TAL1 is observed in 40-60% of human T-ALL cases, frequently together with activation of the NOTCH1 and PI3K-AKT pathways. In this study, we performed chemical screening to identify small molecules that can inhibit the enhancer activity driven by TAL1 using the GIMAP enhancer reporter system. Among approximately 3,000 compounds, PIK- 75, a known inhibitor of PI3K and CDK, was found to strongly inhibit the enhancer activity. Mechanistic analysis demonstrated that PIK-75 blocks transcriptional activity, which primarily affects TAL1 target genes as well as AKT activity. TAL1-positive, AKT-activated T-ALL cells were very sensitive to PIK-75, as evidenced by growth inhibition and apoptosis induction, while T-ALL cells that exhibited activation of the JAK-STAT pathway were insensitive to this drug. Together, our study demonstrates a strategy targeting two types of core machineries mediated by oncogenic transcription factors and signaling pathways in T-ALL.

A Myb enhancer-guided analysis of basophil and mast cell differentiation.

The transcription factor MYB is a crucial regulator of hematopoietic stem and progenitor cells. However, the nature of lineage-specific enhancer usage of the Myb gene is largely unknown. We identify the Myb -68 enhancer, a regulatory element which marks basophils and mast cells. Using the Myb -68 enhancer activity, we show a population of granulocyte-macrophage progenitors with higher potential to differentiate into basophils and mast cells. Single cell RNA-seq demonstrates the differentiation trajectory is continuous from progenitors to mature basophils in vivo, characterizes bone marrow cells with a gene signature of mast cells, and identifies LILRB4 as a surface marker of basophil maturation. Together, our study leads to a better understanding of how MYB expression is regulated in a lineage-associated manner, and also shows how a combination of lineage-related reporter mice and single-cell transcriptomics can overcome the rarity of target cells and enhance our understanding of gene expression programs that control cell differentiation in vivo.

IRF4 as an Oncogenic Master Transcription Factor.

IRF4 is a transcription factor in the interferon regulatory factor (IRF) family. Since the discovery of this gene, various research fields including immunology and oncology have highlighted the unique characteristics and the importance of IRF4 in several biological processes that distinguish it from other IRF family members. In normal lymphocyte development and immunity, IRF4 mediates critical immune responses via interactions with upstream signaling pathways, such as the T-cell receptor and B-cell receptor pathways, as well as their binding partners, which are uniquely expressed in each cell type. On the other hand, IRF4 acts as an oncogene in various mature lymphoid neoplasms when abnormally expressed. IRF4 induces several oncogenes, such as MYC, as well as genes that characterize each cell type by utilizing its ability as a master regulator of immunity. IRF4 and its upstream factor NF-κB form a transcriptional regulatory circuit, including feedback and feedforward loops, to maintain the oncogenic transcriptional program in malignant lymphoid cells. In this review article, we provide an overview of the molecular functions of IRF4 in mature lymphoid neoplasms and highlight its upstream and downstream pathways, as well as the regulatory circuits mediated by IRF4.

Mitochondria transfer mediates stress erythropoiesis by altering the bioenergetic profiles of early erythroblasts through CD47.

Intercellular mitochondria transfer is a biological phenomenon implicated in diverse biological processes. However, the physiological role of this phenomenon remains understudied between erythroblasts and their erythroblastic island (EBI) macrophage niche. To gain further insights into the mitochondria transfer functions, we infused EBI macrophages in vivo into mice subjected to different modes of anemic stresses. Interestingly, we observed the occurrence of mitochondria transfer events from the infused EBI macrophages to early stages of erythroblasts coupled with enhanced erythroid recovery. Single-cell RNA-sequencing analysis on erythroblasts receiving exogenous mitochondria revealed a subset of highly proliferative and metabolically active erythroid populations marked by high expression of CD47. Furthermore, CD47 or Sirpα blockade leads to a decline in both the occurrence of mitochondria transfer events and their mediated erythroid recovery. Hence, these data indicate a significant role of mitochondria transfer in the enhancement of erythroid recovery from stress through the alteration of the bioenergetic profiles via CD47-Sirpα interaction in the early stages of erythroblasts.

Lineage- and Stage-Specific Oncogenicity of IRF4.

Dysregulation of transcription factor genes represents a unique molecular etiology of hematological malignancies. A number of transcription factors that play a role in hematopoietic cell development, lymphocyte activation, or their maintenance have been identified as oncogenes or tumor suppressors. Many of them exert oncogenic abilities in a context-dependent manner by governing the key transcriptional program unique to each cell type. IRF4, a member of the interferon regulatory factor (IRF) family, acts as an essential regulator of the immune system and is a prime example of a stage-specific oncogene. The expression and oncogenicity of IRF4 are restricted to mature lymphoid neoplasms, while IRF4 potentially serves as a tumor suppressor in other cellular contexts. This is in marked contrast to its immediate downstream target, MYC, which can cause cancers in a variety of tissues. In this review article, we provide an overview of the roles of IRF4 in the development of the normal immune system and lymphoid neoplasms and discuss the potential mechanisms of lineage- and stage-specific oncogenicity of IRF4.

Requirement for TP73 and genetic alterations originating from its intragenic super-enhancer in adult T-cell leukemia.

Adult T-cell leukemia/lymphoma (ATL) is a genetically complex hematological malignancy derived from mature T cells. Using an integrative approach, we previously identified genes recurrently associated with super-enhancers in ATL. One of those genes was TP73, a TP53 family gene; however, the roles and function of TP73 and its super-enhancer in ATL pathogenesis are poorly understood. Our study demonstrates that TP73 is highly activated under the control of a super-enhancer in ATL cells but not in normal T cells or other hematological malignancies examined. Full-length TP73 is required for ATL cell maintenance in vitro and in vivo via the regulation of cell proliferation and DNA damage response pathways. Notably, recurrent deletions of TP73 exons 2-3 were observed in a fraction of primary ATL cases that harbored the super-enhancer, while induction of this deletion in cell lines further increased proliferation and mutational burden. Our study suggests that formation of the TP73 intragenic super-enhancer and genetic deletion are likely sequentially acquired in relation to intracellular state of ATL cells, which leads to functional alteration of TP73 that confers additional clonal advantage.