The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription

<jats:title>Summary</jats:title><jats:p>Oncogene activity rewires cellular transcription, creating new transcription networks to which cancer cells become addicted, by mechanisms that are still poorly understood. Using human and mouse models of T cell acute lymphoblastic leukemia (T-ALL), we identify an essential nuclear role for CHMP5, a cytoplasmic endosomal sorting complex required for transport (ESCRT) protein, in establishing and maintaining the T-ALL transcriptional program. Nuclear CHMP5 promoted the T-ALL gene program by augmenting recruitment of the co-activator BRD4 by the histone acetyl transferase p300 selectively at enhancers and super-enhancers, an interaction that potentiated H3K27 acetylation at these regulatory enhancers. Consequently, loss of CHMP5 diminished BRD4 occupancy at enhancers and super-enhancers and impaired RNA polymerase II pause release, which resulted in downregulation of key T-ALL genes, notably<jats:italic>MYC</jats:italic>. Reinforcing its importance in T-ALL pathogenesis, CHMP5 deficiency mitigated chemoresistance in human T-ALL cells and abrogated T-ALL induction by oncogenic NOTCH1<jats:italic>in vivo</jats:italic>. Thus, the ESCRT protein CHMP5 is an essential positive regulator of the transcriptional machinery promoting T-ALL disease.</jats:p><jats:sec><jats:title>Graphical abstract</jats:title><jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="577409v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec><jats:sec><jats:title>Highlights</jats:title><jats:list list-type="bullet"><jats:list-item><jats:p>Identification of a nuclear role for the cytosolic ESCRT protein CHMP5 in transcription</jats:p></jats:list-item><jats:list-item><jats:p>CHMP5 mediates BRD4-dependent Pol II pause release and transcription of T-ALL genes</jats:p></jats:list-item><jats:list-item><jats:p>P300-BRD4 induced enhancer and super-enhancer H3K27 acetylation requires CHMP5</jats:p></jats:list-item><jats:list-item><jats:p>CHMP5 depletion mitigates chemoresistance and abrogates T-ALL initiation<jats:italic>in vivo</jats:italic></jats:p></jats:list-item></jats:list></jats:sec>

section_name: Graphical abstract

section_content: • Identification of a nuclear role for the cytosolic ESCRT protein CHMP5 in transcription • CHMP5 mediates BRD4-dependent Pol II pause release and transcription of T-ALL genes • P300-BRD4 induced enhancer and super-enhancer H3K27 acetylation requires CHMP5
section_name: Highlights:

section_name: • CHMP5 depletion mitigates chemoresistance and abrogates T-ALL initiation in vivo

section_content: Transcriptional dysregulation is a hallmark of cancer cells as oncogenes hijack the transcriptional machinery to promote expression of genes that support their survival and proliferative needs. As "addiction" to the rewired transcriptional machinery is necessary for tumor progression, mediators of transcriptional addiction have emerged as attractive targets against cancers 1, 2. Thus, discovering critical components of the dysregulated transcriptional machinery is essential to understanding cancer biology, identifying therapeutic targets, and improving cancer cell response to drugs. Studies in solid tumors and hematological cancers implicate a crucial role for the bromo and extraterminal (BET) domain protein BRD4 in promoting the cancer gene program especially in MYC-dependent tumors [3] [4] [5] [6]. BRD4 is a pleiotropic transcription factor, with a histone acetyltransferase activity via which it acetylates histones to promote nucleosome decompaction, and a kinase activity by which it can phosphorylate and induce RNA polymerase II (Pol II) pause release and transcription [7] [8] [9] [10] [11]. In BRD4-dependent cancer cells, key pro-tumorigenic genes display marked enrichment for BRD4 at enhancers and super-enhancers, DNA regions defined by histone hyperacetylation modifications, including histone 3 lysine 27 acetylation (H3K27ac) 12, 13. The presence and dependency of cancer gene expression on these hyperacetylated enhancers distinguishes normal from cancer cells 1, 2, and raises the fundamental question how oncogenes, utilizing the same transcription factors as normal cells, selectively modulate only specific regulatory elements in the cancer cell genome. 

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by aberrant development and proliferation of immature thymocytes which populate the peripheral circulation and infiltrate vital organs 14. More than half of all human T-ALL cases are caused by activating NOTCH1 mutations that constitutively generate the intracellular NOTCH1 domain (ICN1) 15, a transcription factor that induces the proto-oncogene MYC to which many T-ALL cells become dependent 16. Importantly, ICN1-driven T-ALL exemplifies a cancer with high BRD4 dependency wherein BRD4, cooperatively with ICN1 and MYC, drive expression of key T-ALL genes in a positive feed-forward loop to establish the leukemogenic program [17] [18] [19]. Accordingly, BET inhibitors which release BRD4 from chromatin downregulate expression of T-ALL genes like MYC and, alone or in combinations with other chemotherapy drugs, suppress T-ALL cell survival in vitro and in pre-clinical models 5, 17, 20. In addition, BRD4-dependent mechanisms, characterized by enriched BRD4 occupancy at super-enhancers of T-ALL genes, have also emerged as an underlying driver of chemoresistance in T-ALL cells 20, 21. Therefore, elucidating cellular factors that modulate BRD4 recruitment and interaction with chromatin and chromatin modifying factors can uncover novel insights into chemoresistance mechanisms in T-ALL. 

Charged multivesicular body protein-5 (CHMP5 or VPS60) is a ~35 kDa coiled-coil protein first identified in yeast 22, 23 as part of the cytosolic endosomal sorting complex required for transport (ESCRT)-III machinery where it promotes activation of the AAA-ATPase VPS4 24, 25. Because its function in membrane remodeling is required for many cellular processes, the ESCRT machinery has long been linked to cancer, but the precise role of individual ESCRT proteins in tumorigenesis remains unclear and often attributed to their membrane scission activity [26] [27] [28]. However, recent studies in osteoclasts and thymocytes, revealed CHMP5 as an adaptor for deubiquitylating enzymes whereby it promoted the stabilization of client proteins required for differentiation and survival 29, 30. 

Intriguingly, in these hematopoietic cell lineages, ESCRT-mediated processes (including membrane receptor recycling, multivesicular body formation) were not impaired by CHMP5 deficiency 29, 30. 

This not only suggested a redundant role for CHMP5 in the ESCRT machinery but also indicated evolved CHMP5 functions beyond the ESCRT machinery. Yet, non-membrane remodeling roles of CHMP5 in either normal or cancer cells remain largely unexplored. 

T-ALL oncogenes usually co-opt the same cellular factors that promote normal thymocyte development to establish leukemia 31. Despite its requirement for normal thymocyte development 30, it is not known whether T-ALL development requires CHMP5. We now report a novel nuclear function for CHMP5 required for T-ALL pathogenesis. Nuclear CHMP5 transcriptionally promoted T-ALL initiation and maintenance by facilitating the recruitment of BRD4 by the histone acetyl transferase p300, an interaction that potentiated H3K27 acetylation selectively at the enhancers and super-enhancers that drive high transcription of key T-ALL genes. Consequently, CHMP5 depletion impaired T-ALL maintenance in vitro and abrogated oncogenic NOTCH1-initiated T-ALL in mice.
section_name: Introduction

section_name: Results

section_content: As more than half of human T-ALL cases have underlying NOTCH1 activating mutations that generate ICN1 15, we set out to define CHMP5 dependent processes and function in T-ALL cells using the CUTLL1 cell line, a CD4 + CD8 + human T-ALL subtype caused by activated NOTCH1 32. Specifically, CUTLL1 cells harbor a t(7;9) (q34;q34) translocation of NOTCH1 into the TCRB loci that results in constitutive cleavage of NOTCH1 into ICN1 by -secretase 32. We depleted CHMP5 by short hairpins RNA (shRNA) and compared shCHMP5 knockdown (KD) cells to non-targeting control shRNA (CT) cells (Figure S1A ). CHMP5 depletion had no impact on T-ALL cell viability (Figure S1B ), but EdU (5-ethynyl-2'-deoxyuridine) incorporation indicated a proliferation defect in KD cells which appeared arrested at S phase with impaired G2/M progression (Figure S1C ). 

To determine the molecular consequences of CHMP5 deficiency in these T-ALL cells, we subjected them to RNA-seq. Comparison of transcriptomes of KD to control T-ALL cells revealed substantial gene expression changes (fold-change  1. 2; adjusted p < 0. 05) due to loss of CHMP5; differentially expressed genes (DEG) included 1057 upregulated and 702 downregulated genes. 

Unbiased pathway analysis of these DEGs revealed significant (p < 0. 05; FDR < 0. 1) upregulation of pathways associated with impaired leukemogenesis including P53, apoptosis, interferon signaling (Figure 1A ). In parallel, CHMP5-deficient T-ALL cells displayed a striking downregulation of "MYC target" genes that included several MYC-regulated genes involved in glycolysis, oxidative phosphorylation, and cell cycle progression (Figures 1A, 1B and S1D ). Given the essential role of MYC in NOTCH1-driven T-ALL [16] [17] [18] [19], these data suggest a role for CHMP5 in promoting the core transcriptional program induced by oncogenic NOTCH1 in T-ALL. 

Using two independent CHMP5 targeting shRNAs we confirmed that MYC transcripts and MYC protein were indeed drastically decreased in CHMP5-deficient T-ALL cells (Figures 1C and 1D ). Control and CHMP5-deficient T-ALL cells showed comparable MYC protein stability in cycloheximide (CHX) chase assays and proteasome inhibition by MG132 failed to restore its MYC levels (Figures S1E to S1G ), implying that CHMP5 likely controlled MYC expression at the level of transcription, in contrast to its post-translational activity in thymocytes and osteoclasts 29, 30. Of note, MYC transcripts and MYC protein levels positively correlated with the amount of CHMP5 proteins present in T-ALL cells (Figure 1E ), suggesting a quantitative effect of CHMP5 on MYC expression in these human T-ALL cells. 

To validate the specificity of CHMP5 loss on MYC expression, we transduced our CHMP5deficient human T-ALL cells with control ("Vector") or lentiviruses encoding murine Chmp5 (mCHMP5) which is 99% identical in amino acid sequence to human CHMP5 33. Murine CHMP5 not only restored MYC mRNA and MYC protein expression (Figures 1F and 1G ) but also rescued downstream MYC-dependent pathways like mitochondria oxidation and endoplasmic reticulum homeostasis in CHMP5-KD cells (Figure S1H ). Interestingly, CHMP5 overexpression (due to transduced mCHMP5) further increased MYC protein levels in CHMP5-sufficient (CT) T-ALL the massive changes in the transcriptome brought about by CHMP5-deficiency raised the possibility that it might regulate gene transcription in the nucleus. Therefore, to understand how it influenced T-ALL gene transcription we first sought to clarify CHMP5's cellular localization in T-ALL cells. 

Remarkably, we found that in addition to its previously reported cytosolic localization CHMP5 also localized to the nucleus in human T-ALL cell lines (CUTLL1, SUPT1) and primary patient-derived T-ALL cells (Figure 2A ) and was also present in normal human T cells (Figure S2A ). Overall, nuclear CHMP5 was ~20-40% of cytosolic CHMP5 levels (Figure 2A and S2A ). 

To determine the basis of its nuclear localization, we examined the amino acid sequence of CHMP5 across various species for a nuclear localization signal (NLS). Notably, the N-terminus of CHMP5 in jawed vertebrates (including mice and humans), in which CHMP5 protein sequence is highly conserved, contained a putative bipartite NLS 36 that was absent in invertebrate eukaryotes (Figure S2B and S2C). To verify this putative NLS we deleted the corresponding amino acid residues (Figure 2B ) and assessed the localization of NLS-deficient (NLS) CHMP5 proteins. Indeed, unlike full-length CHMP5 (FL), NLS-CHMP5 largely failed to localize to the nucleus in CUTLL1 T-ALL cell (Figure 2C ), indicating that, at least in T-ALL cells, CHMP5 proteins are imported into and localize to the nucleus.
section_name: CHMP5 enables expression of a T-ALL gene program exemplified by MYC

section_content: The unexpected nuclear localization of CHMP5 prompted us to hypothesize that CHMP5 directly mediated a mechanism that regulated transcription of T-ALL genes like MYC. Because in activated NOTCH1-induced T-ALL, ICN1 drives transcription in cooperation with BRD4 and MYC [17] [18] [19], we investigated whether nuclear CHMP5 interacted with these transcription factors. Given the lack of suitable immunoprecipitation antibodies for CHMP5, we transduced CUTLL1 T-ALL cells with hemagglutinin (HA)-tagged CHMP5 and performed anti-HA immunoprecipitation from these cells. While CHMP5 did not interact with ICN1 or MYC (Figure S2D ), we found that it associated with endogenous BRD4 in nuclear lysates in T-ALL cells (Figure 2D ). We confirmed this CHMP5-BRD4 interaction in independent reverse immunoprecipitation of endogenous BRD4 in nuclear lysates from human T-ALL cell lines (CUTLL1 and Loucy), primary PDX human T-ALL (Figures 2E, 2F and S2E ), and in HEK293T cells co-transfected with plasmids encoding these proteins (Figure S2F ). Importantly, in a cell-free assay, recombinant BRD4 pull-downed CHMP5 (Figure S2G ), indicating a direct interaction between both proteins. 

To determine whether nuclear CHMP5 bound BRD4 on chromatin we performed anti-BRD4 and anti-HA (CHMP5) chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) on T-ALL cells transduced with HA-tagged CHMP5. We confirmed that both antibodies significantly pulleddown chromatin relative to isotype antibody (Figure S2H ), validating our anti-HA ChIP approach. 

Focusing on the MYC gene locus, ChIP-qPCR revealed CHMP5 binding on chromatin at sites that overlapped with BRD4 binding, including at the MYC enhancer, promoter, and a BRD4-dependent super-enhancer ("BDME") 21 (Figures 2G and 2H ). CHMP5 binding was indistinguishable from isotype (IgG) antibody at the ICN1-specific ("NDME") super-enhancer which is not bound by BRD4 21 (Figures 2G and 2H ), suggesting that CHMP5 bound chromatin at the MYC gene through its association with BRD4. Accordingly, treatment with the BET inhibitor JQ1 which releases BRD4 from chromatin 37 significantly reduced CHMP5 binding across the MYC locus (Figure 2I ). 

That CHMP5 associated with BRD4 on chromatin regulatory elements at the MYC locus raised the possibility that it regulated transcription in T-ALL cells through a BRD4-dependent mechanism. In line with this, we found that the majority (~71%) of DEGs in CHMP5-deficient T-CUTLL1 T-ALL cells have BRD4 bound promoters (GSE51800) 38 and were genes that mediated the topmost perturbed pathways in these CHMP5-deficient T-ALL cells (Figures 2J and 2K ).
section_name: Nuclear CHMP5 interacts with BRD4

section_content: BRD4 has been shown to promote transcriptional elongation by facilitating promoter-proximal Pol II pause release 8, 11, 39, 40. Given the interaction between BRD4 and CHMP5, and that a substantial number of DEGs in CHMP5-deficient T-ALL cells are BRD4-regulated genes (Figure 2J ), we asked whether CHMP5 promoted Pol II pause release. To this end, we performed genome-wide ChIP-seq for Pol II and BRD4 in CHMP5-depleted (KD) or control (CT) CUTLL1 T-ALL cells. 

ChIP-seq analysis showed a modest increase in BRD4 binding at transcriptional start sites (TSS) but largely unchanged binding across, gene bodies, and transcriptional end sites (TES) in CHMP5depleted T-ALL cells (Figures S3A and S3B ). However, in parallel, CHMP5-depleted T-ALL cells displayed decreased Pol II occupancy at the TES with a corresponding increase of Pol II binding at promoters (Figures 3A to 3C and S3C), indicating impaired Pol II pause-release. Importantly, the loss of Pol II at the TES correlated with gene expression, as reduction in Pol II density at the TES was significantly higher in downregulated DEGs compared to upregulated DEGs in KD T-ALL cells (Figure S3D ). In light of this finding, as BRD4 travels with the Pol II during transcription elongation [40] [41] [42], increased BRD4 occupancy at promoters may reflect its binding to stalled Pol II. 

To quantify the extent to which CHMP5 loss impacted Pol II pause release, we calculated genome-wide Pol II traveling ratios (TR), the relative ratio of Pol II density at promoter-proximal regions relative to its density across the gene body (Figure 3D ) 8, 43. Overall, we found higher TRs for BRD4-target genes 38 in CHMP5-deficient T-ALL cells (Figure 3E ) confirming impaired Pol II pause release at promoters and decreased transcriptional elongation of these T-ALL genes. Since BRD4 also transactivates T-ALL genes in cooperation with MYC, another Pol II pause release factor 44, we also determined that the TRs for MYC target genes were higher in CHMP5-deficient T-ALL cells (Figure 3F ). Correlation of the TR of all genes in control (CT) versus CHMP5-deficient (KD) cells highlighted an increased TR of key T-ALL genes including essential pro-leukemogenic genes like MYC, TCF7, PHB2, CDK7, and XBP1 (Figure 3G ). Accordingly, Pol II density at the 3'-ends of these genes (exemplified by MYC and XBP1) was markedly diminished (Figure 3H ). 

Reflecting their BRD4-dependency, mRNA levels for T-ALL genes that displayed Pol II stalling (e. g., MYC, XBP1 and TCF7) were comparably downregulated by loss of CHMP5 and by the BET inhibitor JQ1 (Figure 3I ). In fact, despite intact (or increased) BRD4 binding at promoterproximal and gene body in these cells (Figures S3A and S3B ), JQ1 treatment had no additional effect on downregulating the BRD4-target genes XBP1 and TCF7 in CHMP5-deficient T-ALL cells (Figure 3I ). Altogether, these findings are consistent with a model in which CHMP5 promotes Pol II pause release and transcription of BRD4-dependent genes in NOTCH1-driven T-ALL.
section_name: CHMP5 promotes Pol II pause-release at BRD4-dependent T-ALL genes

section_content: Depletion of CHMP5 did not impact promoter-proximal (TSS) BRD4 binding, which remained largely unchanged in CHMP5-KD T-ALL cells (Figures S3A and S3B ). Therefore, to gain insight into how CHMP5 promoted transcription of BRD4-dependent T-ALL genes like MYC, we wondered if it instead controlled BRD4 binding at distal enhancers defined by hyperacetylated histone modifications, including H3 lysine 27 acetylation (H3K27ac) 45, 46. Notably, recruitment of BRD4 to these hyperacetylated enhancers and super-enhancers promote Pol II pause release and transcriptional elongation 8, 40, 47, 48. We performed H3K27ac specific ChIP-seq and found diminished H3K27ac density at the TSS, gene bodies, and TES of active genes in CHMP5-KD T-ALL cells (Figures 3J, 3K and S3E ). We first confirmed that actively transcribed genes (identified by positive H3K27ac signal), showed impaired Pol II traveling (Figure S3F ). 

Following previously reported methods 45, 46, we then defined enhancers and super-enhancers by ranking H3K27ac signal intensity across the genome. As reported in cancer and stem cells 45, 46, "hockey stick" plots showed that H3K27ac signals were asymmetrically distributed across the genome, with disproportionately higher density at super-enhancers of T-ALL genes like MYC, TCF, ERG, and ETV6 40 (Figure 3L ). Importantly, consistent with their downregulated expression (Figure S3G ), we found drastically diminished H3K27ac density across these key T-ALL genes, with significant loss of H3K27ac and BRD4 at both typical enhancers and super-enhancers in CHMP5-deficient T-ALL cells (Figures 3M and S3H ). Thus, CHMP5 promotes H3K27ac that defines enhancer and super-enhancers to which BRD4 binds to facilitate Pol II pause release and transcription 8, 40, 47, 48. Of note, despite reduced H3K27ac density at TSSs, CHMP5 deficiency did not reduce BRD4 binding at TSS, likely reflecting BRD4 binding to promoter-bound proteins like Pol II. Furthermore, that CHMP5 deficiency largely only impaired BRD4 binding at enhancers and super-enhancers, implied that it functioned selectively to regulate epigenetic events at distal regulatory elements and not promoters.
section_name: Loss of CHMP5 impairs H3K27 acetylation and disrupts super-enhancers

section_content: The histone acetyl transferase (HAT) p300 (with its structural homolog CBP) is a major catalyst of H3K27ac of enhancers and super-enhancers [49] [50] [51]. Studies in normal and cancer cells suggest a model in which p300 recruits BRD4 to hyperacetylated enhancers and super-enhancers and in turn, BRD4 augmented p300's HAT activity, a positive feed-forward mechanism that enriches hyperacetylation of enhancers and super-enhancers [52] [53] [54]. We thus asked if CHMP5 regulated the BRD4-p300 axis to promote H3K27ac and assessed the impact of CHMP5 deficiency on BRD4-p300 interaction. While BRD4's interaction with components of the transcriptional machinery (e. g., MED1, Pol II) were intact, its interaction with p300 was significantly reduced in CHMP5-deficient T-ALL cells (Figure 4A and 4B ). This was despite an overall increase in total p300 expression in CHMP5-deficient cells (Figures 4A and S4A ). 

To directly assess the impact of CHMP5 on the BRD4-p300 interaction, we quantified recombinant BRD4 and p300 binding in the presence and absence of recombinant CHMP5 protein. 

and found that more p300 proteins co-immunoprecipitated with BRD4 when CHMP5 was present (Figure 4C ). Immunoprecipitation studies in HA-CHMP5-expressing T-ALL cells showed p300 association with CHMP5 in nuclear lysates from CUTLL1 T-ALL cells (Figure S4B ) and in cellfree assays, recombinant CHMP5 immunoprecipitated with p300 (Figure 4D ), consistent with a direct binding between both proteins. As it also directly binds BRD4 (Figure S2G ), these findings suggest that CHMP5 promoted H3K27 acetylation at regulatory enhancers and super-enhancers by augmenting the BRD4-p300 interaction, an interaction that potentiates both p300 and BRD4mediated H3K27ac at enhancers and super-enhancers 53. 

Consistent with a model in which CHMP5 selectively regulated epigenetic events at distal regulatory elements, loss of CHMP5 resulted in reduced p300 binding at the MYC enhancer and super-enhancer (BDME), while p300 binding remained unchanged at the promoter (Figure 4E ). Of note, depletion of CHMP5 reduced p300 and H3K27ac at the MYC super-enhancer (BDME) to the same extent as treatment with the BET inhibitor JQ1 (Figure 4F ), establishing CHMP5 upstream of the p300-BRD4 recruitment. Moreover, while JQ1 further decreased super-enhancer-bound BRD4 in CHMP5-deficient T-ALL cells, it had no additional impact on p300 and H3K27ac signals at the super-enhancer in CHMP5-deficient T-ALL cells (Figure 4F ). These findings suggest that CHMP5 mediates the H3K27ac effected by the p300-BRD4 axis at super-enhancers. 

Based on findings from our ChIP-seq and protein interaction studies, we envision a model (Figures 4G and S4C ) in which nuclear CHMP5 functions as a positive regulator of the sequential events that establish distal chromatin modifications, including acetylation of enhancers and superenhancers, which are bound by BRD4 and p300 to facilitate Pol II pause release and transcriptional elongation 51, 55. Specifically, in CHMP5-sufficient T-ALL cells, p300-catalyzed H3K27ac recruits initial BRD4 binding via its bromodomain and in turn, chromatin-bound BRD4 recruits additional p300 proteins, which catalyzes further H3K27ac reactions and enrichment at enhancers and super enhancers. Interaction of factors at such distal regulatory elements and promoter-proximal factors orchestrate productive transcriptional elongation (Figure S4C ). Conversely, in CHMP5-deficient T-ALL cells which have diminished p300-BRD4 interaction and recruitment to chromatin, enhancers and super-enhancers are hypoacetylated and transcriptional elongation is impaired (Figure S4C ).
section_name: Potentiation of BRD4-p300 interaction by CHMP5

section_content: Our findings that CHMP5 mediated the p300-BRD4 interaction that enabled expression of key T-ALL genes such as MYC suggest a significant role for CHMP5 in T-ALL pathogenesis. Thus, we wished to understand the significance of CHMP5 expression in primary human T-ALL disease. 

Interestingly, across hematological and solid cancers, lymphoid leukemias expressed the highest amount of CHMP5 proteins (Figure S5A ). Specific comparison of CHMP5 expression in pediatric T-ALL samples versus normal thymocytes (GSE33470, GSE33469) 56, 57 revealed higher CHMP5 transcripts in T-ALL cells (Figure 5A ) which translated to >5-fold more CHMP5 proteins in primary human T-ALL relative to healthy T-cells (Figures 5B and 5C ; Table 1 ). Human T-ALL cell lines similarly expressed more CHMP5 protein relative to healthy T-cells (Figures S5B and S5C ). Of note, consistent with a unique role for CHMP5 in T-ALL, expression of other ESCRT proteins like CHMP1A (which can also localize to the nucleus 58 ) and VPS4 were comparable in
section_name: High CHMP5 expression is prognostic in human T-ALL

section_content: To understand the implications of CHMP5 expression on human T-ALL disease, we evaluated the prognostic significance of CHMP5 expression levels in the pediatric TARGET T-ALL dataset (dbGaP phs000464) 59. While overall pediatric T-ALL patient survival was high in this cohort, we found that patients with the highest (top 20%) CHMP5 expression had poorer overall survival (Log-rank test, P = 0. 017) compared to patients with the lowest (bottom 20%) CHMP5 expression in this cohort (Figure 5D ). In line with their comparable expression in normal T-cells and T-ALL cells (Figures 5B, S5B and S5C), expression of VPS4A and CHMP1A did not correlate with T-ALL patient survival (Figures S5D and S5E ), supporting the unique role for CHMP5 in T-ALL pathogenesis. Furthermore, CHMP5 expression was significantly higher in T-ALL cells from adult patients that did not achieve complete remission 60 (Figure S5F ). 

Interestingly, although our discovery of the critical role for CHMP5 in promoting T-ALL gene transcription were derived in the NOTCH1-driven human T-ALL model (CUTLL1), CHMP5 expression levels were prognostic in the TARGET T-ALL cohort that comprised patients with T-ALL disease with underlying mutations besides NOTCH1 59. Moreover, high CHMP5 expression marked both NOTCH1 and non-NOTCH1-driven human T-ALL cell lines (Figure S5B ). Together these observations reinforce that CHMP5 promoted expression of the T-ALL gene program by regulating a fundamental transcriptional mechanism -namely, the p300-BRD4 crosstalk-that is probably downstream of other T-ALL oncogenes besides ICN1 and shared across T-ALL subtypes.
section_name: normal T-cells and T-ALL cells (Figures 5B, S5B and S5C).

section_content: The poorer overall survival of T-ALL patients with the highest expression of CHMP5 (Figure 5D ) suggested that CHMP5-regulated mechanisms dictate patient response to chemotherapy. To address this possibility, we assessed the impact of CHMP5 deficiency on T-ALL cell sensitivity to killing by chemotherapy and measured the viability of control (CT) and CHMP5-deficient (KD) human T-ALL cells after cytarabine (AraC) treatment in vitro. CHMP5-deficient CUTLL1 T-ALL cells showed significantly increased sensitivity with >10-fold reduction in IC50 for AraC (Figures 5E and 5F ). Reflecting that the CHMP5-BRD4-p300 axis is a general requirement in T-ALL cells, loss of CHMP5 also synergized with AraC in both activated NOTCH1-driven (CUTLL1 and SUPT1) and 5E and 5F ).
section_name: CHMP5 promotes chemoresistance in T-ALL

section_content: NOTCH1-driven T-ALL cells like the CUTLL1 human T-ALL cell line are highly resistant to single or combination treatment with -secretase inhibitors (GSI) and glucocorticoids including dexamethasone (Dex) as ICN1-induced program represses expression of the glucocorticoid receptor NR3C1 61, 62. Therefore, to delineate CHMP5's contribution to oncogene-specific chemoresistance we assessed the impact of CHMP5 deficiency on CUTLL1 T-ALL cells response to GSI+Dex treatment. Compared to control cells (CT), CHMP5-deficient (KD) CUTLL1 cells were strongly inhibited by GSI+Dex treatment (Figures 5G and 5H ). Increased sensitivity to GSI+Dex treatment was associated with higher expression and increased nuclear translocation of the glucocorticoid receptor NR3C1 (Figures 5I), as well as increased expression of NR3C1-induced genes, including NR3C1 and BIM (Figures 5J and S5G ). 

Together, these drug response studies indicate that CHMP5 is part of the cellular mechanism that promotes chemoresistance in T-ALL cells. Interestingly, in contrast to its synergy with AraC and GSI+Dex that respectively target DNA replication and NOTCH1 processing pathways that are independent of the p300-BRD4 axis, CHMP5 deficiency did not synergize with the BET inhibitor JQ1 to kill T-ALL cells (Figure S5H and S5I ). This is consistent with our model (Figure S4C ) where CHMP5 functions upstream of and promotes p300 recruitment of BRD4 at enhancers and super-enhancers.
section_name: NOTCH1-independent (LOUCY) T-ALL cells (Figures

section_content: To further understand the physiological significance of CHMP5 function in T-ALL disease, we investigated the contribution of CHMP5 to T-ALL development in vivo. We utilized a murine bone marrow chimera T-ALL model in which BM donor cells were transduced with retrovirus encoding oncogenic ICN1 that initiates a CD4 + CD8 + subtype T-ALL disease 63, 64. Bone marrow (BM) cells (CD45. 2 + ) enriched for c-Kit + progenitors from littermate Chmp5 f/f Cd4-Cre -(WT) or Chmp5 f/f Cd4-Cre + (KO) mice 30 were transduced with bi-cistronic IRES retrovirus encoding ICN1, and a truncated nerve growth factor (NGFR) protein used to identify ICN1-transduced donor cells (Figure S6A and S6B). Since Cre recombinase expression is driven by the Cd4 promoter, Chmp5 deletion is restricted to CD4-expressing cells 30 in this murine T-ALL model. Transduction efficiency was confirmed by NGFR expression and transduced BM cells were transplanted into lethally irradiated congenic (CD45. 1 + ) hosts along with congenic (CD45. 1 + ) whole BM cells for hemogenic support (Figure S6B and S6C ). 

As expected 63, 64, chimera mice transplanted with ICN1-transduced WT BM cells (hereafter referred to as "WT" T-ALL mice) developed an aggressive, lethal (median survival, ~5 weeks) and fully penetrant T-ALL disease characterized by marked leukocytosis, splenomegaly with noticeably disrupted white and red pulp zones, and tissue infiltration by leukemic cells (Figures 6A to 6C and S6D). In striking contrast, chimera mice that were transplanted with ICN1-transduced KO donor BM cells (hereafter referred to a "KO" T-ALL mice) survived and did not develop any detectable T-ALL symptoms up to 12 weeks after transplantation (Figures 6A to 6C and S6D). 

To determine T-ALL burden in these animals, we enumerated leukemia cells in the bone marrow (BM), spleen, and peripheral blood by flow cytometry focusing on CD45. 2 + NGFR + donor cells, which corresponded to ICN1-expressing cells. Unlike wildtype T-ALL mice that harbored large numbers of CD45. 2 + NGFR + leukemic cells, KO T-ALL mice contained significantly fewer CD45. 2 + NGFR + cells in BM, spleen and blood (Figures 6D and S6E ). Furthermore, while these CD45. 2 + NGFR + cells were phenotypically all CD4 + CD8 + in WT T-ALL animals, the few KO CD45. 2 + NGFR + cells had reduced frequencies of CD4 + CD8 + cells and instead were also comprised of CD4 -CD8 + and CD4 -CD8 -fraction (Figure S6F ), implying impaired ICN1-induced T-ALL lineage commitment. These results reveal an essential requirement for CHMP5 in ICN1-initiated T-
section_name: CHMP5 is required for T-ALL initiation in vivo

section_content: To understand the molecular basis by which CHMP5 promoted T-ALL initiation by ICN1, we performed RNA-seq on sorted splenic CD45. 2 + NGFR + (i. e., ICN1-transduced) cells from WT and KO T-ALL animals. Analysis of DEGs (fold-change  1. 2; adjusted p < 0. 05) between WT and KO-derived CD45. 2 + NGFR + cells revealed 1157 upregulated genes and 706 downregulated genes. 1A and 1B ), unbiased pathway analysis of these DEGs also revealed marked downregulation of "MYC targets" in CD45. 2 + NGFR + cells from KO T-ALL animals (Figures 6E and 6F ). Accordingly, CD45. 2 + NGFR + cells from KO T-ALL animals expressed significantly less Myc and MYC target genes including C1qbp, Ldha, and Phb2 (Figure 6G and S6G ).
section_name: ALL development in vivo.

section_content: To validate changes in MYC protein expression in our murine T-ALL model, we introduced a MYC-GFP fusion protein knock-in reporter allele 65 into experimental donor mice, allowing us to quantify MYC protein expression by flow cytometry of GFP fluorescence in very few cells. 

Consistent with their Myc gene downregulation, Chmp5-deficient CD45. 2 + NGFR + cells not only had fewer MYC(GFP) + cells but also expressed significantly lower levels of MYC measured by GFP mean fluorescence intensity (MFI) protein (Figure 6H ). These findings confirm that CHMP5 promoted the cellular machinery (namely, the BRD4-p300-MYC axis) [17] [18] [19] that induces MYC gene and protein expression in this murine ICN1-driven T-ALL.
section_name: Similar to CHMP5-deficient human T-ALL cells above (Figures

section_content: to possess leukemia-initiating cell (LIC) potential 5, 17, 66, 67. We thus took advantage of the MYC-GFP reporter to assess the specific impact of CHMP5 deficiency on LICs in this ICN1-initiated murine T-ALL model. Remarkably, CD45. 2 + NGFR + cells from KO T-ALL animals were not only depleted of MYC-GFP hi CD34 + cells, but they also lacked any cells expressing CD34 in their BM and spleen (Figures 6I). properties in activated NOTCH1-driven T-ALL 5, 17, 66, 67. LIC activity is abolished in Myc-deficient T-ALL cells and MYC-GFP lo/-murine T-ALL cells expressing the same MYC-GFP fusion reporter used in our study failed to cause disease in secondary recipients 17. Thus, that CHMP5 deficiency resulted in the severe depletion of all CD34 + cells in primary murine T-ALL, including loss of the LIC-enriched CD34 + MYC-GFP hi fraction, indicated that failure of CHMP5-deficient progenitors to support ICN1-driven T-ALL was likely due to impaired generation of LICs. While we can't exclude that deficiency in other genes besides Myc contributed to the loss of LIC-enriched cells in KO T-ALL mice, our data suggest a mechanism in which CHMP5 promoted T-ALL initiation at least in part through a BRD4-driven Myc transcriptional program. Intriguingly, CHMP5 is dispensable for normal CD4 + CD8 + thymocyte generation 30 (that lack MYC) but required for MYC + CD4 + CD8 + T-ALL cells, implying that oncogenic ICN1 activity created a dependency on CHMP5 as part of the transcriptional rewiring required for T-ALL initiation. 

Whether and how ESCRT proteins contribute to tumorigenesis has become of significant interest given their function in membrane remodeling is required for many key cellular processes. 

Accordingly, studies implicating ESCRT proteins in cancer have largely attributed their activity to membrane repair leading to inhibition of cell death [68] [69] [70] [71]. We show here however that CHMP5 did not control T-ALL cell death but instead promoted leukemia by enabling transcription of T-ALL genes. Indeed, a fundamental insight from our study is the identification of a nuclear, chromatinbound fraction of CHMP5 which, like other ESCRT proteins, was considered to be cytosolic 24, 25, 27. 

In line with its nuclear localization, we found and validated an N-terminal bipartite NLS in CHMP5 that appears to be highly conserved across jawed vertebrates in which it first appeared, suggesting that CHMP5's nuclear function was acquired later in evolution. How cytosolic versus nuclear CHMP5 trafficking is regulated is presently unclear but the presence of an NLS in CHMP5 suggest that it is actively imported into the nucleus. 

To our knowledge our study represents the first report of a nuclear fraction of CHMP5 in mammalian cells. Previous reports from a yeast-two-hybrid screen predicted ESCRT protein interaction with nuclear proteins 72 and some ESCRT proteins have been reported to promote nuclear envelope remodeling [73] [74] [75]. Only ESCRT protein CHMP1A has been previously demonstrated to interact with chromatin binding proteins in the nucleus 58. Importantly however, CHMP5 and CHMP1A appear to play distinct roles in the nucleus and in T-ALL pathogenesis. Whereas mammalian CHMP1A, which also contains a bipartite NLS, functioned to recruit Polycomb-group proteins to silence genes 58, CHMP5 promoted transcription by facilitating BRD4 recruitment to p300-catalyzed histone hyperacetylation at enhancers and super-enhancers. Moreover, we found that CHMP5 expression levels, but not CHMP1A, were prognostic in T-ALL patients. 

Since human T-ALL cells expressed significantly more (>5-fold) CHMP5 proteins than normal T-cells, our results would suggest that one mechanism by which oncogenes in T-ALL might hijack the transcriptional machinery and selectively control transcriptional output is by increasing CHMP5 expression. In turn, the quantity of CHMP5 dictated the amount of target T-ALL gene outputs. Indeed, our biochemical and genomic studies elucidated a mechanism in which nuclear CHMP5 amplified p300 interaction with BRD4 and consequently, improved both p300 and BRD4 binding selectively at enhancer and super-enhancer elements at key T-ALL genes (Figure S4C ). 

Because p300 catalyzes H3K27ac of cis regulatory enhancers [49] [50] [51] and BRD4 promotes p300 HAT activity 53, this p300-BRD4 interaction conceivably operates as a positive feed-forward loop leading to further enriched BRD4 occupancy at enhancers and super-enhancers. At least in T-ALL cells, our data indicate that this process is critically dependent on CHMP5. Surprisingly, while its pro-tumorigenic role has been demonstrated in other hematological cancers, notably acute myeloid leukemia (AML), knowledge of the specific role of p300 in T-ALL pathogenesis is limited. For example, translocations and fusions of p300, or its partner CBP, cause AML disease 76, 77 and small molecule inhibitors of p300/CBP suppress AML progression [78] [79] [80]. That CHMP5 deficiency -which mitigated T-ALL disease initiation and maintenance-impaired p300 recruitment to BRD4 is consistent with a pro-tumorigenic role for p300 in T-ALL pathogenesis. 

Therefore, it would be of interest to evaluate p300 inhibitors as therapeutic targets against T-ALL, especially those with underlying activating NOTCH1 mutations that display BRD4-dependency. 

In conclusion, our study has uncovered a critical role for the ESCRT protein CHMP5 in promoting BRD4-p300-dependent super-enhancer formation that facilitates Pol II pause release and transcription of T-ALL initiation and maintenance genes. These findings provide mechanistic rationale for targeted CHMP5 depletion as a potential strategy against T-ALL. Loss of CHMP5 significantly depleted LICs in vivo suggesting that therapeutic targeting of CHMP5 can achieve more durable T-ALL suppression. Interestingly, high CHMP5 expression has also been reported in hepatocellular carcinoma 81, and AML 82, 83 where its loss correlated with increased apoptosis 84, 85. 

Whether CHMP5 also regulates AML, in which the BRD4-dependent MYC super-enhancer is also a therapeutic vulnerability 52, 86, and other cancers that display a p300-BRD4 dependency remains to be determined. Therefore, future studies to elucidate CHMP5 function in these malignancies have the potential to significantly advance our understanding of transcriptional addiction mechanisms in cancer cells. 

The authors declare no competing interests.
section_name: Murine and human T-ALL cells expressing CD34 and high levels of MYC have been shown

section_name: Methods

section_name: RESOURCE AVAILABILITY

section_content: Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Stanley Adoro (stanley. adoro@nih. gov).
section_name: Lead contact

section_content: Materials generated in this study are available upon request to the lead contact with a completed Materials Transfer Agreement. and Chmp5-deficient ICN1-transduced CD45. 2 + NGFR + splenocytes can be downloaded under GEO number GSE244199. This paper does not report original code.
section_name: Materials availability

section_name: EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

section_content: Six to eight-week-old male or female B6. SJL-Ptprca Pepcb/BoyJ (B6. SJL), NOD. Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG), B6;129-Myc tm1Slek /J (MYC-GFP) 65, and B6. Cg-Tg(Cd4-cre)1Cwi/BfluJ (Cd4-Cre) 87 mice were purchased from Jackson Laboratory. Chmp5 fl/fl mice (with loxP-flanked exons 3-7 of Chmp5) have been previously described 33. All mice were maintained in specificpathogen-free facilities at Case Western Reserve University (Cleveland, OH) or at the NCI campus in Frederick, Maryland under Institutional Animal Care and Use Committee approved protocols.
section_name: Mice

section_content: HEK293T (ATCC) and Plat-E cells (Cell Biolabs #RV-101) were cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, pH 7. 4, 1 × MEM nonessential amino acids, 50 IU/ml penicillin, and 50 μg/ml streptomycin. CUTLL1 (gift from Adolfo Ferrando, Columbia University) and primary T-ALL samples were cultured in complete RPMI (RPMI 1640 supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, pH 7. 4, 1 × MEM nonessential amino acids, 50 IU/ml penicillin, 50 μg/ml streptomycin, 55 μM βmercaptoethanol) with 20% FBS. Jurkat, Loucy, MOLT-3, MOLT-4, CEM, and SUP-T1 were purchased from ATCC; KOPT-K1 and DND-41 cells were provided by W. Pear (University of Pennsylvania) and cultured in complete RPMI with 10% FBS. HSB-2 was purchased from Sigma
section_name: Cell culture

section_content: Normal human T-cells were isolated from peripheral blood collected from the Hematopoietic Biorepository & Cellular Therapy Core at Case Western Reserve University. PBMCs were separated from the blood using a Ficoll density gradient (GE Healthcare #17144002). CD4 or CD8 cells were isolated using negative selection Miltenyi Biotec kits. Primary T-ALL patient samples were generously provided by Martin Carroll at the Stem Cell and Xenograft Core at the University of Pennsylvania. Patient information is provided in Table 1. Patient samples were collected in correspondence with approved protocols.
section_name: Primary human samples

section_content: Primary patient samples were thawed in 30ml IMDM with 2. 5% fetal bovine serum (FBS) and 10 ug/ml DNase, filtered and washed in PBS. At least 1 million live cells were retro-orbitally injected into NSG mice. Mice were bled by the tail vein to monitor leukemia progression by human CD45 expression. T-ALL cells were isolated from the spleen and bone marrow of mice by negative selection with mouse CD45 microbeads (Miltenyi Biotec #130-052-301).
section_name: Xenografts

section_content: Bones were processed with ACK and a mortar and pestle. Cells were then washed with complete RPMI and filtered through a 40μm strainer. Bone marrow (BM) from CD45. 2 mice was subjected to Lineage depletion (Miltenyi Biotec #130-110-470). CD45. 2 BM cells were plated at 1x10 6 cells/ml in a 10 cm plate in 10 ml of complete StemPro-34 SFM (Thermo Fisher #10639011) containing recombinant mouse cytokines (20 ng/ml IL-3, 50 ng/ml IL-6, 10 ng/ml Flt3L and 50 ng/ml SCF, PeproTech), 2 mM L-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin and 55 μM β-mercaptoethanol. After 24 hours, cells were infected with retroviral supernatants on Retronectin coated plates in the presence of 8μg/ml polybrene and centrifugation at 1,000xg for 2 hours at 25°C. After 48 hours, BM from CD45. 1+ mice for hemogenic support were subjected to CD3 depletion (Miltenyi Biotec #130-094-973) and cells were harvested and washed with PBS. 1 million CD45. 2 and 250,000 CD45. 1 cells were injected retro-orbitally into lethally irradiated (1000 rads) CD45. 1 mice. Mice were monitored for engraftment and disease by flow cytometry of the blood and euthanized according to IACUC protocols when mice became too sick (weight loss, hunching, piloerection, lethargy).
section_name: Bone marrow transduction and transplantation

section_name: METHOD DETAILS

section_content: Bone marrow, thymus and spleen were harvested and processed by mechanical dissociation to obtain single cells. Blood was collected either from the tail vein or medial saphenous vein into K2 EDTA coated collection tubes (BD Biosciences #365974). Splenocytes, blood, and bone marrow cells were treated with ACK lysis buffer (Gibco #A1049201) to lyse red blood cells. Single-cell suspensions were filtered through 40-μm strainers and resuspended in complete RPMI (RPMI 1640 supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, pH 7. 4, 1 × MEM nonessential amino acids, 50 IU/ml penicillin, 50 μg/ml streptomycin, 55 μM β-mercaptoethanol, and 10% FBS).
section_name: Murine cell isolation

section_content: For viability assays T-ALL cells were cultured for 3 or 4 days with the indicated concentrations of drugs and analyzed with the Vybrant MTT Cell Proliferation Assay Kit (Thermo #V13154). Cytarabine (AraC; #S1648), and JQ1 (#S7110) were purchased from Selleck Chemicals, and Compound E from Enzo Life Sciences (#ALX-270-415-C250). Where applicable, cells were treated with 1 μM Dexamethasone (Selleck Chemicals #S1322) for 16-18 hours.
section_name: In vitro drug treatments and viability assay

section_content: Single-cell suspensions were washed with flow-cytometry buffer (PBS, 2% FBS and 1 mM EDTA) and stained with fluorochrome-conjugated antibodies for 30 min at 4°C in the dark. After washing, cells were resuspended in a flow-cytometry buffer and DAPI (4′,6-diamidino-2-phenylindole, 0. 5 μg/ml; ThermoFisher #D1306) solution to exclude dead cells. MitoSOX (M36008), MitoTracker (M46753), ER Tracker (E34250) were acquired from ThermoFisher and used as indicated in the manufacturers protocol. Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay kit (Invitrogen #C10458) was used as per manufacturer's instructions. Annexin-V was stained using Annexin-V APC (Biolegend #640941) and Annexin-V Binding buffer (Biolegend #422201) incubated with 7-AAD (Tonbo Bioscience #13-6993-T500) for 20 minutes at RT. Cell cycle analysis was performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen #C10634). 

Cells were incubated for 2 hours with 10uM EdU and stained with LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Invitrogen #L34955) before fixation. After the Click-chemistry was performed, samples were incubated with 7-AAD (BD Biosciences #559925) for 10 minutes before acquisition. Flow cytometry sorting was performed on a BD FACS Aria or Aria-SORP and cell analysis on a BD LSR Fortessa and BD LSR-II. Flow analysis was done using FlowJo software. 

The antibodies used are listed in Table 2.
section_name: Flow cytometry analysis and cell sorting.

section_content: C-terminal Flag-tagged CHMP5 (NP_084090. 1) or N-terminal HA-tagged CHMP5 was amplified by PCR and cloned into pcDNA3. 1 (Thermo #V79020) or pHAGE (Harvard) vectors. BRD4 overexpression plasmid for HEK293T transfection was purchased from Addgene (p6344 pcDNA4-TO-HA-Brd4FL #31351). shRNA constructs in a pLKO. 1 vector targeting human CHMP5 were purchased from Sigma Aldrich along with 2 control non-targeting shRNAs. shRNAs targeting human MYC were a gift from Xi Chen (Baylor College of Medicine). shRNA sequences can be found in Table 3. ICN1-NGFR (pMIG) in which ICN1-transduced cells are marked by NGFR expression, was gifted to us by Lan Zhou (CWRU). The human CHMP5 delta NLS mutant was synthesized by Azenta Life Sciences.
section_name: Plasmids

section_content: Lentiviruses were generated by transfecting HEK293T cells with the plasmid of interest along with VSVg and delta 8. 9 packaging plasmid vectors using Lipofectamine 3000 Transfection Reagent (Thermo #L3000075). After 48 hours, viral supernatants were harvested, filtered through a 0. 45μm filter, concentrated with an Amicon Ultra-15 centrifugal filter (EMD Millipore #UFC903024) and stored at -80°C for future use. 

Retroviruses were produced by transfecting Plat-E cells with the plasmid alone using Lipofectamine 3000. After 48 hours, viral supernatants were harvested and filtered, then concentrated using Retro-X concentrator overnight (Takara Bio #631455). Untreated tissue culture plates were coated with 25mg/ml Retronectin (Takara Bio #T100B) overnight at 4°C to be used for retroviral transduction. Plates were blocked the next day with 2% bovine serum albumin in PBS for 30 minutes at RT. Concentrated retrovirus was spun down on the RetroNectin coated plates for 2 hours before adding the cells. Cells were transduced with lentiviruses or retroviruses by adding 8μg/ml polybrene (Sigma Aldrich #TR-1003-G) and a previously determined amount of virus based on titers in a 6 well plate. The cells were then spun at 1000xg for 2 hours at room temperature and cultured for 48 hours. 

For generation of shRNA knockdown cells, 48 hours after transduction with shRNA lentivirus, cells were selected with 1mg/ml puromycin (Invivogen #ant-pr-1) for 3-5 days. Deletion was validated by western blot and qPCR.
section_name: Virus generation and cell transduction

section_content: Total RNA was isolated from all cells using TRIzol reagent (Thermofisher, #15596026) and 100 to 500 ng of RNA was reverse transcribed using the high-capacity cDNA reverse transcription kit (Thermofisher, #4368814). Real time qPCR was done using Fast SYBR Green Master Mix (Applied Biosystems #43-856-12) and ran on QuantStudio six Flex real-time PCR system (Applied Biosystems # 4485691). Individual gene expression levels were calculated using the change in cycling threshold (ΔCT) method as 2 -ΔCT, where ΔCT is [CT (gene of interest)-CT (housekeeping gene)]. qPCR primer sequences are included in Table 4.
section_name: RNA isolation and quantitative real-time PCR

section_content: Cells are harvested and washed with ice-cold PBS by centrifuging at 5000rpm for 5 minutes at 4°C. Pellets were either stored at -80°C or lysed immediately; for western blots, cells were lysed with RIPA (25 mM Tris-HCl pH 7. 6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0. 1% SDS) buffer. For co-immunoprecipitations, cells were lysed with IP buffer (20 mM Tris-HCl, pH 8. 0, 150 mM NaCl, 5 mM MgCl2, 0. 5% NP-40). All lysis buffers were supplemented with 1X phosSTOP (Sigma Aldrich #4906837001) and 1X EDTA-free Protease Inhibitor Cocktail (Sigma Aldrich #4693159001). Lysates were collected after centrifuging cells at 13,000 rpm for 12 minutes at 4°C. Cell pellets used for fractionation were fractionated using the Nuclear Complex Co-IP kit from Active motif (#54001) according to their protocol. 

Protein concentration was determined using Pierce™ BCA® Protein Assay Kits from Thermo fisher (#23225). 10-40 mg of protein was used for western blots, 200-500 mg of protein was used for immunoprecipitation. Immunoprecipitations were performed using magnetic Protein G Dynabeads (Thermo #10004D) with incubation of the primary antibody done overnight at 4°C with overnight rotation. NuPAGE LDS sample buffer (Thermo #NP0007) and Sample Reducing Agent (Thermo #NP0009) were added to all lysates and boiled at 70°C for 10 minutes. Samples were then run on NuPAGE 4-12% Bis-Tris Protein Gels (Thermo #NP0335BOX) and transferred to a PVDF membrane (EMD Millipore #IPVH00005). Blots were probed with primary antibodies overnight at 4°C in 5% blocking solution. After washing and 1 hour incubation with HRP-linked secondary antibodies, blots were developed using Pico-or Femto-chemiluminescent substrate (ThermoFisher) and visualized with autoradiography or digital imaging. Where indicated, band intensities were determined by densitometry using the ImageJ program (NIH). 

Cell-free protein interaction assays were performed as previously described 11. Recombinant CHMP5 was purchased from Abcam (ab134604), recombinant BRD4 was previously generated 11, 88, recombinant p300 was purchased from Active Motif (#81158). Recombinant proteins were diluted with Buffer D (20 mM HEPES, 100 mM KCl, 0. 2 mM EDTA, and 20% vol/vol glycerol) to 0. 1 mg/ml and 0. 5 mg of each were incubated either individually or mixed in a 1:1 ratio in 500 ml of TBS (50 mM Tris-HCl, 150 mM NaCl) with protease inhibitors. Proteins were incubated overnight at 4°C with gentle rotation. Subsequently, 1ug of anti-FLAG (Sigma #F1804) or anti-BRD4 (Bethyl Laboratories #A301-985A50) was incubated with the proteins for 4 hours at 4°C with gentle rotation, and 40 ml of magnetic Protein G Dynabeads (Thermo #10004D) was added to each condition and incubated for 2 hours at 4°C with rotation. The beads were then washed 3 times with TBS+ 0. 2% NP-40 and boiled at 70°C for 10 minutes in TBS with NuPAGE LDS sample buffer and reducing agent. The samples were run on NuPAGE 4-12% Bis-Tris Protein Gels. All antibodies used in these studies are listed in Table 1.
section_name: Immunoprecipitation and immunoblots

section_content: Cells were cultured in the presence of 20 μM MG132 (Sigma #M7449) for 6 hours unless indicated otherwise. For half-life experiments, cycloheximide (Sigma #01810) was used at 50 μg/ml. Cells were subsequently processed and subjected to immunoblotting as above. Protein expression was quantified by densitometry and half-life was calculated by normalizing protein levels during cycloheximide treatment to DMSO treated cells without cycloheximide.
section_name: Protein stability assays

section_content: Extracellular acidification rate (ECAR) was performed under glycolysis stress test and oxygen consumption rate (OCR) was performed under mitochondrial stress test. Briefly, 1 x 10 5 CUTLL1 were plated per well on poly-D-lysine coated Seahorse XFe96 microplates (Agilent #101085-004) in XF RPMI medium (Agilent #103576-100) supplemented with 1mM Sodium Pyruvate, 2 mM Lglutamine, and 25 mM glucose for the Mito Stress Test (Agilent #103015-100), or 2 mM Lglutamine for the Glycolysis Stress Test (Agilent #103020-100). After an hour incubation at 37°C, OCR and ECAR were measured using a 96 well XF96 Extracellular Flux Analyzer (Agilent Technologies). Measurements were taken under basal conditions and following sequential addition of the drugs provided with the stress test kits. Fluoro-carbonyl cyanide phenylhydrazone (FCCP) was used at 1 mM final concentration. Basal ECAR was calculated from the samples in the Mito Stress Test medium containing glucose, and basal OCR was calculated from the samples in the Glycolysis Stress Test medium at the start of the assay.
section_name: Seahorse metabolic flux assays

section_content: Organs were fixed in 4% paraformaldehyde overnight and transferred to 70% ethanol before embedding. Embedding, sectioning and H&E staining was done at the Cleveland Digestive Diseases Research Core Center. Blood smears were made using Wright-Giemsa stain (Sigma #WG16). Images were taken using an Olympus IX73 microscope.
section_name: Histology

section_content: RNA was isolated from CUTLL1 cells independently transduced with either shControl or shCHMP5 in triplicate using the Qiagen RNeasy kit. RNA was submitted BGI (Hong Kong, China) for sample QC, library preparation, and sequencing. RNA was quantified and integrity was checked using Agilent 2100 Bioanalyzer. Library preparation was done using the NuGEN Trio RNA-seq library preparation kit (Redwood City, CA). Libraries were sequenced with Illumina HiSeq X Ten PE150bp sequencing and 30 million reads per sample.
section_name: CUTLL1 RNA-seq sample processing, library preparation, and sequencing

section_content: CD45. 2 + NGFR(ICN1) + cells were sorted from the spleens of chimera mice at median survival timepoint (4 weeks post-transplantation). RNA was isolated from these cells using the TRIzol reagent and submitted to Azenta Life Sciences LLC (South Plainfield, NJ, USA) for library preparation and Ultra-low RNA sequencing. Total RNA samples were quantified using Qubit 2. 0 Fluorometer (Life Technologies) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies). The SMARTSeq HT Ultra Low Input Kit was used for full-length cDNA synthesis and amplification (Clontech), and Illumina Nextera XT library was used for sequencing library preparation. Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. Sequencing libraries were validated using the Agilent TapeStation and quantified by using Qubit Fluorometer as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were multiplexed and clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq 6000 instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration.
section_name: Murine RNA-seq sample processing, library preparation, and sequencing

section_content: Sequence reads for human samples control (CT) and knock-down (KD) were aligned to GRCh38 reference sequence annotated with Gencode release 39 with Star aligner (v2. 7. 9a) 89 with --sjdbOverhang 100 and default parameters. Mouse wildtype (WT) and knockout (KO) samples were aligned to GRCm39 with Gencode release M27 with Star aligner (v2. 7. 9a) with --sjdbOverhang 150 and default parameters. STAR aligned reads gene counts were calculated with htseq (v0. 11. 4) 90 with default parameters and differential expression analysis performed with DEseq2 (v1. 38. 3) 91 software. Read counts were filtered to retain genes with at least 10 counts in the smallest group. 

Gene set enrichment analysis (GSEA) for the RNA-seq data was performed with GSEA 4. 3. 2 92 and MsigDB Hallmark gene set collection 93. For the comparison of CHMP5-regulated genes in human and mouse T-ALL, genes were extracted for mapping using BioMart R 94 and Ensembl release 105 95. Some DEGs are lost during the overlapping of human and mouse gene IDs.
section_name: RNA-seq data processing and gene set enrichment analysis

section_content: Fixation, isolation of the chromatin, immunoprecipitation and DNA isolation for ChIP analysis was done using the SimpleChIP® Enzymatic Chromatin IP Kit (CST #9003) and ran according to its protocol. Sonication was performed for 15 seconds on, 45 seconds off for 15 minutes. 10 ug of chromatin was isolated with 4 ug of indicated antibodies overnight. Antibodies used in these studies are listed in Table 1. qPCR was performed with 1ul of each per primer and compared to 2% input sample. Library preparation and sequencing of ChIP samples was performed by Azenta Life Sciences on the Illumina with 2 x 150bp, ~350M PE reads.
section_name: Chromatin Immunoprecipitation (ChIP)

section_content: ChIP DNA samples were quantified using Qubit 2. 0 Fluorometer (Life Technologies) and the DNA integrity was checked with 4200 TapeStation (Agilent Technologies). ChIP-Seq library preparation and sequencing reactions were conducted at Azenta US, Inc. (South Plainfield, NJ, USA). NEB NextUltra DNA Library Preparation kit was used following the manufacturer's recommendations (Illumina). Briefly, the ChIP DNA was end repaired and adapters were ligated after adenylation of the 3'ends. Adapter-ligated DNA was size selected, followed by clean up, and limited cycle PCR enrichment. The ChIP library was validated using Agilent TapeStation and quantified using Qubit 2. 0 Fluorometer as well as real time PCR (Applied Biosystems). The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq 6000 instrument according to manufacturer's instructions (Illumina). Sequencing was performed using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the Control Software (NCS). Raw sequence data (. bcl files) generated from the Illumina instrument was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2. 17 software. One mis-match was allowed for index sequence identification.
section_name: ChIP-seq library preparation and sequencing

section_content: Sequencing reads from BRD4, RNA Pol II, and H3K27 acetylation ChIP from control (CT) and knockdown (KD) and the input read samples were aligned to the reference genome UCSC hg38 with Bowtie2 (v2. 4. 5) 90 with -local setting and unique reads were kept by filtering out unmapped, duplicated and multimapped reads with sambamba 0. 8. 2 96 custom filters -F "[XS] == null and not unmapped and not duplicate". MACS2 (v2. 7. 1) 97 with default parameters was used for peak calling over the corresponding background. MACS2 peaks were annotated with ChIPseeker (v1. 34. 1) with TSS defined from -1kb to +1kb 98. Heatmaps and metaplots displaying ChIP-seq occupancy were created with ngsplot 99. ChIP tracks were generated using UCSC genome browser. The ROSE software 12, 45 was used to map typical and super enhancers with a maximum linking distance of 1 Mb for stitching.
section_name: ChIP-seq data processing

section_content: Traveling Ratio (Pausing Index) calculated based on RNA Pol II ChIP-seq utilizing the PIC software (https://github. com/MiMiroot/PIC) as previously described in 43 with default settings,longest option to get the longest isoform, and gene body defined as +300bp to the end of annotated gene. Genes <1kb were removed from the analysis. Traveling Ratio (TR) was calculated for all genes, activated genes (promoters with H3K27ac peaks) as defined before, non-activated genes (no H3K27ac peak), BRD4 target (genes bound by BRD4 at promoters from ChIP-seq), and MYC target genes (HALLMARK MYC_TARGETS _V1 and V2). Traveling Ratio comparison between CT and KD samples was performed on log2 transformed ratios in R 100 using Welch two sample t-Test.
section_name: Traveling Ratio (TR) calculation

section_content: RNA-seq and clinical data from TARGET-ALL-P2 cohort 1 was downloaded from the GDC data portal. Patients were divided into two groups according to the expression level of CHMP5, VPS4A, or CHMP1A; the top 20% were classified as highly-expressed group, and the bottom 20% were classified as lowly-expressed group. The entire group is comprised of the lowly-expressed group and highly-expressed group. Survival analysis is performed on these groups and survival curves are generated by using R-package 'Survival' and 'Survminer'.
section_name: Patient survival curves

section_content: Statistical details for experiments can be found in figure legends. Statistical analysis was performed using GraphPad Prism version 9. 0. Except otherwise indicated, comparison between groups was determined with a two-tailed, unpaired Student's t-test and differences were considered significant if p < 0. 05 as follows: *, p = 0. 01-0. 05; **, p = 0. 001-0. 01; ***, p = 0. 0001-0. 001; ****, p < 0. 0001. No statistical tests were used to predetermine sample size and no exclusion of data points was used.
section_name: QUANTIFICATION AND STATISTICAL ANALYSIS

section_name: TABLES

section_name: Acknowledgments

section_content: We thank A. Tikhonova for critical reading of the manuscript and helpful suggestions, M. Carroll ( University of Pennsylvania ) for providing primary human T-ALL samples, A. Ferrando for CUTLL1 cells, X. Chen for MYC expression plasmids, and W. Pear for KOPT-K1 and DND-41 cell lines. We thank all current and former members of our laboratory for helpful discussions. This work was supported by grants from the US Department of Defense CA180768-W81XWH1910306 (S. A. ); National Cancer Institute (NCI) K22 CA218467 (S. A. ); National Institute of Allergy and Infectious Disease R01 AI1143992 (S. A. ) ; American Cancer Society RSG-19-025-01-DDC (S. A. ) and the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research ZIA BC 012135 (S. A. ). L. Z. is supported by grants from the National Heart, Lung, and Blood Institute R01 HL103827 and NCI R01 CA222064. K. U-W. was supported in part by a Cell and Molecular Biology Training grant NIGMS T32 GM008056 to the Case Western Reserve University School of Medicine. We acknowledge use of the high-performance computational capabilities of the Biowulf Linux cluster at the NIH (http://biowulf. nih. gov). All schematic cartoons were created with BioRender. com.
section_name: Acknowledgments

section_name: Data and code availability

section_content: All sequencing data has been deposited to the Gene Expression Omnibus database (GEO) and are publicly available under the super-series GSE244200 as of the date of publication. RNA-seq on control (CT) and CHMP5 shRNA-depleted (KD) CUTLL1 human T-ALL cells can be downloaded under GEO accession number GSE244198. ChIP-seq of BRD4, Pol II, and H3K27ac in CT and KD CUTLL1 cells can be downloaded under GEO number GSE244197. Murine RNA-seq on wildtype 105 and is also made available for use under a CC0 license.
section_name: Data and code availability

section_name: Author contributions

section_content: Conceptualization, K. U-W., and S. A. ; methodology, K. U-W., and S. A. ; software, S. R., R. W., Q. T., Q. C., and D. M. ; formal analysis, K. U-W., B. N. D., D. S. S., and S. A. ; investigation, K. U-W., B. N. D., and S. A. ; resources, L. Z., A. T., and D. S. S. ; writing -original and final draft, K. U-W., and S. A. ; writing -review & editing, all authors reviewed, edited, or commented on the manuscript; visualization, K. U-W., S. R., and S. A. ; supervision, S. A. ; funding acquisition, S. A.
section_name: Author contributions

section_name: Declaration of interests