Unraveling BCR::ABL1-driven enhancer activation and transcriptional reprogramming in Ph B-ALL

doi:10.21203/rs.3.rs-5326686/v1

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Unraveling BCR::ABL1-driven enhancer activation and transcriptional reprogramming in Ph B-ALL

https://doi.org/10.21203/rs.3.rs-5326686/v1

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B-lineage acute-lymphoblastic leukemia (B-ALL) is driven by genomic lesions and distinct transcriptional programs. Both are often directly linked as most B-ALLs are caused by genetic lesions at transcription factor (TF)-encoding genes. TFs largely mediate their function through gene regulatory ‘enhancer’ elements and enhancer deregulation is known to promote cancer initiation and progression. Consecutively, enhancer-targeting drugs are currently in clinical trials for advanced hematologic cancers such as acute myeloid leukemia and multiple myeloma. However, for B-ALLs not driven by TF-related genetic lesions it is largely unclear how their disease-promoting transcriptional programs are established, if it involves enhancer-deregulation, and if they would be sensitive to therapeutic enhancer-targeting.

We explore this here by focusing on Philadelphia chromosome-positive (Ph+) B-ALL, the most common B-ALL in adults with historically poor prognosis. Ph+B-ALL is driven by the BCR::ABL1 kinase, which has no TF-related function. We report that malignant transformation and associated transcriptional reprogramming by BCR::ABL1 is indeed accompanied by substantial enhancer activation and using Hi-C-based methods we connect enhancers to key genes in Ph+B-ALL. Consequently, Ph+B-ALL-specific enhancer signatures differentiate Ph+B-ALL from other leukemias. We further demonstrate that BCR::ABL1-induced enhancer activation depends on its kinase activity and is executed, at least in part, through the BCR::ABL1-activated TF STAT5. Highlighting the importance of enhancers and the potential of their therapeutic targeting, enhancer inhibition through CBP/P300-specific proteolytic degraders (PROTACs) effectively kills Ph+B-ALL cells.

Hematology

Epigenetics & Genomics

Cancer Biology

BCR::ABL1

transcriptional deregulation

enhancer

P300/CBP

PROTAC

dependency

BCR::ABL1-induced malignant transformation and transcriptional reprogramming towards Ph+B-ALL is defined by genome-wide enhancer activation.
Enhancer-regulation affects key genes in Ph+B-ALL.
Enhancer activation and associated chromatin interactions define Ph+B-ALL identity.
Enhancer activation and associated chromatin interactions are regulated by BCR::ABL1 kinase activity and mediated through BCR::ABL1-activated TFs.
Enhancer inhibition through CBP/P300-specific PROTACs effectively kills Ph+B-ALL cells.

Acute-lymphoblastic leukemia (ALL) is the most common cancer and second most common cause of cancer-related death in children, and mostly arises from B-cell precursors (i.e., B-ALL). B-ALL subtypes are defined and driven by distinct initiating genetic lesions and disease-defining transcriptional programs^1,2. Both are often directly linked, as most initiating genetic lesions in B-ALL affect transcriptions factor (TF)-encoding genes, which cause the disease-defining transcriptional program. Indeed, 13/18 B-ALL subtypes [72%] with defined genetic alterations excluding iAMP21 and hyper/hypodiploidy are caused by lesions that alter one or two TFs². TFs often mediate their function through enhancers, which are distal regulatory DNA elements located from a few kilobases up to 1.7 megabases away of the promoters they regulate^3-5. Through physical interaction by DNA looping, enhancers, associated TFs, and epigenetic cofactors can regulate temporal-, spatial- and cell-type-specific activation of promoters. Importantly, elevated oncogene expression in cancer has been frequently attributed to aberrant enhancer function and many cancers are described to depend on ‘enhancer reprogramming’ or ‘enhancer hijacking’^6-13. Given the importance of transcriptional deregulation in cancer, enhancer-targeting drugs are explored as a therapeutic strategy for cancer treatment for the last two decades, with several improved drug candidates currently being in clinical trials for advanced and high-risk cancers including the hematological malignancies multiple myeloma (MM) and acute myeloid leukemia (AML) (e.g. NCT04068597, NCT03568656, NCT04575766, NCT05488548)^14,15.

Ph+B-ALL is the most common B-ALL in adults and historically a poor prognosis/high-risk leukemia¹⁶. Recent advances in replacing, supplementing, or consolidating chemotherapy with tyrosine kinases inhibitors and immunotherapy has improved its treatment outcome¹⁷. Nevertheless, relapses remain frequent and are associated with poor prognosis. Ph+B-ALL is driven by the oncogenic fusion gene BCR::ABL1, a constitutively active tyrosine kinase that activates its targets by phosphorylation. Multiple studies highlighted that Ph+B-ALL is defined by its distinct transcriptional program^1,2 causing upregulation of genes essential for disease initiation and progression^18-27. However, how BCR::ABL1 induces this disease-defining transcriptional program is little understood. Likewise, it is unknown if disease development is caused by enhancer deregulation, and if Ph+B-ALL cells consecutively are sensitive to enhancer-targeting drugs.

While BCR::ABL1 has no TF-related function, it can induce activation or expression of TFs through phosphorylation. For example, BCR::ABL1 phosphorylates and thereby activates the signal-inducible TFs STAT1/3/5/6^28-30, of which STAT5³¹, particularly STAT5B³², was reported most important for Ph+B-ALL. Through STAT5, BCR::ABL1 further induces expression of MYC^18-20. Additionally, BCR::ABL1 induces the RAS/MEK/ERK signaling pathway, thereby causing the expression of the ETS family TF, ETV5^21,33. Other TFs with important function in B-ALL including Ph+B-ALL are MYB³⁴, CDK6³⁴, CDK8³⁵, FOXM1³⁶ and ERG³⁷. However, it is unclear if they are induced by BCR::ABL1 and thereby shape the Ph+B-ALL-defining transcriptome, or if their importance reflects a general requirement in B-ALL cells. Likewise, it is unclear how BCR::ABL1 would coordinate their action and if it would involve enhancer deregulation.

To better understand how BCR::ABL1 establishes the Ph+B-ALL-defining transcriptional program, we adopted an integrative multi-omics approach to study the transcriptome, enhancer activities, and 3-dimensional interactions of enhancers with target genes in Ph+B-ALL cells. We performed an in-depth analysis of cells from human Ph+B-ALL patients and a murine Ph+B-ALL model using ChIP-Seq, RNA-Seq, and Hi-C-based methods to link enhancers to the promoters they regulate. We further interfered with enhancer function to explore their role in Ph+B-ALL and investigated BCR::ABL1-regulated TFs that are recruited to them using targeted proteasomal degradation and knock-in models. Our results show that enhancer activation is a key characteristic of malignant transformation and transcriptional reprogramming by BCR::ABL1. We demonstrate that BCR::ABL1 itself causes their activation by coopting signal-induced TFs such as STAT5. Most importantly, we show that enhancer function is vital for Ph+B-ALL as their global inhibition using a CBP/P300-specific degrader (PROTAC) effectively kills Ph+B-ALL cells.

Cell lines and primary cells. All cell lines were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). Human samples were deposited into, stored and subsequently retrieved from the Imperial College Healthcare Tissue Bank (ICHTB) under subcollection MEC_AR_16_030. All patients gave specific informed consent for the use of surplus tissue. Murine cells were processed as described^38,39 and experiments performed in agreement with ASPA guidelines & regulations and protocols approved by Home Office UK. Further information on culture conditions, processing, transfections and transduction can be found in the Suppl.Methods.

Drug treatments

Cells were treated with Ponatinib, AK-2292, dTAGV-1, A-485 or dCBP-1 as indicated in the figures/text and described in the Suppl.Methods using 1000X stocks and DMSO (Sigma) as control.

Next generation sequencing. ChIP-Seq, ATAC-Seq, RNA-Seq and PCHi-C were done as previously described^39-41. Libraries were sequenced by paired-end sequencing at the MRC London Institute of Medical Sciences at Imperial College London or at Novogene. Further information on generation, processing and analysis can be found in the Suppl.Methods. Raw and processed data generated here can be found at Gene Expression Omnibus (GEO) under accession numbers GSE279589, GSE279594, GSE279643, GSE279644.

Western blot. Western blot was performed as previously described³⁹ using 4-15% Mini-PROTEAN TGX Precast Gels, the Mini-PROTEAN blotting system (Bio-Rad) and antibodies listed in the Suppl.Methods.

Flow cytometry. Cells were analyzed or sorted by flow cytometry using a BD LSRFortessa or SONY MA900 sorter, respectively, using the antibodies or fluorescent proteins as indicated in the figures and described in the Suppl.Methods.

Statistical analysis: Statistical analysis was performed using R or GraphPad PRISM 9/10 as indicated in the figures and Suppl.Methods.

Data Sharing Statement:

All NGS data generated here is available at GEO. Detailed information on protocols, methods and reagents can be found in the Suppl.Methods section within the Supplementary information. Any tools generated here (plasmids, cell lines etc) will be provided on request.

BCR::ABL1-induced malignant transformation and transcriptional reprogramming is associated with genome-wide enhancer activation

To investigate if BCR::ABL1 causes enhancer deregulation to establish its transcriptional program, we first monitored BCR::ABL1-induced malignant transformation and associated transcriptional reprogramming for changes in enhancer activation. In detail, we used an established murine transformation model based on viral transduction of ex vivo cultured primary B-cell precursors (BCPs) with BCR::ABL1-encoding retrovirus^21,31,39,42 and monitored transcriptional changes and enhancer activation via RNA-Seq and H3K27ac ChIP-Seq. H3K27ac is a widely used histone marker of active regulatory regions including promoters and enhancers⁴³. To differentiate between active promoters and active enhancers, we specifically analyzed H3K27ac signals at non-promoter regions. We first re-assessed RNA-Seq and H3K27ac ChIP-Seq data that we generated for a previous study³⁹ (Suppl.Fig.1A/B). In this study, BCPs from 53BP1^-/- mice were transduced with BCR::ABL1^p210 (Fig.1A). Leukemic transformation was accompanied by substantial transcriptional reprogramming visualized by RNA-Seq (Fig.1B, left; Table S1). BCR::ABL1-induced genes included the known targets Xbp1, Bcl2, Etv5, Dusp6 and Ccnd2, while downregulated genes included many with B-cell specific functions as described before⁴⁴. Changes in gene expression were accompanied by changes in enhancer activation (Fig.1B, middle) including increased signals at the Myc super-enhancer in BCR::ABL1 expressing cells (Fig.1B, right). We repeated our setup using BCPs from C57BL/6 wild-type mice and BCR::ABL1^p190-encoding retrovirus (Suppl.Fig.1C/D; Table S2). This again showed that enhancer-deregulation occurs during BCR::ABL1-mediated transformation (Fig.1C). Of note, besides causing specific changes at defined sites, BCR::ABL1 induced a modest increase in H3K27ac, globally (Fig.1D).

Genes regulated by BCR::ABL1 and/or having critical functions in Ph+B-ALL are defined by long-range chromatin interactions with enhancers

To define which genes are enhancer-regulated in Ph+B-ALL, we performed promoter-capture Hi-C (PCHi-C)^41,45, which profiles long-range chromatin interactions of promoters with distal DNA regions (i.e., other ends/OE) such as enhancers. This is important as enhancers are often distally located to the promoters they regulate, sometimes being separated by megabases of DNA, and often do not regulate the most proximally located gene^3-5. We integrated PCHi-C results with H3K27ac ChIP-Seq data to specifically define interactions between active promoters and active enhancers (Fig.2A). PCHi-C and H3K27ac ChIP-Seq were performed on two Ph+B-ALL cell lines and leukemia cells of one Ph+B-ALL patient that were ex vivo cultured on OP9 feeder cells (Suppl.Fig.2A-E). We identified ~55,000 significant long-range promoter interactions per sample (CHiCAGO score >5) using probes for 18,202 protein-coding and 10,929 non-protein-coding genes. ~19% of these represented interactions of an H3K27ac+ active promoter with another H3K27ac+ distally located site (other end/OE), potentially indicative of an interaction with an enhancer (Fig.2A right, Table S3-8). In line with previous work^3,46, most active promoters were connected via long-range chromatin interactions to such distally located active regions (H3K27ac+ OEs), while the opposite was true for inactive promoters (Fig.2B), and active promoters with H3K27ac+ OE interactions displayed a much higher number of total long-range chromatin interactions compared to active promoters that only interacted with H3K27ac-negative OEs (Fig.2C). Notably, a fraction of active genes with H3K27ac+ OE interactions solely interacted with other active promoters (i.e., Promoter-Promoter Interactions/PPIs; 10.7%). However, most of them displayed interactions with H3K27ac+ non-promoter regions (i.e., potential Enhancer-Promoter Interactions/EPIs; 89.3%) either alone or in combination with PPIs (Suppl.Fig.2G). Interestingly, genes with EPIs, EPIs+PPIs, or PPIs appeared to serve different functions, with EPI+ only genes largely relating to signaling, EPI+PPI+ genes relating to gene expression and RNA processing, and PPI+ only genes associating with protein processing and transport (Suppl.Fig.2F). Importantly, while in total only 43% of all active genes displayed EPIs by PCHi-C (Fig.2D), these included many genes known to be regulated by BCR::ABL1 and/or having critical functions in Ph+B-ALL (Fig.2E), with several of them interacting with enhancers with crucial functions previously reported in other cancers^47-49.

To test whether the PCHi-C-defined genes with EPIs are indeed enhancer-regulated, we next inhibited the enhancer activator CBP/P300⁵⁰ in Ph+B-ALL cells with sub-lethal concentrations of A-485⁵¹ and monitored resulting changes in gene expression by RNA-Seq. A-485 inhibits the histone acetylase activity of CBP/P300 reported crucial for CBP/P300-mediated enhancer activation⁵⁰ and causes dissociation of interacting enhancers and promoters⁵² (Fig.2F) resulting in transcriptional downregulation of enhancer-regulated genes⁵². As such, we expected A-485 to cause preferential downregulation of PCHi-C-defined EPI+ genes compared to EPI- genes. In agreement with A-485 interfering with transcriptional activation, A-485 predominantly caused gene downregulation (~67% of DEGs; Table S9/10). Downregulated genes included EPI+ Ph+B-ALL key genes (Fig.2G and Suppl.Fig.2H) and lineage/cell type-specific genes as previously described⁵³ (Suppl.Fig.2I). A global analysis further showed that genes with PCHi-C-defined EPIs were indeed generally enriched for A-485-induced downregulation when compared to active genes without EPIs or PPIs (Fig.2H). Genes with PPIs rather showed a trend towards upregulation in response to A-485 (Fig.2H). Genes with both EPIs and PPIs showed a trend towards downregulation (Fig.2H), but this only reached statistical significance in one of the two cell lines tested (Suppl.Fig.J). The latter might relate to the opposite effect of A-485 on PPIs described here, which complicates the response of genes with EPIs+PPIs.

Together, the described results show that the transcriptional program of Ph+B-ALL is largely enhancer-associated and that BCR::ABL1-driven malignant transformation towards Ph+B-ALL is associated with defined changes in enhancer activation.

Enhancer activation and associated enhancer-promoter interactions

define Ph+B-ALL identity

We next assessed if the changes in enhancer activity adapted by Ph+B-ALL cells are specific for this leukemia-subtype. We first assessed enhancer activation itself by comparing non-promoter H3K27ac ChIP-Seq signals present in Ph+B-ALL cell lines and primary leukemic cells to those from other leukemias and leukemia sub-types. H3K27ac signals at non-promoter regions clearly separated Ph+B-ALL from Ph-negative B-ALLs and BCR::ABL1-driven chronic myeloid leukemia (CML) by principal component analysis (PCA; Fig.3A). Likewise, we compared our PCHi-C data from Ph+B-ALL cells to PCHi-C data that we generated for healthy CD19+CD10+ BM BCPs and CML cells (Fig.3B/C). Specifically, we defined Ph+B-ALL ‘core interactions’ (i.e., EPIs and PPIs present in all three Ph+B-ALL samples; Suppl.Fig.3; Table S11) and assessed if these chromatin interactions would discriminate Ph+B-ALL cells from Ph-negative cells. Again, both, PCA and cluster analysis showed efficient separation of Ph+B-ALL cells from healthy BCPs and CML (Fig.3C). Notably, healthy BCPs clustered closer to Ph+B-ALL samples than CML and closer to primary Ph+B-ALL than to Ph+B-ALL cell lines, reflecting their similarities in lineage type and cellular state.

To validate these observations with a different method and samples, we performed HiChIP using H3K27ac antibodies⁵⁴ on leukemia cells from three patient-derived xenografts (PDX) each for Ph+B-ALL and KMT2A::AFF1+ B-ALL for comparison (Fig.3D-F). Like PCHi-C, H3K27ac HiChIP visualizes EPIs but, additionally, also visualizes enhancer-enhancer interactions (EEIs). In line with the above, H3K27ac HiChIP-defined EPIs and EEIs efficiently separated Ph+B-ALL from the KMT2A::AFF1+ B-ALL cells (Fig.3E). We further compared H3K27ac HiChIP-derived EPI interaction scores per gene with RNA-Seq expression of the respective genes. Specifically, we used log₂ fold change (log2FC) values from the comparison of Ph+B-ALL and KMT2A::AFF1+ B-ALL cells to assess if their separation by EPIs and enhancer activation relates to respective differences in their gene expression programs. The comparison indeed showed a trend towards positive correlation of EPIs and differential gene expression (Fig.3F).

Enhancer activity and enhancer-promoter interactions of BCR::ABL1-induced genes depend on BCR::ABL1 kinase activity

Of note, BCR::ABL1-induced transcriptional reprogramming and associated enhancer deregulation required a prolonged period of BCR::ABL1 expression in our murine model (Fig.1), potentially indicating a positive selection for changes in enhancer function during malignant transformation instead of a BCR::ABL1-induced active process. To assess if BCR::ABL1 actively regulates enhancer activation, we thus inhibited BCR::ABL1 function for 24 hours using the tyrosine kinase inhibitor, Ponatinib, (Fig.4A) and monitored its effect on enhancer activation through H3K27ac ChIP-Seq. Ponatinib caused substantial loss of H3K27ac signals at H3K27ac+ non-promoter regions in both BCR::ABL1^p190-transformed murine BCPs and human Ph+B-ALL cells (Fig.4B). Ponatinib treatment also increased H3K27ac at other enhancer peaks, possibly reflecting resumed differentiation towards mature B-cells upon BCR::ABL1 inhibition as described previously^44,55. Next, we specifically analyzed genes that display PCHi-C-defined EPIs (Fig.4C), focusing on genes that are downregulated upon Ponatinib treatment by RNA-Seq (Table S12-14). Inspection of the BCR::ABL1 target gene CCND2 showed that PCHi-C interaction as well as H3K27ac signals at both promoters and enhancers were reduced upon Ponatinib treatment (Fig.4D). We consecutively analyzed all genes that are downregulated upon Ponatinib for changes in enhancer-promoter interactions, H3K27ac signals at promoters, and H3K27ac signals at interacting enhancers. PCHi-C scores and H3K27ac signals at both promoters and enhancers were indeed significantly reduced upon Ponatinib treatment in all cases (Fig.4E-G).

BCR::ABL1 induces enhancer activation in part through the BCR::ABL1-induced transcription factor STAT5

To define TFs that BCR::ABL1 employs to induce enhancer activation, we next assessed two published mass spectrometry data sets of BCR::ABL1 phosphorylation targets^56,57. Within TFs identified by both studies, peptides of STAT family TFs dominated, including known phospho-tyrosine (pY) sites of STAT3, 5 and 6 important for their activation (Fig.5A). For the remainder, respective pYs were either not studied yet or shown functionally redundant (e.g. for NFYA^Y266ref.⁵⁸). For functional analysis, we focused on STAT5B as it was reported the most important STAT in Ph+B-ALL³², most enriched in our analysis (Fig.5A), known to recruit CBP/P300 to target genes⁵⁹, and linked by previous studies on murine B- and T-cells to enhancer activation^60,61. Specifically, we generated a Ph+B-ALL cell line model that allows targetable proteasomal degradation (TPD) of STAT5 using the degron (dTAG) system^62,63. In degron cells, a FKBP12^F36V tag is added to a protein of interest, which is a ligand for dTAG compounds that recruit E3 ligases to induce proteasomal degradation of FKBP12^F36V-tagged proteins. Through gene editing, we rendered both STAT5A alleles in Ph+B-ALL BV-173 cells nonfunctional and added an N-terminal FKBP12^F36V tag to STAT5B alleles (Fig.5B). Inclusion of an HA-tag further allow visualization of STAT5 by HA-ChIP-Seq. In parallel, we applied the novel STAT5A/B-specific degrader, AK-2292⁶⁴, on parental BV-173 cells with otherwise germline STAT5A/B. Of note, while dTAGV-1 addition was used in degron cells to induce STAT5 loss, in parental cells, which do not express FKBP12^F36V-tagged proteins, we used dTAGV-1 as a ‘non-specific degrader’ control (Fig.5C). With both systems, we achieved an almost complete loss of STAT5A/B (Fig.5C), though STAT5-degron cells exhibited reduced basal expression of STAT5, which was nevertheless further reduced by dTAGV-1 (Fig.5C). We first determined STAT5 binding through HA-ChIP-Seq using our STAT5-degron cells. STAT5-HA-bound sites included known canonical STAT5 targets (Fig.5D) and were highly enriched for STAT5 DNA-binding motifs (Fig.5E). In line with STAT5 being activated by BCR::ABL1 and phosphorylation being a prerequisite for DNA-binding^28-30, Ponatinib abolished STAT5-HA DNA-binding almost entirely (Fig.5D/F). STAT5 binding was further enriched at promoters of BCR::ABL1-regulated genes (821/1859 [44%] of all Ponatinib DEGs were STAT5-HA bound), which also displayed increased STAT5-HA signals compared to BCR::ABL1-unregulated genes (Suppl.Fig.4A/B). STAT5 loss through AK-2292 in parental cells or dTAGV-1 in degron cells likewise predominantly affected BCR::ABL1-regulated genes (125/187 [67%] of AK-2292 DEGs overlap with Ponatinib DEGs; Fig.5G/H, Suppl.Fig.4C). In agreement with STAT5 also acting as a repressor^65,66, STAT5 loss induced both transcriptional downregulation and upregulation, with AK-2292 largely mirroring Ponatinib-induced transcriptional effects (Fig.5G right). However, notably, only 9% of genes (821/8705) with STAT5 at promoters overlapped with BCR::ABL1-regulated genes (i.e., Ponatinib DEGs, Suppl.Fig.4A), and STAT5 loss through AK-2292 only affected 7% (125/1859) of BCR::ABL1-regulated genes (Fig.5G left, Table S15/16).

Next, we applied these models to investigate STAT5 on BCR::ABL1-induced enhancer activation. Indeed, 69% of STAT5-HA peaks in untreated cells (17,059/24,642) did not overlap with a promoter, supporting a role of STAT5 in enhancer regulation in Ph+B-ALL. As we did not have PCHi-C data on BV-173 cells available, we defined enhancers as H3K27ac+ non-promoter regions within 50 kb proximity of each gene of interest, focusing on STAT5-HA-bound regions at AK-2292/Ponatinib-sensitive genes. AK-2292-induced loss of STAT5 in parental cells and dTAGV-1-induced STAT5 loss in degron cells indeed caused substantial reductions of H3K27ac at enhancers and promoters of the STAT5 target genes CISH and SOCS1 (Fig.5I). Likewise, H3K27ac signals were reduced at STAT5-HA-bound enhancer regions when quantifying all AK-2292/Ponatinib-downregulated genes (Fig.5J). The opposite effect was observed for STAT5-HA-bound enhancers of AK-2292/Ponatinib-upregulated genes (Suppl.Fig.4D/E), and H3K27ac signals at promoters largely followed this trend (Suppl.Fig.4F/G).

Together, these results show that STAT5 function in Ph+B-ALL cells is largely dedicated to the regulation of BCR::ABL1 target genes, including the activation and repression of associated enhancers. However, its contribution to the BCR::ABL1-induced transcriptional program appeared surprisingly small, indicating that BCR::ABL1 employs additional TFs besides STAT5 for transcriptional reprogramming.

Therapeutic targeting of the enhancer-activators CBP/P300 causes cell cycle arrest and consecutive cell death of Ph+B-ALL cells

Given that key genes in Ph+B-ALL cells are enhancer regulated, we next wondered whether Ph+B-ALL cells are especially sensitive to CBP/P300 inhibition, which is essential for enhancer activation⁵⁰ as described before. Of note, CBP/P300 dependency had previously been described for leukemia and lymphoma cells⁶⁷. However, B-ALL itself or individual B-ALL subtypes such as Ph+B-ALL had not been assessed in these studies. We therefore first assessed public DepMap⁶⁸ data, which defines cancer type-specific gene dependencies through negative Chronos/Ceres scores. This analysis showed that B-ALL indeed displays higher dependency for CBP and P300 compared to myeloid leukemia, T-cell leukemia and several B-cell lymphomas (Fig.6A, left), and that CBP/P300 dependencies further vary between individual B-ALL subtypes (Fig.6A, right). However, likely due to the generally low transduction efficiency of Ph+B-ALL cells with large CRISPR/Cas9 vectors that underlies Dependency Mapping⁶⁸, DepMap did not provide information on Ph+B-ALL cells. Therefore, we investigated CBP/P300 dependency of Ph+B-ALL cells ourselves experimentally using the recently developed CBP/P300-specific degrader/PROTAC, dCBP-1⁶⁷. We chose dCBP-1 over A-485 here as we were not able to achieve complete H3K27ac loss with A-485 in Ph+B-ALL cells (Fig.2F), while dCBP-1 almost completely abolished CBP/P300-dependent H3K27 acetylation (Fig.6B). Notably, dCBP-1 caused a marked G1 cell cycle arrest after 2 days of treatment (Fig.6C) and induced apoptosis in almost all Ph+B-ALL cells by day 5 of treatment (Fig.6D/E). Importantly, Ph+B-ALL cells showed similar sensitivity to dCBP-1 as multiple myeloma (MM) cells (Fig.6E), which so far were described the most CBP/P300-inhibition sensitive cancer cells^51,67 and for which CBP/P300-inhibitors are currently in clinical trials.

Changes in enhancer activation are frequently linked to cancer initiation^13,69-71 and progression^72-76 and, as a result, enhancer inhibition is explored in many cancers as a therapeutic strategy, with BET inhibitors explored for the last two decades¹⁵, and more specific CBP/P300 inhibitors currently in phase 1/2 clinical trials for treatment of high-risk cancers including AML, MM and advanced solid tumors¹⁴. We now demonstrate here that enhancer regulation is also a defining feature of the high-risk B-lineage leukemia Ph+B-ALL and that, in line with this, Ph+B-ALL cells are as sensitive to enhancer-targeting drugs as MM cells. Enhancer deregulation appears to be part of the process of malignant transformation towards Ph+B-ALL and is executed by its driving lesion, BCR::ABL1, itself for BCR::ABL1-regulated genes. Enhancer regulation also affects BCR::ABL1-unregulated genes, defining >40% of all active genes in Ph+B-ALL, likely contributing to the sensitivity of Ph+B-ALL cells to enhancer inhibition.

Of note, enhancer function has been studied in hematological malignancies such as AML, T-ALL and MM, and more recently also in B-ALL^75,76, but for B-ALL only as a collective group without differentiating between any subtypes. Thus, B-ALL subtype-specific dependencies on enhancer function remained unclear, in particular regarding B-ALLs not driven by altered TFs such as Ph+B-ALL. While we demonstrate this here now for Ph+B-ALL cells, we further show, by using H3K27ac ChIP-Seq, PCHi-C and H3K27ac HiChIP, that respective enhancer activation signatures and associated chromatin interactions further allow the distinction of Ph+B-ALL to other B-ALLs.

As BCR::ABL1 has no TF-related function, we investigated which TFs BCR::ABL1 uses to deregulate enhancer activation. Based on published mass spectrometry datasets on BCR::ABL1 phosphorylation targets, we focused on STAT5, as it was the most enriched. While STAT5 is an obvious candidate, as it is known to play a role in induction of BCR::ABL1-induced genes and STAT5 DNA-binding by ChIP-Seq was shown to be enriched at super-enhancers in murine B-cells⁶¹. However, the extent of STAT5 contribution to transcriptional deregulation by BCR::ABL1 in Ph+B-ALL remained unclear, in particular regarding BCR::ABL1-induced enhancer activation as the latter was not performed in human Ph+B-ALL cells and only a minority of STAT5-bound genes by ChIP-Seq were STAT5-regulated by RNA-Seq in this study⁶¹. To investigate STAT5 function, we used targeted proteasomal degradation, either through a PROTAC or a dTAG/degron model. In line with reported functions of STAT5 on gene expression in Ph+B-ALL and its association with enhancer function in other cell types, we show that STAT5 indeed mediates BCR::ABL1-induced gene deregulation and activation/suppression of associated enhancers. However, the contribution of STAT5 to BCR::ABL1-mediated changes was surprisingly mild (only 7% of BCR::ABL1-regulated genes were STAT5-regulated), suggesting that BCR::ABL1 largely utilizes other TFs besides STAT5 to mediate its effect. It is worth to mention that most Ph+B-ALLs (83.7%) display deletions of the TF IKZF1⁷⁷, which modulates enhancer accessibility and gene expression^61,78. However, as IKZF1 antagonizes STAT5-induced enhancer activation and gene expression⁶¹, its loss should mostly have STAT5-promoting effects. Noteworthy are also the BCR::ABL1-phosphorylation targets STAT3 and STAT6. We did not investigate them here as previous reports indicated that BCR::ABL1-induced B-ALL primarily depends on STAT5^31,32. However, these studies were done using murine models and mostly investigated dependencies, leaving the possibility that in human Ph+B-ALL, the full induction of the BCR::ABL1-induced transcriptional program requires STAT3/6 in addition to STAT5. Besides STAT proteins, ETS-related TFs such as ERG and ETV5 could further support the Ph+B-ALL-defining transcriptional program as both have been shown essential for Ph+B-ALL^21,33,37 and to modulate gene expression in these cells.

Together, while we did not define the complete set of TFs that mediate BCR::ABL1-induced enhancer activation, we show that therapeutic enhancer-targeting of hematologic malignancies could be extended to Ph+B-ALL. PROTACs against the enhancer activator CBP/P300 induce apoptosis in Ph+B-ALL similar to MM cells, for which CBP/P300 PROTACs are in clinical trials. Thus, while further studies are needed to explore the breadth of TF dependency for Ph+B-ALL, we define enhancer activation as a therapeutic vulnerability that could be explored in future treatments.

Acknowledgements

The work was funded by Blood Cancer UK (BCUK, P77661, to NF) and The Blood Fund of the Imperial College Health Charity (PA2786 and PA6852, to NF). We thank the clinical staff of the Hammersmith hospital involved in providing patient samples, especially The John Goldman Centre for Cellular Therapy.

Authorship Contributions

HLN performed most experiments including PCHi-C, HiChIP, ChIP-Seq, RNA-Seq and respective bioinformatic analyses. TLG performed all dTAG experiments including RNA-Seq and ChIP-Seq and helped with dTAG knock-in generation. MER helped with processing and bioinformatic analyses of HiChIP data and RNA-Seq of mouse BCPs. KNC helped with processing of PDX samples for HiChIP. VM and MS helped with design and guidance for PCHi-C. JZ performed the GO analysis. OD performed ATAC-Seq. NTC provided guidance on dTAG experiments. KH helped with murine RNA-Seq experiments. LS helped with HiChIP analyses. AJI assisted with patient samples. MM supported HiChIP experiments and contributed to the manuscript. NF performed Western blots, flow cytometry, dTAG knock-in generation, mouse work, designed the study and wrote the manuscript.

Disclosure of Conflicts of Interest

The authors have no conflict of interest.

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The authors declare no competing interests.

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Unraveling BCR::ABL1-driven enhancer activation and transcriptional reprogramming in Ph B-ALL

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