As part of canonical Wnt signaling, T cell factor (TCF)–β-catenin complexes promote MYC-dependent proliferation. Lesions of the β-catenin protein degradation machinery are common oncogenic drivers. Here, we show that B cell acute lymphoblastic leukemia (B-ALL) lacks these mutations and critically depends on unencumbered β-catenin protein degradation. Compared to solid tumors, we found that mouse and human B-ALL express β-catenin protein at much lower levels; β-catenin protein was constitutively phosphorylated by glycogen synthase kinase 3B (GSK3β) and poised for proteasomal degradation. Instead of TCF–β-catenin complexes to activate MYC, β-catenin paired with B lymphoid Ikaros and NuRD complex factors, resulting in MYC repression and acute cell death. To leverage β-catenin protein degradation as a previously unrecognized vulnerability in B-ALL, we validated GSK3β inhibition in patient-derived xenograft models in vivo. CRISPR screens confirmed β-catenin protein degradation as a central mechanistic target of established GSK3β inhibitors. As several GSK3β inhibitors achieved favorable safety profiles in clinical trials, our results provide a rationale for repurposing these compounds for persons with refractory B cell malignancies. Cosgun et al. show that, in B cell leukemia, β-catenin expression is maintained at low levels through glycogen synthase kinase 3B (GSK3β)-mediated phosphorylation. Inhibition of GSK3β results in β-catenin–Ikaros–NuRD complex formation, leading to B-ALL cell death through MYC repression.

B-cell acute lymphoblastic leukemia (B-ALL) is driven by transcriptional dysregulation that impairs B-cell differentiation and sustains leukemic growth. A defining feature of high-risk B-ALL is mutations in IKZF1, which encodes the tumor suppressor IKAROS. Here, we map IKAROS gene regulatory networks in IKZF1-mutated Ph B-ALL using an inducible IKAROS system and multi-omic profiling. IKAROS restoration reprograms chromatin accessibility and transcriptional control, shifting regulation from an ETS-dominated state to one enriched for B-cell lineage factors. Among repressed transcription factors, we identify ERG as a key regulatory node directly bound and antagonized by IKAROS. IKAROS binds regulatory elements near ERG and other progenitor-associated genes, coinciding with reduced ERG expression and repression of transcriptional programs linked to early B-cell developmental stages. Analysis of single-cell multiome data from human B-cell progenitors shows that ERG and IKAROS have opposing stage-specific activities and identifies a developmental stage-specific regulatory region in ERG intron 3 which is bound by IKAROS, and functionally important for ERG gene expression. Functional assays using CRISPRi and ETS inhibitors, along with gene dependency data from DepMap, confirm ERG dependency in IKZF1-deficient B-ALL. Our findings identify ERG as a context-specific dependency in IKZF1-deficient B-ALL, providing a mechanistic basis for the observed mitigation of poor prognosis for IKZF1-mutation in patients with co-occurring ERG deletions.

Ikaros is a transcription factor encoded by the Ikzf1 gene, known to be a critical regulator of immune cell development. Ikaros regulates multiple immune cell types, and is found to regulate multiple stages of development within one lineage. The importance of Ikaros to human health is underscored by recurrent IKZF1 mutations found in patients with immunodeficiency, autoimmunity, and leukemia. IKZF1 mutations are also reported in patients with eosinophilia, a condition with elevated levels of eosinophils. However, a role for Ikaros in regulation of eosinophils has not yet been described or experimentally tested. We have found increased levels of eosinophils in the bone marrow of two Ikzf1-mutant mouse models. Furthermore, we have found that these two mouse models display different phenotypes in regards to eosinophils in peripheral tissues, suggesting that Ikaros regulates two or more aspects of eosinophil biology. We study the role of Ikaros in regulation of eosinophil homeostasis through analysis of these Ikzf1-mutant mouse models as well as by using Ikaros-perturbation in human cells. We have found that Ikzf1-mutant bone marrow cells have different response to cytokine-driven in vitro differentiation. Furthermore, degradation of Ikaros, and the family member Aiolos, by IMiDs leads to altered expression of chemokine receptors, which are important for migration. We conclude that Ikaros is an important regulator of eosinophils, and aim to uncover the underlying mechanisms. Hematopoiesis and Immune System Development (HEM)

BACKGROUND:B-cell acute lymphoblastic leukemia (B-ALL) is classified into subgroups based on known driver oncogenes and molecular lesions, including translocations and recurrent mutations. However, the current diagnostic tests do not identify subtypes or oncogenic lesions for all B-ALL samples, creating a heterogeneous B-ALL group of unknown subtypes.METHODS:We sorted primary adult B-ALL cells and performed transcriptome analysis by bulk RNA sequencing (RNA-seq).RESULTS:Transcriptomic analysis of an adult B-ALL cohort allowed the classification of four patient samples with subtypes that were not previously revealed by standard gene panels. The leukemia of two patients were of the DUX4 subtype and two were CRLF2+ Ph-like B-ALL. Furthermore, single nucleotide variant analysis detected the oncogenic NRAS-G12D, KRAS-G12D, and KRAS-G13D mutations in three of the patient samples, presenting targetable mutations. Additional oncogenic variants and gene fusions were uncovered, as well as multiple variants in the PDE4DIP gene across five of the patient samples.CONCLUSION:We demonstrate that RNA-seq is an effective tool for precision medicine in B-ALL by providing comprehensive molecular profiling of leukemia cells, identifying subtype and oncogenic lesions, and stratifying patients for appropriate therapy.

Background and Significance As part of canonical WNT signaling, TCF7-family factors pair with β-catenin to activate MYC and promote survival and proliferation. β-catenin protein degradation is initiated by GSK3B- and CK1a-dependent phosphorylation. Genetic lesions of the β-catenin protein degradation pathway are frequent throughout the spectrum of cancer, but conspicuously absent in B-ALL, CLL and mantle cell lymphoma. Compared to other cell lineages, we found that B-cells express 80-200-fold lower levels of β-catenin protein and critically depend on efficient β-catenin protein degradation. Results Gene expression and proteomic analyses of solid tumor and myeloid leukemia cancer cell lines (n=779; DepMap) and B-ALL and B-cell lymphoma (n=131) revealed that β-catenin protein levels were markedly reduced and barely detectable in the latter despite comparable mRNA expression levels. To address this discrepancy, we engineered 10 B-lymphoid and 9 non-lymphoid cell lines with a dual fluorescence protein stability reporter for simultaneous measurement of β-catenin mRNA and protein levels, which corroborated a B-cell-specific acceleration of β-catenin protein turnover. Unlike other cell types, in B-cells, β-catenin-protein was constitutively phosphorylated by GSK3B and CK1a, neddylated by NAE1 and poised for proteasomal degradation. Combining computational and genetic approaches, we identified constitutive lack of N-cadherin (CDH2) and other adherens junction proteins as the underlying reason for accelerated β-catenin turnover in B-cells (r=0.65; P=3.78E-14). Consistent with their migratory behavior, B-cells lack adherens junctions, which shield β-catenin protein from degradation and promote cell adhesion in tissues. Interestingly, doxycycline induced expression of CDH2 in B-ALL cells enabled rapid accumulation of β-catenin at the cell membrane, while removal of doxycycline caused depletion of both CDH2 and β-catenin protein degradation. To investigate the significance of constitutive β-catenin degradation in B-cell malignancies, we developed genetic models of APC, GSK3B and CK1a haploinsufficiency in B-ALL cells. Induced haploinsufficiency resulted in a moderate accumulation of β-catenin protein, acute cell death and loss of colony formation in B-ALL cells. β-catenin stabilization in B-ALL cells subverted clonal fitness and leukemia initiation capacity. We engineered human B-ALL (n=6), mantle cell lymphoma PDX (n=4) as well as AML (n=4) for inducible expression of degradation resistant β-catenin, which induced acute cell death in B-lymphoid but accelerated proliferation in myeloid cells. In non-lymphoid cells, β-catenin interacts with TCF7 factors to drive transcriptional activation of Myc. Surprisingly, defective protein degradation and accumulation of β-catenin resulted in repression of Myc in B-cell tumors. Flow-cytometry and time-lapse microscopy experiments using mTurqouise-β-catenin and eGFP-Myc double reporter B-ALL cells revealed that 12 hours after disruption of β-catenin protein degradation, B-ALL cells accumulated b-catenin protein and abruptly lost Myc expression. Mass spectrometry-based protein-interactome studies identified that instead of TCF7, β-catenin formed unique with B-lymphoid Ikaros factors (Ikzf1/Ikzf3) and repressive nucleosome remodeling and deacetylase (NuRD) components. ChIP-seq analysis highlighted that binding of β-catenin/Ikaros/NuRD complexes at lymphoid-specific Myc super-enhancer regions led to loss of H3K27Ac active enhancer marks and transcriptional repression of Myc. CRISPR-mediated engineering of a point mutation in a prominent Ikaros binding motif within the Myc-BENC superenhancer region subverted β-catenin-dependent Myc repression and enabled B-ALL to survive defective β-catenin protein degradation. Conclusion Here, we show that B-cell malignancies, including B-ALL and mantle cell lymphoma uniquely depend on constitutive β-catenin degradation. Genetic defects of the β-catenin protein degradation pathway that promote cancer in other lineages are not compatible with B-cell identity. The deleterious outcome of β-catenin accumulation in B-lymphoid cells is predicated on B-cell-specific Ikaros transcription factors and explains the unique dependency of B-lymphoid cells on highly efficient mechanisms of β-catenin protein degradation.

Immune cell development is a tightly controlled process that enables a homeostatic level of innate and adaptive immune cells of the myeloid and lymphoid lineages, respectively. Imbalance in immune cells can lead to diseases due to immune defects or over-activation. such as immunodeficiency, allergies or autoimmunity. Ikaros is a transcription factor known to be critically important for lymphoid development, with recurrent mutations in different human diseases, but less is known about the role of Ikaros in myeloid development. Ikaros is encoded by the Ikzf1gene, and contains four DNA-binding zinc fingers (ZnF) and two ZnFs mediating protein dimerization. The four DNA-binding ZnFs (ZnF1 through ZnF4) are encoded by three exons, with the two central ZnF2 and ZnF3 encoded by exon 5 being required for binding to the core DNA recognition site. The two flanking ZnFs 1 and 4 are encoded by exon 4 and exon 6, and can modulate DNA binding by contacting the flanking DNA. Interestingly, Ikaros is highly alternatively spliced, creating multiple isoforms with different combinations of the DNA-binding ZnFs. We study the role of Ikaros in immune cell development using Ikzf1-mutant mouse models, and analysis of mice with deletions of exon 4 or exon 6 (encoding ZnF1 and ZnF4, respectively) has revealed differential phenotypes in the two mutant mouse models. Here, we describe a new role of Ikaros in regulation of eosinophils. We have found that Ikaros is required for limiting eosinophil production in bone marrow (BM), as both mutants show increased levels of BM eosinophils. Interestingly, the levels of peripheral tissue eosinophils differ between the two mutants, suggesting a second role of Ikaros in regulating migration, tissue recruitment or retention.

β-catenin-signaling promotes proliferation and survival in epithelial, neuronal, and mesenchymal lineages, but is dispensable for B- and T-cell development. β-catenin activation is a common oncogenic driver throughout all types of cancer with the exception of B- and T-lymphoid malignancies. Unlike epithelial tissues, we found that B- and T-cells consistently lack β-catenin expression. Instead, both B- and T-cells were highly sensitive to β-catenin-activation and critically depend on its negative regulation by GSK3β-dependent phosphorylation and degradation. Inhibition of GSK3β induced dramatic nuclear accumulation of β-catenin, anergy and cell death. In contrast, genetic deletion of β-catenin enabled clonal expansion of premalignant B- and T-cells. In epithelial cells, β-catenin and TCF-family factors form complexes for transcriptional activation of MYC. Instead of TCF-factors, our interactome studies in B- and T-cells revealed that, β-catenin formed complexes with lymphoid-specific Ikaros zinc finger (IKZF) transcription factors for transcriptional repressionof MYC at a recently discovered MYC ‘blood enhancer cluster’ (BENC). Hence, activation of β-catenin engages repressive complexes with Ikaros factors to regulate lymphopoiesis through transcriptional control of MYC. We propose that β-catenin-accumulation functions as sensing mechanism of pathological B- and T-cell signaling and that this mechanism can be leveraged for targeted eradication of pathological (autoreactive or malignant) clones. To engage this mechanism, targeted accumulation of β-catenin can be achieved by GSK3β-inhibition and represents a selective vulnerability of pathological B- and T-cell clones.

Background: Small molecule GSK3B inhibitors were developed for solid tumors and neurological conditions, including Alzheimer's and Parkinson disease. Five GSK3B inhibitors achieved favorable safety and PK/PD profiles in a total of 22 Phase 1 and 2 clinical trials but no clinical response for any of these indications. GSK3B is the kinase that phosphorylates β-catenin to initiate its degradation, hence GSK3B small molecule inhibition results in rapid nuclear accumulation of β-catenin protein. Significance: Activating mutations in the Wnt/β-catenin pathway are common oncogenic drivers throughout all main types of cancer (n=64,812) but not found in B- and T-lymphoid malignancies (n=2,137). Studying tissue microarrays by β-catenin immunohistochemistry, lung, colon, breast, and melanoma specimen (n=57) showed consistently high levels of nuclear β-catenin, whereas B- and T-cell lymphomas (n=84) lacked β-catenin signal. Despite comparable mRNA levels, proteomic analyses revealed 60-400-fold lower β-catenin levels in lymphoid compared to epithelial tumors (n=1,389) suggesting that lymphoid malignancies rely on powerful mechanisms for β-catenin degradation, e.g. by GSK3B. Results: To study the effects of β-catenin activation in human cells, we induced expression of stabilized form of β-catenin in 18 lymphoid (B- and T-ALL, MCL, DLBCL, PTCL) and four myeloid leukemia cell lines. In B- and T-lymphoid cells, β-catenin accumulation compromised clonal fitness, colony formation and induced cell death, but increased competitive fitness and colony formation in myeloid leukemia cells. CEBPα-mediated reprogramming of B-ALL cells into a myeloid phenotype reversed the deleterious effects highlighting that effects of β-catenin-accumulation strictly depend on lymphoid lineage-identity. RNA-seq studies upon inducible activation of β-catenin in lymphoid cells, revealed transcriptional repression of Myc and Myc target genes as principal gene expression change. Unlike activating β-catenin:TCF7 complexes in epithelial cells, our global interactome studies in B- and T-ALL and lymphoma cells identified repressive β-catenin complexes with lymphoid-specific Ikaros zinc finger (IKZF1/2/3) proteins and several components of the NuRD complex including Mta1/2, Gatad2a/b, Chd4, HDAC1/2. Interactome and ChIP-seq analyses revealed that β-catenin together with lymphoid-specific Ikaros factors recruited repressive NuRD complex members to deacetylate H3K27 and repress MYC superenhancer (SE) regions. Genetic deletion of both IKZF1 and IKZF3 or HDRT-based mutation of Ikaros binding motifs within the Myc-SE enabled resistance to β-catenin accumulation. To leverage this previously unrecognized vulnerability of lymphoid malignancies, we examined five clinically approved GSK3B small molecule inhibitors that achieved favorable safety profiles at micromolar plasma concentrations in clinical trials for neurological disorders and solid tumors. Strikingly, LY2090314 and CHIR99021 were effective at low nanomolar concentrations in B- and T-cell malignancies that expressed lymphoid Ikaros factors. At IC50 concentrations of <5 nM, GSK3B-inhibitors induced massive accumulation of β-catenin, repression of MYC and acute cell death. In B-ALL cells with IKZF1-deletion, expression of the remaining IKZF3 factor was sufficient to retain full sensitivity to GSK3B-inhibition. Deletion of β-catenin, however, abolished the effects of GSK3B inhibitors and relieved suppression of MYC, demonstrating accumulation of β-catenin represents as central mechanism of action. Preclinical in vivo treatment experiments of refractory B-ALL and mantle cell lymphoma PDX validated small molecule GSK3B-inhibition as strategy to overcome conventional mechanisms of drug-resistance. Conclusions: B- and T-lymphoid malignancies not only lacked expression of β-catenin but critically depend on GSK3B for effective β-catenin degradation. Our interactome studies in lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros and NuRD complex factors. Strikingly, GSK3B small molecule inhibitors engage this new pathway at low nanomolar concentrations and demonstrated favorable safety profiles in Phase 1 and Phase 2 trials. Safety and efficacy of GSK3B-inhibitors opens up an immediate path for repurposing these agents towards refractory B-cell malignancies.

Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator-positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations.

In most cell types, nuclear β-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of β-catenin but critically depended on GSK3β for effective β-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, β-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive β-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3β small molecule inhibitors to subvert β-catenin degradation. Clinically approved GSK3β-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of β-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3β-inhibitors for targeted engagement of lymphoid-specific β-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies.HIGHLIGHTS:Unlike other cell lineages, B-cells express nuclear β-catenin protein at low baseline levels and depend on GSK3β for its degradation.In B-cells, β-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. CRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed β-catenin-dependent Myc repression and induction of cell death. The discovery of GSK3β-dependent degradation of β-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3β-inhibitors for the treatment of refractory B-cell malignancies.GRAPHICAL ABSTRACT:Abundant nuclear β-cateninβ-catenin pairs with TCF7 factors for transcriptional activation of MYCB-cells rely on efficient degradation of β-catenin by GSK3βB-cell-specific expression of Ikaros factors Unique vulnerability in B-cell tumors: GSK3β-inhibitors induce nuclear accumulation of β-catenin.β-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC.

The hematopoietic transcription factor Ikaros (IKZF1) regulates normal B cell development and functions as a tumor suppressor in precursor B cell acute lymphoblastic leukemia (B-ALL). MicroRNAs (miRNAs) are small regulatory RNAs that through post-transcriptional gene regulation play critical roles in intracellular processes including cell growth in cancer. However, the role of Ikaros in the regulation of miRNA expression in developing B cells is unknown. In this study, we examined the Ikaros-regulated miRNA targets using human IKZF1-mutated Ph+ B-ALL cell lines. Inducible expression of wild-type Ikaros (the Ik1 isoform) caused B-ALL growth arrest and exit from the cell cycle. Global miRNA expression analysis revealed a total of 31 miRNAs regulated by IK1, and ChIP-seq analysis showed that Ikaros bound to several Ik1-responsive miRNA genes. Examination of the prognostic significance of miRNA expression in B-ALL indicate that the IK1-regulated miRNAs hsa-miR-26b, hsa-miR-130b and hsa-miR-4649 are significantly associated with outcome in B-ALL. Our findings establish a potential regulatory circuit between the tumor-suppressor Ikaros and the oncogenic miRNA networks in IKZF1-mutated B-ALL. These results indicate that Ikaros regulates the expression of a subset of miRNAs, of which several may contribute to B-ALL growth.

The Ikzf1 gene (encoding Ikaros) is essential for B cell development, but also plays important roles in the development of other hematopoietic lineages. Ikaros is a zinc finger (ZnF) transcription factor, with ZnF1 through ZnF4 involved in DNA binding, and ZnF5-ZnF6 required for dimerization. Previous work in our group has shown that germline deletion of the first or fourth ZnFs (ΔF1 and ΔF4 mice) resulted in different defects in lymphoid development. Here, we present our results from investigation of the roles of ZnF1 and ZnF4 in myeloid cell development. Flow cytometry analysis of bone marrow (BM), peritoneal cavity, lung and spleen cells show that ΔF1 and ΔF4 mice have significantly altered levels of eosinophils, basophils, monocytes, and mast cells. Most importantly, the observed changes differ between the two mutants. Interestingly, we observed a systemic increase in eosinophils in all tissues investigated in one of our ZnF-mutant mice. When culturing BM cells ex-vivo, we see that cells from the mutant mice proliferate at a higher rate than that of wt, and respond more actively to cytokine stimulation. Furthermore, ex vivo differentiation assay displayed a ZnF-dependent defect in mast cell/basophil development, and skewing towards eosinophil development, supporting the finding that ZnF1 and ZnF4 of Ikaros differentially regulate granulocyte development. In addition to studying immune cell subsets and ex-vivo stimulation assays, we aim to sort granulocyte cell populations from the ΔF1 and ΔF4 mice and perform RNA-seq to investigate differential gene regulation. We conclude that Ikzf1 plays a role in the regulation of eosinophils, mast cells and basophils, and will present results to date.

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

Objective Among the different methods to profile the genome-wide patterns of transcription factor binding and histone modifications in cells and tissues, CUT&RUN has emerged as a more efficient approach that allows for a higher signal-to-noise ratio using fewer number of cells compared to ChIP-seq. The results from CUT&RUN and other related sequence enrichment assays requires comprehensive quality control (QC) and comparative analysis of data quality across replicates. While several computational tools currently exist for read mapping and analysis, a systematic reporting of data quality is lacking. Our aims were to 1) compare methods for using frozen versus fresh cells for CUT&RUN and 2) to develop an easy-to-use pipeline for assessing data quality. Results We compared a workflow for CUT&RUN with fresh and frozen samples, and present an R package called ssvQC for quality control and comparison of data quality derived from CUT&RUN and other enrichment-based sequence data. Using ssvQC, we evaluate results from different CUT&RUN protocols for transcription factors and histone modifications from fresh and frozen tissue samples. Overall, this process facilitates evaluation of data quality across datasets and permits inspection of peak calling analysis, replicate analysis of different data types. The package ssvQC is readily available at https://github.com/FrietzeLabUVM/ssvQC.

The zinc-finger (ZnF) transcription factor Ikaros (encoded by Ikzf1) plays an essential role in regulating transcriptional programs required for B cell development, as Ikzf1null/null mice do not develop B cells. Ikaros contains two ZnF domains which function in DNA-binding and protein-protein interactions, respectively. Recently, mutations within the central DNA-binding ZnF domain of the IKZF1 gene has been linked to autoimmunity. Likewise, our murine model containing a germ-line deletion of the fourth ZnF (Ikzf1DF4/DF4) displays a B cell hyper-reactive (HR) phenotype, with B cells activated by anti-IgM without the requirement for a second co-stimulatory signal. Although Ikaros has been well-studied in early developing B cells, how loss-of-function mutations in Ikzf1 impacts downstream effector functions of mature B cells, remains largely unknown. Here, we investigated the role of Ikaros in regulating an epigenetic program required for co-stimulation-restricted activation of B cells. We utilized transcriptomic and epigenomic approaches (RNA-seq and ATAC-seq) to define transcriptional programs which dynamically change upon ex vivo stimulation and investigated their Ikaros-dependency. Interestingly, we found that the largest subset of genes deregulated between wt and Ikzf1DF4/DF4 B cells, occur prior to stimulation. In addition, CUT&RUN performed in wt B cells shows Ikaros binding to a large subset of genes expressed upon stimulation with anti-IgM and CD40L. Together, these results demonstrate that Ikaros regulates the transcriptional landscape of BCR-activated mature B cells. In the future, we will be integrating single cell RNA-seq analysis to investigate B cell heterogeneity in wt and Ikzf1DF4/DF4 B cells.

Novel targeted therapies have substantially improved the prognosis of patients with B-cell malignancies. However, a substantial fraction of patients relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance [EMDR]). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumor cell adhesion enabling the physical displacement from protective niches. In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we antagonized stromal expressed PKC-beta, which we have previously shown to be a stroma-autonomous signaling pathway critical for the survival of malignant B cells (Lutzny et al., Cancer Cell 2013). The dependency on stroma PKC-b was uniformly found for acute (ALL) and chronic (CLL, MCL) B-cell malignancies. In particular, our data demonstrate that stroma PKC-b is of key importance for multidrug resistance of malignant B cells (Park et al., Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B-cell malignancies. We identified that stroma PKC-b drives a transcriptional program, activating TGF-b and BMP-signaling in tumor cells. Our data show that antagonizing stroma signals with TGF-b inhibitors abrogated upregulation of BCL-XL and overcomes stroma-dependent resistance to venetoclax. This activation operates in parallel to the activation of the transcription factor EB (TFEB) as a downstream target of PKC-b. Interference with these signaling pathways impairs plasma membrane integrity of MSCs by downregulation of numerous adhesion and signaling molecules (e.g., ADAM17), required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-b for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EuTCL-1 driven B-cell tumors and treated with venetoclax in combination with or without enzastaurin (PKC-b inhibitor). Combined treatment significantly prolonged survival, based on PKC-b mediated impairment of lysosome biogenesis in vivo. Similarly, concurrent treatment of PKC-b inhibitors with chemotherapy also improved survival in an ALL-PDx model. Our data demonstrate that mitigating EMDR with small-molecule inhibitors of PKC-b or TGF-b signaling enhances the effectiveness of both targeted and nontargeted chemotherapies and, moreover, has the ability to overcome venetoclax resistance in B-cell malignancies in vivo. A clinical trial to test the dual inhibition of stroma and tumor cells in lymphoma patients is in preparation. Citation Format: Eugene Park, Jingyu Chen, Andrew Moore, Maurizio Mangolini, Joseph R. Byod, Hilde Schjerven, James C. Williamson, Paul J. Lehner, Michael Leitges, Alexander Egle, Marc Schmidt-Supprian, Seth Frietze, Ingo Ringshausen. Overcoming venetoclax resistance in B-cell malignancies by antagonism of stromal TGF-beta-mediated drug resistance [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-62.

Inhibition of stromal cell PKC-β mitigates environment-mediated drug resistance in B cell malignancies.

Autoimmunity is thought to arise due to a combination of genetic and environmental factors. IKZF1, encoding the Zinc finger (ZnF) transcription factor Ikaros, is implicated in human autoimmunity through GWAS and recent discovery of germline IKZF1 mutations in patients with autoimmune diseases. We have previously created mouse models with targeted deletions of Ikaros DNA-binding ZnF1 or ZnF4, and recently found that mice lacking Ikaros ZnF4 (Ik-dF4) displayed very high levels of autoreactive antibodies and elevated levels of the autoimmunity-associated cytokine IL-6 in serum at young age, while mice lacking ZnF1 did not. Furthermore, ex vivo stimulation of peripheral B cells with anti-IgM (aIgM) +/− CD40L revealed that Ik-dF4 B cells responded to BCR-stimulation alone (aIgM), while wt B cells required a second co-stimulatory signal (CD40L). To understand the underlying mechanism, we performed transcriptome profiling of these stimulated wt and Ik-dF4 B cells. Overall, the Ik-dF4 B cells displayed the same pattern of gene-expression changes as wt B cell upon activation by both signals (aIgM + CD40L). Further analysis revealed a set of genes selectively altered upon aIgM alone in Ik-dF4 B cells, and another set of genes that were aberrantly expressed in Ik-dF4 B cells at baseline. Pathway analysis suggest candidate mediators, and we have initiated functional studies. Preliminary results indicate that a small molecule inhibitor can block the aberrant activation with aIgM alone in Ik-dF4 B cells, while displaying only partial inhibition of the normal two-signal activation. Studies are actively ongoing to confirm these preliminary findings, and we will present our results to date at the meeting for how Ikaros regulates B cell tolerance.