Markus Muschen - US grants

DESCRIPTION (provided by applicant): B cell lineage acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children and is also common in adults. Compared to patients with other malignancies, cure rates for patients with ALL are in general higher. The ALL subset with the so-called Philadelphia chromosome (Ph) encoding the oncogenic BCR- ABL1 kinase, however, has a particularly poor prognosis. Ph+ ALL is typically treated with BCR-ABL1 kinase inhibitors such as Imatinib. The treatment response to Imatinib, however, is not durable and after a latency of only a few months, Ph+ ALL cells become drug-resistant and ALL relapses. Of note, the oncogenic BCR-ABL1 kinase is not only expressed in Ph+ ALL (mainly p190 BCR-ABL1) but also in >95% of cases of chronic myeloid leukemia (CML;mainly p210 BCR-ABL1). In contrast to Ph+ ALL, long-term treatment of CML with Imatinib is effective and resistance develops only rarely. In a subgroup of patients with CML, however, the disease progresses into B lymphoid blast crisis (CML-LBC), in which treatment responses are as short-lived as in Ph+ ALL. In most cases, acquired resistance to Imatinib in Ph+ ALL and CML-LBC can be attributed to somatic mutations within the BCR-ABL1 kinase domain, which compromise the efficacy of Imatinib. In preliminary experiments for this proposal, we show that AID is specifically expressed in B cell lineage + clones of BCR-ABL1-driven leukemia (Ph ALL and CML-LBC). In these cells, AID functions as a mutator and thereby contributes to the drug-resistance typically observed in Ph+ ALL and CML-LBC. Based on these findings, our proposal addresses the question of + (1) how AID contributes to genetic instability and drug-resistance in Ph ALL (e.g. AID-specific deletions;Aim 1), (2) to which extent AID contributes to the progression of chronic phase CML to CML-LBC (outgrowth of B lymphoid subclones that carry advantageous mutations;Aim 2), (3) which factors cause aberrant expression of AID in Ph+ ALL and CML-LBC (Aim 3), + (4) and whether AID-expressing clones in Ph ALL and CML-LBC can be specifically targeted in a prodrug- based approach that takes advantage of the enzymatic activity of AID (Aim 4). + Together, these four Aims will help to elucidate mechanisms of drug-resistance in Ph ALL and CML-LBC and + propose a novel concept of targeted treatment Ph ALL and CML-LBC for pre-clinical evaluation. Aim 1: Contribution of AID to genetic instability in Ph+ ALL: We have generated BCR-ABL1-transformed B cell lineage leukemia cells with three levels of AID expression based on their genotype, namely Aid-/-, endogenous AID and forced AID-overexpression. We have injected these leukemia cells into congenic mouse recipients and will compare the developing leukemia clones by comparative genomic hybridization (CGH) analysis to identify AID-specific deletions. Deletion breakpoints will be verified by FISH analysis and mapped to AID-related somatic hypermutation hot spots. We will compare development of Imatinib-resistance in Aid-/- and Aid-wildtype leukemias developing in BCR-ABL1 p190-transgenic mice. Aim 2: Contribution of AID-induced mutations to progression of CML into lymphoid blast crisis: To clarify to which extent AID contributes to the progression of CML into lymphoid blast crisis, we will take two approaches. (1) Transformation of hematopoietic stem cells (HSC) by p210 BCR-ABL1 induces CML-like leukemia with subsequent progression into B lymphoid blast crisis. Studying transgenic mice expressing p210 BCR-ABL1 under control of the HSC-specific Scl-promoter on an Aid-/- background, we will investigate whether Aid-function is required for the outgrowth of B lymphoid blast crisis clones. Second, we will cross Scl-BCR-ABL1 p210 transgenic mice with an Aid-Cre reporter strain that carries YFP preceded by a loxP-flanked Stop cassette. Expression of Aid in these cells will lead to permanent genetic labeling with YFP. Based on YFP-labeling, this mouse model will indicate whether or not outgrowth of B lymphoid subclones requires expression of Aid at least at one point in time during the clonal evolution of CML Aim 3: Identification of factors that regulate AID-expression in BCR-ABL1-driven leukemias: We observed that AID expression substantially varies among primary Ph+ ALL cells from the same patient. These findings suggest that besides homogenously expressed BCR-ABL1 and B cell-specific transcription factors, additional AID-regulatory factors are only expressed in a subset of the leukemia population. Using an Aid-GFP reporter system, we will compare AIDhigh and AIDlow Ph+ ALL cells to identify key AID-regulatory factors. Aim 4: Prodrug-based targeting of AID-expressing Ph+ ALL cells: AID can deaminate monomeric deoxycytidine to deoxyuracil. We hypothesize that AID can likewise activate the monomeric prodrugs monomeric 5-FC (Ancobon(R)) and its derivatives 5-DFCR and Capecitabine (Xeloda(R)) into the cytotoxic metabolite 5- fluorouracil (5-FU). Taking advantage of the enzymatic activity of AID in Ph+ ALL and CML-LBC cells, we will target AID-expressing cells using 5-FC, 5-DFCR and Capecitabine for specific targeting of AID-expressing cells in Ph+ ALL and CML lymphoid blast crisis. PUBLIC HEALTH RELEVANCE: The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 kinase, which drives two types of leukemia: Acute lymphoblastic leukemia (Ph+ ALL) is derived from a transformed B lymphocyte and chronic myeloid leukemia (CML) originates from myeloid cells that would otherwise develop into macrophages or monocytes. While Ph+ ALL represents a rapidly progressive disease already at the outset, the course of CML is typically stable over many years and only shows rapid progression in the terminal stage, the so-called "blast crisis". The reasons leading to progression from chronic phase into blast crisis are largely unknown. The treatment of both Ph+ ALL and CML has been revolutionized by the discovery of the BCR-ABL1 kinase-inhibitor Imatinib. However, even though both leukemia types carry the same genetic abnormality, the outcome of Imatinib-treatment is strikingly different: whereas Imatinib is very effective in the treatment of chronic phase CML, treatment success is only transient for patients with Ph+ ALL or blast crisis CML. In these patients, the leukemia recurs after 4 months on average and is typically drug-resistant owing to the acquisition of additional mutations. Therefore, the understanding of the underlying mutation mechanism and its potential inhibition appears to be critical for further improvement of treatment strategies of Ph+ ALL and CML. Recent work by our group demonstrated that the oncogenic BCR-ABL1 kinase in Ph+ ALL and blast crisis, but not chronic phase CML, induces expression of a mutator enzyme, termed AID (Activation-induced Cytidine Deaminase). The mutations that confer drug-resistance in Ph+ ALL and blast crisis CML can indeed be explained by activity of the AID enzyme. Additional experiments showed that engineered expression of AID in AID-negative chronic phase CML cells introduces the same mutations that cause drug-resistance in patients with AID-positive Ph+ ALL and CML blast crisis. Based on these observations, we propose four series of experiments to address the following questions: (1) Is the AID enzyme required for drug-resistance in AID-positive Ph+ ALL? To test this hypothesis, we will investigate whether BCR-ABL1-induced leukemia cells from mice carrying a deletion of the AID-gene fail to develop drug-resistance. (2) Does the AID enzyme play a critical role in the progression of chronic phase CML into blast crisis? Chronic phase CML can be treated very successfully for many years, whereas blast crisis represents a final and often fatal stage of the disease. (3) Which are the factors that induce aberrant expression of the AID enzyme in Ph+ ALL and blast crisis CML? The identification of such factors will likely help to understand how expression of this deleterious mutator enzyme can be prevented. (4) Is it possible to target AID-expressing cells by taking advantage of the enzymatic activity of AID? For this approach, we propose to use a precursor-drug that has no effect as such but will become toxic upon AID-mediated conversion. Given that AID-expressing cells are more likely to be drug-resistant than others, we propose a treatment approach that is focused on the AID-expressing leukemia cells.

DESCRIPTION (provided by applicant): Pre-B cell receptor signaling in acute lymphoblastic leukemia. B cell precursors in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell receptor. For this reason, defects in components of the pre-B cell receptor signaling chain cause a severe block of early B cell development in humans. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at early stages of B cell development. In this proposal, we test the hypothesis that the developmental arrest in B cell lineage ALL predominantly reflects aberrant pre-B cell receptor function. B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at 80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies in addition to cytotoxic drug treatment seem promising to further improve therapy options for ALL patients. In preliminary studies for this proposal on 148 cases of pre-B cell-derived human ALL, we found that ALL cells carrying an E2A-PBX1- or MYC- gene rearrangement are -like normal pre-B cells- highly selected for the expression of a functional pre-B cell receptor. In striking contrast, ALL cells with other cytogenetic abnormalities (e.g. BCR-ABL1- or MLL-AF4) lack expression of a functional pre-B cell receptor in virtually all cases. In a proof-of-principle experiment, we studied pre-B cell receptor function during progressive leukemic transformation of pre-B cells in BCR-ABL1-transgenic mice: Interestingly, signaling from the pre-B cell receptor and the oncogenic BCR-ABL1 kinase are mutually exclusive and only "crippled" pre-B cells that fail to express a functional pre-B cell receptor are permissive to transformation by BCR-ABL1. As opposed to ALL cells with BCR-ABL1- or MLL-AF4-fusion gene, pre-B cell receptor signaling is active in E2A-PBX1- or MYC-transformed ALL, because these cells exhibit a vigorous Ca2+ signal in response to pre-B cell receptor engagement. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell receptor signaling enables (Type I) or suppresses (Type II) leukemic growth. Studying primary human ALL xenografts and transgenic mouse models for Type I and Type II ALL, we propose in Aim 1 to establish characteristic key differences of pre-B cell receptor signaling in the two subgroups. In Aim 2, we propose to identify the requirements for pre-B cell receptor-dependent survival signaling in Type I ALL as potential targets for pharmacological inhibition. Conversely, we propose in Aim 3 to elucidate the mechanisms of pre-B cell receptor-inactivation in Type II ALL and how functional pre-B cell receptor signaling induces apoptosis in Type II ALL cells. Given that pre-B cell receptor signaling in this subgroup of ALL effectively suppresses leukemic growth, our goal in Aim 3 is to interfere with these inactivation mechanisms to restore pre-B cell receptor-dependent apoptosis-signaling in Type II ALL cells. The proposed discrimination between Type I and Type II ALL resembles the classification of mature B cell lymphoma, in which subgroups can be distinguished based on the presence (i.e. Non-Hodgkin's lymphoma) and absence (i.e. Hodgkin's lymphoma) of B cell receptor expression. The central goal of this proposal is to establish the role of pre-B cell receptor signaling during malignant transformation and clonal evolution of ALL and to target individual components of its signaling cascade for the development of novel pathway-specific therapy approaches for ALL. PUBLIC HEALTH RELEVANCE: Pre-B cell receptor signaling in acute lymphoblastic leukemia. B lymphocytes are not only the cells that produce antibodies as part of the human immune system, they are also the cell of origin in most cases of acute lymphoblastic leukemia (ALL). ALL represents by far the most frequent type of cancer in children and is also a common disease in adults. For many years, patients with ALL are treated with chemotherapy and current treatment protocols lead to cure rates of 80 percent for children and 55 percent for adult patients with ALL. Our goal is to better understand the biology of human ALL, namely as a catastrophic aberration of normal B lymphocyte development. During normal B lymphocyte development, the pre-B cell receptor represents a critical signaling unit that guides early B lymphocyte precursors on their path of maturation. If signaling from the pre-B cell receptor is compromised, as for instance in patients with innate immune defects, the B lymphocyte precursors are arrested in their development at a primitive stage -as in ALL cells. Therefore, we propose to investigate the function of the pre-B cell receptor signaling unit (1) as a potential target to disrupt aberrant cell signaling that promotes leukemic growth and (2) to restore normal pre-B cell receptor signaling in the leukemia cells.

DESCRIPTION (provided by applicant): B cell lineage acute lymphoblastic leukemia (ALL) represents by far the most frequent type of malignancy in children and teenagers. Despite significant advances in ALL treatment, many patients still die because of drug- resistance or leukemia relapse. Since recent work implicated leukemia stem cells in both drug-resistance and relapse of the disease, current therapy approaches focus on leukemia stem cell eradication. In contrast to B cell lineage ALL, leukemia stem cells in myeloid lineage leukemia have been extensively characterized. Acute and chronic myeloid leukemias develop hierarchically from a phenotypically distinct stem cell population. However, recent work suggests that no such hierarchy exists in B cell lineage ALL, which precludes an unequivocal phenotypic definition of leukemia stem cells in ALL. Therefore, we propose to functionally characterize leukemia stem cells in B cell lineage ALL based on an operational definition, i.e. the ability to maintain stem cell self-renewal and overcome oncogene-induced senescence. Specifically, we will test the hypothesis that the BCL6 transcriptional repressor represents one of the critical factors in B cell lineage ALL to maintain a pool of functional leukemia stem cells. The BCL6 proto-oncogene is frequently translocated in diffuse large B cell lymphoma (DLBCL) and maintains self-renewal capacity of DLBCL cells by transcriptional repression of p53 in the lymphoma cells. However, an oncogenic function of BCL6 has not been described in other cell types so far. In preliminary studies for this proposal, we have discovered aberrant expression of BCL6 as a central component of a fundamentally novel pathway of leukemia stem cell maintenance: BCL6 prevents leukemia stem cell depletion through negative regulation of the Arf/p53/p21 pathway in B cell lineage ALL. Compared to leukemias from BCL6-/- mice, BCL6 is required for the reactivation of an early embryonic gene expression program, development of drug-resistance, leukemia cell colony formation and leukemia-initiation in serially transplanted NOD/SCID mice in B cell lineage but not myeloid lineage leukemia. A novel retro-inverso BCL6 peptide inhibitor (RI-BPI) strongly synergized with tyrosine kinase inhibitors in the treatment of B cell lineage ALL cells in vitro and in vivo. This proposal will test the hypothesis that BCL6-dependent self-renewal represents a critical requirement for stem cell maintenance in B cell lineage ALL. Based on the discovery of BCL6 as a key component of a fundamentally novel pathway of stem cell self-renewal in various subtypes of ALL, we propose three Aims to develop these findings towards application in patient care: (1) To test the hypothesis that aberrant expression of BCL6 in leukemia cells prevents Arf/p53-mediated senescence and promotes stem cell quiescence, (2) To identify transcriptional targets of BCL6 in primary human leukemia cells and their contribution to self-renewal signaling and (3) To validate BCL6-BPI as a therapy adjuvant for targeted eradication of leukemia stem cells.

DESCRIPTION (provided by applicant): Multiple lines of evidence support a critical role of infections in the etiology of secondary lesions that initiate childhood leukemia. This led to the hypothesis that delayed exposure to common infections predisposes to childhood pre-B ALL. Early exposure to infectious pathogens, e.g. through daycare attendance or vaccinations, leads to mild and typically subclinical immune responses and is associated with a significantly diminished risk to develop childhood ALL. By contrast, delayed infections are often more serious, cause more vigorous immune responses and predispose to childhood pre-B ALL. The central goal of this proposal is to experimentally test the 'delayed infections' hypothesis and to delineate mechanisms of genetic vulnerability of human pre-B cells in the context of infection. Pre-B cells undergo Rag1/2-dependent immunoglobulin V(D)J gene recombination and represent the cell of origin of childhood ALL. Rag1/2-mediated recombinase activity causes DNA double strand breaks and is associated with a low risk to acquire chromosomal translocations. This risk, however, dramatically increases when Rag1/2 enzymes are expressed concomitantly with the B cell-specific mutator enzyme AID. Hence, expression of Rag1/2 and AID is mutually exclusive and temporally separated in early and late B cell development, respectively: Rag1 and Rag2 expression is limited to immunoglobulin V(D)J gene recombination in pro- and pre-B cells. Conversely, AID expression is thought to be restricted to somatic hypermutation and class-switch recombination in mature B cells that have encountered antigen. In preliminary work for this proposal, we found that IL7R?/Stat5/Akt signaling is critical to keep normal pre-B cells in an antigen- unresponsive state. Upon withdrawal of IL7 or conditional deletion of Stat5, pre-B cells become fully responsive to antigen (e.g. LPS) and express AID at similar levels as mature B cells upon LPS stimulation. While high levels of IL7 in the bone marrow secure pre-B cells in an antigen-unresponsive state, we discovered a window of vulnerability during normal pre-B cell differentiation: Pre-B cell receptor signaling downregulates IL7R? expression and induces Rag1/2-mediated immunoglobulin light chain gene recombination in small resting (Fraction D) pre-B cells. Upon antigen encounter, Fraction D pre-B cells express high levels of AID in addition to Rag1/2, which dramatically increases their propensity to chromosomal translocations. Based on these findings, we hypothesize that Fraction D pre-B cells are highly susceptible to genetic lesions in the context of infection. We propose the following three Aims to test the prediction that the size of the Fraction D pre-B cell pool and the frequency and intensity of immune responses to infections will determine the likelihood of a pre-leukemic (e.g. TEL-AML1) pre-B cell clone to acquire critical secondary lesions.

DESCRIPTION (provided by applicant): Tyrosine kinase inhibitors (TKIs) against oncogenic tyrosine kinases (OTK; e.g. BCR-ABL1, EGFRL858R, ERBB2) have created a new era of treatment for OTK leukemia and solid tumors and have substantially improved clinical outcome. Despite their clinical success in chronic myeloid leukemia (CML), resistance to TKIs, such as Imatinib, is a common outcome in most OTK-malignancies. While combination of TKI with conventional chemotherapy results in prolonged survival for most OTK-malignancies, for a large group of patients, no curative treatment option is available. In preliminary work for this proposal we have found that two negative feedback regulators (SPRY2 and DUSP6) are expressed at high levels in OTK-malignancies but not in normal cells or tumor cells lacking an oncogenic tyrosine kinase. The inhibitory linker molecule Sprouty2 (SPRY2) attenuates signal transduction immediately downstream of the oncogenic tyrosine kinase, the Dual Specific Phosphatase DUSP6 interferes with distal signal transduction by dephosphorylation of ERK1/2. Expression levels of these molecules are dependent on the signaling strength of the oncogenic tyrosine kinase and are completely suppressed upon TKI-treatment. Molecules of the Sprouty- and DUSP- families of negative feedback mediators are often inactivated through deletion or promoter hypermethylation in Non- tyrosine kinase tumors but not in OTK-malignancies. Given that SPRY2- and DUSP6-mediated negative feedback signaling was highly active in all OTK-tumor samples studied, we performed experiments that demonstrated that OTK-leukemia cells indeed critically depend on continuous active negative feedback signaling to calibrate otherwise overwhelming tyrosine kinase signaling. In this proposal, we will test the central hypothesis that negative feedback regulators represent a fundamentally novel class of therapeutic targets for the treatment of OTK-leukemias and -solid tumors. The central goal of this proposal is to build a fundamental understanding of how blockade of negative feedback mechanistically leads to cell death in OTK-malignancies (Aims 1-2), and to leverage this mechanistic information towards the development of a new therapy concept based on alternating treatment schedules between TKIs and negative feedback inhibitors (NFIs; Aim 3).

DESCRIPTION (provided by applicant): In 2010, 18,600 adults and children were diagnosed with acute leukemias (ALL and AML) with 10,400 expected deaths (55%). Among acute leukemias, ALL and AML subtypes with an oncogenic tyrosine kinase have a particularly high frequency of relapse and overall poor outcome. For instance, median disease-free survival (DFS) for standard AML is 20 months compared to 4.6 months for patients with FLT3ITD AML. Likewise, DFS for standard risk ALL is at 23.8 months compared to 8.7 months for patients with Ph+ ALL carrying the BCR-ABL1 tyrosine kinase. While initial TKI therapy for patients with FLT3ITD AML and Ph+ ALL is initially successful, TKI fail to eradicate leukemia-initiating cells42 and the leukemias invariably relapses. Therefore, TKD-leukemias represent a frequent unsolved clinical problem. By contrast, the advent of potent TKI has transformed CML into a long-term condition. With 4,870 patients newly diagnosed in 2010, currently 24,800 patients live with CML in the US with a 5-year overall survival of >95%. However, also in CML, TKI fail to eradicate LIC, thus TKI-treatment for CML patients is typically life-long since measurable amounts of LIC persist in the bone marrow and CML re-emerges once TKI-treatment ceases. Since recent work has implicated leukemia initiating cells (LIC) in both initial drug-resistance and relapse of the disease, current therapy approaches need to focus on LIC eradication. Acute and chronic myeloid leukemias develop hierarchically from a phenotypically distinct stem cell population. However, recent work suggests that no hierarchically distinct stem cell population exists in B cell lineage ALL7. In the absence of a stem cell hierarchy, we hypothesize that B cell linage ALL have the ability to temporarily acquire stem cell capabilities, i.e. the ability to initiate leukema in xenograft transplant recipients. We will thus test the hypothesis that TKD-leukemia cells can switch between 'Progenitor-like proliferation' and 'Stem cell-like quiescence'. We are proposing four Aims to (1) validate BCL6 as therapeutic target in TKD-leukemia, (2) define mechanistic elements of BCL6-dependent drug-resistance, (3) validate and prioritize three different approaches of pharmacological inhibition of BCL6 and (4) develop a Phase I clinical trial for BCL6 inhibition in adults with relapse TKD-leukemia.

? DESCRIPTION (provided by applicant): Significance: Outcomes for patients with pre-B ALL have substantially improved over the past decades. However, this is not the case for 15-20% of children who relapse after initially successful treatment. With current algorithms of risk stratification, these patients are undistinguishable from patients who respond well to standard chemotherapy. As a consequence, most patients with pre-B ALL relapse will die from their disease, while others suffer long-term sequelae from unnecessary toxicity. This proposal is based on the central observation that relapse clones in B cell lineage leukemia are resistant to conventional chemotherapy but uniquely vulnerable to perturbations of glucose and energy metabolism. Therefore, agents that specifically target B cell-intrinsic metabolic liabilities may improve outcomes for patients with relapse leukemia without adding substantive toxicity to the treatment regimen. Rationale and Innovation: Unlike other cell types, (pre-) B cells are selected for an intermediate level signaling strength. Critical survival and proliferation signals emanate from the pre-B cell receptor (pre-BCR): Both attenuation below minimum (e.g. non-functional pre-BCR) and hyperactivation above maximum (e.g. autoreactive pre-BCR) thresholds of signaling strength trigger negative selection and cell death. We recently discovered that pre-B ALL, including relapse ALL, is bound by the same rules that also govern normal B cell selection (Swaminathan et al., Nature Medicine 2013). Despite oncogenic transformation, we found that basic mechanisms of negative selection are still functional in pre-B ALL. Recent studies by our group demonstrated that pharmacological hyperactivation of the pre-BCR tyrosine kinase SYK engages a deletional checkpoint, which is functionally equivalent with negative selection of autoreactive B cells. In preliminary studies for this proposal, we found that cell death in response to SYK hyperactivation is caused by acute energy depletion and exhaustion of glycolytic reserves of B cell lineage ALL cells. This proposal is based on the central observation that relapse clones in B cell lineage leukemia are resistant to conventional chemotherapy but uniquely vulnerable to perturbations of glucose and energy metabolism. Hypotheses: Based on these and other findings, we are proposing three new lines of investigation to be developed over the next seven years. This will allow us to test and refine this emerging concept for the treatment of B-lineage ALL relapse in a pre-clinical setting. (1) Relapse pre-B ALL cells frequently acquire deletions of B cell-specific transcription factors PAX5, and IKZF1, the functional significance of which is not known. Here we test the hypothesis that these lesions reduce stringency of B cell lineage commitment and thereby in part mitigate B cell intrinsic liabilities of glucose and energy metabolism. (2) Recent studies by our group revealed that pre-B ALL cells are uniquely addicted to inhibitory regulators of Syk-mTOR signaling (Shojaee et al., Cancer Cell 2014; Chen et al., Nature 2014). Here we test the hypothesis that signaling inhibitors protect pre-B ALL cells from energy depletion and exhaustion of glycolytic reserves and, hence, engagement of a deletional checkpoint, that is functionally equivalent with negative selection of self-reactive B cells. (3) Glucocorticoids are highly effective in killing both pre-B LL cells and self-reactive B cell clones in autoimmune diseases. Compared to any other cell types, B cell lineage cells are >75-fold more sensitive to glucocorticoids. Here we hypothesize that glucocorticoids, by inhibiting glucose uptake and glycolysis, cause acute energy depletion and, hence, exacerbate B cell-intrinsic metabolic liabilities.

PROJECT SUMMARY B cells critically depend on continuous survival and proliferation signals from a functional B cell receptor (BCR). Likewise, in ~50% of B cell malignancies, the tumor clone is driven by an oncogenic BCR-mimic. Oncogenic mimics of BCR-dependent proliferation and survival signals include BCR-ABL1 (Ph+ ALL), viral oncoproteins (e.g. EBV), RAS- and NF-?B-pathway activating lesions (Hodgkin's lymphoma, PMBL, ABC-DLBCL, hairy cell leukemia, Waldenström's macroglobulinemia). In preliminary studies, we found that CD25 is selectively expressed on malignant B cell clones driven by oncogenic BCR-mimics. While CD25 functions as IL2 receptor ?-chain on T cells, we recently discovered that CD25 is a critical feedback regulator of BCR signaling and oncogenic BCR- mimics in human B cell tumors. Genetic experiments demonstrated that CD25 is critical for the initiation of B cell leukemia and lymphoma in transplant recipients. Surface expression is rapidly induced by activity of BTK and PKC? downstream of the BCR and induced by FOXM1 and NF-?B at the transcriptional level. CD25 then recruits an inhibitory complex to the cell membrane to reduce and recalibrate BCR signaling or oncogenic mimicry of BCR-signaling. Analysis of three clinical cohorts revealed that high expression levels of CD25 are associated with poor clinical outcome in various B cell malignancies. While CD25 expression is associated with drug-resistance, inhibition of CD25 or disabling of CD25-dependent feedback control sensitizes multiple B cell malignancies to conventional drug-treatment. Based on these and other findings, we propose three Aims to (1) elucidate mechanisms of CD25 regulation, (2) explore usefulness of pharmacological subversion of CD25-mediated feedback control and (3) targeted eradication of CD25+ cells by CART25 cells and antibody-drug conjugates (ADC) as therapeutic adjuvant.

PROJECT SUMMARY Schistosomiasis affects more than 200 million people worldwide. During infection, the type 2 immune response and M2 macrophages (M?) play a critical role in granuloma formation around the eggs and enable the host to tolerate the tissue damage caused by these eggs, which become embedded in the liver and intestines. We previously identified phenotypic and functional differences between M2 M? derived from either tissue resident M? (M2tiss) or inflammatory monocytes (M2mono). We also determined that the M2 M? in acute liver granulomas formed after infection with Schistosoma mansoni are derived from inflammatory Ly6Chigh monocytes. However, as the granulomas mature and become more organized, M2mono in the granuloma eventually adopt features of M2tiss. This phenotypic conversion into M2tiss is disrupted in mice with vitamin A deficiency, an important micronutrient deficiency in developing countries, which leads to increased morbidity during infection. The key metabolite of vitamin A, retinoic acid (RA), may therefore be essential for the normal function of M2 M? in the liver granulomas. We hypothesize that RA signaling enables M2mono around the S. mansoni egg granulomas to adopt the properties of M2tiss after residence in the local microenvironment. In contrast to S. mansoni, other helminths (e.g. Heligmosoides polygyrus) induce expansion of M2 M? from tissue-resident M?. Hence, different helminth infections induce M2 activation in M? of different lineages. Differences in open chromatin regions may regulate differential activation of M2mono and M2tiss. We have investigated open regions of chromatin in M2mono and M2tiss. By coupling genome-wide gene expression data with sequencing data on chromatin structure, we are uncovering the regulatory networks and identifying key transcription factors that control the differential responses to IL-4 for different lineage of M?. We hypothesize that chromatin structure is reorganized to resemble tissue resident M? when M2mono around the S. mansoni egg granulomas adopt the properties of M2tiss. Failure of this process may increase mortality during infection. In this proposal we will (Aim 1) determine the role and mechanism of action of retinoic acid (RA) in regulating conversion to a M2tiss phenotype and (Aim 2) identify gene regulatory networks mediating differential M2 activation of M2mono and M2tiss, and the phenotypic conversion from M2mono to M2tiss. While our focus is to characterize the biology of M2 M? during helminth infections, our findings should be translatable to the many other sites and physiological processes whereby M2 M? play an important functional role (e.g. in adipose tissues, atherosclerosis, tumor microenvironments and during wound repair). Hence, the impact of these studies should be broader than just helminth infections alone.

PROJECT SUMMARY CD25 was previously identified as ?-chain of the heterotrimeric IL2 receptor (IL2R). High expression levels of CD25 was previously attributed to T-cell activation or transcriptional activation by Foxp3 in regulatory T-cells (Tregs). Our preliminary experiments revealed that CD25 is not only an IL2R chain, but in fact binds the CD3 signal chains of the T-cell receptor (TCR) for feedback control of TCR signaling strength. Reflecting its central role in normal T-cell development, the TCR and its downstream signaling pathway is a target of oncogenic transformation in the majority of peripheral T-cell lymphomas (PTCLs). Oncogenic TCR-mimics promote survival and proliferation even in the absence of a functional TCR. Oncogenic TCR-mimics include activating lesions of the TCR-signaling chain CD3?, proximal tyrosine kinases (PTKs; FYN, LCK, ZAP70) and ITK-SYK, NPM1-ALK fusions. In collaboration with other investigators of this P01, our group recently discovered that lymphoid malignancies are uniquely dependent on feedback regulation of PTKs (Chen et al., Nature 2015), PI3K (Shojaee et al., Nature Med 2016; Chan et al., Nature 2017) and ERK (Shojaee et al., Cancer Cell 2015; Xiao et al., Cell 2018). Here we validate the concept that in multiple PTCL subtypes, CD25 orchestrates feedback control of all three pathways (PTKs, PI3K and ERK). The central goal of this project is to deliver a comprehensive strategy to target CD25-mediated feedback control of oncogenic TCR signaling to overcome drug resistance in refractory PTCL. The following three Aims will (1) elucidate the mechanism of CD25-mediated feedback control of oncogenic TCR-signaling in PTCL subtypes, (2) provide a rationale for combining PI3K inhibitors with CD25 antibody-drug conjugates (ADC) and (3) evaluate efficacy of newly developed CD25 chimeric antigen receptors (CARs) engineered in T- and NK-cells and their effects on endogenous anti-tumor immune responses. Aim 1: Mechanisms of CD25-mediated feedback control of oncogenic TCR-signaling in PTCL. Aim 2: CD25-endocytosis and endosomal recycling as dynamic feedback control of oncogenic TCR-signaling Aim 3: Preclinical development and validation of CD25 CAR-T and CAR-NK cells.