Olivia L. Francis

Regulator of myeloid differentiation and function:The secret life of Ikaros

Ikaros (also known as Lyf-1) was initially described as a lymphoid-specific transcription factor. Although Ikaros has been shown to regulate hematopoietic stem cell renewal, as well as the development and function of cells from multiple hematopoietic lineages, including the myeloid lineage, Ikaros has primarily been studied in context of lymphoid development and malignancy. This review focuses on the role of Ikaros in myeloid cells. We address the importance of post-transcriptional regulation of Ikaros function; the emerging role of Ikaros in myeloid malignancy; Ikaros as a regulator of myeloid differentiation and function; and the selective expression of Ikaros isoform-x in cells with myeloid potential. We highlight the challenges of dissecting Ikaros function in lineage commitment decisions among lymphoid-myeloid progenitors that have emerged as a major myeloid differentiation pathway in recent studies, which leads to reconstruction of the traditional map of murine and human hematopoiesis.

Differences in Mouse and Human Nonmemory B Cell Pools

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ∼8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) stimulates in vitro proliferation of human fetal B-cell precursors. However, its in vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (−T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in −T mice. Patient-derived xenografts generated from +T as compared to −T mice showed a 3–6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from −T mice. +T/−T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

TSLP or IL‐7 provide an IL‐7Rα signal that is critical for human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) and IL‐7 are cytokines that signal via the IL‐7 receptor alpha (IL‐7Rα) to exert both overlapping and unique functions during early stages of mouse B‐cell development. In human B lymphopoiesis, the requirement for IL‐7Rα signaling is controversial and the roles of IL‐7 and TSLP are less clear. Here, we evaluated human B‐cell production using novel in vitro and xenograft models of human B‐cell development that provide selective IL‐7 and human TSLP (hTSLP) stimulation. We show that in vitro human B‐cell production is almost completely blocked in the absence of IL‐7Rα stimulation, and that either TSLP or IL‐7 can provide a signal critical for the production and proliferation of human CD19+ PAX5+ pro‐B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL‐7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro‐B cells under the influence of mouse IL‐7 in a xenograft scenario is reduced by anti‐IL‐7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL‐7Rα mediated signals for normal human B‐cell production.

Fine-tuning patient-derived xenograft models for precision medicine approaches in leukemia

Many leukemias are characterized by well-known mutations that drive oncogenesis. Mice engineered with these mutations provide a foundation for understanding leukemogenesis and identifying therapies. However, data from whole genome studies provide evidence that malignancies are characterized by multiple genetic alterations that vary between patients, as well as inherited genetic variation that can also contribute to oncogenesis. Improved outcomes will require precision medicine approaches–targeted therapies tailored to malignancies in each patient. Preclinical models that reflect the range of mutations and the genetic background present in patient populations are required to develop and test the combinations of therapies that will be used to provide precision medicine therapeutic strategies. Patient-derived xenografts (PDX) produced by transplanting leukemia cells from patients into immune deficient mice provide preclinical models where disease mechanisms and therapeutic efficacy can be studied in vivo in context of the genetic variability present in patient tumors. PDX models are possible because many elements in the bone marrow microenvironment show cross-species activity between mice and humans. However, several cytokines likely to impact leukemia cells are species-specific with limited activity on transplanted human leukemia cells. In this review we discuss the importance of PDX models for developing precision medicine approaches to leukemia treatment. We illustrate how PDX models can be optimized to overcome a lack of cross-species cytokine activity by reviewing a recent strategy developed for use with a high-risk form of B-cell acute lymphoblastic leukemia (B-ALL) that is characterized by overexpression of CRLF2, a receptor component for the cytokine, TSLP.

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Abstract B46: A novel patient-derived xenograft model for evaluating therapies that target the CRLF2 signaling pathway to reduce health disparities for Hispanic children with leukemia

The purpose of the studies described here was to identify drug targets and develop a preclinical model for testing therapies that can reduce health disparities for Hispanic children with high-risk acute lymphoblastic leukemia (ALL). Hispanic children are 1.24 times more likely to develop ALL than non-Hispanic whites and that number rises to 2.09 by adolescence and early adulthood. A major contributor to this health disparity is a type high-risk B-cell ALL called CRLF2 B-ALL. CRLF2 B-ALL occurs 5 times more often in Hispanic children than others, is prevalent in adolescents and young adults, and is associated with a high relapse rate and poor prognosis. CRLF2 B-ALL is caused by genetic alterations that result in over expression of the cytokine receptor, CRLF2. The CRLF2 receptor is activated by the cytokine, TSLP, causing downstream activation of the JAK/STAT5 and PI3/AKT/MTOR pathways. A gene target of activated STAT5 in B cell precursors is Mcl-1, a Bcl2 family pro-survival molecule. In addition, Mcl-1 protein levels are known to be increased through post-transcriptional mechanisms by activation of the mTOR pathway. We hypothesized that the normal level of circulating TSLP cytokine could induce CRLF2 activation leading to increased Mcl-1 expression in CRLF2 B-ALL cells. Our data show that TSLP increases phosphorylation of STAT5, as well as AKT and S6 (downstream of mTOR) in primary CRLF2 B-ALL cells from Hispanic pediatric patients, even when activating JAK mutations are present. When CRLF2 B-ALL cells from Hispanic pediatric patients were cultured for 3 days with and without physiological levels of TSLP, flow cytometry showed that expression of the Mcl-1 protein was significantly increased in cultures with TSLP as compared to cultures without TSLP. CRLF2 B-ALL cells treated in vitro with Mcl-1 inhibitor showed dose-dependent increases in caspase 3 activation and apoptosis as indicated by flow cytometry. These data provide evidence that TSLP can contribute to leukemia cell survival and identify Mcl-1 inhibitor as a candidate therapy for CRLF2 B-ALL. Our next step was to develop a preclinical model for testing therapies that target genes, such as Mcl-1, that are regulated by TSLP-induced CRLF2 signals in this disease. Patient-derived xenograft (PDX) models produced by transplanting leukemia cells from patients into immune deficient mice provide an in vivo model of disease that includes contributions of the background genetic landscape that can influence disease progression or treatment outcome in health disparities diseases. PDX models are possible because most cytokines produced in the mouse are active on human cells, however mouse TSLP is species-specific. Thus classic PDX models do not provide TSLP that can activate the CRLF2 receptor that is overexpressed in CRLF2 B-ALL. To address this issue we engineered PDX mice to express physiological levels of human TSLP (+T PDX mice) and control -T mice that lacked human TSLP. In vivo TSLP activity was validated and +T PDX were successfully generated using leukemia cells from two Hispanic pediatric patients with CRLF2 B-ALL. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than -T PDX mice, we compared RNAseq gene expression profiles of leukemia cells isolated from +T PDX and -T PDX mice to that from the original patient sample. The gene expression pattern in leukemia cells from +T mice was significantly closer to primary patient sample than that from -T mice. The +T PDX mice described here provide a novel in vivo preclinical model for evaluating efficacy of drugs, such as Mcl-1 inhibitor, in context of the background genetic landscape and physiological human TSLP present in patients. Citation Format: Kimberly J. Payne, Cornelia Stoian, Jacqueline S. Coats, Olivia Francis, Terry-Ann M. Milford, Ineavely Baez, Pierce J. McCarthy, George Mambo, Anna V.C. White, Mariah M.Z. Jackson, Juliette M. Personius, Veriah Vidales, Muhammad Omair Kamal, Shadi Farzin Gohar, Sinisa Dovat. A novel patient-derived xenograft model for evaluating therapies that target the CRLF2 signaling pathway to reduce health disparities for Hispanic children with leukemia. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B46.

Abstract 2444: TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations

B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy. A subset of children with B-ALL are at high risk for relapse and death. Gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome positive ALL. Approximately half of these Ph-like B-ALL are characterized by genetic alterations that result in overexpression of CRLF2. CRLF2, together with the IL-7 receptor α chain, forms a receptor complex for the cytokine, TSLP. When TSLP binds, the receptor initiates downstream JAK2/STAT5 and PI3/AKT/mTOR pathway activation. The activating JAK mutations found in some CRLF2 B-ALL led to speculation that TSLP stimulation is not a factor in this disease. However, we find that TSLP increases phosphorylation of STAT5, AKT and S6 (downstream of mTOR) in CRLF2 B-ALL cells, including those with JAK defects. Activation of these pathways has been associated with oncogenesis and chemoresistance and their downstream targets include members of the Bcl2 family. The Bcl2 family pro-survival molecule Bcl-XL is a down stream target of STAT5 in Ph+ B-ALL. Mcl-1, another BCL2 family pro-survival molecule is known to be upregulated by mTOR activation via post-translational mechanisms in B cell lymphoma. We hypothesized that TSLP-induced JAK2/STAT5 and PI3/AKT/mTOR pathway activation contribute to chemoresistance in high risk CRLF2 B-ALL by upregulating the expression of Bcl-XL and Mcl-1. To test this hypothesis we cultured human CRLF2 B-ALL cell lines (MUTZ5 and CALL4) with and without TSLP and evaluated expression of the Bcl2 family pro-survival proteins, Bcl-XL, Mcl-1, and Bcl2. We found that TSLP induced significant increases in Bcl-XL and Mcl-1 proteins, but not Bcl2 in CRLF2 B-ALL cells. These cell lines have activating Jak mutations and thus reflect the ability of TSLP to increase expression of the Bcl2 family proteins in cases where activating JAK mutations are present. Next we evaluated the effect of Mcl-1 inhibitor on MUTZ5 and CALL4 cells. Preliminary data from these experiments show that cell counts in cultures treated with Mcl-1 inhibitor are reduce by >90% and this reduction is maintained in the presence of TSLP. These data provide evidence that TSLP-induced CRLF2 signals increase expression of Bcl2 pro-survival proteins, even in CRLF2 B-ALL cells with activating JAK mutations. These data also suggest that Mcl-1 inhibitors could be an effective treatment for this disease. Ongoing studies will evaluate the effect of TSLP and Mcl-1 inhibitors in primary CRLF2 B-ALL samples. Citation Format: Cornelia Stoian, Muhammad Omair Kamal, Olivia Francis, Rhaya Johnson, Simone Montgomery, Jacqueline Coats, Hannah Choi, Shania Aponte-Paris, Micheal Reed, Shanalee Martinez, Karina Mayagoitia, Evgeny Chirshev, Chunhua Song, Sinisa Dovat, Kimberly J. Payne. TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2444.

Abstract A07: A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL

A subset of high-risk B cell acute lymphoblastic leukemia (ALL) shows a gene expression profile similar to Philadelphia chromosome positive (Ph+) ALL and has been described as Ph-like ALL. Approximately 50% of Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. While activating JAK mutations are associated with CRLF2 B-ALL, over half of CRLF2 B-ALL lack such mutations. Our data show that primary human bone marrow (BM) stromal cells express TSLP. Thus TSLP is present in the tumor microenvironment to provide TSLP-induced CRLF2 signals that could play a role in the initiation, maintenance and/or progression of CRLF2 B-ALL. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) with respect to human TSLP and CRLF2. Further, phospho flow cytometry assays show that human, but not mouse TSLP activates CRLF2 signals in primary human CRLF2 B-ALL cells and cell lines as indicated by increased pSTAT5, pAKT and pS6. These data indicate that the mouse TSLP present in classic patient derived xenograft models (PDX) does not produce the TSLP-induced CRLF2 signals present in the patient. To address this challenge we engineered PDX mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (T) mice were produced by transplantation with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in human CRLF2 B-ALL cells. ELISA assays showed that serum levels of hTSLP in mice was proportional to numbers of stromal cells injected at weekly time points. Normal human serum levels of hTSLP (12-32 pg/ml) could be achieved in +T mice, while hTSLP was undetectable in T mice. Because TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary CRLF2 B-ALL leukemia cells. Human CRLF2 B-ALL cells were isolated from the BM of PDX mice and whole genome microarray was performed. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to T PDX mice, confirming hTSLP activity in the +T PDX mice. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than T PDX mice, we compared RNAseq gene expression profiles of leukemia cells from +T and T PDX mice to that from original patient sample. The gene expression pattern in +T mice was significantly closer to primary patient sample than that from T mice. The +T and T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. Citation Format: Olivia L. Francis, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Christopher L. Morris, Ross Fisher, Ben Van Handel, Ruijun Su, Batul Suterwala, Muhammad Kamal, Shadi Farzin Gohar, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A07.