Sanil Bhatia

Control of AC133/CD133 and impact on human hematopoietic progenitor cells through nucleolin.

AC133 is a prominent surface marker of CD34+ and CD34− hematopoietic stem/progenitor cell (HSPC) subsets. AC133+ HSPCs contain high progenitor cell activity and are capable of hematopoietic reconstitution. Furthermore, AC133 is used for prospective isolation of tumor-initiating cells in several hematological malignancies. Nucleolin is a multifunctional factor of growing and cancer cells, which is aberrantly active in certain hematological neoplasms, and serves as a candidate molecular target for cancer therapy. Nucleolin is involved in gene transcription and RNA metabolism and is prevalently expressed in HSPCs, as opposed to differentiated hematopoietic tissue. The present study dissects nucleolin-mediated activation of surface AC133 and its cognate gene CD133, via specific interaction of nucleolin with the tissue-dependent CD133 promoter P1, as a mechanism that crucially contributes to AC133 expression in CD34+ HSPCs. In mobilized peripheral blood (MPB)-derived HSPCs, nucleolin elevates colony-forming unit (CFU) frequencies and enriches granulocyte–macrophage CFUs. Furthermore, nucleolin amplifies long-term culture-initiating cells and also promotes long-term, cytokine-dependent maintenance of hematopoietic progenitor cells. Active β-catenin, active Akt and Bcl-2 levels in MPB-derived HSPCs are nucleolin-dependent, and effects of nucleolin on these cells partially rely on β-catenin activity. The study provides new insights into molecular network relevant to stem/progenitor cells in normal and malignant hematopoiesis.

Infection exposure promotes ETV6-RUNX1 precursor B cell leukemia via impaired H3K4 demethylases

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365-77. ©2017 AACR.

Lmo2 expression defines tumor cell identity during T‐cell leukemogenesis

The impact of LMO2 expression on cell lineage decisions during T‐cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T‐cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human‐like T‐ALL. In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T‐ALL. The resulting T‐ALLs lacked LMO2 and its target‐gene expression, and histologically, transcriptionally, and genetically similar to human LMO2‐driven T‐ALL. We next found that during T‐ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T‐ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre‐mediated activation of Lmo2 at different stages of B‐cell development induces systematically and unexpectedly T‐ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T‐ALL to current therapies.

GEMMs addressing Pax5 loss-of-function in childhood pB-ALL.

Germline mutations in transcription factors, which are implicated in hematopoiesis in general or specifically in B-cell differentiation have recently been described to confer an inherited risk to pB-ALL with often reduced penetrance. Predicting leukemia development, therapy response and long term follow up of mutation carriers is challenging because experience from large patient cohorts and their long term follow up are not available. Genetically Engineered Murine Models (GEMMs) represent a promising approach to create individualized and precise models reproducing the molecular makeup of the human disease. This review focuses on PAX5 loss-of-function and summarizes techniques of murine model generation, available GEMMs, which mimic Pax5 loss-of-function in leukemia development and discusses the challenges and drawbacks of these models. These aspects are discussed in the context of creating a robust model, which serves not only for validation of the relevance of a genomic alteration in pB-ALL but at the same time as a valid preclinical model.

Mouse models for pre-clinical drug testing in leukemia

ABSTRACT Introduction: The development of novel drugs which specifically target leukemic cells, with the overall aim to increase complete remission and to reduce toxicity and morbidity, is the most important prerequisite for modern leukemia treatment. In this regard, the current transition rate of potential novel drugs from bench to bedside is remarkably low. Although many novel drugs show promising data in vitro and in vivo, testing of these medications in clinical phase I trials is often sobering with intolerable toxic side effects leading to failure in FDA approval. Areas covered: In this review, the authors discuss the development of murine model generation in the context of targeted therapy development for the treatment of childhood leukemia, aiming to decrease the attrition rate of progressively complex targeted therapies ranging from small molecules to cell therapy. As more complex therapeutic approaches develop, more complex murine models are needed, to recapitulate closely the human phenotype. Expert opinion: Combining xenograft models for efficacy testing and GEMMs for toxicity testing will be a global approach for pre-clinical testing of complex therapeutics and will contribute to the clinical approval of novel compounds. Finally, this approach is likely to increase clinical approval of novel compounds.

T-cell leukemogenesis is an inappropriate lineage decision-making process: implications for precision oncology

ABSTRACT Genetic lineage tracing in cell type-specific mouse models of T-cell acute lymphoblastic leukemia (T-ALL) have revealed that tumor cell identity is imposed by expression of the oncogene Lim Domain Only 2 (LMO2), rather than by the target cell phenotype. This approach allowed to identify that secondary genomic alterations, like Notch1 mutations, appeared late and only took place within the thymus during T-ALL development. These concepts are therefore critical for the development of modern therapies aimed at curing T-ALL.

Nucleolin promotes execution of the hematopoietic stem cell gene expression program

Nucleolin is a multifunctional protein of growing and cancer cells, and serves as a candidate molecular target for cancer therapy (reviewed in refs. [1–3]). Nucleolin is directly involved in regulation of gene transcription and RNA metabolism, and also in DNA damage repair and cellsurface signaling [1–5]. It is preferentially expressed in hematopoietic stem/progenitor cells (HSPCs), as opposed to differentiated hematopoietic tissue, while its aberrant activity is associated with certain hematological malignancies [1, 3, 6] (and references in [1, 3, 6, 7]). Nucleolin activates CD34 and AC133/CD133 expression through defined mechanisms, and promotes long-term, cytokinedependent maintenance of HSPCs [6, 7]. Working out the relationship of nucleolin to stem cell gene expression in hematopoiesis will improve understanding of hematopoietic stem/progenitor cells at the molecular level. This study describes that, in human HSPCs, nucleolin promotes execution of gene expression programs of hematopoietic stem cells (HSCs) and stemness shared by HSCs and AML LSCs. Furthermore, nucleolin affects transcription factor activity and upregulates a number of HSCenriched transcriptional regulators, including homeobox A9 (HOXA9) and the ETS transcription factor ERG. Thus, nucleolin drives an expression program that involves HOXA9 and ERG. The study provides new insights into molecular networks relevant to stem/progenitor cells in normal and malignant hematopoiesis. Nucleolin-dependent transcriptional changes in hematopoietic stem/progenitor cells were analyzed using mobilized peripheral blood (MPB) HSPCs, that are exploited in hematological transplantology for hematopoietic reconstitution [8]. HSPCs, with CD34 cell purity >90%, were transduced with lentivirus expressing full-length human nucleolin (HSPC-NCL), or with no cDNA (HSPCmock). Transduced cells were ΔLNGFR-immunopurified, and RNA from ~90% pure ΔLNGFR+ viable cells was subsequently employed for expression profiling using microarrays (Supplementary Methods and Supplementary Table S1). Out of a total of 14910 detectably expressed genes, 2090 were consistently and significantly upregulated in HSPC-NCL, versus HSPC-mock, cells while 1480 were downregulated (adjusted p-value ≤0.05 and fold change cutoff ≥2 or ≤−2). The expression trends of >20 differentially expressed genes were validated by qRT-PCR (Supplementary Figure S1). Nucleolin protein levels were monitored by immunoblotting and were ~4-fold higher in HSPC-NCL, versus HSPC-mock, cells from all patients employed (ref. 7 and data not shown). Indicative of nucleolin activity [7], CFU-GEMM assays revealed significantly increased individual CFU counts with HSPCNCL, versus HSPC-mock, cells and a difference in CFU pattern (Supplementary Figure S2). Functional enrichment analysis of biological processes associated with differentially expressed genes suggested that the nucleolin-upregulated gene set in HSPCs was primarily associated with transcription and nucleotide metabolism (Fig. 1a, Supplementary Table S2A). On the other hand, the nucleolin-downregulated gene set was associated with inflammation, innate immunity, cell differentiation, cell death and B cell immunity (Fig. 1a, Supplementary Table S2B). Furthermore, gene set enrichment analysis (GSEA) revealed that HSPC-NCL cells were enriched in gene expression signatures reported as upregulated in HSCs. These gene signatures, upregulated in HSCs, were: (i) from cord blood (CB) [9], (ii) from CB and MPB [10] and (iii) common to CB, BM and MPB [11] (Fig. 1b, Supplementary Figures S3, S4). Moreover, an upregulated expression program common to HSCs and AML LSCs, underlying stemness properties [9], was enriched, as was * Edgar Grinstein Edgar.Grinstein@uni-duesseldorf.de

Discovery of the first-in-class dual histone deacetylase-proteasome inhibitor

Dual- or multitarget drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin degradation and aggresome pathways. Here, we present the design, synthesis, binding modes, and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy refractory primary patient-derived leukemia cells demonstrated that RTS-V5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.

The homeobox transcription factor HB9 induces senescence and blocks differentiation in hematopoietic stem and progenitor cells

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53–p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated β-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia.

Interleukin-2-Inducible T-Cell Kinase Deficiency—New Patients, New Insight?

Patients with primary immunodeficiency can be prone to severe Epstein–Barr virus (EBV) associated immune dysregulation. Individuals with mutations in the interleukin-2-inducible T-cell kinase (ITK) gene experience Hodgkin and non-Hodgkin lymphoma, EBV lymphoproliferative disease, hemophagocytic lymphohistiocytosis, and dysgammaglobulinemia. In this review, we give an update on further reported patients. We believe that current clinical data advocate early definitive treatment by hematopoietic stem cell transplantation, as transplant outcome in primary immunodeficiency disorders in general has gradually improved in recent years. Furthermore, we summarize experimental data in the murine model to provide further insight of pathophysiology in ITK deficiency.