TET2-mediated mRNA demethylation regulates leukemia stem cell homing and self-renewal.

TET2 is recurrently mutated in acute myeloid leukemia (AML) and its deficiency promotes leukemogenesis (driven by aggressive oncogenic mutations) and enhances leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. Here, we show that Tet2 deficiency significantly facilitates leukemogenesis in various AML models (mediated by aggressive or less aggressive mutations) through promoting homing of LSCs into bone marrow (BM) niche to increase their self-renewal/proliferation. TET2 deficiency in AML blast cells increases expression of Tetraspanin 13 (TSPAN13) and thereby activates the CXCR4/CXCL12 signaling, leading to increased homing/migration of LSCs into BM niche. Mechanistically, TET2 deficiency results in the accumulation of methyl-5-cytosine (m5C) modification in TSPAN13 mRNA; YBX1 specifically recognizes the m5C modification and increases the stability and expression of TSPAN13 transcripts. Collectively, our studies reveal the functional importance of TET2 in leukemogenesis, leukemic blast cell migration/homing, and LSC self-renewal as an mRNA m5C demethylase.

METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.

N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.

In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.

N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects in vitro and in vivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Opioid receptor signaling suppresses leukemia through both catalytic and non-catalytic functions of TET2.

Acute myeloid leukemia (AML) is a genetically heterogeneous and frequently fatal malignancy. The ten-eleven translocation (TET)-mediated DNA demethylation is known to be critically associated with AML pathogenesis. Through chemical compound screening, we find that the opioid receptor agonist, loperamide hydrochloride (OPA1), significantly suppresses AML cell viability. The potential therapeutic effects of opioid receptor agonists, especially OPA1, are verified in AML cells in vitro and mouse and human AML models in vivo. OPA1-induced activation of OPRM1 signaling enhances the transcription of TET2 and thus activates both catalytic-dependent and -independent functions of TET2. Notably, AMLs with TET2 mutations or chemotherapy resistance are sensitive to OPA1 as well. Our results reveal the OPRM1-TET2 regulatory axis in AML and suggest that opioid agonists, particularly OPA1, a US Food and Drug Administration (FDA)-approved antidiarrheal drug, have therapeutic potential in AML, especially in TET2-mutated and chemotherapy-resistant AMLs, which have a poor prognosis.

ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells.

Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX.

Current classes of cancer therapeutics have negative side effects stemming from off-target cytotoxicity. One way to avoid this would be to use a drug delivery system decorated with targeting moieties, such as an aptamer, if a targeted aptamer is available. In this study, aptamers were selected against acute myeloid leukemia (AML) cells expressing the MLL-AF9 oncogene through systematic evolution of ligands by exponential enrichment (SELEX). Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and three candidate sequences were identified. These sequences consisted of two 23-base primer regions flanking a 30-base central domain. Binding studies were performed using flow cytometry, and the lead sequence had a binding constant of 37.5 + / - 2.5 nM to AML cells, while displaying no binding to fibroblast or umbilical cord blood cells at 200 nM. A truncation study of the lead sequence was done using nine shortened sequences, and showed the 5' primer was not important for binding. The lead sequence was tested against seven AML patient cultures, and five cultures showed binding at 200 nM. In summary, a DNA aptamer specific to AML cells was developed and characterized for future drug-aptamer conjugates.

PD-1 Inhibition Enhances Blinatumomab Response in a UCB/PDX Model of Relapsed Pediatric B-Cell Acute Lymphoblastic Leukemia.

Immune therapies such as blinatumomab, CD19-directed bispecific CD3 T-cell Engager (BiTE), have resulted in significant improvements in outcomes for relapsed B-cell acute lymphoblastic leukemia (B-ALL). However, up to half of blinatumomab treated patients do not respond completely or relapse after therapy. As a result, there is a need to identify potential strategies to improve the efficacy of BiTE therapy. The anti-PD-1 antibody pembrolizumab has been shown to successfully activate T cells against a wide range of cancer types. Here, we tested the ability of umbilical cord blood (UCB) reconstituted mice to respond to blinatumomab therapy with or without concurrent pembrolizumab treatment. Humanized mice were engrafted with patient-derived xenograft (PDX) cells derived from pediatric and adolescent/young adult (AYA) B-ALL patients who had either failed to achieve remission with negative minimum residual disease (MRD negative) or experienced a relapse. Mock-treated humanized mice engrafted with PDX cells efficiently developed overt disease within 30 days of engraftment of B-ALL. However, single agent therapy with either blinatumomab or pembrolizumab reduced disease burden in engrafted mice, with some mice observed to be MRD negative after the 28-day treatment course. Combination therapy significantly improved the percentage of MRD negative mice and improved long-term survival and cure rates as compared to mice that were given blinatumomab alone. Importantly, no benefits were observed in treated mice that lacked human immune cell reconstitution. These results indicate that UCB-humanized NRGS mice develop activatable immune function, and UCB-humanized PDX leukemia models can be used in preclinical studies to evaluate specificity, efficacy, and cooperativity of immune therapies in B-ALL.

Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion.

Fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, plays oncogenic roles in various cancers, presenting an opportunity for the development of effective targeted therapeutics. Here, we report two potent small-molecule FTO inhibitors that exhibit strong anti-tumor effects in multiple types of cancers. We show that genetic depletion and pharmacological inhibition of FTO dramatically attenuate leukemia stem/initiating cell self-renewal and reprogram immune response by suppressing expression of immune checkpoint genes, especially LILRB4. FTO inhibition sensitizes leukemia cells to T cell cytotoxicity and overcomes hypomethylating agent-induced immune evasion. Our study demonstrates that FTO plays critical roles in cancer stem cell self-renewal and immune evasion and highlights the broad potential of targeting FTO for cancer therapy.

Rapid desensitization of humanized mice with anti-human FcεRIα monoclonal antibodies.

BACKGROUND: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. OBJECTIVE: This study sought to use humanized mice to extend these results to humans. METHODS: We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. RESULTS: Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed ∼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. CONCLUSIONS: Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.

Therapeutic targeting of the E3 ubiquitin ligase SKP2 in T-ALL.

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Improved chemotherapy modeling with RAG-based immune deficient mice.

We have previously characterized an acute myeloid leukemia (AML) chemotherapy model for SCID-based immune deficient mice (NSG and NSGS), consisting of 5 days of cytarabine (AraC) and 3 days of anthracycline (doxorubicin), to simulate the standard 7+3 chemotherapy regimen many AML patients receive. While this model remains tractable, there are several limitations, presumably due to the constitutional Pkrdcscid (SCID, severe combined immune deficiency) mutation which affects DNA repair in all tissues of the mouse. These include the inability to combine preconditioning with subsequent chemotherapy, the inability to repeat chemotherapy cycles, and the increased sensitivity of the host hematopoietic cells to genotoxic stress. Here we attempt to address these drawbacks through the use of alternative strains with RAG-based immune deficiency (NRG and NRGS). We find that RAG-based mice tolerate a busulfan preconditioning regimen in combination with either AML or 4-drug acute lymphoid leukemia (ALL) chemotherapy, expanding the number of samples that can be studied. RAG-based mice also tolerate multiple cycles of therapy, thereby allowing for more aggressive, realistic modeling. Furthermore, standard AML therapy in RAG mice was 3.8-fold more specific for AML cells, relative to SCID mice, demonstrating an improved therapeutic window for genotoxic agents. We conclude that RAG-based mice should be the new standard for preclinical evaluation of therapeutic strategies involving genotoxic agents.

Overcoming adaptive therapy resistance in AML by targeting immune response pathways.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course; however, most patients will relapse because of target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. Here, we describe mechanisms of adaptive resistance in FMS-like receptor tyrosine kinase (FLT3)-mutant acute myeloid leukemia (AML) by examining integrative in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). We identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i and showed that innate immune pathway activation via the interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) complex contributes to adaptive resistance in FLT3-mutant AML cells. To overcome this adaptive resistance mechanism, we developed a small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The multikinase FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cells in vitro and in vivo and displayed superior efficacy as compared to current targeted FLT3 therapies. These findings uncover a polypharmacologic strategy for overcoming adaptive resistance to therapy in AML by targeting immune stress response pathways.

Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia.

FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.

Alternative translation initiation generates the N-terminal truncated form of RUNX1 that retains hematopoietic activity.

Transcription factor RUNX1 plays a crucial role in hematopoiesis and its activity is tightly regulated at both the transcriptional and posttranslational levels. However, translational control of RUNX1 expression has not been fully understood. In this study, we demonstrated that RUNX1b mRNA is translated from two alternative initiation sites, Met-1 and Met-25, giving full-length RUNX1b and a shorter protein lacking the first 24 amino acids (RUNX1ΔN24). Presence/absence of strong Kozak consensus sequences around Met-1 determines which initiation site is mainly used in RUNX1b cDNA. Selective disruption of either Met-1 or Met-25 abrogates expression of the corresponding protein while facilitating the production of another protein. The RUNX1b cDNA containing 65bp natural promoter sequences mainly produces full-length RUNX1b in human cord blood cells, but disruption of Met-1 in this cDNA also induced translation from Met-25. Consistent with these data, disruption of endogenous RUNX1b around Met-1 using CRISPR/Cas9 induced selective expression of RUNX1ΔΝ24 in several leukemia cell lines. RUNX1ΔN24 protein is more stable than full-length RUNX1b and retains hematopoietic activity. We also found that FLAG-tagged full-length RUNX1 showed altered activity, indicating the influence of N-terminal FLAG-tag on RUNX1 function. The alternative translation initiation of RUNX1b may participate in fine tuning RUNX1 activity.

Improved multilineage human hematopoietic reconstitution and function in NSGS mice.

Genetic manipulation of NOD/SCID (NS) mice has yielded numerous sub-strains with specific traits useful for the study of human hematopoietic xenografts, each with unique characteristics. Here, we have compared the engraftment and output of umbilical cord blood (UCB) CD34+ cells in four immune-deficient strains: NS, NS with additional IL2RG knockout (NSG), NS with transgenic expression of human myeloid promoting cytokines SCF, GM-CSF, and IL-3 (NSS), and NS with both IL2RG knockout and transgenic cytokine expression (NSGS). Overall engraftment of human hematopoietic cells was highest in the IL2RG knockout strains (NSG and NSGS), while myeloid cell output was notably enhanced in the two strains with transgenic cytokine expression (NSS and NSGS). In further comparisons of NSG and NSGS mice, several additional differences were noted. NSGS mice were found to have a more rapid reconstitution of T cells, improved B cell differentiation, increased levels of NK cells, reduced platelets, and reduced maintenance of primitive CD34+ cells in the bone marrow. NSGS were superior hosts for secondary engraftment and both strains were equally suitable for experiments of graft versus host disease. Increased levels of human cytokines as well as human IgG and IgM were detected in the serum of humanized NSGS mice. Furthermore, immunization of humanized NSGS mice provided evidence of a functional response to repeated antigen exposure, implying a more complete hematopoietic graft was generated in these mice. These results highlight the important role that myeloid cells and myeloid-supportive cytokines play in the formation of a more functional xenograft immune system in humanized mice.

Comparative utility of NRG and NRGS mice for the study of normal hematopoiesis, leukemogenesis, and therapeutic response.

Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdcscid Il2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ-/-, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2-/-IL2Rγ-/-) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed.

Cell Polarity and Division Symmetry Analyses in Transformed Blood Cells.

Cdc42 was originally discovered as a key regulator of bud site assembly and polarity in S. cerevisiae. Recent genetic studies have shown that the function of Cdc42 in regulating cell polarity appears highly conserved from budding yeast to humans. The role of Cdc42 in hematopoietic cell transformation and leukemia progression has been studied in an acute myeloid leukemia model using the MLL-AF9 oncogene-induced transformation and a Cdc42 conditional gene-targeted mouse model. Here we describe the leukemia cell polarity and division symmetry assays in the context of leukemia cell fate determination.