Murine Leukemia Model Research Articles

Histological and In Vivo Microscopic Analysis of the Bone Marrow Microenvironment in a Murine Model of Chronic Myelogenous Leukemia.

Imaging of the leukemic bone marrow microenvironment, also called the leukemic bone marrow niche, is an essential method to determine and to evaluate the progression of chronic myelogenous leukemia (CML) and other leukemias in murine models. In this chapter we introduce the murine model of CML primarily used in our laboratory by describing blood and bone marrow analysis as well as the method of histological sectioning and immunohistochemistry in combination with various stainings that can help to understand the complex interaction between leukemic cells, their normal hematopoietic counterparts, and the bone marrow microenvironment. We conclude with describing how to image the bone marrow niche using in vivo microscopy.

Drug-DNA Conjugated Gold Nanoparticles for the Treatment of Acute Myeloid Leukemia

INTRODUCTIONNovel therapies are needed for acute myeloid leukemia as only ~60% of children are cured despite maximally intensive cytotoxic chemotherapy. Functionalized gold nanoparticles (AuNP) are utilized for many biomedical applications and represent a potentially novel therapeutic approach in leukemia. We enhanced this technology by developing an AuNP system that selectively releases molecularly targeted drugs in leukemia cells. METHODSAuNPs were functionalized with short, double-stranded oligonucleotides with sequence complementarity to genes overexpressed in or unique to a leukemia cell (e.g. survivin or AML/ETO). Only the anti-sense oligonucleotide is covalently bound to the nanoparticle via a thiol linker. Thus, after entering leukemia cells, the endogenous targeted oncogene mRNA can bind to its complementary sequence on the AuNP and displace the non-covalently bound oligonucleotide which, in our system, is conjugated to the multi-tyrosine kinase inhibitor dasatinib. As a binary reaction, the amount of dasatinib-conjugated oligonucleotide released from the nanoparticle is directly proportional to both the presence and abundance of the complementary mRNA present in a cell. We evaluated AuNP uptake into multiple AML cell lines, as well as normal hematopoietic cells. The effect of dasatinib-AuNPs on dasatinib-sensitive leukemia cell lines was evaluated using proliferation assays, annexin V staining, and cell colony assays. Toxicity in T-cells and CD34+ cells was assessed with T-cell activation and p-SRC assays, respectively. RESULTSConjugation of dasatinib to an oligonucleotide complementary to a region of the survivin gene did not perturb its ability to inhibit SRC and c-KIT kinases in vitro. Leukemia cells demonstrate highly efficient AuNP uptake when cultured alone or with up to a 100-1000 fold excess of normal bone marrow cells. Treatment of K562 leukemia cells, containing a BCR/ABL translocation and high levels of survivin mRNA, with dasatinib-AuNPs resulted in dose-dependent p-SRC and p-CRKL inhibition. Furthermore, K562 cells also showed significantly impaired proliferation, increased apoptosis, and formed fewer colonies in methylcellulose. Conversely, normal T-cells and CD34+ cells, which express less survivin than leukemia cells, were significantly less affected by dasatinib-AuNPs than dasatinib alone as measured by T-cell activation assays and p-SRC levels, respectively. CONCLUSIONSThis method of using functionalized AuNPs to deliver and activate dasatinib in leukemia cells augments drug efficacy while minimizing toxicity and thus represents a novel therapeutic strategy for AML. Ongoing work is underway to characterize the mechanism of preferential AuNP uptake in leukemia cells and assessing in vivo activity using a murine xenotransplantation model of leukemia. DisclosuresNo relevant conflicts of interest to declare.

IKZF2, a Novel Target of MSI2 RNA-Binding Protein Plays an Oncogenic Role in Myeloid Leukemia

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known.Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2f/f and Ikzf2D/D mice. Colony assays of isolated Lin- Sca1+ c-Kit+ (LSK) cells from Ikzf2f/f and Ikzf2D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2f/f and Ikzf2D/D bone marrow cells showed similar chimerism after 34 months indicating that I kzf2D/D mice have normal HSC function and hematopoiesis.To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model. Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs. We then transduced Ikzf2f/f and Ikzf2D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2f/f cells. Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2D/D compared to the Ikzf2f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance.To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA.To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2f/f and Ikzf2D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines.In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Targeting of Spliceosomal Mutant Myeloid Leukemias through Modulation of Splicing Catalysis

Mutations in the spliceosomal genes SRSF2, U2AF1 and SF3B1 are commonly found in patients with myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations occur at highly restricted amino acid residues, are always heterozygous, and rarely co-occur with one another. These data suggest that splicing factor mutations confer an alteration of splicing function and/or that cells may only tolerate a certain degree of splicing modulation. Consistent with this, it has been recently shown that mice expressing the heterozygous Srsf2P95H mutation develop MDS-like features due to altered mRNA recognition. To begin to identify therapeutic vulnerabilities created by spliceosomal gene mutations, we first generated Mx1-Cre Srsf2P95H/KO mice to study the effects of wildtype Srsf2 deletion with concomitant activation of the Srsf2P95H mutant allele. In noncompetitive transplantation, hemizygous Mx1-Cre Srsf2P95H/KO mutant mice exhibited severe bone marrow failure due to complete loss of hematopoietic stem/progenitor cells (HSPCs), suggesting that Srsf2P95H cells depend on wildtype Srsf2 for survival (Fig. 1A). RNA-seq analyses revealed an increase in mis-splicing due to altered mRNA recognition in Srsf2P95H/KO hemizygous mutant HSPCs compared with heterozygous Srsf2P95H/+ mice, which resulted in strong repression of genes involved in hematopoietic self-renewal, most notably Runx1, Erg, and the entire HoxA cluster.Based on these observations, we hypothesized that spliceosomal-mutant leukemias might display greater sensitivity to pharmacologic modulation of splicing than their wildtype counterparts. To address this, we tested the efficacy of a known spliceosome modulator, E7107, in myeloid leukemias with or without splicing factor mutations in vivo. We created a murine leukemia model using MLL-AF9 overexpression on either Srsf2+/+ or Srsf2P95H/+ background, a mutational combination which represents ~10% of adult MLL-rearranged leukemias (LavallŽe et al., 2015). In vivo E7107 treatment resulted in decreased disease burden and ultimately survival benefit in Srsf2P95H/+ mice, whereas the same treatment regimen had no impact on overall survival of Srsf2+/+ mice (Fig. 1B). RNA-seq analysis of leukemic cells after 5 consecutive days ofE7107 treatment in vivo revealed massive global splicing inhibition in response to E7107 in both genotypes, most notably global cassette exon skipping and retention of both constitutive and alternative introns. E7107-induced splicing inhibition was more severe in Srsf2P95H/+ versus Srsf2+/+ mice, consistent with its differential effect on survival in these 2 genotypes. Interestingly, among the most differentially spliced genes in Srsf2P95H/+ mice in response to E7107 are genes involved in maintaining the leukemogenic programs in MLL-rearranged leukemias, including Dot1l, Meis1 and Lsd1 (Fig. 1C).To confirm that preferential sensitivity of spliceosomal-mutant myeloid leukemias to splicing modulation is directly translatable to a human context, we next generated patient-derived xenografts (PDX) from AML patients wildtype (n=2) or mutant (n=3) for splicing factors. 10 mice were successfully engrafted per patient via tail-vein injection in NSG mice and treated with vehicle or E7107 for 10 days once human bone marrow chimerism was >50%. In each PDX model, targeted genomic analysis of human leukemic cells confirmed faithful engraftment of the major leukemic clones as found in patients pre-engraftment and that spliceosomal mutations were present in the major leukemic clone engrafted in mice. All spliceosomal mutant AML PDXs showed significant reductions in human leukemic burden in response to E7107 while the response in splicing wildtype AML cells was less robust (Fig. 1D).Collectively, these data provide both genetic and pharmacologic evidence that splicing factor mutant leukemias are preferentially sensitive to splicing modulation in vivo compared with spliceosomal wildtype counterpart cells. This has important therapeutic implications for the wide range of patients with MDS and AML bearing these alterations. [Display omitted] DisclosuresThomas:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Smith:H3 Biomedicine Inc.: Employment.

Abstract PL07-01: Altered metabolism in leukemic microenvironment

Abstract Interactions of leukemia cells and their bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells. It has been postulated that specific niches provide a sanctuary where subpopulations of leukemic cells evade chemotherapy-induced death and acquire a drug-resistant phenotype. Understanding the cellular and molecular biology of the leukemia stem cell (LSC) niche and of microenvironment/leukemia interactions may provide new targets that allow destruction of LSCs without adversely affecting normal stem cell self-renewal. Key emerging therapeutic targets include chemokine receptors such as CXCR4 and hypoxia-related proteins, as well as the metabolic abnormalities of the leukemia-associated stroma. We have recently reported that CXCR4 inhibition causes leukemia cell dislodgement from CXCL12-producing marrow niches, reduced proliferation and induction of differentiation of AML cells in an in vivo model of AML, translating into pronounced anti-leukemia effects. Studies in murine leukemia models using the hypoxia probe pimonidazole demonstrated extensive areas of hypoxia within leukemic, but not healthy normal, bone marrow. Time-course analysis of bone marrow spaces within calvaria and femurs by multiphoton intravital microscopy (MP-IVM) demonstrated lodging of p190-Bcr/Abl tdTomato cells in close proximity to blood vessels, followed by accumulation of leukemia cells localized within the sinusoidal and marrow spaces resulting in the demise of the animals within 3 weeks. In this model, pimonidazole detected hypoxic areas despite abundant vascular supply in the marrow cavities. In vivo magnetic resonance imaging with hyperpolarized pyruvate showed higher pyruvate-lactate conversion (high glycolytic flux) in leukemic marrows. These findings were supported by significant pimonidazole uptake by the diseased bone marrow in patients with acute leukemia, causing stabilization of HIF-1α in 55% (76/138) of primary AML patients and of its target CA9. Paradoxically, AML cells become highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for their survival, and inhibition of OXPHOS with the novel small molecule complex I inhibitor IACS-10759 inhibits oxygen consumption, eliminates hypoxia in vivo and inhibits AML growth. These findings suggest that altered tumor metabolism underlies the hypoxia observed in leukemias. We postulate that the altered tumor microenvironment within the hypoxic niche cells will influence leukemia development and response to therapy. Hence, targeting key metabolic alterations should be considered in the armamentarium of anti-AML therapies. IACS-10759 is presently completing IND enabling safety/toxicity studies with first in human studies targeting relapsed/refractory AML planned for 2016. Citation Format: Marina Y. Konopleva, Tomasz Zal, Niki M. Zacharias Millward, Byoung-Sik Cho, Karine Harutyunyan, Anna Zal, Hong Mu, Sergej Konoplev, Juliana Benito, Juliana Velez, Carlos Bueso-Ramso, Jennifer Molina, Pratip K. Bhattacharya, Maria Emilia Di Francesco, Joseph Marszalek, Michael Andreeff. Altered metabolism in leukemic microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-01.

Chemopreventive Effects of Dietary Eicosapentaenoic Acid Supplementation in Experimental Myeloid Leukemia.

Current therapies for treatment of myeloid leukemia do not eliminate leukemia stem cells (LSC), leading to disease relapse. In this study, we supplemented mice with eicosapentaenoic acid (EPA, C20:5), a polyunsaturated omega-3 fatty acid, at pharmacologic levels, to examine whether the endogenous metabolite, cyclopentenone prostaglandin delta-12 PGJ3 (Δ(12)-PGJ3), was effective in targeting LSCs in experimental leukemia. EPA supplementation for 8 weeks resulted in enhanced endogenous production of Δ(12)-PGJ3 that was blocked by indomethacin, a cyclooxygenase (COX) inhibitor. Using a murine model of chronic myelogenous leukemia (CML) induced by bone marrow transplantation of BCR-ABL-expressing hematopoietic stem cells, mice supplemented with EPA showed a decrease in the LSC population, and reduced splenomegaly and leukocytosis, when compared with mice on an oleic acid diet. Supplementation of CML mice carrying the T315I mutation (in BCR-ABL) with EPA resulted in a similar effect. Indomethacin blocked the EPA effect and increased the severity of BCR-ABL-induced CML and decreased apoptosis. Δ(12)-PGJ3 rescued indomethacin-treated BCR-ABL mice and decreased LSCs. Inhibition of hematopoietic-prostaglandin D synthase (H-PGDS) by HQL-79 in EPA-supplemented CML mice also blocked the effect of EPA. In addition, EPA supplementation was effective in a murine model of acute myeloid leukemia. EPA-supplemented mice exhibited a decrease in leukemia burden and a decrease in the LSC colony-forming unit (LSC-CFU). The decrease in LSCs was confirmed through serial transplantation assays in all disease models. The results support a chemopreventive role for EPA in myeloid leukemia, which is dependent on the ability to efficiently convert EPA to endogenous COX-derived prostanoids, including Δ(12)-PGJ3.

The molecular basis of leukemogenesis caused by MLL gene rearrangements

Chromosomal translocations at the MLL gene generate chimeric genes of MLL and its partner, thereby causing aggressive acute leukemia. The sequence of the MLL gene was revealed in the early 1990s. Several years later, murine leukemia models using genetic engineering or retroviral gene transfer techniques were established, which spurred basic research on this disease. Since then, a series of technological breakthroughs such as DNA arrays, shRNA library screening, and deep sequencing have provided us with a much deeper understanding of the molecular basis of leukemogenesis caused by MLL mutations. Based on the understanding of these molecular mechanisms, several small molecules that inhibit critical processes of leukemogenesis have been developed as molecularly-targeted drug candidates. I herein review the rapid progress in this research on the molecular mechanisms of MLL-associated leukemia.

Stat5-deficient hematopoiesis is permissive for Myc-induced B-cell leukemogenesis.

Despite being an attractive molecular target for both lymphoid and myeloid leukemias characterized by activated tyrosine kinases, the molecular and physiological consequences of reduced signal transducer and activator of transcription-5 (Stat5) during leukemogenesis are not well known. Stat5 is a critical regulator of mouse hematopoietic stem cell (HSC) self-renewal and is essential for normal lymphocyte development. We report that pan-hematopoietic deletion in viable adult Vav1-Cre conditional knockout mice as well as Stat5ab(null/null) fetal liver transplant chimeras generated HSCs with reduced expression of quiescence regulating genes (Tie2, Mpl, Slamf1, Spi1, Cited2) and increased expression of B-cell development genes (Satb1, Dntt, Btla, Flk2). Using a classical murine B-cell acute lymphoblastic leukemia (B-ALL) model, we demonstrate that these HSCs were also poised to produce a burst of B-cell precursors upon expression of Bcl-2 combined with oncogenic Myc. This strong selective advantage for leukemic transformation in the background of Stat5 deficient hematopoiesis was permissive for faster initiation of Myc-induced transformation to B-ALL. However, once established, the B-ALL progression in secondary transplant recipients was Stat5-independent. Overall, these studies suggest that Stat5 can play multiple important roles that not only preserve the HSC compartment but can limit accumulation of potential pre-leukemic lymphoid populations.

Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21–mediated tumor suppressor Pdcd4 repression

AbstractThe modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage-tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4.

Abstract A10: Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model.

Abstract Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Until recently, the tumor-derived signals which stimulate host type I IFN production have remained elusive. However, emerging data suggest that tumor-derived DNA is sufficient to induce IFN-β production in responding cells through activation of a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes). STING-mediated IFN-β production, in turn, leads to functional priming of antigen-specific T cells which mediate tumor rejection in pre-clinical solid tumor models. However, because of their disseminated growth pattern, hematological cancers, such as acute leukemia, may not release sufficient quantities of DNA upon apoptosis to activate the STING pathway in innate immune cells, such as dendritic cells (DCs). The failure of acute leukemias to induce a type I IFN response may contribute to their ability to generate T cell tolerance in the host. Thus, we hypothesized that activating the STING pathway would promote a type I IFN response sufficient to facilitate DC-mediated priming of leukemia-specific T cells and to prolong survival of mice with acute myeloid leukemia (AML). Murine C1498 AML cells engineered to express a model peptide antigen called SIY (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which were then treated with a selective murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control. Preliminary experiments confirmed that DMXAA administration resulted in robust IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control-treated spleen cells). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control-treated animals) of endogenous SIY antigen-specific T cells as determined by SIY/Kb pentamer analysis. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated, leukemia-bearing animals, revealed that the expanded SIY-specific CD8+ T cells were functionally active, and produced high-levels of IFN-γ. To determine if this enhanced immune response translated to a benefit in survival, C1498.SIY AML cell-challenged mice were treated with a single dose of DMXAA or vehicle control five days after tumor inoculation. Among mice treated with DMXAA, 80% of mice rejected the leukemia and survived long-term, while control-treated mice succumbed to AML within approximately 3 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting the DMXAA treatment led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to enhanced survival compared to control-treated animals. This effect was T and or B cell-dependent, as DMXAA treatment of RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway upstream of type I IFN potently enhances adaptive immunity to AML which culminates in prolonged survival or even disease cure in treated animals. It will be important to determine whether the STING pathway may be successfully manipulated in other murine leukemia models. If so, then STING may be a promising therapeutic target to enhance endogenous immunity in AML patients. Citation Format: Emily K. Curran, Xiufen Chen, Leticia Corrales, Justin Kline. Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A10.

Antileukemic Efficacy of Continuous vs Discontinuous Dexamethasone in Murine Models of Acute Lymphoblastic Leukemia.

Osteonecrosis is one of the most common, serious, toxicities resulting from the treatment of acute lymphoblastic leukemia. In recent years, pediatric acute lymphoblastic leukemia clinical trials have used discontinuous rather than continuous dosing of dexamethasone in an effort to reduce the incidence of osteonecrosis. However, it is not known whether discontinuous dosing would compromise antileukemic efficacy of glucocorticoids. Therefore, we tested the efficacy of discontinuous dexamethasone against continuous dexamethasone in murine models bearing human acute lymphoblastic leukemia xenografts (n = 8 patient samples) or murine BCR-ABL+ acute lymphoblastic leukemia. Plasma dexamethasone concentrations (7.9 to 212 nM) were similar to those achieved in children with acute lymphoblastic leukemia using conventional dosages. The median leukemia-free survival ranged from 16 to 59 days; dexamethasone prolonged survival from a median of 4 to 129 days in all seven dexamethasone-sensitive acute lymphoblastic leukemias. In the majority of cases (7 of 8 xenografts and the murine BCR-ABL model) we demonstrated equal efficacy of the two dexamethasone dosing regimens; whereas for one acute lymphoblastic leukemia sample, the discontinuous regimen yielded inferior antileukemic efficacy (log-rank p = 0.002). Our results support the clinical practice of using discontinuous rather than continuous dexamethasone dosing in patients with acute lymphoblastic leukemia.

Abstract B44: Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton's tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. The PI3K p110δ inhibitor, idelalisib, was granted a breakthrough therapy designation by the FDA in relapsed CLL and the BTK inhibitor, ibrutinib, has received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, IPI-145, which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of IPI-145 in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM IPI-145 for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to IPI-145, displaying about 20% decrease in viability at concentrations greater than 0.5μM, however the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM IPI-145 for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, IPI-145 is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, IPI-145. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, as IPI-145 can continue to inhibit the PI3K/AKT pathway in the setting of a C481S BTK mutation, it provides justification for clinical studies of this agent in this subset of patients. Citation Format: Amy J. Johnson, Shuai Dong, Daphne Guinn, Jason A. Dubovsky, Yiming Zhong, Amy Lehman, Jeff Kutok, Jennifer A. Woyach, John C. Byrd. Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B44.

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1+DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Designed to Kill: Novel Menin-MLL Inhibitors Target MLL-Rearranged Leukemia

The interaction between menin and oncogenic mixed lineage leukemia (MLL) fusion proteins is required for leukemic transformation and may represent a therapeutic opportunity. In this issue of Cancer Cell, Borkin and colleagues describe the development of highly potent small-molecule inhibitors of this interaction that reverse the leukemic phenotype and prolong survival in murine models of MLL-rearranged leukemia.

The Zinc Finger Transcription Factor Mds1‐Evi1 Forms a Novel Protein Complex in MLL leukemia

The dual transcription factor‐methyltransferase Mds1‐Evi1 is frequently overexpressed in high‐risk leukemias. Mds1‐Evi1 contributes to leukemias induced by the mixed lineage leukemia (MLL) fusion protein MLL‐AF9, although the mechanism is unknown. MLL contains chromatin‐targeting and histone lysine methyltransferase domains, and fusion to AF9 can cause aberrant protein recruitment to target genes. Although Mds1‐Evi1 contains a methyltransferase domain, it does not appear to act on histones. Therefore, we are interested in how protein‐protein interactions involving Mds1‐Evi1 contribute to leukemogenesis. Many known interactors of MLL and Mds1‐Evi1 include chromatin modifying enzymes, suggesting that coordination between epigenetic modulators is key in leukemia pathogenesis.Here, we report the biochemical purification of a novel Mds1‐Evi1‐containing protein complex using a unique murine model of leukemia. To isolate the complex, we induced acute MLL‐AF9 leukemia in mice containing a tandem affinity purification (TAP) tag knocked into the endogenous Mds1‐Evi1 gene. Leukemic spleen tissue from these mice was used to purify the TAP‐tagged protein complex. Proteomic analysis revealed several members of the SWI/SNF chromatin remodeling complex, which is involved in epigenetic control of a variety of gene programs. Association between Mds1‐Evi1 and SWI/SNF may allow the activation of cell‐cycle and differentiation programs, contributing to transformation. SWI/SNF also coordinates with MLL fusion proteins to activate the Hox family of leukemia oncogenes. Our results provide a link between Mds1‐Evi1 and the chromatin remodeling machinery, where abnormal coordination of Mds1‐Evi1, MLL‐AF9 and SWI/SNF may lead to gene activation and the development of leukemia.

Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model.

Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers.

N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule destabilizer: Synthesis, cytotoxicity, inhibition of cell migration and in vivo activity against acute lymphoblastic leukemia

Tubulin-interacting agents, like vinca alkaloid and taxanes, play a fundamental role in cancer chemotherapy, making cellular microtubules (MT), one of the few validated anticancer targets. Cancer resistance to classical MT inhibitors has motivated the development of novel molecules with increased efficacy and lower toxicity. Aiming at designing structurally-simple inhibitors of MT assembly, we synthesized a series of thirty-one 3,4,5-trimethoxy-hydrazones and twenty-five derivatives or analogs. Docking simulations suggested that a representative N-acylhydrazone could adopt an appropriate stereochemistry inside the colchicine-binding domain of tubulin. Several of these compounds showed anti-leukemia effects in the nanomolar concentration range. Interference with MT polymerization was validated by the compounds' ability to inhibit MT assembly at the biochemical and cellular level. Selective toxicity investigations done with the most potent compound, a 3,4,5-trimethoxy-hydrazone with a 1-naphthyl group, showed remarkably selective toxicity against leukemia cells in comparison with stimulated normal lymphocytes, and no acute toxicity in vivo. Finally, this molecule was as active as vincristine in a murine model of human acute lymphoblastic leukemia at a weekly dose of 1 mg/kg.

Musashi2 sustains the mixed-lineage leukemia-driven stem cell regulatory program.

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 (MSI2) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2-deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2-deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc, and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia.

Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies.

Signaling via the MyD88/IRAK pathway in T cells is indispensable for cell survival; however, it is not known whether this pathway functions in the progression of T acute lymphoblastic leukemia (T-ALL). Here, we determined that compared with thymic and peripheral T cells, T-ALL cells from patients have elevated levels of IRAK1 and IRAK4 mRNA as well as increased total and phosphorylated protein. Targeted inhibition of IRAK1 and IRAK4, either with shRNA or with a pharmacological IRAK1/4 inhibitor, dramatically impeded proliferation of T-ALL cells isolated from patients and T-ALL cells in a murine leukemia model; however, IRAK1/4 inhibition had little effect on cell death. We screened several hundred FDA-approved compounds and identified a set of drugs that had enhanced cytotoxic activity when combined with IRAK inhibition. Administration of an IRAK1/4 inhibitor or IRAK knockdown in combination with either ABT-737 or vincristine markedly reduced leukemia burden in mice and prolonged survival. IRAK1/4 signaling activated the E3 ubiquitin ligase TRAF6, increasing K63-linked ubiquitination and enhancing stability of the antiapoptotic protein MCL1; therefore, IRAK inhibition reduced MCL1 stability and sensitized T-ALL to combination therapy. These studies demonstrate that IRAK1/4 signaling promotes T-ALL progression through stabilization of MCL1 and suggest that impeding this pathway has potential as a therapeutic strategy to enhance chemotherapeutic efficacy.

Pathophysiological Scenario of Hematopoietic Disorders: A Comparative Study of Aplastic Anemia, Myelodysplastic Syndrome and Leukemia in Experimental Animals

The conversion of physiology to pathophysiology in hematological disorders viz: aplastic anemia, myelodysplastic syndrome (MDS) and leukemia in murine models was the subject of study in the present programme. Peripheral blood hemogram, spleno-somatic index, bone marrow smear study, cytochemical staining of marrow, cell release kinetics study during marrow explants culture, hematopoietic niche assessment, chromosomal aberration study, plasma membrane stability study of marrow cells, lysosomal membrane and mitochondrial membrane stability study and innate immune parameters were performed in the aplastic anemia, leukemia and MDS mouse model. In bone marrow aplasia, peripheral blood pancytopenia, marrow hypocellularity, decreased marrow cellular viability, deterioration of bone marrow hematopoiesis as well as hematopoietic microenvironment and extramedullary hematopoiesis were noticed. In addition, disruption of mitochondrial and lysosomal membrane integrity along with reduction of innate immune parameters were found in the hematopoietic suppressed condition. Surprisingly, no noticeable chromosomal aberration was found in the aplastic condition. Ineffective marrow hematopoiesis together with the disruption of hematopoietic microenvironment was observed in MDS. Also, extramedullary hematopoiesis, increased marrow cellular death, chromosomal aberration and loss of innate immunity were the common events. During leukemia, the number of functionally and structurally immature cells in the peripheral blood and bone marrow was increased together with malignant conversion of hematopoietic cells in the presence of malignancy supportive stromal microenvironment. Chromosomal aberration, decrease of cell mediated immunity with least mitochondrial apoptotic damage were also found in leukemic condition as well.