Murine Acute Myeloid Leukemia Model Research Articles

Phase IB/II Study of Nivolumab in Combination with Azacytidine (AZA) in Patients (pts) with Relapsed Acute Myeloid Leukemia (AML)

Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by unleashing T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition alone demonstrated limited activity in AML (Berger et al, Clin Cancer Res 2008). Rational combination strategies to enhance the anti-tumor and immunogenic effects of PD1 inhibitors in AML are needed. AZA up-regulates PD-1 and PD-L1 in AML and the up-regulation of these genes has been associated with emergence of resistance to AZA (Yang et al., Leukemia 2013).These could be blocked with the PD-1 inhibitor nivolumab.Methods: Pts were eligible if they had AML and failed prior therapy, had adequate performance status (ECOG ² 2), and organ function. The first six pts received AZA 75mg/m2 Days 1-7 with nivolumab 3mg/kg on Day 1 and 14. Courses were repeated approximately every 4-5 weeks indefinitely. Only one of six pts had a dose limiting toxicity (grade 3 pneumonitis) and this dose was established as RP2D. 45 additional pts have been treated at the RP2D. Responses were evaluated at the end of 3 courses of therapy.Results: 51 pts with a median age of 69 years (range, 45 - 90), secondary AML (55%), poor risk cytogenetics (47%), median number of prior regimens 2 (range, 1-7) have been enrolled. All 51 pts had baseline next generation sequencing and most frequently detected mutations included DNMT3A (n=12), TP53 (n=11), TET2 (N=9), ASXL1 (n=7), and RAS (n=7). 35 pts are evaluable for response: 6 (18%) achieved complete remission (CR)/ complete remission with insufficient recovery of counts (CRi) (3 CR, 3 CRi), 5 (15%) had hematologic improvement (HI), 9 (26%) had ³50% BM blast reduction,3 pts (9%) had stable disease > 6 months, and 12 (34%) had progression. 16 pts are early for response assessment (<3 courses). The median number of courses to CR/CRi/HI was 3 (1 - 9). The CR/CRi/HI (n=11) have been durable with no relapses among pts who achieved CR/CRi/HI [CR duration; not reached (NR)]. The 4- and 8-week mortalitywere 0 and 6%, respectively. The median overall survival for the 35 evaluable pts was 9.3 months (range, 1.8 - 14.3) (Fig 1) and this compares favorably to historical survival with AZA-based salvage protocols in similar pts treated at MDACC.Grade 3/4 and Grade 2 immune mediated toxicities were observed in 7 (14%) and 6 (12%) pts, respectively. These included 8 episodes of pneumonitis, 2 nephritis, 2 transaminitis, and 1 skin rash. 1 pt died from grade 4 pneumonitis/epiglottitis. In the remaining 12 cases the toxicities responded rapidly to steroids and all 12 pts were successfully rechallenged with nivolumab. Time to onset of toxicities ranged from 4 days to 3.5 months. Four pts were postSCT and one had a grade 3 flare of GVHD of the skin and gut.Multicolor flow-cytometry studies and Mass-cytometry (CyTOF) studies are conducted by the Immunotherapy Platform on baseline and on-treatment BM aspirate (end of cycle 1, 2, 4, 8) and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T cell subsets and leukemic blasts respectively. Baseline and end of cycle 1 BM was evaluated on 25 of the 35 evaluable patients, including 6 responders (CR/CRi/HI) and 19 non-responders. Pts who achieved a CR/CRi/HI had a baseline higher live total CD3 (P=0.10), CD8+ T-cells (P=0.02), and lower live CD4+Foxp3+PD1+ T-regulatory (T-reg) cells (P=0.01) infiltrate in the BM (Figure 2). The ratio of PD1+CD8+ T-effector cells to PD1+CD4+Foxp3+ T-reg cells was significantly higher in the CR/CRi/HI pts vs non-responders (208.4 vs 8.91, P<0.05) in baseline BM. On IHC evaluation OX40 appeared to increase post-treatment in non-responders but not in responders.Conclusion: Full dose AZA and nivolumab are tolerable and produce an encouraging response rate with durable responses in relapsed AML with poor risk features. Immune mediated toxicities occur and may be adequately managed with early recognition and systemic steroids. On prelim analysis responders had higher baseline BM CD8+ T-effectors, lower T-regs and higher PD1+CD8/ PD1+CD4+Foxp3+. [Display omitted] [Display omitted] DisclosuresDaver:Kiromic: Research Funding; Pfizer: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Karyopharm: Honoraria, Research Funding; BMS: Research Funding; Ariad: Research Funding; Sunesis: Consultancy, Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

The Dose-Dependent Effects of Microrna-155 in Acute Myeloid Leukemia

MicroRNAs are a class of non-coding, regulatory RNAs that control several critical cellular processes. Subsets of microRNAs are dysregulated in cancer, and can act as oncogenes or tumour suppressors.MicroRNA-155 (miR-155) has a well-established role as an oncogene in B cell lymphoma but has a more enigmatic role in acute myeloid leukemia (AML), in which there is evidence that miR-155 may promote or repress the development and progression of AML. We have used enforced expression of miR-155 in murine AML cell lines and AML models to explore the biology of miR-155 in AML.We show that the capacity of miR-155 to promote or repress the ability of AML cells to form colonies and to proliferate is dependent on miR-155 expression levels. Enforced high expression of miR-155 in AML cell lines results in reduced proliferation and colony formation. However, critical long-term assays of cells transduced with miR-155 resulted in selection in favour of an intermediate miR-155 expression level accompanied by a restoration in clonogenic potential. In vivo, enforced expression of miR-155 in murine AML models showed no differences in disease latency compared to controls, but resulted in an increased tumour burden. Most interestingly, RNA-Sequencing analysis demonstrated that the contrasting levels of miR-155 regulate a substantially different set of gene targets, with downstream consequences on transcription that are consistent with the contrasting effects of high and intermediate miR-155 levels. The intermediate levels of miR-155 we observe are the same as that seen in human AML, whereas the high levels of miR-155 have a completely different inflammatory counterpart. Our data shows that that the levels of miR-155 powerfully influences that gene targets it controls and the resultant phenotypes observed. MiR-155 expressed within a specific range promotes AML disease progression. DisclosuresNo relevant conflicts of interest to declare.

The Role of MSCs in the Development of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow. The prognosis of AML is still poor with only 25% of patients living longer than 5 years. Thus, new therapeutic approaches are warranted to improve patients' survival. The growth of AML cells not only depends on cell intrinsic factors, but is also supported by stroma cells, among them mesenchymal stem cells (MSCs). We reported here that MSCs derived from human AML patients or murine models of human AML better supported the in vitro growth of leukemic cells than MSCs from non-leukemic patients or mice and this is dependent on cell-to-cell contact. Similarly, immortalized MSCs lines derived from AML patients supported the growth of human AML cell lines better than a MSC line originating from a healthy donor. In addition, the frequency of MSCsin the BM of leukemic mice was increased. On a molecular level, the polarization of MSCs towards an AML-supporting state depended on the expression of the transcription factor Growth factor independence 1 (Gfi1). Loss of Gfi1 abrogated the tumor-supporting state of AML-associated MSCs in vitro and in vivo. In summary, MSCs from AML patients support the growth of AML cells in vitro in a Gfi1-dependent manner which could open the path to new therapeutic approaches. DisclosuresDührsen:Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Khandanpour:Hospital of Essen university: Research Funding; Max-Eder: Research Funding.

PRL2 Phosphatase Is a Key Mediator of Oncogenic Cytokine Signaling in Leukemia Stem Cells

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple signaling pathways contributing to its pathogenesis. Mutations in receptor tyrosine kinase KIT and FLT3 are found in approximately 40% of AML patients and targeted therapies for inhibiting KIT and FLT3 have failed, thus new targets for therapeutic intervention need to be identified. The phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL1, PRL2, and PRL3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in human cancer. While PRL2 is highly expressed in some subtypes of human AML, including AML1-ETO+ AML and AML with mixed lineage leukemia (MLL) translocations, its role in AML is largely unknown. To determine the role of PRL2 in the pathogenesis of AML, we utilized two murine models of human AML induced by transducing mouse HSCs with AML1-ETO or MLL-AF9. We found that PRL2 is important for the progression and maintenance of leukemia induced by AML1-ETO or MLL-AF9 through enhancing leukemia stem cell (LSC) self-renewal. To elucidate the mechanisms by which PRL2 promotes LSC maintenance, we performed genome wide RNA-seq analysis of MLL-AF9+ LSCs. Gene Set Enrichment Analysis (GESA) indicates that PRL2 deficiency alters the MLL-AF9 signature essential for LSC self-renewal.We have recently identified PRL2 to be important for the proliferation and self-renewal of hematopoietic stem cells (HSCs) through the regulation of KIT signaling. Notably, PRL2 null hematopoietic progenitor cells showed decreased KIT phosphorylation as well as ERK phosphorylation following SCF stimulation, suggesting that PRL2 is important for KIT activation. Given that KIT inactivation could be mediated by removal from the cell surface and intracellular degradation, we reasoned that PRL2 may regulate KIT receptor internalization and stability. That was indeed the case. We found that the KIT protein half-life in PRL2 null hematopoietic progenitor cells (Kit+) was significantly decreased compared to WT cells. Furthermore, PRL2 null progenitor cells showed enhanced KIT ubiquitination compared to WT cells and less KIT was found on the surface of PRL2 null progenitor cells compared to WT cells following SCF stimulation. We also found that loss of PRL2 in human AML cells resulted in enhanced internalization of KIT. These observations demonstrate that PRL2 deficiency results in less KIT on the cell surface and a lower global KIT level in the cell.Upon SCF stimulation, KIT binds to and induces the phosphorylation of CBL proteins, which in turn act as E3 ligases, mediating the ubiquitination and degradation of KIT. To understand how PRL2 modulates the turnover of KIT in hematopoietic cells, we performed GST-pulldown assays and found that the substrate-trapping mutant PRL2/CS-DA showed an increased association with KIT and CBL compared to wild-type PRL2 in Kasumi-1 cells, suggesting that KIT and CBL may be PRL2 substrates. Furthermore, we found that PRL2/CS-DA mutant showed enhanced association with FLT3 and CBL compared to wild-type PRL2 in MV4-11 cells. Our data suggest that PRL2 dephosphorylates CBL and inhibits CBL activity toward KIT and FLT3, leading to sustained activation of downstream signaling pathways.To determine the functional significance of PRL2 in human AML with KIT and FLT3 mutations, we utilized two well-established murine model of myeloproliferative neoplasms (MPN) induced by KITD814V or FLT3-ITD. We found that loss of Prl2 decreased the ability of oncogenic KITD814V and FLT3-ITD to promote mouse hematopoietic stem and progenitor cell (HSPC) proliferation in vitro andthe development of MPN in vivo. Furthermore, we found that genetic and pharmacological inhibition of PRL2 decreased the proliferation and survival of human AML cells bearing KIT or FLT3 mutations.Taken together, we demonstrate that PRL2 promotes leukemia stem cell (LSC) self-renewal and maintenance through sustaining the activity of oncogenic KIT and FLT3 signals. Our findings suggest that pharmacological inhibition of PRL2 holds potential as a novel therapy for acute myeloid leukemia, and might also be applicable to the treatment of other human cancers. DisclosuresNo relevant conflicts of interest to declare.

Evasion of Necroptosis and Inflammasome Activation Promotes Myeloid Leukemogenesis

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic neoplasms driven partly by the loss of differentiation and theblockade of cell death. AML is sustained by leukemia-initiating cells (LICs) that arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML.While the regulatory factors that maintain HSPC proliferation and differentiation under normal conditions are well understood, significantly less is known about how LIC fate is regulated. As many hematopoietic disorders are characterized by the overproduction of pro-inflammatory cytokines, we hypothesized that necroptosis controlled cytokine secretion and inflammatory cell death might influence AML development. We therefore addressed the role of MLKL and XIAP in AML and tested whether deletion of Mlkl or Xiap would affect disease progression.Here we show that MLKL limits oncogene-mediated leukemogenesis by promoting the inflammatory cell death of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (HSCs) in experimental mice. Upon oncogenic stress MLKL-dependent necroptosis and subsequent inflammasome activation were triggered, promoting the production of IL-1β, a potent stimulator of HSPC differentiation and maturation, thus, suppressing the emergence of LICs and limiting leukemogenesis.In a murine bone marrow transplantation model of AML the absence of MLKL accelerated AML development significantly. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartments to contain LICs. The survival advantage of Mlkl-/- HSPCs became apparent in colony-forming assays and liquid cultures specifically within the CMP and ST-HSC compartments. Sorted ST-HSCs from Mlkl-/- produced more GEMM colonies than WT, the colony type harboring the multipotential myeloid progenitor cells, and both ST-HSCs and CMPs retained significantly more lineage-negative cells in liquid culture. In addition, Mlkl-/- colonies showed a reduction in propidium iodide (PI)-positive dead cells compared with WT colonies. Importantly, WT cells showed caspase activation and produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Mlkl deficiency. We also observed reduced expression of MLKL in leukemic cells on both mRNA and protein level, implying that suppression of cell death was beneficial for the survival of LICs.In contrast, deletion of Xiap did not alter survival or differentiation of leukemic cells when compared with WT cells. Furthermore, XIAP was not differentially expressed on mRNA or protein level compared with WT, indicating that XIAP does not play a critical role in leukemogenesis.In agreement with the murine data, gene expression analysis from primary leukemia cells from two large patient cohorts newly diagnosed with AML showed significantly lower expression of MLKL, but not XIAP, in a variety of AML subtypes compared to healthy controls.Overall, our data demonstrate a key role for MLKL-mediated cell death and activation of the inflammasome in AML and represents a novel tumor-suppressive mechanism. DisclosuresPeschel:MophoSys: Honoraria.

Targeting Innate and Adaptive Immune Responses for the Treatment of Acute Myeloid Leukemia

Background: Successful immunotherapeutic approaches for acute myeloid leukemia (AML) have yet to be developed. We hypothesized that targeting both the innate and adaptive immune responses in leukemic hosts would elicit significant anti-tumor activity with lesser toxicities than chemotherapy. To test this, we evaluated the efficacy of immune checkpoint inhibition (murine anti-PD-1 antibody (ab)) alone and in combination with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an innate immune agonist and anti-vascular agent, in an immunocompetent model of murine AML.Methods: Expression of PD-L1 was assessed by flow cytometry on the murine AML cell line, C1498, alone and following treatment with vehicle, DMXAA or interferon-gamma (positive control). A LEGEND MAX mouse ELISA kit was utilized to measure IL-6 and IFN-β. C57BL/6 mice were inoculated with stably transfected C1498 murine AML cells expressing luciferase and the fluorescent protein DSRed2. Once disease was established, animals were treated with vehicle, DMXAA (20 mg/kg every four days x 7 weeks), anti-murine PD-1 antibody (10 mg/kg every 3 days x 4 doses) or DMXAA + anti-PD-1 antibody (same doses). Animals underwent weekly clinical assessments, weights, and bioluminescent imaging for disease burden. Overall study endpoints were time to morbidity and differences in leukemia disease burden as compared with vehicle-treated controls. Mice were euthanized on day 15 after injection of C1498 cells (8 days following treatment) for collection of plasma, bone marrow, liver and spleen samples for tumor burden, activated T-cells.Results: DMXAA doses (ranging from 1-100 μg/ml) inhibited C1498 in vitro cell growth at 48 hours (48h) in a dose dependent manner. Treatment of C1498 cells in culture with escalating doses of DMXAA (1-100μg/ml) or IFN-gamma (positive control) induced higher PD-L1 expression on these AML cells consistent with direct immunomodulatory effects. Furthermore, C1498 cells exposed to higher doses of DMXAA (10-100μg/ml) for 48h produced measurably higher levels of IL-6 and IFN-β expression in cell supernatants.We then examined the effects of DMXAA, anti-PD-1 ab, or the combination of DMXAA + anti-PD-1 ab treatment in vivo in C57BL/6 mice systemically engrafted with C1498-luciferase AML cells. Treatment overall was well tolerated and resulted in significantly decreased disease burden as measured by total body bioluminescence vs. vehicle controls (p<0.05). Median time to morbidity was significantly decreased in all treatment arms as compared with controls: vehicle = 28 days, DMXAA = 32 days, anti-PD-1 ab = 39 days, and combination DMXAA + anti-PD-1 ab = 53 days (p<0.05). Combination therapy resulted in significantly longer overall survival than single agent therapy (DMXAA vs. DMXAA+anti-PD-1 ab, p=0.032; anti-PD1 ab vs. DMXAA+antii-PD-1 ab p=0.038)(n=total 13-16 mice per group) (representative data shown in Figure 1). Therapy with DMXAA alone and in combination with anti-PD-1 ab was associated with markedly higher PD-1, PD-L1, and PD-L2 expression levels in bone marrow cells harvested from leukemic mice 48h after treatment. Significantly higher numbers of activated T cells were also identified in the bone marrow and spleen of leukemic mice following two weeks of DMXAA therapy alone or in combination with anti-PD-1 ab. Additional in vivo measurements of systemic cytokine levels following therapy are underway.Conclusions: Here we demonstrate that the combination of an innate immune agonist (DMXAA) with an immune checkpoint inhibitor (anti-PD-1 ab) improved anti-leukemic effects in a preclinical AML model. In vitro DMXAA therapy inhibited murine AML growth in a dose dependent manner, enhanced PD-L1 expression, and induced leukemic production of cytokines (IL-6, IFN-β). In vivo combination DMXAA and anti-PD-1 ab therapy in an immunocompetent murine AML model increased activated host T cell numbers and marrow PD-1/L1/L2 expression in conjunction with decreased tumor burden and prolonged overall survival. These studies may pave the way for future clinical trials evaluating this novel immunomodulatory strategy in AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the Enzyme Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia

BackgroundThe prognosis for adults diagnosed with acute myeloid leukemia (AML) remains poor, with a five-year survival of 25%. This prognosis is even more dismal in older patients, and those who are not well enough to receive standard induction chemotherapy. Speaking to the need for new therapies is the fact that our therapeutic backbone - a combination of cytarabine and an anthracycline - remains unchanged for 40 years.Differentiation therapy (e.g. ATRA and Arsenic trioxide) revolutionized care for the small subset of patients diagnosed with acute promyelocytic leukemia (APL). Where APL was once the worst form of myeloid leukemia, it now carries the best prognosis, with a five-year survival exceeding 85%. Replicating the success of differentiation therapy in the remaining 90% of patients with AML has remained elusive, and was the motivation behind our studies.HypothesisAML comprises disparate genetic sub-types, but one shared hallmark is that the leukemic blasts are frozen at an immature and self-renewing stage of development. Despite AML's heterogeneity, 70% of cases overexpress HoxA9, a gene that must be downregulated in the process of normal myeloid cell differentiation. We hypothesized that the process through which HoxA9 maintains the undifferentiated state represented a commonly dysregulated node, and that therapies that bypass this node would be useful in the treatment of AML. Thus, we asked the question: 'can we identify small molecules that overcome myeloid differentiation arrest?'A Phenotypic Differentiation ScreenWe established a HoxA9-based AML screening system that permitted an unbiased flow cytometry phenotypic differentiation screen. We screened 330,000 small molecules for those with myeloid differentiation activity. Target identification of our lead compounds led to the unexpected discovery that inhibitors of dihydroorotate dehydrogenase (DHODH) can overcome myeloid differentiation arrest. DHODH is a mitochondrial enzyme involved in the endogenous synthesis of pyrimidines. The modulation of DHODH had not previously been shown to affect myeloid differentiation.DHODH Inhibitors in the Treatment of LeukemiaThe small molecule brequinar is a potent DHODH inhibitor (Panel A). Brequinar triggered myeloid differentiation in vitro (Panel B) and in vivo. Brequinar was highly active in vivo, as demonstrated in syngeneic murine AML models (HoxA9+Meis1 and MLL/AF9) as well as xenotransplant AML models (THP1, HL60, MOLM13, OCI/AML3).In an aggressive MLL/AF9 model of AML, treatment of mice with brequinar caused myeloid differentiation (Panel D), reduced leukemic cell burden, and improved overall survival (Panel C). Furthermore, treatment with brequinar reduced the number of leukemia stem cells, reduced colony-formation activity, and depleted the number of leukemia initiating cell activity (Panel E). These findings support the hypothesis that the myeloid differentiation resulting from DHODH inhibition is both phenotypic and functional, such that the matured leukemia cells lose their self-renewing capability.DHODH Inhibitors and Normal HematopoiesisTreatment with brequinar was better-tolerated and more effective than treatment with cytotoxic chemotherapy. Unlike cytarabine and doxorubicin, brequinar could be given for many weeks without cumulative toxicity. To assay the effect of DHODH inhibition on normal cells, we performed competitive bone marrow transplantation assays. Mice were treated with brequinar, 5-fluorouracil or induction chemotherapy. Their bone marrow was transplanted in competition (1:1) with normal (untreated) bone marrow, to gauge the effect of therapy on hematopoietic stem cell (HSC) function. Treatment with 5-FU and induction chemotherapy led to a dramatic loss of HSC fitness. In contrast, HSCs from mice treated with brequinar were functionally equivalent to those of untreated mice (Panel F) supporting the notion that DHODH inhibition does not lead to detrimental differentiation of the HSC compartment.DiscussionThe mechanism for the selective vulnerability of leukemia cells to DHODH inhibition remains under investigation. Our studies point towards DHODH as a new metabolic target; DHODH inhibitors may provide differentiation therapy for patients with AML. [Display omitted] DisclosuresSykes:Bayer Pharma AG: Research Funding. Meyer:Bayer Pharma AG: Employment. Stoeckigt:Bayer Pharma AG: Employment. Janzer:Bayer Pharma AG: Employment. Scadden:GlaxoSmithKline: Research Funding; Dr. Reddy’s: Consultancy; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Teva: Consultancy; Apotex: Consultancy; Bone Therapeutics: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

The Role of Gata2 in Murine Models of Acute Myeloid Leukemia

The importance of stem cell and self-renewal programs in Acute Myeloid Leukemia (AML) is generally accepted, but the molecular details are incompletely understood. The master transcriptional regulator GATA2 is highly expressed in hematopoietic stem cells (HSCs) and has critically important roles in the hematopoietic system. Gata2 is required for murine HSC development and maintenance, and heterozygous loss of Gata2 compromises murine HSC- and progenitor cell-function. High levels of GATA2-expression have been correlated with adverse prognosis in human AML. GATA2 is also overexpressed in human chronic myeloid leukemia. These data suggest an important role for GATA2 in normal stem cells and in leukemia. However, genetic lesions resulting in compromised GATA2 function can lead to MDS and in some cases AML. In a murine AML model driven by Flt3-ITD and inactivation of Tet2, Gata2 is strongly downregulated. Furthermore, mouse models of leukemia suggest that high-level forced expression of Gata2 can have a tumor suppressor role. To clarify the role of Gata2in AML we used homozygous genetic inactivation in established murine models of leukemia, using a a conditional allele.We initially tested the role of Gata2 in a murine leukemia mediated by forced expression of Meningioma1 (MN1). This model has a HoxA9/Meis1 transcriptional program. We recently found that MN1-driven leukemia depends on the histone methyltransferase Dot1l (J Clin Invest. 2016 Feb 29. pii: 80825). Lineage marker negative (Lin-), Sca1+, Kit-positive (LSK) bone marrow cells from mice with a floxed exon 5 in the Gata2 gene, and a ROSA26-YFP Cre-reporter allele were transduced with an MSCV-based ecotropic retroviral vector expressing MN1 and linked via an internal ribosomal entry site (IRES) the selectable marker GFP. Floxed Gata2-sequences were excised using transduction with a self-excising Cre-expressing vector (HR-Cre). Cells were sorted and plated in methylcellulose. The GFP/YFP double positive Gata2ko cells showed a replating defect compared to GFP single positive Gata2-floxed cells, both with regard to colony number and colony size. Next, we tested the role of Gata2 in disease maintenance in vivo. We established MN1 Gata2ff leukemias in primary recipients. Primary leukemias were transduced with Cre-expressing vector and Gata2ko and Gata2ff MN1 cells were transplanted. While all mice in the Gata2ff group developed leukemia with a median survival of 35 days, the mice in the Gata2ko cohort developed leukemia with incomplete penetrance with a latency of 249 days (p=0.0005). These data suggest an important role for Gata2in MN1 leukemia in vitro and in vivo. Genetic inactivation of Gata2 resulted in increased protein levels of p53 in vitro as detected by Western blot. Furthermore, MN1-transduced cells showed accentuated p53 stabilization and apoptosis in response to the Mdm2-antagonist and p53 stabilizer Nutlin3.We next tested the role of Gata2 in leukemia driven by the oncogenic fusion MLL-AF9. In contrast to the MN1 model, recipients of Gata2ko leukemias only showed a trend towards prolonged latency in secondary recipients (median survival Gata2ff=48 days vs. Gata2ko=62 days, p=0.09). In this model, we also did not observe a substantial effect of Gata2-inactivation on p53 activation. We are currently characterizing the underlying molecular mechanisms.Our data document an important role for Gata2 in AML mediated by MN1 and to a lesser degree, MLL-AF9. The role of Gata2 in leukemia is complex and depends on expression levels and cellular context. A more detailed understanding of leukemic self-renewal, including the role of Gata2, will inform the development of more efficacious and less toxic therapies for this difficult-to-treat malignancy. DisclosuresBernt:Epizyme: Patents & Royalties: patent filed. Neff:Epizyme: Patents & Royalties: patent filed; Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel.

Interferon-γ Promotes Antibody-mediated Fratricide of Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

Targeting Chromatin Regulators Inhibits Leukemogenic Gene Expression in NPM1 Mutant Leukemia.

MLL1 and DOT1L are chromatin regulators that control HOX, MEIS1, and FLT3 expression and are therapeutic targets in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on-target activity and constitutes a novel therapeutic concept for this common AML subtype. Cancer Discov; 6(10); 1166-81. ©2016 AACR.See related commentary by Hourigan and Aplan, p. 1087This article is highlighted in the In This Issue feature, p. 1069.

Cytoprotective autophagy maintains leukemia-initiating cells in murine myeloid leukemia

AbstractDespite advances in the treatment of acute myeloid leukemia (AML), relapse and drug resistance frequently occur. Therefore, detailed mechanisms of refractoriness, including leukemia-initiating cell (LIC) biology, should be elucidated to treat AML. The self-degradative property of cytosolic macromolecules is central to autophagy and can contribute to homeostasis and stress response. Recent reports suggest the importance of autophagy in hematopoietic stem cells and various tumors. Thus, this study investigated the functional role of autophagy in AML maintenance and drug resistance using tamoxifen-inducible conditional knockout mice of Atg5 or Atg7, which are essential genes for autophagy, combined with an mixed lineage leukemia–eleven nineteen leukemia–induced murine AML model. Inactivation of autophagy by deletion of Atg5 or Atg7 prolonged survival in leukemic mice and reduced functional LICs. Atg7-deficient LICs displayed enhanced mitochondrial activity and reactive oxygen species production together with increased cell death. In addition, Atg7 deletion markedly decreased peripheral blood leukemia cells, concurrent with increased apoptosis, suggesting a higher dependency on autophagy compared with bone marrow leukemia cells. Finally, cytarabine (AraC) treatment activated autophagy in LICs, and Atg7 deletion potentiated the therapeutic effects of AraC, which included decreased LICs and prolonged survival, suggesting that autophagy contributes to AraC resistance. Our results highlight the intratumoral heterogeneity related to autophagy in AML and the unique role of autophagy in leukemia development and drug resistance.

Targeting Innate and Adaptive Immune Responses for the Treatment of Acute Myeloid Leukemia

Abstract Background: Successful immunotherapeutic approaches for acute myeloid leukemia (AML) have yet to be developed. We hypothesized that targeting both the innate and adaptive immune responses in leukemic hosts would elicit significant anti-tumor activity with lesser toxicities than chemotherapy. To test this, we evaluated the efficacy of immune checkpoint inhibition (murine anti-PD-1 antibody (ab)) alone and in combination with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an innate immune agonist and anti-vascular agent, in an immunocompetent model of murine AML. Methods: Expression of PD-L1 was assessed by flow cytometry on the murine AML cell line, C1498, alone and following treatment with vehicle, DMXAA or interferon-gamma (positive control). A LEGEND MAX mouse ELISA kit was utilized to measure IL-6 and IFN-β. C57BL/6 mice were inoculated with stably transfected C1498 murine AML cells expressing luciferase and the fluorescent protein DSRed2. Once disease was established, animals were treated with vehicle, DMXAA (20 mg/kg every four days x 7 weeks), anti-murine PD-1 antibody (10 mg/kg every 3 days x 4 doses) or DMXAA + anti-PD-1 antibody (same doses). Animals underwent weekly clinical assessments, weights, and bioluminescent imaging for disease burden. Overall study endpoints were time to morbidity and differences in leukemia disease burden as compared with vehicle-treated controls. Mice were euthanized on day 15 after injection of C1498 cells (8 days following treatment) for collection of plasma, bone marrow, liver and spleen samples for tumor burden, activated T-cells. Results: DMXAA doses (ranging from 1-100 μg/ml) inhibited C1498 in vitro cell growth at 48 hours (48h) in a dose dependent manner. Treatment of C1498 cells in culture with escalating doses of DMXAA (1-100μg/ml) or IFN-gamma (positive control) induced higher PD-L1 expression on these AML cells consistent with direct immunomodulatory effects. Furthermore, C1498 cells exposed to higher doses of DMXAA (10-100μg/ml) for 48h produced measurably higher levels of IL-6 and IFN-β expression in cell supernatants. We then examined the effects of DMXAA, anti-PD-1 ab, or the combination of DMXAA + anti-PD-1 ab treatment in vivo in C57BL/6 mice systemically engrafted with C1498-luciferase AML cells. Treatment overall was well tolerated and resulted in significantly decreased disease burden as measured by total body bioluminescence vs. vehicle controls (p Conclusions: Here we demonstrate that the combination of an innate immune agonist (DMXAA) with an immune checkpoint inhibitor (anti-PD-1 ab) improved anti-leukemic effects in a preclinical AML model. In vitro DMXAA therapy inhibited murine AML growth in a dose dependent manner, enhanced PD-L1 expression, and induced leukemic production of cytokines (IL-6, IFN-β). In vivo combination DMXAA and anti-PD-1 ab therapy in an immunocompetent murine AML model increased activated host T cell numbers and marrow PD-1/L1/L2 expression in conjunction with decreased tumor burden and prolonged overall survival. These studies may pave the way for future clinical trials evaluating this novel immunomodulatory strategy in AML patients. Download : Download high-res image (50KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

RALB provides critical survival signals downstream of Ras in acute myeloid leukemia.

Mutations that activate RAS proto-oncogenes and their effectors are common in acute myeloid leukemia (AML); however, efforts to therapeutically target Ras or its effectors have been unsuccessful, and have been hampered by an incomplete understanding of which effectors are required for AML proliferation and survival. We investigated the role of Ras effector pathways in AML using murine and human AML models. Whereas genetic disruption of NRAS(V12) expression in an NRAS(V12) and Mll-AF9-driven murine AML induced apoptosis of leukemic cells, inhibition of phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) signaling did not reproduce this effect. Conversely, genetic disruption of RALB signaling induced AML cell death and phenocopied the effects of suppressing oncogenic Ras directly – uncovering a novel role for RALB signaling in AML survival. Knockdown of RALB led to decreased phosphorylation of TBK1 and reduced BCL2 expression, providing mechanistic insight into RALB survival signaling in AML. Notably, we found that patient-derived AML blasts have higher levels of RALB-TBK1 signaling compared to normal blood leukocytes, supporting a pathophysiologic role for RALB signaling for AML patients. Overall, our work provides new insight into the specific roles of Ras effector pathways in AML and has identified RALB signaling as a key survival pathway.

Targeting the RAS/MAPK pathway with miR-181a in acute myeloid leukemia.

Deregulation of microRNAs' expression frequently occurs in acute myeloid leukemia (AML). Lower miR-181a expression is associated with worse outcomes, but the exact mechanisms by which miR-181a mediates this effect remain elusive. Aberrant activation of the RAS pathway contributes to myeloid leukemogenesis. Here, we report that miR-181a directly binds to 3′-untranslated regions (UTRs); downregulates KRAS, NRAS and MAPK1; and decreases AML growth. The delivery of miR-181a mimics to target AML cells using transferrin-targeting lipopolyplex nanoparticles (NP) increased mature miR-181a; downregulated KRAS, NRAS and MAPK1; and resulted in decreased phosphorylation of the downstream RAS effectors. NP-mediated upregulation of miR-181a led to reduced proliferation, impaired colony formation and increased sensitivity to chemotherapy. Ectopic expression of KRAS, NRAS and MAPK1 attenuated the anti-leukemic activity of miR-181a mimics, thereby validating the relevance of the deregulated miR-181a-RAS network in AML. Finally, treatment with miR-181a-NP in a murine AML model resulted in longer survival compared to mice treated with scramble-NP control. These data support that targeting the RAS-MAPK-pathway by miR-181a mimics represents a novel promising therapeutic approach for AML and possibly for other RAS-driven cancers.

Supraphysiologic levels of the AML1-ETO isoform AE9a are essential for transformation

Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34(+) cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.

Abstract 3284: Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model

Abstract FLT3 internal tandem duplication (ITD) mutations are the most common alterations in acute myeloid leukemia (AML) and are associated with poor disease prognosis. Targeted therapy using FLT3-ITD inhibitors showed limited effect in reducing leukemia blasts in bone marrow (BM) than that in peripheral blood. The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents. Therefore, blocking CXCR4 and E-selectin concomitantly with FLT3 inhibition could theoretically eliminate the protection in FLT3-mutant AML patients. We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 showed significant prolongation of survival of mice engrafted with FLT3-ITD mutant MV4-11 leukemia cells. In the present study, we further investigated anti-leukemia effects of dual CXCR4/E-selectin inhibition. Results indicate efficient mobilization of leukemia cells into the circulation by GMI-1359 in a MOLM14-engrafted murine model 2h after drug treatment, which was 3.3-fold (± 0.3) higher compared with the CXCR4 antagonist plerixafor, and 7.4-fold (± 2.7) higher compared with controls. In addition, GMI-1359 also mobilized normal murine leukocytes from the BM, suggesting that GMI-1359 may block interactions of leukemia cells with various BM components. Combination therapy of GMI-1359 and sorafenib significantly reduced leukemia burden (1.9e7 vs. 2.3e9, 1.0e9 and 8.5e7 photons/sec in the combination group versus control, GMI-1359 and sorafenib groups, respectively, at day 20 as determined by Xenogen IVIS bioluminescence Imaging; p &amp;lt; 0.001). Moreover, the combination treatment profoundly reduced leukemia burden in the BM of leukemic mice after a 15-day treatment (1.7e7 vs. 1.6e9, 6.3e8 and 7.0e7 photons/sec, p &amp;lt; 0.01; and 1.76% vs. 70.96%, 72.8% and 33.8%.leukemia cell engraftment measured as GFP positive leukemic cells in BM by flow cytometry, in the combination group vs. vehicle, GMI-1359 and Sorafenib groups, respectively, p &amp;lt; 0.01). Of note, the combination therapy profoundly extended the median survival of the leukemia-bearing mice from 27 days in the control group to 46.5 days in the combination treatment groups (p &amp;lt; 0.001). Mechanistically, we demonstrated that direct blockade of the interaction of E-selectin-CXCR4/SDF-1α with GMI-1359 was critical for leukemic cell mobilization via the disruption of adhesion to stromal cells. Furthermore, the presence of GMI-1359 enhanced sorafenib-induced apoptosis in FLT3-ITD mutated leukemic cells co-cultured with stromal cells. This effect occurred even under hypoxia, which is characteristic for the leukemic BM microenvironment. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3-mutant AML. Citation Format: Weiguo Zhang, Hong Mu, Qi Zhang, Nalini B. Patel, William E. Fogler, John L. Magnani, Michael Andreeff. Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model. [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 3284.

Inhibition of CREB signaling through antagonism of vasoactive intestinal peptide enhances CD8 T cell function in a murine model of AML

Abstract Signaling pathways that activate CREB may inhibit the potency of T cell responses following stimulation. One of the major mechanisms by which this occurs is the sequestration of CBP, which is also an NF-κB binding partner. One of the major molecules that signals through this pathway is vasoactive intestinal peptide (VIP). VIP is a 28 amino acid peptide that is secreted throughout the body by a variety of cell types including immune cells. VIP has shown to be a potent anti-inflammatory molecule with pleiotropic effects on T cells including cell cycle arrest and inhibition of cytokine secretion. The VIP signaling pathway represents a therapeutic target in disease settings in which a more robust T cell response would be beneficial. Acute myeloid leukemia (AML) is a hematological malignancy that has been explored as a candidate for a variety of T cell-based immunotherapies due to weak CD8 responses to AML blasts. We demonstrate here that administration of a hybrid peptide antagonist that blocks VIP signaling enhances CD8 T cell responses in a murine model of AML. CD8 T cells from treated mice showed enhanced secretion of IFN-γ and TNF-α as well as reduced expression of PD-1. These enhanced effector functions lead to a significant survival enhancement, which was lost when treated mice lacked T cells. Additionally, CD8 T cells from antagonist-treated survivors made up the majority of CD3+ cells following transfer to Rag1 knockout recipients. The phenotype of the cells was consistent with enhanced effector functions as transferred CD8+ T cells were predominately CD44+CD62L−. Taken together, these results demonstrate the potential for VIP antagonism as an immunotherapy strategy in settings of hematological malignancies.

Rheb1 promotes tumor progression through mTORC1 in MLL-AF9-initiated murine acute myeloid leukemia.

BackgroundThe constitutive hyper-activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways has frequently been associated with acute myeloid leukemia (AML). While many inhibitors targeting these pathways have been developed, the anti-leukemic effect was not as robust as expected. As part of the molecular link between PI3K/Akt and mTOR kinase, the role of Rheb1 in AML remains unexplored. Our study aims to explore the role of Rheb1 in AML and estimate whether Rheb1 could be a potential target of AML treatment.MethodsThe expressions of Rheb1 and other indicated genes were analyzed using real-time PCR. AML mouse model was established by retrovirus transduction. Leukemia cell properties and related signaling pathways were dissected by in vitro and in vivo studies. The transcriptional changes were analyzed via gene chip analysis. Molecular reagents including mTOR inhibitor and mTOR activator were used to evaluate the function of related signaling pathway in the mouse model.ResultsWe observed that Rheb1 is overexpressed in AML patients and the change of Rheb1 level in AML patients is associated with their median survival. Using a Rheb1-deficient MLL-AF9 murine AML model, we revealed that Rheb1 deletion prolonged the survival of AML mice by weakening LSC function. In addition, Rheb1 deletion arrested cell cycle progression and enhanced apoptosis of AML cells. Furthermore, while Rheb1 deletion reduced mTORC1 activity in AML cells, additional rapamycin treatment further decreased mTORC1 activity and increased the apoptosis of Rheb1Δ/Δ AML cells. The mTOR activator 3BDO partially rescued mTORC1 signaling and inhibited apoptosis in Rheb1Δ/Δ AML cells.ConclusionsOur data suggest that Rheb1 promotes AML progression through mTORC1 signaling pathway and combinational drug treatments targeting Rheb1 and mTOR might have a better therapeutic effect on leukemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0264-3) contains supplementary material, which is available to authorized users.

Analysis of telomerase target gene expression effects from murine models in patient cohorts by homology translation and random survival forest modeling.

Acute myeloid leukemia (AML) is an aggressive and rapidly fatal blood cancer that affects patients of any age group. Despite an initial response to standard chemotherapy, most patients relapse and this relapse is mediated by leukemia stem cell (LSC) populations. We identified a functional requirement for telomerase in sustaining LSC populations in murine models of AML and validated this requirement using an inhibitor of telomerase in human AML. Here, we describe in detail the contents, quality control and methods of the gene expression analysis used in the published study (Gene Expression Omnibus GSE63242). Additionally, we provide annotated gene lists of telomerase regulated genes in AML and R code snippets to access and analyze the data used in the original manuscript.

The target cell of transformation is distinct from the leukemia stem cell in murine CALM/AF10 leukemia models

The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.