Administration Of pIpC Research Articles

RAF-Independent MEK Mutations Drive Histiocytic Neoplasms In Vivo and Are Sensitive to Single-Agent ERK Inhibition in Patients

Histiocytic neoplasms are clonal disorders of the monocyte/macrophage lineage that include Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD). Histiocytoses are defined by recurrent, mutually exclusive mutations activating MAP kinase signaling, including BRAFV600E mutations in ~50% of LCH and ECD patients, which confer robust lasting clinical responses to BRAF inhibition. Mutations in MEK1/2, ARAF, and a diverse array of other kinases are present in BRAF wild-type histiocytoses patients and are broadly responsive to the allosteric MEK1/2 inhibitor cobimetinib. Following genomic analysis of ~300 pediatric and adult patients with diverse histiocytoses, the single most common genomic alteration following BRAFV600E mutations are MEK1 E102_I103 in-frame deletions. In contrast to BRAFV600E, the biological impact of activating mutations in MEK1/2 kinases are unknown, and no cancer associated MEK1/2 mutation has ever been modeled in vivo. MEK1/2 mutations are recurrent across a wide variety of human cancers and RASopathies and are now often found at resistance to emerging KRAS-mutant selective inhibitors. These findings motivated us to generate Map2k1E102_I103del conditional knock-in mice within the endogenous locus of Map2k1 (encoding MEK1 kinase), which represent the first genetically engineered mouse model of MEK1/2 mutations. We first crossed Map2k1E102_I103del mice to inducible Mx1-cre driver mice. Interestingly, Mx1-cre Map2k1E102_I103del knock-in mice developed a 100% penetrant lethal myelomonocytic disorder by 5-6 weeks of age while their cre-negative Map2k1E102_I103del littermates did not develop disease (Figure A). Notably, recombination of the Map2k1E102_I103del alleleoccurred spontaneously without pIpC administration and resulted in strong activation of phosphorylated ERK1/2 in bone marrow and spleen mononuclear cells. Mx1-cre Map2k1E102_I103del mice developed a myelomonocytic neoplasm with suppression of B-lymphopoiesis and expansion of classical and non-classical monocyte subsets, macrophages, and Cd11b+ conventional dendritic cells. Monocyte, macrophage, and dendritic cells infiltrated hematopoietic organs (bone marrow, spleen, and peripheral blood), as well as liver and skin, each of which are common sites of LCH and ECD disease involvement. The Mx1-cre Map2k1E102_I103del disease was hematopoietic cell autonomous and could be propagated by serial hematopoietic reconstitution in irradiated recipient mice, as well as using Vav1-cre Map2k1E102_I103del knock-in mice (where Cre is expressed only in hematopoietic cells from earliest hematopoietic development). Finally, restricting MEK1 mutant expression to monocyte and dendritic cell precursors using CD11c-cre resulted in similar lethal expansions of monocyte, macrophage, and dendritic cells as seen in the Mx1-cre and Vav1-cre models. Prior in vitro work has demonstrated that MEK1 p.E102_I103del activates MEK and ERK without requiring activated GTP-bound RAS and is associated with resistance to allosteric MEK inhibitors. Consistent with this, we identified nine histiocytosis patients with this specific MEK1 mutation, three of which had de novo resistance to cobimetinib. These three patients and one treatment-naïve patient were treated with an oral ERK1/2 inhibitor ulixertinib under the FDA single-patient compassionate use program. Three of 4 patients had a clinical or radiological partial or complete response to ulixertinib (Figure B). Overall, these data identify the impact of oncogenic MEK kinase mutations in vivo as found in children and adults with histiocytoses and RASopathies. Expression of a RAF-independent MEK1 mutation in vivo gave rise to disease reminiscent of human systemic histiocytic neoplasms. Importantly, this molecular entity is resistant to MEK inhibition in some patients, a phenomenon previously undocumented, but responsive to ERK inhibition, which may be a promising therapeutic approach to histiocytic neoplasms unresponsive to allosteric MEK inhibition. The clinical observation of efficacy of ERK inhibition in MEK inhibitor resistant histiocytoses patients has resulted in a soon-to-be initiated focused phase 2 clinical trial of ulixertinib in adults with histiocytoses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Contrasting Stage Dependent Requirements for the Histone Methyltransferase EZH2 during the Evolution of Acute Myeloid Leukemia

Epigenetic regulators function in a context-dependent manner and are commonly mutated in tumors. Activating mutations and overexpression of the histone-methyltransferase, enhancer of zeste, homolog 2 (EZH2), occur in lymphoma and other malignancies, whilst loss-of-function mutations are predicted in myeloid malignancies. We study this apparent contradiction, examining the importance of cellular context and disease stage for Ezh2 loss, genetically deleting Ezh2 during the induction or maintenance of a single malignancy, Acute Myeloid Leukemia (AML).We initially focused on the role of Ezh2 in AML maintenance, using conditional Ezh2fl/fl mice. Following in vitro retroviral immortalization of haematopoietic stem and progenitor cells (HSPCs) from these with AML-fusion oncogenes: MLL-AF9 or AML1-ETO9a (reflecting two separate models of AML clinically associated with good and poor prognosis, markedly differing in mechanism of leukemic transformation and representing AML heterogeneity), these were then transduced with p-babe-Cre-puro vector (or empty vector) to delete Ezh2 . Strikingly, Ezh2 loss completely abolished cellular growth and colony formation, whilst empty vector transduced cells maintained their clonogenic output. To interrogate Ezh2 as a target in AML maintenance in vivo, we generated secondary MLL-AF9 or AML1-ETO9a leukemias through transplantation of bulk leukemia derived from Ezh2fl/fl ; Mx1-Cre primary leukemic mice. Following engraftment, deletion of Ezh2 was achieved by pIpC administration. Loss of Ezh2 in vivo markedly impaired both secondary MLL-AF9 and AML1-ETO9a leukemia progression. These data suggest that Ezh2 is an oncogene across multiple disparate subtypes of established AML and propose it as a possible therapeutic target in this context .Addressing this hypothesis, we examined the role of pharmacological inhibition of EZH2 in AML, utilizing GSK343, an S-Adenosyl Methionine competitive EZH2 inhibitor. Through colony formation and functional assays, we demonstrated in vitro sensitivities of MLL-AF9 transformed murine BM HSPCs, primary murine Ezh2+/+ MLL-AF9 tumours , the AML1-ETO fusion driven human cell line Kasumi-1 and primary leukemic cells isolated from patients with various AML genotypes to GSK343 at similar IC50 (~10µM) across all cells. In contrast, minimal effects of GSK343 were seen on normal primary CD34+ HSCs clonogenic function. Together these data provide evidence of therapeutic potential, validating EZH2 as a target in AML.To characterise the role of Ezh2 during AML induction in vivo, we utilized Ezh2fl/fl; Mx1-Cre mice to generate Ezh2-/- mice and compared them to Ezh2+/+ controls. HSPCs from these were retrovirally transduced with MLL-AF9 or AML1-ETO9a and transplanted into lethally irradiated wild-type recipients, and leukemias allowed to develop. Remarkably in the context of disease induction, and in stark contrast to the maintenance experiments, Ezh2 functioned as a tumor suppressor gene, with its loss significantly accelerating leukemia generation across both subtypes of AML (compared to control).To interrogate the molecular mechanisms of these findings we performed integrated genomic analysis with differential global gene expression analysis via RNA-sequencing (in Ezh2+/+ vs Ezh2-/- non-transformed HSPCs, Ezh2+/+ vs Ezh2-/- AML1-ETO9a and Ezh2+/+ vs Ezh2-/- MLL-AF9 leukemias) and ChIP-sequencing of the histone modifications H3K27me3 and H3K4me3. These revealed that during disease induction, Ezh2 represses a subset of bivalent promoters that resolve towards gene activation upon Ezh2 loss, inducing a number of genes including the transcription factor Plag1 (with known oncogenic function in CBFB-MYH11 murine leukemogenesis). Of note, overexpression of Plag1 phenocopied Ezh2 loss i n vivo to significantly accelerate MLL-AF9 leukemia induction.In addition, we demonstrated virtual mutual exclusivity of genes de-repressed in established MLL-AF9 leukemias following pharmacological Ezh2 inhibition compared to those de-repressed in the induction of Ezh2-/- MLL-AF9 leukemias. This de-repression of entirely separate gene programmes explains the starkly contrasting phenotypic outcomes of targeting Ezh2 in AML induction and maintenance, and further provides reassurance that EZH2 inhibition in established AML is a safe strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inducible SBDS Deficiency and Germline FANCC/FANCG Deficiency Differentially Impact the Capacity of Bone Marrow Niches to Engraft Hematopoietic Stem Cells after Transplantation

Strategies to optimize interactions between the recipient bone marrow (BM) microenvironment and donor hematopoietic stem cells (HSC) could improve hematopoietic stem cell transplantation (HSCT) outcomes by reducing graft failure rates, improving blood and immune reconstitution efficiency, and reducing intensive cytotoxic conditioning requirements. Such methods are particularly relevant for patients with inherited bone marrow failure syndromes (iBMFs), rare genetic disorders for which HSCT is required to cure bone marrow failure (BMF), but for which HSCT is also associated with a high incidence of delayed donor engraftment, graft failure and morbidity due to chemotherapy and radiation sensitivity.In previous studies of the MPL-/- model of congenital amegakaryocytic thrombocytopenia, we discovered that decreased numbers of megakaryocytes and/or long-term (LT)-HSC caused by absent thrombopoietin/MPL signaling resulted in diminished capacity of the MPL-/- BM niche microenvironment to efficiently engraft wildtype (WT) donor HSC after HSCT. This observation led us to hypothesize that pre-existing BMF, regardless of cause, may generally alter HSC niche elements critical for engraftment after HSCT. We now present data testing this hypothesis in mouse models of Fanconi Anemia (FA) and Shwachman-Diamond Syndrome (SDS).To model FA, we used double mutant FANCC-/-;FANCG-/- mice, which have previously been demonstrated to develop manifestations of BMF. In our facility, FANCC-/-;FANCG-/- mice did not develop evidence of BMF, as measured by blood counts, BM cellularity, and HSC quantitation out to 1 year of age even with induction of HSC cycling by polyinosinic-polycytidilic acid (pIpC). Using SCT assays in which GFP+ WT donor BM was transplanted into FA recipients (FANCC-/-, FANCG-/-, FANCC-/-;FANCG-/-) or WT controls after 800-1100 cGy total body irradiation (TBI), efficiency and durability of donor engraftment was assessed at 3 weeks and 5 months, respectively, after primary SCT by competitive secondary HSCT. Unexpectedly, all FA recipients demonstrated more rapid BM reconstitution and enhanced early hematopoietic progenitor engraftment of WT donor BM compared with WT controls. However, there was no significant difference in LT-HSC engraftment efficiency or durability in the FA recipient groups versus WT controls.To model SDS, we first attempted to generate a model of osteoblast-specific SBDS deletion by crossing Col1A1Cre mice with mice carrying floxed SBDS alleles (SBDSfl/fl). However, this Col1A1CreSBDSExc model proved to be embryonic lethal at E12-E15 due to profound developmental abnormalities. We thus generated an inducible model by crossing SBDSfl/fl mice with Mx1Cre+ mice, and inducing SBDS deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by pIpC administration. SBDS deletion induced by pIpC was confirmed with PCR methods.Within 4 weeks of pIpC induction, Mx1CreSBDSExc mice show evidence of BMF, including a 40% reduction in platelet counts, inverted myeloid/lymphoid cell ratio in blood, and up to 80% reduction in immunophenotypic BM LT-HSC. When we attempted HSCT with GFP+ WT donor BM in Mx1CreSBDSExc and littermate control recipients following 1100 cGy TBI, 90% of Mx1CreSBDSExc recipients died by day 9 after HSCT, whereas all littermate controls survived. BM sections from Mx1CreSBDSExc recipients demonstrated persistent aplasia out to 1 week post-HSCT, suggesting that Mx1CreSBDSExc died from primary graft failure. Competitive secondary HSCT performed from BM harvested 1 week after primary HSCT demonstrated severe deficits in GFP+ WT donor HSC engraftment in Mx1CreSBDSExc recipients. Our initial studies as to why donor engraftment fails in Mx1CreSBDSExc recipients have identified that compared to WT, Mx1CreSBDSExc mice show significantly reduced expansion of osteolineage niche cells after TBI, a response that we have previously shown to be critical for efficient donor engraftment after HSCT.Taken together, our data demonstrate that pre-existing BMF and SBDS deficiency in BM osteolineage cells, but not FANCC or FANCG deficiency without co-existent BMF, significantly reduce the capacity of the BM niche to engraft donor HSC. Future studies defining specific niche deficits in our inducible SBDS deficiency model will provide critical insights into cellular and molecular pathways required for efficient engraftment after HSCT. DisclosuresNo relevant conflicts of interest to declare.

The RNA Splicing Factor U2AF1 Controls Hematopoietic Stem Cell Survival and Function

Somatic mutations in U2AF1 have been identified in ~11% cases of MDS. U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. Most U2AF1 mutations are found in two hotspots (S34 and Q157) within the first and second zinc finger domains. Transgenic and knock-in mice expressing U2AF1 S34F mutant exhibit impaired hematopoiesis. However, the role of wild-type U2AF1 in regulating hematopoietic stem cell (HSC) function and normal hematopoiesis has remained unknown. To determine the role of U2AF1 in normal hematopoiesis, we generated a new conditional U2af1 knockout (floxed) mouse. We crossed U2af1 floxed mouse with Mx1-Cre mouse and the expression of Cre recombinase was induced with polyinosine-polycytosine (pI-pC) injection at 5 to 6 weeks after birth. We observed that deletion of U2af1 significantly reduced white blood cell, neutrophil, red blood cell and platelet counts in their peripheral blood compared with control animals within 10-14 days after pI-pC injection. Histopathologic analysis of the BM sections from U2af1-deficient mice showed severe BM aplasia. Flow cytometric analyses revealed a marked decrease in myeloid, erythroid and megakaryocytic precursors in the BM of U2af1-deficient mice compared with control animals. We also observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2af1-deleted mice. Hematopoietic progenitor colony assays showed a significant decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2af1-deficient mice.Together, these data suggest that loss of U2af1 causes severe defects in hematopoiesis. We performed both non-competitive and competitive BM transplantation assays using U2af1-deficient BM to determine the role of U2af1 in HSC function. There was marked reduction of HSC, progenitors and all types of blood and BM cell precursors upon U2af1 deletion (by pI-pC administration) in the transplanted animals. Also, U2af1-deficient HSCs were unable to compete with WT HSCs and there was rapid loss of hematopoietic progenitors/precursors derived from the U2af1-deficient HSCs. Since U2af1 deletion resulted in rapid decrease of hematopoietic progenitors in the BM, we asked whether deletion of U2af1 insulted the genome and induced apoptosis to hematopoietic cells in the BM. We observed significantly increased apoptosis in the total BM as wells as in c-kit+, Gr1+, Ter119+and CD41+cells suggesting that hematopoietic progenitors and precursors of multiple cell lineages underwent apoptosis upon U2af1 deletion. We also performed gamma-H2AX assay using imaging flow cytometry to evaluate DNA damage in total BM, Gr1+(myeloid) and CD71+(erythroid) cells in control and U2af1-deleted mice. We observed markedly elevated gamma-H2AX in total BM, Gr1+and CD71+cells from U2af1-deficient mice compared with control mice.In addition, we observed increased Chk1 phosphorylation (ser345), a hallmark for activation of the ATR pathway, and increased histone H2A K119 ubiquitination (H2AK119Ub), a marker for DNA damage response, in the BM of U2af1-deficient mice. Thus, depletion of U2af1 causes insult to the genome and induces DNA damage and increased cell death. To gain insights into severe hematopoietic defects observed in U2af1-deficient mice, we performed transcriptome profiling of sorted LSK cells from U2af1 wild type (control) and U2af1-deleted mice. GSEA analysis of RNA sequencing data revealed significant downregulation of genes related to HSC maintenance in U2af1-deficient LSK. GSEA also revealed enrichment for cell cycle and DNA damage response-related genes, consistent with decreased proliferation and increased DNA damage and apoptosis observed in U2af1-deficient hematopoietic progenitors. We also determined the effects of U2af1 deletion on RNA splicing. Interestingly, we observed significant changes in gene expression as well as splicing alterations in several genes important for HSC survival and function. In conclusion, our results suggest a crucial role for U2af1 in the survival and function of HSC. Disclosures Mohi: Tolero Pharmaceuticals Inc.: Research Funding.

CXCL12 Knock-out Enhances Leukemia Stem Cell Response to Combination Chemotherapy Plus Tyrosine Kinase Inhibition in Flt3-ITD Acute Myeloid Leukemia

One of the most common mutations in adult AML is a constitutively activating internal tandem duplication in the juxtamembrane domain of the Flt3 receptor (Flt3-ITD), which portends poor prognosis due to high recurrence rates and defines a distinct subtype of disease with unique features and biology. Although several FLT3 TKIs have been developed for clinical use, responses to these drugs, especially as single agents, are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML, as well as primary Flt3-ITD TET2 mutant AML patient samples, to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. In the animal model, administration of pIpC leads to deletion of TET2 and development of AML characterized by leukocytosis, accumulation of blasts, splenomegaly, anemia, thrombocytopenia, and lethality. Limiting dilution transplantation of FACS-sorted ST-HSC, MPP and GMP populations revealed that LIC were absent from GMPs and almost exclusively limited to the phenotypic ST-HSC population (calculated stem cell frequencies: <1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD TET2 mutant AML patients LIC capacity was restricted to primitive HSPC populations (Lin-CD34+CD38-), and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We characterized bone marrow stromal cells in Flt3-ITD AML mice by flow cytometry on collagenase digested bone fragments. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found expansion of several stromal populations in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+) and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). CXCL12 expression, however, was greater than 2-fold higher in osteoprogenitors and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing niche in supporting Flt3-ITD AML LSC. To assess the effect of CXCL12 deletion from the marrow microenvironment on AML TKI response, we transplanted murine AML cells into CXCL12flox/flox UBC-cre mice and control Cre-ve mice. We found that AML developing in Cre-ve control mice was resistant to single agent Flt3 TKI (AC220, Quizartinib) treatment, but that CXCL12 deletion modestly improved response to TKI. We next tested a combination of standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and AC220, and found that this approach resulted in more effective and selective, but only partial, reduction of leukemia cells in this model. We found that control AML mice showed an initial response to combination chemo + TKI, but developed disease recurrence by 3 weeks of treatment. In contrast, CXCL12-deleted AML mice maintained peripheral blood response for up to 3 weeks, and showed enhanced suppression of LIC-containing populations compared to control mice. We are now performing secondary transplants using BM cells harvested from these treated mice to assess long-term effects on leukemia stem cell capacity. We are also testing the effect of the combination of chemotherapy and TKI following osteoblast-specific deletion of CXCL12, using CXCL12flox/flox BGLAP-cre mice, to assess whether osteoblastic cells are the source of CXCL12 responsible for this effect. In conclusion, our results suggest that LSC in Flt3-ITD AML are found within a primitive phenotypic ST-HSC population as opposed to GMP populations as seen in some other types of AML. Furthermore, CXCL12-expressing bone marrow microenvironmental cells contribute to drug resistance in AML LSC and global knockout of CXCL12 enhances drug response in these populations. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: <1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

The Cold-Shock Protein Ybx1 Is Required for Development and Maintenance of Acute Myeloid Leukemia (AML) in Vitro and In Vivo

The family of cold shock proteins (CSPs) is highly conserved and consists of 8 members, including Ybx1-3, Csde1 and Lin28. Ybx1 is a multifunctional DNA/RNA binding protein that modulates gene transcription and translation during inflammation and malignant transformation. Recently, our group identified Ybx1 as a mediator of Jak2 signaling in MPN that protects Jak2-mutated cells from Jak-inhibitor induced apoptosis.In a recently published genome wide CRISPR-Cas9 dropout screen in AML cell lines, depletion of Ybx1 resulted in the highest dropout indices compared to other CSP members, with strongest dependencies in cell lines harboring MLL-rearrangements. Protein expression of Ybx1 in healthy individuals (n=10), primary MDS (n=54) and AML (n=58) bone marrow (BM) biopsies, revealed high protein expression in the majority of AML and MDS cases. Consistently, gene expression data revealed high mRNA expression of Ybx1 in AML samples compared to normal controls.Genetic inactivation of Ybx1 in human AML cell lines by RNAi resulted in reduced proliferative capacity. Therefore, we sought to investigate the requirement for Ybx1 in malignant transformation. We used BM cells from a previously published conventional knockout (ko) mouse model (Lu et al., 2005) in which homozygous deletion is embryonically lethal due to brain malformation. We sorted Lineage-Sca1+Kit+ (LSK-) cells from the BM of heterozygous (Ybx1+/-) and wildtype (Ybx1+/+) mice. Cells were retrovirally infected with either MLL-AF9 (MA9) or HoxA9 and Meis1a (HA9M1) to assess for disease development by serial plating in methylcellulose. Haploinsufficiency for Ybx1 in MA9- or HA9M1 transformed cells limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-Ybx1+/- cells (n=8) and MA9-Ybx1+/+ (n=10) showed development of AML. However, recipients of MA9-Ybx1+/- cells had a significant delay in AML development (median survival 67.5 days for Ybx1+/+ versus 101.5 days for Ybx1+/- animals, p=0.0078**). This effect appeared even more pronounced when 1x 106 whole BM cells were transplanted into sublethally irradiated secondary recipients. Besides a significant delay in AML development (median survival 37.5 days for recipients of MA9-Ybx1+/+ versus 79 days for MA9-Ybx1+/- BM, p=0.0042**), disease penetrance was reduced by 40%, indicating that haploinsufficiency for Ybx1 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of stem- and progenitor cells in Ybx1+/+ versus Ybx1+/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of Ybx1+/+ and Ybx1+/- BM into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of Ybx1+/- cells.To address the question whether Ybx1 may also be essential for maintenance of AML, we used RNAi to deplete Ybx1 in already established MA9 driven AML. LSK cells from BL/6 mice transformed with MA9 were injected into primary recipient mice. After AML onset, MA9-LSK cells were sorted and infected with either one of 3 shRNAs against Ybx1 or non-targeting (NT-) control. Lentiviral knockdown of 40% reduced colony formation by more than 50% but did not limit the re-plating capacity in vitro. When injected into sub-lethally irradiated recipient mice, lentiviral knockdown (kd) of Ybx1 resulted in a significant delay in AML development (median survival 39.5 days for NT-control versus 53 days for Ybx1 kd, p=0.0446*).To validate our findings, we used a newly generated conditional ko mouse model for Ybx1, in which exon 3 coding for the cold-shock domain is deleted by activation of an Mx1-Cre-recombinase following pIpC administration. Preliminary results provide first evidence that genetic deletion of Ybx1 after onset of MA9 driven leukemia resulted in improved survival of primary recipient (median survival 73 versus 83 days) and a reduced penetrance in secondary recipient mice.Taken together our results may provide first evidence for a functional role of Ybx1 in MLL-AF9 driven AML. As Ybx1 seems to be dispensable for normal hematopoietic cells, these findings may offer a potential therapeutic index. Experiments to assess for the requirement for Ybx1 in maintenance of murine and human AML as well as analysis on proteomic and transcriptional changes following Ybx1 deletion are currently under way. DisclosuresNo relevant conflicts of interest to declare.

THU0529 The Mouse Model of Human Autoinflammatory Syndromes – Human IL-1 Alpha Conditional Transgenic Mice

BackgroundRecently, IL-1 is implicated in the pathogenesis of autoinflammatory syndromes, which characterized by rash, fever, systemic polyarthritis and neurological comorbidity. Recently, IL-1 is being accepted as a therapeutic target of...

Nephrotoxicity induced by piperacillin–tazobactam in late elderly Japanese patients with nursing and healthcare associated pneumonia.

This study aimed to clarify the efficacy, safety, and pharmacokinetics of piperacillin–tazobactam (PIPC– TAZ) in late elderly Japanese patients. This is the first antimicrobial pilot study in late elderly patients with nursing and healthcare associated pneumonia. After PIPC–TAZ administration, PIPC concentrations in plasma were measured chromatographically and the pharmacokinetic parameters were estimated. Efficacy, safety, and bacteriological evaluations were also carried out. The mean age was 85.0 years old and most of the patients were late elderly. Chest X-rays, body temperature, white blood cell count, and C reactive protein all improved significantly, and a high efficacy ratio of 90.9% was observed. Serious nephrotoxicity was observed in 4 cases (18.2%) after administration of PIPC–TAZ. Creatinine clearance (meanS.D.) measured before PIPC–TAZ therapy was significantly lower in the nephrotoxicity group (32.54.4 mL/min) than in the non-nephrotoxicity group (46.116.7 mL/min), although the ages were not different between the 2 groups. In the pharmacokinetic parameters for PIPC, total clearance was slightly lower in the nephrotoxicity group than in the non-nephrotoxicity group. However, no significant difference was observed in plasma PIPC levels between the 2 groups. In patients with renal impairment, especially with a creatinine clearance of <40 mL/ min, renal impairment was found to be an influencing factor for severe nephrotoxicity following PIPC–TAZ administration. In conclusion, the results suggest that physicians should pay close attention in order to avoid possible toxicity, and that deliberate administration planning and careful follow-up are required in late elderly patients with comprised organ dysfunction.

Inactivation of the Cell Fate Determinate Llgl1 (Lethal Giant Larvae Homolog 1) Leads to Enhanced HSC Fitness and Predicts a Poor Prognosis in AML.

AbstractAbstract 2293Regulation of the balance between symmetric and asymmetric cell division is considered to be important for hematopoietic stem cell (HSC) division and homeostasis. Recently, several polarity regulators have been shown to influence HSC function using RNAi-screening approaches. Llgl1 is a known regulator of cell polarity in drosophila and epithelial cells, however, its role in HSC remains elusive. Using a previously published conditional knockout mouse model with exon 2 of Llgl1 being flanked by loxP-sites we generated Llgl1loxP/loxP Mx-Cre+ animals and induced deletion of Llgl1 (Llgl1−/−) by administration of pIpC as determined by PCR.After deletion of Llgl1 no significant abnormalities in hematopoiesis were evident for 16 weeks in the peripheral blood. However, bone marrow of Llgl1−/− animals revealed up to a 2-fold increase in HSC (CD34lo Flk2− LSK). When transplanted competitively into recipient animals Llgl1−/− cells showed a significant competitive advantage compared to Llgl1+/+ controls, and that competitive advantage increased over time in serial transplants. In tertiary recipient mice the bone marrow was dominated by Llgl1−/− cells with a chimerism of above 90%. Llgl1 deleted animals show increased numbers of cycling HSC (CD34lo LSK) when compared to their wildtype counterparts as detected by Ki-67 staining (64.94% ± 2.537 Llgl1+/+ HSC vs 50.81% ± 4.213 Llgl1−/− HSC in G0; p=0.0184*) and BrdU incorporation and this effect was restricted to the HSC compartment. The Llgl1 deleted cells proved to be better able to reconstitute hematopoiesis after being stressed in ex vivo culture, and protected the survival of recipient mice to a higher extent than wildtype cells. Gene-expression analysis on sorted Llgl1−/− versus Llgl1+/+ steady state HSC revealed known regulators of HSC self-renewal capacity (such as Hox-genes and self-renewal associated transcription factors) to be among the top regulated downstream targets.The observed phenotype suggests that Llgl1 expression might influence leukemogenesis and its close homologue Llgl2 has been described recently as an early hit in malignant transformation of chronic neutropenia. Thus, we analyzed Llgl1 gene-expression in human acute myeloid leukemia (AML). Llgl1 was the top polarity regulator to be down-regulated in leukemia initiating cells of different phenotype (L-MPP or L-GMP), when compared to their normal counterparts (MPP or GMP). Moreover, reduced Llgl1 expression was associated with inferior overall survival in two independent patient cohorts treated for AML: Low expression of Llgl1 as measured by Affymetrix gene arrays was associated with decreased survival in 83 karyotypic normal (CN-) AML patients below the age of 60 (p=0.0092**). This finding could be confirmed in an independent CN-AML cohort (n=80, p=0.0056**) by qRT-PCR. Moreover, the Llgl1−/− GEP signature obtained from murine HSC was not associated with a specific genetic subgroups of a human AML dataset, but AML cases matching this signature showed again decreased overall survival (p=0.0089**).Genetic inactivation of Llgl1 contributes to fitness and viability of hematopoietic stem cells in vivo. Given its impact on survival in patients treated for AML it is tempting to speculate on its role in development and maintenance of leukemia stem cells (LSC). Disclosures:No relevant conflicts of interest to declare.

Genetic Deletion of Bcl-x Reveals a Potential Role in Governing Stem Cell Homeostasis.

Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x deficient embryos failed to thrive due to massive apoptosis of immature hematopoietic and neural cells. Deletion of this gene in the adult has uncovered its requirement for the final maturation of erythroid cells, the transition of pre-B cells to the pro-B cell stage and recently, we showed an important role in the development of natural killer cells. While the role of bcl-x in hematopoietic stem cells (HSC) has yet to be addressed, a role for other members of anti-apoptotic bcl-2 family of proteins has been established. Overexpression of bcl-2 led to accumulation of HSC, whereas genetic deletion of mcl-1 resulted in ablation of HSC. In the present study, we determined by quantitative Real-Time PCR, that in addition to mcl-1, bcl-x is also highly expressed in hematopoietic stem cells, whereas the level of bcl-2 is threefold lower. Moreover, we found the expression of bcl-x to be down regulated with differentiation of HSC into progenitor populations (i.e. GMP, CMP and MEP). Therefore, we established a model to delete bcl-x in the stem cells of adult mice. We bred the Mx1-cre transgenic line with mice that carry the bcl-x gene flanked by loxP sites. Deletion of bcl-x occurred following administration of pIpC. A total of 10 bcl-x deficient mice and 7 age-matched control animals (Mxi-cre/wildtype bcl-x) were examined. Quantitative real-time PCR revealed a 55 – 95% reduction of bcl-x mRNA in Lin−, Sca-1+, c-kit+ cells 8 days post the first administration of pIpC. This population consists of phenotypically and functionally defined long-term HSC (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitors. We used FACS analysis to identify LT-HSC versus ST-HSC populations and to further assess the effects of bcl-x deletion. LT-HSC were defined by staining, for the following cell surface markers as Lin−, Sca-1+, c-kit+, Flt3−, CD34−; ST-HSC were characterized as Lin−, Sca-1+, c-kit+, Flt3−, CD34+. We found a 2 fold shift in the ratio of LT-HSC versus ST-HSC in bcl-x deficient mice compared to the ratio in control animals. This result raises the hypothesis that bcl-x regulates entry of HSC into the cell cycle. A notion that is supported by the observation that overexpression of bcl-x in fibroblasts delays transition from the Go/G1 to the S phase of the cell cycle. Future experiments will determine if the observed phenomenon is mediated through the anti-apoptotic function or cell cycle activity of the bcl-x protein.

Bcl-x Is Indispensable for the Development of Natural Killer Cells.

The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC.As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen.To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.

1042. RNA as a Source of Transposase for Sleeping Beauty -Mediated Transposition and Long-Term Expression in Somatic Cells and Tissues

The Sleeping Beauty (SB) transposon system has been demonstrated to mediate gene insertion and long-term expression, suggesting the potential application of this plasmid based system for non-viral gene therapy. All reported studies of SB-mediated gene transfer to somatic cells have supplied the transposase by expression of co-injected plasmid DNA. Although successful for both in vitro and in vivo transposition, there is a lack of control with respect to the transient and long-term levels of transposase expressed in a target cell. There is also the potential for genomic integration of the transposase encoding DNA, resulting in sustained transposase expression and potential destabilizing effects on the integrated transgene or host genome integrity. We address these problems by supplying the transposase-encoding molecule in the form of mRNA. Rapid, high pressure injection of m7G-capped luciferase (Luc) mRNA through the tail vein of mice resulted in efficient luciferase expression with respect to a DNA element where the Caggs promoter controlled luciferase transcription. Using this system as a model for efficient gene transfer in the liver, we have taken steps to optimize delivery/expression of the mRNA by generating constructs to promote stability and translation. Real-time whole body in vivo imaging of Luc mRNA translation was conducted to measure the effect of an IRES and UTR sequences on luciferase expression. These studies determined that incorporation of 5' and 3' UTR elements from the Xenopus -globin gene were essential to achieve peak luciferase expression levels from an otherwise unprotected transcript. Co-delivery of these mRNAs with an inhibitor of or a substrate for endogenous RNases provided luciferase expression sufficient to mimic levels achieved using promoters known to provide optimum transposase expression for in vivo transposition. Using this genetic arrangement, we have co-delivered SB transposase mRNA with a transposon encoding the Luc gene (driven by the Caggs promoter) to the liver of transgenic mice in which transcription of Cre recombinase is under the control of an interferon response element. To distinguish between plasmid-mediated expression and expression mediated by transposition, we have engineered this transposon encoding plasmid with lox-P sites positioned such that Cre-mediated recombination excises a sequence which includes the Caggs promoter, unless it has been segregated away from the plasmid by the SB transposase (Score et al., Mol. Ther. 7: S9, 2003). We found that pIpC induction of Cre recombinase caused a 1600-fold reduction in luciferase expression in animals infused with the transposon alone. While co-delivery of transposase mRNA resulted in a moderate degree of inadvertent Cre induction, we observed only a 45-fold reduction in luciferase expression following analogous pIpC administration. This remaining luciferase expression is most likely attributable to SB mediated transposition. These results provide the first evidence for use of SB transposase mRNA to mediate transposition and long-term expression in an in vivo setting.