Murine Bone Marrow Progenitor Research Articles

Abstract 102: Trained Immunity Impairs Inflammation Resolution And Dampens Efferocytosis

Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent findings suggest innate immune cells maintain ‘memory’ of prior exposure to inflammatory stimuli, a phenomenon known as ‘trained immunity’. In response to inflammatory stimuli, macrophages undergo metabolic and epigenetic rewiring that primes them to mount an augmented response upon a second exposure. Oxidized low-density lipoproteins (oxLDL) have recently been shown to be potent triggers of trained immunity. While trained immunity has been shown to promote inflammation, little is known about how immune training impacts efferocytosis. Therefore, we hypothesize that trained immunity in macrophages promotes inflammation by impairing efferocytosis. We treated murine bone marrow progenitors with oxLDL for 24 hours, then washed and differentiated them into macrophages (BMDMs). Upon assessing efferocytosis, trained BMDMs were able to ingest a first apoptotic cell (AC) better than untrained BMDMs yet had an impaired ability to take up additional ACs, reflecting a defect in continual efferocytosis. Using an in vivo approach, we transplanted donor bone marrow from Ldlr -/- mice fed a chow or Western diet into naïve C57BL/6 recipients. After recovery, we elicited peritoneal macrophages to assess efferocytosis and found that recipients receiving marrow from Western diet fed Ldlr -/- mice not only displayed impaired efferocytosis, but also significantly upregulated PGE 2 production, suggesting impaired resolution. To determine whether PGE 2 is a mediator of trained immunity, we primed bone marrow progenitors with PGE 2 and differentiated them into BMDMs. We found that macrophages primed with PGE 2 elaborated higher levels of inflammatory cytokines in response to LPS stimulation than controls. Overall, these findings demonstrate that oxLDL/Western diet-training impinges on the resolution program by impairing efferocytosis and are durable effects that demonstrate heritability. Future directions include determining the impact of these findings on the development of atherosclerosis.

The CSF-1R inhibitor pexidartinib affects FLT3-dependent DC differentiation and may antagonize durvalumab effect in patients with advanced cancers.

Tumor-associated macrophages (TAMs) are a critical determinant of resistance to PD-1/PD-L1 blockade. This phase 1 study (MEDIPLEX, NCT02777710) investigated the safety and efficacy of pexidartinib, a CSF-1R-directed tyrosine kinase inhibitor (TKI), and durvalumab (anti-PD-L1) in patients with advanced colorectal and pancreatic carcinoma with the aim to enhance responses to PD-L1 blockade by eliminating CSF-1-dependent suppressive TAM. Forty-seven patients were enrolled. No unexpected toxicities were observed, one (2%) high microsatellite instability CRC patient had a partial response, and seven (15%) patients experienced stable disease as their best response. Increase of CSF-1 concentrations and decrease of CD14lowCD16high monocytes in peripheral blood mononuclear cells (PBMCs) confirmed CSF-1R engagement. Treatment decreased blood dendritic cell (DC) subsets and impaired IFN-λ/IL-29 production by type 1 conventional DCs in ex vivo TLR3-stimulated PBMCs. Pexidartinib also targets c-KIT and FLT3, both key growth factor receptors of DC development and maturation. In patients, FLT3-L concentrations increased with pexidartinib treatment, and AKT phosphorylation induced by FLT3-L ex vivo stimulation was abrogated by pexidartinib in human blood DC subsets. In addition, pexidartinib impaired the FLT3-L- but not GM-CSF-dependent generation of DC subsets from murine bone marrow (BM) progenitors in vitro and decreased DC frequency in BM and tumor-draining lymph node in vivo. Our results demonstrate that pexidartinib, through the inhibition of FLT3 signaling, has a deleterious effect on DC differentiation, which may explain the limited antitumor clinical activity observed in this study. This work suggests that inhibition of FLT3 should be considered when combining TKIs with immune checkpoint inhibitors.

A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.

Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated 'gut-like' DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/- (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These 'gut-like' DCs extended transepithelial dendrites across the intact epithelium of enteroids. 'Gut-like' DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in 'gut-like' DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.

The Protein Interactome of DNMT3A, and Altered Interactions Caused By Missense Mutations Associated with AML and/or the DNMT3A Overgrowth Syndrome

DNA methylation mediated by the de novo DNA methyltransferases (DNMT3A and DNMT3B) is essential for normal embryonic development. Somatic loss-of-function or mutations in DNMT3A are common in individuals with clonal hematopoiesis, and are found in nearly 30% of normal karyotype AML patients; mutations at amino acid R882 represent a hotspot in AML, accounting for ~60% of DNMT3A mutations in AML. Acquired germline DNMT3A mutations are associated with the DNMT3A Overgrowth Syndrome (aka Tatton-Brown-Rahman Syndrome/TBRS), a leukemia susceptibility syndrome. Mutations at R882 have a dominant negative effect on WT DNMT3A, and cause a distinctive, focal, canonical DNA hypomethylation phenotype. Many non-R882 DNMT3A missense mutations also have a canonical hypomethylation phenotype in hematopoietic cells that is similar to that of haploinsufficiency caused by gene deletion (Smith et al, Nat. Comms, 2021), suggesting that they are also loss-of-function mutations. Although some non-R882 missensemutations may cause DNMT3A protein instability to cause loss-of-function (Huang et al, Can Disc 2022), others do not yet have a mechanism described. For this study, we investigated the hypothesis that missense mutations in DNMT3A may inactivate its methyltransferase activity by altering its ability to interact with proteins that influence its function. To better understand mechanisms involving altered protein interactions, we used a proximity labeling system using a highly active biotin ligase (TurboID/"TID"), fused in-frame to full length human DNMT3A1 at either the N-or C-terminus in an MSCV-based vector tagged with GFP. Vectors with either WT or mutant DNMT3A cDNAs were transduced into murine bone marrow progenitors, which were allowed to grow in culture for 2 days, enriched for GFP+ cells, and then labelled with biotin for 4 hours. Proteins within 10 angstroms of the TurboID-DNMT3A fusions were tagged with biotin, enriched with a streptavidin bead pulldown, and identified using mass spectrometry. We evaluated protein interactions with TurboID alone (n=9 independent experiments), wildtype DNMT3A (n=9), or with 21 different, recurrent missense mutations (7 TBRS mutations, 4 AML mutations, and 10 mutations found in both AML and TBRS patients, n=2-4 per mutation, with 53 total, independent samples) in mouse hematopoietic stem/progenitor cells (HSPCs). ETV3, RUNX1, DNMT3B, MBD1, GATA1, KDM5A, RB1, and 428 other proteins interacted specifically with wild type DNMT3A, compared to TurboID alone. Although some of these proteins had previously been identified as DNMT3A interactors, the vast majority were previously unknown (see Fig. 1A for the 50 strongest interactors). Altered DNMT3A protein interactions caused by missense mutations are shown for 10 selected proteins in Fig. 1B. Three mutations located in the PWWP domain of DNMT3A (G298W, I310N, and R326C) caused a loss of nearly all interactions, which was found to be due to mislocalization of DNMT3A to the cytoplasm by these variants (data not shown). Mutations known to cause DNMT3A instability (Huang et al, Can Disc 2022) including C583Y, R635W, Y660H, R736H, R749C, F902S and P904L, had increased interactions with ubiquitin ligases (Hectd3, Rnf31, Ube2v1, Smurf1, Trim8) which may target them for accelerated degradation (data not shown). The normal interaction between DNMT3A and DNMT3B was essentially eliminated by several mutations (I310N, R635W, Y660H, R736H) and decreased in several more (G298W, F902S and P904L) (Fig.1B). In AML cells, the dominant isoform of DNMT3B is DNMT3B3, which is missing exons that are essential for its methyltransferase activity. However, DNMT3B3 is known to interact with DNMT3A and, in doing so, increase its activity by acting as a chaperone. Therefore, the disruption of DNMT3B3 binding to DNMT3A may represent a novel mechanism for missense mutations to reduce DNMT3A activity. Finally, we used the same system to define DNMT3A protein interactions in human CD34+ cord blood cells (with DNMT3A WT, R736H, and R882H, and others in progress), which revealed high concordance with the mouse data. DNMT3A missense mutations can alter many protein interactions with DNMT3A, which can clearly reduce its methyltransferase activity by a number of mechanisms. These findings may suggest new approaches for restoring DNMT3A activity in patients with myeloid malignances. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

CSF3R Splicing Regulates Granulopoiesis Via Splice Variant Specific Responses to G-CSF

The granulocyte colony stimulating factor receptor (CSF3R) is a critical regulator of neutrophil production with multiple alternatively spliced variants. The truncated CSF3R-V4 splice variant confers enhanced growth signals, and changes in its expression levels relative to the canonical V1 (wild type) isoform have been implicated in chemotherapy resistance and relapse of AML. We previously demonstrated that the CSF3R-V3 isoform, a variant of V1 with an insertion in the cytoplasmic domain, produces hypoproliferative signals in lymphoid cells in response to G-CSF. We also reported that expression of all three splice variants is significantly altered in AML, suggesting that aberrant CSF3R splicing is involved in the pathogenesis of some myeloid malignancies. The functional signaling capabilities of the different CSF3R isoforms in regulating granulopoiesis remain largely unknown. Herein, we describe a novel myeloid model system and show that the V3 and V4 isoforms generate opposing proliferative signals without effects on myeloid cell differentiation.ER-HoxB8 cells are murine bone marrow progenitor cells ectopically expressing an ER-HoxB8 fusion protein, and in the presence of estradiol (E2) the fusion protein dimerizes producing a functional HoxB8 dimer which enforces self-renewal. Thus, in the presence of E2 these cells continually proliferate; however, when E2 is withdrawn they differentiate into mature granulocytes. Addition of G-CSF to culture medium of E2 ER-HoxB8 cells increases progenitor cell proliferation in a dose dependent manner (Figure 1A). Using CRISPR/Cas9, we knocked-out the endogenous murine Csf3r. As expected, ER-HoxB8-Csf3r-/- cells still produced mature neutrophils with E2 withdrawal and no increase in differentiation or proliferation of the knock-out cells (KO) was observed in response to G-CSF. The functional behavior of our ER-HoxB8-Csf3r-/- cells recapitulates the published phenotype of the Csf3r knock-out mouse, which exhibits severe neutropenia but has circulating neutrophils. ER-HoxB8 KO cells were transduced with human CSF3R splice variants and expression confirmed by immunoblot analysis using splice-variant specific antibodies. KO cells expressing the CSF3R-V3 demonstrated a hypoproliferative response to G-CSF with an ~40-fold increase in the EC50 relative to cells expressing CSF3R-V1 (Figure 1B), confirming our prior observations in the lymphoid BaF3 cell line. In contrast, KO cells expressing the truncated CSF3R-V4 variant hyperproliferated in response to G-CSF consistent with our previously published data in lymphoid cells.Using multi-color flow cytometry with antibodies against CD117, CD11b, and Ly6G to identify progenitor, intermediately differentiated cells (NeuP), and mature neutrophils, we found that KO cells (like parental ER-HoxB8 cells) produced significant numbers of CD11b+/LyGG- NeuP cells upon E2 withdrawal and addition of G-CSF had no effect on differentiation. Transduction of ER-HoxB8 KO cells with the wild type human CSF3R-V1 restored their capacity to respond to G-CSF in a dose dependent manner. KO cells transduced with CSF3R-V3 displayed normal production of NeuP cells with E2 withdrawal, and addition of G-CSF produced a substantial increase in the numbers of mature neutrophils (CD11b+, Ly6G+) after 5 days in culture, relative to KO cells (Figure 2C). Thus, we have demonstrated that CSF3R-V3 is able to support the production of fully mature neutrophils. Notably, a G-CSF induced increase in the numbers of mature neutrophils was also evident in CSF3R-V4 transduced cells. Previous work by others indicated that CSF3R-V4 was not able to drive myeloid differentiation. We hypothesize that this difference in phenotype is due to a V4-dependent hyperproliferation of the neutrophil progenitors. On-going work is focused on the determination of the specific effects these CSF3R splice variants have on each stage of granulopoiesis.In conclusion, using our novel engineered CSF3R model system, we confirm differential effects of CSF3R splice variants on myeloid cell proliferation and show sustained differentiation capacity of each isoform. Additional studies using this model system provide the opportunity for identification of new therapeutic targets for treatment of disorders of granulocyte production. [Display omitted] DisclosuresAvalos: JUNO: Membership on an entity's Board of Directors or advisory committees.

Synthetic lethal targeting of TET2-mutant hematopoietic stem and progenitor cells (HSPCs) with TOP1-targeted drugs and PARP1 inhibitors.

Inactivating mutations in TET2 serve as an initiating genetic lesion in the transformation of hematopoietic stem and progenitor cells (HSPCs). Thus, effective therapy for this subset of patients would ideally include drugs that are selectively lethal in TET2-mutant HSPCs, at dosages that spare normal HSPCs. In this study, we tested 129 FDA-approved anticancer drugs in a tet2-deficient zebrafish model and showed that topoisomerase 1 (TOP1)-targeted drugs and PARP1 inhibitors selectively kill tet2-mutant HSPCs. We found that Tet2-deficient murine bone marrow progenitors and CRISPR-Cas9-induced TET2-mutant human AML cells were more sensitive to both classes of drugs compared with matched control cells. The mechanism underlying the selective killing of TET2-mutant blood cells by these drugs was due to aberrantly low levels of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that is important for removing TOP1 cleavage complexes (TOP1cc). Low TDP1 levels yield sensitivity to TOP1-targeted drugs or PARP1 inhibitors and an inability to remove TOP1 cleavage complexes, leading to DNA double-strand breaks and cell death. The finding that TET2 mutations render HSPCs uniquely vulnerable to disruption of TOP1 and PARP1 activity may therefore represent a unique opportunity to use relatively low dosages of these drugs for the "precision therapy" of TET2-mutant myeloid malignancies.

Investigating the role of the innate immune response in relapse or blast crisis in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by expression of the tyrosine kinase oncogene, Bcr–abl. Tyrosine kinase inhibitors (TKI) induce prolonged remission in CML, and therapy discontinuation is an accepted approach to patients with reduction in Bcr–abl transcripts of four logs or greater. Half such individuals sustain a therapy free remission, but molecular mechanisms predicting relapse are undefined. We found relative calpain inhibition in CML cells with stabilization of calpain substrates, including βcatenin and Xiap1. Since the Survivin gene is activated by βcatenin, this identified two apoptosis-resistance mechanisms. We found that Survivin impaired apoptosis in leukemia stem cells (LSCs) and Xiap1 in CML granulocytes. Consistent with this, we determined treatment with an inhibitor of Survivin, but not Xiap1, prevented relapse during TKI treatment and after therapy discontinuation in a murine CML model. By transcriptome profiling, we identified activation of innate immune response pathways in murine CML bone marrow progenitors. This was increased by TKI treatment alone, but normalized with addition of a Survivin inhibitor. We found that activation of the innate immune response induced rapid blast crisis in untreated CML mice, and chronic phase relapse during a TKI discontinuation attempt. These results suggest that extrinsic stress exerts adverse effects on CML-LSCs.

Characterization of a Novel JAK1 Pseudokinase Mutation in the First Case of Trisomy 21-Independent GATA1-Mutated Transient Abnormal Myelopoiesis

Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.

Neutrophil Maturation and Survival Is Controlled by IFN-Dependent Regulation of NAMPT Signaling.

Granulocyte-colony stimulating factor (G-CSF)/nicotinamide phosphoribosyltransferase (NAMPT) signaling has been shown to be crucial for the modulation of neutrophil development and functionality. As this signaling pathway is significantly suppressed by type I interferons (IFNs), we aimed to study how the regulation of neutrophil differentiation and phenotype is altered in IFN-deficient mice during granulopoiesis. The composition of bone marrow granulocyte progenitors and their Nampt expression were assessed in bone marrow of type I IFN receptor knockout (Ifnar1-/-) mice and compared to wild-type animals. The impact of NAMPT inhibition on the proliferation, survival, and differentiation of murine bone marrow progenitors, as well as of murine 32D and human HL-60 neutrophil-like cell lines, was estimated. The progressive increase of Nampt expression during neutrophil progenitor maturation could be observed, and it was more prominent in IFN-deficient animals. Altered composition of bone marrow progenitors in these mice correlated with the dysregulation of apoptosis and altered differentiation of these cells. We observed that NAMPT is vitally important for survival of early progenitors, while at later stages it delays the differentiation of neutrophils, with moderate effect on their survival. This study shows that IFN-deficiency leads to the elevated NAMPT expression in the bone marrow, which in turn modulates neutrophil development and differentiation, even in the absence of tumor-derived stimuli.

PF151 RAS/MAPK ACTIVATION COOPERATES WITH GAIN OF CHROMOSOME 21 IN B CELL LEUKEMIA AND IS AN ATTRACTIVE TARGET TO IMPROVE THE OUTCOME OF DS CHILDREN WITH B‐ALL

Background:B cell precursors acute lymphoblastic leukemia (B‐ALL) is the most commonly diagnosed childhood cancer. Major advances in treatments protocols, allowing for the 5‐year overall survival rate to reach 90%. However, many children still undergo intensive therapy and will develop relapse after a complete remission. Children with Down syndrome (DS) have a 27‐fold increased risk of developing B‐ALL and face a dismal prognosis due to treatment‐related mortality, and an increased risk of infections and relapse rate.Aims:The goal of the study was to better understand the molecular bases of DS‐ALL and develop specific therapies to improve outcomes.Methods:Here, we comprehensively characterized DS‐ALL samples, extended our study to other groups of childhood B‐ALL carrying gains of chromosome 21 (B‐ALL+21) and developed Patient Derived Xenograft (PDX) models suitable for preclinical studies. We performed exome and RNA sequencing to characterize the genetic and transcriptomic landscape of DS‐ALL (n = 8), iAMP21‐ALL (n = 7), HeH‐ALL (n = 14) compared to other pediatric B‐ALL (n = 12).Results:Somatic mutations leading to RAS/MAPK pathway activation (affecting the N/KRAS, JAK2, FLT3 and CBL genes) were present in 75% (22/29) of B‐ALL +21 samples, confirming recent observations by other group, and strengthening the link between RAS/MAPK pathway activation and +21. To gain insights into the molecular bases of this cooperation, we ectopically expressed the constitutively active mutant KRASG12D in murine bone marrow progenitors. We observed that KRASG12D expression in trisomic progenitors led to an increased number of CFU‐preB colonies that presented increased self‐renewal capacities compared to wild‐type, trisomy 21 or KRASG12D alone. RNA sequencing showed that 261 genes were synergistically deregulated by KRASG12D+Trisomie 21 in murine B cell progenitors, including 24 genes that were transcriptionally deregulated in B‐ALL+21 with RAS/MAPK mutations patient samples as compared to healthy B‐cell progenitors. Together, tshese results indicated that constitutive RAS activation and +21 functionally cooperates at the cellular and molecular level to impose an altered differentiation of B‐cell progenitors.From this cohort, we also developed 18 preclinical PDX models (13 B‐ALL+21 and 5 Others) to test the efficacy of inhibitors of the RAS/MAPK pathway. We showed that the most potent compound, Trametinib (GSK1120212), significantly decreased viability of B‐ALL+21 blasts presenting a constitutive RAS/MAPK activation in vitro (IC50 = 0,1 ± 0,25 μM compared to 3,95 ± 4,7 μM for B‐ALL samples without RAS/MAPK activation). In vivo treatments with Trametinib significantly decreased leukemia burden in the bone marrow and spleen compared to vehicle controls, and increased survival of DS‐ALL, iAMP21‐ALL and HeH‐ALL PDX models. Finally, we assessed the efficacy of Trametinib in combination with conventional chemotherapy agents. We observed that Trametinib synergised with Vincristine in vitro, and delayed leukemia progression in vivo to enhance survival of DS‐ALL PDXs.Summary/Conclusion:Altogether, we built a comprehensive cohort of preclinical models of DS‐ALL and B‐ALL+21, and suggested that inhibiting RAS/MAPK pathway activation in combination with current chemotherapy may represent a promising strategy to improve the outcome of Down syndrome children with B‐ALL.

Eosinophil Differentiation and Viability in Hypereosinophilia Is Impaired By Genetic and Pharmacologic Targeting of LCP1 and PKC Beta

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Massive overproduction of hyperactive eosinophils leads to enhanced tissue infiltration and, in some cases, severe organ damage. Clonal malignancies with HE involve the FIP1L1-PDGFRA (F/P)-positive neoplasms and other neoplasms harboring PDGFRA and PDGFRB rearrangements, all of which are sensitive to imatinib. However, most subtypes do not carry a defined genetic aberration and, therefore, lack specific treatment.In eosinophils, modulation of the actin cytoskeleton is mandatory for priming and migratory function. In this study, we aimed to target the actin-binding protein LCP1, also termed L-plastin, to reduce eosinophilic burden in HE. LCP1 is a direct target of the serine/threonine kinase PKCß, which phosphorylates LCP1 at serine 5, essential for its full activity. We confirmed high LCP1 expression and Ser5 phosphorylation dependent on F/P activity in the HES cell line Eol-1 and 32D-F/P cells. The PKCβ selective inhibitor enzastaurin led to dephosphorylation of LCP1 in Eol-1 cells, and this was associated with significantly reduced proliferation (p < 0.0001), metabolic activity (p < 0.0001), and colony formation (11.8 fold reduced) as well as enhanced apoptosis (p < 0.0001) and impaired migration to SDF-1 (p < 0.001). Apoptosis was associated with ß-Catenin and c-Jun protein stabilization and increased expression of p73. Reduced surface expression of CD11b was detected (1.5 fold reduction; p=0.0043), which may explain the impaired migration capacity to SDF-1. While enzastaurin did not alter F/P-induced STAT3, STAT5, and ERK1/2 phosphorylation, it specifically inhibited STAT1Tyr701 and AKTSer473 but not AKTThr308 phosphorylation, and shRNA knock-down experiments confirmed that this process was mediated by LCP1.The mTORC2 complex triggers AKTSer473 phosphorylation, and PDK1 is the main Ser/Thr kinase responsible for AKTThr308 phosphorylation. Knockdown of LCP1 protein or reduction of its phosphorylation by enzastaurin treatment resulted in mTORC2 activity loss, while PDK1 activity remained unchanged, suggesting differential phosphorylation of AKTSer473 and AKTThr308. Importantly, AKT substrate activity was significantly reduced upon loss ofAKTSer473 phosphorylation, and SIN1T86 phosphorylation as an indicator of mTORC2 activity was dependent on LCP1 protein, suggesting that targeting of LCP1 reduces mTORC2 activity.Biologically, both enzastaurin treatment and LCP1 knockdown led to impaired IL5-induced eosinophil differentiation of HoxB8-immortalized murine bone marrow progenitor cells (10.1 fold decrease; p=0.021). Moreover, enzastaurin reduced eosinophil differentiation (1.38 fold decrease; p=0.005) and survival (1.6 fold decrease; p=0.0078) of primary peripheral blood-derived samples from HE patients in vitro.In conclusion, our data demonstrate that HE involves active LCP1, which triggers mTORC2 activity and eosinophil migration, and that the PKCβ inhibitor enzastaurin may provide a novel treatment approach for hypereosinophilic disorders. DisclosuresReiter:Incyte: Consultancy, Honoraria. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy.

Menin Is a Therapeutic Target in MN1 High Leukemia

Meningioma-1 (MN1) has been found overexpressed in acute myeloid leukemia (AML). High MN1 expression levels are associated with poor prognosis and limited therapeutic options. Overexpression of MN1 in murine bone marrow progenitor cells causes leukemia as a single hit. Interestingly, MN1 induces a gene expression program that is reminiscent of KMT2a-rearranged leukemia, with high expression of the KMT2a (MLL-1) target genes HOXA7-13 and MEIS1. However, MN1 oncogenic functions remain unclear and no targetable therapies are available for MN1 high leukemia. We have previously shown, using a conditional knock-out mouse, that deletion of the histone methyltransferase Kmt2a increased latency and decreased penetrance of MN1 driven leukemia. We sought to determine what specific function of KMT2A is relevant to MN1 leukemogenesis in order to identify new therapeutic targets.KMT2a is a H3K4 histone methyltransferase that can perform mono, di and tri-methylation through its C-terminal catalytic site, the conserved SET domain. We first asked whether the enzymatic activity of Kmt2a was required in MN1 driven leukemia. Mice with a constitutive germline deletion of the SET domain of Kmt2a (∆SET) present some reduction of Hox gene expression during development but survive into adulthood with normal hematopoiesis. We established MN1 leukemia on a ∆SET background and found no difference in disease latency. Similarly, deletion of the SET domain did not influence colony forming potential in vitro.Menin, the product of the MEN1 suppressor gene, interacts with the N-terminal of KTM2a. The KMT2a-Menin interaction has been shown to be critical for leukemic transformation by KMT2a fusion proteins. Interestingly, this is a unique feature of leukemic cells since Menin is dispensable for steady state hematopoiesis. Since Kmt2a is required for MN1 leukemogenesis, we next investigated whether Menin was also necessary. We established MN1 leukemia on a conditional Menin knock-out background. In primary transplants, upon Cre-mediated excision of Menin, we found a slight decrease in colony forming potential as well as a modest but significant increase in survival of injected recipients. The effect of Menin deletion on MN1 leukemogenesis became much more dramatic in secondary transplants. In vitro, replating efficiency was severely impaired with increased apoptosis and cell cycle arrest. In vivo, we noted a significant increase in disease latency. Finally, we asked if Menin was a potentially relevant therapeutic target in MN1 high leukemia. In vitro, pharmacologic inhibition of the KMT2a-Menin interaction resulted in reduced growth and increased apoptosis of MN1 murine leukemia cells, at micromolar concentrations. We have identified a human leukemia cell line with high MN1 and high HOXA9 expression, Mutz3. To ensure that our findings translate to human AML, we will address the effect of Menin inhibition in Mutz3 cells as well as in patient samples with confirmed MN1 high leukemia.In summary, our data suggest that the role of Kmt2a in MN1 driven leukemia is mediated by the KMT2a-Menin interaction, identifying Menin as a druggable target in MN1 high leukemia. DisclosuresLibbrecht:Institut Servier: Research Funding. Bernt:GSK: Other: husband works at GSK.

High-throughput single-cell fate potential assay of murine hematopoietic progenitors in vitro

The advent of single-cell transcriptomics has led to the proposal of a number of novel high-resolution models for the hematopoietic system. Testing the predictions generated by such models requires cell fate potential assays of matching, single-cell resolution. Here we detail the development of an in vitro high-throughput single-cell culture assay using flow cytometrically sorted single murine bone marrow progenitors, which measures their differentiation into any of five myeloid lineages. We identify critical parameters for single-cell culture outcome, including the choice of sorter nozzle size and pressure, culture media, and the coating of culture dishes with extracellular matrix proteins. Further, we find that accurate assay readout requires the titration of antibodies specifically for their use under low-cell-number conditions. Our approach may be used as a template for the development of single-cell fate potential assays for a variety of blood cell progenitors.

ΔNp73 overexpression promotes resistance to apoptosis but does not cooperate with PML/RARA in the induction of an APL-leukemic phenotype.

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.

Preclinical Characterization of G1T28: A Novel CDK4/6 Inhibitor for Reduction of Chemotherapy-Induced Myelosuppression.

Chemotherapy-induced myelosuppression continues to represent the major dose-limiting toxicity of cytotoxic chemotherapy, which can be manifested as neutropenia, lymphopenia, anemia, and thrombocytopenia. As such, myelosuppression is the source of many of the adverse side effects of cancer treatment including infection, sepsis, bleeding, and fatigue, thus resulting in the need for hospitalizations, hematopoietic growth factor support, and transfusions (red blood cells and/or platelets). Moreover, clinical concerns raised by myelosuppression commonly lead to chemotherapy dose reductions, therefore limiting therapeutic dose intensity, and reducing the antitumor effectiveness of the treatment. Currently, the only course of treatment for myelosuppression is growth factor support which is suboptimal. These treatments are lineage specific, do not protect the bone marrow from the chemotherapy-inducing cytotoxic effects, and the safety and toxicity of each agent is extremely specific. Here, we describe the preclinical development of G1T28, a novel potent and selective CDK4/6 inhibitor that transiently and reversibly regulates the proliferation of murine and canine bone marrow hematopoietic stem and progenitor cells and provides multilineage protection from the hematologic toxicity of chemotherapy. Furthermore, G1T28 does not decrease the efficacy of cytotoxic chemotherapy on RB1-deficient tumors. G1T28 is currently in clinical development for the reduction of chemotherapy-induced myelosuppression in first- and second-line treatment of small-cell lung cancer. Mol Cancer Ther; 15(5); 783-93. ©2016 AACR.

The Homeobox Gene VENTX Collaborates with AML1-ETO in Inducing an Acute Leukemia in Mice

Homeobox genes are key factors in the development of acute leukemias. So far, little is known about the role of non-clustered homeobox genes such as VENTX. The Vent-like homeobox gene VENTX is a member of the Vent gene family in mammals and is the mammalian homolog of the Xenopus xvent gene. Our group has previously shown that VENTX promotes myeloid differentiation when overexpressed in normal CD34+ human stem - and progenitor cells and shows high and aberrant expression in human acute myeloid leukemia (AML) characterized by the AML1-ETO (AE) fusion (Rawat et al., PNAS 2010). To prove the functional relevance of aberrant VENTX expression in this AML genotype, we set up in vitro assays and a murine bone marrow transplantation model, mimicking high expression of VENTX by retrovirally engineered expression of the gene in murine bone marrow progenitor cells. In the CFU-S assay coexpression of both genes significantly increased the frequency of spleen colonies 14-fold and 7-fold compared to VENTX and AE, respectively (p<0.02), and more than 7-fold compared to the control (p<0.001). Most importantly and in contrast to AE alone, coexpression of AE and VENTX induced an acute leukemia after a median latency of 11.3 months post transplant. All leukemias evaluated were rapidly transplantable with a median latency until leukemia induced death of 40 days. The median blast percentage in primary recipient mice was 66% (range 35-100%, n=24). Of note, massive expansion of the red pulpa was documented with the appearance of erythroblasts in the spleen. Furthermore, blasts in the bone marrow expressed CD19, partly in combination with Sca1 and Gr1, pointing to an aberrant expression of this antigen in the AE/VENTX leukemias. Ex vivo, leukemic cells grew permanently generating AE/VENTX positive cell lines. RNA-Seq analyses from CD34+ cord blood transduced with VENTX documented 279 differentially expressed genes compared to the GFP control, hitting 7 pathways (Hematopoietic cell lineage, Cytokine-cytokine receptor pathway, Jak-STAT signaling pathway, Porphyrin and Chlorophyll Metabolism, Asthma, Thyroid cancer and Endocytosis) in the KEGG-Analysis and showed a downregulation of genes necessary for terminal erythroid differentiation such as the EPO-receptor and GATA1. Taken together, these data characterize VENTX as an important contributing factor to AE positive leukemias. Importantly, coexpression of AE and VENTX allows the generation of a murine AE model, which in contrast to all other published mouse models mimics aberrant expression of CD19 in human AE positive AML, a hallmark of this AML genotype. DisclosuresBuske:CELLTRION, Inc.: Consultancy, Honoraria.

Tet2-Deficient Bone Marrow Progenitors Have a Proliferative Advantage in the Presence of TNF-Alpha and IFN-Gamma: Implications for Clonal Dominance in Inflammaging and MDS

Background:TET2 is a hematopoietic tumor suppressor gene that has been implicated in DNA demethylation and the epigenetic regulation of gene expression. Inactivating TET2 mutations are common in aging-associated clonal hematopoiesis of indeterminate potential (CHIP) and myelodysplastic syndromes (MDS). TET2 mutations may contribute to early clonal dominance and myeloid transformation, although the exact mechanisms remain to be elucidated. Emerging evidence suggests that the MDS bone marrow niche may be abnormal and this abnormal niche may act as fertile ground for expansion of neoplastic cells in vivo. Common to the environment of MDS and “inflammaging” are elevations in cytokines, such as TNFa and IFNg. We hypothesized that TET2 mutant clones may thrive in an inflammatory environment and further condition this environment to promote their own survival.Methods: Adult (10-14 weeks-old) Tet2 wild type and Tet2 mutant C57BL/6 mice strains (JAX) were chosen as a model system. The floxed Tet2 allele was deleted by targeting exon 3 with Vav1-cre mediated, hematopoietic-specific excision. We isolated lineage negative cells (Lin-), enriched for hematopoietic stem and progenitor cells (HSPC), from Tet2 wild type and -/- and bone marrow (BM) (EasySep; StemCell Technologies) and cultured these in the absence or presence of TNFα (0.1, 1, or 10 ng/ml) and IFN-γ (0.01, 0.1 or 1 ng/ml) in a methylcellulose colony formation assay (MethoCult; StemCell) or liquid culture media, and then examined their colony growth, cell count and phenotypic characterization over a period of 12 days. Where indicated, serial re-plating was performed.Results: We found an increased proportion of Lin- cells in Tet2 -/- BM compared to wild type, suggesting in vivo HSPC expansion. In triplicate experiments starting with equal numbers of wild type and Tet2 -/- Lin- cells (104 cells/MethoCult well), we found no significant difference in colony counts on days 3, 6, 9, or 12, when cultured in the absence or presence of increasing TNFα concentrations. As expected, TNFα dose-dependently reduced colony counts in both genotypes (up to 3 to 4-fold at 10 ng/ml). However, Tet2 -/- Lin- cells displayed a proliferative advantage over wild type in serial re-plating assays. In the presence of TNFα, this Tet2 -/- re-plating advantage was striking. As exemplified by day 12 colony counts at first re-plating (Fig. 1), while wild type colonies declined with increasing TNFα, Tet2 -/- colony counts increased with TNFα concentration (i.e. average 20-fold higher than wild type at 10 ng/ml TNFα; p<0.05).We next shifted our analysis to IFNg, and found significantly increased day 6, 9 and 12 Tet2 -/- methylcellulose colonies at first plating. Upon re-plating in IFNg, Tet2 -/- cells demonstrated significantly increased (1.5 to 2-fold; p<0.05) mean day 12 colony counts at 0.01, 0.1 and 1 ng/ml IFNg. To gain some insight into the nature of the cells emerging under IFNg stress, we performed flow cytometry upon re-plating. Preliminary experiments revealed increases (1.5 to 2-fold) in Mac1+Gr1+ and Sca1+Kit1+ populations in Tet2 -/-, as compared to wild type, in the presence of IFNg.We are currently comparing apoptosis in wild type and Tet2 -/- cells in the MethoCult system +/- TNFα and IFNg (and a more amenable liquid culture system), using Annexin V/Propidium Iodide-based flow cytometry. These results will be reported. Future directions include the characterization of differential: a) gene expression signatures in Tet2 wild type and -/- Lin- cells under TNFα and IFNg stress, and b) TNFα and IFNg signatures in TET2 -mutant and non-mutant human MDS.Conclusion:Tet2 -deficient murine bone marrow progenitors demonstrate a proliferative advantage, as compared to their wild type counterparts, under TNFα and IFNg stress. Given that these inflammatory cytokines have been associated with inflammaging and myelodysplasia, it is worth exploring whether TET2 -mutant human clones may emerge under inflammatory stress, leading to CHIP and/or MDS, and presenting a novel therapeutic target for clone eradication. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity.

Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype.