Competitor Cells Research Articles

Assembly of a unique membrane complex in type VI secretion systems of Bacteroidota

The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis (tssNQOPR) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.

Cellular Barcoding of JAK2-V617F Hematopoietic Stem Cells Reveals No Substantial Preferences in the Contribution of Individual Stem Cell Clones to Erythroid Versus Megakaryocytic Lineages

JAK2-V617F is the most frequent driver gene mutation in patients with myeloproliferative neoplasms (MPN) but the phenotypic manifestation is heterogeneous with some patients presenting as polycythemia vera (PV), whereas others with essential thrombocythemia (ET) or myelofibrosis (MF). JAK2-V617F is acquired in a hematopoietic stem cell (HSC) and leads to clonal expansion of HSCs and progenitor cells that gain dominance over unmutated hematopoiesis. We tested the hypothesis that the preferential expansion of megakaryopoiesis in ET versus erythropoiesis in PV could be due to the acquisition of JAK2-V617F in different subsets of HSCs with inherent bias towards megakaryopoiesis or erythropoiesis. In addition, we addressed the question of whether sensitivity or resistance to interferon-α (IFNα), currently the only treatment that can induce a deep molecular remission in some MPN patients, could be also due to heterogeneity in HSC subpopulations. We used a mouse model of JAK2-V617F driven MPN in combination with molecular barcoding that allowed us to monitor expansion and lineage contribution of individual HSC subclones. First, we genetically barcoded HSCs from our tamoxifen-inducible SclCre;JAK2-V617F ( VF) mice with a lentiviral vector, and transplanted 2'000 HSCs into lethally irradiated recipient mice. After 12 weeks we sequenced the barcodes in bone marrow (BM) progenitors by next generation sequencing (NGS). We observed a strong selection of 1-2 HSCs clones per recipient mouse, which alone contributed to > 80% of all the myeloid cells. Since cytokine storm after irradiation and aplasia during the reconstitution with lentivirally transduced BM may favor such oligo-clonal dominance observed in this experiment, we characterized the clonal composition of JAK2-mutant HSCs in non-transplanted MPN mice. To this end, we crossed a CRISPR-based genetic barcoding mouse line ( CARLIN) (Bowling, S. et al, Cell 2020) with our VF mice to obtain VF;CARLIN mice. Cas9-mediated barcoding in VF;CARLIN or in CARLIN mice with wildtype Jak2(WT;CARLIN) was induced with doxycycline and immediately followed by the activation of the JAK2-V617F by tamoxifen (Figure 1A). After 12 weeks, when the mice developed full PV phenotype (hemoglobin 195 g/L; platelets 4.3x10 12 /L and neutrophils 10.5x10 9/L), we performed scRNAseq on lin- cKit+ BM cells using the 10X-platform to retrieve the cellular barcodes and transcriptomic profiles. MPN hematopoiesis in these non-transplanted VF;CARLIN mice was polyclonal (Figure 1B). A total of > 500 barcoded clones per mouse were detected and in both VF;CARLIN and WT;CARLIN mice, but ~5 individual barcodes accounted for 25% of the cells in all progenitor subsets analyzed (Figure 1B). Overall, clonal composition was similar between WT;CARLIN and VF;CARLIN mice, indicating that clonal selection was not more prominent in JAK2-mutant mice. Importantly, in the offspring of HSCs that accounted for 25% of the cells, we did not observe preferential presence of some barcodes in MkP versus EryP and other barcodes in EryP versus MkP, arguing against the model predicting that erythrocytosis and thrombocytosis originated in different subsets of biased HSCs. To investigate the question of whether sensitivity or resistance to IFNα could be also due to heterogeneity in HSC subpopulations, we transplanted BM cells from these barcoded VF;CARLIN mice together with an excess of WT competitor cells into irradiated WT recipient mice, and treated them with IFNα or vehicle for 16 weeks. IFNα normalized hemoglobin (122 g/L) compared to vehicle (177 g/L) and reduced VF;CARLIN chimerism in granulocytes from 85% to 44%. BM cells were harvested and the clonal composition is currently being analyzed by NGS in order to identify IFNα resistant and sensitive subclones. Our barcoding analyses of individual HSCs showed that, during recovery after BM transplantations, a few VF clones dominated, whereas induction of VF expression in situ without transplantation resulted in a polyclonal disease that showed a similar number of HSCs actively contributing to hematopoiesis as in a control experiment with WT mice. The lack of substantial preference in the clonal contribution of individual HSCs to erythropoiesis versus megakaryopoiesis in VF mice suggests that PV and ET are not caused by acquiring JAK2-V617F in HSCs with a pre-existing lineage bias.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Maternal Obesity Causes Sex Specific Long-Term Hematopoietic and Metabolic Defects

Maternal obesity is increasingly common and can negatively impact offspring health. Children born to obese mothers are at higher risk of developing diseases involved with metabolic health (e.g., type 2 diabetes). Many of these diseases are associated with abnormalities within the hematopoietic system such as atypical immune profiles or hematopoiesis. Interestingly, disease incidence and/or severity is often dependent on the offspring's gender. This suggests that there may be sex-specific reprogramming of hematopoietic stem and progenitor cells (HSPCs) during immune cell development. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health. To test this, C57BL/6 (CD45.2+) dams were fed standard chow (2018SX, Envigo, IN) or western-style diet (TD.88137, Envigo, IN) for four weeks prior to mating, through pregnancy, and the lactating period. At postnatal day 21, HSPCs (Lineage negative, Sca1 positive, cKit positive cells) were flow-sorted from bone marrow (BM) of offspring from dams fed chow (Con) or western-style diet (MatOb) and used for competitive transplant (Boy/J BM competitor cells; CD45.1+) into same-sex lethally irradiated C57BL/6 x Boy/J F1 (CD45.1+CD45.2+) recipient mice (8-10 weeks of age) or for RNA-sequencing analysis. Secondary transplants were performed to look at long-term engraftment potential of HSPCs. Body weight, body adiposity, fasting insulin, glucose tolerance test (GTT), and pancreatic β and α-cell area were assessed on male and female recipients of both primary and secondary transplants. Compared to mice that received male Con HSPCs, recipient mice that received male MatOb HSPCs had significantly lower donor chimerism in the blood and BM post-secondary transplantation. This was associated with a significant reduction in donor hematopoietic stem cell (HSC) numbers in the BM 16 weeks post-secondary transplant, indicating male MatOb HSPCs had decreased long-term engraftment capability due to an exhaustion of the HSC pool. This reduction in HSPC engraftment seen between male MatOb versus Con HSPCs was not seen when using HSPCs from female offspring. Next, metabolic parameters of our recipient mice was examined. Sixteen weeks after primary transplant, there were no differences detected in body weight and adiposity in the Con and MatOb HSPC recipient mice. Compared to sex-matched controls, we found that recipients transplanted with male MatOb HSPCs developed glucose intolerance, increased fasting serum insulin concentrations, and decreased β-cell area, supporting the presence of impaired glucose metabolism due to a combination of insulin resistance and lower β-cell mass. There was no difference in recipients of HSPCs from female Con and female MatOb pups. RNA-seq of BM HSPCs at time of transplant (p21) from Con or MatOb revealed 100 differentially expressed genes (DEG) in male MatOb HSPCs and 1788 DEG in female MatOb HSPCs. There were more and higher magnitude of differential gene expression in female mice than male, despite the long-term phenotypic differences being evident in males but not females, and the majority of genes changed in males were also altered in females. This suggests that the gene programs responsible for the observed phenotypes may be altered by maternal obesity in both male and female offspring, but that female offspring have a transcriptomic profile that confers protection on the HSPC population. One of the most striking findings from the transcriptomic analysis was a marked enrichment in gene programs associated with immune response, interferon signaling, and cytokine production in female MatOb offspring compared to sex matched controls that is not seen in male MatOb offspring. Pathway analysis identified upregulation of programs involving both IFNγ and IL-10 in the female MatOb HSPCs when compared to sex-mated Con HSPCs which correlated with an increase in IL-10 concentrations in the serum of p21 female MatOb mice. These pathways were downregulated or unaltered in males with no change in systemic IL-10 concentration.Our results demonstrate that maternal obesity results in sex-specific changes in offspring HSPC function and a novel role HSPCs may play in regulating glucose metabolism in the offspring of obese dams.

Lack of evidence that Pseudomonas aeruginosa AmpDh3-PA0808 constitute a type VI secretion system effector-immunity pair.

Type VI secretion systems (T6SSs) are cell envelope-spanning protein complexes that Gram-negative bacteria use to inject a diverse arsenal of antibacterial toxins into competitor cells. Recently, Wang et al. reported that the H2-T6SS of Pseudomonas aeruginosa delivers the peptidoglycan recycling amidase, AmpDh3, into the periplasm of recipient cells where it is proposed to act as a peptidoglycan degrading toxin. They further reported that PA0808, the open reading frame downstream of AmpDh3, encodes an immunity protein that localizes to the periplasm where it binds to and inactivates intercellularly delivered AmpDh3, thus protecting against its toxic activity. Given that AmpDh3 has an established role in cell wall homeostasis and that no precedent exists for cytosolic enzymes moonlighting as T6SS effectors, we attempted to replicate these findings. We found that cells lacking PA0808 are not susceptible to bacterial killing by AmpDh3 and that PA0808 and AmpDh3 do not physically interact in vitro or in vivo. Additionally, we found no evidence that AmpDh3 is exported from cells, including by strains with a constitutively active H2-T6SS. Finally, subcellular fractionation experiments and a 1.97 Å crystal structure reveal that PA0808 does not contain a canonical signal peptide or localize to the correct cellular compartment to confer protection against a cell wall targeting toxin. Taken together, these results cast doubt on the assertion that AmpDh3-PA0808 constitutes an H2-T6SS effector-immunity pair.

Hybrids of thiazolidinone with 1,2,3-triazole derivatives: design, synthesis, biological evaluation, in silico studies, molecular docking, molecular dynamics simulations, and ADMET profiling

A new series of thiazolidinone linked 1,2,3-triazole hybrids 5a-h was designed and synthesized using the copper-catalyzed Huisgen azide-alkyne cycloaddition (CuAAC) between thiazolidinone linked alkyne and aromatic azides. The structures of the newly synthesized compounds were established by NMR (1H and 13C) and HRMS. The targeted thiazolidinone-1,2,3-triazole hybrids were evaluated for their cytotoxic activity against four human cancer cell lines, including fibrosarcoma (HT-1080), lung carcinoma (A-549), and breast carcinoma (MCF-7 and MDA-MB-231) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide (MTT). The obtained data showed that most of these compounds have moderate anti-proliferative activity with IC50 values between 10.26 ± 0.71 and 53.93 ± 1.20 μM. The compound 5a exhibited higher activity with an IC50 value of 10.26 ± 0.71 µM, compared to 5d with an IC50 value of 11.56 ± 1.98 µM for the HT-1080 and MCF-7 cancer cells line, respectively. Moreover, Annexin-V apoptosis was assessed by flow cytometry for hybrid compounds 5a and 5d against HT-1080 and MCF-7 competitor cell lines, as they increase the level of active caspase 3/7. The experimental results were further confirmed by docking studies followed by molecular dynamic simulations. Both the potent derivatives i.e. 5a and 5d have comparable docking scores and MD simulations results showed that the docked complex of 5a is somewhat more stable than 5d primarily for protein p53. The ADMET profile of both derivatives established their safety zone and drug-like potential. Communicated by Ramaswamy H. Sarma

Calcium Promotes T6SS-Mediated Killing and Aggregation between Competing Symbionts.

Bacteria use a variety of strategies to exclude competitors from accessing resources, including space within a host niche. Because these mechanisms are typically costly to deploy, they are often tightly regulated for use in environments where the benefits outweigh the energetic cost. The type VI secretion system (T6SS) is a competitive mechanism that allows inhibitors to kill competing microbes by physically puncturing and translocating cytotoxic effectors directly into neighboring competitor cells. Although T6SSs are encoded in both symbiotic and free-living taxa where they may be actively secreting into the extracellular milieu during growth in liquid culture, there is little evidence for bacteria engaging in T6SS-mediated, contact-dependent killing under low-viscosity liquid conditions. Here, we determined that calcium acts as a pH-dependent cue to activate the assembly of an antibacterial T6SS in a Vibrio fischeri light organ symbiont in a low-viscosity liquid medium. Moreover, competing V. fischeri isolates formed mixed-strain aggregates that promoted the contact necessary for T6SS-dependent elimination of a target population. Our findings expand our knowledge of V. fischeri T6SS ecology and identify a low-viscosity liquid condition where cells engage in contact-dependent killing. IMPORTANCE Microbes deploy competitive mechanisms to gain access to resources such as nutrients or space within an ecological niche. Identifying when and where these strategies are employed can be challenging given the complexity and variability of most natural systems; therefore, studies evaluating specific cues that conditionally regulate interbacterial competition can inform the ecological context for such competition. In this work, we identified a pH-dependent chemical cue in seawater, calcium, which promotes activation of a contact-dependent interbacterial weapon in the marine symbiont Vibrio fischeri. This finding underscores the importance of using ecologically relevant salts in growth media and the ability of bacterial cells to sense and integrate multiple environmental cues to assess the need for a weapon. Identification of these cues provides insight into the types of environments where employing a weapon is advantageous to the survival and propagation of a bacterial population.

The Long Non-Coding RNA MALAT1 Is Necessary for Hematopoietic Stem and Progenitor Cell Expansion and Preleukemic Myeloproliferation in TET2 Deficient Myeloid Neoplasms

Ten Eleven Translocation 2 (TET2) is a dioxygenase which regulates gene expression by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine. Loss of function mutations in TET2 occur in up to 58% and 30% of myeloid malignancies and clonal hematopoiesis respectively. Loss of TET2 function also occurs with isocitrate dehydrogenase 2 (IDH2) mutations in Acute Myeloid Leukemia (AML) via 2-hydroxyglutarate (2-HG). Genetically engineered murine models (GEMM) of Tet2 deficiency produce a Chronic Myelomonocytic Leukemia (CMML)-like phenotype with splenomegaly, granulomonocytic expansion and increased bone marrow hematopoietic stem and progenitors (LSK). Further, bone marrow mononuclear cell (BMNC) transplant models of Tet2-/- clonal hematopoiesis suggest that this myeloid expansion leads to enhanced inflammation and increased cardiovascular disease risk. However, the downstream effects of TET2 loss of function leading to this phenotype are unknown. Here we show that TET2-deficient granulomonocytic expansion and inflammation are mediated via MALAT1, a long non-coding RNA overexpressed in several malignancies. MALAT1 expression was increased in BMNCs from IDH2 mutated AML patients (n=152 WT n=17 mutant IDH2, p=0.04). Increased MALAT1 expression was also seen in human and murine models of TET2 deficiency including isogenic IDH2 mutated TF-1 cells (5 biological replicates p=0.005), 2-HG treated leukemia cell lines (2 biological replicates p<0.0001) and BMNCs from Tet2f/f mice (n= 7/grp p=0.005). To evaluate the impact of Malat1 on normal and Tet2 deficient hematopoiesis we utilized the Malat1tg overexpression and Tet2-/-Malat1-/-GEMM models. Similar to Tet2-/- mice, bone marrow (n=4/grp p=0.02) and spleen (n=8/grp p=0.004) LSK numbers were increased in Malat1tg mice compared to control. However, unlike Tet2-/-LSKs, MALAT1tgLSKs did not show increased self-renewal in serial in vivo competitive transplants. Further, depleting Malat1 in Tet2-/- mice did not rescue the increased self-renewal capacity seen in Tet2-/- LSKs. Collectively, these data suggest that Malat1 overexpression is sufficient for LSK expansion but does not impact LSK function. Splenomegaly, myeloproliferation and extramedullary hematopoiesis is a phenotype termed Preleukemic Myeloproliferation (PMP) and is predictive of myeloid neoplasm development in Tet2-/- models (Meisel M et al, Nature 2018). Consistent with a role of Malat1 in PMP (Fig A), Malat1 overexpression recapitulated PMP at 8 (n=10/grp p<0.0001) and 20 (n=10/grp p=0.0005) weeks. Malat1 overexpression was also associated with an increase in classical monocytes in the peripheral blood- a hallmark of CMML (8 weeks n= 7/grp p<0.0001, 20 weeks n=10/grp p=0.0007). This was recapitulated in Tet2-/-mice (n=3/grp p=0.001) and CMML patients with TET2 mutations (n=27 mutant TET2 v 109 WT, odds ratio 5.6 p= 0.001). Finally, depleting Malat1 in a Tet2-deficient GEMM rescued PMP (n=4 Tet2-/-Malat1-/-v 5 Tet2-/-p=0.01) and classical monocytosis (n=2/grp p=0.002) suggesting that Malat1 overexpression is both sufficient and necessary for PMP in Tet2 deficient leukemia. This impact was also observed in recipients in BMNC competitive transplant experiments (Fig B). Initial insights into the molecular mechanism underlying the role of Malat1 in PMP were derived from analyzing competitor and host cells during in vivo competitive transplants. Interestingly, transplant recipients with Malat1tg LSK demonstrated myeloid and granulomonocytic expansion in both donor and non-donor cells suggesting that the effects of Malat1 overexpression are, in part, cell extrinsic. Consistent with this hypothesis and the impact of NF-kB on inflammatory cytokine secretion and Tet2-/- PMP, we show that MALAT1 deletion in human leukemia attenuates NF-kB activity by enhancing the phosphatase activity of PP2A. Further, Interleukin 6 (IL-6), a major downstream target of NF-kB and potent activator of myelopoiesis in response to inflammatory stimuli, was increased in the plasma of MALAT1tg mice (n=8/grp p=0.01) suggesting a possible cell-extrinsic mechanism for Malat1 dependent PMP. Ongoing studies are focused on demonstrating that IL-6 is necessary for Malat1 dependent PMP. Our current data provides hitherto unknown insights establishing MALAT1 as a major effector of TET2 loss of function which may lead to novel therapeutic options in TET2 deficient myeloid neoplasms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of DHX15 Function Impairs Hematopoiesis in Vav-Dhx15-KO Mice and Cause DNA Damage-Induced Cell Cycle Arrest and Apoptosis in Leukemia Cell Lines

DHX15, a member of the DEAD-box RNA helicase family, acts as splicing factor in pre-messenger RNA splicing, pre-ribosomal RNA, and RNA metabolism. Our previous studies showed that DHX15 overexpression is associated with poor prognosis in acute myeloid leukemia (AML), and DHX15 regulates cell apoptosis via the NF-kB signaling pathway(Pan et al., 2017). However, its molecular function remains obscure in the blood system. First, with the Cre-loxp knockout system, Dhx15fl/fl male mice were crossed with vav-icre female mice (specifically expressed in blood system), we found that Dhx15fl/fl; vav-icre homozygous knockout mice (vav-Dhx15-KO) are lethal by E17.5-E18.5. Then, to explore the inner difference in blood system, the hematopoietic stem cells (HSC) were analyzed by flow cytometry in fetal liver (FL) (E13.5, E15.5). Compared with Dhx15fl/fl (wild-type, WT) mice, the lineage-negative cells (Lin- cells; Lin+ Cocktail, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4 and Ter-119) significantly increased in vav-Dhx15-KO FL cells (P<0.0001) and the short-term HSC(ST-HSC) proportion compensatively increased in LSK (Lin-c-Kit+Sca1+cells) population (P<0.01), while the proportion of LK(Lin-c-Kit+Sca1-cells) (P<0.0001 in E13.5, P<0.001 in E15.5), LSK(Lin-c-Kit+Sca1+cells)(P<0.1 in E15.5), and multipotent progenitor (MPP) (P<0.0001 in E15.5) all witnessed decrease in the Lin-/LSK population respectively. Meanwhile, vav-Dhx15-KO mice exhibited impaired multi-lineage hematopoiesis especially in erythroid, where the precursors and immature erythrocytes increased, mature erythrocytes decreased (P<0.001 in E13.5). Next, in vivo competitive repopulation assay were performed to further examine any potential cell-intrinsic defect of HSC function caused by Dhx15-inactivation. Thus, 1,000,000 E13.5/E15.5 total FL donor cells (CD45.2, vav-Dhx15-KO or WT) together with 1,000,000 CD45.1 unfractionated BM competitor cells were intra-venous transplanted into lethally irradiated (9Gy) B6.SJL (CD45.1) recipient mice. Recipient mice were sacrificed 8-weeks after transplantation, flow cytometry was conducted to analysis the ratio of CD45.2 in mice, the results showed that vav-Dhx15-KO FL was severely deficient compared to WT FL(0.1±0.04%vs 62.2±2.8% in BM, 0.2±0.05% vs 64.5±6.1% in SP, 0.1±0.05% vs 45.1±5.5% in PB). These results indicate that Dhx15 is crucial in mice blood system, loss of its function may block cell differentiation, leading to the reduction of mature cells. At the same time, to further explore the molecular mechanism of DHX15 in blood system, MOLM-13 and NB4 leukemia cell lines were selected for shRNA-KD (knockdown) experiments due to the lethality of vav-Dhx15-KO mice. We found that Dhx15-KD significantly impaired the proliferation ability of these cell lines in vitro and in vivo, by experiments of survival competition, clone formation, subcutaneous and intra-venous tumorigenesis. Besides, our data demonstrate DHX15-KD-mediated G0/G1 cell cycle arrest and increase in early-late apoptotic cells by Hoechest 33342-EdU cell cycle and DAPI-Annexin V cell apoptosis assays. Mechanistically, we found that UV_RESPONSE signaling pathway which is mainly related to DNA damage was up-regulated by RNAseq and bioinformatics analysis (DHX15-KD cell line vs WT cell line). Correspondingly, the DNA damage indicators γH2AX and 53BP1 were also increased in DHX15-KD cell lines detected by WB and immunofluorescence experiments, suggesting that the deletion of DHX15 could lead to DNA damage-mediated cell cycle arrest and apoptosis in leukemia cell lines. In summary, these results show that DHX15 deficiency leads to DNA damage-induced cell cycle arrest and apoptosis which further impairs the proliferative capacity of leukemia cell lines and lost function of Dhx15 impairs multi-lineage hematopoiesis in mouse embryos, which may lead to its perinatal death.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time pointspost-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6×BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

S191: CALRETICULIN-MUTATED HEMATOPOIETIC CELLS ARE VULNERABLE TO THE COMBINED INHIBITION OF THE PROTEASOME AND THE IRE1A-XBP1 AXIS OF THE UNFOLDED PROTEIN RESPONSE

Background: Mutations in the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). These mutant CALR proteins have impaired chaperone function. Concordant with this, transcriptional upregulation of the unfolded protein response (UPR) has been reported in patients with CALR-mutated MPN. However, it is not understood how CALR-mutated cells counter-balance ER stress resulting from impaired chaperone function. Despite the frequency of CALR mutations in MPN, there are currently no treatment strategies to preferentially target CALR-mutant cells over healthy cells. Aims: To determine the mechanisms by which mutant CALR cells alleviate ER stress and to exploit these mechanisms selectively by pharmacological intervention in vivo. Methods: Long-term HSCs (LT-HSCs) from CalrΔ52 knockin mice were isolated for RNA-Seq. CalrΔ52 bone marrow (BM) was differentiated ex vivo, subsequently enriched for megakaryocytes, and subjected to quantitative proteomic analysis. Pathway analyses of CALR-mutant megakaryocyte progenitors (MkPs) were performed on Genotyping of Transcriptome (GoT) data from patients with Essential Thrombocytosis (ET). CalrΔ52 VAVCre mice and chimeric transplanted CalrΔ52 mice were treated with a proteasome inhibitor, an IRE1a inhibitor, or both. Results: To identify dysregulated pathways in CalrΔ52/Δ52 LT-HSCs, we performed RNA-seq and found that Xbp1s downstream of IRE1a and the proteasome pathway was significantly upregulated in CalrΔ52/Δ52 animals. We confirmed elevated XBP1s protein level in heterozygous CalrΔ52 knockin mice by flow cytometry. Since elevated and abnormal megakaryopoiesis are hallmarks of CALR-mutant MPN, we performed quantitative proteomics on megakaryocyte-enriched cells from CalrΔ52 mice. We confirmed upregulation of both proteasome and ER chaperone proteins in Calr-mutant cells. To verify these findings in human MPN, we interrogated a published GoT data set and found that in addition to XBP1s, the proteasome pathway was also differentially upregulated in CALR-mutated patient MkPs compared to the wildtype MkPs (p<2e-16). To investigate the functional consequences of IRE1a or proteasome inhibition, we treated CALR-mutant hematopoietic cell lines with KIRA6 (IRE1a inhibitor) and/or bortezomib (proteasome inhibitor) and found CALR-mutant cells to be differentially sensitive as compared to isogenic controls. We subsequently found that treatment with bortezomib leads to the accumulation of misfolded and ubiquitinylated proteins in mutant Calr BM, resulting in pro-apoptotic priming. In vivo treatment of CalrΔ52/+ mice with bortezomib resulted in a significant reduction in platelets. Finally, we combined IRE1a and proteasome inhibition in a chimeric BM transplantation in vivo model using CD45.2 CalrΔ52/+ MxCre GFP and CD45.1 wild-type competitor cells. Following combination therapy, we found a significant reduction in Calr-mutant donor chimerism, specifically in LT-HSCs and platelets, accompanied by reductions in LT-HSC frequency and platelet count. Summary/Conclusion: In summary, we have found that disrupted proteasis in CALR-mutated MPN cells results in a dependency on the IRE1-XPB1 axis of the UPR and proteasome activity. Using pre-clinical MPN models, we found that combined inhibition of both pathways in vivo normalizes important features of MPN and preferentially targets CalrΔ52 over wild-type cells. These findings highlight combined inhibition of IRE1 and the proteasome as a promising therapeutic strategy in CALR-mutant MPN.

A Putative Lipoprotein Mediates Cell-Cell Contact for Type VI Secretion System-Dependent Killing of Specific Competitors.

ABSTRACTInterbacterial competition is prevalent in host-associated microbiota, where it can shape community structure and function, impacting host health in both positive and negative ways. However, the factors that permit bacteria to discriminate among their various neighbors for targeted elimination of competitors remain elusive. We identified a putative lipoprotein (TasL) in Vibrio species that mediates cell-cell attachment with a subset of target strains, allowing inhibitors to target specific competitors for elimination. Here, we describe this putative lipoprotein, which is associated with the broadly distributed type VI secretion system (T6SS), by studying symbiotic Vibrio fischeri, which uses the T6SS to compete for colonization sites in their squid host. We demonstrate that TasL allows V. fischeri cells to restrict T6SS-dependent killing to certain genotypes by selectively integrating competitor cells into aggregates while excluding other cell types. TasL is also required for T6SS-dependent competition within juvenile squid, indicating that the adhesion factor is active in the host. Because TasL homologs are found in other host-associated bacterial species, this newly described cell-cell attachment mechanism has the potential to impact microbiome structure within diverse hosts.

CaMKK2 Knockout Bone Marrow Cells Collected/Processed in Low Oxygen (Physioxia) Suggests CaMKK2 as a Hematopoietic Stem to Progenitor Differentiation Fate Determinant.

Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2-/- and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2-/- cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk-/- mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.

Interleukin-1/Toll-like Receptor Inhibition Can Restore the Disrupted Bone Marrow Microenvironment in Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice.Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors.Results: Transplant of NHD13 BM cells into aged IL1R1 wt recipients (NHD13→IL1R1 wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ko recipients (NHD13→IL1R1 ko). IL1R1 wt developed macrocytic anemia compared to IL1R1 ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p<0.05). In CFU-C assays, NHD13→IL1R1 wt and NHD13→IL1R1 ko demonstrated similar levels of CFU-activity whereas CFU-fibroblast (CFU-F) assays of IL1R1 wt recipients demonstrated lower numbers of large colonies (reported to contain highly proliferative mesenchymal stem cells (MSC)). Flow cytometry analysis of hematopoietic stem and progenitor cells (HSPC) population in IL1R1 wt recipients showed increased myeloid progenitors and decreased long-term HSC (LT-HSC) compared to IL1R1 ko recipients. Cytokine/chemokine profiles revealed that the inflammatory cytokines (IL-6 and TNFα) and macrophage activating cytokines (MCP-1 and M-CSF) were significantly decreased in NHD13→IL1R1 ko compared to NHD13→IL1R1 wt. Next, we treated MDS model mice with inhibitors. The pharmacological targeting of IL1R1 signaling in vivo was associated with decreased NHD13 cell burden and improvement of macrocytic anemia. CFU-C assays demonstrated some decreases in NHD13 CFU capacity post-treatment. Interestingly, non-NHD13 HSPCs assessed by CFU capacity increased in IRAK4 inhibitor treated mice. Consistent with this, flow cytometric analyses of HSPC pools demonstrated decreased NHD13 HSPCs (LT-HSC and granulocyte-monocyte progenitor cells) and increased non-NHD13 HSCs and myeloid progenitors compared to vehicle group. BMME cell populations showed that arteriolar endothelial cells and MSCs were also affected by drug treatment and both IL1R antagonist and MCC950 increased the number of large CFU-F colonies. Analysis of BMEF revealed the decreased IFNγ and IL-18 and upregulated M-CSF in MCC950 treated group. The cytokines of human BM serum revealed higher concentrations of soluble formed IL1R1, IL-18, CXCL1, and osteopontin in MDS compared to young or aged normal donors.Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME. DisclosuresNo relevant conflicts of interest to declare.

Reverting to Quiescence Preserves Hematopoietic Stem Cells Following Genome Editing

Hematopoietic stem cells (HSCs) in steady-state are quiescent in cell cycle. CRISPR-Cas9 genome editing has revolutionized the HSC research and therapeutic application of HSCs for hematological diseases. Although these methods and clinical results are promising, keeping HSC function after highly efficient genome editing is still challenging because HSCs gradually lose their repopulation capacity following cell cycle activation. Preserving the function of HSCs after genome editing is an urgent issue. In this study, we interrogated the culture method following genome editing to reverse the cell cycle status of HSCs into a quiescent state to test how cell cycle status affects the genome editing efficiency and the HSC potential.To assess the relationship between genome editing efficiency and cell cycle status, we compared genome editing efficiency among the freshly-isolated or cultured HSCs and progenitors (HSPCs) at various time points. Ribonucleoprotein (RNP) complex was delivered into cells by electroporation. Genome-editing efficiency was evaluated by CD45 knockout rate comparing sgRNA for Rosa and CD45. All the HSPC fractions cultured displayed higher genome-editing efficiency following over-night preculture. Genome-editing efficiency of fresh HSC was lower than 20% while fresh granulocyte/monocyte progenitor (GMP) showed higher than 40%. Cell cycle analysis using EdU or Ki67 and Hoechst 33342 staining confirmed that genome-editing efficiency positively correlated with cell cycle activation. We further tested the effect of pre- and post-culture conditions for genome editing. While preculture with high cytokine concentration for a long period of time (> 16 hours) is required for the efficient genome editing, post-culture condition did not compromise the genome-editing efficiency.Given the quiescent nature of HSCs, we hypothesized that reverting activated HSCs back to quiescent state may improve the function of HSCs following genome editing. To test this, genome-edited HSCs were cultured in the quiescence-maintaining condition (SCF 1.5 ng/mL and TPO 1.0 ng/mL) under 1% O 2 atmosphere. After 7-day culture, more than 30% of cells kept the surface marker phenotype of CD150 + CD48 - LSK, and over 60% of cells were successfully underwent genome editing. Less than 10% of HSCs were EdU +, suggesting that HSCs reverted to cell cycle quiescence after genome editing. By contrast, HSCs cultured in a conventional high cytokine condition (proliferative condition) lost the surface marker phenotypes and highly incorporated EdU. To assess the long-term reconstitution potential of edited HSCs, the Evi1 expression level was evaluated using Evi1-IRES-GFP mice. The expression level of Evi1 was significantly higher in quiescent HSCs than proliferating HSCs after editing. These results suggest that, as post-electroporation culture, quiescence-maintaining condition reverts precultured HSCs back to a quiescent state in cell cycle. This protocol maintains phenotypic HSCs without compromising the genome-editing efficiency.To further determine the function of genome-edited HSCs, single cell colony assay was performed. Clonally sorted CD45 knockout HSCs cultured in the quiescence-maintaining condition after gene editing fully maintained colony-forming capacity, but HSCs cultured in the proliferating condition lost their capacity. We then performed transplantation assays using Ubc-GFP mice. GFP + HSCs were genome-edited for Rosa and transplanted into lethally-irradiated recipient mice with competitor cells. The donor-derived chimerism of edited HSCs with quiescence-maintaining condition in peripheral blood and bone marrow was generally superior to that of edited HSCs with proliferative condition. These data demonstrates that edited HSCs cultured in quiescence-maintaining condition maintain stem cell potential in vitro and in vivo.Altogether, we established an HSC-optimized, highly efficient genome-editing protocol. This study demonstrated that effectiveness of keeping HSC in a quiescent state even in the setting of genome editing. Our protocol is suitable for unveiling the function of genes distinguishing cycling and quiescent HSCs. DisclosuresKataoka: Celgene: Honoraria; Eisai: Honoraria; Astellas Pharma: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; MSD: Honoraria; Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Asahi Genomics: Current equity holder in publicly-traded company; Otsuka Pharmaceutical: Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding; Eisai: Other: Scholarship; Otsuka Pharmaceutical: Other: Scholarship; Ono Pharmaceutical: Other: Scholarship; Kyowa Kirin: Other: Scholarship; Shionogi: Other: Scholarship; Takeda Pharmaceutical: Other: Scholarship; Summitomo Dainippon Pharma: Other: Scholarship; Chugai Pharmaceutical: Other: Scholarship; Teijn Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Genetic Alterations: Patents & Royalties: PD-L1 abnormalties .

SRSF2-P95Hdelays Myelofibrosis Development through Altered JAK/STAT Signaling in JAK2-V617F Megakaryocytes

Background: The gain-of-function JAK2 V617F mutant is the most common driver mutation identified in myeloproliferative neoplasms (MPNs). Additional somatic variants, also found in other malignant hemopathies, are detected in primary myelofibrosis (MF) and supposed to contribute to fibrosis or leukemia development. One of these mutations affects SRSF2, a gene encoding a component of the splicing machinery. SRSF2 heterozygous mutation mainly affects the proline 95 residue of the protein. Its association with JAK2 V617F correlates with a reduced leukemia free survival. Whether and how SRSF2 P95 variants could favor fibrosis development in JAK2 V617F cells remained unknown.Methods & Results: To investigate how homozygous Jak2 V617F and heterozygous Srsf2 P95H could interact in the hematopoietic tissue, we generated conditional knock-in mice in which the CreERt recombinase expression was driven by the HSC-Scl promoter leading to Jak2 V617F and/or Srsf2 P95H hematopoietic-specific expression upon tamoxifen induction.Srsf2 P95H mutation initially exhibited limited effect on Jak2 V617F-induced polycythemia vera (PV) only slightly reducing erythrocytosis and leukocytosis (through a previously described decrease in B220 + B cell number). The expansion of hematopoietic stem cells (SLAM), multipotent progenitors (MPP) and megakaryocyte progenitors (MKP) observed in Jak2 V617F mice was not affected by Srsf2 status. However, while platelet count was decreasing in Jak2 V617F alone mice at later time point due to fibrosis development, Srsf2 P95H/Jak2 V617F combination further increased platelet counts correlating with a significant delay in the development of myelofibrosis. Bone marrow cells (BM) were transplanted into lethally irradiated recipient mice together with GFP-positive wild-type competitor cells and tamoxifen was administrated after transplantation. Double mutant cells initially demonstrated a limited competitive advantage over wild-type cells as compared to Jak2 V617F-only cells. However, serial transplantation revealed a rapid exhaustion of Jak2 V617F single mutant cells leading to lethal pancytopenia, which was not observed in animals transplanted with Jak2 V617F/Srsf2 P95H double mutant cells.As both monocytes and megakaryocytes (MK) were involved in fibrosis development, we further explored the role of these two cell populations. Spectral flow analysis of monocyte subsets in peripheral blood and BM failed to detect any significant change in double compared to single mutant animals. In contrast, double-mutant mice presented a significant delay in MK maturation with normalized expression of c-Mpl and ploidy. Using mass cytometry, we found ex vivo a higher proportion of MKP and MK expressing high levels of P-Stat5 in Jak2 V617F mice, which the addition of Srsf2 P95H tend to reduce, suggesting an altered Mpl/Jak2 signaling pathway.To validate the hypothesis that Srsf2 P95H negatively interfere with Jak2-mediated signaling in MK, we injected high dose of the thrombopoietin-mimetic romiplostim in mice transplanted with wild-type or Srsf2 P95H BM. Both thrombocytosis and myelofibrosis were significantly reduced in Srsf2 P95H transplanted animals.To further decipher the mechanism by which Srsf2 P95H could alter cell signaling, we performed bulk RNA sequencing on sorted MK. Pathway analysis using gene set enrichment analysis identified mostly a down-regulation of signaling pathways, including JAK/STAT signaling, in Jak2 V617F/Srsf2 P95H compared to Jak2 V617F single mutant cells. Further analysis of splicing events in Srsf2 P95H mutant cells identified an increased exon 14 skipping in Jak2, which was validated by RT-qPCR.Summary: Contrary to EZH2 mutation that promotes JAK2 V617F-induced myelofibrosis in mouse models, heterozygous Srsf2 P95H delays myelofibrosis development in Jak2 V617F-transgenic mice. Srsf2 P95H co-mutation prevents the clonal exhaustion induced by serial transplantation of JAK2 V617F BM cells. This effect is associated with a reduced signaling in MK, which may involve abnormal splicing of signaling components including Jak2 exon 14 skipping. DisclosuresAbdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Clonal Competition of Cancer Cells During Acute Lymphoblastic Leukemia Treatment

The importance of cancer cell heterogeneity is increasingly recognized. However, the interaction of different cancer cells remains poorly understood, particularly in response to therapeutic treatment. Past studies used naturally occurring genetic mutations to examine the heterogeneity and evolutionary trajectories of cancer cells. As these mutations occur at different times, clones marked by these mutations cannot be directly compared to assess cellular interactions. In this study, we use engineered genetic barcodes to track patient derived acute lymphoid leukemia (ALL) cells in a novel mouse model that closely mimics the ‘intensive’ and ‘maintenance’ chemo-therapy regiments used in clinical treatment.Intensive and maintenance chemotherapy are commonly combined together in ALL treatment. After the combination treatment, ALL patients who relapse early (<36 months from diagnosis) are particularly resistant to additional chemotherapy and have a very poor prognosis. In contrast, a fraction of ALL patients who relapse late (>36 months from diagnosis) have a much better prognosis, and remain chemo-sensitive. It is unclear why ALL patients who relapse late are chemo-sensitive while patients who relapse early are chemo-resistant. We hypothesize that low intensity maintenance therapy allows “beneficial competitor cells” to survive and outcompete chemo resistant cancer clones. These beneficial competitor cells expand slowly during the maintenance treatment. They are chemo sensitive and are easily eradicated by intensive therapy.To test our hypothesis, we took primary human B-ALL cells and genetically barcoded them before transplantation into immune deficient NSG mice. We established a chemotherapy treatment protocol in mice that closely resembles the ‘intensive’ and ‘maintenance’ phase of ALL treatment currently used in clinic. In our experiment, mice were first treated with a four-week ‘intensive’ phase of chemotherapy consisting of Vincristine, Dexamethasone and PEG L-Asparaginase. This intensive phase was followed by a long-term ‘maintenance’ chemotherapy phase consisting of Methotrexate.We found that ‘intensive’ therapy did not significantly change the clonal composition in mice. However, ‘new’ leukemia clones arose during ‘maintenance’ treatment. These clones might have been suppressed during the undisturbed progression of the disease, and they may play a crucial role in the relapse of the disease during the long-term ‘maintenance’ treatment.To investigate the growth phenotype of chemotherapy surviving ALL clones, we used a competitive transplantation assay. We took surviving ALL clones from ‘intensive’ only and ‘intensive + maintenance’ treated xenografts and co-transplanted these cells with untreated ALL cells. As predicted by our hypothesis, we found that ALL clones that survived ‘intensive’ only treatment exhibited a more aggressive growth phenotype compared to untreated ALL clones. Conversely, ALL clones that survived ‘intensive + maintenance’ treatment grew more slowly than untreated clones. More importantly, the growth of the co-transplanted untreated ALL clones was significantly suppressed by the presence of ALL clones that had survived intensive treatment. In contrast, the growth of the co-transplanted untreated ALL clones was significantly accelerated in the presence of ALL clones that survive the ‘intensive + maintenance’ treatment, suggesting that new clones that expanded during the long-term maintenance treatment were outcompeted by other clones in the absence of maintenance treatment.Together, our findings suggest that the clonal competition of cancer cells plays an important role in determining treatment outcome. In particular, the benefit of maintenance therapy to ALL patients may arise from cellular competition. Further characterization of the beneficial competitor cells may help to improve ALL treatment and may also help to improve the treatment of other types of cancer. DisclosuresMerchant:Pfizer: Consultancy, Research Funding.

BET-Inhibition By JQ1 Promotes Proliferation and Self-Renewal Capacity of Hematopoietic Stem Cells

Background/significance: Bromodomain and extra terminal domain (BET) proteins have been shown to be critical for maintaining the function of leukemia stem cells. Although BET-inhibitors are in clinical development and show promising activity in different hematologic malignancies, a systematic analysis of the consequences of pharmacological BET-inhibition on healthy hematopoietic (stem) cells is outstanding.Methods: C57BL/6J (WT) or B6.SJL-Ptprca Pepcb/BoyJ mice, which carry the congenic pan-leukocyte marker CD45.1 were treated for 3 weeks with the BET-Inhibitor JQ1 or a placebo. For competitive repopulation transplantations, bone marrow from treated CD45.1 mice was transplanted together with WT competitor cells (CD45.2) into lethally irradiated WT mice (9 Gy) in different ratios. Engraftment of all major hematopoietic lineages was monitored in peripheral blood (PB) and BM using differential blood counting and flow cytometry. For the determination of HSC frequency in vivo, extreme limiting dilution analysis (ELDA) was carried out by transplanting 2x103, 2x104 or 2x105 JQ1- or placebo-treated bone marrow cells from WT mice together with 2x105 bone marrow cells from CD45.2 mice. Engraftment was assumed when CD45.1+ myeloid cells were present in the peripheral blood in frequencies > 1 %.Colony formation assays were used to measure the abundance of colony forming progenitor cells (M3434) as well as megakaryocytic progenitors (MegaCult-C). For analysis of recovery after myelosuppression, JQ1- or placebo-treated WT mice received sublethal irradiation (5 Gy) and hematopoietic repopulation was monitored. For analysis of HSC proliferation, 1 mg BrdU was injected 24 h, 12 h, and 6 h before sacrifice.Results: As a first step, we analyzed the mRNA expression patterns of Brd2-4 in all major hematopoietic subpopulations, because BET-family members represent the main molecular targets of JQ1. Expression of Brd4 mRNA is most abundant in megakaryocytes (MK), followed by B cells, T cells and hematopoietic stem cells (HSC) and expression levels of Brd2+3 show similar distribution.JQ1-treatment decreases the numbers of pre-, immature and mature B cells while numbers of early pro-B cells remain constant (pre-B cells: 125 ± 7 vs. 52 ± 5 and immature B cells: 121 ± 9 vs. 26 ± 5 x104 cells/tibia; n=4; *p<0.05). Furthermore, it increases apoptosis in all T cell subsets, all together leading to reduced cellularity in thymus, BM and spleen. Intriguingly, JQ1 induces a 3-fold increase in the number of hematopoietic stem and progenitor cells (25 ± 2 vs. 70 ± 5 x103 LSK/femur; n=5; *p<0.05) and mobilizes HSC into the peripheral blood (4 ± 1 vs. 28 ± 2 x106 CFU/ml blood; n=5; *p<0.05). Consistently, extreme limiting dilution analysis (ELDA) showed a 3-fold increase in stem cell frequencies in the bone marrow upon JQ1-treatment (1/19480 vs. 1/6284; n=8-12; *p<0.05).We found that the elevated HSC numbers arise from increased cycling of LT-HSC, as these cells incorporate more BrdU upon JQ1-treatment (29 ± 2 vs. 41 ± 3 % BrdU+ LT-HSC in bone marrow; n=5; *p<0.05). Moreover and in contrast to placebo, JQ1-treated bone marrow cells preserve their colony-forming properties during several rounds of replating in vitro, indicating sustained stemness (18 ± 4 vs. 77 ± 10 CFU/1x104 bone marrow cells after third replating; n=3-8; *p<0.05). Due to increased numbers of HSC, JQ1-treated bone marrow engrafts better after competitive stem cell transplantation and repopulates the hematopoietic system significantly faster after sublethal myeloablation. Six weeks after irradiation, total leukocyte counts of JQ1-treated mice fully recover to levels before irradiation (94 ± 7 % recovery; n=5; *p<0.05), whereas recovery is slower in the control group (47 ± 5 % recovery; n=5; *p<0.05).Conclusions: JQ1 induces proliferation and mobilization of HSC, thereby increasing their number in bone marrow and peripheral blood. Therefore, BET-inhibition might benefit patients after myelosuppression or stem cell transplantation. DisclosuresLoges:BerGenBio ASA: Research Funding.

Normal Hematopoiesis Is Dependent on the Expression of U2af1, a Spliceosome Gene Commonly Mutated in MDS

Recently, somatic mutations in genes encoding factors involved in pre-mRNA splicing have been identified in up to 50% of myelodysplastic syndrome (MDS) patients. U2AF1, a spliceosome gene, has recurrent missense mutations in 11% of MDS patients. This gene is a key component of the spliceosome that recognizes the AG dinucleotide in the 3' splice acceptor site of pre-mRNA. U2AF1 mutations are always heterozygous and the wild-type allele is expressed, suggesting that cells may not tolerate biallelic mutations. Mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients but the role of wild-type U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse by flanking exon 2 with LoxP sites.We confirmed deletion at the genomic DNA and mRNA level in hematopoietic cells by crossing mice to various Cre-recombinase expressing models. We first generated U2af1flox/flox , U2af1flox/wt and U2af1wt/wt littermate control mice all carrying ERT2- Cre (and U2af1flox/flox without ERT2- Cre as a control). To study the hematopoietic cell-intrinsic effects of U2af1 deletion, we performed a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice. Four weeks after engraftment, we treated mice with tamoxifen to active ERT2- Cre and monitored their survival and blood counts. Homozygous U2af1 KO mice (ERT2- Cre/ U2af1flox/flox ), but not other genotypes, became moribund (and were sacrificed) as early as only 6 doses of tamoxifen (Fig. 1A). The bone marrow cellularity in homozygous U2af1 KO mice was reduced (Fig. 1B). Analysis of mice at day 24 (from 1st tamoxifen dose) revealed anemia and a reduction in blood and bone marrow neutrophils, monocytes, and B-cells (p ≤ 0.01 ).We also generated mice carrying the interferon inducible Mx1- Cre to verify the observed phenotypes using an independent model. After a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice, deletion of U2af1 was induced by polyinosinic:polycytidylic acid (poly I:C). The homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and a reduction in platelets (>9 fold) and white blood cell counts (>4.7 fold) in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5) . Peripheral blood neutrophils, monocytes, B-cells, and T-cells were reduced (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils, monocytes, and B-cells as well as a significant depletion hematopoietic progenitor cells [ KL (Lin-, cKit+, Sca1-), and KLS (Lin-, cKit+, Sca1+)], (Fig. 1C). In order to study the hematopoietic cell-intrinsic effects of U2af1 deletion on stem cell function, we mixed equal numbers of whole bone marrow test cells (Ly5.2) with congenic control wild-type bone marrow competitor cells (Ly5.1/5.2) and transplanted them into lethally irradiated congenic recipient mice (Ly5.1). As early as 10 days following Cre-induction we observed a significant decrease in white blood cell count chimerism (Fig. 1D) and complete loss of homozygous U2af1 KO neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001 ). Heterozygous U2af1 KO bone marrow had a modest competitive disadvantage compared to controls after 120 days (Figure 1D, p ≤ 0.05 ).Collectively, the data indicate that stem cell function, multilineage hematopoiesis and hematopoietic cell survival are dependent on U2af1 expression. In ongoing studies to understand why U2AF1 mutations are always heterozygous, we will cross the U2af1 KO mouse to existing transgenic mutant U2AF1(S34F)-expressing mice and determine whether mutant U2AF1-expression is sufficient to rescue cell survival and normal hematopoiesis when both endogenous U2af1 wild-type alleles are deleted. Proving that U2AF1 mutant cells are vulnerable to loss of the wild-type U2AF1 allele could provide a therapeutic vulnerability to mutant cells by selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Beyond JAK-STAT: Contribution of Abi-1, STAT3, and NF-κB Signaling to Pathogenesis of Myeloproliferative Neoplasms

Activation of the JAK-STAT pathway is essential for the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs), but in the absence of curative treatment options, a major need remains to identify other targetable mechanisms in those diseases. We have previously shown that mice with conditional, inducible bone marrow-specific knockout of Abelson interactor 1 (Abi-1 KO) develop progressive myeloid hyperplasia, splenomegaly and bone marrow fibrosis leading to weight loss and decreased survival (Chorzalska, ASH 2016). Our data also indicated that hematopoietic progenitors from PMF patients show decreased levels of ABI1 transcript independent of the presence of JAK2 mutations. Abi-1 is one of several interactors of the Abelson kinase involved in Ras, PI3K and integrin signaling, and its deficiency in embryonic development leads to lethal defects of the heart and placenta. Despite suggestions that Abi-1 may act as a tumor suppressor, its involvement in hematopoiesis has not been elucidated.We have now characterized mechanisms that underlie the progressive PMF-like phenotype in our Abi-1 KO mice model. Colony-forming unit (CFU) assays indicated a significant decrease in the number of BFU-E colonies, a significant increase in CFU-GM colonies, and a nearly 3-fold increase in CFU-Mk colonies in the Abi-1 KO animals (Fig. 1A). Gene expression profiling studies showed a significant increase in the frequency of long-term hematopoietic stem cells (LT-HSCs) and HSCs enriched lineage- Sca-1+ cKit+ (LSK) cells in Abi-1 KO mice, associated with increased cell cycle activity in those cells: 3- to 4-fold higher expression of Mcm2 and Mki67 genes (specific to S phase and DNA replication), >6-fold upregulation of cMyb (specific for the G1/S phase transition), a 5-fold upregulation of M-phase specific Ccna1 and Ccnb1 and >15-fold upregulation of Cdk2, compared with wild-type (WT) animals. We also observed a 60 to 70% decrease in Cdkn1a, Cdkn1b, Cdkn2b (G1- or G2-phase-specific cell cycle inhibitors) transcripts in Abi-1 KO LT-HSCs (Fig. 1B). Investigation of cell cycle phases in HSC-enriched LSK cells using bromodeoxyuridine (BrdU) incorporation showed significantly more BrdU staining in cells from Abi-1 KO compared with WT mice, with a higher proportion of cells in S phase (Fig. 1C). Bone marrow transplantation assays done in C57BL/6 WT mice in the absence of competitor cells showed progressive loss of engraftment (decreased donor chimerism) of Abi-1 KO bone marrow cells over 6 months post transplant (Fig. 1D). Similarly, in competitive repopulation assays (C57BL/6 wild type mice engrafted with the whole bone marrow from Abi-1 KO or WT mice in 1:1 ratio with competitor, congenic CD45.2 WT cells), we observed a progressive decrease in donor chimerism with Abi1 inactivation (Fig. 1E).Using the Abi-1 KO model we then evaluated signaling pathways critical for the MPN pathophysiology, detecting only a modest increase in JAK2 and STAT5 activities (Fig. 1F). Immunoblotting analyses of the activities of Src Family Kinases (SFKs) showed increased phosphorylation on Tyr 416, consistent with activation of SFKs as well as increased activity of STAT3 and NF-κB (Fig. 1G). We confirmed these findings in CD34+ cell isolates from PMF patients (n=2), showing a decrease in Abi-1 protein levels and significant upregulation of SFKs, STAT3 and NF-κB activities (Fig. 1H).In summary, bone marrow-specific inactivation of Abi1 appears to be sufficient to induce a MPN-like phenotype, replicating features of human PMF. Results from gene expression profiling, immunoblotting, and proteomics consistently show that activation of SFKs, STAT3 and NF-κB may be involved in the genesis of this phenotype. We continue to explore the mechanisms responsible for the downregulation of ABI1 in hematopoietic progenitor cells from patients with PMF/MPN, as well as use of murine models with mutations in JAK2, CALR or MPL to determine mechanisms responsible for ABI1 loss and its association with classical MPN-associated pathways. Our work provides further evidence supporting a role for Abi-1 in pathogenesis of MPNs and a possible link to the SFK-STAT3-NF-κB signaling axis. Full elucidation of these mechanisms may couple the proliferative and pro-inflammatory phenomena that characterize PMF/MPNs, and provide a new therapeutic strategy for these diseases. [Display omitted] DisclosuresReagan:Teva: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Shire: Speakers Bureau; Novartis: Honoraria, Speakers Bureau.