Deficient Clones Research Articles

HLA-lacking clones in aplastic anaemia: Adaptive or maladaptive?

HLA loss represents the result of immune forces shaping bone marrow clonal dynamics in immune aplastic anaemia. Human leukocyte antigen (HLA)-deficient clones may rescue haematopoiesis by evading immune attacks, potentially guiding treatment strategies. Commentary on: Zaimoku etal. Haematopoietic regeneration by HLA-A*0206-deficient clones in severe aplastic anaemia without definitive immunosuppressive treatment. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19712.

RAD50 Deficient in a Breast Cancer Model Predicts Sensitivity to PARP Inhibitors.

Breast and ovarian tumors with pathogenic variants in BRCA1 or BRCA2 genes are more sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) treatment than wild-type tumors. Pathogenic variants in non-BRCA1/2 homologous recombination repair genes (HRR) also concede sensitivity to PARPi treatment. RAD50 participates in the Mre11-Rad50-Nbn (MRN) complex of the HRR pathway and plays an important role in DNA repair. The objective of this study is to evaluate whether RAD50 protein deficiency modulates the PARPi response in breast cancer cell lines. T47D breast cancer cell line was modified using small interfering RNA and CRISPR/Cas9 technology, to knockout the RAD50 gene. PARPi response (niraparib, olaparib and rucaparib alone or in combination with carboplatin), in T47D and T47D-edited clones, was evaluated by cell viability, cell cycle, apoptosis and protein expression analyses. Treatment with niraparib and carboplatin exerted a synergistic effect on T47D-RAD50 deficient cells and an antagonistic effect on T47D cells parental. Cell cycle analysis demonstrated an increase in the G2/M population in cells treated with niraparib or rucaparib alone or in combination with carboplatin. T47D-RAD50 deficient cells treated with rucaparib and carboplatin exhibited twofold levels in late apoptosis, also showing differences in PARP activation. All T47D RAD50 deficient clones treated with niraparib or rucaparib combined with carboplatin, or rucaparib alone showed increased levels of H2AX phosphorylation. T47D RAD50 deficient cells treated with PARP inhibitors alone or in combination with carboplatin showed cell cycle arrest in the G2/M phase, leading to death by apoptosis. Thus, RAD50 deficiency may be a good biomarker for predicting PARPi response.

The XPO7/Npat Axis Inactivation Is a Therapeutic Vulnerability for TP53-Mutated AML

In acute myeloid leukemia (AML), TP53 mutations are the most prognostically unfavorable genetic alterations, as they confer resistance to various therapeutic agents and form relapsing clones, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes or pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. We employed two distinct KO screen libraries, through which we identified Xpo7, a putative nuclear/cytoplasmic transporter, as a common factor necessary for Trp53-KO AML survival. Interestingly, Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. Further probing this phenomenon, we performed a CRISPR-Cas9 saturation mutagenesis scan on all Xpo7 exons. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT AMLs. In stark contrast, these same sgRNAs were mostly depleted in Trp53-KO AMLs. To determine the role of Xpo7 depletion on the expansion of Trp53 deficient clones, we prepared AML cells containing a small fraction of Trp53-KO cells both in the Xpo7-WT and KO backgrounds and assessed the proportion over time in vitro or in vivo. Remarkably, the expansion of Trp53-KO clones, noticeable in the Xpo7-WT setting, was significantly reduced under the Xpo7-KO background, prolonging the mice's survival. These data suggest that Xpo7 depletion effectively impedes Trp53-KO clone expansion. Since Xpo7 is presumed to mediate the nuclear import and export of proteins, we hypothesized that Xpo7 contributes to retaining Trp53 protein within the nucleus in Trp53-WT AMLs. As expected, nuclear Trp53 protein and mRNA levels of Trp53-targeting genes decreased significantly upon Xpo7 depletion. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AMLs. Next, to elucidate the underlying mechanisms causing the toxic effects in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose localization was potentially regulated by Xpo7 in the Trp53-KO state. Employing mass spectrometry on an immunoprecipitate using anti-Xpo7 and cytoplasmic/nuclear protein extracts from both Xpo7-WT and KO AML cells, we found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and showed a significant shift towards cytoplasmic localization upon Xpo7 depletion in Trp53-KO AMLs. Strikingly, Npat protein levels displayed a notable upregulation by Trp53 depletion in AML cells. To evaluate the relevance of the XPO7/NPAT axis in human TP53-mutated AML cells, we examined the correlation between XPO7 and NPAT expression levels and AML subtypes and genetic background using publicly available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets. Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018; Iacobucci et al. Nat Genet. 2019). The Xpo7 and Npat protein expression evaluation using primary AML samples corroborated these findings, revealing overexpression in TP53-mutated AML cases. Finally, we assessed the therapeutic effect of Npat depletion in Trp53-deficient AML cells, using the dTAG system to induce Npat protein degradation by knock-in FKBP12 F36V into Npat loci. dTAG v-1 treatment immediately degraded endogenous Npat protein and led to delayed G1/S transition progression. Moreover, Npat degradation induced DNA damage, particularly in the presence of a WEE1 inhibitor or etoposide, and subsequent apoptosis. Transplantation of these AML cells into immunocompromised mice followed by dTAGv1 treatment for one week demonstrated suppressing leukemia progression in vivo. Furthermore, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed the proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. We unveiled the novel regulatory mechanism of protein localization by XPO7, depending on TP53 mutational state. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML.

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

TP53 Null AML Line Does Not Selectively Expand Under Lenalidomide Treatment

Lenalidomide is an immunomodulatory therapeutic (IMiD) with known anti-proliferative and anti-angiogenic effects, and is an approved therapy for both 5q- Myelodysplastic Syndromes and Multiple Myeloma (MM). This thalidomide analog is known to retarget the E3 ubiquitin ligase cereblon to differential ubiquitination, and therefore degradation, of specific substrates. While lenalidomide maintenance is known to promote progression-free survival, increased rates of therapy-related myeloid neoplasms (TMNs) have been observed in MM patients on IMiD maintenance therapy following autologous stem cell transplant. Of note, TP53 is the most commonly mutated gene in TMN arising after lenalidomide therapy (Mouhieddine et al. 2020). TP53 is also recurrently mutated in TMNs following chemotherapy, suggesting that genotoxic stress results in the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations in TP53 (TN Wong et al. 2018). Based on these observations, we hypothesize that lenalidomide may contribute to the development of TMN by providing a fitness advantage to HSPCs carrying TP53 mutations.To test this hypothesis, we generated TP53 -/- deficient MOLM13 AML cells using CRISPR-Cas9 gene editing. We confirmed guide editing efficiency by next generation sequencing of a bulk edited pool of cells and generated isogenic clones via outgrowth of singly sorted cells, in an accepted approach for the field (Boettcher et al. 2019). We validated clones via sequencing and western blotting, and identified no differences in basal proliferation characteristics. Next, we further compared TP53 -/- deficient clones' with TP53 +/+ clones' response to cisplatin treatment (0.05-10µM) and observed a 2.8 fold increase in IC50 for TP53 -/- deficient clones. Satisfied that TP53 -/- isogenic clones successfully recapitulated this known resistance phenotype, we examined the response to lenalidomide by mixing TP53 -/- and TP53 +/+(AAVS1 edited control) clones and measuring expansion under lenalidomide treatment. However, we observed substantial inter-clone heterogeneity, with widely varying cellular proliferation characteristics following clone mixing, despite our prior validation. Therefore, we shifted to using bulk edited MOLM-13 cells in order to mitigate the potential impact of off-target effects and strong selective pressure of singly sorted clones. We edited a pool of cells at either TP53 or the safe harbor locus AAVS1 , allowed cells to recover, and mixed the two edited populations before treating with PBS, vehicle (DMSO), 1µm or 10µM lenalidomide for up to 28 days. We sequenced both AAVS1 and TP53 loci to measure cellular expansion by tracking the fraction of unedited, safe harbor edited, and TP53-/- genomic DNA at 0, 3, 7, and 28 days. Contrary to our hypothesis, TP53 -/- cells did not expand over time in response to lenalidomide treatment, and we did not detect a difference in TP53 -/- cell expansion with lenalidomide treatment compared to vehicle control (Figure 1). While we did not detect a cell intrinsic effect of TP53 mutations on lenalidomide response, it is possible that there are cell extrinsic effects, such as alterations in the bone marrow microenvironment, that contribute to the development of TP53-mutant TMN. It is also possible that a more prolonged exposure of lenalidomide is needed to see a significant effect of TP53-mutant cell expansion. This is relevant, since patients may be maintained on lenalidomide therapy for years. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Glycosylphosphatidylinositol Anchor Regulates T Cell Antigen Receptor Induced IL-2 Production

Differential contributions of the glycosylphosphatidylinositol (GPI)-anchor and GPI-anchored proteins (GPI-AP) to signalling remain poorly understood. Here we show that GPI-AP deficient murine clones produce on average 18 and 181-fold more IL-2 mRNA and protein, respectively, upon T cell receptor (TCR) stimulation, in a cell-intrinsic fashion. This phenotype is formally attributed to a mutation within the transferase complex that predicates the initial step in GPI-anchor biosynthesis. Conditional disruption of the transferase complex enabled the generation of primary GPI-AP deficient CD4+ T cells, which produce on average 10- and 23-fold more IL-2 mRNA and protein, respectively, upon TCR stimulation. Conditional disruption of the transamidase complex yields GPI-sufficient, GPI-AP deficient primary CD4+ T cells. TCR stimulation of these cells yields levels of IL-2 mRNA and protein ranging from 1 - 3 and 3-fold, respectively, of controls. These results provide the first evidence of a profound impact of GPI in the regulation of TCR signalling.

Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL

The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion.

Baseline clinical characteristics and disease burden in patients with paroxysmal nocturnal hemoglobinuria (PNH): updated analysis from the International PNH Registry

The International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry (NCT01374360) was initiated to optimize patient management by collecting data regarding disease burden, progression, and clinical outcomes. Herein, we report updated baseline demographics, clinical characteristics, disease burden data, and observed trends regarding clone size in the largest cohort of Registry patients. Patients with available data as of July 2017 were stratified by glycosylphosphatidylinositol (GPI)-deficient granulocyte clone size (< 10%, ≥ 10%–< 50%, and ≥ 50%). All patients were untreated with eculizumab at baseline, defined as date of eculizumab initiation or date of Registry enrollment (if never treated with eculizumab). Outcomes assessed in the current analysis included proportions of patients with high disease activity (HDA), history of major adverse vascular events (MAVEs; including thrombotic events [TEs]), bone marrow failure (BMF), red blood cell (RBC) transfusions, and PNH-related symptoms. A total of 4439 patients were included, of whom 2701 (60.8%) had available GPI-deficient granulocyte clone size data. Among these, median clone size was 31.8% (1002 had < 10%; 526 had ≥ 10%–< 50%; 1173 had ≥ 50%). There were high proportions of patients with HDA (51.6%), history of MAVEs (18.8%), BMF (62.6%), RBC transfusion (61.3%), and impaired renal function (42.8%). All measures except RBC transfusion history significantly correlated with GPI-deficient granulocyte clone size. A large proportion of patients with GPI-deficient granulocyte clone size < 10% had hemolysis (9.7%), MAVEs (10.2%), HDA (9.1%), and/or PNH-related symptoms. Although larger GPI-deficient granulocyte clone sizes were associated with higher disease burden, a substantial proportion of patients with smaller clone sizes had history of MAVEs/TEs.

RanBP3L is a NFAT5 target gene and its deficiency in principal cells promotes epithelial mesenchymal transition through MAPK signaling

Inner medullary kidney cells are exposed to an increasing hyperosmolality altering several intracellular processes. As a key regulator involved in adaption to hyperosmotic environment the transcription factor nuclear factor of activated T cells‐5 (NFAT5) was identified. Its nuclear translocation results in activation of different genes to restore homeostasis. By using primarily cultured rat renal inner medullary collecting duct cells (IMCD) and microarray analysis we identified hundreds of genes that showed differences in expression level in a time‐ and osmolality‐dependent manner. The kidney specific Ran binding protein 3 like (RanBP3L) showed the highest changes in expression level and is located in IMCDs. Within the collecting duct its expression is specific to the principal cells. Since studies describing the renal function are missing, we further characterized the cellular and physiological role of RanBP3L in a renal mouse cell line.We used the murine kidney collecting duct cell line mpKCCD creating a RanBP3L deficient cell clone by using CRISPR/Cas9. To investigate its physiological role the gene expression profiles were analyzed by Next Generation Sequencing. We further analyzed the migration and colony forming capability of the cells and used immunofluorescence to study differences in cell morphology and specific signaling pathways. These experiments were performed under isoosmolar (300 mosmol/kg) and hyperosmolar (600 mosmol/kg) conditions. Since RanBP3L expression is induced by hyperosmolality, the NFAT5 dependent expression of RanBP3L was examined through CRISPR/Cas9 mediated NFAT5 knock out.Loss of RanBP3L under 300 mosmol/kg showed no significant changes in the morphology while under 600 mosmol/kg massive changes were induced resulting in a loss of epithelial structures. This correlates with our functional analysis showing a higher migration and invasiveness capacity. Furthermore RanBP3L deficiency is associated with a massive change in gene expression, indicating a dysregulation of stress associated pathways. Immunofluorescence showed that TGF‐β stimulation in wild type induced a similar phenotype to RanBP3L knock out cells. Downstream analysis of MAPK pathway showed that the key proteins JNK, c‐JUN and Smad2 are deregulated in RanBP3L deficient cells resulting in an epithelial mesenchymal transition (EMT) like phenotype, which is a hallmark in cancer development. This matches with the data from the cancer genome atlas from patients with renal clear cell carcinoma (RCC) where low RanBP3L expression level is associated with shorter overall survival. It seems as if the expression level of RanBP3L in kidney cells is crucial for cell protection. Here we could show that RanBP3L expression is reduced in NFAT5 deficient cells, indicating that RanBP3L is a NFAT5 target gene.Taken together we have shown that RanBP3L is expressed NFAT5 dependent and prevents a MAPK associated EMT in renal cells and should be used as a prognostic marker for patients with RCC. Further experiments, like in‐vivo studies of a conditional RanBP3L knock out mouse will give us more insights to resolve the underlying pathway.Support or Funding Informationgerman research foundation

Modeling the Function of TATA Box Binding Protein in Transcriptional Changes Induced by HIV-1 Tat in Innate Immune Cells and the Effect of Methamphetamine Exposure.

Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.

Selective Pharmacologic Inhibition of Paroxysmal Nocturnal Hemoglobinuria Clones

Paroxysmal nocturnal hemoglobinuria (PNH) is usually associated with reduced bone marrow (BM) capacity caused by acquired idiopathic aplastic anemia (AA). PIGA mutations lead to a partial or total deficiency of glycosylphosphatidyl-inositol (GPI) anchor proteins (AP). AA is characteristically accompanied by the presence of often tiny GPI-AP deficient clones, which in a significant proportion of patients (10-15%), irrespective of the initial success of immunosuppressive therapy, will evolve to produce manifest hemolytic PNH. Indeed in our cohort of BM failure patients (n=319), 41% of AA patients had a PNH clone present (0.02-20% of granulocytes) (AA/PNH), 14% of patients had primary PNH (primary PNH), and 8% had a history of PNH post AA (secondary PNH).To date, drug development for PNH has focused on designing supportive therapies to prevent transfusions due to hemolysis or thrombotic complications. In addition to the current FDA approved C5 inhibitor eculizumab, new, more convenient and effective complement blockers are under development. Apart from hematopoietic stem cell (HSC) transplantation, no direct strategies targeting basic pathophysiologic mechanisms of PNH have been ventured to prevent evolution of PNH clones and cure the disease. In early AA/PNH syndrome, the PIGA mutant HSCs are rare and unlikely contribute to significant blood cell production. While in later stages of manifest hemolytic PNH, hematopoiesis relies most frequently on mutant HSCs and thus elimination of these cells would result in AA.We hypothesized that if a selective inhibitor of GPI-AP-deficient [GPI-AP (-)] cells can be developed, it could be used primarily in AA/PNH patients with a small clone size. The hope would be to prevent both later expansion of GPI-AP d(-) cells and development of manifest PNH.To discover compounds acting selectively against GPI-AP (-) cells, we subjected wild type (WT) and GPI-AP (-) cell lines (K562, TF-1) to a high-throughput screen using a platform of 3000 bio-active molecules to identify hits and chemical compounds capable of selectively eliminating GPI-AP (-) cells. Our robotic screen yielded several top hits including GR -89696 fumarate, D-cycloserine and CGS-15943. Dose-response experiments confirmed CGS-15943 as a candidate growth inhibitor of GPI-AP (-) cells. CGS-15943 is an adenosine receptor antagonist and non-phosphodiesterase inhibitor which has previously been shown to inhibit cancer cell growth via PI3K/Akt pathway.Low range dose CGS-15943 (1uM) induced cell growth inhibition in K562 and TF-1 GPI-AP (-) cells by 4.7 fold and 3.2 fold, respectively. No cell growth arrest was observed in K562 WT and TF-1 WT cells, as the percentage of alive cells was >95% upon drug treatment. Mixed competition assays were conducted in vitro using equal ratios of K562 and TF-1 WT and GPI-AP (-) cells exposed to CGS-15943 (1uM). Six days after culture, flow cytometric analysis of CD59 surface expression revealed that CGS-15943 allowed for preferential survival of WT cells (84.7 % K562, 96.3% TF-1) vs. GPI-AP (-) cells (15.3% K562, 3.7% TF-1). CGS-15943 induced an increase in the % of AnnexinV+/PI- and AnnexinV+/PI+ in TF-1 GPI-AP (-) cells (12.04% and 44.82, respectively). Similar results were obtained in K562 GPI-AP (-) cells (15.84% and 21.08%). Mononuclear cells of a PNH patient were stimulated with CD3/28 beads in presence of CGS-15943. Flow cytometric analysis indicates a dose dependent growth inhibition effect on GPI-AP (-) lymphocytes after 3 days of culture. Previous reported observations from our group identified that the survival differences between GPI-AP (-) and WT cells largely depend on active PI3K signaling pathway. Our pilot investigation of CGS-15943 - indicates that CGS-15943 induces an decrease in the protein expression of the PI3K isoform - p110γ - exclusively in GPI-AP (-) cells possibly suggesting that CGS-15943 inhibits the catalytic subunit of- p110γ.In sum, we describe that the small molecule compound CGS-15943 selectively eliminates GPI-AP (-) cells in vitro, in both cell lines and in primary PNH cells most likely interfering with the PI3K/AKT survival pathway. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

PO-229 The fatty acid desaturase SCD1 as crucial factor for tumour progression in colon cancer

IntroductionAberrant activation of lipogenesis is a hallmark of cancer metabolism. However, the functional contribution of lipids to tumorigenesis is still uncovered. We analysed the clinical implication of key enzymes of fatty acid synthesis and processing, including FASN (fatty acid synthase), fatty acid elongase 6 (ELOVL6), the master transcription factor of lipid synthesis, SREBP1 (sterol regulatory element binding protein 1) and steaoryl-CoA desaturase 1 (SCD1). SCD1 is essential for the production of mono-unsaturated fatty acids (MUFAs) and crucially involved in colon cancer, suggested by our previous expression profiling data. To investigate its functional role, we generated SCD1 deficient colon cancer cells.Material and methodsSCD1, SREBP1, FASN and ELOVL6 were quantified by qPCR in 78 patients with stage III colon cancer, and clinical and prognostic significance was assessed by Kaplan-Meier analysis. SCD1 and SREBP1 were further analysed on protein level. SCD1-deficiency was implemented in colon cancer cells HCT116 by the CRISPR-Cas9 system. Mass spectrometry (MS) analysis was used to analyse the activity of SCD1; cell proliferation and survival of the SCD1 deficient clones were compared to parental cells.Results and discussionsExpression of SCD1 and FASN was highly significantly increased in tumours, whereas SREBF1 and ELOVL6 were significantly reduced. However, SCD1 and SREBP1 were upregulated in tumour tissue on protein level and their mRNA was significantly co-expressed. SCD1 was significantly associated with tumour grading and worse post-operative survival, and elevated expression of FASN and ELOVL6 with increased metastatic relapse.SCD1 deficiency was verified by DNA sequencing and immunoblotting, and MS analysis confirmed the deprivation of MUFAs. KO of SCD1 led to significantly decreased cell proliferation and survival under normal growth conditions, which was even more pronounced under reduced serum. This was fully rescued by addition of the SCD1 product oleate, suggesting that SCD1 is necessary for growth and survival especially in the absence of exogenous lipids.ConclusionSCD1, FASN and ELOVL6 are negative prognostic markers in stage III colon cancer. Functionally, SCD1 is essential for cell survival under serum depletion. Together, these data suggest that the cell-autonomous synthesis of fatty acid species in cancer cells is due to reduced access to exogenous lipids. Enhanced lipogenesis rises the requirement for the production of MUFAs by SCD1, e.g., to maintain survival and circumvent lipotoxicity.

Role of miR-203 in estrogen receptor-mediated signaling in the rat uterus and endometrial carcinoma.

The role of microRNAs (miRNA) in estrogen receptor (ER) signaling in the uterus and in endometrial cancer is not well understood. We therefore analyzed miRNA expression in uterine samples from a standard 3-day uterotrophic assay using young female adult rats to identify E2-regulated miRNAs. Microarray analysis identified 47 E2 down-regulated miRNAs including miR-30a, and 25 E2up-regulated miRNAs including miR-672, miR-203, and miR-146b. The strongly E2-upregulated miR-203 was selected for further analysis. miR-203 was deleted in the rat endometrial adenocarcinoma cell line, RUCA-I, using CRISPR/CAS9. Five clones devoid of miR-203 expression were generated. Proliferation was reduced and G2-arrest was observed in all miR-203 deficient RUCA-I clones. Transfection with a miR-203-3p mimic partially rescues this effect. Comparison of mRNA expression in three miR-203 knockout clones to wild type RUCA-I cells reveals 566 miR-203-upregulated and 592 miR-203-downregulated genes. 43 of the genes that are upregulated by miR-203 knockout in vitro are downregulated in the uterus by E2. Of these Acer2, Zbtb20, Ptn, Rcbtb2, Mum1l1, Hmgn3, and Nfat5 possess one or more seed sequence matches in their 3'-UTR that are predicted to be targets of miR-203. These data demonstrate the importance of E2 regulated miRNAs in general, and miR-203 in particular, for E2 regulated gene expression and physiological processes including proliferation and cell migration, in the uterus as well as in the etiology of endometrial carcinomas.

Abstract 2476: Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models

Abstract Rucaparib (CO-338) is an oral small molecule inhibitor of the poly(ADP-ribose) polymerase (PARP) enzymes PARP-1, PARP-2 and PARP-3 that is being developed for cancer patients with evidence of homologous recombination (HR) deficiency, including BRCA1 and BRCA2 mutations. PARP inhibitors have demonstrated clinical activity in BRCA1, BRCA2, and ATM mutant prostate cancer patients (Mateo et al. N Engl J Med. 2015;373:1697-708); however, limited preclinical validation of PARP inhibitors in prostate cancer has been reported to date, since no BRCA mutant nor HR deficient prostate cell lines have been identified. In this study, we modelled the functional inactivation of DNA repair genes using siRNA and CRISPR/Cas9, and examined the impact on PARP inhibition, in the DU145 and PC-3 prostate cancer cell lines. These cell lines were chosen based on transfection efficiency, lack of obvious genetic defects in DNA repair pathways, and baseline insensitivity to rucaparib (IC50 &amp;gt; 10 µM). A panel of 26 DNA repair genes were individually knocked down by siRNA, and changes to rucaparib potency were examined in a 7-day cell viability assay. The knockdown of BRCA1 or BRCA2 increased rucaparib cytotoxicity in both prostate cell lines. For example, the IC50 of rucaparib in DU145 and PC-3 cells with reduced BRCA2 levels was 275 and 297 nM, representing a 36- and 37-fold increase in rucaparib sensitivity, respectively. In addition to BRCA1 and BRCA2, the knockdown of several other genes including BARD1, CDK12, FANCA, PALB2, RAD51, RAD51C, RAD51D, and RAD54L increased rucaparib efficacy ≥ 2-fold in at least 1 cell line examined. To further investigate HR deficiency in prostate cancer, BRCA2 was knocked out in the human 22Rv1 prostate cancer cell line using CRISPR/Cas9. Clone 1-50 displayed biallelic BRCA2 inactivation due to the introduction of a deleterious premature stop codon, and immunoblot analysis verified lack of the full-length BRCA2 protein. The IC50 of rucaparib in the parental and clone 1-50 cells was 9,504 and 287 nM, respectively, demonstrating a 33-fold increase in rucaparib cytotoxicity in this BRCA2 deficient model. Androgen receptor (AR) signaling and proliferation were also examined in clone 1-50. In contrast to published reports (Schiewer et al. Cancer Discov. 2012;2:1134-49), no impact of PARP inhibition on dihydrotestosterone (DHT) induced gene expression was observed in the parental 22Rv1 and BRCA2 deficient clone 1-50 cell lines. In vivo model development and efficacy studies with the 22Rv1 and clone 1-50 cell lines are ongoing. Taken together, these findings support the hypothesis that deficiencies in BRCA1 and BRCA2, as well as additional HR genes, may sensitize prostate cancer cells to rucaparib. A phase 2 study investigating rucaparib in patients with metastatic castration-resistant prostate cancer and HR gene deficiency (TRITON2) is ongoing (NCT02952534). Citation Format: Minh Nguyen, Andrew D. Simmons, Thomas C. Harding. Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2017-2476

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Abstract 1396: Unraveling the cellular hierarchywithin human tonsillar crypt epithelium and oropharyngeal squamous cellcarcinoma tumors

Abstract OPSCCs commonly arise from the epithelium lining the lingual and palatine tonsillar crypts. The stratified epithelium at the surface gives way to complex reticulated epithelium in the crypts which contain a diverse cell population with varying cytokeratin expression patterns. How such a diverse cell population is maintained has not yet been explored. It has been observed that HPV+ HNSCC consistently arise from the tonsillar crypts. In addition, somatic stem and progenitor cells have been identified as the likely targets of tumorigenesis in many tissues. To further evaluate whether tonsillar crypts contain unique multi-potent stem cells that are targets of tumorigenesis, and similar stem cells are maintained in OPSCC tumors of tonsillar origin, we propose the following studies: Aim 1: Characterize the cellular populations of crypt epithelium and compare to oral cavity epithelium. Human tissue obtained from the IRB approved EVMS Biorepository will be evaluated. We will stain for purported markers of stem cell activity including LGR5/6, CD34, p63, Bmi-1, cytokeratins 15, 19, and hTert using tyramide signal amplification when necessary. Cytokeratins 5, 8, 14 and 17 (previously identified in subsets of crypt epithelium) will be evaluated and co-stained with the above markers. Ki67 will be used to identify proliferative regions within the crypt epithelium. Aim 2: Characterize the cellular population of oropharyngeal squamous cell carcinoma and compare to normal tonsillar crypt epithelium. Human tumor obtained from above biorepository will be evaluated. HPV+/- OPSCC cell lines will be quantified for the markers of stem cell activity listed in Aim 1. Similarly, the same markers will be evaluated in HPV+/- human OPSCC tumors and compared to normal tonsil. The cell cycle regulator, p16, which is up-regulated in HPV-induced OPSCC and has previously been identified in a subset of normal tonsillar crypt epithelial cells will also be co-stained with any identified stem cell markers to determine if it is associated with any stem/progenitor cell function. Patient data will be correlated to identify prognostic value of these markers. Aim 3: Evaluate clonal expansions of cells in tonsillar crypts and OPSCC tumors by mitochondrial lineage tracing. Acquired deficiencies in the mitochondrial gene for the respiratory chain enzyme cytochrome c oxidase (CCO) can be exploited to identify cellular lineages within human tissues and tumors. This aim will evaluate if such CCO deficient (CCO-) clones exist within normal tonsillar crypts and OPSCC tumors, which will allow for careful lineage tracing in future experiments to fully understand the cellular hierarchy underlying tonsillar crypt and OPSCC tumor morphology. Elucidating the cellular hierarchy within tonsillar crypts, and identifying whether multi-potent stem cells exist within the tissue is imperative to understanding OPSCC, including those that are HPV-induced. Citation Format: Vivian F. Wu, Tennison Yu, Brette Harding, Robert Bruno. Unraveling the cellular hierarchywithin human tonsillar crypt epithelium and oropharyngeal squamous cellcarcinoma tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2015-1396

Resilient immortals, characterizing and utilizing Bax/Bak deficient Chinese hamster ovary (CHO) cells for high titer antibody production

Cell death due to apoptosis is frequently observed in large-scale manufacturing of therapeutic proteins, and can reduce product accumulation in bioreactors. Several different strategies that involve overexpression of antiapoptotic or downregulation of proapoptotic proteins have been designed in attempt to curb this problem in Chinese hamster ovary (CHO) cell culture. However, each of these designs has their own shortcomings and limits, rendering them ineffective for large-scale protein production. Recently, we have reported generation of a Bax and Bak deficient dhfr(-/-) CHO cell line using zinc-finger nucleases. Here we demonstrate that puromycin, but not methotrexate, selection can be used to generate antibody-expressing Bax and Bak deficient clones that are not only resistant to apoptosis, but that can also achieve higher titers relative to parental CHO cells due to higher cell density. Additionally, we show that Bax and Bak deficient cells have more mitochondria with healthy membrane potential, an attribute that perhaps contributes to their more potent growth compared to parental cells. Bax and Bak deficient cells do not readily apoptose, as shown by the ability to withstand high concentrations of apoptosis inducing agents, such as sodium butyrate, without a reduction in viability, growth, or titer. These traits render Bax and Bak deficient cells a potentially attractive host for production of therapeutic proteins at industrial scale.

Overwhelming Paroxysmal Nocturnal Haemoglobinuria in a Patient with Low-Risk Myelodysplastic Syndrome and Long-Term Anticoagulation for Sick Sinus Syndrome

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare form of acquired Coombs negative haemolytic anaemia manifested by the clinical triad of intravascular haemolysis, venous thrombosis and cytopenia. At the molecular level PNH is defined by a clonal expansion of hematopoietic stem cells having undergone somatic mutation of the X-chromosome gene PIG-A. Here we report the case of an 80-year old female patient known for sick sinus syndrome for more than 30 years and low-risk myelodysplastic syndrome (MDS) with uneventful course over the past two years. In good health she underwent her fifth lead replacement under short-term reversal of anticoagulation. Two weeks later she presented at the emergency room for epigastric pain, vomiting and fever. Work up revealed extensive right jugular vein thrombosis, Coombs-negative haemolytic anaemia and acute renal failure. Paroxysmal nocturnal haemoglobinuria was suspected and confirmed by flow cytometric FLAER-assay, which detects clonal deficiency of glycosyl-phosphatidyl-inositol linked surface proteins on monocytes and granulocytes. Thus, search of a PNH clone with FLAER was reliable in the presence of RBC-transfusions and ongoing intravascular haemolysis. Though stabilization of haemolysis was achieved, renal failure progressed and the patient deceased suddenly at the 11th day of hospitalization. Short-term reversal of anticoagulation and functionless retained pacing leads may have catalyzed thrombosis in our MDS patient with a large glycosyl-phosphatidyl-inositol (GPI) deficient clone. In MDS patients under long-term anticoagulation any short-term reversal of anticoagulation for surgical procedures should be preceded by FLAER analysis to uncover an emerging GPI-deficient clone since recent developments in the treatment of this condition allow prevention of intravascular haemolysis and thrombosis by halting the complement cascade at the C5 level with targeted immunotherapy.

Prevalence of paroxysmal nocturnal hemoglobinuria in Chinese patients with Budd‐Chiari syndrome or portal vein thrombosis

Routine screening for paroxysmal nocturnal hemoglobinuria (PNH) in patients with Budd-Chiari syndrome (BCS) or portal vein thrombosis (PVT) has been recommended in Western countries. However, little is known about whether the routine screening test should be necessary in Chinese patients with BCS or PVT. We conducted a prospective observational study to examine the prevalence of PNH in these patients. Patients with primary BCS or non-malignant PVT who were consecutively admitted to our department or regularly followed up between September 2009 and December 2011 were eligible for the study and detected the expression of CD55 and CD59 on erythrocytes and granulocytes. The CD55 or CD59 deficiency was considered as the proportion of erythrocytes or granulocytes with normal expression of CD55 or CD59 was less than 90%. PNH was diagnosed by both CD55 and CD59 deficient clone at flow cytometry of peripheral blood cells. CD55 and/or CD59 deficiencies were found in 1.6% (2/127) of patients with primary BCS, 1.0% (1/100) of non-malignant and non-cirrhotic patients with PVT, and 4.7% (4/85) of cirrhotic patients with PVT. Only one patient had both CD55 and CD59 deficiencies on granulocytes. But he had been diagnosed with PNH before BCS. Paroxysmal nocturnal hemoglobinuria was very rare in Chinese patients with BCS or PVT, suggesting that routine screening for PNH should not be indiscriminately performed in such patients.

Thrombotic complications without evidence of hemolysis in paroxysmal nocturnal hemoglobinuria: is eculizumab indicated?

Dear Editor, We would like to discuss the treatment of paroxysmal nocturnal hemoglobinuria (PNH) by presenting the fatal outcome of one of our patients with this disease. A 30-year-old Caucasian man with a history of auto-immune hepatitis developed pancytopenia. He had no complaints and did not take any medication. On physical examination, a pale man was seen without any other remarkable findings. His hemoglobin was 8.3 g/dL (13.8–16.9 g/dL), leucocytes 0.30 (neutrophils 0.2) × 109/L (3.5–10 × 109/L), platelet count 16 × 109/L (150–350 × 109/L) and reticulocytes 8.4 × 109/L (30–95 × 109/L) without signs of hemolysis. Bone-marrow biopsy showed reduced cellularity (10 %) without any other (including cytogenetic) abnormalities. In peripheral blood, glycosylphosphatidylinositol (PI)-deficient clones were detected in 34, 27, and 1 % of the granulocyte, monocyte, and erythroid lineages, respectively. Severe aplastic anemia (SAA) was diagnosed secondary to PNH. Lacking an HLA-identical family donor, standard treatment with immunosuppression was started: rabbit anti-thymocyte globuline, methylprednisolone, and cyclosporine. This resulted in a partial response 3 months later: persistent transfusion-dependent anemia without signs of intravascular hemolysis, thrombocytopenia, and a normal leucocyte count. An expansion of the PI-deficient clones up to 86, 86, and 7 % of the granulocyte, monocyte, and erythroid lineages, respectively (Fig. 1) was noted. Fig. 1 PNH clones at diagnosis (a) and their expansion (b) after treatment for SAA. Comparison of the PI-deficient clones at diagnosis of severe aplastic anemia (a) and after triple immunosuppression (b). Monocytes and granulocytes were gated based on scatter ... Three months after initial presentation, he was re-admitted to hospital because of abdominal pain and bloody stools. Thrombosis of the superior mesenteric vein was diagnosed and anticoagulant therapy was instituted with nadroparin 15,200 IU/day subcutaneously. Eculizumab treatment was considered, but because of the complete absence of intravascular hemolysis, the effect of the anticoagulant therapy was awaited. Subsequently, he developed headache and a left hemiparesis. Repeated CT scans of the brain showed expanding ischemic areas parieto-occipital with secondary bleeding, and the patient finally died. Thrombosis is the most feared and life-threatening complication in PNH patients, because after onset, it is frequently progressive and refractory to anticoagulant therapy [1, 2]. The mechanism of thrombotic complications in PNH is not completely understood, but it has mainly been attributed to intravascular hemolysis [1]. Experimental studies show that the free plasma hemoglobin liberated during hemolysis scavenges nitric oxide (NO). As a result, NO depletion produces dystonia and spasm of the smooth muscle, suppresses the anti-inflammatory effect on the endothelium and promotes platelet activation and thrombosis [3]. Eculizumab is a monoclonal antibody that binds complement factor C5, thereby inhibiting complement activation on erythrocytes and reducing hemolysis. As a result, eculizumab decreases thrombotic risks in PNH patients [2, 4, 5]. Interestingly, our patient suffered from progressive abdominal and cerebral thrombotic complications without any signs of intravascular hemolysis. It is also possible that the pathogenesis of thrombosis in PNH might be caused by other mechanisms associated to complement activation and not directly related to hemolysis [1, 6]. We speculate that the mechanism of thrombosis could be associated to platelet activation caused by the absence of CD59 and CD55 on the platelet surface [7, 8]. Internationally accepted indications for eculizumab treatment include transfusion-dependent hemolysis (four or more transfusions in 12 months) and PNH-related complications (i.e., thrombosis or renal failure) regardless of transfusion history [3]. Although no clinical data are available, eculizumab could be indicated in patients with thrombotic events in the absence of hemolysis [5, 9, 10].