Methylcellulose Colony Formation Assay Research Articles

Clinical Significance of USP5 Expression Level in Acute Myeloid Leukemia and Its Regulatory Effects on AKT/mTOR/4EBP1 Signaling Pathway

To investigate the clinical significance, functional role and potential downstream mechanism of USP5 in acute myeloid leukemia (AML). The expression of USP5 in AML and normal tissues and its correlation with patients' survival were analyzed based on TCGA database. USP5 was knocked down and overexpressed in Jurkat and HL-60 cells using lentivirus. USP5 mRNA and protein expression were detected by RT-qPCR and Western blot, respectively. Cell proliferation and growth were measured by CCK-8 and methylcellulose colony-forming assay. Flow cytometry was used to analyze cell cycle and apoptosis. USP5 was highly expression in AML compared with normal tissues. Up-regulation of USP5 was negatively correlated with the survival of AML patients. USP5 knockdown and overexpression inhibited and promoted the proliferation and colony growth of AML cells, respectively. Cell cycle arrest and apoptosis were induced in USP5 knockdown Jurkat and HL-60 cells. Furthermore, USP5 knockdown inhibited the phosphrylation of AKT, mTOR and 4EBP1. Overexpression of USP5 predicts poor survival of AML patients. Targeting USP5 suppresses AKT/mTOR/4EBP1 signaling and reduces the proliferation and growth of AML cells.

Interleukin-17A Directly Signals to Bone Marrow-Derived Hematopoietic Stem and Progenitor Cells to Promote Their Expansion and Differentiation in Vitro

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.

Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation

Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.

Abstract LB095: A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer

Abstract INTRODUCTION: A growing body of evidence suggests that increased expression of the vascular endothelial growth factor (VEGF) co-receptor Neuropilin-2 (NRP2) is associated with aggressive breast cancers and that VEGF/NRP2 signaling contributes to clinical resistance to chemotherapy and tumor recurrence, making NRP2 a promising therapeutic target. A major limitation that has hampered the development of such a therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that specifically block VEGF/NRP2 signaling. aTyr has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of diseases that involve NRP2 signaling. Among them, ATYR2810 has been characterized to bind to the b1 domain of NRP2 that encompasses the VEGF binding sites. It completely blocks the binding of VEGF to NRP2, and VEGF-induced NRP2/VEGFR dimerization. Importantly, ATYR2810 had no effect on Semaphorin 3F (Sema3F) induced NRP2/PlexinA1 dimerization, demonstrating its specificity for blocking the VEGF/NRP2 pathway. We have shown that ATYR2810, but not a Sema3F-blocking mAb has tumor-inhibitory effects on triple negative breast cancer (TNBC) cell lines or patient-derived organoids. RESULTS: We further evaluated the efficacy of ATYR2810 in combination with standard-of-care therapeutics including Cisplatin and Bevacizumab (anti-VEGF-A blocking antibody). In in vitro 3D methylcellulose colony formation assays, ATYR2810 sensitized TNBC cells to Cisplatin or Bevacizumab and considerably reduced colony formation in combination with these treatments. In an in vivo TNBC xenograft cancer model (MDA-MB-231), ATYR2810 augmented the anti-tumor effects of Cisplatin or Bevacizumab. To explore the underlying molecular mechanism, we performed gene expression profiling with samples treated by ATYR2810 alone and the combo therapy with Cisplatin or Bevacizumab. A number of gene markers of cancer stem cells (CSC) and/or epithelial-mesenchymal transition (EMT) were found to be down-regulated by ATYR2810 treatment in TNBC patient-derived organoids, including a key EMT transcription factor ZEB1. We also confirmed the reduction of ZEB1 protein expression by ATYR2810 treatment in TNBC cells. CONCLUSIONS: These results demonstrate the efficacy of ATYR2810 in combination with standard-of-care therapeutics in in vitro and in vivo TNBC models, and suggest its activity is mediated through inhibiting both EMT and cellular dedifferentiation that renders tumors more sensitive to the treatment regimes. The targeting of VEGF/NRP2 signaling by ATYR2810 may provide a new therapeutic option, and lead to the identification of new treatment biomarkers, which could offer improved efficacy and reduced toxicity in aggressive breast cancers. Citation Format: Zhiwen Xu, Christoph Burkart, Hira L. Goel, Justin Rahman, Clara Polizzi, Matt Seikkula, Luke Burman, Arthur M. Mercurio, Leslie A. Nangle. A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB095.

TET2 Deficiency Led to Expansion of Dysfunctional Erythroid Progenitors

Erythrocyte development is a complex process involving multiple developmental states that include erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells and erythroid precursors, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts in the bone marrow. Orthochromatic erythroblasts expel nucleus to become reticulocytes which mature into erythrocytes in blood stream. Defects at any of these developmental stages will lead to anemia. Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenia and ineffective hematopoiesis. Anemia is the defining cytopenia in the majority of patients with MDS, yet the molecular mechanisms for dyserythropoiesis in MDS remain largely unclear. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is the most common mutation in MDS.In the present study, we explored shRNA-mediated knockdown approach in human CD34+ cell to study the role of TET2 in human erythropoiesis. We show that TET2 is downregulated starting at the CFU-E stage. Growth curves of purified CFU-E cells show no difference between control and TET2 knockdown cells until day 6. An increased proliferation of TET2 knockdown-CFU-E cells is noted starting at day 7. Interestingly, while the growth of control CFU-E cells plateaued on day 11 with completion of terminal erythroid differentiation and enucleation, TET2 knockdown CFU-E cells continued their growth for additional 13 days. Flow cytometry and cytospin morphologic analyses revealed that by day 11 all control cells differentiated into GPA+ late stage erythroblasts or reticulocytes. In marked contrast, about 30 % of TET2 knockdown cells are still GPA- but CD71+, IL-3R-, CD34-, CD36+, phenotypically resembling erythroid progenitor CFU-E cells. Surprisingly, in contrast to freshly purified control and TET2 knockdown CFU-E cells that generate colonies in methylcellulose colony forming assays, these cells fail to give rise to CFU-E colonies. We thus termed these late proliferating cells as “TET2 knockdown marker CFU-E”. Further characterization of the “TET2 knockdown marker CFU-E” cells revealed that in contrast to control CFU-E, the “TET2 knockdown marker CFU-E” exhibit following distinct features: 1) they had lower proliferative potential; 2) their proliferation required both EPO and SCF (while control CFU-E cells only require EPO); 3) they underwent extensive apoptosis in the absence of SCF; 4) they failed to undergo terminal erythroid differentiation. These findings demonstrate that TET2 knockdown led to expansion of dysfunctional erythroid progenitors.To explore the underlying molecular mechanisms, we performed RNA-seq analysis on control CFU-E, TET2 knockdown CFU-E and “TET2 knockdown marker CFU-E” cells. Bioinformatics analysis revealed that while only about 50 genes were differentially expressed between control CFU-E and TET2 knockdown CFU-E, about 400 genes were differentially expressed between control CFU-E and “TET2 knockdown marker CFU-E”. Gene set enrichment analysis revealed upregulation of pathways associated with apoptosis/cell death. Notably, FAS (CD95), the cell surface death receptor previously shown to be upregulated in MDS bone marrow cells, was among the top five upregulated apoptosis associated genes. The increased surface expression of FAS was also noted in TET2-knockdown cells as assessed by flow cytometry. Furthermore, the increased surface expression of FAS was detected in primary bone marrow erythroblasts of MDS patients with TET2 mutation. Our findings uncovered previously unknown roles of TET2 and its potential underlying molecular mechanisms. As the phenotypic changes upon TET2 knockdown resemble the characteristic pathological changes of MDS, our findings suggest the involvement of TET2 deficiency in the pathophysiology of MDS. DisclosuresAli:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. Raza:Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau; Genoptix: Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Janssen R&D: Research Funding.

MMSET I Acts As an Oncoprotein and Regulates GLO1 Expression in T(4;14) Multiple Myeloma Cells

Recurrent chromosomal translocations are central to the pathogenesis of multiple myeloma (MM). The translocation t(4;14)(p16;q32) is one of the most common translocation and associated with poor prognosis. T(4;14) MM universally overexpresses MMSET that exists in three major isoforms, full length MMSET II, and short isoforms REIIBP and MMSET I. MMSET II has been suggested to play an important tumorigenic role in t(4;14) MM, but little is yet known about whether and how the MMSET short isoforms contribute to MM tumorigenesis. Survival analysis indicated there was no significant difference in outcomes between t(4;14) positive patients expressing MMSET II (breakpoint MB4-1) or not expressing MMSET II (breakpoint MB4-2/MB4-3). Therefore overexpression of MMSET II is not necessary for the poor outcome of t(4;14) MM. The truncated or short isoforms of MMSET might also be important for t(4;14) MM. The aim of this study is to characterize the role of short isoform MMSET I and determine its downstream targets in t(4;14) MM.MMSET I was knocked down by shRNAs in four t(4;14) MM cell lines (KMS11, KMS18, KMS28BM and KMS34). MMSET I knockdown (KD) reduced cell cycle S phase and induced cell apoptosis. MMSET I KD significantly inhibited the capacity to produce colonies in t(4;14) MM cells on methylcellulose colony formation assay. MMSET I KD inhibited KMS11 tumor formation or reduced tumor size (ShLuc vs ShMMSET I, 8/8 vs 2/8) in xenograft studies. Gene array analysis indicated 46 genes down-regulated, and 22 genes up-regulated upon MMSET I KD in t(4;14) MM cell lines. Glyoxalase I (GLO1) was one of the top 5 down-regulated genes and was down-regulated in all 4 cell lines upon MMSET I KD. qPCR and western blot (WB) analysis confirmed GLO1 expression was down-regulated upon MMSET I KD. When MMSET I was transfected into U266 cells, a non-t(4;14) MM cell line, the ectopic MMSET I decreased cell apoptosis, promoted U266 cell colony formation and tumorigenicity in vivo (VCon vs MMSET I, 1/7 vs 4/7). qPCR and WB analysis indicated ectopic MMSET I also increased GLO1 expression. ChIP analysis showed binding of MMSET I at upstream of the GLO1 transcription start site (-350bp). When U266 cells were transfected with MMSET I mutant vectors, WB analysis showed that both the MMSET I N and C-terminus participated in regulating GLO1 expression, but only the C-terminus HMG-b domain was necessary to regulate GLO1 expression. These observations confirm that MMSET I is a bona fide oncogene and promotes GLO1 expression.GLO1 belongs to the glyoxalase (GLO) system, which catalyzes the conversion of deleterious methylglyoxal (MG) to non-toxic D-lactate in cytoplasm. GLO1 KD induced apoptosis and reduced colony formation greatly in t(4;14) MM cells. WB analysis showed MMSET I or GLO1 KD increased MG-modified protein significantly, and reduced the levels of anti-apoptosis factors such as MCL1 and BCL2. In cell growth media, ascorbate is oxidized to dehydroascorbate (DHA). DHA is transported across cellular membrane and reduced to ascorbate in the cytoplasm by depleting glutathione (GSH), which is required for GLO detoxification pathway. Therefore we hypothesize that ascorbate will inhibit GLO pathway through decreasing GSH level, result in MG accumulation and inhibition of cell growth. As expected, ascorbate reduced GSH level and increased MG-modified protein level in t(4;14) MM cells. CTG assays indicated ascorbate less than 0.2 mM inhibited t(4;14) MM cell growth significantly in 48 h. Apoptosis analysis showed that ascorbate was cytotoxic instead of cytostatic to t(4;14) MM cells. The effect of ascorbate was then investigated in a NOD/scid KMS11 xenograft model. Compared with vehicle control, ascorbate extended survival of the mice significantly (on day 54 after cell injection, 0/8 vs 6/8, P < 0.01). Flow cytometric analysis indicated that IV injection of KMS11 led to spleen and bone marrow infiltration, which was inhibited by ascorbate treatment. Femur weight showed ascorbate treatment also reduced bone lesion greatly.Taken together, our data indicated that MMSET I acts as an oncoprotein and regulates GLO1 expression in t(4;14) multiple myeloma cells. We found that ascorbate inhibits GLO pathway through depleting GSH and is cytotoxic to t(4;14) MM cells. Furthermore ascorbate alone inhibits tumor formation and extends mouse survival in MM xenograft model. Our study suggested that ascorbate might be exploited therapeutically for t(4;14) MM. DisclosuresChng:Janssen China R&D: Research Funding.

Myeloid Progenitors of Defined Features Provide Permissiveness to Leukemic Transformation

The conventional view of oncogenesis posits that driver mutations, when present in appropriate cell types, initiate malignant transformation. At the apex of the differentiation hierarchy, stem cells, such as hematopoietic stem cells, have been suggested to be the cell-of-origin for a variety of hematopoietic malignancies. However, since the mutation bearing hematopoietic stem cells can differentiate to give rise to downstream progeny, it suggests the possibility that some of the downstream cells be the relevant cell-of-origin permitting malignant transformation. Given that cancer cell-of-origin has been a product of logic deduction evading rigorous experimentation, whether and how their unique cellular properties, if any, contribute to transformation remain undetermined.To experimentally define the key properties of the cell-of-origin permissive to oncogenic transformation, we constructed a doxycycline (Dox) inducible MLL-AF9 knock-in mouse model, which allows precise temporal control of oncogene expression in desired hematopoietic cells, in vitro and in vivo . Whole body induction by Dox led to 100% lethality due to acute myeloid leukemia (AML) within 6-8 weeks, while the un-induced littermates remained healthy, confirming the inducibility and potency of the MLL-AF9 oncogene. To minimize the differences contributed by cellular differentiation stages and to focus solely on the contributions by specific cellular properties, we narrowed our analysis on a phenotypically homogeneous population, the granulocyte-macrophage progenitors (GMPs), which have been shown to be critically important in supporting leukemic development (Ugale et al ., 2014, Cell Reports; Ye et al ., 2015, Cell Stem Cell). We found that although MLL-AF9 expression can be induced in all GMPs, only a subset (~25%) of them is permissive to transformation. These results prompted us to investigate the underlying cellular and molecular properties that enable oncogenic transformation.Inspired by our earlier work demonstrating that the cell fate plasticity of GMPs is related to their rapid cell cycle behavior (Guo et al ., 2014, Cell), we tested whether the rapid proliferation of GMPs underlies their permissiveness to oncogenic transformation. If so, transformation potential should be enriched in the faster-proliferating GMP subsets. To test this possibility, we designed single cell assays to investigate the transformation efficiencies of GMPs with regard to their cell cycle speeds. Specifically, the intrinsic cell cycle speed was measured within 24 hours of Dox addition, and transformation status of hundreds of individual GMPs was determined by methylcellulose colony formation assay. Within the first 24 hours of Dox addition, the cell cycle speed remained unchanged, reflecting the intrinsic cell cycle kinetics, rather than a proliferative response to oncogene expression. Our results show that the faster a GMP divides, the more likely it transforms in response to MLL-AF9 expression. Strikingly, most GMPs that can divide more than 3 times within 24 hours are transformed. These data indicate the fastest cycling GMPs provide the oncogene with a permissive cellular context to exert its function. Furthermore, prospectively isolated faster cycling GMPs enriched for transformed colony formation in vitro, and induced earlier lethal AML in vivo . Importantly, transient deceleration of GMPs for 48 hours by low-dose CDK4/6 inhibitor, Palbociclib, significantly reduced transformation efficiency by ~ 60%. Taken together, these results demonstrate that MLL-AF9 initiated transformation is dependent on specific cellular properties which are associated with a rapid cell cycle. Genomic analyses of the transformation-permissive GMPs, using non-permissive GMPs as controls, are being performed to define the permissiveness in molecular details, on both transcriptional and epigenetic levels. DisclosuresNo relevant conflicts of interest to declare.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2− CD34− Lin− Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2− CD34− LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2− CD34− LSK-HSCs.

Glucocorticoids Induce the Maintenance and Expansion of an Immature CFU-E Erythroid Progenitor Population in Humans

Glucocorticoids are widely used to increase red cell production in a number of hematological disorders including Diamond Blackfan anemia (DBA). Despite their clinical effectiveness we do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. To improve the clinical management of these patients, we studied the mechanisms of dexamethasone (Dex) on human erythroid progenitors. We employed an erythroid culture system to differentiate primary human CD34+ cells isolated from patients with DBA and healthy controls as well as cord blood samples. We found that Dex increases the proliferation of CD34+ cells derived from peripheral blood but surprisingly not of CD34+ cells from cord blood. Furthermore, by mass spectrometry proteomic analysis, we found that Dex specifically altered the induction of several targets unique to peripheral blood including the transcription factor and cell cycle regulator NR4A1. When examining the cell populations expanded with Dex treatment by immunophenotyping, we detected a specific expansion of CFU-Es. Dex increased the average size of CFU-E colonies in methylcellulose colony forming assays in the presence of Epo alone, suggesting that CFU-Es are the most Dex responsive progenitor in humans. In support of this hypothesis, reticulocyte counts from patients with DBA treated with prednisone were found to increase approximately one week after treatment, consistent with the time course of differentiation of CFU-E to reticulocytes in bone marrow. In order to further resolve the progenitor target of steroid responsive progenitor we developed a new gating scheme that allows for increased resolution of BFU-E and CFU-E subpopulations by monitoring surface expression of CD71 and CD105. Using this system, we found that Dex treatment led to the maintenance of a novel immature transitional progenitor population in peripheral blood-derived cells that we term immature CFU-E. In methylcellulose colony forming assays, these peripheral blood-derived immature CFU-E treated with Dex produced larger colonies than the untreated controls and mature CFU-E. The expansion and proliferation of Dex treated immature CFU-Es derived from peripheral blood was associated with a decreased percentage of cells in the S phase of the cell cycle. No change was noted for either Dex treated BFU-E from peripheral blood or for BFU-E or CFU-E from cord blood. Importantly, Dex responsive peripheral blood-derived CFU-Es divided at a faster rate than untreated CFU-Es, revealing a dissociation in the link between differentiation and proliferation. Mechanistically, we found that Dex treated peripheral blood CFU-E exhibited increased expression of the negative cell cycle regulator p57Kip2. Moreover, this protein was also found to be dysregulated in CD34+ cells from transfusion-dependent patients with DBA, wherein p57Kip2 expression was not significantly altered following Dex treatment, indicating a role for p57Kip2 in the erythroid progenitor defects seen in these patients. To further evaluate the role of p57Kip2 in the Dex dependent alteration in CFU-E proliferation, peripheral blood-derived CD34+ cells were transduced with p57Kip2-specific shRNA lentiviral constructs. shRNA knockdown of p57Kip2 abrogated the effects of Dex on growth and induced a premature EPO-mediated differentiation. In summary, our findings provide new insights into functional heterogeneity of human erythroid progenitors and reveal mechanistic insights into the action of Dex in normal and disordered erythropoiesis. Disclosures Vlachos: Novartis: Other: Steering committee member.

NUP98-KDM5A Fusion Induces Hematopoietic Cell Proliferation and Alters Myelo-Erythropoietic Differentiation

Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied. In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p&lt;0.01). In addition to a growth advantage, expression of NUP98-KDM5A led to differences in differentiation. NUP98-KDM5A expressing cells showed a 4.9-fold decrease (p&lt;0.05) in myeloid terminal differentiation (defined as the CD33+, CD11B+ population) and 2.8-fold increase (p&lt;0.05) in early stem cell progenitors (defined as the CD117+ population). Interestingly, NUP98-KDM5A expressing cells showed a 3.3-fold increase (p&lt;0.01) in the erythroid population (defined by the CD235a+, CD71+ population). The increase in erythroid differentiation was confirmed by RNA sequencing, which showed an enrichment in genes involved in heme metabolism (Hallmark Heme Metabolism, FDR q-value 7.33x10-8) after only 72 hours post-transduction. Furthermore, RNA sequencing demonstrated upregulation of HOXA genes, including HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10 and HOXA11 (p 3.36x10-2 - 1.16x10-7), which has been previously described in other high-risk leukemic fusions (including other NUP98 fusions) as a primary mechanism of stem-cell renewal. To further elucidate NUP98-KDM5A effects on differentiation, cytokine supplements known to drive myeloid differentiation (SCF, TPO, G-CSF, and GM-CSF) were added to culture media, which demonstrated the same disruption in myelo-erythropoiesis based on a 4.0-fold decrease (p&lt;0.001) in the terminal myeloid population, a 3.2-fold increase in CD117+ (p&lt;0.001), and 2.6-fold increase in erythroid markers (p&lt;0.001). Furthermore, NUP98-KDM5A recapitulated this phenotype in methylcellulose colony forming assays, with increased BFU-E and reduced CFU-GM colonies compared to control (p&lt;0.01), despite no difference in overall colony number. Taken together, these in vitro studies demonstrate increased proliferation and altered differentiation in HSPCs by NUP98-KDM5A, with the pattern of differentiation by NUP98-KDM5A paralleling the M6 (erythroleukemia) AML subtype observed in patient samples harboring this fusion, supporting the clinical observation that NUP98-KDM5A is often a monogenic driver. To evaluate the leukemic potential of NUP98-KDM5A, cells were injected into NRG-SGM3 mice with NUP98-KDM5A inducing at 12 weeks a rapidly lethal myeloid disease in vivo (97.8% human CD45+ cells vs. 2.3% in control, p&lt;0.001). Furthermore, spleens of the NUP98-KDM5A mice were markedly enlarged, weighing on average 926.7g vs. 47.3g spleens seen in control, a 19.8-fold increase in weight (p&lt;0.05). In conclusion, the expression of NUP98-KDM5A in HSPCs drives proliferation and alters differentiation to maintain stem cell markers while driving an erythroid phenotype in vitro. Furthermore, this fusion is sufficient to engraft in the bone marrow and spleen of NRG-SGM3 mice in vivo and cause marked splenomegaly. Taken together, these data suggest this model properly recapitulates the human disease phenotype seen in patients expressing NUP98-KDM5A and future functional assays and drug screens may provide important insights into understanding the pathophysiology and pharmacologic vulnerabilities of this fusion class. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.

A Distinct Metabolic Signature in DNMT3A-Mutated Leukemia

Background: Acute myeloid leukemia (AML) is a type of cancer that consists of a group of hematological malignancies with high heterogeneity. AML patients with DNMT3A mutations often have a poor prognosis. Metabolic alterations have long been recognized in cancer cells. Metabolic homeostasis is a fundamental property of cells that becomes dysregulated in cancer to meet the altered, often heightened, demand for metabolism for increased growth and proliferation. Oncogenic mutations can directly change the cellular metabolism, priming cells for malignancy. It is well known that 2-hydroxyglutarate (2-HG) is an oncometabolite resulting from mutations of the isocitrate dehydrogenase 1 and 2 (IDH1and IDH2) genes and isa strong prognostic predictor independent of other clinical and molecular features. Alpha-ketoglutarate-dependent dioxygenase encodedby the TET2 gene could catalyze 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), thereby regulating DNA methylation and inducing hematopoietic malignancies. Methods: Metabolomic profiles of all serum samples were achieved using the GC-TOF MS platform. CCK-8 assays were used to examine cell viability and cytotoxic activity of 2-ketoisocaproic acid (2-KA) to leukemia cells. Methyl-Cellulose colony-forming cell assays and flow cytometry were performed to assess cell proliferation and cell differentiation. The pIpC was administrated in the Dnmt3aR878H/WTMx1Cre+mice at the time of weaning (4 weeks old) to activate transgene expression in vivo. The expressions of genes/proteins were detected by RNA sequencing, quantitative RT-PCR and western blot. Results: We report a metabolomics study with a total of 171 newly diagnosed AML patients and identified a distinct leucine metabolic signature in AML patients with DNMT3A mutations. The prognostic value of 2-KA was demonstrated in cytogenetically normal AML patients, and a low 2-KA level predicted a poor overall patient survival. We further compared the gene expression patterns of the blast cells in bone marrow (BM) between AML groups with and without a DNMT3A mutation; these patterns correlated well with those of the leucine metabolic pathways and exhibited enhanced ACAA1 and decreased ALDH7A1 gene expression in DNMT3A-mutated leukemic cells. In vitro results demonstrated that a high 2-KA level could inhibit the proliferation of DNMT3A-mutated cells but did not affect the differentiation of these leukemic cells. Conclusions:Our data suggest that 2-KA isa unique feature of AML with a DNMT3A mutation and might be a metabolic marker of early-stage hematopoietic malignances.This study also suggests that ACAA1 and ALDH7A1 were aberrantly expressed in DNMT3A mutant leukemic cells and may be potential targetsfor AML therapy. We also hypothesized that a combination of the metabolic inhibitor and chemotherapeutic agent may be a possible treatment strategy for DNMT3A-mutated AML. Disclosures No relevant conflicts of interest to declare.

Abstract 2953: Selective activity of XPO1 inhibitors in TET2-mutant myeloid malignancies

Abstract TET2 is among the most frequently mutated genes in hematopoietic malignancies. Inactivating mutations in TET2 are found in ∼30% of patients with myelodysplastic syndrome (MDS), ∼19% with de novo acute myeloid leukemia (AML), and ~15% with myeloproliferative neoplasm (MPN). TET2 mutations are also identified in a subset of individuals over 50 years of age with clonal hematopoiesis of indeterminate potential (CHIP), a condition that predisposes affected individuals to progression to myeloid malignancy and atherosclerotic heart disease with heart attack or stroke. TET2 mutations represent an early genetic lesion in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state of clonal dominance that predisposes to the acquisition of additional mutations. Thus, effective therapy for patients with TET2 mutations in HSPCs will require identifying drugs that are selectively lethal to TET2 mutant HSPCs but spare normal HSPCs, a property analogous to the widely studied genetic relationship called “synthetic lethality”. Using a tet2-mutant zebrafish model created in our laboratory, we screened for drugs from libraries of FDA-approved compounds and drugs in Phase I/II testing. We found that nuclear exporter XPO1 inhibitors, selinexor (KPT-330) and eltanexor (KPT-8602), were among the most promising, in that they are selectively lethal to HSPCs in tet2-mutant compared to wild-type fish. Moreover, we treated HSPCs of wild-type and Tet2-deficient mice with both selinexor and eltanexor in a methylcellulose colony formation assay. WT HSPCs lose their replating capacity at passage 3 (P3), while Tet2-mutant clones demonstrate aberrant sustained self-renewal capacity over multiple replatings. We found that both drugs selectively kill primary Tet2-mutant murine HSPCs and also block the aberrant self-renewal of these cells. By transplanting WT and Tet2-mutant CD45.2 donor cells into CD45.1 recipient mice, we monitored peripheral blood cells before and after treatment with selinexor or eltanexor to explore synthetic lethality of these drugs in vivo. Moreover, the human AML cell line K562 with TET2 homozygous mutations generated with CRISPR-Cas9, was more sensitive to selinexor and eltanexor than the parental cell line. To elucidate the mechanism behind the selective targeting of Tet2-mutant blood stem cells, we will use PRO-Seq and START-Seq methods to identify transcriptional elongation and initiation sites at single-nucleotide resolution. These preclinical studies must be done to show a therapeutic efficacy and mechanism of action of these new drugs before they can be translated to improve therapy of patients who have TET2 inactivating mutations. Citation Format: Nicole Prutsch, Chang-Bin Jing, Julia Etchin, Alla Berezovskaya, Hong Tiv, Michael J. Poitras, Prafulla Gokhale, Yosef Landesman, A. Thomas Look. Selective activity of XPO1 inhibitors in TET2-mutant myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2953.

Dexamethasone Accelerates the Transition of Human BFU-E to CFU-E and Enhances CFU-E Proliferation through Cell Cycle Regulation

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is frequently managed with glucocorticoids to increase red cell mass. Despite sustained effective clinical use for many decades in DBA and other anemias, we still do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. In order to improve the clinical management of patients with DBA, we studied the mechanisms of action of dexamethasone (Dex) on erythroid progenitors.To study the effects of Dex on erythroid progenitor biology, we employed a serum-free erythroid culture system to differentiate primary human CD34+ cells isolated from the peripheral blood of adults or DBA patients as well as cord blood samples. We found that Dex increases the total proliferation of CD34+ cells from adult peripheral blood but not in cord blood over 14 days in culture. Dex treatment also led to an acceleration of the BFU-E to CFU-E transition in peripheral blood with a minimal effect in cord blood. In methylcellulose colony forming assays, peripheral blood derived CFU-E treated with Dex for 24hrs produced more colonies or larger colonies than the untreated controls while a lesser effect was seen in cord blood derived CFU-E. Both assays revealed that Dex treatment enhanced self-renewal of peripheral blood derived CFU-E.We also noted that BFU-E and CFU-E differentially express the glucocorticoid receptor (GR) isoforms, GRα and GRβ. BFU-E expressed comparable levels of GRα and the dominant-negative GRβ, whereas only the GRα transcript was identified in CFU-E. We observed similar expression patterns for GR between peripheral and cord blood derived progenitors. Importantly, following Dex treatment, GRα was shown to translocate to the nucleus of erythroid progenitors derived from peripheral blood derived CD34+ cells but not in the cord blood derived progenitors.When we examined the cell cycle progression of sorted human erythroid progenitors, CFU-E and BFU-E, we observed a G0/G1 arrest of peripheral blood CFU-E treated with Dex compared to untreated CFU-E. In marked contrast, no such change was noted for either Dex treated BFU-E from peripheral blood or for CFU-E or BFU-E from cord blood. In addition, we found an increase in the expression of cell cycle regulators p57Kip2 and p27Kip1 in Dex treated peripheral blood CFU-E but no such increased expression was seen in cord blood CFU-E.When peripheral blood derived erythroid progenitors were treated with olomoucine (Olo), a small molecule CDK inhibitor, we observed an acceleration of the BFU-E to CFU-E transition similar to that induced by Dex. Upon co-treatment with Dex and Olo, there was a synergistic effect in the accelerated formation of CFU-E population from BFU-Es. To validate this finding of the dependence of CFU-E on cell cycle regulators, we transduced peripheral blood derived CD34+ cells with a lentiviral shRNA construct to knockdown p57 expression. Following successful knockdown of the expression levels of p57, the numbers of CFU-E indeed decreased implying a role for cell cycle in defective transition of BFU-E to CFU-E.Erythroid progenitors derived from cultures of primary CD34+ cells from DBA patients were also studied. Importantly, we noted increased baseline expression of p57Kip2 and p27Kip1 in erythroid progenitors from steroid resistant DBA patients and in contrast to controls, their expression did not increase following Dex treatment.Our findings provide novel mechanistic insights into the regulation of human erythropoiesis by Dex. We have shown that alterations in cell cycle regulation of erythroid progenitors plays a key role in the effects of glucocorticoids on red blood cells. Furthermore, this work has direct relevance for the development of improved therapeutic treatment strategies for patients with bone marrow failure syndromes such as DBA. DisclosuresNo relevant conflicts of interest to declare.

Leukemia Risk Gene ARID5B is a Crucial Regulator of B-Cell Development

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Genome wide association studies by us and others have identified germline genetic variants at the ARID5B locus that strongly influence susceptibility to ALL (Nat Genet 2009, J Clin Oncol 2012). Despite compelling results from these genetic association studies, the molecular mechanisms by which ARID5B contributes to ALL pathogenesis remains largely unknown. ARID5B is a member of the ARID transcription family characterized by a conservative AT rich binding domain, but the physiological functions of ARID5B are poorly understood. Therefore we sought to develop mouse models to comprehensively characterize the roles of ARID5B in normal and malignant hematopoiesis. To this end, we first established a Vav-1 specific Arid5b overexpression (AOE) mouse model. Mice were designed with a tetO element knocked in before the start codon of Arid5b to create an Arid5b tetO mouse. Arid5b tetO mice were then crossed with Vav-1-tTA mice and, overexpression of Arid5b confirmed in the hematopoietic system of progenies with the desired genotype.At 6-8 weeks old complete blood counts (CBC) analysis revealed that AOE mice had a large decrease in white blood cells (WBCs) and a slight reduction in red blood cells (RBCs) and hemoglobin (Hb). We also noted a significant reduction in the bone marrow cellularity of our Arid5b overexpressing mice. To further characterize our AOE mouse model we used flow cytometry to quantify the proportions of mature as well as various stem and progenitor cell populations during hematopoiesis. We found that AOE mice showed an increase in the MegE biased MPP2 (LSK flt3−/CD150+/CD48+) population and a decrease in the lymphoid biased MPP4 (LSK (Flt3+/CD150−/CD48+/−) population. Overexpression of Arid5b resulted in a significant reduction in the proportion of all bone marrow B cell populations (Hardy Fraction A-F) (Figure 1A) and B220+ cells in the spleen. In vitro methylcellulose colony forming assays further revealed a loss of functional pre B lymphoid progenitor in AOE bone marrow. To determine if the hematopoietic defects seen in younger AOE mice was due to a delay in hematopoiesis we aged AOE mice. We found that as we aged AOE mice there was still a reduction in bone marrow cellularity and all bone marrow B cell populations. Surprisingly, as mice were aged we found that AOE mice were prone to sudden death and displayed a dramatic reduction in overall survival when compared to wildtype littermates with a mean survival age of 8 months (Figure 1B). At the time of death, we found that AOE mice presented with severely enlarged spleens. To predict the death of AOE mice we performed ultrasounds to track spleen volume. Based on ultrasounds performed on AOE mice and wildtype littermates, AOE mice spleens become larger than those of their littermates as early as 6 months old, however spleen volume did not predict sudden death. In aged AOE mice, while the spleens were grossly enlarged and contained larger amounts of red pulp and increased Ter119+ cells, there was still a reduction in the proportion of B cells and no increase in the proportions of other white blood cells (Figure 1D). Older AOE mice exhibited anemia, hypergammaglobulinemia, and splenomegaly. By 30 weeks, defects in B cell proportions were seen in the lymph nodes of AOE mice via immunohistochemistry (Figure 1C). Compared to wildtype littermates, AOE mice displayed reduced white blood cells, red blood cells, and platelets (Figure 1D). The anemia was associated with higher reticulocyte numbers and increased serum erythropoietin concentration. The life span of erythrocytes from AOE mice was less than that of wildtype littermates. Together, these results indicated that Arid5b plays an important role in B lymphopoiesis and erythropoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Roles of Cyclin A, Myc, Jun and Ppm1l in tumourigenic transformation of NIH3T3 cell

To analyse the mechanism of tumourigenic transformation of NIH3T3 cells at the transcriptional level, we used cancerogen 3-methylcholanthrene (3-MCA) and cancerogenic substance phorbol-12-myristate-13-acetate (PMA) to transform NIH3T3 cells and the assessment of transformation was performed using Giemsa staining and methylcellulose colony formation assay. Changes in gene expression profile were detected by Mouse Genome 430 2.0 microarray; and quantitative real-time polymerase chain reaction and Western blotting were used to verify the expression changes of mRNAs and proteins, respectively. With the aid of bioinformatics method, five signalling pathways were identified to participate in different stages of NIH3T3 cell transformation. Further, our study suggested that oncogenes Cyclin A, Myc, Jun and the tumour suppressor gene Ppm1l may play important roles in these pathways.

Abstract 2499: Identification of functional proteins interacting with NSD2 in multiple myeloma

Abstract Multiple myeloma (MM), characterized by the uncontrolled proliferation of malignant plasma cells, occurs mainly in the elderly population. Recurrent chromosomal translocations are central to the pathogenesis of MM, with t(4;14) being the second most common and associated with poor prognosis. The histone methyltransferase (HMTase) NSD2 is overexpressed in MM due to the t(4;14) translocation and has been suggested to play an important tumorigenic role in t(4;14) MM. However, the detailed molecular mechanism or signaling pathways describing how deregulation of NDS2 contributes to MM cell growth and survival are still uncertain. In this study, stable isotope labeling of amino acid in cell culture (SILAC)-based mass spectrometry analysis was used to determine NSD2-interacting proteins. Mass spectrometry analysis indicated that there were 75 proteins identified in the immunoprecipitation of NSD2 protein complexes. Gene ontology (GO) enrichment analysis showed that these proteins were enriched in processes related to chromatin organization, RNA processing and translation. Five high potential MMSET-interacting proteins from the most enriched functions were selected and verified by Immunoprecipitation and Western blot assays. The results indicated that there were interactions between NSD2 and SMARCA2, topoisomerase IIa, ADAR1, DDX5 and RPS6. In silico analysis with STRING database showed that SMARCA2 interacts with NSD2. SMARCA2 is the ATPase subunit of the SWI/SNF chromatin remodeling complex, which harnesses the energy from ATP to remodel the position of nucleosomes, making the DNA accessible during transcription, replication and DNA repair. Double immunofluorescence showed NSD2 and SMARCA2 co-localized in the cell nucleus. SMARCA2 was knocked down (KD) by shRNAs in t(4;14) MM cells. CellTiter-Glo Luminescent Cell Viability Assay (CTG assay) indicated that SMARCA2 KD by shRNA reduced t(4;14) MM cell growth. SMARCA2 KD also significantly inhibited the capacity to form colonies in t(4;14) MM cells on methylcellulose colony formation assay. Inhibiting SMARCA2 with active DNA-dependent ATPase A domain inhibitor (ADAADi) also reduced t(4;14) MM cell growth. In addition, SMARCA2 KD reduced cell cycle S phase. Western blot analysis showed downregulation of PRL3 and CCND1 protein level upon NSD2 or SMARCA2 KD. This study reveals a protein-protein interaction between NSD2 and SMARCA2. SMARCA2 KD showed SMARCA2 might be a novel therapeutic target for t(4;14) MM. Citation Format: Jing Yuan Chooi, Zhigang Xie, Hannah Lee-Foon Swa, Jayantha Gunaratne, Dennis Kappei, Wee Joo Chng. Identification of functional proteins interacting with NSD2 in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2499.

Effects of Triptolide Combined with Homoharringtonine on Proliferation and Apoptosis of KG-1α Cells

To investigate the effect and possible mechanism of low concentration of triptolide (TPL) combined with homoharringtonine (HHT) on the proliferation and apoptosis of KG-1α cells. CCK-8 method was used to detect the antiproliferating effects of different concentrations of TPL and HHT single-use and combined use on KG-1α cells, and the combined index (CI) was calculated. The colony formation ability was also determined by methylcellulose colony formation assay, cell surface molecules, apoptosis rate and cell cycle changes were detected by flow cytometry. Westerrn blot was used to detect the expression of Akt signaling pathway related proteins before and after low dose TPL combined with HHT using. High expression of CD34 and CD123 were on KG-1a cells, which being lack expression of CD38. TPL and HHT dose-dependently inhibited the proliferation of KG-1α cells. Compared with low dosage TPL and HHT single-use groups, the cell proliferation and colony formation efficiency were lower, and the cell apoptosis rate was higher in the combined group. CI values also indicated that low concentration TPL combined with HHT possessed highly synergistic effect. After the combination of the 2 drugs, the expressions of P-Aktser473, P-Aktthr308, BCL-2, PARP and survivin protein were down-regulated and the cleavage of PARP protein was increased. Low concentration of TPL combined with HHT can synergistically inhibit KG-1α cell proliferation and induce its apoptosis through the PI3K/Akt signaling pathway and downstream protein.

MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia

The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.

SETD2 Functional Loss through Mutation or Genetic Deletion Promotes Expansion of Normal and Malignant γδ T Cells through Loss of Tumor Suppressor Function and Upregulation of Oncogenic Pathways

Peripheral T cell lymphomas (PTCL) of γδ T cell origin including subsets of hepatosplenic T cell lymphoma (HSTL) and enteropathy associated T cell lymphoma (EATL) are characterized by short survival with multiagent chemotherapy programs compared to other PTCLs. Additionally, the molecular and genetic events underlying the development of HSTL and EATL are poorly understood, thus hampering development of more effect novel therapies in these lymphoma subtypes. Given this problem, we sought to define and characterize genetic events associated with HSTL/EATL.Through whole exome sequencing of 150 HSTL/EATL cases along with paired normal control tissues we found the histone methyltransferase SETD2 to be the most frequently mutated tumor suppressor in HSTL and EATL (>40% of cases in both entities). Conversely, SETD2 mutation was found to be rare in other PTCL subtypes. HSTL/EATL SETD2 mutations frequently occurred as nonsense and frameshift mutations and more than half had evidence of biallelic mutation, suggesting these mutations constitute a loss of function phenotype. Accordingly, we found that overexpression of representative mutant SETD2 (c.T4361C/F1544S and c.7571_7572delGT/C2524*) in HEK293 cells resulted in decreased downstream H3K36me3 compared to wild type SETD2 controls. We further investigated the ramifications of SETD2 functional loss in the only known HSTL cell line, DERL2. DERL2 cells were transduced with lentiviral constructs bearing inducible SETD2 (N=2) or non-silencing control shRNAs. SETD2 shRNA induction in DERL2 cells resulted in >75% loss of SETD2 mRNA expression compared to non-silencing controls by quantitative PCR and resulted in potent depletion of both SETD2 and H3K36me3 protein by Western blotting. We then performed RNA sequencing of DERL2 cells after SETD2 knockdown compared to non-silencing controls. Gene expression analysis and gene set enrichment analysis (GSEA) revealed that SETD2 knockdown was associated dysregulation of multiple oncogenic pathways involved in cell cycle, mitotic, G2/M and NF-kB pathways (FDR <0.1 for each). DERL2 cells post SETD2-knockdown also revealed increased cellular proliferation in in vitro culture and in methylcellulose colony forming assays as compared to non-silencing controls (p<0.01). This effect was not noted in T lymphoblast Jurkat cells engineered in similar fashion for SETD2 loss, suggesting SETD2 loss favors γδ T cell expansion specifically in a context-specific manner. Overall these experiments thus confirm an association between the acquisition of oncogenic gene expression pathways with SETD2/H3K36me3 loss and enhanced cell survival and proliferation in malignant γδ T cells.To understand effects of SETD2 loss in vivo, we quantitated progenitor and mature T cell subsets in T cell specific SETD2 (Setd2fl/fl, lck-cre) knockout mice. Sorted T cells from 6-8 week old mice but not other tissues derived from Setd2fl/fl, lck-cre mice showed lack of SETD2 protein and low H3K36me3/2 ratios consistent with lineage-specific SETD2 loss. In this model, we found 2.2 fold expansion (p=0.016) of γδ T cells in mesenteric lymph nodes and Peyer patches of Setd2fl/fl, lck-cre compared to controls. Analysis of gene expression in murine T cell progenitors in normal mice without SETD2 loss revealed upregulation of SETD2 mRNA in thymic double positive cells, prior to αβ lineage commitment. Taken together with the results of our in vitro experiments, this suggests that SETD2/H3K36me3 may control normal T cell progenitor fate and γδ T cell expansion through regulation of specific gene expression programs.In summary, we describe SETD2 functional loss resulting in attenuation of H3K36 trimethylation as a frequent event in gamma delta T cell derived lymphomas. By gene expression analysis and functional studies in murine and human cancer cell line models, we show that SETD2 is important both in the regulation of γδ versus αβ T lymphocyte developement and has a direct role in fostering proliferation of malignant cells derived from the γδ T cell compartment. This appears in part due to loss of epigenetic control of diverse gene expression programs. This work has direct translational importance as we define a novel role of the SETD2/H3K36me3 axis in controlling global T cell differentiation and we identify a potential target for synthetic lethal approaches in malignancies such as HSTL and EATL. DisclosuresNo relevant conflicts of interest to declare.

PTPN11 Mutations in JMML Confer Dasatinib Sensitivity in a TNK2-Dependent Manner

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare disease characterized by an overproduction of granulocytes and monocytes, with cells displaying hypersensitivity to stimulation by the cytokine GM-CSF in culture. The only curative approach for JMML is allogeneic hematopoietic stem cell transplant, and disease recurrence is the leading cause of death in these patients, occurring within two years in 50% of patients. New therapeutic strategies are needed to improve treatment and survival.Methods: In order to better identify therapeutic strategies, our lab has developed ex vivo functional assays with small-molecule inhibitors and RNAi for the profiling of actionable targets in primary patient samples. In these assays, mononuclear cells from patient bone marrow or blood are cultured in the presence of small molecule inhibitor or RNAi libraries and cell viability is measured by colorimetric assay. To investigate targets identified in these assays, gene constructs are transfected into HEK293 T17 cells for immunoblots or transduced into mouse bone marrow cells for methylcellulose-based colony formation assays.Results: Mononuclear white blood cells from a patient with recurrent JMML were found to be sensitive to the multi-family kinase inhibitor dasatinib as well as siRNA targeting the dasatinib target tyrosine kinase non-receptor 2 (TNK2, also known as ACK1), suggesting an over-reliance on TNK2 for cell viability. TNK2 was recently demonstrated by our lab and others to regulate signaling downstream of signaling pathways including EGFR, ER, Src, and CSF3R. Based on the ex vivo functional data for this JMML specimen, the patient was placed on daily dasatinib and achieved marked improvement, which bridged the patient to an additional bone marrow transplant, resulting in disease-free survival for another year.This patient had a mutation in PTPN11 (also known as SHP2), which encodes a protein tyrosine phosphatase that is mutated in approximately 35% of JMML patients. PTPN11 is an auto-inhibited phosphatase that dephosphorylates targets in many signaling pathways including RAS-MAPK, with an overall positive effect on pathway activity and cellular proliferation. In JMML, mutations in residues of the N-terminal SH2 domain of PTPN11 result in a constitutively active form of the protein, which increases proliferation and cell survival and is linked to dysregulation of RAS/MAPK.Results of in vitro experiments demonstrate, for the first time, an interaction of TNK2 with mutant PTPN11. Co-expression of mutant-PTPN11 and TNK2 results in increased phospho-PTPN11 at two sites associated with activation of PTPN11 (Y542 and Y580), and co-immunoprecipitation experiments indicate direct interaction of TNK2 with PTPN11. These phosphorylation events coincide with significantly increased MEK/ERK signaling in the context of TNK2/mutant PTPN11 co-expression relative to expression of either gene alone. Expression of a TNK2 mutant harboring a loss-of-function mutation at a key activating residue (TNK2 Y284F) reduces PTPN11/MAPK activation, whereas expression of a gain-of-function, drug-resistant TNK2 gatekeeper mutant increases PTPN11/MAPK activation when compared to wild-type TNK2. Inhibition of TNK2 with either dasatinib or the TNK2-specific inhibitor AIM100 also reduces PTPN11/MAPK activation. Interestingly, phospho-TNK2 levels are significantly reduced in the presence of mutant-PTPN11, suggesting a feedback mechanism that has yet to be fully elucidated.Functional experiments suggest that mutant-PTPN11 and TNK2 work in a synergistic fashion to increase cell transformation and cytokine hypersensitivity. Mouse bone marrow cells transduced with both mutant PTPN11 and TNK2 have significantly more colonies in methylcellulose colony formation assays than with mutant- PTPN11 alone or with wild-type PTPN11 and TNK2. Treatment of cells in colony formation assays with either dasatinib or AIM100 inhibits colony formation, suggesting a dependence on TNK2 kinase activity for cell transformation.Conclusions: Taken together, the preliminary data suggest that PTPN11 mutations may be a functional marker for dasatinib sensitivity, and that the underlying mechanism for this sensitivity may be through the inhibition of TNK2 activity by dasatinib. Our aim is further validate the role of PTPN11 mutations as a marker for efficacy of dasatinib or other TNK2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.