THP-1 Acute Myeloid Leukemia Cells Research Articles

Venetoclax Inhibition of Pyrimidine Synthesis Guides Methods for Integration with Decitabine or 5-Azacytidine That Are Non-Myelosuppressive

Venetoclax (Ven) administered daily with pulse-cycled parenteral decitabine (Dec) or 5-azacytidine (5Aza) is standard therapy for acute myeloid leukemia (AML) in the elderly. In practice, toxicity/myelosuppression is frequent, and prompts Ven dose reductions, but by guess-work, because the mechanism downstream of BCL2-inhibition by which Ven augments Dec/5Aza activity is unclear. For the first time, we show that Ven inhibits de novo pyrimidine synthesis, an effect that can guide its integration with Dec/5Aza in a way that enhances anti-AML activity without suppressing normal myelopoiesis. Dec and 5Aza are pro-drugs processed by pyrimidine metabolism into a deoxycytidine analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1), a pharmacodynamic effect that terminates malignant but not normal self-replication. We recently demonstrated that Dec- and 5Aza-resistance emerges automatically from adaptive responses of the pyrimidine metabolism network to Dec/5Aza-induced nucleotide perturbations, such that Dec/5Aza processing into DNMT1-depleting nucleotide is forestalled (Leukemia - https://rdcu.be/b58pS). A key element in this auto-resistance is upregulated de novo pyrimidine synthesis, that out-competes salvaged Dec/5Aza. De novo pyrimidine synthesis has an electron-transport dependent mitochondrial step executed by dihydroorotate dehydrogenase (DHODH): BCL2-inhibition by Ven depolarizes mitochondrial membranes - we examined for the first time Ven impact on DHODH/pyrimidine synthesis (others have focused on apoptosis and other metabolic consequences of mitochondrial depolarization). Consistent with Ven inhibiting DHODH/pyrimidine synthesis, both Ven and the direct DHODH inhibitor teriflunomide, at non-apoptotic concentrations, significantly decreased cytidine- and deoxycytidine triphosphate (CTP, dCTP) in AML cells (Fig1A). To see if this effect of Ven can counter Dec/5Aza resistance, we selected for THP1 AML cells double-resistant to Dec 0.5 μM and 5Aza 5 μM - these cells upregulated expression of de novo pyrimidine synthesis enzymes and contained uridine, cytidine and deoxycytidine at levels up to 7-fold higher than parental cells. The double-resistant AML cells were significantly cytoreduced by Ven at a clinically relevant concentration of 1 μM, even though parental THP1 AML cells were minimally sensitive (Fig1B). Consistent with inhibition of de novo pyrimidine synthesis as the mechanism, this action was significantly abrogated by cytidine supplementation (Fig1B). We previously showed that timed alternation of Dec with 5Aza, and incorporation of the cytidine deaminase inhibitor tetrahydrouridine (THU), counters metabolic Dec or 5Aza-resistance to extend non-cytotoxic DNMT1-depletion and survival in vivo (https://rdcu.be/b58pS). However, AML still eventually progresses, via upregulated de novo pyrimidine synthesis. In considering use of Ven to counter this mode of resistance, we reasoned that concurrent administration risks antagonism, because Ven can cause transient cytostasis, and DNMT1-depletion by Dec/5Aza is S-phase dependent. Prior Ven administration, however, creates effect-time sufficient to deplete endogenous nucleotides from AML cells and hence upregulate pyrimidine salvage (that uptakes Dec/5Aza) (Fig1A), as well as resumed cell cycle. Therefore, in vivo in a patient-derived xenotransplant model (PDX) of AML, we introduced Ven (at human equivalent dose), parsimoniously 2X/week, the day before each Dec or 5Aza administration, and at the time of overt AML progression on the base THU-Dec/5Aza regimen. This minimalist Ven application significantly extended survival (time-to-distress) even though it was initiated at progression (Fig1C). As expected, use of this regimen upfront in a PDX of Dec/5Aza-resistant AML produced even greater (several-fold) survival extension (treatment ongoing) vs the base regimen (Fig1D). A non-cytotoxic/non-myelosuppressive mechanism-of-action was confirmed by serial blood counts on-therapy. In practice, Ven dose in combination with Dec or 5Aza to treat AML is frequently empirically reduced because of toxicity/myelosuppression. Instead, Ven inhibition of de novo pyrimidine synthesis can guide mechanism-based, intermittent administration that avoids toxicity/myelosuppression yet enhances Dec/5Aza anti-AML activity. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago.

Triple Gene-Modified iPSC-Derived NK Cells Combined with Daratumumab for Targeted Immunotherapy Against AML

Triple Gene-Modified iPSC-Derived NK Cells Combined with Daratumumab for Targeted Immunotherapy Against AML

Therapeutic targeting of TP53-mutated acute myeloid leukemia by inhibiting HIF-1α with echinomycin.

TP53 mutation in acute myeloid leukemia (AML) is associated with poor prognosis. Since no targeted therapy is available to restore p53 function, it is of great interest to test whether other pathways activated by TP53 mutations can be therapeutically targeted. Here we showed HIF-1α target genes are enriched in TP53-mutated vs TP53-wild type AML. To determine the role of this activation, we tested efficacy of HIF-1α inhibitor echinomycin in TP53-mutated AML samples in vitro and in vivo. Echinomycin was broadly effective against a panel of primary AML blast cells, with low nanomolar IC50s and, based on colony-forming unit assay, was 10-fold more effective in eliminating AML stem cells. Echinomycin selectively eliminated CD34+CD38- AML cells. To test the therapeutic efficacy of echinomycin, we established a xenograft model of TP53-mutated AML. Echinomycin was broadly effective against xenografts from multiple AML samples in vivo, and more effective than cytarabine + daunorubicin chemotherapy. Importantly, while cytarabine + daunorubicin enriched for AML stem cells, echinomycin nearly eliminated this population. Using TP53-mutated AML cell line THP1 and patient-derived AML cells, we tested a new echinomycin formulation with longer half-life and significantly improved therapeutic effect. Our data suggest a novel approach to treat AML with TP53 mutations.

LAG3 Inhibition Decreases AML-Induced Immunosuppression and Improves T Cell-Mediated Killing

Background: Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy with known immune dyregulation. In addition to their capacity to rapidly divide, AML cells directly inhibit the activation and proliferation of immune cells in culture. Immunosuppressive features observed in the bone marrow of AML patients include upregulation of Tregs and production of immunosuppressive cytokines (e.g., TGFβ). Irradiating AML cells diminishes their immunosuppressive capacity while maintaining antigen presentation, leading to increased activation of T cells in co-culture. We subsequently identified the immune checkpoint LAG3 as an important mediator of AML-induced immunosuppression and LAG3 modulation as potential treatment strategy. Methods: Normal PBMC were isolated from healthy donors. PBMC were co-cultured with non-irradiated and irradiated (40 Gy) human AML cell lines (Kasumi1 (K1), THP1) separately at a 1:2 ratio. On day 3 of co-culture, immunophenotypic characterization of T cells was performed on a flow cytometer using the following surface markers: CD3, CD4, CD8, CD25, CD137, CD154, PD-1, TIM3, TIGIT, and LAG3 and intracellular IFNg and FOXP3. Supernatant from co-culture media were analyzed for cytokine (IL-2, IL-6, IL-10 & TGFβ) secretion by ELISA. CFSE-labeled AML cells were incubated with healthy donor PBMCs in the presence or absence of LAG3, then viability was measured by 7-ADD on flow cytometry. PBMCs were also isolated from AML patients' peripheral blood and mononuclear cells were isolated from their respective bone marrow samples. Primary AML cultures were established in RPMI complete media with 20% FBS. CFSE-7-ADD killing assay was conducted after incubation of AML cells with autologous PBMCs. Results: Healthy donor PBMC co-cultured with irradiated K1 AML cells showed higher intracellular IFNg expression (11.8% ± 3.1 v. 7% ± 3.3; n=7, P=0.012) and higher CD137 expression (9.3% ± 1.21 v. 5.7% ± 3.4; n=7, P<0.001) on CD8+ T cells, and higher CD154 expression on CD4+ cells (44.7% ± 20.3 v. 26.3% ± 14.2; n=5, P=0.002) when compared to the non-irradiated K1-PBMC co-cultures. There were fewer Tregs (CD4+ CD25+ FOXP3+) in the PBMC co-cultured with irradiated K1 cells (1.96% ± 0.37 v. 3.39% ± 0.58; n=4, P=0.03) compared to the non-irradiated K1-PBMC co-cultures. LAG3 expression on CD8+ T cells co-cultured with irradiated K1 was decreased (11.8% ± 2.4 v. 17.5% ± 2.5; n=4, P=0.002) compared to the PBMC co-cultured with non-irradiated K1 cells. No other changes in checkpoint expression on CD8+ T cells were observed. No changes were observed in MHCI or PDL1 expression on non-irradiated K1 AML cells before or after co-culture with PBMC. We observed similar findings with healthy donor PBMC co-cultured with a different AML cell line, THP1; CD137 expression was higher on CD8+ T cells (17.6% v. 6.5%; P=0.02, n=3). ELISA of the supernatant of culture media showed higher mean OD for secreted TGFβ in the non-irradiated AML co-cultures compared to the irradiated AML co-cultures at 6 hours (2.5 v. 2.0, P=0.03, n=3) and 72 hours (7.9 v. 5.3, P=0.04, n=3). Adding anti-LAG3 antibody (3DS223H; 100 µg/ml) to PBMC co-cultured with non-irradiated AML cells resulted in higher IFNg (16.3% v. 6.6%, P=0.01, n=4) and CD137 expression (6.5% v. 4.1%, p=.007, n=4) on CD8+ cells and fewer Tregs (1.7% v. 3.8%, P=.04, n=4) compared to no antibody added. Healthy donor PBMC (n=3) were incubated with CFSE labeled AML cells (K1 and THP1) separately at an effector:target ratio of 5:1. The addition of anti-LAG3 antibody lead to increased killing of both K1 and THP1 AML cells at 4 and 24 hours (Figure 1A). To eliminate the HLA mismatch effect, we incubated PBMC from AML patients with autologous AML cells in the presence or absence of anti-LAG3 (Figure 1B). MHC-I blocking (W6/32, 30 µg/ml) lead to inhibition of cell mediated killing in the presence of anti-LAG3 (Figure 1B). Conclusion: In this in vitro model, AML cells showed immunosuppressive features with decreased activation of CD8+ T cells, upregulation of Tregs, increased secretion of TGFβ and higher expression of LAG3 on CD8+ T cells. Antibody blocking of LAG3 mitigated this effect, resulting in increased activation of T cells, fewer T regs and improved MHC-I-mediated killing against AML cells. These results demonstrate that the immunosuppressive effects of AML cells can be modulated through inhibition of LAG-3, suggesting a potential strategy for future combination therapy in AML. Disclosures Lin: Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Knockdown of IARS2 Inhibited Proliferation of Acute Myeloid Leukemia Cells by Regulating p53/p21/PCNA/eIF4E Pathway.

IARS2 encodes mitochondrial isoleucine-tRNA synthetase, which mutation may cause multiple diseases. However, the biological function of IARS2 on acute myeloid leukemia (AML) has not yet been identified. In the present study, qRT-PCR was used to determine the expression of IARS2 in K562, THP1, and HL-60 leukemia cells. Additionally the mRNA levels of IARS2 in CD34 cells and AML cells obtained from patients were detected by qRT-PCR. IARS2-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. qRT-PCR and Western blot analysis were employed to assess the knockdown effect of IARS2. The proliferation rate and cell cycle phase of HL-60 cells after IARS2 knockdown were evaluated by CCK-8 assay and flow cytometry. The PathScan Antibody Array was used to determine the expression of cell cycle-related proteins in HL-60 cells after IARS2 knockdown. The expression of proliferation-related proteins in HL-60 cells after IARS2 knockdown was determined by Western blot analysis. Results showed that IARS2 expression was stable and much higher in HL-60, THP-1, and K562 leukemia cells and AML cells obtained from patients than that of human CD34 cells. Compared with cells of the shCtrl group, IARS2 was markedly knocked down in cells that were transfected with lentivirus encoding shRNA of IARS2 in HL-60 cells (p < 0.05). IARS2 knockdown significantly inhibited the proliferation and induced cycle arrest at the G1 phase in HL-60 cells. Additionally IARS2 knockdown significantly increased the expression of p53 and p21, and decreased the expression of PCNA and eIF4E in HL-60 cells. In conclusion, IARS2 knockdown can inhibit acute myeloid leukemia HL-60 cell proliferation and cause cell cycle arrest at the G1 phase by regulating the p53/p21/PCNA/eIF4E pathways.

Rapamycin downregulates NKG2D ligands in acute myeloid leukemia cells via an activation of the STAT3 pathway: a potential mechanism for rapamycin-induced immune escape in leukemia.

BackgroundThe constitutive activation of the mammalian target of rapamycin (mTOR) is involved in the pathogenesis of many cancers. Rapamycin (RAPA), a specific inhibitor of mTOR, has been applied to the clinical treatment of tumors, and its anti-leukemia effect has also been confirmed.MethodsWe detected apoptosis and the NKG2D ligands expression in acute myeloid leukemia (AML) cells using flow cytometry and investigated the cytotoxicity of AML cells that had been co-cultured with natural killer (NK) cells using CFSE staining. We evaluated the signal pathways with a western blot assay.ResultsIn this study, we found that RAPA can significantly inhibit the proliferation of AML cells. Further studies showed that the use of RAPA alone reduced the expression of NKG2D ligands on the membranes of HL-60 and THP-1 AML cells. Also, RAPA blocked the upregulation of the NKG2D ligand when AML cells were cultured with the demethylation drug decitabine (DAC). We found that RAPA decreased the expression of the NKG2D ligands by inducing the STAT3 phosphorylation of AML cells.ConclusionsThe discovery of this mechanism might further optimize the clinical use of RAPA for the treatment of AML.

Genome-Wide CRISPR-Cas9 Screen Identifies Sensitizers to LSD1 Inhibition in MLL-Translocated Human AML Cells

Lysine-specific demethylase (LSD1, also known as KDM1A) is an epigenetic regulator that has recently emerged as a potential therapeutic target in acute myeloid leukemia (AML). It is a flavin dependent monoamine oxidase which can demethylate monomethyl or dimethyl lysine 4 of histone H3. Pharmacological inhibition of LSD1 induces differentiation of blast cells in MLL-translocated AML and has shown significant promise in pre-clinical studies. With LSD1 inhibitors advancing through early-phase clinical trials, there is a strong pre-clinical rationale for the identification of genes whose protein products collaborate with LSD1 to retard differentiation in cancer and which could potentially be targeted in combination therapies for enhanced therapeutic benefit.To identify potential drug-gene synthetic lethal interactions, we performed a genome wide loss-of-function CRISPR-Cas9 screen in human THP1 AML cells treated with a potent and selective tranylcypromine-derivative LSD1 inhibitor trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (OG86). THP1 cells exhibit a t(9;11) MLL gene rearrangement and display similar phenotypic and functional responses to those observed in primary MLL-translocated AML cells following LSD1 inhibition. The cells were transduced with lentiviral human CRISPR knockout (hGeCKOv2) library containing 122,411 sgRNAs targeting 19,050 protein coding genes and 1864 miRNA precursors in the human genome. The transduced cells were divided into two groups, treated with either DMSO or 250nM OG86 and maintained in culture for ~10 population doublings. To investigate sgRNA representation in the cell population harvested at different stages of the screen, sgRNA cassettes were PCR amplified from genomic DNA and deep sequenced; negatively selected genes were identified with the MAGeCK computational algorithm. After successful initial quality assessment of the screen, we next searched for genes selectively depleted in OG86-treated versus DMSO-treated THP1 cells in samples collected on Day 15 and Day 18. At a false discovery rate of 7.5% there were 10 expressed genes whose sgRNA representation was depleted at both time points. In particular, these included two genes coding for components of the MTOR signaling pathway: MTOR associated protein, LST8 homolog (MLST8) and Ras-related GTP-binding protein A (RRAGA). In the Day 18 comparison, an additional MTOR pathway gene LAMTOR2 scored among the ten most depleted. MLST8 is a core component of TORC1/TORC2 complex and RRAGA is involved in the activation of TORC1 by amino acids. Recruitment of both RAG proteins and TORC1 to lysosomal membranes in response to amino acids, and the consequent activation of TORC1 signalling, requires the trimeric Ragulator complex, of which LAMTOR2 is a member.Based on our screen, we hypothesized that THP1 AML cells exposed to pharmacologic inhibition of LSD1 exhibit increased sensitivity to concomitant inhibition of the amino acid sensing component of the TORC1 pathway.Using genetic knockdown and pharmacological inhibition strategies, we then validated our screen hits in combination with LSD1 inhibition in targeting human AML cells. RNAi based knockdown of RRAGA, LAMTOR2 and MLST8 in combination with LSD1 inhibition were found to promote myeloid differentiation and reduce cell proliferation in THP1 cells. Interestingly the mTORC1 pathway inhibitor everolimus (RAD001) showed at least additive effect in combination with OG86 to decrease THP1 cell proliferation and promote immunophenotypic differentiation. Comparison of transcription changes in combined versus single treatment conditions by RNA-seq analysis further confirmed a more extensive and wide-ranging upregulation of a myeloid differentiation program upon concomitant inhibition of LSD1 and mTORC1 pathway. In vitro studies performed in primary patient AML cells gave similar results. Finally, in vivo studies using AML patient-derived xenograft mouse model confirmed that combination treatment promotes a strong myeloid differentiation program. In conclusion, we report here that inhibition of mTORC1 sensitizes human MLL-translocated AML cells to LSD1 inhibitor-mediated differentiation therefore highlighting a novel combination approach for evaluation in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Regulated Natural Killer Cell Expansion and Anti-Tumor Activity with Inducible MyD88/CD40

▪BackgroundNatural Killer (NK) lymphocytes possess innate anti-tumor activity that has the potential to be used as an allogeneic cell therapy due to reduced GvHD risk relative to αβ T cells. Despite their potential, adoptive NK cell immunotherapies have been limited by poor expansion in vivo. Using our previously developed Chimeric Antigen Receptor-T cell (CAR-T) strategy that relies on rimiducid-based dimerization of inducible MyD88/CD40 (iMC) to regulate T cell expansion and survival, we demonstrate that iMC can also be applied to NK cell growth and anti-tumor efficacy in vitro and in vivo. Moreover, a rapamycin-inducible Caspase-9 (iRC9) was used to provide an orthogonally regulated safety switch.Methods and ResultsCD56+ NK cells were isolated from peripheral blood of human donors, stimulated overnight with IL-15 then activated by seeding with K562 erythroleukemia target cells. NK cells were then transduced with γ-retrovirus encoding control iRC9-2A-ΔCD19, iRC9-2A-ΔCD19-2A-iMC (dual-switch NK) or iRC9-2A-IL-15-2A-ΔCD19-2A-iMC (dual-switch/IL-15 NK). ΔCD19 marked transduced cells in 50:50 cocultures with untransduced NK cells. NK cells containing only iRC9 grew at the same rate as untransduced cells, but iMC-expressing NK cells displayed enhanced growth that was further augmented by 1 nM rimiducid treatment. In cocultures with THP1 acute myeloid leukemia cells at increasing Target:Effector (T:E) ratios, presence (P < 0.001, two way ANOVA) and activation (P <0.001) of iMC increased tumor killing activity. Inflammatory cytokine and chemokine production was also dramatically (10 to 1000-fold) elevated by the expression and activation of iMC in NK cells in the presence and absence of THP1 tumor target.To study in vivo anti-tumor activity, immunodeficient NSG mice were engrafted with dual-switch NK cells with or without autocrine IL-15 expression in the presence or absence of THP-1 tumor targets. When tumor was present, unstimulated iMC with IL-15 or activation of iMC without IL-15 expression supported modest NK cell expansion, but rimiducid stimulation of iMC plus autocrine IL-15 showed enhanced NK expansion in vivo. Furthermore, in tumor-free animals only dual-switch/IL-15 NK cells with weekly rimiducid stimulation expanded and persisted in vivo (up to 7 weeks). Cotransduction of a first generation CD123-targeted CAR to produce dual-switch/IL-15 CD123CAR-NK cells led to rimiducid-dependent control of THP1 tumor outgrowth in vivo beyond 40 days. Conversely, temsirolimus-mediated activation of the iRC9 safety switch rapidly (< 24 hours) ablated dual-switch NK cells in vivo.ConclusionsInducible MyD88/CD40 is an activation switch that supports NK cell expansion, persistence and anti-tumor activity. When paired with autocrine IL-15 expression, this platform supports NK expansion and persistence in vivo, and AML tumoricidal activity that can be further activated by target-specific CAR expression. Moreover, the fast-acting, orthogonally regulated proapoptotic switch, iRC9, mitigates the risk of off-tumor targeting. Therefore, we describe a novel, regulated NK cell platform that solves many of the challenges of NK cell-based therapy and should be amenable to a readily translatable off-the-shelf cellular therapy for malignancies. [Display omitted] DisclosuresWang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Chang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Jasinski:Bellicum Pharmaceuticals: Employment, Equity Ownership. Medina:Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Functional Analysis of CD99 Upregulation in Acute Myeloid Leukemia

Background: Acute Myeloid Leukemia (AML) is a hematological malignancy with a 5-yr survival rate of 27%. This indicates an urgent need to identify better therapies. We previously analyzed various gene expression data sets of normal hematopoietic vs AML cells and reported that CD99 is upregulated in AML. CD99 loss of function by siRNA or monoclonal antibody decreased proliferation and migration of AML cells (Vaikari et al, ASH abstract, 2016). Recently, we have also shown that AML blasts transduced with CD99 overexpressing lentivirus exhibit a significant increase in cell proliferation (Vaikari et al, ASH abstract, 2017). Here we further expand our preclinical investigation to study the functional role of CD99 in AML.Methods: We performed lenti-viral transduction to overexpress CD99 (CD99 OE) or empty vector (EV) in THP-1, U937, and MOLM-13 AML cell lines. Proliferation assay was performed by seeding 1X105 cells/mL and measuring cell proliferation using trypan blue at 72 hours. Aggregation assay was performed by seeding 1X105CD99 OE (or EV) cells in a 6 well plate and images for cell aggregation were taken 6 hours later. For the differentiation and apoptosis assays, CD99 OE (or EV) (5X105 cells/mL) were starved overnight. Flow cytometry analyses for CD11b and Annexin-V PI were performed 24 hours later. To determine the effect of CD99 overexpression on cell migration, THP-1, U937, and MOLM-13 (1X105 cells) CD99 OE or EV cells were seeded in a transwell chamber for 4 hours and migration towards SDF-1a was analyzed. For the THP-1 and MOLM-13 murine model, 2.5 X106 cells overexpressing CD99 or EV (n=3 for each) were engrafted into NOD-scid /Il2rg-/- (NSG) mice. Bone marrow (BM) and peripheral blood (PB) were collected to determine engraftment by hCD45 staining through flow cytometry.Results: Transducing cells with CD99 resulted in increased cell proliferation as compared with their respective controls in THP-1 (CD99 OE vs EV: 1.78 fold, p<0.0001), U937 (CD99 OE vs EV: 1.56 fold, p<0.0001), and in MOLM-13 cells (CD99 OE vs EV: 1.87 fold, p<0.0001). THP-1, MOLM-13 and U937 cells stably overexpressing CD99 displayed higher cell aggregation capacity compared to EV cells. Cells stably overexpressing CD99 showed an increase in CD11b expression in THP-1 (CD99 OE vs EV: 2.055-fold, p=0.0027), U937 (CD99 OE vs EV: 1.56-fold, p=0.01), and MOLM-13 cells (CD99 OE vs EV: 1.89- fold, p<0.0001). Cell aggregation was also accompanied by an increase in cell apoptosis of CD99 OE cells in THP-1(CD99 OE vs EV: 3.48-fold, p=0.001), U937 (CD99 OE vs EV: 3.68-fold, p=0.11), and MOLM-13 cells (CD99 OE vs EV: 6.56-fold, p=0.0001).Additionally, CD99 overexpression decreased cell migration compared with EV cells in THP-1(CD99 OE vs EV: 67% decrease, p<0.0001), U937 (CD99 OE vs EV: 75% decrease, p<0.0001), and MOLM-13 cells (CD99 OE vs EV: 73% decrease, p=0.0003). Based on these results, we hypothesize that homotypic interaction of CD99 could play a role in its signaling process in AML.In both the THP-1 and MOLM-13 murine model, mice engrafted with CD99 OE cells had smaller spleens compared with EV mice. In the THP-1 murine model, CD99 OE mice had significantly less engraftment compared with the EV mice in the BM (6.69 vs 14 %, p=0.047), and PB (3.61 vs 91.67%, p<0.0001). Similarly, in the MOLM-13 murine model, hCD45 flow analysis revealed that CD99 OE mice have significantly less engraftment compared with the EV mice in the BM (39.83 vs 71.43%, p=0.0022), and PB (19.43 vs 67.13 %, p=0.018).Conclusion: In summary, our results suggests that even though CD99 enhances AML cell proliferation, it also enhances homotypic cell interaction and cell aggregation, which results in increased cell apoptosis as well as a decrease in cell migration and possibly responsible for the decrease leukemia engraftment. Further investigations are ongoing to determine the effect of homotypic interaction of CD99 in AML. DisclosuresNo relevant conflicts of interest to declare.

CircPAN3 mediates drug resistance in acute myeloid leukemia through the miR-153-5p/miR-183-5p–XIAP axis

The contribution and role of circular RNAs (circRNAs) in mediating chemoresistance in acute myeloid leukemia (AML) are still poorly understood and need further investigation. In this study, we established a doxorubicin (ADM)-resistant THP-1 AML cell line (THP-1/ADM). A high-throughput microarray was used to identify circRNA expression profiles of THP-1/ADM cells and naive THP-1 cells. The identified potential functional circRNA molecule was further validated in THP-1/ADM cells and bone marrow (BM) specimens from 42 AML patients. The interactions with target microRNAs (miRNAs) and downstream messenger RNAs (mRNAs) were also explored. As a result, 49 circRNAs that are significantly differentially expressed between THP-1/ADM and THP-1 cells were identified. Of these circRNAs, downregulation of circPAN3 by small interfering RNA significantly restored ADM sensitivity of THP-1/ADM cells. Furthermore, BM samples from patients with refractory and recurrent AML showed increased expression of circPAN3. A detailed circRNA/miRNA/mRNA interaction network was predicated for this circRNA. Subsequent mechanistic experiments showed that downregulation of circPAN3 could decrease the expression of X-linked inhibitor of apoptosis protein (XIAP), but this effect was counteracted by miR-153-3p or miR-183-5p specific inhibitors. Luciferase experiments further demonstrated that these molecules are involved in the circPAN3 regulatory network. Our results revealed that circPAN3 may be a key mediator for chemoresistance of AML cells, which may depend on the circPAN3-miR-153-5p/miR-183-5p-XIAP axis. Our findings provide evidence that circPAN3 can be a valuable indicator for predicting clinical efficacy of chemotherapy in AML patients and also can serve as a potential target for reversing drug resistance in AML.

Targeted Inhibition of STAT/TET1 Axis As a Potent Therapeutic Strategy for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. Over 70% of patients with AML cannot survive over 5 years. Many AML subtypes, such as the MLL -rearranged AMLs, are often associated with unfavorable outcome. Current treatment frequently involves intensive chemotherapy, which impairs the quality of life of the patients. While the incidence of AML is continually rising due to aging, most elder patients cannot bear intensive chemotherapy and are associated with very poor survival. Thus, improved therapeutic strategies with less intensive treatment but a higher cure rate are urgently needed.The Ten-eleven translocation (TET) proteins (including TET1/2/3) are known to be able to convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), leading to DNA demethylation. In contrast to the repression and tumor-suppressor role of TET2 observed in hematopoietic malignancies, we recently showed that TET1, the founding member of the TET family, was significantly up-regulated in MLL -rearranged AML and played an essential oncogenic role. An independent study by Zhao et al. confirmed the essential oncogenic role of Tet1 in the development of myeloid malignancies. Thus, TET1 is an attractive therapeutic target for AML.In order to identify chemical compounds that may target TET1 signaling, we searched the drug-sensitivity/gene expression database of a total of 20,602 chemical compounds in the NCI-60 collection of cancer cell samples. The expression levels of endogenous TET1 showed a significant positive correlation with the responsiveness of cancer cells across the NCI-60 panel to 953 compounds (r > 0.2; P <0.05). We selected the top 120 with the highest r values and tested their effects on cell viability of TET1 -high and TET1 -low AML cell lines. Finally we got two top candidate compounds, NSC-X1 and especially, NSC-X2, with the highest potency of inhibiting cell viability of TET1 -high AML cell, but not TET1 -low cells. Both compounds significantly suppressed cellular TET1 expression levels as well as 5hmC levels in TET1 -high AML cells.Further, secondary bone marrow transplantation followed by drug treatment was carried out to test the in vivo therapeutic effects of NSC-X1 and NSC-X2. Both candidate compounds significantly inhibited MLL-AF9 -induced AML, by prolonging the median survival from 49 days (control) to 94 (NSC-X1) or >200 (NSC-X2) days. Notably, 57% of the NSC-X2 treated mice were cured. In another AML model induced by AML-ETO9a (AE9a), NSC-X1 and NSC-X2 also exhibited remarkable therapeutic effects, with an elongated median survival from 46 days (control) to 95 (NSC-X1) and 122 (NSC-X2) days, respectively.To decipher the molecular mechanism by which NSC-X2 represses TET1 expression, we treated THP-1 AML cells with moderate to high concentration of NSC-X2 for over 100 days and then isolated a set of individual drug-resistant THP-1 single clones. Through RNA-seq of 6 of the NSC-X2-resistant clones, recurrent mutations were found in 14 genes, including JAK1 . Ingenuity pathway analysis (IPA) was used to analyze biological relationships amongst the 14 mutated genes. The top one network identified by IPA involving all of the 14 genes is closely associated with the JAK/STAT5 pathway. Results of chromatin immunoprecipitation (ChIP) assays showed TET1 is one of the direct downstream gene targets of STAT3 and STAT5. Through NMR chemical shift perturbation (CSP) and electrophoretic mobility shift assays (EMSAs), we showed a strong direct association between STAT3/5 DNA binding domain and the TET1 promoter, which could be severely interrupted by NSC-X2. Compared to currently available JAK/STAT inhibitors (e.g., Pacritinib, KW-2449, Stattic, and sc-355979), our compounds (NSC-370284 and UC-514321) exhibit a much higher selectivity and also a higher efficacy in targeting TET1 -high AML.Taken together, we identified chemical compounds NSC-X1 and especially, NSC-X2, as potent inhibitors that significantly and selectively suppress the viability of AML cells with high level of TET1 expression, and dramatically repress the progression of TET1 -high AML in mice. NSC-X2 directly binds STAT3/5 as STAT inhibitors and thereby suppress TET1 transcription and TET1 signaling, leading to potent anti-leukemic effects. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Anti-leukemia activity of NSC-743380 in SULT1A1-expressing acute myeloid leukemia cells is associated with inhibitions of cFLIP expression and PI3K/AKT/mTOR activities.

Our recent study showed that acute myeloid leukemia (AML) cells expressing SULT1A1 are highly sensitive to NSC-743380, a small molecule that inhibits STAT3 activity and induces SULT1A1-dependent apoptosis of various cancer cell lines. In this study, we characterized the molecular mechanisms of NSC-743380–mediated anti-leukemia activity in AML cell lines and antileukemia activity of NSC-743380 in patient-derived primary leukemia cells from AML patients. Our results showed that treatment with NSC-743380 triggered robust apoptosis in SULT1A1-positive AML cells. Treatment with NSC-743380 did not increase intracellular reactive oxygen species or change of STAT3 activity in AML cells, but did dramatically and rapidly decrease cFLIP expression. Proteomic analysis with reverse phase protein microarray revealed that treatment of U937 and THP-1 AML cells with NSC-743380 led to drastic and time-dependent suppression of phosphorylation of several key nodes in the PI3K/AKT/mTOR pathway, including AKT and mTOR. Moreover, primary AML cells expressed SULT1A1 were highly sensitive to treatment with NSC-743380, which was not affected by co-culture with bone marrow mesenchymal stem cells. Thus, our results provide proof-of-concept evidence that AML cells expressing SULT1A1 can be targeted by small molecules that induce apoptosis through inhibiting the expression or activities of multiple targets.

Effect of NPM1 type B mutation on the proliferation, invasion and chemosensitivity of THP-1 leukemia cells.

Acute myeloid leukemia (AML) is the most malignant myeloid disorder in adults. AML with mutated nucleophosmin (NPM1) is regarded as an independent leukemia subtype. According to previous studies, the role of NPM1 gene A mutation in AML has been well established; however, another major type, NPM1 gene B type mutation (NPM1 MutB) has been rarely reported. In the present study, we found that overexpression of NPM1 MutB enhanced the proliferation and invasion of THP-1 AML cells through the regulation of TIMP-2, MMP-2, Ang-1, c-myc and CCND1; led to no significant change of apoptosis rate with the absence of chemotherapy agents, while enhanced the chemosensitivity of THP-1 AML cells to chemotherapy agents DNR and Ara-C through the regulation of Bax, Bcl-2 and caspase-3. Further, we revealed that NPM1 MutB overexpression reduced the NF-κB activity of THP-1 cells upon drug treatment. Taken together, we demonstrated the detailed functions of NPM1MutB in THP-1 proliferation, invasion, apoptosis and chemo-sensitivity. We provided a novel understanding of prognosis of patients carrying the NPM1 B mutation.

MUC1-mediated induction of myeloid-derived suppressor cells in patients with acute myeloid leukemia

AbstractMyeloid-derived suppressor cells (MDSCs) play a critical role in promoting immune tolerance and disease growth. The mechanism by which tumor cells evoke the expansion of MDSCs in acute myeloid leukemia (AML) has not been well described. We have demonstrated that patients with AML exhibit increased presence of MDSCs in their peripheral blood, in comparison with normal controls. Cytogenetic studies demonstrated that MDSCs in patients with AML may be derived from leukemic or apparently normal progenitors. Engraftment of C57BL/6 mice with TIB-49 AML led to an expansion of CD11b+ Gr1+ MDSCs in bone marrow and spleen. Coculture of the AML cell lines MOLM-4, THP-1 or primary AML cells with donor peripheral blood mononuclear cells elicited a cell contact–dependent expansion of MDSCs. MDSCs were suppressive of autologous T-cell responses as evidenced by reduced T-cell proliferation and a switch from a Th1 to a Th2 phenotype. We hypothesized that the expansion of MDSCs in AML is accomplished by tumor-derived extracellular vesicles (EVs). Using tracking studies, we demonstrated that AML EVs are taken-up myeloid progenitor cells, resulting in the selective proliferation of MDSCs in comparison with functionally competent antigen-presenting cells. The MUC1 oncoprotein was subsequently identified as the critical driver of EV-mediated MDSC expansion. MUC1 induces increased expression of c-myc in EVs that induces proliferation in the target MDSC population via downstream effects on cell cycle proteins. Moreover, we demonstrate that the microRNA miR34a acts as the regulatory mechanism by which MUC1 drives c-myc expression in AML cells and EVs.

Balance of Anti-CD123 Chimeric Antigen Receptor (CAR) Binding Affinity and Density for the Treatment of Acute Myeloid Leukemia

Chimeric Antigen Receptors (CARs)-redirected T lymphocytes are a promising novel immunotherapeutic approach, nowadays object of accurate preclinical evaluation also for the treatment of Acute Myeloid Leukemia (AML). In this context, we recently developed a CAR against CD123 (IL-3 receptor α subunit), a tumor associated antigen over-expressed on AML blasts and leukemic stem cells. However, the potential recognition by anti-CD123 CAR-T cells of low CD123 positive healthy tissues, i.e. endothelium and monocytes, through the known "on-target-off-organ" effect, warrants careful preclinical investigation for a safe employment of this approach in the clinic. Therefore, in search for an optimization of this strategy, we evaluated the effect of several variables implicated in the CAR design, known to modulate CAR T-cell functional profiles in a context-dependent manner, such as CAR binding affinity, CAR expression and target antigen density.Computational structural biology tools have been exploited to design rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity. To this aim, Cytokine-Induced Killer (CIK) effector cells have been genetically modified with four Chimeric Affinity Mutants (CAMs), represented by CAM-H1 (High Affinity 1), CAM-H2, which maintain the same binding affinity of the wild type (wt) anti-CD123 CAR (10-9 M), CAM-M (Medium affinity) and CAM-L (Low affinity), displaying a 10- and 100-fold affinity reduction, respectively.The functional characterization of CAM-CIK cells revealed both their specificity and effectiveness in early cytotoxic responses against both the high CD123+ THP-1 AML cell line and primary AML cells, reaching more than 70% of killing activity as the wt CAR-CIK cells. However, in this context, among all the variables analyzed, a CAR expression level up to ≈40% proved to be necessary for inducing effective later effector functions towards a high CD123+target, such as proliferation and cytokine production. To mimic the CD123 expression on leukemic and healthy tissues, a panel of cell lines with different surface levels of CD123 was introduced. In particular, a number of ≈1600 CD123 molecules was sufficient to induce a good cytotoxic response by all the CARs tested, with the CAM-L being the less powerful against both the AML U937 cell line and healthy TIME endothelial cell line, sharing the same target antigen density, and allowing for the identification of ≈1600 CD123 molecules as the "lytic threshold" in our setting. At the same time, this antigen density was not enough to determine a good proliferative capability for all the CAR-CIK cells tested, which instead occurred with leukemic cells expressing ≈5000 or more CD123 molecules. Cytokine release induced by all CAM-CIK cells was instead directly proportional to target antigen density, starting from ≈1600 molecules/cell, indicating that both these "activation thresholds" depend on different target antigen densities for being respectively triggered. As a matter of fact, a proportional CAR down-modulation in response to increasing target antigen densities was observed, implying this mechanism as one of the principal determinant for CAR-CIK cell related functional outcomes, independently of CAR binding affinity. Furthermore, with the aim of evaluating the response of CAM-CIK cells against the endothelium in a more physiological context, we set up a co-culture of Matrigel-embedded endothelial cells and CIK cells. The same and limited impairment of vessel formation was observed, with no differences encountered between un-manipulated and CAR-redirected CIK cells, implying that anti-CD123 CAR CIK cells are not activated more than the un-manipulated counterpart towards this target, independently of CAR binding affinity.In conclusion, we were able to define both "lytic" and "activation" antigen thresholds (Figure 1), showing that whereas the early T-cell cytotoxic activity is not affected either by CAR expression or CAR affinity tuning, later effector functions are impaired by low CAR expression. Moreover, a promising balance in the efficacy and safety profiles of CAR-CIK cells was observed in the lowest affinity mutant in response to targets with low CD123 densities. Overall, the full dissection of all these variables offers additional knowledge for the proper design of a suitable anti-CD123 CAR for the treatment of AML. [Display omitted] DisclosuresBiondi:Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Single-Cell Gene Expression Analysis Identifies Alcam As a Novel Candidate Therapeutic Target in AML

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, characterized by high rate of relapse even in recent days. AML is organized in a hierarchy of heterogeneous cell populations and relapse is generally attributable to chemoresistant leukemia stem cells (LSCs). Anthracyclins and cytarabine, which cannot eliminate LSCs, have remained the mainstay of AML therapy for almost 40 years and novel drugs targeting LSCs more efficiently are urgently needed. Toward that end, a potential strategy is to identify cell surface markers that can be differentially regulated in LSCs within the bulk of leukemic cells. Here in this study, focusing on heterogeneity of AML cells, we exploited single-cell gene expression analysis to find a novel cell surface antigen differentially expressed in LSCs, which might be missed by analyses as a whole.Firstly, we proposed "single-cell leukemia initiating cell (LIC) score", defined as sum of mRNA expression levels of 15 genes specifically expressed in functionally-determined LICs according to public transcriptome datasets (GSE30375), and graded primary AML samples on a single-cell basis. Single CD34+ CD38- cells (n = 66) and CD34+ CD38+ cells (n = 30) from bone marrow (BM) of two AML patients were analyzed. Single-cell gene expression analysis using Fluidigm technology revealed that within 50 candidate cell surface molecules highly expressed in LICs according to public datasets, expression of 19 genes were positively related to the LIC score. Among them, we focused on ALCAM (Activated Leucocyte Cell Adhesion, CD166), a cell adhesion molecule implicated in tumorigenesis, because survival analyses using public datasets showed that high expression of ALCAM was associated with shorter overall survival in AML.When we examined cell-surface expression of ALCAM in undifferentiated AML cells, ALCAM was highly expressed on CD34+ AML cells compared with CD34+ normal hematopoietic cells. We separated BM cells from AML patients into CD34+ ALCAM high fraction and CD34+ ALCAM low fraction. Colony-forming activities were enriched in CD34+ ALCAM high fraction in vitro (n = 5). And then, we did xenotransplantation model by intravenous injection of human AML BM cells to NSG mice. We show that LICs tended to be enriched within CD34+ ALCAM high fraction in some primary AML samples by using xenotransplantation model.To elucidate whether ALCAM exerts any biological functions and could be a novel therapeutic target in AML, we established human THP-1 AML cells in which expression of ALCAM was silenced by short hairpin RNA (shRNA). Knockdown of ALCAM reduced proliferation and enhanced chemosensitivity of THP-1 cells to cytarabine by curbing genotoxic stress-induced apoptosis in vitro. THP-1 cells expressing shRNA against ALCAM also showed decreased leukomogenic capacity in a xenotransplantation model. When we suppressed activities of ALCAM using a soluble isoform of ALCAM (soluble ALCAM), which binds to ALCAM and inhibits intercellular ALCAM-ALCAM hemophilic interactions, THP-1 cells expressing soluble ALCAM also showed decreased proliferation, enhanced chemosensitivity and decreased leukomogeneic capacity. Furthermore, we transplanted human primary AML cells transduced with a tetracycline-inducible lentiviral construct encoding soluble ALCAM into NSG mice. After engraftment of human cells, expression of soluble ALCAM was induced. BM engraftment of human primary AML cells expressing soluble ALCAM was reduced, which indicated that decreased BM engraftment was not due to impaired homing, but to leukemogenic ability.These findings implicate that LICs with high clonogenic capacity were enriched within CD34+ ALCAM high fraction and ALCAM could be a novel therapeutic target of AML cells. Furthermore, our results also suggests single-cell gene expression analysis and a scoring method such as "LIC score" could be applicable to other hematological malignancies and other cancers to identify and exploit a property of heterogenous cancer cells. DisclosuresNo relevant conflicts of interest to declare.

Nucleophosmin Mutants Promote Adhesion, Migration and Invasion of Human Leukemia THP-1 Cells through MMPs Up-regulation via Ras/ERK MAPK Signaling.

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a unique subgroup in the new classification of myeloid neoplasm, and the AML patients with mutated NPM1 frequently present extramedullary infiltration, but how NPM1 mutants regulate this process remains elusive. In this study, we found that overexpression of type A NPM1 gene mutation (NPM1-mA) enhanced the adhesive, migratory and invasive potential in THP-1 AML cells lacking mutated NPM1. NPM1-mA had up-regulated expression and gelatinolytic matrix metalloprotease-2 (MMP-2)/MMP-9 activity, as assessed by real-time PCR, western blotting and gelatin zymography. Following immunoprecipitation analysis to identify the interaction of NPM1-mA with K-Ras, we focused on the effect of NPM1-mA overexpression on the Ras/Mitogen-activated protein kinase (MAPK) signaling axis and showed that NPM1-mA increased the MEK and ERK phosphorylation levels, as evaluated by western blotting. Notably, a specific inhibitor of the ERK/MAPK pathway (PD98059), but not p38/MAPK, JNK/MAPK or PI3-K/AKT inhibitors, markedly decreased the cell invasion numbers in a transwell assay. Further experiments demonstrated that blocking the ERK/MAPK pathway by PD98059 resulted in reduced MMP-2/9 protein levels and MMP-9 activity. Additionally, NPM1-mA overexpression had down-regulated gene expression and protein production of tissue inhibitor of MMP-2 (TIMP-2) in THP-1 cells. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that MMP-2 was overexpressed in AML patient samples with NPM1 mutated and high MMP-2 expression associated with leukemic skin infiltration. Taken together, our results reveal that NPM1 mutations contribute to the invasive potential of AML cells through MMPs up-regulation via Ras/ERK MAPK signaling pathway activation and offer novel insights into the potential role of NPM1 mutations in leukemogenesis.

Clinical Significance of WDR5 High Expression and Its Effect on Tumorigenesis in Adult Leukemia

Background: Mixed-lineage leukemia (MLL) functions within the context of a large multiprotein complex including MLL, WDR5, RbBP5, and ASH2L that is required for maximal enzymatic activity. Each component is required for full histone H3 trimethyl Lysine 4 (H3K4me3) methyltransferase activity of the complex. The structure-function analysis shows that WDR5 mediates interaction between the MLL catalytic unit and the core complex, as well as the histone H3 substrate. Recently it is found that blocking MLL1-WDR5 interaction by an inhibitor could result in the complex disassembly, inhibition methyltransferase activity and suppression of proliferation of leukemia cells. Despite our growing knowledge about MLL1 fusion proteins and leukemia, very little is known about the role of WDR5 in leukemia.Methods: The WDR5 expression was determined by qPCR in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients. The genome-wide binding profiling of WDR5 and H3K4me3 was obtained by ChIP-seq. The effect of WDR5 on its target gene expression, cell proliferation and apoptosis was observed by qPCR, WST-1 cell proliferation assay and Annexin V-PE staining following the flow cytometry analysis, respectively, in leukemic cells with WDR5 shRNA knockdownResults: WDR5 expression is significantly increased in adult ALL and AML compared to that of normal bone marrow control. WDR5 high expression is associated with high risk factors in the patients. Also, its high expression is associated with MLL1 high expression; particularly the patients with WDR5 high expression plus MLL1 high expression has poor complete remission (CR) rate. We further identified the global genomic binding of WDR5 in RS4:11 ALL and THP-1 AML cells by ChIP-seq and detected more than 2000 binding peaks in the two leukemia cell lines. We also examined global H3K4me3 peaks, analyzed the correlation of WDR5 peaks with H3K4me3 peaks and found the genomic co-localization of WDR5 binding with H3K4me3 enrichment. Moreover, WDR5 knockdown by shRNA suppressed the cell proliferation, induced apoptosis, inhibited the expression of WDR5 targets on oncogenesis and anti-apoptosis, blocked the H3K4me3 enrichment on the promoter of these targets. We also observed the positive correlation of WDR5 expression with its targets in B-ALL and AML cohort.Conclusion: WDR5 has oncogenic effect and WDR5-mediated H3K4 methylation plays important role in the leukemogenesis DisclosuresNo relevant conflicts of interest to declare.

Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

SUMOylation of sPRDM16 promotes the progression of acute myeloid leukemia.

BackgroundIn addition to genetic and epigenetic alteration, post-translational modification of proteins plays a critical role in the initiation, progression and maturation of acute myeloid leukemia (AML).MethodsThe SUMOylation site of sPRDM16 at K568 was mutated to arginine by site-directed mutagenesis. THP-1 acute myeloid leukemia cells were transduced with a lentivirus containing wild type or K568 mutant sPRDM16. Proliferation, self-renewal and differentiation of transduced THP-1 cells were analyzed both in vitro cell culture and in mouse xenografts. Gene expression profiles were analyzed by RNA-seq.ResultsOverexpression of sPRDM16 promoted proliferation, enhanced self-renewal capacity, but inhibited differentiation of THP-1 acute myeloid leukemia cells. We further confirmed that K568 is a bona fide SUMOylation site on sPRDM16. Mutation of the sPRDM16 SUMOylation site at K568 partially abolished the capacity of sPRDM16 to promote proliferation and inhibit differentiation of acute myeloid leukemia cells both in vitro and in mouse xenografts. Furthermore, THP-1 cells overexpressing sPRDM16-K568R mutant exhibited a distinct gene expression profile from wild type sPRDM16 following incubation with PMA.ConclusionsOur results suggest that K568 SUMOylation of sPRDM16 plays an important role in the progression of acute myeloid leukemia.